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Nuclear aggregates formed by proteins containing expanded poly-glutamine (poly-Q) tracts have been linked to the pathogenesis of poly-Q neurodegenerative diseases. Here, we show that a protein (GFP170*) lacking poly-Q tracts forms nuclear aggregates that share characteristics of poly-Q aggregates. GFP170* aggregates recruit cellular chaperones and proteasomes, and alter the organization of nuclear domains containing the promyelocytic leukemia (PML) protein. These results suggest that the formation of nuclear aggregates and their effects on nuclear architecture are not specific to poly-Q proteins. Using GFP170* as a model substrate, we explored the mechanistic details of nuclear aggregate formation. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses show that GFP170* molecules exchange rapidly between aggregates and a soluble pool of GFP170*, indicating that the aggregates are dynamic accumulations of GFP170*. The formation of cytoplasmic and nuclear GFP170* aggregates is microtubule-dependent. We show that within the nucleus, GFP170* initially deposits in small aggregates at or adjacent to PML bodies. Time-lapse imaging of live cells shows that small aggregates move toward each other and fuse to form larger aggregates. The coalescence of the aggregates is accompanied by spatial rearrangements of the PML bodies. Significantly, we find that the larger nuclear aggregates have complex internal substructures that reposition extensively during fusion of the aggregates. These studies suggest that nuclear aggregates may be viewed as dynamic multidomain inclusions that continuously remodel their components.

We show that even in the complete absence of potential energies among the atoms in a protein-aqueous solution system, there is a physical factor that favors the folded state of the protein. It is a gain in the translational entropy (TE) of water originating from the translational movement of water molecules. An elaborate statistical-mechanical theory is employed to analyze the TE of water in which a protein or peptide with a prescribed conformation is immersed. It is shown that if the number of residues is sufficiently large, the TE gain is powerful enough to compete with the conformational-entropy loss upon folding. For protein G we have tested over 100 compact conformations generated by a computer simulation with the all-atom potentials as well as the native structure. A significant finding is that the largest TE is attained in the native structure. The translational movement of water molecules is quite effective in achieving the tight packing in the interior of a natural protein. These results are true only when the solvent is water whose molecular size is the smallest among the ordinary liquids in nature.

No abstract available.

Dendrimers are emerging as a relatively new class of polymeric biomaterials with applications in drug delivery, and imaging. Achieving a high drug payload in dendrimers, and understanding the therapeutic effect of the dendrimer-drug conjugates are receiving increasing attention. A high drug payload nanodevice was obtained by covalent conjugation of ibuprofen to a polyamidoamine (PAMAM-G4-OH) dendrimer. Using DCC as a coupling agent, 58 molecules of ibuprofen were covalently conjugated to one molecule of generation 4 PAMAM-OH dendrimer. Cellular entry of the fluoroisothiocynate (FITC)-labeled dendrimer-drug conjugate was evaluated in vitro by using human lung epithelial carcinoma A549 cells by flow cytometry, confocal microscopy and UV/Visible spectroscopy. The pharmacological activity of the dendrimer-ibuprofen conjugate was compared to pure ibuprofen at various time points by measuring the suppression of prostaglandin E2. Significant amounts of the conjugate entered the cells rapidly within 15 min. Suppression of prostaglandin was noted within 30 min for the dendrimer-drug conjugates versus 1 h for the free ibuprofen. The results suggest that dendrimers with high drug payload improve the drug's efficacy by enhanced cellular delivery, and may produce a rapid pharmacological response. These dendrimer-drug conjugates can potentially be further modified by attaching antibodies and ligands for targeted drug delivery.

Gene-expression profiling has identified 3 major subgroups of diffuse large B-cell lymphoma (DLBCL): germinal center B-cell-like (GCB), activated B-cell-like (ABC), and primary mediastinal DLBCL (PMBCL). Using comparative genomic hybridization (CGH), we investigated the genetic alterations of 224 cases of untreated DLBCL (87 GCB-DLBCL, 77 ABC-DLBCL, 19 PMBCL, and 41 unclassified DLBCL) previously characterized by gene-expression profiling. The DLBCL subgroups differed significantly in the frequency of particular chromosomal aberrations. ABC-DLBCL had frequent trisomy 3, gains of 3q and 18q21-q22, and losses of 6q21-q22, whereas GCB-DLBCL had frequent gains of 12q12, and PMBCL had gains of 9p21-pter and 2p14-p16. Parallel analysis of CGH alterations, locus-specific gene-expression profiles, and global gene-expression signatures revealed that DNA amplifications and gains had a substantial impact on the expression of genes in the involved chromosomal regions, and some genes were overexpressed in a DLBCL subgroup-specific fashion. Unexpectedly, specific chromosomal alterations were associated with significant changes in gene-expression signatures that reflect various aspects of lymphoma cell biology as well as the host response to the lymphoma. In addition, gains involving the chromosomal region 3p11-p12 provided prognostic information that was statistically independent of the previously defined gene-expression-based survival model, thereby improving its predictive power.

<h4>Background</h4>It has been reported that some persons with hemochromatosis have low total blood lymphocyte counts, but the reason for this is unknown.<h4>Methods</h4>We measured total blood lymphocyte counts using an automated blood cell counter in 146 hemochromatosis probands (88 men, 58 women) with HFE C282Y homozygosity who were diagnosed in medical care. Univariate and multivariate analyses of total blood lymphocyte counts were evaluated using these variables: sex; age, transferrin saturation, and serum ferritin concentration at diagnosis; units of blood removed by phlebotomy to achieve iron depletion; and human leukocyte antigen (HLA)-A and -B alleles and haplotypes.<h4>Results</h4>The mean age at diagnosis was 49 +/- 14 years (range 18 - 80 years) in men and 50 +/- 13 years (range 22-88 years) in women. The correlations of total blood lymphocyte counts with sex, age, transferrin saturation, and serum ferritin concentration at diagnosis, and units of blood removed by phlebotomy to achieve iron depletion were not significant at the 0.05 level. Univariate analyses revealed significant associations between total blood lymphocyte counts and presence of the HLA-A*01, -B*08, and -B*14 alleles, and the A*01-B*08 haplotype. Presence of the A*01 allele, B*08 allele, or A*01-B*08 haplotype were associated with a lower total blood lymphocyte count, whereas presence of the B*14 allele was associated with a greater total blood lymphocyte count. There was an inverse association of total blood lymphocyte count with units of phlebotomy to achieve iron depletion, serum ferritin concentration, and with presence of the A*01-B*08 haplotype.<h4>Conclusion</h4>We conclude that there is a significant inverse relationship of total blood lymphocyte counts and severity of iron overload in hemochromatosis probands with HFE C282Y homozygosity. The presence of the HLA-A*01 allele or the -B*08 allele was also associated with significantly lower total blood lymphocyte counts, whereas presence of the -B*14 allele was associated with significantly higher total blood lymphocyte counts. In univariate and multivariate analyses, total blood lymphocyte counts were significantly lower in probands with the HLA-A*01-B*08 haplotype than in probands without this haplotype.

The multifunctional Nef protein of HIV-1 is important for the progression to AIDS. One action of Nef is to down-regulate surface MHC I molecules, helping infected cells to evade immunity. We found that Nef also down-regulates the macrophage-expressed MHC 1b protein HFE, which regulates iron homeostasis and is mutated in the iron-overloading disorder hemochromatosis. In model cell lines, Nef reroutes HFE to a perinuclear structure that overlaps the trans-Golgi network, causing a 90% reduction of surface HFE. This activity requires a Src-kinase-binding proline-rich domain of Nef and a conserved tyrosine-based motif in the cytoplasmic tail of HFE. HIV-1 infection of ex vivo macrophages similarly down-regulates naturally expressed surface HFE in a Nef-dependent manner. The effect of Nef expression on cellular iron was explored; iron and ferritin accumulation were increased in HIV-1-infected ex vivo macrophages expressing wild-type HFE, but this effect was lost with Nef-deleted HIV-1 or when infecting macrophages from hemochromatosis patients expressing mutated HFE. The iron accumulation in HIV-1-infected HFE-expressing macrophages was paralleled by an increase in cellular HIV-1-gag expression. We conclude that, through Nef and HFE, HIV-1 directly regulates cellular iron metabolism, possibly benefiting viral growth.

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin (Htt) protein. N-terminal Htt peptides with polyglutamine tracts in the pathological range (51-122 glutamines) form high-molecular-weight protein aggregates with fibrillar morphology in vitro, and they form discrete inclusion bodies in a cell-culture model. However, in some studies, formation of discrete Htt inclusions does not correlate well with cell death. We coexpressed N-terminal Htt fragments containing 91 glutamines fused to different affinity tags in HEK293 cells, and we isolated small aggregates by double sequential-affinity chromatography to assure the isolation of multimeric molecules. Transmission electron microscopy and atomic force microscopy revealed the isolated aggregates as globules or clusters of globules 4-50 nm in diameter without any detectable fibrillar species. Because small nonfibrillar oligomers, not mature fibrils, recently have been suggested to be the principal cytotoxic species in neurodegenerative disease, these Htt globular aggregates formed in cells may represent the pathogenic form of mutant Htt.

<h4>Background</h4>Lowering of brain serotonin by acute tryptophan depletion (TD) frequently leads to transient symptoms of depression in vulnerable individuals but not in euthymic healthy subjects with a negative family history of depression. The effects of TD on regional serotonin transporter binding potential (5-HTT BP), an index of 5-HTT density and affinity, were studied in healthy individuals using 3-(11)C-amino-4-(2-dimethylaminomethylphenylsulfanyl)benzonitrile ([11C]DASB) positron emission tomography (PET). Adaptive decreases in 5-HTT density and/or affinity during TD would be a possible compensatory mechanism to maintain sufficient extracellular serotonin levels during TD, thereby preventing a depressive relapse.<h4>Methods</h4>Regional noninvasive 5-HTT BP was found in 25 healthy subjects using [11C]DASB PET. Fourteen subjects were scanned twice, once after TD and once after sham depletion, and 11 other healthy subjects were scanned twice to measure test-retest reliability of the method.<h4>Results</h4>None of the healthy subjects experienced depressive symptoms during TD and there was no difference in regional 5-HTT BP during TD as compared with sham depletion.<h4>Conclusions</h4>Acute changes in 5-HTT density or affinity are unlikely to play a role in protecting healthy subjects against mood symptoms during TD. Other mechanisms that may be associated with greater resilience against acute lowering of extracellular serotonin should be explored to gain further insight into the neurochemical basis of different vulnerabilities to short-term depressive relapse.

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-gamma, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.

<h4>Background</h4>Oligodendroglioma (ODG) and oligoastrocytoma (OAC) are diffusely infiltrating primary brain tumors whose pathogenesis remains unclear. We previously identified a group of genes whose expression was inversely correlated with survival in a cohort of patients with glioblastoma (GBM), and some of these genes are also reportedly expressed in ODG and OAC. We examined the expression patterns and localization of these survival-associated genes in ODG and OAC in order to analyze their possible roles in the oncogenesis of these two tumor types.<h4>Methods</h4>We used UniGene libraries derived from GBM and ODG specimens to examine the expression levels of the transcripts for each of the 50 GBM survival-associated genes. We used immunohistochemistry and cDNA microarrays to examine expression of selected survival-associated genes and Id4, a gene believed to control the timing of oligodendrocyte development. The expression of FABP7 and Id4 and the survival of patients with ODG and OAC were also analyzed.<h4>Results</h4>Transcripts of most survival-associated genes as well as Id4 were present in both GBM and ODG tumors, whereas protein expression of Id4 and one of the survival-associated genes, brain-type fatty acid-binding protein (FABP7), was present in cells with astrocytic features, including reactive and neoplastic astrocytes, but not in neoplastic oligodendrocytes. Id4 was co-expressed with FABP7 in microgemistocytes in ODG and in neoplastic astrocytes in OAC. Id4 and FABP7 expression, however, did not correlate with the clinical outcome of patients with ODG or OAC tumors.<h4>Conclusion</h4>Expression of Id4 and some of our previously identified GBM survival-associated genes is present in developing or mature oligodendrocytes. However, protein expression of Id4 and FABP7 in GBM, ODG, and OAC suggests that this group of functionally important genes might demonstrate two patterns of expression in these glioma subtypes: one group is universally expressed in glioma cells, and the other group of genes is expressed primarily in neoplastic astrocytes but not in neoplastic oligodendrocytes. Differential protein expression of these two groups of genes in ODG and OAC may be related to the cellular origins and the histological features of the neoplastic cells.

Mutant mice bearing a targeted disruption of the heparan sulfate (HS) modifying enzyme GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) exhibit severe developmental defects of the forebrain and forebrain-derived structures, including cerebral hypoplasia, lack of olfactory bulbs, eye defects and axon guidance errors. Neural crest-derived facial structures are also severely affected. We show that properly synthesized heparan sulfate is required for the normal development of the brain and face, and that Ndst1 is a modifier of heparan sulfate-dependent growth factor/morphogen signalling in those tissues. Among the multiple heparan sulfate-binding factors potentially affected in Ndst1 mutant embryos, the facial phenotypes are consistent with impaired sonic hedgehog (Shh) and fibroblast growth factor (Fgf) interaction with mutant heparan sulfate. Most importantly, the data suggest the possibility that defects in heparan sulfate synthesis could give rise to or contribute to a number of developmental brain and facial defects in humans.

Several trials have indicated that selective serotonin reuptake inhibitors (SSRIs) are most efficient in the treatment of social phobia (SP). The activity of the serotonin transporter (5-HTT), as determined by [3H]5-HT uptake to blood lymphocytes, was measured in 15 drug-free patients with generalized SP (7M/8F, aged 21-37 years) and compared to 18 healthy control subjects (10M/8F, aged 21-32 years). The maximum uptake velocity (Vmax) of [3H]5-HT to fresh lymphocytes and the affinity (Km) of the 5-HTT were similar in the two groups (295+/-155 versus 227+/-117 pmol/min/mg protein and 767+/-425 versus 709+/-408 nM, respectively). This study suggests that the functioning of lymphocyte 5-HTT is unaltered in SP.

Correlations exist between the incidence of depression, irritable bowel syndrome (IBS) and overactive bladder [Masand, P.S., Kaplan, D.S., Gupta, S., Bhandary, A.N., Nasra, G.S., Kline, M.D., Margo, K.L., 1995. Major depression and irritable bowel syndrome: is there a relationship? J. Clin. Psychiatry 56, 363-367.; Cukier, J.M., Cortina-Borja, M., Brading, A.F., 1997. A case-control study to examine any association between idiopathic detrusor instability and gastrointestinal tract disorder, and between irritable bowel syndrome and urinary tract disorder. Br. J. Urol. 79, 865-878.; Monga, A.K., Marrero, J.M., Stanton, S.L., Lemieux, M.C., Maxwell, J.D., 1997. Is there an irritable bladder in the irritable bowel syndrome? Br. J. Obstet. Gynaecol. 104, 1409-1412.; Zorn, B.H., Montgomery, H., Pieper, K., Gray, M., Steers, W.D., 1999. Urinary incontinence and depression. J. Urol. 162, 82-84.]. Furthermore, alterations in serotonergic neurotransmission may play a common role in the etiology of these disorders. Serotonin reuptake transporter knockout mice (5-HTT(-/-)) display phenotypes consistent with clinical features of mood and bowel disorders including anxiety and abnormal gastrointestinal motility [Holmes, A., Murphy, D.L., Crawley, J.N., 2003. Abnormal behavioral phenotypes of serotonin transporter knockout mice: parallels with human anxiety and depression. Biol. Psychiatry 54, 953-959.]. In the present study, we evaluated bladder function in 5-HTT(-/-) mice. We have found that female 5-HTT(-/-) mice exhibit bladder dysfunction, characterized by significant increases in the frequency of spontaneous non-voiding bladder contractions and decreases in void volume compared to control female mice. These differences were not observed in male knockout mice. These studies provide significant supporting data for a mechanistic link between alterations in 5-HT, depression, IBS and overactive bladder in women.

Five different silica gels containing two chiral ferrocenyl ligands were prepared by various synthetic routes and tested in an enantioselective Pd(0)-catalyzed allylic substitution and Rh-catalyzed hydrogenation. All the prepared anchored ligands were characterized by porosimetry data, DRIFTS spectra, thermal data and AAS. The aim of the work was to compare the influence of the carrier, surface properties and immobilization strategy on the performance of the catalyst.

In eukaryotes, the nuclear export of mRNA is mediated by nuclear export factor 1 (NXF1) receptors. Metazoans encode additional NXF1-related proteins of unknown function, which share homology and domain organization with NXF1. Some mammalian NXF1-related genes are expressed preferentially in the brain and are thought to participate in neuronal mRNA metabolism. To address the roles of NXF1-related factors, we studied the two mouse NXF1 homologues, mNXF2 and mNXF7. In neuronal cells, mNXF2, but not mNXF7, exhibited mRNA export activity similar to that of Tip-associated protein/NXF1. Surprisingly, mNXF7 incorporated into mobile particles in the neurites that contained poly(A) and ribosomal RNA and colocalized with Staufen1-containing transport granules, indicating a role in neuronal mRNA trafficking. Yeast two-hybrid interaction, coimmunoprecipitation, and in vitro binding studies showed that NXF proteins bound to brain-specific microtubule-associated proteins (MAP) such as MAP1B and the WD repeat protein Unrip. Both in vitro and in vivo, MAP1B also bound to NXF export cofactor U2AF as well as to Staufen1 and Unrip. These findings revealed a network of interactions likely coupling the export and cytoplasmic trafficking of mRNA. We propose a model in which MAP1B tethers the NXF-associated mRNA to microtubules and facilitates their translocation along dendrites while Unrip provides a scaffold for the assembly of these transport intermediates.

Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC) proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide-binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I-binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8-10-mer class I-binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC-binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.

<h4>Background</h4>A secreted peptide Pep27 initiates the cell death program in S. pneumoniae through signal transduction. This study was undertaken to evaluate the relation between the structure and cytotoxic activity of Pep27 and its analogues on cancer cells.<h4>Results</h4>Pep27anal2 characterized substituting (2R-->W), (4E-->W), (11S-->W) and (13Q-->W) in native Pep27, exhibited greater hydrophobicity and anticancer activity than Pep27 and other analogues. The IC50 values of Pep27anal2 were approximately 10 - 30 microM in a number of cell lines (AML-2, HL-60, Jurkat, MCF-7 and SNU-601). Confocal microscopy showed that Pep27anal2-FITC was localized in the plasma membrane, and then moving from the membrane to subcellular compartments with the initiation of membrane blebbing. Flow cytometric analysis using propidium iodide and Annexin V also revealed that Pep27anal2 induced apoptosis with minor membrane damage. Electron microscopy revealed that Pep27 induced apoptosis in Jurkat cells. The anticancer activity of Pep27anal2 was neither abrogated by pan-caspase inhibitor (Z-VAD-fmk) nor related to cytochrome c release from mitochondria. The 3D solution structures of these two Pep27 peptides revealed that both form a random coil conformation in water; however, they adopted stable alpha-helical conformations in solutions.<h4>Conclusion</h4>The results indicate that Pep27anal2 can penetrate the plasma membrane, and then induce apoptosis in both caspase-and cytochrome c-independent manner. The hydrophobicity of Pep27anal2 appears to play an important role in membrane permeabilization and/or anticancer properties. The structure-functional relationships of these peptides are also discussed. It is proposed that Pep27anal2 is a potential candidate for anticancer therapeutic agents.

A lentiviral vector expressing a mutant huntingtin protein (htt171-82Q) was used to generate a chronic model of Huntington's disease (HD) in rat primary striatal cultures. In this model, the majority of neurons expressed the transgene so that Western blot analysis and flow cytometry measurement could complement immunohistological evaluation. Mutant huntingtin produced a slowly progressing pathology characterized after 1 month by the appearance of neuritic aggregates followed by intranuclear inclusions, morphological anomalies of neurites, loss of neurofilament 160, increased expression in stress response protein Hsp70, and later loss of neuronal markers such as NeuN and MAP-2. At 2 months post-infection, a significant increase in TUNEL-positive cells confirmed actual striatal cell loss. Interestingly, cortical cultures infected with the same vector showed no sign of neuronal dysfunction despite accumulation of numerous inclusions. We finally examined whether the trophic factors CNTF and BDNF that were found neuroprotective in acute HD models could prevent striatal degeneration in a chronic model. Results demonstrated that both agents were neuroprotective without modifying inclusion formation. The present study demonstrates that viral vectors coding for mutant htt provides an advantageous system for histological and biochemical analysis of HD pathogenesis in primary striatal cultures.

Many endocytic proteins shuttle between the nucleus and the cytoplasm; however, their putative function in the nucleus is unclear. Now, new data demonstrate that huntingtin interacting protein 1 (HIP1), an endocytic protein, modulates the transcriptional activity of nuclear hormone receptors. In network theory, therefore, HIP1 can be regarded as a hub connecting heterogeneous functional "territories:" a possibility with important physiological and pathological implications.

Gene-specific expansion of polyglutamine-encoding CAG repeats can cause neurodegenerative disorders, including Huntington's disease. It is believed that part of the pathological effect of the expanded protein is due to transcriptional dysregulation. Using Drosophila as a model, we show that cAMP-response element-binding protein (CREB) is involved in expanded polyglutamine-induced toxicity. A mutation in the Drosophila homolog of CREB, dCREB2, enhances lethality due to polyglutamine peptides (polyQ), and an additional copy of dCREB2 partially rescues this lethality. Neuronal expression of expanded polyQ attenuates in vivo CRE-mediated transcription of a reporter gene. As reported previously, overexpression of heat-shock protein 70 (Hsp70) rescues polyglutamine-dependent lethality. However, it does not rescue CREB-mediated transcription. The protective effects of CREB and heat-shock protein 70 against polyQ are additive, suggesting that targeting multiple pathways may be effective for treatment of polyglutamine diseases.

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the approximately 1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.

The fusion peptide (FP) in the N terminus of the HIV envelope glycoprotein, gp41, functions together with other gp41 domains to fuse the virion with the host cell membrane. We now report that FP colocalizes with CD4 and TCR molecules, coprecipitates with the TCR, and inhibits antigen-specific T cell proliferation and proinflammatory cytokine secretion in vitro. These effects are specific: T cell activation by PMA/ionomycin or mitogenic antibodies is not affected by FPs, and FPs do not interfere with antigen-presenting cell function. In vivo, FPs inhibit the activation of arthritogenic T cells in the autoimmune disease model of adjuvant arthritis and reduce the disease-associated IFN-gamma response. Hence, FPs might play 2 roles in HIV infection: mediating membrane fusion while downregulating T cell responses to itself that could block infection. Disassociated from HIV, however, the FP molecule provides a novel reagent for downregulating undesirable immune responses, exemplified here by adjuvant arthritis.

<h4>Purpose</h4>Using a web-based tool to screen the human proteome for potential mimecan/osteoglycin interacting proteins, we found that the leucine-rich B7 protein may have functional associations with several small leucine-rich proteoglycans (SLRP), including mimecan. The purpose of this study was to determine the expression of leucine-rich B7 protein in human eye tissues and its subcellular localization in MG-63 cells.<h4>Methods</h4>Primers were synthesized to amplify the two known differentially spliced B7 mRNA transcripts. Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to determine the expression of B7 mRNAs in human ocular and nonocular tissues. A rabbit anti-human B7 antibody was generated that specifically immunostained B7 proteins. The expression of B7 proteins in the human eye was determined by immunohistochemistry (IHC). Intracellular localization of leucine-rich B7 and mimecan proteins were determined using transient co-transfections, cell immunostaining, and laser scanning confocal microscopy.<h4>Results</h4>RT-PCR analysis showed moderate expression of B7, transcript variant 2, in human cornea, iris, sclera, and retina. In contrast, B7, transcript variant 1, was strongly expressed only in the cornea. The two B7 mRNAs were highly expressed in human brain, blood, peripheral mononuclear cells, and other human tissues. By IHC, immunostaining for leucine-rich B7 protein was found in epithelial and endothelial layers of the cornea, epithelial and fiber cells of the lens, in sclera, and in the rod and cone layer of the retina of adult human eye. Leucine-rich B7 protein was found to localize to both nucleus and cytoplasm of MG-63 cells, whereas mimecan was found only in the cytoplasm of these cells. Merged images obtained by confocal microscopy revealed certain cytoplasmic regions in MG-63 cells where B7 and mimecan proteins appeared to co-localize.<h4>Conclusions</h4>The present work is the first to demonstrate the expression and localization of leucine-rich B7 protein in human eye and other human tissues. The results reported here are an essential prerequisite for future studies aimed at understanding the biological roles of leucine-rich B7 proteins in health and disease.

Mutations in a recently identified gene HJV (also called HFE2, or repulsive guidance molecule C, RgmC) are the major cause of juvenile hemochromatosis (JH). The protein product of HJV, hemojuvelin, contains a C-terminal glycosylphosphatidylinositol anchor, suggesting that it can be present in either a soluble or a cell-associated form. Patients with HJV hemochromatosis have low urinary levels of hepcidin, the principal iron-regulatory hormone secreted by the liver. However, neither the specific role of hemojuvelin in maintaining iron homeostasis nor its relationship to hepcidin has been experimentally established. In this study we used hemojuvelin-specific siRNAs to vary hemojuvelin mRNA concentration and showed that cellular hemojuvelin positively regulated hepcidin mRNA expression, independently of the interleukin 6 pathway. We also showed that recombinant soluble hemojuvelin (rs-hemojuvelin) suppressed hepcidin mRNA expression in primary human hepatocytes in a log-linear dose-dependent manner, suggesting binding competition between soluble and cell-associated hemojuvelin. Soluble hemojuvelin was found in human sera at concentrations similar to those required to suppress hepcidin mRNA in vitro. In cells engineered to express hemojuvelin, soluble hemojuvelin release was progressively inhibited by increasing iron concentrations. We propose that soluble and cell-associated hemojuvelin reciprocally regulate hepcidin expression in response to changes in extracellular iron concentration.

Inhibitors of PPII (pyroglutamyl-peptidase II) (EC 3.4.19.6) have potential applications as investigative and therapeutic agents. The rational design of inhibitors is hindered, however, by the lack of an experimental structure for PPII. Previous studies have demonstrated that replacement of histidine in TRH (thyrotropin-releasing hormone) with asparagine produces a competitive PPII inhibitor (Ki 17.5 microM). To gain further insight into which functional groups are significant for inhibitory activity, we investigated the effects on inhibition of structural modifications to Glp-Asn-ProNH2 (pyroglutamyl-asparaginyl-prolineamide). Synthesis and kinetic analysis of a diverse series of carboxamide and C-terminally extended Glp-Asn-ProNH2 analogues were undertaken. Extensive quantitative structure-activity relationships were generated, which indicated that key functionalities in the basic molecular structure of the inhibitors combine in a unique way to cause PPII inhibition. Data from kinetic and molecular modelling studies suggest that hydrogen bonding between the asparagine side chain and PPII may provide a basis for the inhibitory properties of the asparagine-containing peptides. Prolineamide appeared to be important for interaction with the S2' subsite, but some modifications were tolerated. Extension of Glp-Asn-ProNH2 with hydrophobic amino acids at the C-terminus led to a novel set of PPII inhibitors active in vitro at nanomolar concentrations. Such inhibitors were shown to enhance recovery of TRH released from rat brain slices. Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4-methylcoumarin displayed a Ki of 1 nM, making it the most potent competitive PPII inhibitor described to date. PPII inhibitors with this level of potency should find application in exploring the biological functions of TRH and PPII, and potentially provide a basis for development of novel therapeutics.

Cartilaginous fish are very ancient organisms. In the Antarctic sea, the modern chondrichthyan genera are poorly represented, with only three species of sharks and eight species of skates; the paucity of chondrichthyans is probably an ecological consequence of unusual trophic or habitat conditions in the Southern Ocean. In the Arctic, there are 26 species belonging to the class Chondrichthyes. Fish in the two polar regions have been subjected to different regional histories that have influenced the development of diversity: Antarctic marine organisms are highly stenothermal, in response to stable water temperatures, whereas the Arctic communities are exposed to seasonal temperature variations. The structure and function of the oxygen-transport haem protein from the Antarctic skate Bathyraja eatonii and from the Arctic skate Raja hyperborea (both of the subclass Elasmobranchii, order Rajiformes, family Rajidae) is reported in the present paper. These species have a single major haemoglobin (Hb 1; over 80% of the total). The Bohr-proton and the organophosphate-binding sites are absent. Thus the haemoglobins of northern and southern polar skates appear functionally similar, whereas differences were observed with several temperate elasmobranchs. Such evidence suggests that, in temperate and polar habitats, physiological adaptations have evolved along distinct pathways, whereas, in this case, the effect of the differences characterizing the two polar environments is negligible.

PRDX6, a member of the peroxiredoxins (PRDXs) family, is a key player in the removal of reactive oxygen species (ROS). Using targeted inactivation of the Prdx6 gene, we present evidence that the corresponding protein offsets the deleterious effects of ROS on lens epithelial cells (LECs) and regulates gene expression by limiting its levels. PRDX6-depleted LECs displayed phenotypic alterations and elevated alpha-smooth muscle actin and betaig-h3 expression (markers for cataractogenesis), indistinguishable from transforming growth factor beta (TGFbeta)-induced changes. Biochemical assays disclosed enhanced levels of ROS, as well as high expression and activation of TGFbeta1 in Prdx6-/- LECs. A CAT assay revealed transcriptional repression of lens epithelium-derived growth factor (LEDGF), HSP27, and alphaB-crystallin promoter activities in these cells. A gel mobility shift assay demonstrated the attenuation of LEDGF binding to heat shock or stress response elements present in these genes. A supply of PRDX6 toPrdx6-/- LECs reversed these changes. Based on the above data, we propose a rheostat role for PRDX6 in regulating gene expression by controlling the ROS level to maintain cellular homeostasis.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are strong and independent risk factors for hepatocellular carcinoma (HCC) development. Patients with hereditary hemochromatosis (HH) are considered at risk of developing cancer. However, the interaction between HFE gene mutations and hepatitis viruses for HCC development has not been systematically searched for. To assess the interaction between HFE gene mutations and exogenous risk factors in the risk of HCC occurrence, a case-only approach, in which just a series of patients is enrolled, was used. Three hundred three cirrhotic patients (231 males, 72 females) from five liver units in different geographic areas of Italy, who developed HCC during regular follow-up between January 1999 and March 2003, and whose blood DNA was available, were analyzed. In all subjects, hepatitis B surface antigen (HBsAg), anti-HCV and HFE gene mutations were assayed; alcohol intake was recorded by history. The interaction between HFE genotypes and hepatitis viruses for HCC was estimated by multivariate analysis adjusting for the confounding effect of alcohol intake, area of residence and months of follow-up. Of the 303 HCC cases, 12 (4.0%) were heterozygous for the C282Y mutation, 93 (30.7%) for the H63D, and 198 (65.3%) homozygous for the wild allele. Multivariate analysis showed that C282Y heterozygous males were 3.8-fold (95% CI=1.0-15.2) more likely to be HBV positive and that H63D heterozygous females were 6.0-fold (95% CI=1.2-113.8) more likely to be HCV positive than wild type subjects. In conclusion, given the association between C282Y mutation and HBV infection in male patients with HCC, a careful evaluation and follow-up should be considered in the C282Y-positive subjects with hepatitis B virus related liver disease. The interaction between the H63D mutation and HCV, observed only in women, may reflect a higher sensitivity to H63D-induced iron metabolism abnormalities and a reduced antioxidant capability in the presence of an even minor increase of iron which may occur as a consequence of the coexistence of hepatitis C infection and heterozygosity for HH.

Microtubules are primarily responsible for facilitating long-distance transport of both proteins and organelles. Given the critical role of this process in cellular function, it is not surprising that perturbation of microtubule-based transport can lead to diverse phenotypes in humans, including cancer and neurodegenerative disorders such as Alzheimer or Huntington disease. Recent investigations have also indicated that defects in specialized microtubule-based transport systems, such as mutations affecting the transport of protein particles along the length of cilia (intraflagellar transport) can cause retinal dystrophy, polycystic kidney disease or more complex syndromic phenotypes, such as Bardet-Biedl syndrome. In this review, we discuss recent findings implicating defects in microtubule-associated transport and motor proteins in a variety of diseases, particularly the role of defective microtubular transport in neurological and ciliary disease. These defects frequently display phenotypic consequences that manifest as human disease yet do not cause organismal lethality.

The cellular iron exporter ferroportin 1 is expressed in both the duodenum and in cells of the mononuclear phagocyte system. Expression of ferroportin 1 protein on the cell surface is regulated by the interaction of ferroportin 1 with hepcidin. Hepcidin treatment of cells results in internalization and lysosomal degradation of cell surface ferroportin 1. Recently, ferroportin 1 mutations leading to hemochromatosis (HFE4) have been identified. HFE4 differs from classical hemochromatosis in that there is a greater amount of macrophage iron sequestration. The data presented here demonstrate that HFE4 mutations are heterogeneous in their effects on protein function. Some mutations result in loss of function with partial protein sequestration in the ER. Others are indistinguishable from native ferroportin 1 and have a similar ability to deplete transfected cells of iron as evidenced by activation of the iron-response proteins and cellular ferritin depletion. Significantly, all mutants appear to be unresponsive to hepcidin and do not demonstrate the expected internalization on exposure to hepcidin. The clinical phenotypes observed in patients may be secondary to cell-type-specific defects in hepcidin-mediated inhibition of ferroportin 1 expression.

<h4>Background</h4>Transferrin receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in humans cause type 3 haemochromatosis and a targeted mutation in mice leads to iron overload with a similar phenotype. We have previously described the generation of a complete TfR2-knockout (KO) mouse.<h4>Aims</h4>The aims of this study were to determine the phenotype and analyse expression of iron related molecules in the liver, duodenum, and spleen of homozygous TfR2-KO, heterozygous, and wild-type mice.<h4>Methods</h4>Serum and tissue iron levels were determined in 10 week old male mice. Expression of iron related mRNA transcripts were analysed in the liver, duodenum, and spleen using real time polymerase chain reaction. Expression of iron related proteins in the liver were analysed by immunoblotting and immunohistochemistry.<h4>Results</h4>Homozygous TfR2-KO mice had no TfR2 protein expression and developed significant iron overload typical of TfR2 associated haemochromatosis. In the liver of TfR2-KO mice there was no upregulation of hepcidin mRNA or prohepcidin protein in response to iron loading.<h4>Conclusions</h4>Our results suggest that TfR2 is required for iron regulated expression of hepcidin and is involved in a pathway related to Hfe and hemojuvelin.

Although cancer is a multifaceted disease, all cancer types share identical molecular and cellular mechanisms. These mechanisms involve a collection of alterations critical to the normal physiological functioning of cells, such as alterations of growth factor signalling pathways, angiogenesis, cell adhesion signals, DNA replication and apoptotic cell death. Many genes involved in the processes enumerated above are functionally inactive in tumour cells, designating them as putative 'tumour suppressor genes'. Back in the early 1990s, Vogelstein and colleagues suggested that a gene called DCC (for Deleted in Colorectal Cancer) could be a tumour 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 15 years, controversial data have failed to firmly establish whether DCC is indeed a tumour suppressor gene. However, the recent observations that DCC triggers cell death and is a receptor for netrin-1, a molecule recently implicated in colorectal tumorigenesis, have prompted a renewal of interest in the role of DCC in tumorigenesis and suggest that the netrin-1/receptor pairs act as novel negative regulators of tumour development.

Using chromatin immunoprecipitation assays, we studied the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated recruitment of the aryl hydrocarbon receptor (AhR) and several co-regulators to the CYP1A1 promoter. AhR displayed a time-dependent recruitment, reaching a peak at 75 min and maintaining promoter occupancy for the remainder of the time course. Recruitment of AhR was followed by TIF2/SRC2, which preceded CBP, histone H3 acetylation, and RNA polymerase II (RNAPII). Simultaneous recruitment to the enhancer and the TATA box region suggests the formation of a large multiprotein complex bridging the two promoter regions. Interestingly, estrogen receptor alpha (ERalpha) displayed a TCDD- and time-dependent recruitment to the CYP1A1 promoter, which was increased by co-treatment with estradiol. Transfection in HuH7 human liver cells confirmed previously reported ERalpha enhancement of AhR activity. In contrast, TCDD did not induce the recruitment of ERalpha to the estrogen-responsive pS2 promoter, and after 120 min of co-treatment with estradiol, ERalpha is still present on the CYP1A1 promoter but no longer at pS2. RNA interference studies with T47D cells support a role for ERalpha in TCDD-dependent CYP1A1 expression. Our data suggest that ERalpha acts as a coregulator of AhR-mediated transcriptional activation and that the recruitment of ERalpha by AhR represents a novel mechanism AhR-ERalpha cross talk.

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.

<h4>Background</h4>Epidemiologists have observed that blood donation is associated with decreased risk of type 2 diabetes and cardiovascular disease.<h4>Methods</h4>We investigated the relationship between iron stores and insulin sensitivity, after controlling for known confounding factors, and compared insulin sensitivity between blood donors and individuals who had never donated blood (nondonors). In 181 men, insulin sensitivity and insulin secretion were evaluated through frequently sampled intravenous glucose tolerance tests with minimal model analysis. Men who donated blood between 6 months and 5 years before inclusion (n = 21) were carefully matched with nondonors (n = 66) for age, body mass index, waist-to-hip ratio, and cardiovascular risk profile, including blood lipids, blood pressure, and smoking status.<h4>Results</h4>Frequent blood donors (2-10 donations) had increased insulin sensitivity [3.42 (1.03) vs 2.45 (1.2) x 10(-4) x min(-1) x mIU/L; P = 0.04], decreased insulin secretion [186 (82) vs 401.7 (254) mIU/L x min; P <0.0001], and significantly lower iron stores [serum ferritin, 101.5 (74) vs 162 (100) microg/L; P = 0.017] than nondonors, but the 2 groups had similar blood hematocrits and blood hemoglobin concentrations.<h4>Conclusions</h4>Blood donation is simultaneously associated with increased insulin sensitivity and decreased iron stores. Stored iron seems to impact negatively on insulin action even in healthy people, and not just in classic pathologic conditions associated with iron overload (hemochromatosis and hemosiderosis). According to these observations, it is imperative that a definition of excessive iron stores in healthy people be formulated.

The male-specific-lethal (MSL) proteins in Drosophila melanogaster serve to adjust gene expression levels in male flies containing a single X chromosome to equal those in females with a double dose of X-linked genes. Together with noncoding roX RNA, MSL proteins form the "dosage compensation complex" (DCC), which interacts selectively with the X chromosome to restrict the transcription-activating histone H4 acetyltransferase MOF (males-absent-on-the-first) to that chromosome. We showed previously that MSL3 is essential for the activation of MOF's nucleosomal histone acetyltransferase activity within an MSL1-MOF complex. By characterizing the MSL3 domain structure and its associated functions, we now found that the nucleic acid binding determinants reside in the N terminus of MSL3, well separable from the C-terminal MRG signatures that form an integrated domain required for MSL1 interaction. Interaction with MSL1 mediates the activation of MOF in vitro and the targeting of MSL3 to the X-chromosomal territory in vivo. An N-terminal truncation that lacks the chromo-related domain and all nucleic acid binding activity is able to trigger de novo assembly of the DCC and establishment of an acetylated X-chromosome territory.

<h4>Aim</h4>To assess the prevalence of the two mutations, C282Y and H63D of HFE gene, in healthy subjects, patients with chronic hepatitis C (CHC), and patients with nonalcoholic fatty liver disease (NAFLD) in Taiwan and to explore the contribution of the HFE mutation on serum iron stores in CHC and NAFLD groups.<h4>Methods</h4>We examined C282Y and H63D mutations of HFE gene in 125 healthy subjects, 29 patients with CHC, and 33 patients with NAFLD. The serum iron markers, including ferritin, iron, and total iron binding capacity (TIBC), were assessed in all patients.<h4>Results</h4>All of the healthy subjects and patients were free from C282Y mutation. The prevalence of H63D heter-ozygosity was 4/125 (3.20%) in healthy subjects, 2/29 (6.90%) in CHC group, and 1/33 (3.03%) in NAFLD group. The healthy subjects showed no significant difference in the prevalence of H63D mutation as compared with the CHC or NAFLD group. Increased serum iron store was found in 34.48% of CHC patients and 36.36% of NAFLD patients. In three patients of H63D heterozygosity, only one CHC patient had increased serum iron store. There was no significant difference in the prevalence of HFE mutations between patients with increased serum iron store and those without in CHC or NAFLD group.<h4>Conclusion</h4>The HFE mutations may not contribute to iron accumulation in the CHC or NAFLD group even when serum iron overload is observed in more than one-third of these patients in Taiwan.

Autism is a spectrum of neurodevelopmental disorders with a primarily genetic etiology exhibiting deficits in (1) development of language and (2) social relationships and (3) patterns of repetitive, restricted behaviors or interests and resistance to change. Elevated platelet serotonin (5-HT) in 20%-25% of cases and efficacy of selective 5-HT reuptake inhibitors (SSRIs) in treating anxiety, depression, and repetitive behaviors points to the 5-HT transporter (5-HTT; SERT) as a strong candidate gene. Association studies involving the functional insertion/deletion polymorphism in the promoter (5-HTTLPR) and a polymorphism in intron 2 are inconclusive, possibly because of phenotypic heterogeneity. Nonetheless, mounting evidence for genetic linkage of autism to the chromosome 17q11.2 region that harbors the SERT locus (SLC6A4) supports a genetic effect at or near this gene. We confirm recent reports of sex-biased genetic effects in 17q by showing highly significant linkage driven by families with only affected males. Association with common alleles fails to explain observed linkage; therefore, we hypothesized that preferential transmission of multiple alleles does explain it. From 120 families, most contributing to linkage at 17q11.2, we found four coding substitutions at highly conserved positions and 15 other variants in 5' noncoding and other intronic regions transmitted in families exhibiting increased rigid-compulsive behaviors. In the aggregate, these variants show significant linkage to and association with autism. Our data provide strong support for a collection of multiple, often rare, alleles at SLC6A4 as imposing risk of autism.

<h4>Aim</h4>This report gives a comprehensive overview of ultrasonography of splenic abnormalities. Certain ultrasonic features are also discussed with pathologic correlation.<h4>Methods</h4>We review the typical ultrasonic characteristics of a wide range of splenic lesions, illustrating them with images obtained in our institution from 2000 to 2003. One hundred and three patients (47 men, 56 women), with a mean age of 54 years (range 9-92 years), were found to have an abnormal ultrasonic pattern of spleen.<h4>Results</h4>We describe the ultrasonic features of various splenic lesions such as accessory spleen, splenomegaly, cysts, cavernous hemangiomas, lymphomas, abscesses, metastatic tumors, splenic infarctions, hematomas, and rupture, based on traditional gray-scale and color Doppler sonography.<h4>Conclusion</h4>Ultrasound is a widely available, noninvasive, and useful means of diagnosing splenic abnormalities. A combination of ultrasonic characteristics and clinical data may provide an accurate diagnosis. If the US appearance alone is not enough, US may also be used to guide biopsy of suspicious lesions.

A role for apoptotic mediators in neurodegenerative disease has long been considered, but the identity of the putative effectors and the basis of their actions in neurodegeneration remain ill-defined. Huntington's disease (HD) is a polyglutamine disorder whose pathogenesis likely involves mitochondrial dysfunction. In this issue of Neuron, Bae et al. present data implicating p53 in HD pathogenesis. This intriguing study may thus provide a link between nuclear transcription dysregulation and mitochondrial abnormalities in HD.

We present evidence for a specific role of p53 in the mitochondria-associated cellular dysfunction and behavioral abnormalities of Huntington's disease (HD). Mutant huntingtin (mHtt) with expanded polyglutamine (polyQ) binds to p53 and upregulates levels of nuclear p53 as well as p53 transcriptional activity in neuronal cultures. The augmentation is specific, as it occurs with mHtt but not mutant ataxin-1 with expanded polyQ. p53 levels are also increased in the brains of mHtt transgenic (mHtt-Tg) mice and HD patients. Perturbation of p53 by pifithrin-alpha, RNA interference, or genetic deletion prevents mitochondrial membrane depolarization and cytotoxicity in HD cells, as well as the decreased respiratory complex IV activity of mHtt-Tg mice. Genetic deletion of p53 suppresses neurodegeneration in mHtt-Tg flies and neurobehavioral abnormalities of mHtt-Tg mice. Our findings suggest that p53 links nuclear and mitochondrial pathologies characteristic of HD.

A novel rotavirus strain (Dhaka6) isolated from a 21-year-old Bangladeshi male patient was characterized by sequence analysis of its VP7 and VP4 gene segments. Phylogenetic analysis of the VP7 gene of the Dhaka6 strain revealed a common evolutionary lineage with porcine G11 rotavirus strains. This isolate is the first reported G11 rotavirus strain infecting a human host. Comparison of the VP4 gene sequences with all currently recognized 24 different P genotypes revealed only low nucleotide (54 to 71%) and amino acid (52 to 76%) sequence identities. This lack of high sequence similarity in the VP4 gene indicates that the Dhaka6 isolate represents a new group A rotavirus P genotype, to which we propose assignment of the designation P[25].

Asthma and lead poisoning are prevalent among urban children in the United States. Lead exposure may be associated with excessive production of immunoglobulin E, possibly increasing asthma risk and contributing to racial disparities. The objective of this study was to examine racial differences in the association of blood lead level (BLL) to risk of developing asthma. We established and followed a cohort prospectively to determine asthma onset, using patient encounters and drug claims obtained from hospital databases. Participants were managed care enrollees with BLL measured and documented at 1-3 years of age. We used multiple variable analysis techniques to determine the relationship of BLL to period prevalent and incident asthma. Of the 4,634 children screened for lead from 1995 through 1998, 69.5% were African American, 50.5% were male, and mean age was 1.2 years. Among African Americans, BLL > or = 5 and BLL > or = 10 microg/dL were not associated with asthma. The association of BLL > or = 5 microg/dL with asthma among Caucasians was slightly elevated, but not significant [adjusted hazard ratio (adjHR) = 1.4; 95% confidence interval (CI), 0.7-2.9; p = 0.40]. Despite the small number of Caucasians with high BLL, the adjHR increased to 2.7 (95% CI, 0.9-8.1; p = 0.09) when more stringent criteria for asthma were used. When compared with Caucasians with BLL < 5 microg/dL, African Americans were at a significantly increased risk of asthma regardless of BLL (adjHR = 1.4-3.0). We conclude that an effect of BLL on risk of asthma for African Americans was not observed. These results demonstrate the need for further exploration of the complex interrelationships between race, asthma phenotype, genetic susceptibilities, and socioenvironmental exposures, including lead.

The production of polysaccharide-derivatized surfaces, polymers, and biomaterials has been shown to be a useful strategy for mediating the biological properties of materials, owing to the importance of polysaccharides for the sequestration and protection of bioactive proteins in vivo. We have therefore sought to combine the benefits of polysaccharide derivatization of polymers with unique opportunities to use these polymers for the production of bioactive, noncovalently assembled hydrogels. Accordingly, we report the synthesis of a heparin-modified poly(ethylene glycol) (PEG) star copolymer that can be used in the assembly of bioactive hydrogel networks via multiple strategies and that is also competent for the delivery of bioactive growth factors. A heparin-decorated polymer, synthesized by the reaction of thiol end-terminated four-arm star PEG (M(n) = 10 000) with maleimide functionalized low molecular weight heparin (LMWH, M(r) = 3000), has been characterized via (1)H NMR spectroscopy and size-exclusion chromatography; results indicate attachment of the LMWH with at least 73% efficiency. Both covalently and noncovalently assembled hydrogels can be produced from the PEG-LMWH conjugate. Viscoelastic noncovalently assembled hydrogels have been formed on the basis of the interaction of the PEG-LMWH with a PEG polymer bearing multiple heparin-binding peptide motifs. The binding and release of therapeutically important proteins from the assembled hydrogels have also been demonstrated via immunochemical assays, which demonstrate the slow release of basic fibroblast growth factor (bFGF) as a function of matrix erosion. The combination of these results suggests the opportunities for producing polymer-polysaccharide conjugates that can assemble into novel hydrogel networks on the basis of peptide-saccharide interactions and for employing these materials in delivery applications.

Binding interactions between low molecular weight heparin (LMWH) and heparin-binding peptides (HBP) have been applied as a strategy for the assembly of hydrogels that are capable of sequestering growth factors and delivering them in a controlled manner. In this work, the assembly of four-arm star poly(ethylene glycol) (PEG)-LMWH conjugate with PEG-HBP conjugates has been investigated. The interactions between LMWH and the heparin-binding regions of antithrombin III (ATIII) or the heparin interacting protein (HIP) have been characterized via heparin affinity chromatography and surface plasmon resonance (SPR); results indicate that the two peptides have slightly different affinities for heparin and LMWH, and bind LMWH with micromolar affinity. Solutions of the PEG-LMWH and of mixtures of the PEG-LMWH and PEG-HBP were characterized via both bulk rheology and laser tweezer microrheology. Interestingly, solutions of PEG-LMWH (2.5 wt % in PBS) form hydrogels in the absence of PEG-ATIII or PEG-HIP, with storage moduli, determined via bulk rheological measurements, in excess of the loss moduli over frequencies of 0.1-100 Hz. The addition of PEG-ATIII or PEG-HIP increases the moduli in direct proportion to the number of cross-links introduced. Characterization of the hydrogels via microrheology shows the gel microstructure is composed of polymer-rich fibrillar structures surrounded by polymer-depleted buffer. Potential applications of these hydrogels are discussed.

New type of N-glycosylated peptides having periodic sequence of -[X-Gln(beta-D-GlcNAc)-Aib]- [X = L-Glu(OMe), L-Lys(Ac), L-Ala; Aib = alpha-aminoisobutyric acid] were synthesized by polymerization of glycosylated tripeptides with an active ester methods using Cl(-+)H(3)N-L-Glu(OMe)-Gln[beta-D-GlcNAc(Ac)(3)]-Aib-ONp (Np=p-nitrophenyl) (13a), Cl(-+)H(3)N-L-Lys(Ac)-Gln[beta-D-GlcNAc(Ac)(3)]-Aib-ONp (13b), and Cl(-+)H(3)N-L-Ala-Gln[beta-D-GlcNAc(Ac)(3)]-Aib-ONp (13c) as the monomers. Polymerizable glycosylated tripeptides were prepared by stepwise N,N-dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) method. Polymerizations of 13a-c were initiated by triethylamine and proceeded in DMSO at 50 degrees C for 5 days in the presence of 1-hydroxy-7-azabenzotriazole (HOAt) as the activator (conversions were 25-75%). The glycopeptides were deacetylated by hydrazine monohydrate in methanol to afford periodic glycopeptides 14 (12-27 residues) without racemization (yield, 35-89%). CD spectra in methanol, trifluoroethanol, and water of deacetylated glycopolymers 14a, 14b, and 14c showed double minima (206 and 222 nm) of negative Cotton effect indicating that N-glycoside (N-acetyl-d-glucosamine) was arranged three-dimensionally along the alpha-helical peptides in water as well as in organic protic solvents. The helix content depends on the solvent, peptide sequence, and spacer between peptide backbone and sugar. Interaction of the glycopeptides with wheat germ agglutinin (WGA) lectin was investigated by fluorescence measurement.

"Evidence-based" recommendations for warfarin prescription in patients with history of ischemic stroke limit its use to prevention of stroke due to atrial fibrillation. Warfarin is also prescribed by the authors to prevent thrombosis in stroke patients with thrombophilia and potential cardiac or arterial source for thromboembolism. These potential conditions, in the face of thrombophilia, include, but may not be limited to, dilated cardiomyopathy, decreased left ventricular function, atrial septal aneurysm with or without patent foramen ovale (PFO), PFO with evidence of pelvic or lower extremity deep venous thrombosis or with clear thrombophilia, spontaneous echocardiographic contrast, intracardiac or intra-arterial thrombus, intra-aortic arch thrombus, high degree of stenosis of large- and medium-sized cerebrovascular arteries, and arterial dissection. Commonly diagnosed thrombophilic states in our population currently include protein S or C deficiency, antiphospholipid antibodies, and less commonly ATIII deficiency, factor V Leiden mutation, G20210A PT mutation, and plasminogen activator inhibitor-1 mutation. Thrombophilic states often occur in combination. The occurrence of combined arterial, cardiac, and thrombophilic sources of thromboembolism poignantly describes the complexity of causation of ischemic stroke in any one patient. Our practice of treating the complex interaction of thromboembolic sources is based on scientific evidence, which is not arbitrarily limited to probability-based statistics. Warfarin is well known in the clinical setting to interact with many different contextual factors of the individual patient, making its dosing and response unique to that patient. We have shown why the indications for warfarin use and its dosing cannot be directly extrapolated to the individual patient from the results of large, double-blind, randomized trials. In practice, the unique patient and his or her context must be considered by the expert physician who makes the therapeutic decision. The context includes, but is not limited to, known pathologies that contribute to thrombus formation according to the accepted pathophysiologic model of thrombosis based on Virchow's triad of altered flow, endothelium, and blood components.

In humans, eight types of histone H1 exist (H1.1-H1.5, H1 degrees , H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails. Although the precise functions of these isoforms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific functions may be assigned to certain individual H1 subtypes. Moreover, microsequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins. This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes. Two deviations from wild-type H1 sequences were found. In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position 173 in H1.4 was replaced by arginine. We confirmed these findings by DNA sequencing of the corresponding gene segments. In K562 cells, a homozygous GCC-->GTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones. Raji cells showed a heterozygous AAA-->AGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCC-->GTC shift, indicating that this is a relatively frequent polymorphism. The AAA-->AGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt's lymphoma. The significance of these sequence variants is unclear, but increasing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions.

Miniaturization of genetic tests represents the convergence of molecular biology and engineering and is leading to a new class of small analyzers and test systems for genetic testing with improved analytical characteristics. Miniaturization initially focused on devices that contained micrometer-sized features designed for a particular analytical purpose (e.g., filters for cell isolation and chips for capillary electrophoresis). Now, the focus is shifting to analytical applications based on nano-sized objects such as nanotubes, nanochannels, nanoparticles, nanopores and nanocapacitors. These nanofabricated objects provide new tools for sequencing of nucleic acids and rapid, multiplexed, nucleic acid detection.

Thalamic innervation of each neocortical area is vital to cortical function, but the developmental strategies that guide axons to specific areas remain unclear. We took a new approach to determine the contribution of intracortical cues. The cortical patterning molecule fibroblast growth factor 8 (FGF8) was misexpressed in the cortical primordium to rearrange the area map. Thalamic axons faithfully tracked changes in area position and innervated duplicated somatosensory barrel fields induced by an ectopic source of FGF8, indicating that thalamic axons indeed use intracortical positional information. Because cortical layers are generated in temporal order, FGF8 misexpression at different ages could be used to shift regional identity in the subplate and cortical plate either in or out of register. Thalamic axons showed strikingly different responses in the two different conditions, disclosing sources of positional guidance in both subplate and cortical plate. Unexpectedly, axon trajectories indicated that an individual neocortical layer could provide not only laminar but also area-specific guidance. Our findings demonstrate that thalamocortical axons are directed by sequential, positional cues within the cortex and implicate FGF8 as an indirect regulator of thalamocortical innervation.

Two cases of community-acquired septicemia caused by serotype-O1 Yersinia pseudotuberculosis were diagnosed in middle-aged, HIV-positive, immunodeficient patients during an 8-month period. Bacterial isolates were genetically indistinguishable, but no epidemiologic link between the 2 patients was established. HIV-related immunosuppression should be regarded as a risk factor for Y. pseudotuberculosis septicemia.

<h4>Background</h4>Cigarette smoking has been associated with mood enhancing properties and modulating effects on serotonin activity. The loudness dependence (LD) of the auditory-evoked N1/P2-component has been related to serotonergic neurotransmission, i. e. the allelic variants in the promoter of the 5-hydroxytryptamine-transporter (5-HTT) gene (SCL6A4). Moreover, smoking behavior has been associated to the 5-HTT-genotype. It was hypothesized that cigarette smoking modulates the LD and this effect was expected to interact with the 5-HTT-genotype.<h4>Methods</h4>5-HTT-genotype and LD were determined in 63 healthy smokers and 114 nonsmokers.<h4>Results</h4>LD was significantly affected by smoking status (p = 0.008) and 5-HTT-genotype (p = 0.045) but not by smoking*genotype-interaction or daily cigarette consumption. Current smokers exhibited a significantly weaker LD compared to nonsmokers. 5-HTT-genotype showed no significant effect on smoking behavior.<h4>Discussion</h4>The results indicate a higher serotonergic activity in smokers as compared to nonsmokers independent of 5-HTT-genotype. Since former smokers and never smokers showed similar LDs, the serotonin enhancing effect of smoking seems to be a characteristic state, which may contribute to the maintenance of smoking behavior.

The intestinal absorption of the essential trace element iron and its mobilization from storage sites in the body are controlled by systemic signals that reflect tissue iron requirements. Recent advances have indicated that the liver-derived peptide hepcidin plays a central role in this process by repressing iron release from intestinal enterocytes, macrophages and other body cells. When iron requirements are increased, hepcidin levels decline and more iron enters the plasma. It has been proposed that the level of circulating diferric transferrin, which reflects tissue iron levels, acts as a signal to alter hepcidin expression. In the liver, the proteins HFE, transferrin receptor 2 and hemojuvelin may be involved in mediating this signal as disruption of each of these molecules decreases hepcidin expression. Patients carrying mutations in these molecules or in hepcidin itself develop systemic iron loading (or hemochromatosis) due to their inability to down regulate iron absorption. Hepcidin is also responsible for the decreased plasma iron or hypoferremia that accompanies inflammation and various chronic diseases as its expression is stimulated by pro-inflammatory cytokines such as interleukin 6. The mechanisms underlying the regulation of hepcidin expression and how it acts on cells to control iron release are key areas of ongoing research.

The aim of this study was to characterize the reactivity of monoclonal antibodies (mAbs) that had been submitted to the HLDA8 Workshop. The lineage specificity of target molecules was tested by analyzing their expression patterns on blood cells, leukocytes, and lymphocyte subsets. The expression of target molecules during B cell development, ranging from early precursors to plasma cells, was analyzed using a large panel of B cell lines. Our results have permitted us to characterize the expression of 10 new CD molecules: CD316 (HM1.24, BST2), CD268 (BAFF-R, TNFRSF13C), CD269 (BCMA, TNFRF17), CD267 (TACI, TNFRSF13B), CD275 (ICOSL, B7H2), CD254 (TRANCE, TNFSF11), CD252 (OX40L TNFSF4), CD315 (CD9-P), CD316 (EWI-2, PGRL), and CD307 (IRTA-2 or FcRH5). Three of these new CDs, CD267, CD269, and CD307 presented a B cell-restricted expression pattern. MAbs against these novel cell-surface molecules may offer new tools for research, diagnosis, and therapy.

The article presents current knowledge on skeleton development, which is initiated by mesenchymal cells condensation. The mesenchymal to epithelial transformation, vascular regression, cartilage formation and ossification are involved in normal skeletogenesis. All the processes are influenced by various genes and regulatory molecules, e.g., N-cadherin, vascular endothelial growth factor, transforming growth factor-beta, syndecan-3 and others. The chondrogenesis depends on the Sox genes expression - Sox5, Sox6 and especially Sox9, which continued action produce persistent cartilage. The essential role in osteogenesis is played by the transcription factor Runx2 that directly activates expression of genes coding typical bone proteins like collagen type I, osteocalcin, osteopontin and osteonectin. Development of fibrous, cartilaginous and synovial joints, bone maturation and remodeling, and mechanisms leading to skeleton dysmorphogenesis are also discussed.

Neonatal periventricular hemorrhage (PVH) is a devastating complication of prematurity in the human infant. Based upon observations made primarily in adult rodents and the fact that the immature brain uses proteolytic systems for cell migration and growth, we hypothesized that thrombin and plasmin enzyme activities contribute to the brain damage after PVH. The viability of mixed brain cells derived from newborn rat periventricular region was suppressed by whole blood and thrombin, but not plasmin. Following injection of autologous blood into the periventricular region of newborn rat brain, proteolytic activity was detected in a halo around the hematoma using membrane overlays impregnated with thrombin and plasmin fluorogenic substrates. Two-day old rats received periventricular injection of blood, thrombin, and plasminogen. After 2 days, thrombin and blood were associated with significantly greater damage than saline or plasminogen. Two-day old mice received intracerebral injections of blood in combination with saline or the proteolytic inhibitors hirudin, alpha2macroglobulin, or plasminogen activator inhibitor-1. After 2 days, hirudin significantly reduced brain cell death and inflammation. Two-day-old mice then received low and high doses of hirudin mixed with blood after which behavioral testing was conducted repeatedly. At 10 weeks there was no statistically significant evidence for behavioral or structural brain protection. These results indicate that thrombin likely plays a role in neonatal periventricular brain damage following PVH. However, additional factors are likely important in the recovery from this result.

<h4>Aim</h4>The aim of this study was to evaluate haemostatic variables in women with anorexia nervosa --AN.<h4>Material and methods</h4>23 obese female patients, and 39 female anorexic patients diagnosed acc. to DSM-IV and ICD-10 criteria were recruited. The studied group was compared to a control group--37 healthy female volunteers. BMI was calculated for AN, obese, and the control group. The following measurements were taken: prothrombin index, prothrombin time, thrombin time, fibrinogen, factor VII, ATIII, D-dimers, INR, APTT, protein C and PAI-1 activity, t-PA activity, PAI-1 antigen, t-PA antigen, glucose level, insulin level, E2 and total fat.<h4>Results</h4>In the AN group the INR, prothrombin time, PAI-1 activity, t-PA activity, protein C level were increased, whereas PAI-1 antigen, t-PA antigen, prothrombin index, fasting insulin level and fasting glucose level were decreased.<h4>Conclusions</h4>We conclude that, in the AN group, an impaired fibrinolysis and coagulation are observed and there is a correlation between fibrinolytic and coagulation parameters and carbohydrate metabolism.

Over the last two decades, a number of mutations have been identified that give rise to neurodegenerative disorders, including familial forms of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Although in most cases sporadic cases vastly outnumber familial forms of such diseases, study of such inherited forms has the potential to provide powerful clues regarding the pathophysiological basis of neurodegeneration. One powerful approach to analyzing disease mechanisms is the development of transgenic animal models, most notably in the mouse. However, development and analysis of such models can be costly and time consuming. Development of improved transgenic technologies have contributed to the development of Drosophila models of a number of neurodegenerative disorders that have shown striking similarities to the human diseases. Moreover, genetic screens using such models have begun to unravel aspects of the pathophysiological basis of neurodegenerative disorders. Here, we provide a general overview of fly models pertinent to trinucleotide repeat expansion disorders, Alzheimer's, and Parkinson's diseases, and highlight key genetic modifiers that have been identified to date using such models.

Attention-deficit hyperactivity disorder (ADHD) is a common behavioral disorder observed during childhood, detected in 3% to 5% of school-age children. The disorder is characterised by marked inattention, hyperactivity, and impulsiveness. In most cases, symptoms can be treated by catecholamine-releasing drugs, such as methylphenidate. Children with ADHD are at higher risk for substance abuse and oppositional, conduct and mood disorders. Familial and adoption studies shed light on the genetic vulnerability of ADHD. Twin studies estimated the broad heritability to range between 40% and 90%. The mode of transmission is yet unknown, but is likely polygenic. Molecular genetic studies in ADHD should contribute to a greater understanding of the pathophysiology of the disorder (genetics of the vulnerability), and could help to select a more rational type of treatment (pharmacogenetic). Family-based association studies already performed are reviewed in this manuscript. Association studies, using haplotype relative risk (HRR) or transmission disequilibrium test (TDT) have focused on candidate genes which code for proteins potentially involved in the etiopathogenesis of the disorder. Genes involved in dopamine, serotonin, and noradrenalin systems have thus been assessed for their role in core features of ADHD, such as motor overactivity, inattention, and impulsiveness. According to a meta-analysis, the DAT1 gene, an obvious candidate gene in ADHD vulnerability, does not appear to be involved (OR = 1.13, p = 0.21). On the other hand, DRD4 (OR = 1.26, p = 0.01) and DRD5 (OR = 1.4, p = 0.01) are significantly associated to ADHD according to the present meta-analysis, confirming previous ones. Recent studies showed a trend for an association between one allele of the 5-HTT (considering case-control studies) and DBH (OR = 1.27, p = 0.06) genes and ADHD, but these positive findings have to be replicated. ADHD is a complex disorder with potentially many different risk factors. Genetic and phenotypic heterogeneity could explain why some association studies are positive, whereas others are negative. For instance, different developmental pathways are likely to lead to similar clinical outcomes. More clear-cut phenotypes, such as ADHD with conduct disorder, or ADHD with bipolar disorder, could be more homogenous, the genes involved being therefore more easy to detect. These phenotypes are beginning to be specifically studied in molecular genetics. In addition, the development of pharmacogenetics could help to identify predictors of clinical response for a specific type of treatment, which would be clearly helpful in clinical practice.

Vibrational spectroscopy (Infrared and Raman), and in particular micro-spectroscopy and micro-spectroscopic imaging has been used to characterize developmental changes in bone and other mineralized tissues, to monitor these changes in cell cultures, and to detect disease and drug-induced modifications. Examples of the use of infrared micro-spectroscopy and micro-spectroscopic imaging are discussed in this review.

SUMO (Small Ubiquitin-related Modifier) is a small protein that covalently attaches to a lysine residue of another protein in a reversible fashion. SUMO attachment to its substrate proteins causes changes in the localization, activity, or binding partners of the substrate. SUMO has been shown to play a role in a multitude of processes; these include chromosome segregation, cell cycle progression, and DNA damage recovery. Defects in the SUMO pathway have been demonstrated to affect tumorigenesis and the inflammatory response as well as other human diseases.

No abstract available.