No abstract available

To clarify the contribution of Cblb to the development of type1 diabetes (T1D), we investigated Japanese younger-onset T1D patients. We sequenced the cblb gene in 10 T1D patients and screened the identified mutations in 109 Japanese T1D patients and 100 normal subjects. In addition to four previously reported synonymous single nucleotide polymorphisms (SNPs), we identified two novel nonsynonymous variants (786 C>T (A155V) and 1718 A>G (N466D)). The A155V mutation was found in one subject with Basedow's disease whose mother also carried both the mutation and Basedow's disease. The N466D mutation was found in 6 T1D cases including a subject who was classified as fulminant T1D. We found no significant differences in the allele frequency of these SNPs among T1D and control subjects, suggesting that the contribution of cblb to the genetic susceptibility to T1D might not be high for Japanese younger-onset T1D.

Despite many claims of broad benefits, especially for in utero development, derived from the consumption of fish as a source of omega-3 fatty acids, individual species of fish and shellfish provide substantially varied levels of these fatty acids. Likewise, mean methylmercury (MeHg) concentrations for fish and shellfish species differ by greater than an order of magnitude. Consideration of within-species variability would increase this variation farther. Exposures to both MeHg and to the omega-3 fatty acids reflect dietary choices including species consumed, frequency of consumption, and portion size. In view of these sources of variability, data on dietary patterns and blood mercury (microg/L) among women of child-bearing age (e.g., 16-49 years) provided an indication of exposures in the United States. Utilizing data from the National Health and Nutrition Examination Survey (NHANES) for survey years 1999--2002, calculated consumption of MeHg and omega-3 fatty acids from fish and shellfish have been estimated based on results from 3614 women who provided 30-day dietary recall and 24-hours records. Statistics from NHANES when appropriately weighted are representative of the US population. The association between dietary MeHg from fish and shellfish and dietary fish intake yielded a Pearson correlation of 0.68. The Pearson correlation between estimated 30-day intake from fish/shellfish consumption for omega-3 fatty acids and MeHg was 0.66. Evaluation of the most commonly consumed fish and shellfish species as sources of MeHg and omega-3 fatty acids indicated that salmon followed by shrimp are principal sources of omega-3 fatty acids and are lesser sources of MeHg, in contrast with tuna which provides omega-3 fatty acids, but considerably higher levels of MeHg. These data can be used to guide selection of individual fish and shellfish species that are higher in omega-3 content and low in MeHg concentrations. This more refined dietary approach contrasts with generic recommendations that simply advise increasing fish consumption as a path toward improving cardiovascular health and providing benefits for in utero development or avoiding fish altogether.

FBN1, the gene mutated in Marfan syndrome, encodes fibrillin-1, a large glycoprotein component of the extracellular microfibrils. Human FBN1 has three untranslated upstream exons, and homologous sequences can be identified in a number of mammalian species. In this work, we have used functional assays to characterize the FBN1 upstream region. Sequences upstream of exon 1 and at least two of the upstream untranslated exons were shown to possess promoter activity in vitro. The strongest activity in luciferase assays was shown for sequences upstream of the untranslated exon A. Sequence analysis of the sequences in and upstream of exon A in humans and six other mammalian species demonstrated several highly conserved potential cis-acting sequences as well as a 66-basepair (bp) ultraconserved sequence with nearly perfect conservation in the seven species. The ultraconserved sequence contains an initiator element (Inr), a downstream promoter element (DPE), and a 10-bp palindromic element. Mutational assays showed that both the Inr and the DPE are critical for full promoter activity. A mutation of the 10-bp palindromic element completely abolished basal promoter activity. The element was shown to bind specifically to an unknown nuclear protein by electrophoretic mobility shift assay. Ultraconservation within an alternate promoter has not been previously reported. We suggest that the ultraconservation may reflect the importance of finely tuned regulation of alternate transcription of FBN1 and that the sequences involved have been under negative selective pressure for at least the last 180 million years of mammalian evolution.

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.

Trypanosoma brucei harbors two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. We previously found that recombinant TbHK1 (rTbHK1) has hexokinase activity, while rTbHK2 has not, a finding attributed to differences in the C-termini of the proteins. Sequence analysis suggests that the C-termini of TbHKs are part of a newly identified conserved motif found in other eukaryotic hexokinases. Here, we have explored the role of tail residues in the differences in catalytic activity between TbHK1 and TbHK2. Our studies reveal that tail residues D454, F462, M466, and N469 are essential for HK activity while both I458 and V468 are required for catalysis and substrate specificity. To activate rTbHK2, all of the residues important for activity in rTbHK1 (D454, V458, F462, M466, V468, and N469) were required. These results indicate that the overall structure of the C-terminal tail influences the HK activity of rTbHK1.

Slice preparations isolate functional networks, permitting single unit recording under visual control, and the use of fluorescent indicators. Circuits of interest often lie at a tilt in both the rostrocaudal and ventrodorsal axis, thus exposing circuits of interest at the cut surface of a slice would require a device for tilting a preparation along two orthogonal axes relative to the blade. Such a device, designed to be used in conjunction with a vibrating microtome, permitting the isolation of slice preparations at reproducible angles, is described here. Because the two orthogonal axes of tilt can be independently and continuously adjusted, it is possible to use this device to successively refine tilt parameters from preparation to preparation for optimal exposure of circuits of interest, facilitating the development of new slice preparations. Its use in cutting a thick medullary slab preparation, isolated from the neonate rat, which exposes respiratory networks at the cut surface is described.

Ongoing efforts for sequencing the genome of the protozoan parasite Perkinsus marinus, together with functional genomic initiatives, have continued to provide invaluable information about genes and metabolic pathways that not only will increase our understanding of its biology, but also have the potential to reveal useful targets for intervention. The lack of molecular tools for the functional characterization of genes of interest, however, has hindered progress in this regard. Here we report the development and validation of transfection methodology for this parasite. We first selected from our P. marinus EST collection a highly expressed gene, which we designated "MOE" (PmMOE), to which we fused at the C-terminus the enhanced green fluorescent protein (GFP) as a reporter gene (pPmMOE-GFP). The exogenous DNA was introduced into the trophozoite stage of the parasite by electroporation using the Nucleofector technology. The transfection efficiency was 37.8% with fluorescence detected as early as 14 h after electroporation, with the transfectants still remaining fluorescent after 8 months even in the absence of drug selection. The 5' flanking region was essential for transcription; constructs with 100 and 204 bp flanking the transcription start site also drove transcription effectively. Polymerase chain reaction (PCR) and Southern blot analyses was consistent with integration by non-homologous recombination. This transfection technique, the first one reported for a member of the Perkinsozoa, provides a new tool for studies of gene regulation and expression, protein targeting, and protein-protein interactions, and should significantly contribute to gain further insight into the biology of Perkinsus spp.

Influenza B viruses were predominant in Taiwan during the 2004-2005 epidemic and both Victoria and Yamagata lineage viruses co-circulated. A reassortant influenza B virus that contained a Victoria lineage hemagglutinin (HA) gene and Yamagata lineage neuraminidase (NA) gene appeared first in 2002 and became predominant during the 2004-2005 epidemic. During the 2006-2007 epidemic, an influenza B outbreak occurred in Taiwan and only Victoria lineage viruses circulated. We characterized the viruses isolated in the 2006-2007 epidemic and found that the HA genes of influenza B viruses from that epidemic were highly similar to those from the 2004-2005 epidemic. We also analyzed the NA genes of isolates from the 2006-2007 epidemic and found that they all belonged to the Yamagata lineage and formed a new genetic subclade. Comparison of isolates from the 2004-2005 and 2006-2007 epidemics revealed four substitutions, N220K, E320D, K343R and E404K in NA genes. Although the HA sequences from the 2006-2007 epidemic were similar to those from the 2004-2005 epidemic, the NA sequences differed, suggesting distinct patterns of evolution of the HA and NA genes from 2004-2007 in Taiwan. This study emphasizes that the evolution of the NA genes may contribute to reemergence of influenza B viruses.

Flexibly adapting behavior in dynamic environments relies on fronto-limbic networks that include the amygdala, orbitofrontal cortex, and striatum. Animal work demonstrates that interactions among these regions are critical for flexible feedback-guided learning, but it remains unknown to what extent such anatomical-functional interactions operate in humans. Here, we use connectivity analyses in both structural and functional MRI to further our understanding of how brain circuits work in conjunction to promote goal-directed behavior. In particular, fiber tracking based on diffusion-weighted imaging provides information about anatomical connectivity between brain structures, and functional MRI provides estimates of functional connectivity between structures. We found that, during a feedback-guided reversal learning task, the strength of estimated white matter tracts from the amygdala to the hippocampus, orbitofrontal cortex, and ventral striatum predicted both how subjects adapted their behavior following positive and negative feedback, and the functional connectivity (estimated from functional MRI time series) between the amygdala and these regions. In addition, we identified a dissociation between an amygdala-hippocampus circuit that predicted response switching, and an amygdala-orbitofrontal cortex circuit that predicted learning following rule reversals. These findings provide novel insights into how the anatomy and functioning of amygdala-related brain circuits mediate different aspects of feedback-guided learning behavior.

We previously demonstrated that some fish have unique response in the form of reactive oxygen species (ROS) production. In the present study, we cloned and sequenced the full-length cDNAs for carp (Cyprinus carpio) phagocyte NADPH oxidase components: gp91phox, p22phox, p47phox, p67phox and p40phox. These amino acid sequences were compared with other teleost and mammalian homologues, to elucidate the features of ROS production of fish neutrophils. The phylogeny analysis clearly demonstrates that the radiation of phagocyte oxidase components took place in the common ancestor of teleosts and mammals. Thereafter, the overall structure and expression pattern of phagocyte oxidase have been highly conserved in two different strains. However the amino acid identity of p67phox and p47phox was relatively lower than the amino acid identities of other components. Moreover, a synteny analysis supports the hypothesis that there was strong selective pressure in the p67phox and p47phox genes. Thus, it is likely that the higher divergence of p67phox and p47phox are responsible for the difference of ROS responses between different species of teleosts.

The phonological deficit hypothesis of dyslexia was studied by analyzing language-related lateralization of theta (4-8 Hz) and beta rhythms (13-30 Hz) during various phases of word processing in a sample of 14 dyslexics and 28 controls. Using a word-pair paradigm, the same words were contrasted in three different tasks: Phonological, Semantic and Orthographic. Compared with controls, dyslexic children showed a delay in behavioral responses which was paralleled by sustained theta EEG peak activity. In addition, controls showed greater theta and beta activation at left frontal sites specifically during the Phonological task, whereas dyslexics showed a dysfunctional pattern, as they were right-lateralized at these sites in all tasks. At posterior locations, and reversed with respect to controls' EEG responses, dyslexics showed greater left lateralization during both Phonological and Orthographic tasks--a result which, in these children, indicates an altered and difficult phonological transcoding process during verbal working memory phases of word processing. Results point to a deficit, in phonological dyslexia, in recruitment of left hemisphere structures for encoding and integrating the phonological components of words, and suggest that the fundamental hierarchy within the linguistic network is disrupted.

Activation of steroid receptors results in global changes of gene expression patterns. Recent studies showed that steroid receptors control only a portion of their target genes directly, by promoter binding. The majority of the changes are indirect, through chromatin rearrangements. The mediators that relay the hormonal signals to large-scale chromatin changes are, however, unknown. We report here that APRIN, a novel hormone-induced nuclear phosphoprotein has the characteristics of a chromatin regulator and may link endocrine pathways to chromatin. We showed earlier that APRIN is involved in the hormonal regulation of proliferative arrest in cancer cells. To investigate its function we cloned and characterized APRIN orthologs and performed homology and expression studies. APRIN is a paralog of the cohesin-associated Pds5 gene lineage and arose by gene-duplication in early vertebrates. The conservation and domain differences we found suggest, however, that APRIN acquired novel chromatin-related functions (e.g. the HMG-like domains in APRIN, the hallmarks of chromatin regulators, are absent in the Pds5 family). Our results suggest that in interphase nuclei APRIN localizes in the euchromatin/heterochromatin interface and we also identified its DNA-binding and nuclear import signal domains. The results indicate that APRIN, in addition to its Pds5 similarity, has the features and localization of a hormone-induced chromatin regulator.

The health hazards and prevalence of ecstasy use have been documented in two decades of research, but no review reporting on potentially modifiable antecedents of use is available. The aim of this study was to integrate systematically research identifying cognitive correlates of ecstasy use. Such research has the potential to identify targets for evidence-based interventions designed to discourage use.

The aim of the study was to investigate if an abnormal brain response to pain exists in patients with myofascial pain syndrome (MPS) when stimulated in a hypersensitive myofascial trigger point (MTP). Event-related functional magnetic resonance imaging was used to characterize the brain response to pain evoked from an MTP. Activation patterns from patients were compared with those evoked from an equivalent site in healthy controls with stimulus intensity matched and pain intensity matched stimuli. Compared to healthy controls at matched stimulus intensity, patients experienced significantly higher pain intensity (hyperalgesia). The corresponding brain response revealed significantly enhanced somatosensory (SI, SII, inferior parietal, mid-insula) and limbic (anterior insula) activity and suppressed right dorsal hippocampal activity in patients compared with controls. At matched pain intensity, enhanced activity was found in the same somatosensory areas but not in limbic areas. Our results show that the hyperalgesic state observed in MPS patients was associated with abnormal hyperactivity in regions processing stimulus intensity and negative affect. We speculate that suppressed hippocampal activity might reflect stress-related changes in relation to chronic pain as an effective physical and emotional stressor.

Traumatic brain injury (TBI) is one of the most common causes of long-term disability. Despite the importance of identifying neuropathology in individuals with chronic TBI, methodological challenges posed at the stage of inter-subject image registration have hampered previous voxel-based MRI studies from providing a clear pattern of structural atrophy after TBI. We used a novel symmetric diffeomorphic image normalization method to conduct a tensor-based morphometry (TBM) study of TBI. The key advantage of this method is that it simultaneously estimates an optimal template brain and topology preserving deformations between this template and individual subject brains. Detailed patterns of atrophies are then revealed by statistically contrasting control and subject deformations to the template space. Participants were 29 survivors of TBI and 20 control subjects who were matched in terms of age, gender, education, and ethnicity. Localized volume losses were found most prominently in white matter regions and the subcortical nuclei including the thalamus, the midbrain, the corpus callosum, the mid- and posterior cingulate cortices, and the caudate. Significant voxel-wise volume loss clusters were also detected in the cerebellum and the frontal/temporal neocortices. Volume enlargements were identified largely in ventricular regions. A similar pattern of results was observed in a subgroup analysis where we restricted our analysis to the 17 TBI participants who had no macroscopic focal lesions (total lesion volume >1.5 cm(3)). The current study confirms, extends, and partly challenges previous structural MRI studies in chronic TBI. By demonstrating that a large deformation image registration technique can be successfully combined with TBM to identify TBI-induced diffuse structural changes with greater precision, our approach is expected to increase the sensitivity of future studies examining brain-behavior relationships in the TBI population.

The Ensembl project (http://www.ensembl.org) is a comprehensive genome information system featuring an integrated set of genome annotation, databases and other information for chordate and selected model organism and disease vector genomes. As of release 47 (October 2007), Ensembl fully supports 35 species, with preliminary support for six additional species. New species in the past year include platypus and horse. Major additions and improvements to Ensembl since our previous report include extensive support for functional genomics data in the form of a specialized functional genomics database, genome-wide maps of protein-DNA interactions and the Ensembl regulatory build; support for customization of the Ensembl web interface through the addition of user accounts and user groups; and increased support for genome resequencing. We have also introduced new comparative genomics-based data mining options and report on the continued development of our software infrastructure.

The Escherichia coli RNA chaperone Hfq is involved in riboregulation of target mRNAs by small trans-encoded non-coding (ncRNAs). Previous structural and genetic studies revealed a RNA-binding surface on either site of the Hfq-hexamer, which suggested that one hexamer can bring together two RNAs in a pairwise fashion. The Hfq proteins of different bacteria consist of an evolutionarily conserved core, whereas there is considerable variation at the C-terminus, with the gamma- and beta-proteobacteria possessing the longest C-terminal extension. Using different model systems, we show that a C-terminally truncated variant of Hfq (Hfq(65)), comprising the conserved hexameric core of Hfq, is defective in auto- and riboregulation. Although Hfq(65) retained the capacity to bind ncRNAs, and, as evidenced by fluorescence resonance energy transfer assays, to induce structural changes in the ncRNA DsrA, the truncated variant was unable to accommodate two non-complementary RNA oligonucleotides, and was defective in mRNA binding. These studies indicate that the C-terminal extension of E. coli Hfq constitutes a hitherto unrecognized RNA interaction surface with specificity for mRNAs.

HOCl-modified low-density lipoprotein (LDL) has proinflammatory effects, including induction of inflammatory cytokine production, leukocyte adhesion, and ROS generation, but the components responsible for these effects are not completely understood. HOCl and the myeloperoxidase-H(2)O(2)-halide system can modify both protein and lipid moieties of LDL and react with unsaturated phospholipids to form chlorohydrins. We investigated the proinflammatory effects of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine (SOPC) chlorohydrin on artery segments and spleen-derived leukocytes from ApoE(-/-) and C57 Bl/6 mice. Treatment of ApoE(-/-) artery segments with SOPC chlorohydrin, but not unmodified SOPC, caused increased leukocyte-arterial adhesion in a time- and concentration-dependent manner. This could be prevented by pretreatment of the artery with P-selectin or ICAM-1-blocking antibodies, but not anti-VCAM-1 antibody, and immunohistochemistry showed that P-selectin expression was upregulated. However, chlorohydrin treatment of leukocytes did not increase expression of adhesion molecules LFA-1 or PSGL-1, but caused increased release of ROS from PMA-stimulated leukocytes by a CD36-dependent mechanism. The SOPC chlorohydrin-induced adhesion and ROS generation could be abrogated by pretreatment of the ApoE(-/-) mice with pravastatin or a nitrated derivative, NCX 6550. These findings suggest that phospholipid chlorohydrins formed in HOCl-treated LDL could contribute to the proinflammatory effects observed for this modified lipoprotein in vitro.

We studied the immune compartment in patients with myelodysplastic syndromes. We show increased surface expression of activation markers (HLA-DR(+), CD57(+), CD28(-), CD62L(-)) on T lymphocytes in blood and bone marrow (n=131). T cell activation was not restricted to any relevant clinical subgroup (FAB, IPSS, cytogenetics) and did not correlate with blood counts or need for treatment. In vitro clonogenic growth of marrow mononuclear cells (n=18) was not influenced by T cells expressing these markers. In addition, using X-chromosome inactivation analysis (n=12) we demonstrate clonal involvement of NK and B cells in half of these patients. We conclude that although activated T lymphocytes can be found in MDS, their role in disease pathogenesis remains unclear in the majority of patients.