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A critical issue in understanding Huntington's disease (HD) pathogenesis is how the ubiquitously expressed mutant huntingtin (mhtt) with an expanded polyglutamine repeat can cause selective toxicity of striatal and cortical neurons. Two potential cellular models may contribute to such specificity: expression of mhtt in these vulnerable neurons alone may be sufficient to result in their dysfunction and/or degeneration (cell-autonomous model); or mhtt in other cell types can elicit pathological cell-cell interactions to cause the vulnerable neurons to become dysfunctional and be at risk for degeneration (cell-cell interaction model). To distinguish between these two models, we have selectively expressed a neuropathogenic fragment of mhtt-exon1 in striatal medium spiny neurons (MSNs) by crossing a conditional mouse model of HD with a striatal-specific Cre mouse line. In this striatal model of HD, we observed progressive and cell-autonomous nuclear accumulation of mhtt aggregates in MSNs. Surprisingly, unlike the mouse model expressing mhtt-exon1 in all the neurons in the brain, the striatal model lacks significant locomotor deficits and striatal neuropathology including gliosis and dark degenerating neurons. Electrophysiological findings from acutely dissociated MSNs revealed a cell-autonomous deficit in N-methyl-d-aspartate (NMDA) receptor sensitivity to Mg2+, a deficit also present in other mouse models of HD. In conclusion, this study provides the first in vivo genetic evidence that pathological cell-cell interactions are necessary for striatal pathogenesis in a conditional mouse model of HD, and suggests a ''two-hit'' hypothesis in which both cell-autonomous toxicity and pathological cell-cell interactions are critical to HD pathogenesis.

Drosophila melanogaster telomeres have two DNA domains: a terminal array of retrotransposons and a subterminal repetitive telomere-associated sequence (TAS), a source of telomere position effect (TPE). We reported previously that deletion of the 2L TAS array leads to dominant suppression of TPE by stimulating in trans expression of a telomeric transgene. Here, we compared the transcript activities of a w transgene inserted between the retrotransposon and TAS arrays at the 2L telomere in genotypes with different lengths of the 2L TAS. In contrast to individuals bearing a wild-type 2L homologue, flies with a TAS deficiency showed a significant increase in the level of telomeric w transcript during development, especially in pupae. Moreover, we identified a read-through w transcript initiated from a retrotransposon promoter in the terminal array. Read-through transcript levels also significantly increased with the presence of a 2L TAS deficiency in trans, indicating a stimulating force of the TAS deficiency on retrotransposon promoter activity. The read-through transcript contributes to total w transcript, although most w transcript originates at the w promoter. While silencing of transgenes in nonhomologous telomeres is suppressed by 2L TAS deficiencies, suggesting a global effect, the overall level of HeT-A transcripts is not increased under similar conditions.

Recent evidence suggests that low oxygen tension (hypoxia) may control fetal development and differentiation. A crucial mediator of the adaptive response of cells to hypoxia is the transcription factor Hif-1alpha. In this study, we provide evidence that mesenchymal condensations that give origin to endochondral bones are hypoxic during fetal development, and we demonstrate that Hif-1alpha is expressed and transcriptionally active in limb bud mesenchyme and in mesenchymal condensations. To investigate the role of Hif-1alpha in mesenchymal condensations and in early chondrogenesis, we conditionally inactivated Hif-1alpha in limb bud mesenchyme using a Prx1 promoter-driven Cre transgenic mouse. Conditional knockout of Hif-1alpha in limb bud mesenchyme does not impair mesenchyme condensation, but alters the formation of the cartilaginous primordia. Late hypertrophic differentiation is also affected as a result of the delay in early chondrogenesis. In addition, mutant mice show a striking impairment of joint development. Our study demonstrates a crucial, and previously unrecognized, role of Hif-1alpha in early chondrogenesis and joint formation.

Although intensively researched, the fundamental mechanism of protein misfolding that leads to protein aggregation and associated diseases remains somewhat enigmatic. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. Over 20 different human diseases have now been designated as 'conformational diseases' and include neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD) and Creutzfeldt Jakob disease (CJD) that are becoming increasingly prevalent in an ageing human population. Such diseases are usually characterised by the deposition of specific misfolded proteins as amyloid fibrils and hence are often referred to as the amyloidoses.

Polyadenylation plays an important role in regulating RNA stability in Trypanosoma brucei mitochondria. To date, little is known about the enzymes responsible for the addition of mRNA 3' tails in this system. In this study, we characterize a trypanosome homolog of the human mitochondrial poly(A) polymerase, which we term kPAP2. kPAP2 is mitochondrially localized and expressed in both bloodstream and procyclic form trypanosomes. Targeted gene depletion using RNAi showed that kPAP2 is not required for T. brucei growth in either bloodstream or procyclic life stages, nor is it essential for differentiation from bloodstream to procyclic form. We also demonstrate that steady state abundance of several mitochondrial RNAs was largely unaffected upon kPAP2 down-regulation. Interestingly, mRNA 3' tail analysis of several mRNAs from both life cycle stages in uninduced kPAP2 RNAi cells demonstrated that tail length and uridine content are both regulated in a transcript-specific manner. mRNA-specific tail lengths were maintained upon kPAP2 depletion. However, the percentage of uridine residues in 3' tails was increased, and conversely the percentage of adenosine residues was decreased, in a distinct subset of mRNAs when kPAP2 levels were down-regulated. Thus, kPAP2 apparently contributes to the incorporation of adenosine residues in 3' tails of some, but not all, mitochondrial mRNAs. Together, these data suggest that multiple nucleotidyltransferases act on mitochondrial mRNA 3' ends, and that these enzymes are somewhat redundant and subject to complex regulation.

Milk quality is measured by a variety of parameters and one of these is the number of somatic cells in the milk. Within the bovine dairy industry there are maximum acceptable levels on bulk milk cell counts and these are being introduced into caprine dairy production. The reference method for cell counting in goats is the direct microscope (DM) method using pyronin Y-methyl green stain, which stains cell DNA. The use of the DeLaval cell counter (DCC) on goats' milk was compared with this reference method. Samples from 102 udder halves were cell counted using the direct microscope method and the DeLaval cell counter (DCC). Each sample was counted twice by two different observers by the DM method and once using DCC. DCC showed large coefficients of regression (1.03) and correlation (0.95) when compared with the DM cell count (F1,199=1080.0; P<0.001). Goat, udder half and reader type were significant factors.

We conducted a genome-wide association study for type 2 diabetes (T2D) in Icelandic cases and controls, and we found that a previously described variant in the transcription factor 7-like 2 gene (TCF7L2) gene conferred the most significant risk. In addition to confirming two recently identified risk variants, we identified a variant in the CDKAL1 gene that was associated with T2D in individuals of European ancestry (allele-specific odds ratio (OR) = 1.20 (95% confidence interval, 1.13-1.27), P = 7.7 x 10(-9)) and individuals from Hong Kong of Han Chinese ancestry (OR = 1.25 (1.11-1.40), P = 0.00018). The genotype OR of this variant suggested that the effect was substantially stronger in homozygous carriers than in heterozygous carriers. The ORs for homozygotes were 1.50 (1.31-1.72) and 1.55 (1.23-1.95) in the European and Hong Kong groups, respectively. The insulin response for homozygotes was approximately 20% lower than for heterozygotes or noncarriers, suggesting that this variant confers risk of T2D through reduced insulin secretion.

Identifying the genetic variants that increase the risk of type 2 diabetes (T2D) in humans has been a formidable challenge. Adopting a genome-wide association strategy, we genotyped 1161 Finnish T2D cases and 1174 Finnish normal glucose-tolerant (NGT) controls with >315,000 single-nucleotide polymorphisms (SNPs) and imputed genotypes for an additional >2 million autosomal SNPs. We carried out association analysis with these SNPs to identify genetic variants that predispose to T2D, compared our T2D association results with the results of two similar studies, and genotyped 80 SNPs in an additional 1215 Finnish T2D cases and 1258 Finnish NGT controls. We identify T2D-associated variants in an intergenic region of chromosome 11p12, contribute to the identification of T2D-associated variants near the genes IGF2BP2 and CDKAL1 and the region of CDKN2A and CDKN2B, and confirm that variants near TCF7L2, SLC30A8, HHEX, FTO, PPARG, and KCNJ11 are associated with T2D risk. This brings the number of T2D loci now confidently identified to at least 10.

The molecular mechanisms involved in the development of type 2 diabetes are poorly understood. Starting from genome-wide genotype data for 1924 diabetic cases and 2938 population controls generated by the Wellcome Trust Case Control Consortium, we set out to detect replicated diabetes association signals through analysis of 3757 additional cases and 5346 controls and by integration of our findings with equivalent data from other international consortia. We detected diabetes susceptibility loci in and around the genes CDKAL1, CDKN2A/CDKN2B, and IGF2BP2 and confirmed the recently described associations at HHEX/IDE and SLC30A8. Our findings provide insight into the genetic architecture of type 2 diabetes, emphasizing the contribution of multiple variants of modest effect. The regions identified underscore the importance of pathways influencing pancreatic beta cell development and function in the etiology of type 2 diabetes.

It is well established that protein aggregation is associated with many neurodegenerative disorders including polyglutamine diseases, but a mechanistic understanding of the role of protein aggregates in the disease pathogenesis remains elusive. Previously thought to be the cause of cellular toxicity such as cellular dysfunction and cell death, protein aggregation is now proposed to serve a protective role by sequestering toxic oligomers from interfering with essential physiological processes. To investigate the relationship between protein aggregation and cellular toxicity, we have characterized and compared the effects of two GFP-fusion proteins that form aggregates in Saccharomyces cerevisiae, one with a polyasparagine repeat (GFP(N104)) and one without (GFP(C)). Although both proteins can form microscopically visible GFP-positive aggregates, only the GFP(N104)-containing aggregates exhibit morphological and biochemical characteristics that resemble the aggregates formed by mutant huntingtin in yeast cells. Formation of both the GFP(C) and GFP(N104) aggregates depends on microtubules, while only the GFP(N104) aggregate requires the chaperone Hsp104 and the prion Rnq1 and is resistant to SDS. Although no microscopically visible GFP(N104) aggregates were observed in the hsp104Delta and rnq1Delta mutant cells, SDS-insoluble aggregates can still be detected by the filter trap assay. These observations argue that the GFP(N104)-containing aggregates can exist in at least two distinct states in vivo. We also show that a nucleus-targeted GFP(N104) interferes with transcription from two SAGA-dependant promoters and results in a decrease in cell viability. Overall, the results imply that the GFP(N104) protein behaves similarly to the mutant huntingtin in yeast cells and provides a new model for investigating the interplay between protein aggregates and the associated phenotypes.

<h4>Objective</h4>To investigate the prevalence of antibodies to cyclic citrullinated peptide (anti-CCP) and rheumatoid factor in patients with hereditary haemochromatosis (HHC) and to evaluate their diagnostic reliability in distinguishing HHC-associated arthropathy from rheumatoid arthritis.<h4>Methods</h4>Anti-CCP antibodies and rheumatoid factor levels were determined by ELISA in sera from 87 patients with HHC homozygous for the C282Y mutation of the HFE gene, 31 patients with rheumatoid arthritis and 162 healthy controls.<h4>Results</h4>Of the 87 patients with HHC, 32 (36.8%) had joint involvement. Anti-CCP antibodies were detected in only 1 patient (1.1%) with HHC, who had no joint disease, and in (1.2%) healthy controls. In total, 18 (58.1%) patients with rheumatoid arthritis displayed anti-CCP reactivity (p<0.001). Rheumatoid factor was detected in 10 (11.5%) patients with HHC compared with 7 (4.3%) healthy control subjects (p = 0.03) and 21 of 31 (65.6%) patients with rheumatoid arthritis.<h4>Conclusions</h4>Testing for anti-CCP antibodies discriminates HHC arthropathy from rheumatoid arthritis, as these patients were consistently anti-CCP negative. Thus, HHC arthropathy should be considered in the differential diagnosis of CCP-negative arthritis.

Choline is an essential nutrient for humans, though some of the requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Premenopausal women are relatively resistant to choline deficiency compared with postmenopausal women and men. Studies in animals suggest that estrogen treatment can increase PEMT activity. In this study we investigated whether the PEMT gene is regulated by estrogen. PEMT transcription was increased in a dose-dependent manner when primary mouse and human hepatocytes were treated with 17-beta-estradiol for 24 h. This increased message was associated with an increase in protein expression and enzyme activity. In addition, we report a region that contains a perfect estrogen response element (ERE) approximately 7.5 kb from the transcription start site corresponding to transcript variants NM_007169 and NM-008819 of the human and murine PEMT genes, respectively, three imperfect EREs in evolutionarily conserved regions and multiple imperfect EREs in nonconserved regions in the putative promoter regions. We predict that both the mouse and human PEMT genes have three unique transcription start sites, which are indicative of either multiple promoters and/or alternative splicing. This study is the first to explore the underlying mechanism of why dietary requirements for choline vary with estrogen status in humans.

Aging is associated with a decline of immune competence and an increase in markers of inflammation. There is considerable evidence that inflammatory processes play a role in aging and the determination of lifespan. Hypopituitary Ames dwarf mice have extended longevity and exhibit many symptoms of delayed aging, although various aspects of immune function are suppressed in the mutants. In the present study, the expression of genes related to immunity and inflammation was compared in peripheral blood leukocytes (PBL) from Ames dwarf and normal mice using Affymetrix GeneChip arrays. Among the more than 3000 probe sets that were differentially expressed, 273 were identified as being associated with immunity and/or inflammation. Pathway analysis revealed interactions among 91 of these probe sets, centered on casp3, bcl2, il4, prkca, mapk14 and TGFbeta1. Ames dwarf mice had reduced leukocyte expression of casp3 and TGFbeta and increased expression of Bcl2. Alterations in the expression of these genes suggest likely functional changes in apoptosis, B and T cell homeostasis, prostaglandin synthesis, humoral immunity, chemokine activity, complement activation, hemostasis and wound healing pathways. Collectively, these results suggest that activation of both anti-inflammatory pathways and an anti-clotting mechanism combined with reduced turnover of leukocytes may contribute to delayed aging and extended longevity of Ames dwarf mice. We are also aware that alterations in gene expression in PBLs can be due to different composition of PBL populations when comparing Ames dwarf to WT animals, and it will be interesting to investigate these genes in particular PBL populations in the future. However, whole leukocytes population represents the function of immune system in these organisms.

Modifying serotonin (5-HT) abundance in the embryonic mouse brain disrupts the precision of sensory maps formed by thalamocortical axons (TCAs), suggesting that 5-HT influences their growth. We investigated the mechanism by which 5-HT influences TCAs during development. 5-HT(1B) and 5-HT(1D) receptor expression in the fetal forebrain overlaps with that of the axon guidance receptors DCC and Unc5c. In coculture assays, axons originating from anterior and posterior halves of the embryonic day 14.5 dorsal thalamus responded differently to netrin-1, reflecting the patterns of DCC and Unc5c expression. 5-HT converts the attraction exerted by netrin-1 on posterior TCAs to repulsion. Pharmacological manipulation of 5-HT(1B/1D) receptors and intracellular cAMP showed the signaling cascade through which this modulation occurs. An in vivo correlate of altered TCA pathfinding was obtained by transient manipulation of 5-HT(1B/1D) receptor expression abundance in the dorsal thalamus by in utero electroporation. These data demonstrate that serotonergic signaling has a previously unrecognized role in the modulation of axonal responsiveness to a classic guidance cue.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder linked to a mutation in the huntingtin gene leading to protein aggregation in neurons. The generation of new neurons in neurogenic regions, such as the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus, is affected by these aggregation processes. In particular, hippocampal neurogenesis is reduced in the R6/2 transgenic mouse model of HD. Since physical activity stimulates adult hippocampal neurogenesis, we examined whether running is capable to rescue the impaired hippocampal neurogenesis in R6/2 mice. Proliferation of hippocampal cells measured by proliferating cell nuclear antigen (PCNA) marker was reduced in R6/2 animals by 64% compared to wild type mice. Accordingly, newly generated neurons labeled with doublecortin (DCX) were diminished by 60% in the hippocampus of R6/2 mice. Furthermore, the number of newly generated mature neurons was decreased by 76%. Within the hippocampus of wild type animals, a four-week running period resulted in a doubling of PCNA-, DCX-, and bromo-deoxyuridine (BrdU)-labeled cells. However, physical exercise failed to stimulate proliferation and survival of newly generated neurons in R6/2 transgenic mouse model of HD. These findings suggest that mutant huntingtin alters the hippocampal microenvironment thus resulting in an impaired neurogenesis. Importantly, this adverse microenvironment impeded neurogenesis upregulation such as induced by physical exercise. Future studies need to decipher the molecular pathways involved in repressing the generation of new neurons after physical activity in huntingtin transgenic rodents.

Identifying genomic locations that have experienced selective sweeps is an important first step toward understanding the molecular basis of adaptive evolution. Using statistical methods that account for the confounding effects of population demography, recombination rate variation, and single-nucleotide polymorphism ascertainment, while also providing fine-scale estimates of the position of the selected site, we analyzed a genomic dataset of 1.2 million human single-nucleotide polymorphisms genotyped in African-American, European-American, and Chinese samples. We identify 101 regions of the human genome with very strong evidence (p < 10(-5)) of a recent selective sweep and where our estimate of the position of the selective sweep falls within 100 kb of a known gene. Within these regions, genes of biological interest include genes in pigmentation pathways, components of the dystrophin protein complex, clusters of olfactory receptors, genes involved in nervous system development and function, immune system genes, and heat shock genes. We also observe consistent evidence of selective sweeps in centromeric regions. In general, we find that recent adaptation is strikingly pervasive in the human genome, with as much as 10% of the genome affected by linkage to a selective sweep.

No abstract available.

Beta-2 microglobulin (B2M) plays a pivotal role in the biology of mammals, including its association with major histocompatibility complex (MHC) Class I gene products. The latter molecules have been shown to affect reproduction in both mice and humans, although the exact mechanism is still unknown. Here we report the results of a longitudinal study of the reproductive performance of a genetically modified B2m deficient mouse strain with low MHC Class I expression. Our data show that this mouse strain has an impaired reproductive performance. However, the mice superovulate well and show a normal estrous cycle. Breeding studies from crosses between the transgenic mice and the wild-type parental strain show that B2m deficient mice have a significantly lower frequency of mating than the control B2m+/+ mice. In addition, the litter size and weaning success of B2m deficient mice were lower than the control. Perinatal lethality of the B2m deficient offspring was also inflicted by cannibalism of the young pups by the B2m deficient female. The impaired breeding phenotype (IBP) can be reversed by reintroducing the B2m gene in F1 heterozygous B2m+/- animals; thus the presence of B2M confers a normal breeding pattern. The acquisition of an impaired breeding phenotype (IBP) as a result of the knockout of B2m directly implicates B2M in the reproductive cycle of mice and raises the possibility of an effect of B2M on the reproduction of other mammals.

<h4>Background</h4>Enolase (2-Phospho-D-glycerate hydrolase; EC 4.2.1.11) is one of the glycolytic enzymes, whose levels are highly elevated in malaria parasite infected red blood cells. In several organisms, enolases have been shown to have diverse non glycolytic (moonlighting) biological functions. As functional diversity of a protein would require diverse sub-cellular localization, the possibility of involvement of Plasmodium enolase in moonlighting functions was examined by investigating its sub-cellular distribution in the murine malarial parasite, Plasmodium yoelii.<h4>Methods</h4>Cellular extracts of P. yoelii were fractionated in to soluble (cytosolic) and particulate (membranes, nuclear and cytoskeletal) fractions and were analysed by one and two-dimensional gel electrophoresis. These were probed by Western blotting using antibodies raised against recombinant Plasmodium falciparum enolase. Immunofluorescence assay was used for in situ localization. Fe+3 based metal affinity chromatography was used to isolate the phospho-proteome fraction from P. yoelii extracts.<h4>Results</h4>Apart from the expected presence of enolase in cytosol, this enzyme was also found to be associated with membranes, nuclei and cytoskeletal fractions. Nuclear presence was also confirmed by in situ immunofluorescence. Five different post translationally modified isoforms of enolase could be identified, of which at least three were due to the phosphorylation of the native form. in situ phosphorylation of enolase was also evident from the presence of enolase in purified phosphor-proteome of P. yoelii. Different sub-cellular fractions showed different isoform profiles.<h4>Conclusion</h4>Association of enolase with nuclei, cell membranes and cytoskeletal elements suggests non-glycolytic functions for this enzyme in P. yoelii. Sub-cellular fraction specific isoform profiles indicate the importance of post-translational modifications in diverse localization of enolase in P. yoelii. Further, it is suggested that post-translational modifications of enolase may govern the recruitment of enolase for non-glycolytic functions.

In the developing mouse brain, the highest Bcl-X(L) expression is seen at the peak of neurogenesis, whereas the peak of Bax expression coincides with the astrogenic period. While such observations suggest an active role of the Bcl-2 family proteins in the generation of neurons and astrocytes, no definitive demonstration has been provided to date. Using combinations of gain- and loss-of-function assays in vivo and in vitro, we provide evidence for instructive roles of these proteins in neuronal and astrocytic fate specification. Specifically, in Bax knockout mice, astrocyte formation was decreased in the developing cortices. Overexpression of Bcl-X(L) and Bax in embryonic cortical precursors induced neural and astrocytic differentiation, respectively, while inhibitory RNAs led to the opposite results. Importantly, inhibition of caspase activity, dimerization, or mitochondrial localization of Bcl-X(L)/Bax proteins indicated that the differentiation effects of Bcl-X(L)/Bax are separable from their roles in cell survival and apoptosis. Lastly, we describe activation of intracellular signaling pathways and expression of basic helix-loop-helix transcriptional factors specific for the Bcl-2 protein-mediated differentiation.

The common feature of many neurodegenerative diseases is emergence of protein aggregates. Identifying their composition can provide valuable insights into the cellular mechanisms of protein aggregation and neuronal death. No reliable method for identification of the aggregate-associated proteins has been available. Here we describe a method for characterization of protein aggregates based on sedimentation of immunocomplexes without involvement of a solid support. As a model, we used the aggregates formed in yeast by a polyglutamine-containing segment of mutant huntingtin. Sixteen proteins associated with the isolated aggregates were identified with 2-D gel electrophoresis followed by mass spectrometry. We found that the aggregates in cells lacking Rnq1 prion recruited lesser amounts of chaperones than those in the wild-type cells. The method can be utilized for characterization of various types of aggregates, prions and very large protein complexes under mild conditions that preserve associated proteins.

The Drosophila BEAF-32A and BEAF-32B proteins bind to the scs' insulator and to hundreds of other sites on Drosophila chromosomes. These two proteins are encoded by the same gene. We used ends-in homologous recombination to generate the null BEAF(AB-KO) allele and also isolated the BEAF(A-KO) allele that eliminates production of only the BEAF-32A protein. We find that the BEAF proteins together are essential, but BEAF-32B alone is sufficient to obtain viable flies. Our results show that BEAF is important for both oogenesis and development. Maternal or zygotic BEAF is sufficient to obtain adults, although having only maternal BEAF impairs female fertility. In the absence of all BEAF, a few fertile but sickly males are obtained. Using both a chromosomal position-effect assay and an enhancer-blocking assay, we find that BEAF is necessary for scs' insulator function. Lack of BEAF causes a disruption of male X polytene chromosome morphology. However, we did not find evidence that dosage compensation was affected. Position-effect variegation of the w(m4h) allele and different variegating y transgenes was enhanced by the knockout mutation. Combined with the effects on male X polytene chromosomes, we conclude that BEAF function affects chromatin structure or dynamics.

The linker histones H1 are a family of lysine-rich proteins that associate with the stretch of DNA that enters and exits the nucleosome. The linker histones facilitate the compaction and condensation of chromatin. The globular domain of histone H1(0), a specific subtype of histone H1, was crystallized at 288 K using the microbatch under silicone oil method with potassium phosphate as a precipitating agent. Diffraction data were collected to a resolution of 1.98 A. The crystal belongs to the trigonal space group P3(1)21, with unit-cell parameters a = 54.13, b = 54.13, c = 71.99 A, and contains one molecule per asymmetric unit. The V(M) value and solvent content were calculated to be 3.04 A3 Da(-1) and 59.6%, respectively.

Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, with a 5-year survival rate of less than 4%. Effective early detection and screening are currently not available, and tumors are typically diagnosed at a late stage, frequently after metastasis. Existing clinical markers of pancreatic cancer lack specificity, as they are also found in inflammatory diseases of the pancreas and biliary tract. In the work described here, naturally occurring glycoproteins were enriched by using lectin affinity chromatography and then further resolved by nonporous reversed-phase chromatography. Glycoprotein microarrays were then printed and probed with a variety of lectins to screen glycosylation patterns in sera from normal, chronic pancreatitis, and pancreatic cancer patients. Ten normal, 8 chronic pancreatitis, and 6 pancreatic cancer sera were investigated. Data from the glycoprotein microarrays were analyzed using bioinformatics approaches including principal component analysis (PCA) and hierarchical clustering (HC). Both normal and chronic pancreatitis sera were found to cluster close together, although in two distinct groups, whereas pancreatic cancer sera were significantly different from the other two groups. Both sialylation and fucosylation increased as a function of cancer on several proteins including Hemopexin, Kininogen-1, Antithrombin-III, and Haptoglobin-related protein, whereas decreased sialylation was detected on plasma protease C1 inhibitor. Target alterations on glycosylations were verified by lectin blotting experiments and peptide mapping experiments using microLC-ESI-TOF. These altered glycan structures may have utility for the differential diagnosis of pancreatic cancer and chronic pancreatitis and identify critical differences between biological samples from patients with different clinical conditions.

<h4>Background</h4>Prostate cancer is characterized by heterogeneity in the clinical course that often does not correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer, and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets.<h4>Methods</h4>Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors.<h4>Results</h4>The metastatic samples are highly heterogenous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodelling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1).<h4>Conclusion</h4>We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxic effects of halogenated aromatic hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and other structurally diverse ligands. While HAHs are several orders of magnitude more potent in producing AhR-dependent biochemical effects than PAHs or other AhR agonists, only the HAHs have been observed to produce AhR-dependent toxicity in vivo. Here we have characterized the dissociation of a prototypical HAH ligand ([(3)H] 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) and PAH-like ligand ([(3)H] beta-naphthoflavone [betaNF]) from the guinea pig, hamster, mouse, and rat hepatic cytosolic AhR in order to elucidate the relationship between the apparent ligand-binding affinities and the divergent potency of these chemicals. Both compounds dissociated very slowly from the AhR with the amount of specific binding remaining at 96 h ranging from 53% to 70% for [(3)H]TCDD and 26% to 85% for [(3)H] betaNF, depending upon the species examined. The rate of ligand dissociation was unaffected by protein concentration or incubation temperature. Preincubation of cytosol with 2,3,7,8-tetrachlorodibenzofuran, carbaryl, or primaquine, prior to the addition of [(3)H]TCDD, shifted the apparent IC(50) of these compounds as competitive AhR ligands by approximately 10- to 50-fold. Our results support the need for reassessment of previous AhR ligand-binding affinity calculations and competitive binding analysis since these measurements are not carried out at equilibrium binding conditions. Our studies suggest that AhR binding affinity/occupancy has little effect on the observed differences in the persistence of gene expression by HAHs and PAHs.

The zinc-finger transcription factor GLI3 is a key regulator of development, acting as a primary transducer of Sonic hedgehog (SHH) signaling in a combinatorial context dependent fashion controlling multiple patterning steps in different tissues/organs. A tight temporal and spatial control of gene expression is indispensable, however, cis-acting sequence elements regulating GLI3 expression have not yet been reported. We show that 11 ancient genomic DNA signatures, conserved from the pufferfish Takifugu (Fugu) rubripes to man, are distributed throughout the introns of human GLI3. They map within larger conserved non-coding elements (CNEs) that are found in the tetrapod lineage. Full length CNEs transiently transfected into human cell cultures acted as cell type specific enhancers of gene transcription. The regulatory potential of these elements is conserved and was exploited to direct tissue specific expression of a reporter gene in zebrafish embryos. Assays of deletion constructs revealed that the human-Fugu conserved sequences within the GLI3 intronic CNEs were essential but not sufficient for full-scale transcriptional activation. The enhancer activity of the CNEs is determined by a combinatorial effect of a core sequence conserved between human and teleosts (Fugu) and flanking tetrapod-specific sequences, suggesting that successive clustering of sequences with regulatory potential around an ancient, highly conserved nucleus might be a possible mechanism for the evolution of cis-acting regulatory elements.

<h4>Background</h4>Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (ARA, 20:4n-6) are the major long chain polyunsaturated fatty acids (LCPUFA) of the central nervous system (CNS). These nutrients are present in most infant formulas at modest levels, intended to support visual and neural development. There are no investigations in primates of the biological consequences of dietary DHA at levels above those present in formulas but within normal breastmilk levels.<h4>Methods and findings</h4>Twelve baboons were divided into three formula groups: Control, with no DHA-ARA; "L", LCPUFA, with 0.33%DHA-0.67%ARA; "L3", LCPUFA, with 1.00%DHA-0.67%ARA. All the samples are from the precentral gyrus of cerebral cortex brain regions. At 12 weeks of age, changes in gene expression were detected in 1,108 of 54,000 probe sets (2.05%), with most showing <2-fold change. Gene ontology analysis assigns them to diverse biological functions, notably lipid metabolism and transport, G-protein and signal transduction, development, visual perception, cytoskeleton, peptidases, stress response, transcription regulation, and 400 transcripts having no defined function. PLA2G6, a phospholipase recently associated with infantile neuroaxonal dystrophy, was downregulated in both LCPUFA groups. ELOVL5, a PUFA elongase, was the only LCPUFA biosynthetic enzyme that was differentially expressed. Mitochondrial fatty acid carrier, CPT2, was among several genes associated with mitochondrial fatty acid oxidation to be downregulated by high DHA, while the mitochondrial proton carrier, UCP2, was upregulated. TIMM8A, also known as deafness/dystonia peptide 1, was among several differentially expressed neural development genes. LUM and TIMP3, associated with corneal structure and age-related macular degeneration, respectively, were among visual perception genes influenced by LCPUFA. TIA1, a silencer of COX2 gene translation, is upregulated by high DHA. Ingenuity pathway analysis identified a highly significant nervous system network, with epidermal growth factor receptor (EGFR) as the outstanding interaction partner.<h4>Conclusions</h4>These data indicate that LCPUFA concentrations within the normal range of human breastmilk induce global changes in gene expression across a wide array of processes, in addition to changes in visual and neural function normally associated with formula LCPUFA.

Human immunodeficiency virus type 1 (HIV-1) requires the sequential activities of virus-encoded proteins during replication. The activities of several host cell proteins and machineries are also critical to the completion of virus assembly and the release of infectious virus particles from cells. One of these proteins, the double-stranded RNA-binding protein Staufen1 (Stau1), selectively associates with the HIV-1 genomic RNA and the viral precursor Gag protein, pr55Gag. In this report, we tested whether Stau1 modulates pr55Gag assembly using a new and specific pr55Gag oligomerization assay based on bioluminescence resonance energy transfer (BRET) in both live cells and extracts after cell fractionation. Our results show that both the overexpression and knockdown of Stau1 increase the pr55Gag-pr55Gag BRET levels, suggesting a role for Stau1 in regulating pr55Gag oligomerization during assembly. This effect of Stau1 on pr55Gag oligomerization was observed only in membranes, a cellular compartment in which pr55Gag assembly primarily occurs. Consistently, expression of Stau1 harboring a vSrc myristylation signal led to a 6.5-fold enrichment of Stau1 in membranes and a corresponding enhancement in the Stau1-mediated effect on pr55Gag-pr55Gag BRET, demonstrating that Stau1 acts on assembly when targeted to membranes. A role for Stau1 in the formation of particles is further supported by the detection of membrane-associated detergent-resistant pr55Gag complexes and the increase of virus-like particle release when Stau1 expression levels are modulated. Our results indicate that Stau1 influences HIV-1 assembly by modulating pr55Gag-pr55Gag interactions, as shown in a live cell interaction assay. This likely occurs when Stau1 interacts with membrane-associated assembly intermediates.

There are limited data on nonalcoholic fatty liver disease (NAFLD) from India. The clinicopathological profile of Indian patients with NAFLD may be different from that of Western patients. One hundred NAFLD patients with increased liver enzymes were prospectively evaluated for clinical presentation, associated diseases, overweight/obesity, central obesity (n=54), presence of diabetes mellitus, lipid abnormalities, insulin resistance (n=39), metabolic syndrome (n=54), serum iron, serum ferritin, and transferrin saturation (n=60), and HFE gene mutations (n=30). Risk factors for the grade and stage of the disease on histology were studied in 38 biopsy-proven patients. Patients were treated with lifestyle modifications and ursodeoxycholic acid (UDCA). Seventeen nonresponder patients were treated with metformin. The majority of patients were males (n=70). Twenty percent of patients were overweight, 68% had obesity, and 78% had central obesity. Abnormal cholesterol, HDL, and triglycerides were present in 36%, 66%, and 53% of patients, respectively. Twelve percent of patients had diabetes mellitus and 16% patients had various associated diseases. All 22 (100%) patients studied by ITT and all but 1 (98%) studied by HOMA-IR were found to have reduced insulin sensitivity and 50% were found to have metabolic syndrome by the modified ATP III criteria. Two (3%) patients were found to have high serum iron, 4 (7%) patients had high ferritin, 5 (8%) patients had increased transferrin saturation, and 4 (13%) patients were found to be heterozygotes for H63D HFE gene mutation. Twenty patients of 38 (53%) had histological evidence of NASH (class 3=6, class 4=14). The other 18 (47%) qualified for class I (n=1) or class II (n=17) NAFLD. Four (10.5%) patients had bridging fibrosis and none had evidence of cirrhosis liver. Seventy-four (74%) patients achieved a biochemical response to lifestyle modification and UDCA. All 17 patients treated with metformin had a reduction in ALT level and 10 (59%) of them had normalization of their enzymes. We conclude that the clinicopathological profile of NAFLD in Indian patients is different from that in the West.

<h4>Background</h4>Microorganisms and plants are able to produce tryptophan. Enzymes catalysing the last seven steps of tryptophan biosynthesis are encoded in the canonical trp operon. Among the trp genes are most frequently trpA and trpB, which code for the alpha and beta subunit of tryptophan synthase. In several prokaryotic genomes, two variants of trpB (named trpB1 or trpB2) occur in different combinations. The evolutionary history of these trpB genes is under debate.<h4>Results</h4>In order to study the evolution of trp genes, completely sequenced archeal and bacterial genomes containing trpB were analysed. Phylogenetic trees indicated that TrpB sequences constitute four distinct groups; their composition is in agreement with the location of respective genes. The first group consisted exclusively of trpB1 genes most of which belonged to trp operons. Groups two to four contained trpB2 genes. The largest group (trpB2_o) contained trpB2 genes all located outside of operons. Most of these genes originated from species possessing an operon-based trpB1 in addition. Groups three and four pertain to trpB2 genes of those genomes containing exclusively one or two trpB2 genes, but no trpB1. One group (trpB2_i) consisted of trpB2 genes located inside, the other (trpB2_a) of trpB2 genes located outside the trp operon. TrpA and TrpB form a heterodimer and cooperate biochemically. In order to characterise trpB variants and stages of TrpA/TrpB cooperation in silico, several approaches were combined. Phylogenetic trees were constructed for all trp genes; their structure was assessed via bootstrapping. Alternative models of trpB evolution were evaluated with parsimony arguments. The four groups of trpB variants were correlated with archeal speciation. Several stages of TrpA/TrpB cooperation were identified and trpB variants were characterised. Most plausibly, trpB2 represents the predecessor of the modern trpB gene, and trpB1 evolved in an ancestral bacterium.<h4>Conclusion</h4>In archeal genomes, several stages of trpB evolution, TrpA/TrpB cooperation, and operon formation can be observed. Thus, archeal trp genes may serve as a model system for studying the evolution of protein-protein interactions and operon formation.

Liver is the primary target organ of Hereditary Hemochromatosis Type I, with the HFE mutations C282Y and H63D recognized as markers of this iron-overload disease. Hepatocytes are also the main site of synthesis of HFE. However, most early studies of overexpression of HFE were done in non-hepatic, non-HFE-expressing, cell lines. Here we report the setting up of a stable transfection model of wt- and mutant-HFE (H63D and C282Y) proteins in a hepatic cell line (HepG2), the analysis of its intracellular distribution and the effect of diferric transferrin on HFE localization. The C282Y mutant is retained in the ER, whereas HFE-wt and H63D co-localize with TfR1 exclusively in early recycling endosomes. Holotransferrin induces a re-localization of wt- and H63D-HFE, from early recycling endosomes to the cytoplasmic membrane. In conclusion our results establish the HepG2 cell line as a valuable model for the study of HFE.

In addition to their established functions in programmed cell death, there is increasing evidence that caspases contribute to several other cellular processes beside of apoptosis. So-called "dependence receptors" represent a group of receptors, which derive from different protein families, but are functionally linked by their capability to regulate cell survival in presence of their respective ligands thereby preserving cellular homeostasis. In the absence of their ligands these receptors are cleaved by caspases thereby releasing pro-apoptotic receptor fragments (e.g. rearranged during transfection [RET]) or permitting the exposure of death domains, which were masked before through other receptor domains (e.g. deleted in colorectal carcinoma [DCC]). Apart from these, there are other plasma membrane receptors such as the epidermal growth factor receptor, which have been identified as substrates of caspases. In terms of signal-transduction, caspase-mediated cleavage of these receptors blocks ligand-induced activation of their intracellular signalling. It is hypothesized that this might be another mechanism, whereby caspases trigger cell toxicity through shut-down of survival signals.

In the present study, turpentine oil was injected in the hind limb muscle of the rat to stimulate an acute-phase response (APR). The changes in the gene expression of cytokines and proteins known to be involved in the iron regulatory pathway were then studied in the liver and in extra-hepatic tissue. In addition to the strong upregulation of interleukin-6 (IL-6) and IL-1 beta observed in the inflamed muscle, an upregulation of the genes for IL1-beta and tumor necrosis factor-alpha, but not IL-6, were detectable in the liver. Hepatic Hepc gene expression increased to a maximum at 6 h after the onset of APR. An upregulation of transferrin, transferrin receptor 1 (TfR1), TfR2, ferritin-H, iron responsive element binding protein-1 (IRP1), IRP2 and divalent metal transporter gene expression was also found. Hemojuvelin (Hjv)-, ferroportin 1-, Dcytb-, hemochromatosis-gene- and hephaestin gene expression was downregulated. Hepcidin (Hepc) gene expression was not only detectable in extra-hepatic tissues such as heart, small intestine, colon, spleen and kidney but it was also upregulated under acute-phase conditions, with the Hjv gene being regulated antagonistically. Fpn-1 gene expression was downregulated significantly in heart, colon and spleen. Most of the genes of the known proteins involved in iron metabolism are expressed not only in the liver but also in extra-hepatic tissues. Under acute-phase conditions, acute-phase cytokines (eg IL-6) may modulate the gene expression of such proteins not only in the liver but also in other organs.

The human mixed lineage leukemia (MLL) gene is frequently involved in genetic rearrangements with more than 55 different translocation partner genes, all associated with acute leukemia. Reciprocal chromosomal translocations generate two MLL fusion alleles, where 5'- and 3'-portions of MLL are fused to gene segments of given fusion partners. In case of t(4;11) patients, about 80% of all patients exhibit both reciprocal fusion alleles, MLL.AF4 and AF4.MLL, respectively. By contrast, 20% of all t(4;11) patients seem to encode only the MLL.AF4 fusion allele. Here, we analyzed these 'MLL.AF4(+)/AF4.MLL(-)' patients at the genomic DNA level to unravel their genetic situation. Cryptic translocations and three-way translocations were found in this group of t(4;11) patients. Reciprocal MLL fusions with novel translocation partner genes, for example NF-KB1 and RABGAP1L, were identified and actively transcribed in leukemic cells. In other patients, the reciprocal 3'-MLL gene segment was fused out-of-frame to PBX1, ELF2, DSCAML1 and FXYD6. The latter rearrangements caused haploinsufficiency of genes that are normally expressed in hematopoietic cells. Finally, patients were identified that encode only solitary 3'-MLL gene segments on the reciprocal allele. Based on these data, we propose that all t(4;11) patients exhibit reciprocal MLL alleles, but due to the individual recombination events, provide different pathological disease mechanisms.

We aimed to determine the relationships between iron overload and HFE gene mutation in chronic liver disease in Turkey. One hundred thirteen chronic liver disease patients and 138 healthy controls were evaluated regarding their clinical, biochemical, and genetic parameters. Each group was divided into two subgroups according to transferrin saturation (TS) (45% and >45%). HFE gene mutation was analyzed by the PCR-RFLP method. C282Y homozygote, heterozygote, and wild-type mutation rates were 1.7%, 0%, and 98.3% in patients and 0%, 1.4%, and 98.6% in controls, respectively. H63D homozygote, heterozygote, and wild-type mutation rates were 1.8%, 24.7%, and 73.5% in patients and 1.4%, 24%, and 74.6% in controls, respectively. Mutation rates were not statistically different in patients with high and normal TS. Iron overload was positively correlated with biochemical activity and Child-Pugh score (P < 0.05). In multivariate analysis, H63D homozygotic mutation was an independent factor for the development of hepatocellular carcinoma (P = 0.004). We conclude that C282Y mutation is very rare in Turkey. Iron overload is not related to H63D mutation but is positively correlated with biochemical activity and Child-Pugh score in chronic liver diseases.

Gene silencing through aberrant CpG island methylation is a frequent epigenetic defect in hepatocellular carcinoma (HCC). However, nothing is known as yet whether aberrant hypermethylation occurs already in non-neoplastic liver cells from patients with hereditary haemochromatosis who have a clearly elevated risk for developing HCC. Therefore, quantitative real-time PCR-based methylation analysis of six genes frequently hypermethylated in HCC (RASSF1A, cyclinD2, p16(INK4a), GSTpi1, SOCS-1, APC) was performed for liver biopsies from patients with hereditary haemochromatosis. For genotyping of the HFE gene restriction enzyme analysis and Pyrosequencing were used. Transcriptional repression of hypermethylated genes was assessed using real-time RT-PCR. Eighty-four percent of all samples with severe hepatic iron overload and a mutated HFE gene (but without HCC) had at least one gene hypermethylated. All six genes tested were affected by aberrant hypermethylation, albeit to a different extent: RASSF1A 55%, cyclinD2 45%, p16(INK4a) 32%, GSTpi1 10%, SOCS-1 6%, APC 8%. Concomitant transcriptional down-regulation was shown for RASSF1A, cyclinD2, GSTpi1 and SOCS-1. Biopsies from haemochromatosis patients showed significantly more aberrant hypermethylation than normal liver tissue or benign liver tumours (P < 0.001) and also to a higher degree. This effect is independent of patient age, cirrhosis or hepatitis infection. This is the first report demonstrating that longstanding severe iron overload is frequently associated with epigenetic defects characteristic of HCC, which reflects the increased risk of these lesions to progress to HCC. Thus, changes in DNA methylation patterns are an early event preceding morphological alterations of malignant transformation and represent promising targets for early detection.

<h4>Introduction</h4>In mammals, iron balance is maintained by regulating intestinal iron absorption and iron recycling by macrophages.<h4>Exegesis</h4>The central role of hepcidin in the establishment of iron deficiency or overload has been revealed by the study of murine models.<h4>Conclusion</h4>Many hopes have emerged from the discovery of hepcidin, pointing to potential treatments for hemochromatosis and anemia of inflammation (anemia of chronic disease).

Ketoprofen-polyethylene glycol (PEG) conjugates (KPEG) were prepared and their potential as a prolonged release system was investigated. Three KPEG conjugates were synthesized from ketoprofen and methoxy PEG with three different molecular weights by esterification in the presence of DCC. The KPEG conjugates were characterized by FT-IR and (1)H NMR spectroscopy. The rate of hydrolysis profile showed a specific acid-base catalysis pattern with a minimum at pH 4-5. The pharmacokinetic study after the intravenous and intramuscular administration of KPEG750 showed that the plasma levels of KP increased slowly and reached a maximum concentration at later time. The AUC of KPEG750 was higher than that after administering an equivalent dose of ketoprofen except 40mg/kg dose of intramuscular administration. The tail-flick experiment and paw edema test after intramuscular administration showed that KPEG750 had extended analgesic and anti-inflammatory effects compared with ketoprofen. These results suggest that KPEG could be a promising NSAID prodrug with an extended pharmacological effect owing to delayed-release of parent drug.

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)(s843) transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.

UDP-glucose (UDPG) and derivatives are naturally occurring agonists of the Gi protein-coupled P2Y14 receptor, which occurs in the immune system. We synthesized and characterized pharmacologically novel analogues of UDPG modified on the nucleobase, ribose, and glucose moieties, as the basis for designing novel ligands in conjunction with modeling. The recombinant human P2Y14 receptor expressed in COS-7 cells was coupled to phospholipase C through an engineered Galpha-q/i protein. Most modifications of the uracil or ribose moieties abolished activity; this is among the least permissive P2Y receptors. However, a 2-thiouracil modification in 15 (EC50 49 +/- 2 nM) enhanced the potency of UDPG (but not UDP-glucuronic acid) by 7-fold. 4-Thio analogue 13 was equipotent to UDPG, but S-alkylation was detrimental. Compound 15 was docked in a rhodposin-based receptor homology model, which correctly predicted potent agonism of UDP-fructose, UDP-mannose, and UDP-inositol. The hexose moiety of UDPG interacts with multiple H-bonding and charged residues and provides a fertile region for agonist modification.

Our investigation of bivalent ligands at mu, delta, and kappa opioid receptors is focused on the preparation of ligands containing kappa agonist and mu agonist/antagonist pharmacophores at one end joined by a chain containing the mu antagonist pharmacophores (naltrexone, naloxone, or nalbuphine) at the other end. These ligands were evaluated in vitro by their binding affinity at mu, delta, and kappa opioid receptors and their relative efficacy in the [35S]GTPgammaS assay.

Huntington disease is caused by the expansion of a CAG repeat encoding an extended glutamine tract in a protein called huntingtin. Here, we provide evidence supporting the hypothesis that somatic increases of mutation length play a role in the progressive nature and cell-selective aspects of HD pathogenesis. Results from micro-dissected tissue and individual laser-dissected cells obtained from human HD cases and knock-in HD mice indicate that the CAG repeat is unstable in all cell types tested although neurons tend to have longer mutation length gains than glia. Mutation length gains occur early in the disease process and continue to accumulate as the disease progresses. In keeping with observed patterns of cell loss, neuronal mutation length gains tend to be more prominent in the striatum than in the cortex of low-grade human HD cases, less so in more advanced cases. Interestingly, neuronal sub-populations of HD mice appear to have different propensities for mutation length gains; in particular, smaller mutation length gains occur in nitric oxide synthase-positive striatal interneurons (a relatively spared cell type in HD) compared with the pan-striatal neuronal population. More generally, the data demonstrate that neuronal changes in HD repeat length can be at least as great, if not greater, than those observed in the germline. The fact that significant CAG repeat length gains occur in non-replicating cells also argues that processes such as inappropriate mismatch repair rather than DNA replication are involved in generating mutation instability in HD brain tissue.

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Expression profiles of primary breast tumors were investigated in relation to disseminated tumor cells (DTCs) in bone marrow (BM) in order to increase our understanding of the dissemination process. Tumors were classified into five pre-defined molecular subtypes, and presence of DTC identified (at median 85 months follow-up) a subgroup of luminal A patients with particular poor outcome (p=0.008). This was not apparent for other tumor subtypes. Gene expression profiles associated with DTC and with systemic relapse for luminal A patients were identified. This study suggests that DTC in BM differentially distinguishes clinical outcome in patients with luminal A type tumors and that DTC-associated gene expression analysis may identify genes of potential importance in tumor dissemination.

Transcriptional dysregulation and aberrant chromatin remodeling are central features in the pathology of Huntington's disease (HD). In order to more fully characterize these pathogenic events, an assessment of histone profiles and associated gene changes were performed in transgenic N171-82Q (82Q) and R6/2 HD mice. Analyses revealed significant chromatin modification, resulting in reduced histone acetylation with concomitant increased histone methylation, consistent with findings observed in HD patients. While there are no known interventions that ameliorate or arrest HD progression, DNA/RNA-binding anthracyclines may provide significant therapeutic potential by correcting pathological nucleosome changes and realigning transcription. Two such anthracyclines, chromomycin and mithramycin, improved altered nucleosome homeostasis in HD mice, normalizing the chromatin pattern. There was a significant shift in the balance between methylation and acetylation in treated HD mice to that found in wild-type mice, resulting in greater acetylation of histone H3 at lysine 9 and promoting gene transcription. Gene expression profiling in anthracycline-treated HD mice showed molecular changes that correlate with disease correction, such that a subset of downregulated genes were upregulated with anthracycline treatment. Improved nucleosomal dynamics were concurrent with a significant improvement in the behavioral and neuropathological phenotype observed in HD mice. These data show the ability of anthracycline compounds to rebalance epigenetic histone modification and, as such, may provide the rationale for the design of human clinical trials in HD patients.

The present study attempted to identify and characterize the embryonic promoter of Sox6, a determinant regulator of chondrogenic differentiation. A common transcription start region for human and mouse Sox6 was initially identified, which contained a highly conserved sequence, A-box. Tandem repeats of A-box had a strong transcriptional activity both at the basal level and in response to Sox9. Cells carrying the 4xA-box-DsRed2 reporter fluoresced only upon chondrogenic differentiation. The 46-bp core enhancer region (CES6) was then identified in the 3' half of A-box, within which a C/EBP-binding motif was identified. Overexpressed C/EBPbeta activated the Sox6 promoter, and mutant 4xCES6 constructs lacking the C/EBP motif lost their basal activity. CES6 and nuclear extracts formed a specific complex, which was supershifted by anti-C/EBPbeta antibody, and in vitro translated C/EBPbeta specifically bound to CES6. Thus, we successfully identified the Sox6 promoter and its core enhancer and characterized the interactions with regulatory transcription factors.

Mounting evidence exists for the operation of a functional serotonin (5-HT) system in osteoclasts and osteoblasts, which involves both receptor activation and 5-HT reuptake. In previous work we showed that the serotonin transporter (5-HTT) is expressed in osteoclasts and that its activity is required by for osteoclast differentiation in vitro. The purpose of the current study was to determine the effect of treatment with fluoxetine, a specific serotonin reuptake inhibitor, on bone metabolism in vivo. Systemic administration of fluoxetine to Swiss-Webster mice for 6 weeks resulted in increased trabecular BV and BV/TV in femurs and vertebrae as determined by micro-computed tomography (microCT). This correlated with an increase in trabecular number, connectivity, and decreased trabecular spacing. Fluoxetine treatment also resulted in increased volume in vertebral trabecular bone. However, fluoxetine-treated mice were not protected against bone loss after ovariectomy, suggesting that its anabolic effect requires the presence of estrogen. The effect of blocking the 5-HTT on bone loss following an LPS-mediated inflammatory challenge was also investigated. Subcutaneous injections of LPS over the calvariae of Swiss-Webster mice for 5 days resulted in increased numbers of osteoclasts and net bone loss, whereas new bone formation and a net gain in bone mass was seen when LPS was given together with fluoxetine. We conclude that fluoxetine treatment in vivo leads to increased bone mass under normal physiologic or inflammatory conditions, but does not prevent bone loss associated with estrogen deficiency. These data suggest that commonly used anti-depressive agents may affect bone mass.

Solution-phase, DNA melting analysis for heterozygote scanning and single nucleotide polymorphism (SNP) genotyping was performed in 10 nl volumes on a custom microchip. Human genomic DNA was PCR amplified in the presence of the saturating fluorescent dye, LCGreen Plus, and placed within microfluidic channels that were created between two glass slides. The microchip was heated at 0.1 degrees C/s with a Peltier device and viewed with an inverted fluorescence microscope modified for photomulitiplier tube detection. The melting data was normalized and the negative first derivative plotted against temperature. Mutation scanning for heterozygotes was easily performed by comparing the shape of the melting curve to homozygous standards. Genotyping of homozygotes by melting temperature (T(m)) required absolute temperature comparisons. Mutation scanning of ATM exon 17 and CFTR exon 10 identified single base change heterozygotes in 84 and 201 base-pair (bp) products, respectively. All genotypes at HFE C282Y were distinguished by simple melting analysis of a 40-bp fragment. Sequential analysis of the same sample on the gold-standard, commercial high-resolution melting instrument HR-1, followed by melting in a 10 nl reaction chamber, produced similar results. DNA melting analysis requires only minutes after PCR and is a simple method for genotyping and scanning that can be reduced to nanoliter volumes. Microscale systems for performing DNA melting reduce the reagents/DNA template required with a promise for high throughput analysis in a closed chamber without risk of contamination.

Few studies on pharmacogenetics of Attention-Deficit/Hyperactivity Disorder (ADHD) have been conducted. Most of them evaluated dopaminergic genes resulting in positive and negative findings. We assessed effects of polymorphisms in candidate dopaminergic (DRD4, DAT1) and serotonergic genes (HTR1B, HTR2A, and 5-HTT) on the response to treatment in 111 patients for whom methylphenidate (MPH) was prescribed. Outcome measures (Swanson, Nolan, and Pelham scale-version IV, Children Global Assessment Scale, Barkley's Stimulants Side Effects Rating Scale) were assessed at baseline and 1 month after the intervention. No significant association was detected between polymorphisms assessed and both response and side effects to MPH. Prospective multi-site controlled studies with larger sample sizes are needed in order to disentangle the role of candidate genes in response to ADHD treatment.

To explore the role of axon guidance molecules during regeneration in the lamprey spinal cord, we examined the expression of mRNAs for semaphorin 3 (Sema3), semaphorin 4 (Sema4), and netrin during regeneration by in situ hybridization. Control lampreys contained netrin-expressing neurons along the length of the spinal cord. After spinal transection, netrin expression was downregulated in neurons close (500 mum to 10 mm) to the transection at 2 and 4 weeks. A high level of Sema4 expression was found in the neurons of the gray matter and occasionally in the dorsal and the edge cells. Fourteen days after spinal cord transection Sema4 mRNA expression was absent from dorsal and edge cells but was still present in neurons of the gray matter. At 30 days the expression had declined to some extent in neurons and was absent in dorsal and edge cells. In control animals, Sema3 was expressed in neurons of the gray matter and in dorsal and edge cells. Two weeks after transection, Sema3 expression was upregulated near the lesion, but absent in dorsal cells. By 4 weeks a few neurons expressed Sema3 at 20 mm caudal to the transection but no expression was detected 1 mm from the transection. Isolectin I-B(4) labeling for microglia/macrophages showed that the number of Sema3-expressing microglia/macrophages increased dramatically at the injury site over time. The downregulation of netrin and upregulation of Sema3 near the transection suggests a possible role of netrin and semaphorins in restricting axonal regeneration in the injured spinal cord.

Huntington disease (HD) is a progressive neurodegenerative disorder characterized by emotional, cognitive, and motor dysfunctions. Aggregation of huntingtin is a hallmark of HD and, therefore, a crucial parameter for the evaluation of HD animal models. We investigated here the regional, cellular, and subcellular distribution of N-terminal huntingtin aggregates and associated neuropathological changes in the forebrain of a rat transgenic for HD (tgHD). The tgHD rat brain showed enormously enlarged lateral ventricles and a similar atrophy of cortical and subcortical areas as known in HD patients. Huntingtin aggregates of varying size and forms were regionally identified in neuronal nuclei, cytoplasm, dendrites, dendritic spines, axons, and synaptic terminals, closely resembling the results described earlier for human HD brains and in established HD mouse models. Huntingtin aggregates in mitochondria support mitochondrial dysfunction as contributing to the disease pathogenesis. Dark cell degeneration was reminiscent of results in HD individuals and HD mouse models. Interestingly, huntingtin aggregates were especially well accumulated in two interacting limbic forebrain systems, the ventral striatopallidum and the extended amygdala, which may contribute to the early onset of emotional changes observed in the tgHD rat. In conclusion, the tgHD rat model reflects to a remarkable extent the cellular and subcellular neuropathological key features as observed in human HD and HD mouse brains and hints of changes in limbic forebrain systems, which may elucidate the emotional dysfunction in the tgHD rat and affective disturbances in HD patients.

Angelman syndrome (AS) is a neurogenetic disorder characterized by severe mental retardation, ataxia, seizures, EEG abnormalities and bouts of inappropriate laughter. AS individuals fail to inherit a normal active maternal copy of ubiquitin protein ligase E3A (UBE3A). UBE3A is subject to genomic imprinting, with predominant transcription of the maternal allele in brain. The known genetic causes of AS are maternal deletion of chromosome 15q11-q13, paternal chromosome 15 uniparental disomy, UBE3A mutation and an abnormality of the imprinting process, termed imprinting defect. There remain major questions concerning the molecular pathogenesis of AS, including: 1) the mechanisms underlying the imprinting defect class of AS, 2) the identity of proteins targeted by UBE3A, 3) the role of a noncoding antisense transcript in regulating UBE3A imprinting and 4) the contribution of other genes such as methyl-binding CpG-binding protein 2 and gamma-aminobutyric acid A receptor, subunit beta3 to the AS phenotype.

Prolonged neurotoxicity of the recreational drug, MDMA (3,4-methylenedioxymethamphetamine) on serotoninergic axon terminals has been suggested. The effect of a single (15 mg/kg) dose of intraperitoneally administered MDMA on serotoninergic fibre density, defined by tryptophan hydroxylase (TpH) and serotonin transporter (5-HTT) immunoreactivity, has been evaluated in the spinal cord and brain areas in Dark Agouti rats, 7 and 180 days after MDMA applications. Immunostaining for amyloid precursor protein (APP) has been performed to examine possible defects of the fast axonal transport, and 5-HTT mRNA expressions were quantified in neurones of medullary raphe nuclei. Seven days after MDMA treatment, a substantial decrease in the density of TpH-immunoreactive fibres was detectable in the frontal cortex, the caudate-putamen, the CA1 region of the hippocampus, and marked decreases were found in the spinal cord. These changes in TpH density showed a high correlation with 5-HTT densities. In contrast, APP-immunoreactive axonal bulbs were not detected in any of the brain regions studied. Seven days after MDMA administrations, significantly elevated 5-HTT mRNA expressions were found in the raphe pallidus and obscurus. Our results suggest that a single dose of MDMA elicits widespread depletion of TpH and 5-HTT immunoreactivity in serotoninergic axons without morphological sign of the blockage of the fast anterograde axonal transport. Our results do not support the notion of MDMA-induced axotomy of serotoninergic neurones. The up-regulation of 5-HTT mRNA expressions 1 week after MDMA injections might indicate the potential recovery of the serotonin system.

No abstract available.

No abstract available.

Mitochondrial dysfunction is believed to participate in Huntington's disease (HD) pathogenesis. Here we compare the bioenergetic behavior of forebrain mitochondria isolated from different transgenic HD mice (R6/2, YAC128 and Hdh150 knock-in) and wild-type littermates with the first determination of in situ respiratory parameters in intact HD striatal neurons. We assess the Ca2+-loading capacity of isolated mitochondria by steady Ca2+-infusion. Mitochondria from R6/2 mice (12-13 weeks) and 12 months YAC128, but not homozygous or heterozygous Hdh150 knock-in mice (15-17 weeks), exhibit increased Ca2+-loading capacity when compared with respective wild-type littermates. In situ mitochondria in intact striatal neurons show high respiratory control. Moreover, moderate expression of full-length mutant huntingtin (in Hdh150 knock-in heterozygotes) does not significantly impair mitochondrial respiration in unstimulated neurons. However, when challenged with energy-demanding stimuli (NMDA-receptor activation in pyruvate-based media to accentuate the mitochondria role in Ca2+-handling), Hdh150 neurons are more vulnerable to Ca2+-deregulation than neurons from their wild-type littermates. These results stress the importance of assessing HD mitochondrial function in the cellular context.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the major risk factors include chronic infections with the hepatitis B (HBV) or C (HCV) virus, and exposure to dietary aflatoxin B(1) (AFB(1)) or alcohol consumption. Multiple genetic and epigenetic changes are involved in the molecular pathogenesis of HCC, for example, somatic mutations in the p53 tumor suppressor gene (TP53) and the activation of the WNT signal transduction pathway. AFB(1) frequently induces G:C to T:A transversions at the third base in codon 249 of TP53 and cooperates with HBV in causing p53 mutations in HCC. The detection of TP53 mutant DNA in plasma is a biomarker of both AFB(1) exposure and HCC risk. Chronic infection with HBV and HCV viruses, and oxyradical disorders including hemochromatosis, also generate reactive oxygen/nitrogen species that can both damage DNA and mutate cancer-related genes such as TP53. Certain mutant p53 proteins may exhibit a 'gain of oncogenic function'. The p53 biological network is a key responder to this oxidative and nitrosative stress. Depending on the extent of the DNA damage, p53 regulates the transcription of protective antioxidant genes and with extensive DNA damage, transactivates pro-oxidant genes that contribute to apoptosis. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC and the integrated HBx is frequently mutated. Mutant HBx proteins still retain their ability to bind to p53, and attenuate DNA repair and p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology of TP53 mutations during the molecular pathogenesis of HCC.

Tap/NXF1, the founding member of the evolutionarily conserved NXF (Nuclear RNA export Factor) family of proteins, is required for the nuclear export of bulk poly(A)+ RNAs. In mice, three additional NXF family genes (NXF2, NXF3, NXF7) have been identified and characterized to date. Cumulative data suggest that NXF family members play roles, not only in nuclear mRNA export, but also in various aspects of post-transcriptional mRNA metabolism. In order to better understand the functional role of NXF2, we searched for its binding partners by yeast two-hybrid screening and identified several cytoplasmic motor proteins, including KIF17. The interaction of NXF2 with KIF17, which was confirmed by GST pull-down and co-immunoprecipitation assays, is mediated by the N-terminal domain of NXF2, which is required for the punctate localization patterns in dendrites of primary neurons. We also found that the NXF2-containing dendritic granules, which were co-localized with KIF17, mRNA and Staufen1, a known component of neuronal mRNA granules, moved bidirectionally along dendrites in a microtubule-dependent manner. These results suggest that NXF2, a nucleo-cytoplasmic mRNA transporter, plays additional roles in the cytoplasmic localization of mRNAs through interactions with cytoplasmic motor proteins.

Endogenous cannabinoids (endocannabinoids) mediate retrograde signals for short- and long-term suppression of transmitter release at synapses of striatal medium spiny (MS) neurons. An endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is synthesized from diacylglycerol (DAG) after membrane depolarization and Gq-coupled receptor activation. To understand 2-AG-mediated retrograde signaling in the striatum, we determined precise subcellular distributions of the synthetic enzyme of 2-AG, DAG lipase-alpha (DAGLalpha), and its upstream metabotropic glutamate receptor 5 (mGluR5) and muscarinic acetylcholine receptor 1 (M1). DAGLalpha, mGluR5, and M1 were all richly distributed on the somatodendritic surface of MS neurons, but their subcellular distributions were different. Although mGluR5 and DAGLalpha levels were highest in spines and accumulated in the perisynaptic region, M1 level was lowest in spines and was rather excluded from the mGluR5-rich perisynaptic region. These subcellular arrangements suggest that mGluR5 and M1 might differentially affect endocannabinoid-mediated, depolarization-induced suppression of inhibition (DSI) and depolarization-induced suppression of excitation (DSE) in MS neurons. Indeed, mGluR5 activation enhanced both DSI and DSE, whereas M1 activation enhanced DSI only. Importantly, DSI, DSE, and receptor-driven endocannabinoid-mediated suppression were all abolished by the DAG lipase inhibitor tetrahydrolipstatin, indicating 2-AG as the major endocannabinoid mediating retrograde suppression at excitatory and inhibitory synapses of MS neurons. Accordingly, CB1 cannabinoid receptor, the main target of 2-AG, was present at high levels on GABAergic axon terminals of MS neurons and parvalbumin-positive interneurons and at low levels on excitatory corticostriatal afferents. Thus, endocannabinoid signaling molecules are arranged to modulate the excitability of the MS neuron effectively depending on cortical activity and cholinergic tone as measured by mGluR5 and M1 receptors, respectively.

Overactivation of NMDA receptors (NMDARs) is believed to play a role in degeneration of striatal medium-sized spiny neurons (MSNs) in Huntington's disease (HD). This hereditary disorder is caused by an expansion >35 in the polyglutamine (polyQ) region of the protein huntingtin (htt). Previous work has shown that NMDAR current, calcium signaling, and/or toxicity are enhanced in striatal MSNs in a variety of transgenic mice and cellular models of HD, but whether the enhancement is specific for MSNs or correlated with mutant htt (mhtt) polyQ length is not known. Furthermore, the mechanism underlying the increase in NMDAR activity has not been elucidated. Here we report polyQ length-dependent enhancement of peak NMDAR current density by mhtt in cultured MSNs, but not cortical neurons, from the yeast artificial chromosome (YAC) transgenic HD mouse model. We also observed a shift of NMDAR subunits NR1 and NR2B from internal pools to the plasma membrane and a significantly faster rate of NMDAR insertion to the surface in YAC72 MSNs. In comparing YAC72 with wild-type striatal tissue, subcellular fractionation revealed a relative enrichment of NR1 C2'-containing NMDARs in the vesicle/microsome-enriched fraction, and coimmunoprecipitation experiments demonstrated an increased proportion of NR1 C2' isoforms associated with NR2 subunits, which may contribute to faster forward trafficking of these receptors. Our results suggest that altered NMDAR trafficking may underlie potentiation of NMDAR-mediated current and toxicity in the YAC72 HD mouse model. This polyQ length-dependent, neuronal-specific change in NMDAR activity induced by mhtt may contribute to selective neuronal degeneration in HD.

Previous studies using dominant-mutant constructs have implicated Rac1 GTPase in neuritogenesis and neuronal migration. However, overexpression of dominant mutants generally blocks Rho-GTPase activity; thus, it may not reveal the specific or physiological functions of Rac1. To address this issue, we have applied a conditional gene-targeting strategy, using Foxg1-Cre mice to delete Rac1 in the ventricular zone (VZ) of telencephalon and Dlx5/6-Cre-IRES (internal ribosomal entry site)-EGFP (enhanced green fluorescent protein) (Dlx5/6-CIE) in the subventricular zone (SVZ) of ventral telencephalon, respectively. Surprisingly, the deletion of Rac1 in VZ progenitors did not prevent axonal outgrowth of telencephalic neurons. However, the anterior commissure was absent, and the corpus callosal as well as hippocampal commissural axons failed to cross the midline in Rac1/Foxg1-Cre knock-out embryos. The thalamocortical and corticothalamic axons also showed defasciculation or projection defects. These results suggest that Rac1 controls axon guidance rather than neuritogenesis. In addition, although Rac1/Foxg1-Cre knock-out embryos showed delayed radial migration of cortical projection neurons and severe impairment of tangential migration by the ventral telencephalon-derived interneurons, deletion of Rac1 in the SVZ by Dlx5/6-CIE mice produced no discernible defects in tangential migration. These contrasting effects of Rac1 deletion on tangential migration suggest that Rac1 is dispensable for cellular motility per se during neuronal migration. Together, these results underscore the challenge of deciphering the biological functions of Rac1, and Rho-GTPases in general, during mammalian brain development. Moreover, they indicate that Rac1 has a critical role in axon guidance and in acquisition of migratory competency during differentiation of the progenitors for the ventral telencephalon-derived interneurons.

Huntington disease (HD) is caused by the expansion of a glutamine (Q) repeat near the N terminus of huntingtin (htt), resulting in altered conformation of the mutant protein to produce, most prominently in brain neurons, nuclear and cytoplasmic inclusion pathology. The inclusions and associated diffuse accumulation of mutant htt in nuclei are composed of N-terminal fragments of mutant protein. Here, we used a panel of peptide antibodies to characterize the htt protein pathologies in brain tissues from human HD, and a transgenic mouse model created by expressing the first 171 amino acids of human htt with 82Q (htt-N171-82Q). In tissues from both sources, htt pathologic features in nuclei were detected by antibodies to htt peptides 1-17 and 81-90 but not 115-129 (wild-type huntingtin numbering with 23 repeats). Human HEK 293 cells transfected with expression vectors that encode either the N-terminal 233 amino acids of human htt (htt-N233-82Q) or htt-N171-18Q accumulated smaller N-terminal fragments with antibody-binding characteristics identical to those of pathologic peptides. We conclude that the mutant htt peptides that accumulate in pathologic structures of human HD and httN171-82Q in mice are produced by similar, yet to be defined, proteolytic events in a region of the protein near or within amino acids 90-115.

<h4>Background</h4>Asians and Pacific Islanders in the Hemochromatosis and Iron Overload Screening (HEIRS) Study had the highest prevalence of elevated serum ferritin (SF) and transferrin saturation (TS) levels, but to our knowledge, the reasons for this have not been investigated.<h4>Methods</h4>Using multiple linear regression, we compared TS and SF distributions for 42 720 Asian, Pacific Islander, and white HEIRS Study participants recruited through 5 field centers in North America who did not have HFE C282Y or H63D alleles.<h4>Results</h4>Compared with their white counterparts, Asian men had a 69-ng/mL (155-pmol/L) higher adjusted mean SF level and a 3% higher TS level (P<.001); Asian women had 23-ng/mL (52-pmol/L) higher adjusted mean SF level and a 3% higher TS level (P<.001). The mean TS level of Asian women was higher than that of Pacific Islander women, and the mean SF level of Pacific Islander men was significantly higher than that of white men. These differences remained significant after adjusting for self-reported history of diabetes or liver disease. Additional information for selected participants suggested that these differences are largely unrelated to mean corpuscular volume less than 80 fL, body mass index, or self-reported alcohol intake. Available liver biopsy and phlebotomy data indicated that iron overload is probably uncommon in Asian participants.<h4>Conclusion</h4>Higher TS and SF levels in persons of Asian or Pacific Island heritage may need to be interpreted differently than for whites, although the biological basis and clinical significance of higher levels among Asians and Pacific Islanders are unclear.

The inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are a family of Ca2+ release channels localized predominately in the endoplasmic reticulum of all cell types. They function to release Ca2+ into the cytoplasm in response to InsP3 produced by diverse stimuli, generating complex local and global Ca2+ signals that regulate numerous cell physiological processes ranging from gene transcription to secretion to learning and memory. The InsP3R is a calcium-selective cation channel whose gating is regulated not only by InsP3, but by other ligands as well, in particular cytoplasmic Ca2+. Over the last decade, detailed quantitative studies of InsP3R channel function and its regulation by ligands and interacting proteins have provided new insights into a remarkable richness of channel regulation and of the structural aspects that underlie signal transduction and permeation. Here, we focus on these developments and review and synthesize the literature regarding the structure and single-channel properties of the InsP3R.

Recent research has yielded many interesting and potentially important therapeutic targets in sepsis. Specifically, the effects of antagonistic anti-cytokine therapies (tumor necrosis factor-alpha [TNF-alpha], interleukin-1 [IL-1]) and anti-endotoxin strategies utilizing antibodies against endotoxin or endotoxin receptor/carrier molecules (anti-CD14 or anti-LPS-binding protein) have been studied. Unfortunately, these approaches often failed clinically, and in many cases, the efficacy of these treatments was dependent on the severity of sepsis. Recently, clinical trials using insulin to lock blood glucose levels and activated protein C treatment have showed that while they provided some survival benefit, their efficacy does not appear to be predicated solely upon anti-inflammatory effects. Here, we will review work done in animal models of polymicrobial sepsis and clinical findings that support the hypothesis that apoptosis in the immune system is a pathologic event in sepsis that can be a therapeutic target. In this respect, experimental studies looking at the septic animal suggest that loss of lymphocytes during sepsis may be due to dysregulated apoptosis and that this appears to be brought on by a variety of mediators effecting 'intrinsic' as well as 'extrinsic' cell death pathways. From a therapeutic perspective this has provided a number of novel targets for clinically successful current, as well as future therapies, such as caspases (caspase inhibition/protease inhibition), pro-apoptotic protein-expression (via administration and/or over-expression of Bcl-2) and the death receptor family Fas-FasL (via. FasFP [fas fusion protein] and the application of siRNA against a number pro-apoptotic factors).

There is increasing evidence that heparan sulfate (HS) plays an essential role in various axon guidance processes. These observations, however, have not addressed whether HS is required cell autonomously as an axonal coreceptor or as an environmental factor that modulates the localization of guidance molecules in the terrain in which growing axons navigate. Here we demonstrate that netrin-1-mediated commissural axon guidance requires cell-autonomous expression of HS in commissural neurons in vivo. We used the Wnt1-Cre transgene to drive region-specific ablation of Ext1, which encodes an enzyme essential for HS synthesis, in the dorsal part of the spinal cord. Remarkably, Wnt1-Cre-mediated ablation of Ext1 causes commissural axon pathfinding defects that share similarities with those of Netrin-1-deficient and DCC (deleted in colorectal cancer)-deficient mice. Neither Ext1-deficient dorsal spinal cord explants nor wild-type explants in which HS expression was ablated could extend axons in response to netrin-1. Intracellular signaling downstream of netrin-1 and DCC was defective in Ext1-deficient commissural neurons and in DCC-transfected HEK293T cells from which HS was removed. These results demonstrate that the expression of HS by commissural neurons is essential for these neurons to transduce netrin-1 signals, thus providing evidence for a cell-autonomous role of HS in netrin-1/DCC-mediated axon guidance.

<h4>Background</h4>Necrotizing enterocolitis (NEC), the devastating enteric process of premature neonates, is marked by severe intravascular abnormalities and disseminated intravascular coagulation. Treatment to date remains historical and continues to be merely supportive without attempts to ameliorate progress within the inflammatory or coagulation cascades. Antithrombin III (ATIII) supplementation has been shown to favorably alter the process of disseminated intravascular coagulation and sepsis in adults. However, no reported use of this treatment exists in neonates. Therefore, we analyze the efficacy of our recent experience with ATIII replacement therapy in neonates with NEC.<h4>Methods</h4>Age and diseased-matched controls with NEC were identified before the introduction of ATIII in our institution and compared against neonates with NEC undergoing ATIII replacement for diminished ATIII levels. Data collected included demographics, course of treatment parameters, and outcomes. Course of treatment parameters included hemoglobin, platelet count, prothrombin time, and partial thromboplastin time over the first 10 consecutive days of treatment. Outcome variables included packed red blood cell, platelet, fresh frozen plasma, and cryoprecipitate transfusions, as well as transfusion cost, length of stay, and survival.<h4>Results</h4>Over a 5-year period, 19 neonates with NEC received ATIII and were compared to 17 historical controls. Treatment hematologic profiles were not worsened in the ATIII-treated patients. The control patients received less overall transfusions and had a shorter length of stay.<h4>Conclusion</h4>Antithrombin III appears to be safe in neonates with NEC, and its impact on reversing intravascular pathology in these patients warrants more thorough investigation.

A novel way of seeing cellular receptors has recently emerged. While a receptor used to be considered as inactive until bound by its ligand, it has been proposed that some receptors may also be active in the absence of their ligand. These so-called dependence receptors induce a specific death signal when the ligand is absent from the cell: Therefore, the expression of one of these receptors leads to the cell becoming dependent on the presence of the ligand for its survival. We have hypothesized that such a trait is a mechanism that allows inhibition of tumor growth, by inducing apoptosis "abnormal" cells that would usually grow in settings of ligand unavailability, i.e. local growth of tumor cells or growth beyond primary tumor site. Along this line, back in the early 90s, Vogelstein and colleagues suggested that a gene called DCC (for deleted in colorectal cancer) could be a tumor suppressor gene because it was found to be deleted in more than 70% of colorectal cancers, as well as in many other cancers. During the last fifteen years, controversial data have failed to firmly establish whether DCC is indeed a tumor suppressor gene. However, our observation that DCC behaves as a dependence receptor that induces cell death unless its ligand netrin-1 is present, together with the fact that mice engineered to block DCC-induced cell death by overexpressing netrin-1 are predisposed to develop colorectal tumors, strengthen the role of dependence receptors as tumor suppressors. In this review, we will describe the implication of the netrin-1/receptor pairs as novel negative regulators of tumor development.

Liver fibrosis and cirrhosis are predisposing factors for the development of hepatocellular carcinoma (HCC). Hemosiderosis has also been described to trigger carcinogenesis. A significant iron overload, as found in hereditary hemochromatosis (HHC), is a risk factor for HCC and may also promote the symptoms of porphyria cutanea tarda (PCT). A 68-year old male patient presented to our clinic with a suspected HCC, elevated alpha-fetoprotein but normal liver function tests. He reported a 25 year-old history of vitiligo upon exposure to sunlight. The patient underwent an extended left hemihepatectomy, and the recovery was uneventful, with the exception of a persistent hyperbilirubinemia. Perfusion problems and extrahepatic cholestasis were ruled out by CT-scan with angiography and MR-cholangiopancreatography. However, MRI showed an iron overload. Histology confirmed the HCC (pT3, pN0, G3, R0) and revealed a portal fibrosis and hemosiderosis. Based on the skin lesions we suspected a PCT that was confirmed by laboratory tests showing elevated porphyrin, uroporphyrin, coproporphyrin and porphobilinogen. Concurrently, molecular diagnostics revealed homozygosity for the C282Y mutation within the hemochromatosis HFE gene. After phlebotomy and normalization of liver function tests the patient was discharged. This is the first case ever showing the unusual combination of HCC in a fibrotic liver with HHC and PCT. This diagnosis not only warrants oncological follow-up but also symptomatic therapy to normalize iron metabolism and thereby improve liver function and alleviate the symptoms of HHC and PCT. Thus progression of fibrosis may be prevented and liver regeneration supported.

<h4>Objective</h4>To identify altered gene expression in the dorsolateral prefrontal cortex obtained after death from subjects with schizophrenia.<h4>Method</h4>Restriction fragment differential display (RFDD) was used to measure levels of mRNA in Brodmann area (BA) 46 from schizophrenia and control subjects. Findings on specific mRNA identified with RFDD were further investigated using real-time polymerase chain reaction (real-time PCR), PCR and western blotting.<h4>Results</h4>Levels of mRNA for 63 of approximately 12,500 genes differed in BA 46 in schizophrenia. Subsequent real-time PCR has shown that mRNA for muscleblind protein 1 (MBNL1) and protocadherin 17 (PCDH17) are increased in BA 46 from subjects with schizophrenia of short, but not long, duration. Altered levels of mRNA for neither gene were present in BA 9 from subjects with schizophrenia or in either cortical area from subjects with bipolar 1 disorder. By contrast, both RFDD and real-time PCR failed to show altered expression of the schizophrenia candidate gene disrupted in schizophrenia 1 (DISC1) BA46 from any diagnostic cohort.<h4>Conclusion</h4>The present study has identified genes that are differentially expressed in BA 46 in schizophrenia. Initial studies have shown that there is a need for a careful validation of genes shown to be affected in schizophrenia using high-throughput technologies. In addition the present study has shown that gene expression may vary considerably depending on the duration of schizophrenia. This raises the hypothesis that changing gene expression may be underlying the change in symptom profile that occurs with disease progression in some subjects with schizophrenia.

<h4>Aim</h4>To investigate the histopathological and genetic differences between polypoid growth (PG) and non-polypoid growth (NPG) submucosal invasive colorectal carcinoma (CRC).<h4>Methods</h4>A total of 96 cases of submucosal CRC were divided into two groups according to their growth type; 60 cases of PG and 36 cases of NPG. The size, histological degree of dysplasia, depth of submucosal invasion and lymph node metastasis were compared between the two groups. Furthermore, expression of p53 was detected by immunohistochemical staining, and K-ras gene mutation was examined by polymerase chain reaction based single-strand conformation polymorphism (SSCP).<h4>Results</h4>The average size of the lesions in the NPG group was significantly smaller than those in the PG group (7.5 mm vs 13.8 mm, P<0.001). The histological degree of dysplasia tended to be more severe in NPG group, while the incidence of submucosal massive invasion and the lymph node metastasis were both significantly higher in the NPG type than in the PG group (64.3% vs 43.3%, P=0.004; 43% vs 7%, P=0.008, respectively). In addition, K-ras gene mutations were detected in 67% of lesions in the PG group, but none in the NPG group, while no difference in p53 immunohistochemical expression was found between the two groups.<h4>Conclusion</h4>Compared with PG submucosal CRC, NPG type demonstrates more frequent submucosal massive invasion, more lymph node metastasis and a higher degree dysplasia. Genetically, NPG type shows much less frequent K-ras mutation.

<h4>Aim</h4>To compare and identify the differences in expression of retinal proteins between normal and diabetic rats, and to analyze the molecular pathogenetic mechanisms of retinal diseases caused by diabetes.<h4>Methods</h4>Changes in protein expression of retinal tissues from diabetic and normal rats were observed using 2-dimensional polyacrylamide gel electrophoresis (2-DE). Some protein spots exhibiting statistically significant variations (P<0.05) were selected randomly and identified by tandem mass spectrometry and analyzed by bioinformatics.<h4>Results</h4>2-DE showed that the expression was up-regulated in 5 retinal proteins, down-regulated in 23 retinal proteins, and disappeared in 8 retinal proteins. Eight spots were identified from the 36 spots by tandem mass spectrometry (MS/MS) and analyzed by bioinformatics. Guanylate kinase 1, triosephosphate isomerase 1, ATP synthase subunit d, albumin and dimethylarginine dimethylaminohydrolase 2 played an important role in signal transduction. Triosephosphate isomerase 1, crystallin alpha B, ATP synthase subunit d and peroxiredoxin 6 were involved in energy metabolism of retinal tissues. Guanylate kinase 1 played an important role in photoexcitation of retinal rod photoreceptor cells. Whether crystallin beta A1 plays a role in diabetic retinas is unknown so far.<h4>Conclusion</h4>There are differences in expression of retinal proteins between diabetic and normal rats. These proteins may be involved in the mechanisms and prognosis of retinal diseases caused by diabetes.

While selective neuronal death has been an influential theme in Huntington's disease (HD), there is now a preponderance of evidence that significant neuronal dysfunction precedes frank neuronal death. The best evidence for neuronal dysfunction is the observation that gene expression is altered in HD brain, suggesting that transcriptional dysregulation is a central mechanism. Studies of altered gene expression began with careful observations of postmortem human HD brain and subsequently were accelerated by the development of transgenic mouse models. The application of DNA microarray technology has spurred tremendous progress with respect to the altered transcriptional processes that occur in HD, through gene expression studies of both transgenic mouse models as well as cellular models of HD. Gene expression profiles are remarkably comparable across these models, bolstering the idea that transcriptional signatures reflect an essential feature of disease pathogenesis. Finally, gene expression studies have been applied to human HD, thus not only validating the approach of using model systems, but also solidifying the idea that altered transcription is a key mechanism in HD pathogenesis. In the future, gene expression profiling will be used as a readout in clinical trials aimed at correcting transcriptional dysregulation in Huntington's disease.

A 47-year-old white male patient who manifested biochemical evidence of iron overload was found not to be a carrier of the three most common mutations, C282Y, H63D and S65C, of the HFE gene. Sequencing of the patient's entire HFE-coding region revealed a presence of a previously undescribed frameshift deletion c.471del in exon 3 resulting in a premature termination of a nonsense HFE protein. Interestingly, the patient was a homozygous carrier of this novel mutation and his hemochromatosis phenotype can be explained by the fact that he has no intact HFE protein. To the best of our knowledge, this is the first description of a complete loss of function of the HFE gene because of a homozygous mutation. The patient's son was found to be a heterozygous carrier of the mutation and has so far exhibited no indications of iron overload. Similarly, A*02-B*40/A*02-B*40 homozygous human leukocyte antigen (HLA) genotype was determined in the patient and heterozygous A*02-B*40/A*03-B*35 HLA genotype in his son. Thus, the novel HFE frameshift deletion c.471del was linked to the HLA-A*02-B*40 haplotype.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (htt) gene. Emergence and progression of HD depend on continuous expression of mutant Huntingtin protein (Htt). Therefore, blocking expression of mutant Htt might be a promising therapeutic strategy. We generated a high-capacity adenoviral (HC-Ad) vector expressing a short hairpin RNA (shRNA) targeted to exon 1 of the htt gene. In vitro, this vector efficiently inhibited Htt expression in neuronal and nonneuronal cell lines. In addition, the number of Htt-immunoreactive (IR) aggregates, a hallmark of HD pathology, was significantly reduced after gene transfer with this vector. Importantly, the attenuation of aggregate formation by shRNA was observed in vivo after stereotaxic injection into the striatum of mouse models of HD. The vector was tested in two models: the R6/2 transgenic mouse model and a mouse model based on the local injection of an adenoviral vector expressing a truncated version of mutant Htt. In both models an efficient reduction in mutant Htt aggregate load measured by decreased Htt-IR aggregate formation was observed. Our results support the further development of shRNA for HD therapy.

The completely assembled human genome has made it possible for modern medicine to step into an era rich in genetic information and high-throughput genomic analysis. These novel and readily available genetic resources and analytical tools may be the key to unravel the molecular basis of hepatocellular carcinoma (HCC). Moreover, since an efficient treatment for this disease is lacking, further understanding of the genetic background of HCC will be crucial in order to develop new therapies aimed at selected targets. We report on the current status and recent developments in HCC genetics. Special emphasis is given to the genetics and regulation of major signalling pathways involved in HCC such as p53, Wnt-signalling, TGFbeta, Ras, and Rb pathways. Furthermore, we describe the influence of chromosomal aberrations as well as of DNA methylation. Finally, we report on the rapidly developing field of genomic expression profiling in HCC, mainly by microarray analysis.

<h4>Objective</h4>Provide an overview of the demographics and pathology of breast cancer in the female population of Karachi South during a 3 year period, 1995-1997.<h4>Methods</h4>Epidemiological data for 709 incident breast cancer cases, ICD-10 category C50 registered at Karachi Cancer Registry during 1st January 1995 to 31st December 1997 were reviewed.<h4>Results</h4>Breast cancer accounted for approximately one-third of the cancers in females. The age standardized incidence rate (ASR) world per 100,000 was 53.8, the crude incidence rate was 30.9. In KS 60% of the newly diagnosed breast cancers were observed in women below 50 years. The age-specific curves showed a gradual increase in risk from the third up till the seventh decade, followed by an actual/apparent decrease in risk. The socio-economic distribution was 24.9% in category I the financially deprived class, 38.9% in category II the middle class and 35.9% in category III, the affluent class. Microscopic confirmation of malignancies was 99%. Invasive breast cancers predominated with 99.4%, with in-situ cancers contributing to 0.6% of the malignancies. The morphology of cancers was tilted towards duct cell carcinoma (DCC), pure DCC (92%), combinations of DCC /Paget's disease (0.6%) and lobular carcinoma (0.4%). Approximately 45% of duct cell carcinoma were seen in the premenopausal age group (<45 years). All bilateral breast cancers were duct cell carcinoma with a family history of first degree relative with breast cancer. The majority of the cases presented as moderately differentiated or grade 2 lesions (59.0%). Approximately 56% cancers had spread to the regional lymph nodes and 8.3% to a distant site at the time of diagnosis. A family history of first degree relative with breast cancer was present in 3% and second degree relatives in 7% of the cases. Odds ratio (OR) for 680 breast cancer cases with complete demographic information was calculated with 675 gender matched controls. A slightly higher risk was observed in non-Muslims and migrant ethnicities: two to three fold elevation in the Indian migrants (Gujrati speaking Mohajirs OR 3.86 (95% CI 2.51; 5.92) Urdu speaking Mohajirs OR 2.85 (95% CI 2.05; 3.96), Memon Mohajirs OR 2.21 (95% CI 1.48; 3.29) and Afghan migrants [OR 2.99 (95% CI 11.20; 7.44)]. The risk was also high in the females of Punjabi ethnicity settled in KS [OR 2.73 (95% CI 1.87; 3.99)]. The risk seems much less for the ethnicities belonging to North Western Pakistan i.e. Pathans [OR 1.684 (95% CI 0.89; 3.17)] and Baluchs [OR 0.90 (95% CI 0.58; 1.39)]. A marginally higher risk was observed in the higher socio-economic categories. The risk of developing breast cancer increased gradually for each age category from illiterate [OR 1.2 (95% CI 0.94; 1.55)] to college graduates [OR 13.12 (95% CI 7.31; 23.73)].<h4>Conclusions</h4>The incidence of breast cancer in Karachi South (KS) for the period 1995-1997 was the third highest in Asia. The hallmarks were a high reproductive age malignancy involving a higher socio-economic class, an invasive duct cell carcinoma diagnosed at an advanced stage, in younger more educated females and a low in-situ malignancy. More studies are required to obtain a deeper insight into this breast cancer epidemic in Karachi. Implementation of breast cancer screening with stress on public health education is today a major responsibility of the government.