Gene Literature Dashboard

hStaufen is the human homolog of dmStaufen, a double-stranded (ds)RNA-binding protein involved in early development of the fly. hStaufen-containing complexes were purified by affinity chromatography from human cells transfected with a TAP-tagged hStaufen gene. These complexes showed a size >10 MDa. Untagged complexes with similar size were identified from differentiated human neuroblasts. The identity of proteins present in purified hStaufen complexes was determined by mass spectrometry and the presence of these proteins and other functionally related ones was verified by western blot. Ribosomes and proteins involved in the control of protein synthesis (PABP1 and FMRP) were present in purified hStaufen complexes, as well as elements of the cytoskeleton (tubulins, tau, actin and internexin), cytoskeleton control proteins (IQGAP1, cdc42 and rac1) and motor proteins (dynein, kinesin and myosin). In addition, proteins normally found in the nucleus, like nucleolin and RNA helicase A, were also found associated with cytosolic hStaufen complexes. The co-localization of these components with hStaufen granules in the dendrites of differentiated neuroblasts, determined by confocal immunofluorescence, validated their association in living cells. These results support the notion that the hStaufen-containing granules are structures essential in the localization and regulated translation of human mRNAs in vivo.

<h4>Purpose</h4>This study was conducted to explore whether CDCA derivatives induce apoptosis in a stomach cancer cell line, and to dissect the detailed mechanism underlying apoptosis.<h4>Materials and methods</h4>The human stomach cancer cell line, SNU-1, cells were treated with the synthetic CDCA derivatives, HS-1199 and HS-1200. DNA and mitochondrial stains were used to detect apoptotic cells by fluorescence imaging or flow cytometry. The caspase-3 activity was measured by Western blotting.<h4>Results</h4>Both the HS-1199 and HS-1200 induced decreased viabilities of the SNU-1 cells, in time-dependent manners. The CDCA derivatives demonstrated various apoptosis hallmarks, such as mitochondrial changes (reduction of MMP, cytochrome c release, and Smac/ DIABLO translocation), activation of caspase-3 (resulting in the degradation of PARP and DFF45), DNA fragmentation and nuclear condensation.<h4>Conclusion</h4>The CDCA derivatives, HS-1199 and HS-1200, both induced apoptosis of the SNU-1 gastric cancer cells in caspase- and mitochondria-dependent fashions. Many important issues relating to their therapeutic applications remain to be elucidated.

Efficient and highly stereoselective syntheses of cryptophycin B and arenastatin A, potent cytotoxic agents, are described. An ester-derived titanium enolate mediated <i>syn</i>-aldol reaction was employed to generate the stereocenters C-5 and C-6. The route is convergent and provides a convenient access to the synthesis of structural variants of cryptophycins as well as members of its family.

In most human populations, the ability to digest lactose contained in milk usually disappears in childhood, but in European-derived populations, lactase activity frequently persists into adulthood (Scrimshaw and Murray 1988). It has been suggested (Cavalli-Sforza 1973; Hollox et al. 2001; Enattah et al. 2002; Poulter et al. 2003) that a selective advantage based on additional nutrition from dairy explains these genetically determined population differences (Simoons 1970; Kretchmer 1971; Scrimshaw and Murray 1988; Enattah et al. 2002), but formal population-genetics-based evidence of selection has not yet been provided. To assess the population-genetics evidence for selection, we typed 101 single-nucleotide polymorphisms covering 3.2 Mb around the lactase gene. In northern European-derived populations, two alleles that are tightly associated with lactase persistence (Enattah et al. 2002) uniquely mark a common (~77%) haplotype that extends largely undisrupted for >1 Mb. We provide two new lines of genetic evidence that this long, common haplotype arose rapidly due to recent selection: (1) by use of the traditional F(ST) measure and a novel test based on p(excess), we demonstrate large frequency differences among populations for the persistence-associated markers and for flanking markers throughout the haplotype, and (2) we show that the haplotype is unusually long, given its high frequency--a hallmark of recent selection. We estimate that strong selection occurred within the past 5,000-10,000 years, consistent with an advantage to lactase persistence in the setting of dairy farming; the signals of selection we observe are among the strongest yet seen for any gene in the genome.

Protein p6 of Bacillus subtilis bacteriophage Phi29 is essential for phage development. In vitro it activates the initiation of DNA replication and is involved in the early to late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA forms a right-handed superhelix wrapping around a multimeric protein core. However, there was no evidence of p6 binding to Phi29 DNA in vivo. By crosslinking, chromatin immunoprecipitation and real-time PCR we show that protein p6 binds to most, if not all, the viral genome in vivo, although with higher affinity for both DNA ends, which contain the replication origins. In contrast, the affinity for plasmid DNA is negligible, but greatly increases when the negative supercoiling decreases, as shown in vivo by treatment of cells with novobiocin and in vitro by fluorescence quenching with plasmids with different topology. In conclusion, binding of protein p6 all along the Phi29 genome strongly suggests that its functions in replication and transcription control could be local outcomes of a more global role as a histone-like protein. The p6 binding dependence on DNA topology could explain its preferential binding to viral with respect to bacterial DNA, whose level of negative supercoiling is presumably higher than that of Phi29 DNA.

The mechanisms controlling axon guidance are of fundamental importance in understanding brain development. Growing corticospinal and somatosensory axons cross the midline in the medulla to reach their targets and thus form the basis of contralateral motor control and sensory input. The motor and sensory projections appeared uncrossed in patients with horizontal gaze palsy with progressive scoliosis (HGPPS). In patients affected with HGPPS, we identified mutations in the ROBO3 gene, which shares homology with roundabout genes important in axon guidance in developing Drosophila, zebrafish, and mouse. Like its murine homolog Rig1/Robo3, but unlike other Robo proteins, ROBO3 is required for hindbrain axon midline crossing.

Epithelial-mesenchymal transformation is a critical developmental process reiterated in multiple organs throughout embryogenesis. Formation of endocardial cushions, primordia of valves and septa, is a classic example of epithelial-mesenchymal transformation. Several gene mutations are known to affect cardiac valve formation. Sox9 is activated when endocardial endothelial cells undergo mesenchymal transformation and migrate into an extracellular matrix, called cardiac jelly, to form endocardial cushions. In Sox9-null mutants, endocardial cushions are markedly hypoplastic. In these mutants, Nfatc1 is ectopically expressed and no longer restricted to endothelial cells. Further, Sox9-deficient endocardial mesenchymal cells fail to express ErbB3, which is required for endocardial cushion cell differentiation and proliferation. Our results reveal a succession of molecular steps in the pathway of endocardial cushion development. We propose that loss of Sox9 inhibits epithelial-mesenchymal transformation after delamination and initial migration, but before definitive mesenchymal transformation.

Inflammation influences iron balance in the whole organism. A common clinical manifestation of these changes is anemia of chronic disease (ACD; also called anemia of inflammation). Inflammation reduces duodenal iron absorption and increases macrophage iron retention, resulting in low serum iron concentrations (hyposideremia). Despite the protection hyposideremia provides against proliferating microorganisms, this 'iron withholding' reduces the iron available to maturing red blood cells and eventually contributes to the development of anemia. Hepcidin antimicrobial peptide (Hamp) is a hepatic defensin-like peptide hormone that inhibits duodenal iron absorption and macrophage iron release. Hamp is part of the type II acute phase response and is thought to have a crucial regulatory role in sequestering iron in the context of ACD. Mice with deficiencies in the hemochromatosis gene product, Hfe, mounted a general inflammatory response after injection of lipopolysaccharide but lacked appropriate Hamp expression and did not develop hyposideremia. These data suggest a previously unidentified role for Hfe in innate immunity and ACD.

Simple and reliable genotyping technology is a key to success for high-throughput genetic screening in the post-genome era. Here we have developed a new real-time PCR genotyping approach that uses displacement hybridization-based probes: displacing probes. The specificity of displacing probes could be simply assessed through denaturation analysis before genotyping was implemented, and the probes designed with maximal specificity also showed the greatest detection sensitivity. The ease in design, the simple single-dye labeling chemistry and the capability to adopt degenerated negative strands for point mutation genotyping make the displacing probes both cost effective and easy to use. The feasibility of this method was first tested by detecting the C282Y mutation in the human hemochromatosis gene. The robustness of this approach was then validated by simultaneous genotyping of five different types of mutation in the human beta-globin gene. Sixty-two human genomic DNA samples with nine known genotypes were accurately detected, 32 random clinical samples were successfully screened and 114 double-blind DNA samples were all correctly genotyped. The combined merits of reliability, flexibility and simplicity should make this method suitable for routine clinical testing and large-scale genetic screening.

Fgf-3 is expressed in a dynamic and complex spatiotemporal pattern during mouse development. Previous studies identified GATA-4 as a transcription factor that binds the key regulatory element PS4A of the Fgf-3 promoter and stimulates transcription. Here we show that members of the SOX family of transcription factors also bind PS4A and differentially modulate transcription. At least five SOX genes, Sox2, Sox6, Sox7, Sox13, and Sox17, were expressed in F9 cells, and of these, Sox7 and Sox17 were dramatically induced in parallel with Fgf-3 following differentiation into parietal endoderm-like cells with retinoic acid and dibutyryl cAMP. Complexes could be detected on PS4A with SOX2, SOX7, and SOX17 by using nuclear extracts from differentiated F9 cells. However, only Sox7 expression markedly activated the Fgf-3 promoter in these cells. By contrast, SOX2 was a poor activator of Fgf-3 transcription, and when Sox2 was coexpressed with Gata4, it negatively modulated the strong activation mediated by GATA-4. More detailed analyses showed that SOX7 competes with GATA-4 for PS4A occupancy and to activate the Fgf-3 promoter. In situ hybridization analysis showed that Sox7 is co-expressed with Fgf-3 and Gata4 in the parietal endoderm of E7.5 mouse embryos. In culture, GATA-4-deficient embryonal stem cells were shown to express Fgf-3 upon differentiation into embryoid bodies, although at lower levels than were found in wild type embryonal stem cells. This Fgf-3 expression was virtually abolished when Sox7 expression was suppressed by RNA interference. These results show that SOX7 is a potent activator of Fgf-3 transcription.

<h4>Background</h4>Increased serotonin (5-hydroxytryptamine, 5-HT) transporter activity has been observed in human familial pulmonary hypertension.<h4>Methods and results</h4>We investigated pulmonary hemodynamics and the development of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in mice overexpressing the gene for the 5-HT transporter (5-HTT+ mice). Right ventricular pressure was elevated 3-fold in normoxic 5-HTT+ mice compared with their wild-type controls. Hypoxia-induced increases in right ventricular hypertrophy and pulmonary vascular remodeling were also potentiated in the 5-HTT+ mice. 5-HTT-like immunoreactivity, protein, and binding sites were markedly increased in the lungs from the 5-HTT+ mice. Hypoxia, however, decreased 5-HT transporter immunoreactivity, mRNA transcription, protein, and binding sites in both wild-type and 5-HTT+ mice.<h4>Conclusions</h4>Increased 5-HT transporter expression causes elevated right ventricular pressures, and this occurs before the onset of right ventricular hypertrophy or pulmonary arterial remodeling. Hypoxia-induced remodeling is, however, increased in 5-HTT+ mice, whereas hypoxia inhibits 5-HTT expression. This provides a unique model that demonstrates differential mechanisms for familial pulmonary arterial hypertension and pulmonary arterial hypertension with hypoxemia.

Several lines of evidence suggest that patterns of genetic variability in the human angiotensinogen gene (AGT) contribute to phenotypic variability in human hypertension. The A(-6) promoter variant of AGT is associated with higher plasma angiotensinogen levels and increased risk of essential hypertension. The geographic distribution of the A(-6) variant leads to the intriguing hypothesis that the G(-6) promoter variant has been selectively advantageous outside Africa. To test these hypotheses, we investigated the roles of population history and natural selection in shaping patterns of genetic diversity in AGT, by sequencing the entire AGT gene (14400 bp) in 736 chromosomes from Africa, Asia, and Europe. We found that the A(-6) variant is present at higher frequency in African populations than in non-African populations. Neutrality tests found no evidence of a departure from selective neutrality, when whole AGT sequences were compared. However, tests restricted to sites in the vicinity of the A(-6)G polymorphism found evidence of a selective sweep. Sliding-window analyses showed that evidence of the sweep is restricted to sites in tight linkage disequilibrium (LD) with the A(-6)G polymorphism. Further, haplotypes carrying the G(-6) variant showed elevated levels of LD, suggesting that they have risen recently to high frequency. Departures from neutral expectation in some but not all regions of AGT indicate that patterns of diversity in the gene cannot be accounted for solely by population history, which would affect all regions equally. Taken together, patterns of genetic diversity in AGT suggest that natural selection has generally favored the G(-6) variant over the A(-6) variant in non-African populations. However, important localized effects may also be present.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

It is now thought that NO* (nitric oxide) and its redox congeners may play a role in the physiological regulation of mitochondrial function. The inhibition of cytochrome c oxidase by NO* is characterized as being reversible and oxygen dependent. In contrast, peroxynitrite, the product of the reaction of NO* with superoxide, irreversibly inhibits several of the respiratory complexes. However, little is known about the effects of HNO (nitroxyl) on mitochondrial function. This is especially important, since HNO has been shown to be more cytotoxic than NO*, may potentially be generated in vivo, and elicits biological responses with some of the characteristics of NO and peroxynitrite. In the present study, we present evidence that isolated mitochondria, in the absence or presence of substrate, convert HNO into NO* by a process that is dependent on mitochondrial concentration as well as the concentration of the HNO donor Angeli's salt. In addition, HNO is able to inhibit mitochondrial respiration through the inhibition of complexes I and II, most probably via modification of specific cysteine residues in the proteins. Using a proteomics approach, extensive modification of mitochondrial protein thiols was demonstrated. From these data it is evident that HNO interacts with mitochondria through mechanisms distinct from those of either NO* or peroxynitrite, including the generation of NO*, the modification of thiols and the inhibition of complexes I and II.

Family and twin studies have supported a strong genetic factor in the etiology of obsessive-compulsive disorder (OCD), although the precise mechanism of inheritance is unclear. Clinical and pharmacological studies have implicated the serotonergic and dopaminergic systems in disease pathogenesis. In this cross-sectional study, we have examined the allelic and genotypic frequencies of a Val-158-Met substitution in the COMT gene, a 44-base pair (bp) length variation in the regulatory region of the serotonin transporter gene (5-HTTLPR) and the T102C and C516T variants in the serotonin receptor type 2A (5HT2A) gene in 79 OCD patients and 202 control subjects. There were no observed differences in the frequencies of allele and genotype between patients and control groups for the COMT, the 5HTTLPR and the T102C 5HT2A gene polymorphisms. In contrast, a statistically significant difference between OCD patients and controls was observed on the genotypic distribution (chi(2) = 16.7, 2df, P = 0.0002) and on the allelic frequencies (chi(2) = 15.8, 1df, P = 0.00007) for the C516T 5HT2A gene polymorphism. The results suggest that the C516T variant of the 5HT2A gene may be one of the genetic risk factors for OCD in our sample. However, further studies using larger samples and family based methods are recommended to confirm these findings.

Drosophila Guanylate-kinase holder (Gukh) is an adaptor molecule bridging Discs large (Dlg) and Scribble (Scrib), which are implicated in the establishment and maintenance of epithelial polarity. Here, we searched for human homologs of Drosophila gukh by using bioinformatics, and identified GUKH1 and GUKH2 genes. GUKH1 was identical to Nance-Horan syndrome (NHS) gene, while GUKH2 was a novel gene. FLJ35425 (AK092744.1), DKFZp686P1949 (BX647246.1) and KIAA1357 (AB037778.1) cDNAs were derived from human GUKH2 gene. Nucleotide sequence of GUKH2 cDNA was determined by assembling 5'-part of FLJ35425 cDNA and entire region of DKFZp686P1949 cDNA. Human GUKH2 gene consists of 8 exons. Exon 5 (132 bp) of GUKH2 gene was spliced out in GUKH2 cDNA due to alternative splicing. GUKH2-REPS1 locus at human chromosome 6q24.1 and GUKH1-REPS2 locus at human chromosome Xp22.22-p22.13 are paralogous regions within the human genome. Mouse Gukh2 and zebrafish gukh2 genes were also identified. N-terminal part of human GUKH2, mouse Gukh2 and zebrafish gukh2 proteins were completely divergent from human GUKH1 protein. Human GUKH2 and GUKH1, consisting of eight GUKH homology (GKH1-GKH8) domains and Proline-rich domain, showed 28.5% total-amino-acid identity. GKH1, GKH4, GKH5, GKH7 and GKH8 domains were conserved among human GUKH1, human GUKH2 and Drosophila Gukh. Because human homologs of Drosophila dlg (DLG1-DLG7) as well as human homologs of Drosophila scrib (SCRIB, ERBB2IP and Densin-180) are cancer-associated genes, human homologs of Drosophila gukh (GUKH1 and GUKH2) are predicted cancer-associated genes.

<h4>Background</h4>Pathological brain iron deposition has been implicated as a source of neurotoxic reactive oxygen species in Alzheimer disease (AD). Recent reports suggest that heterozygosity for the two common hfe mutations responsible for hereditary hemochromatosis (HH) may be a risk factor for AD, possibly by accelerating brain iron accumulation.<h4>Methods</h4>To test this hypothesis, we genotyped 213 sporadic AD, 106 MCI, and 63 normal elderly control (NEC) individuals for the H63D and C282Y hfe mutations by polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis. We determined the relationship of these mutations to the demographic, clinical, and neuropsychological features of AD and MCI, and evaluated whether an interaction existed between hfe and apolipoprotein E (apoE) status in these patients.<h4>Results</h4>We observed no significant impact of H63D or C282Y heterozygosity on age at AD symptoms onset or diagnosis, age at onset of cognitive symptoms (AD and MCI combined), rates of MCI-to-AD conversion or specific neuropsychological deficits. No interactions between hfe zygosity and apoE status were discerned. Patients homozygous for H63D exhibited trends towards accelerated MCI-to-AD conversion rates and a subset of younger individuals (aged 55-75) exhibited earlier onset of cognitive symptoms relative to wild-type hfe and H63D heterozygotes.<h4>Conclusions</h4>Contrary to earlier reports, the results of the present study do not implicate the common hfe mutations as genetic modifiers of sporadic AD and MCI. Trends towards accelerated cognitive dysfunction in H63D homozygotes warrant further study.

No abstract available.

Staufen is a host protein that is selectively incorporated into human immunodeficiency virus type 1 (HIV-1) particles in a poorly defined process that involves the selection of HIV-1 genomic RNA for encapsidation and the activity of its third double-stranded RNA-binding domain (dsRBD3). To better understand this, we characterized its interactions with pr55(Gag), the principal mediator of HIV-1 genomic RNA encapsidation. Chimeric proviruses harboring wild-type or mutant forms of Staufen were expressed in 293T cells. Cell fractionation analyses demonstrated that Staufen cosedimented with pr55(Gag) within detergent-resistant, trypsin-sensitive complexes that excluded mature capsid and matrix proteins. Coimmunoprecipitation and bioluminescence resonance energy transfer assays demonstrated a specific and direct interaction between Staufen and the nucleocapsid domain of pr55(Gag) in vitro and in live cells. This interaction is shown here to be mediated by Staufen's dsRBD3, with a contribution from its C-terminal domain. Immunoprecipitation and reverse transcription-PCR analyses showed that the 9-kb genomic RNA was found within Staufen-containing immune complexes. Spliced HIV-1 RNAs were not detected in these Staufen complexes, indicating a preferential association of Staufen with the 9-kb species. These results substantiate that Staufen and pr55(Gag) interact directly during HIV-1 expression. Knockdown of Staufen expression by small interfering RNAs in HIV-1-expressing cells demonstrated that this cellular protein was important for the generation of infectious virus. These data show that Staufen, pr55(Gag), and genomic RNA are part of the same intracellular complex and support a role for Staufen in pr55(Gag) function in viral assembly, genomic RNA encapsidation, and the generation of infectious viral particles.

The acute myeloid leukemia (AML)-associated translocation products AML1-ETO, PML-retinoic acid receptor alpha (RARalpha), and PLZF-RARalpha encode aberrant transcription factors. Several lines of evidence suggest similar pathogenetic mechanisms for these fusion proteins. We used high-density oligonucleotide arrays to identify shared target genes in inducibly transfected U937 cells expressing AML1-ETO, PML-RARalpha, or PLZF-RARalpha. All three fusion proteins significantly repressed the expression of 38 genes and induced the expression of 14 genes. Several of the regulated genes were associated with Wnt signaling. One of these, plakoglobin (gamma-catenin), was induced on the mRNA and protein level by all three fusion proteins. In addition, primary AML blasts carrying one of the fusion proteins significantly overexpressed plakoglobin. The plakoglobin promoter was cloned and shown to be induced by AML1-ETO, with promoter activation depending on the corepressor and histone deacetylase binding domains. The induction of plakoglobin by AML fusion proteins led to downstream signaling and transactivation of TCF- and LEF-dependent promoters, including the c-myc promoter, which was found to be bound by plakoglobin in vivo after AML1-ETO expression. beta-Catenin protein levels and TCF and LEF target genes such as c-myc and cyclin D1 were found to be induced by the fusion proteins. On the functional level, a dominant negative TCF inhibited colony growth of AML1-ETO-positive Kasumi cells, whereas plakoglobin transfection into myeloid 32D cells enhanced proliferation and clonal growth. Injection of plakoglobin-expressing 32D cells into syngeneic mice accelerated the development of leukemia. Transduction of plakoglobin into primitive murine hematopoietic progenitor cells preserved the immature phenotype during colony growth, suggesting enhanced self-renewal. These data provide evidence that activation of Wnt signaling is a common feature of several balanced translocations in AML.

Constitutive activation of the Wnt/beta-catenin signaling pathway is a notable feature of a large minority of cases of malignant melanoma, an aggressive and increasingly common cancer. The identification of target genes downstream from this pathway is therefore crucial to our understanding of the disease. The POU domain transcription factor Brn-2 has been implicated in control of proliferation and melanoma survival, and its expression is strongly upregulated in melanoma. We show here that in vivo Brn-2 is expressed in melanocytes but not in embryonic day 11.5 melanoblasts and that its expression is directly controlled by the Wnt/beta-catenin signaling pathway in melanoma cell lines and in transgenic mice. Moreover, silent interfering RNA-mediated inhibition of Brn-2 expression in melanoma cells overexpressing beta-catenin results in significantly decreased proliferation. These results, together with the observation that BRAF signaling also induces Brn-2 expression, reveal that Brn-2 is a focus for the convergence of two key melanoma-associated signaling pathways that are linked to cell proliferation.

Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation.

Hepcidin is a recently identified hormone peptide involved in regulation of iron homeostasis. HAMP gene mutations have been described to date in five families with iron overload. We have identified the c.208T>C (p.C70R) mutation in the HAMP gene in a patient affected by a severe form of hereditary hemochromatosis. The variant, occurring in a highly conserved amino acid, disrupts one of the 4 intramolecular disulphide bonds present in hepcidin molecules of all vertebrates, and is presumably able to destabilize the peptide structure. The investigated patient was also found to harbor a heterozygous HFE c.845G>A (p.C282Y) mutation that may have contributed in increasing his iron burden.

A recently developed strip-assay for hemochromatosis provides a rapid method for simultaneous detection of multiple mutations, which among others includes the HFE gene mutations V53M, V59M, H63D, H63H, S65C, Q127H, E168Q, and C282Y, previously detected in the general South African population using gel-based mutation-screening methods. The objective of the study was to determine the frequency of the relatively rare mutations in samples selected for altered iron parameters or a family history of hereditary hemochromatosis (HH) as part of the validation process of the assay for routine diagnostic purposes. The study population consisted of 451 individuals previously screened for mutations C282Y and H63D by restriction enzyme analysis in order to confirm or possibly exclude a diagnosis of HH. These individuals were subjected to mutation screening using the commercially available hemochromatosis strip-assay. Previous positive results for mutations C282Y and H63D in 233 individuals confirmed the accuracy of the reverse-hybridization assay. Mutation S65C was detected in 13 Caucasians, including three compound heterozygotes. These constituted 2% (13/600) of the chromosomes without mutations C282Y or H63D. The African-specific HFE mutation V53M was detected in one out of 11 (9%) African subjects screened. Mutation E168Q was detected in a single Caucasian individual together with mutation H63D. Our data demonstrate the value of the strip-based technology in providing a rapid and reliable comprehensive test for simultaneous analysis of multiple mutations.

<h4>Background</h4>We studied serum/Plasma iron, ferritin, transferrin concentrations, and total iron binding capacity (TIBC) in addition to highly sensitive C-reactive protein (hs-CRP), low-density lipoprotein (LDL) cholesterol and magnesium concentrations in a population of 800 in- and outpatients and defined new 95% reference intervals for pediatrics using the Dade Behring RxL Dimension Clinical Chemistry System.<h4>Methods</h4>Plasma/serum concentrations of the above analytes were determined on patient samples accrued from January to June 2003 and the data were analyzed employing a computer adapted Hoffmann approach.<h4>Results and conclusions</h4>New pediatric reference intervals on the Dade RxL Dimension were obtained for serum iron, ferritin, transferrin, TIBC, hs-CRP, LDL-cholesterol and magnesium. This work represents the first hs-CRP data for children <3 years old. The values for all the analytes were established on new data (n=800). These values are new for hs-CRP and LDL cholesterol, while for iron, ferritin and magnesium these intervals are more reliable than those previously published by our group which had used regression equations on samples measured by the old (Vitros/Immuno1) methods for which intervals were available.

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-alpha in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-gamma-producing Th cells depending on their capacity to residually produce IFN-alpha. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-alpha-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.

Bleomycin-induced pulmonary fibrosis is characterized by inflammation, emergence of myofibroblasts, and deposition of extracellular matrix. In an attempt to identify genes that may be involved in fibrosis, we used a 10,000 element (10 K) rat cDNA microarray to analyze the lung gene expression profiles in this model in the rat. Cluster analysis showed 628 genes were more than or equal to twofold up- or down-regulated, many of which were known to be involved in fibrosis. However, the most dramatic increase was observed with FIZZ1 (found in inflammatory zone; also known as RELM-alpha or resistin-like molecule-alpha), which was induced 17-fold to approximately 25-fold at the peak of expression. In situ hybridization analysis revealed FIZZ1 expression to localize primarily to alveolar and airway epithelium, which was confirmed in vitro by analysis of isolated type II alveolar epithelial cells. However FIZZ1 expression was not detected in isolated lung fibroblasts. Co-culture of FIZZ1-expressing type II cells with fibroblasts stimulated alpha-smooth muscle actin and type I collagen expression independent of transforming growth factor-beta. Transfection of a FIZZ1-expressing plasmid into fibroblasts or treatment with glutathione S-transferase-FIZZ1 fusion protein stimulated alpha-smooth muscle actin and collagen I production. These results suggest a novel role for FIZZ1 in myofibroblast differentiation in pulmonary fibrosis.

Calcification of necrotic tissue is frequently observed in chronic inflammation and atherosclerosis. A similar response of myocardium to injury, referred to as dystrophic cardiac calcinosis (DCC), occurs in certain inbred strains of mice. We now examined a putative inhibitor of calcification, osteopontin, in DCC after transdiaphragmal myocardial freeze-thaw injury. Strong osteopontin expression was found co-localizing with calcification in DCC-susceptible strain C3H/HeNCrlBr, which exhibited low osteopontin plasma concentrations otherwise. Osteopontin mRNA induction was 20-fold higher than in resistant strain C57BL/6NCrlBr, which exhibited fibrous lesions without calcification and little osteopontin expression. Sequence analysis identified several polymorphisms in calcium-binding and phosphorylation sites in osteopontin cDNA. Their potential relevance for DCC was tested in congenic mice, which shared the osteopontin locus with C57BL/6NCrlBr, but retained a chromosomal segment from C3H/HeNCrlBr on proximal chromosome 7. These mice exhibited strong osteopontin expression and DCC comparable to C3H/HeNCrlBr suggesting that a trans-activator of osteopontin transcription residing on chromosome 7 and not the osteopontin gene on chromosome 5 was responsible for the genetic differences in osteopontin expression. A known osteopontin activator encoded by a gene on chromosome 7 is the transforming growth factor-beta1, which was more induced (3.5x) in C3H/HeNCrlBr than in C57BL/6NCrlBr mice.

Leishmania spp. are protozoans that survive and replicate intracellularly in mammalian macrophages. Antileishmanial immunity requires gamma interferon (IFN-gamma)-mediated macrophage activation and generation of microbicidal effector molecules. The presence of intracellular Leishmania sp. impairs macrophage responses to IFN-gamma, which has led to the description of macrophages as deactivated. It has recently become apparent that in addition to classical activation, macrophages can be activated by distinct triggers to express noninflammatory or anti-inflammatory genes. These nonclassical activation programs have been called alternative or type II pathways. We hypothesized that during initial contact with a phagocyte, leishmaniae activate one of these nonclassical pathways, resulting in expression of genes whose products suppress microbicidal responses. Using DNA microarrays, we studied gene expression in RNAs from BALB/c bone marrow macrophages with and without Leishmania chagasi infection. Some changes were verified by an RNase protection assay, reverse transcription-PCR, immunoblotting, or a bioassay. The pattern of genes activated by leishmania phagocytosis differed from the pattern of genes activated by bacteria or lipopolysaccharide and IFN-gamma. Genes encoding some proinflammatory cytokines, receptors, and Th1-type immune response genes were down-modulated, and some genes associated with anti-inflammatory or Th2-like immune responses were up-regulated. Nonetheless, some markers of alternative (arginase) or type II activation (interleukin-10, tumor necrosis factor alpha) were unchanged. These data suggest that macrophages infected with L. chagasi exhibit a hybrid activation profile that is more characteristic of alternative or type II activation than of classical activation but does not strictly fall into either of these categories. We speculate that the pattern of genes upregulated by leishmania phagocytosis optimizes the chance of parasite survival in this hostile environment.

The putative linker histone in Saccharomyces cerevisiae, Hho1p, has two regions of sequence (GI and GII) that are homologous to the single globular domains of linker histones H1 and H5 in higher eukaryotes. However, the two Hho1p "domains" differ with respect to the conservation of basic residues corresponding to the two putative DNA-binding sites (sites I and II) on opposite faces of the H5 globular domain. We find that GI can protect chromatosome-length DNA, like the globular domains of H1 and H5 (GH1 and GH5), but GII does not protect. However, GII, like GH1 and GH5, binds preferentially (and with higher affinity than GI) to four-way DNA junctions in the presence of excess linear DNA competitor, and binds more tightly than GI to linker-histone-depleted chromatin. Surprisingly, in 10 mM sodium phosphate (pH 7.0), GII is largely unfolded, whereas GI, like GH1 and GH5, is structured, with a high alpha-helical content. However, in the presence of high concentrations of large tetrahedral anions (phosphate, sulphate, perchlorate) GII is also folded; the anions presumably mimic DNA in screening the positive charge. This raises the possibility that chromatin-bound Hho1p may be bifunctional, with two folded nucleosome-binding domains.

Hereditary haemochromatosis is the most common inherited disorder in white populations, whereas non-alcoholic steatohepatitis (NASH) is becoming the most common reason for referral for investigation of abnormal liver function tests (LFTs). This report describes two sisters, from similar environments, who were referred to the clinic after being found to be C282Y homozygotes and to have abnormal LFTs. One sister had developed features of haemochromatosis and the other had developed NASH. These cases illustrate the potential non-penetrance of HFE gene mutations and the need to investigate abnormal LFTs fully, even when there is a positive genetic test at the outset.

A role for the serotonin transporter (5-HTT) gene polymorphism in mental illnesses and anxiety-traits has been implicated. The contribution of genetic factors in personality traits and the manifestation of specific symptoms in psychiatric illnesses have yet to be elucidated. Anxious-depressive symptoms are a significant component in a pattern of schizophrenic symptoms. This study focused on the relation between 5-HTT polymorphism and clinical presentations of schizophrenia, specifically those related to the affective spectrum. Using clinical and psychological analyses, we tested the genetic association between the 5-HTTLPR polymorphism (5-HTT gene-linked polymorphic region) and anxiety- and depressive-related symptoms emerged in schizophrenia. In 260 patients with an ICD-10 diagnosis of schizophrenia (broad definition), we studied the 5-HTTLPR genotype (insertion-deletion polymorphism), the Positive and Negative Syndrome Scale (PANSS), and self-rated inventories (EPI, MMPI, STAI) scores. Patients with the "ss" genotype (deletion variant) scored significantly higher on "Guilt feelings" and "Depression" items, as compared with those of the "ll" genotype (insertion variant) (P = 0.016, 0.039, respectively). The frequency of the "ss" genotype was reduced in patients with no depression or guilt feelings, or in those patients exhibiting questionable symptoms. In contrast, the "ss" genotype carriers prevailed among the patients with mild, moderate, or severe ratings of the symptoms. The scores on all anxiety- and depression-related traits, self-rated by the patients, did not significantly differ by genotype. Our finding may contribute to understanding of molecular genetic features underlying an appearance of psychopathological symptoms emerged in schizophrenia.

In previous studies, serotonin (5-HT) system disturbance was found involved in a variety of behavioral disorders, psychopathologies, and substance use disorders. A functional polymorphism in the promoter region of the human serotonin transporter gene (5-HTTLPR) was recently identified and the presence of the short (S) allele found to be associated with a lower level of expression of the gene, lower levels of 5-HT uptake, type 2 alcoholism, violence and suicidal behavior. In the present study, 101 heroin addicts (males, West European, Caucasians) and 101 healthy control subjects matched for race and gender, with no history of substance use disorder, have been genotyped. Aggressiveness levels were measured in both heroin addicts and controls utilizing Buss-Durkee-Hostility-Inventory (BDHI). Data about suicide attempt and violent criminal behavior in subject history have been collected. The short-short (SS) genotype frequency was significantly higher among heroin dependent individuals compared with control subjects (P = 0.025). The odds ratio for the SS genotype versus the long-long (LL) genotype frequency was 0.69, 95% Cl (0.49-0.97), when heroin addicts were compared with healthy controls. The SS genotype frequency was significantly higher among violent heroin dependent individuals compared with addicted individuals without aggressive behavior (P = 0.02). BDHI mean total scores and suspiciousness and negativism subscales scores were significantly higher in SS individuals, in comparison with LL subjects, among heroin addicts. No association was found between SS genotype and suicide history. Our data suggest that a decreased expression of the gene encoding the 5-HTT transporter, due to "S" promoter polymorphism, may be associated with an increased risk for substance use disorders, particularly in the subjects with more consistent aggressiveness and impulsiveness.

There is strong biological evidence relating alterations in the serotonergic system with mental disorders. These alterations may be originated at the DNA level by sequence mutations that alter the functioning of serotonin receptors and transporter. To test this hypothesis we investigated three genetic variants of the 5-HT2A receptor (-1438G/A, 102T/C and His452Tyr) and two variants of the serotonin transporter (a VNTR in the second intron and a 44 bp insertion/delition in the promoter region of the gene) in a clinical sample recruited in a human isolate and in surrounding areas in Northern Spain (N = 257) and in ethnically matched controls (N = 334). No clear association was found between 5-HT2A variants and psychosis. However, marginal associations were observed between the 5-HTT LPR and VNTR variants and psychosis (P < or = 0.05) indicating a minor contribution to psychosis of genetic alterations in this gene.

Iron uptake and storage are tightly regulated to guarantee sufficient iron for essential cellular processes and to prevent the production of damaging free radicals. A non-classical class I MHC molecule, the hemochromatosis factor HFE, has been shown to regulate iron metabolism, potentially via its direct interaction with the transferrin receptor (TfR). In this study, we demonstrate that a soluble beta2microglobulin-HFE monochain (sHFE) folds with beta2microglobulin (beta2m) and associates with the TfR, indicating that the transmembrane and cytoplasmic domains are not necessary for assembly and trafficking through the ER-Golgi network. We also demonstrate human TfR-specific uptake and accumulation of extracellular sHFE by treated cells. The sHFE localized to the endosomal compartment albeit we observed variation in the time taken for endosomal trafficking between different cell types. The sHFE monochain was effective in reducing Tf uptake into cells, however this did not correlate to any changes in TfR or ferritin synthesis, in contrast to the HFE-induced increase and decrease of TfR and ferritin, respectively. These findings of incongruent sHFE activity suggest that either variation in affinity binding of sHFE to TfR prevents efficient modulation of iron-regulated proteins or that HFE has multiple functions some of which may be independent of TfR but dependent on interactions within the endosomal compartment for effective modulation of iron metabolism.

We report that administration of paroxetine, a widely prescribed antidepressant drug that acts by inhibiting reuptake of the neurotransmitter serotonin, suppresses the neurodegenerative process and increases the survival of huntingtin mutant mice, an animal model of Huntington's disease (HD). Paroxetine attenuated motor dysfunction and body weight loss and improved glucose metabolism in the HD mice. Paroxetine was beneficial when treatment was initiated before or after the onset of motor dysfunction, suggesting a potential for such antidepressant drugs in the treatment of presymptomatic and symptomatic HD patients.

<h4>Objectives</h4>To investigate whether the expression of protein from the "deleted in colorectal cancer" (DCC) gene, which predicts a poor outcome for patients with colorectal carcinoma, can also serve as a prognostic factor in renal cell carcinoma (RCC).<h4>Patients and methods</h4>The expression of DCC was evaluated immunohistochemically in 94 paraffin-embedded tumour samples from patients with stage T1, T2, and T3 clear cell RCC. The mean follow-up was 52.3 months. The endpoints of the study were recurrence of disease and death from disease.<h4>Results</h4>The under-expression of DCC protein was detected in 63% of patients who died from the disease and in 36% with no evidence of disease. DCC protein under-expression was detected in all patients with T1 tumours who died from the disease, in half the T2 tumours and in two-thirds of T3 tumours.<h4>Conclusion</h4>DCC protein under-expression correlated with more aggressive tumour behaviour and a greater risk of death from RCC. However, a larger cohort of patients should be assessed before drawing definitive conclusions.

Epidemiological data suggest that some genetic determinants of Alzheimer's disease (AD) might reside in those polymorphisms for the immune system genes that regulate immune inflammatory responses, such as the major histocompatibility complex (MHC). Therefore, MHC polymorphisms have been the focus of a large number of AD association studies. Class Ia, Ib (hemochromatosis gene (HFE)), class II and class III (complement, tumour necrosis factor and heat shock proteins) alleles have been studied. Nearly every positive result has been followed by several studies that have failed to replicate it or that have contradicted it. Several factors, including methodological biases, might explain these discordant results. However, the discordant results obtained with the same alleles in the various populations might also indicate linkage with another nearby locus, different in the diverse populations. In fact, the non-random assortment of alleles at neighbouring loci, i.e. ancestral haplotypes (AH), has been claimed to be maintained as the result of directional selection, i.e. molecular cooperation during the immune response. Thus, AH studies might contribute to explaining why discordant results are obtained with the same alleles in different populations. Hence, it has been suggested that the overall chance of a subject to develop AD might be profoundly affected by a 'susceptibility profile' reflecting the combined influence of inheriting multiple high-risk alleles. Discordant results may be due to other genetic factors not determined in these MHC studies and multivariate analysis in large patient cohorts considering both MHC and non-MHC genes are therefore necessary.

<h4>Aim</h4>To study the sequential changes of serum ferritin levels in lamivudine-treated patients with chronic viral hepatitis B and the clinical implications.<h4>Methods</h4>Thirty-eight patients with chronic viral hepatitis B were prospectively studied during their treatment with lamivudine. Each patient received 100 mg oral lamivudine daily for 12 mo, and was observed and tested for blood biochemistry and hepatitis B virus (HBV) DNA levels and serum ferritin levels at baseline and at 3, 6 and 12 mo during the treatment. Serum HBV DNA levels were quantitatively determined using fluorescent quantitative polymerase chain reaction (FQ-PCR), and serum ferritin levels were measured by radioimmunoassay. The sequential changes of serum ferritin levels and their relationships with virological, serological and biochemical responses in the patients were analyzed.<h4>Results</h4>All the patients had a baseline HBV DNA level higher than 1 x 10(7) copies/L as determined by FQ-PCR and positive HBsAg and HBeAg and abnormal ALT levels. At the end of the 12-mo treatment, 19 of the 38(50.00%) patients had undetectable serum HBV DNA levels by FQ-PCR, and 12(31.58%) became negative for serum HBeAg and 10(26.32%) had seroconversion from HBeAg to HBeAb. Nineteen out of the 38(50.00%) patients had biochemically normal ALT levels after 12-mo lamivudine treatment. Sequential determination showed that lamivudine treatment significantly reduced ferritin levels in chronic hepatitis B patients. When the patients were divided into different groups according to their post-treatment virological, serological and biochemical responses for analysis of the sequential changes of ferritin levels, it was found that the decrease of ferritin levels in HBV DNA-negative group was significantly more obvious than that in HBV DNA-positive group at 6 mo during the treatment (P=0.013). Consecutive comparisons showed that ferritin levels at 3 mo of treatment were obviously decreased as compared with the baseline levels (P<0.05) in HBeAg-negative group, and the decrease of serum ferritin levels in patients with normalized ALT was more significant than that in patients with abnormal ALT at the end of the 12-mo treatment (P=0.048).<h4>Conclusion</h4>Lamivudine treatment can reduce the serum ferritin levels in chronic viral hepatitis B patients and decreases of ferritin levels can be more significant in patients exhibiting virological, serological and biochemical responses, indicating that dynamic observation of serum ferritin levels in patients with chronic viral hepatitis B during lamivudine treatment might be helpful for monitoring and predicting patients' responses to the therapy.

Dynamic combinatorial chemistry (DCC) has emerged as an efficient approach to receptor/ligand identification based on the generation of combinatorial libraries by reversible interconversion of the library constituents. In this study, the implementation of such libraries on carbohydrate-lectin interactions was examined with the plant lectin Concanavalin A as a target species. Dynamic carbohydrate libraries were generated from a pool of carbohydrate aldehydes and hydrazide linker/scaffold components through reversible acylhydrazone exchange, resulting in libraries containing up to 474 constituents. Dynamic deconvolution allowed the efficient identification of the structural features required for binding to Concanavalin A and the selection of a strong binder, a tritopic mannoside, showing an IC(50)-value of 22 microM.

The functional coupling of transcription and splicing has been reported both in vivo and in vitro, but the molecular mechanisms governing these interactions remain largely unknown. Here we show that p54(nrb), a transcription/splicing factor, associates with the 5' splice site (SS) within large complexes present in HeLa cell nuclear extracts, in which the hyperphosphorylated form of RNA polymerase II (RNAPIIO) is associated with U1 or U1 and U2 snRNPs. These RNAPIIO-snRNP complexes also contain other transcription/splicing factors, such as PSF and TLS, as well as transcription factors that interact with RNAPIIO during elongation, including P-TEFb, TAT-SF1 and TFIIF. The presence of these factors in functional elongation complexes, demonstrated using an immobilized DNA template assay, strongly suggests that the RNAPIIO-snRNP complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Our finding that both p54(nrb) and PSF, which bind the C-terminal domain of the largest subunit of RNAPII, can interact directly with the 5' SS indicates that these factors may mediate contacts between RNAPII and snRNPs during the coupled transcription/splicing process.

<h4>Background</h4>Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact.<h4>Methods</h4>A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy.<h4>Results</h4>Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin.<h4>Conclusions</h4>Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.

GW112 is a novel gene that has little homology to other known genes. It is overexpressed in a number of human tumor types, especially in those of the digestive system. We show here that GW112 is associated with GRIM-19, a protein known to be involved in regulating cellular apoptosis. Functionally, GW112 could significantly attenuate the ability of GRIM19 to mediate retinoic acid-IFN-beta-mediated cellular apoptosis and apoptosis-related gene expression. In addition, GW112 demonstrated strong antiapoptotic effects in tumor cells treated with other stress exposures such as hydrogen peroxide. Finally, forced overexpression of GW112 in murine prostate tumor cells led to more rapid tumor formation in a syngeneic host. Taken together, our data suggest that GW112 is an important regulator of cell death that plays important roles in tumor cell survival and tumor growth.

Hyperplasia of pulmonary artery smooth muscle cells (PA-SMCs) is a hallmark pathological feature of pulmonary hypertension (PH). Serotonin (5-HT) is involved in the hyperplasia through its interactions with specific receptors and internalization by a specific plasma membrane transporter. We investigated the expression and role of the 5-HT transporter (5-HTT) and 5-HT1B, 5-HT2A, and 5-HT2B receptors in lungs and isolated PA-SMCs from patients with primary PH (n=14), pulmonary veno-occlusive disease (n=4), or secondary PH (SPH, n=8) and nonpulmonary hypertensive control subjects. Whereas strong immunostaining for the three receptor types and 5-HTT was seen in remodeled pulmonary vessels from patients in all PH categories, only 5-HTT expression was increased in lungs and cultured PA-SMCs from patients versus controls. The increased growth response of PA-SMCs from patients with primary PH, pulmonary veno-occlusive disease, or SPH to 5-HT or serum was entirely attributable to 5-HTT overexpression, because 5-HTT inhibitors but not 5-HT receptor antagonists abolished 5-HT mitogenic activity and reduced the serum-induced growth response to similar levels in patients as in controls. The L-allelic variant of the 5-HTT gene promoter, which is associated with 5-HTT overexpression, was present homozygously in 14 of 25 (56%) lung transplantation patients with SPH but in only 27% of controls. Polymorphism of the 5-HTT gene promoter was only partly responsible for the increased 5-HTT expression in PH, because PA-SMCs from patients exhibited higher 5-HTT levels than same-genotype cells from controls and no additional promoter sequence alterations were found. We conclude that 5-HTT overexpression is a common pathogenic mechanism in various forms of PH.

<h4>Background</h4>There is evidence that iron may play a role in the pathology of Alzheimer's disease (AD). There may be genetic factors that contribute to iron deposition resulting in tissue damage thus exacerbating AD.<h4>Methods</h4>We have genotyped 269 healthy elderly controls, 191 cases with definite or probable AD, and 69 with mild cognitive impairment (MCI) from the OPTIMA cohort.<h4>Results</h4>We have examined the interaction between the C2 variant of the transferrin (TF) gene and the C282Y allele of the haemochromatosis (HFE) gene as risk factors for developing AD. Our results showed that each of the two variants was associated with an increased risk of AD only in the presence of the other. Neither allele alone had any effect. Carriers of both variants were at 5 times greater risk of AD compared with all others. The interaction was significant by logistic regression (p = 0.014) and by synergy factor analysis (p = 0.015, synergy factor = 5.1). Further, carriers of these two alleles plus apolipoprotein E epsilon4 (APOE4) were at still higher risk of AD: of the 14 tri-carriers of the three variants, identified in this study, 12 had AD and two MCI.<h4>Conclusion</h4>We suggest that the combination of TF C2 and HFE C282Y may lead to an excess of redox-active iron and the induction of oxidative stress in neurones, which is exacerbated in carriers of APOE4. Since 4% of Northern Europeans carry the two iron-related variants and since iron overload is a treatable condition, these results merit replication.

No abstract available.

The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication.

We propose a thermodynamic model that includes the non-specific binding of the lambda phage regulatory proteins CI and Cro. By fitting the model to experimental in vivo data on activities of the two promoters P(RM) and P(R) versus concentration, we estimate the free energy upon non-specific binding to be -4.1+/-0.9 kcal/mol for CI and -4.2+/-0.8 kcal/mol for Cro. For concentrations >100 nM of CI or Cro, we find that >50% of these proteins are non-specifically bound. In particular, in a lysogen (approximately 250 CI monomeric equivalents per cell) nearly 90% of CI is non-specifically bound.

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.

Huntington's disease (HD) is characterized by the accumulation of a pathogenic protein, Huntingtin (Htt), that contains an abnormal polyglutamine expansion. Here, we report that a pathogenic fragment of Htt (Httex1p) can be modified either by small ubiquitin-like modifier (SUMO)-1 or by ubiquitin on identical lysine residues. In cultured cells, SUMOylation stabilizes Httex1p, reduces its ability to form aggregates, and promotes its capacity to repress transcription. In a Drosophila model of HD, SUMOylation of Httex1p exacerbates neurodegeneration, whereas ubiquitination of Httex1p abrogates neurodegeneration. Lysine mutations that prevent both SUMOylation and ubiquitination of Httex1p reduce HD pathology, indicating that the contribution of SUMOylation to HD pathology extends beyond preventing Htt ubiquitination and degradation.

Normal intestinal motility requires orderly development of the complex nerve plexuses and smooth muscular layers in the gut wall. Organization of these structures results, in part, from cell autonomous programmes directed by transcription factors, which orchestrate appropriate temporal and spatial expression of specific target genes. Hox proteins appear to function in combination to dictate regional codes that establish major structural landmarks in the gut such as sphincters and muscle layers. These codes are translated in part by intercellular signals, which allow populations of cells in the embryonic gut wall to alter the developmental fate of their neighbours. Some of the best characterized intercellular signalling pathways involved in enteric neurodevelopment are mediated by GDNF/GFRa1/RET, EDN3/ENDRB, and NETRINS/DCC. These signals affect enteric neural precursors as they colonize the gut, and perturbations of these molecules are associated with various types of intestinal neuropathology.

Axon guidance requires signal transduction of extracellular cues through the plasma membrane for directional motility. Here we present evidence that cholesterol- and sphingolipid-enriched membrane microdomains (lipid rafts) mediate specific guidance responses of nerve growth cones. Disruption of lipid rafts by various approaches targeting cholesterol or gangliosides selectively abolished growth cone attraction and repulsion in BDNF and netrin-1 gradients, respectively, without affecting glutamate-induced attraction. Interestingly, local raft disruption on one side of the growth cone in bath BDNF or netrin-1 produced opposite turning responses to that induced by the gradients. Raft manipulation also blocked Semaphorin 3A-induced growth cone repulsion, inhibition, and collapse. Finally, guidance responses appeared to involve raft-dependent activation of p42/p44 MAPK and ligand-induced receptor recruitment to lipid rafts. Together with the observation of asymmetric receptor-raft associations at the growth cone in guidance gradients, our findings indicate that localized signaling through membrane rafts plays a role in mediating guidance actions of extracellular cues on developing axons.

Many but not all ammonia-oxidizing bacteria (AOB) produce urease (urea amidohydrolase, EC 3.5.1.5) and are capable of using urea for chemolithotrophic growth. We sequenced the urease operons from two AOB, the beta-proteobacterium Nitrosospira sp. strain NpAV and the gamma-proteobacterium Nitrosococcus oceani. In both organisms, all seven urease genes were contiguous: the three structural urease genes ureABC were preceded and succeeded by the accessory genes ureD and ureEFG, respectively. Green fluorescent protein reporter gene fusions revealed that the ure genes were under control of a single operon promoter upstream of the ureD gene in Nitrosococcus oceani. Southern analyses revealed two copies of ureC in the Nitrosospira sp. strain NpAV genome, while a single copy of the ure operon was detected in the genome of Nitrosococcus oceani. The ureC gene encodes the alpha subunit protein containing the active site and conserved nickel binding ligands; these conserved regions were suitable primer targets for obtaining further ureC sequences from additional AOB. In order to develop molecular tools for detecting the ureolytic ecotype of AOB, ureC genes were sequenced from several beta-proteobacterial AOB. Pairwise identity values ranged from 80 to 90% for the UreC peptides of AOB within a subdivision. UreC sequences deduced from AOB urease genes and available UreC sequences in the public databases were used to construct alignments and make phylogenetic inferences. The UreC proteins from beta-proteobacterial AOB formed a distinct monophyletic group. Unexpectedly, the peptides from AOB did not group most closely with the UreC proteins from other beta-proteobacteria. Instead, it appears that urease in beta-proteobacterial autotrophic ammonia oxidizers is the product of divergent evolution in the common ancestor of gamma- and beta-proteobacteria that was initiated before their divergence during speciation. Sequence motifs conserved for the proteobacteria and variable regions possibly discriminatory for ureC from beta-proteobacterial AOB were identified for future use in environmental analysis of ureolytic AOB. These gene sequences are the first publicly available for ure genes from autotrophic AOB.

The persistence of functional CD8 T cell responses is dependent on checkpoints established during priming. Although naive CD8 cells can proliferate with a short period of stimulation, CD4 help, inflammation, and/or high peptide affinity are necessary for the survival of CTL and for effective priming. Using OX40-deficient CD8 cells specific for a defined Ag, and agonist and antagonist OX40 reagents, we show that OX40/OX40 ligand interactions can determine the extent of expansion of CD8 T cells during responses to conventional protein Ag and can provide sufficient signals to confer CTL-mediated protection against tumor growth. OX40 signaling primarily functions to maintain CTL survival during the initial rounds of cell division after Ag encounter. Thus, OX40 is one of the costimulatory molecules that can contribute signals to regulate the accumulation of Ag-reactive CD8 cells during immune responses.

In this study, we evaluated the impact of mutations of the HFE and ferroportin gene on iron overload in thalassemia intermedia and betas/betathal patients. Neither HFE (C282Y and H63D) nor ferroportin(Val162del) mutations were determinants of total body iron status, as assessed by ferritin levels, in either group of patients.

We compared the nutritional status of children aged 1-5 years from slums attending two day-care centres (DCCs) in Nepal (one in an urban slum area) with that of non-attendees to evaluate the impact of supplementary feeding. We measured the anthropometrics of 23 children attending two DCCs and 23 matched controls from the same neighbourhood and interviewed their mothers. We found a better nutritional status, particularly the height-for-age Z-score, among the attendees of the DCC with children's longer attendance, but no difference at the other, although attendees who had been fed for longer tended to show better nutrition. Our study indicates that in the least developed countries good quality day-care of sufficient duration has the potential to improve child nutrition.

In a previous study, we isolated a novel gene, LRRC8 (leucine-rich repeat-containing 8), in a girl with congenital agammaglobulinemia. We have now identified four unknown LRRC8-like genes, named TA-LRRP, AD158, LRRC5, and FLJ23420. Their predicted structures are very similar to each other, and highly conserved between humans and the mouse. All five genes encode proteins consisting of 16 extracellular leucine-rich repeats (LRRs), all of which have four transmembrane regions except for FLJ23420. These genes belong to a novel family, designated the LRRC8 family, within the superfamily of LRR proteins. TA-LRRP, AD158, and LRRC5 might be implicated in proliferation and activation of lymphocytes and monocytes.

The vertebrate retina has been widely used as a model to study the development of the central nervous system. Its accessibility and relatively simple organization allow analysis of basic mechanisms such as cell proliferation, differentiation and death. For this reason, it could represent an ideal place to solve the puzzle of Hh signaling during neural development. However, the extensive wealth of data, sometimes apparently discordant, has made the retina one of the most complicated models for studying the role of the Hh cascade. Given the complexity of the field, a deep analysis of the data arising from different animal models is essential. In this review, we will compare and discuss all reported roles of Hh signaling in eye development to shed light on its multiple functions.

A previously introduced technique of cycling gradient capillary electrophoresis (CGCE) was applied to monitoring of molecular changes during adenoma-carcinoma transition in progression of sporadic colorectal cancer. The purpose of this work was optimization of separation parameters for selected mutation regions in tumor suppressor genes involved in the early stages of colorectal carcinogenesis, followed by scanning for these mutations in clinical tissue samples from patients with adenomatous polyps and early carcinomas. A total of 47 colorectal tumors in various stages of progression were examined. Main emphasis was given to evaluation of mutation detection sensitivity and specificity required for effective early disease detection. A total of 7 different somatic mutations was identified among 32 K-ras mutant samples, 1 inherited mutation and 5 somatic mutations were identified among 15 adenomatous polyposis coli (APC) mutated samples. None of the two previously reported "deleted in colorectal carcinomas" (DCC) mutations was found in any of the clinical samples. In addition to simple optimization of running conditions, CGCE has demonstrated sensitivity and selectivity allowing detecting small mutant fractions as well as combination of multiple mutants within a single target sequence.

The irritable bowel syndrome (IBS) is a complex disorder that is associated with altered gastrointestinal motility, secretion, and sensation. Serotonin (5-HT) is an important neurotransmitter and paracrine signalling molecule in the gastrointestinal tract. 5-HT release from enterochromaffin (EC) cells initiates peristaltic, secretory, vasodilatory, vagal and nociceptive reflexes. The enteric nervous system (ENS) comprises a semiautonomous effector system that is connected to the central autonomic network. Parasympathetic and sympathetic nerves modulate the ENS via afferent and efferent communications. Ongoing, bidirectional brain-gut interactions involving 5-HT pathways occur that significantly influence the effector systems. Altered 5-HT signalling may lead to both intestinal and extraintestinal symptoms in IBS. 5-HT directly and indirectly affects intestinal motor and secretory function and abnormalities may lead to either constipation or diarrhea. 5-HT modulates sensation and perception of visceral stimulation at peripheral and central sites. Therapeutic agents targeting altered 5-HT signalling may provide new, effective treatments for patients with IBS.

<h4>Background</h4>The airway smooth muscle (ASM) cell, originally thought of as a passive structural cell, is now well recognized as an active participant in the pathologic events that occur during persistent asthma. Cell-surface molecules play an important role in the development of an immune response. A number of cell-surface molecules are expressed on ASM cells, and these might contribute to the inflammatory reaction.<h4>Objective</h4>The purpose of this study was to determine whether OX40 ligand (OX40L), a molecule known to be involved in T-cell activation, was present on the ASM cell surface.<h4>Methods</h4>We used real-time RT-PCR to detect mRNA expression and flow cytometry, ELISA, and immunoprecipitation to detect the presence of cell-surface protein on ASM cells isolated from asthmatic and nonasthmatic individuals. ELISAs and Western blotting were used to determine the functional outcomes of engagement of OX40L.<h4>Results</h4>OX40L was present on both asthmatic and nonasthmatic ASM cells. Engagement of OX40L with recombinant OX40:Fc resulted in a significantly greater increase in release of IL-6 from ASM cells of asthmatic patients than from ASM cells of nonasthmatic patients (P<.01). Ligation of OX40L resulted in a rapid translocation of protein kinase C beta2 to the cell membrane.<h4>Conclusion</h4>Because the receptor for OX40L, OX40, is expressed on CD4+ T cells within 48 hours of stimulation through the T-cell receptor, elucidation of the cross-talk between OX40 and OX40L could be very important in understanding the interaction of cells present in the inflamed airways of an asthmatic patient.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, modulates neuronal survival, differentiation, and synaptic function. Reduced BDNF expression in the cortex caused by mutation of the huntingtin gene has been suggested to play a role in the striatal degeneration observed in Huntington's disease. BDNF expression rises dramatically in the cortex during the first few weeks of postnatal life in mice. Previously, it has been impossible to study the specific long-term effects of BDNF absence on CNS structures because of the early postnatal lethality of BDNF-/- mice. Mice harboring a floxed BDNF gene were bred with Emx1(IREScre/+) mice to generate Emx-BDNF(KO) mice that lack cortical BDNF but are viable. Adult Emx-BDNF(KO) mice display a hindlimb clasping phenotype similar to that observed in mouse models of Huntington's disease. The striatum of postnatal Emx-BDNF(KO) mice was reduced in volume compared with controls, and the most abundant neuron type of the striatum, medium spiny neurons (MSNs), had shrunken cell somas, thinner dendrites, and fewer dendritic spines at 35 d of age. Although significant striatal neuron losses were not detected at 35 or 120 d postnatal, 35% of striatal neurons were missing in Emx-BDNF(KO) mice aged beyond 1 year. Thus, cortical BDNF, although not required for the generation or near-term survival of MSN, is necessary for normal striatal neuron dendrite morphology during the period when BDNF expression rises in the cortex. Furthermore, a long-term in vivo requirement for cortical BDNF in supporting the survival of MSNs is revealed.

A recent publication shows that two integrins, alpha6beta4 and alpha3beta1, interact with netrin-1, a protein best known for its role in axon guidance. Consistent with a role in pancreatic development in vivo, netrin is expressed together with these integrins in fetal pancreatic epithelium. The paper presents strong evidence that pancreatic epithelial cells adhere, spread and migrate on netrin-coated substrata through alpha6beta4. Data also indicate that alpha3beta1 mediates HGF-stimulated cell migration on netrin-coated substrata. These results indicate that netrins might have much broader functions than previously suspected.

Analysis of metastatic prostate cancers has identified the Raf kinase inhibitory protein (RKIP) as a suppressor of metastases. Previous studies demonstrated that RKIP binds to Raf-1 and prevents the activation of the extracellular regulated kinase (ERK) cascade. New work shows that phosphorylation of RKIP by protein kinase C disassociates RKIP from Raf-1 and stimulates its binding to, and inhibition of G-protein-coupled receptor kinase 2 (GRK2). This switching enhances signaling by activation of the ERK pathway and by decreased receptor desensitization.

Condensins are pentameric complexes that were initially described as being involved in the dynamics of chromosomes during mitosis. It has been recently established that two related complexes (Condensin I and Condensin II) contribute to this process. An increasing sum of studies, using different approaches in various organisms, leads to the paradigm that Condensins are required for the correct segregation of replicated chromosomes by cooperating somehow with Topoisomerase II in sister chromatid resolution. Depending on species and/or experimental studies, these complexes also contribute to some aspects of the assembly and compaction of mitotic chromosomes. Recent studies provided evidences that Condensins and related complexes also function in non-mitotic processes such as replication and transcription. Biochemical studies have highlighted mechanistic aspects of Condensin function and initiated a fine functional dissection of core and regulatory subunits. However, the exact contribution of each subunit remains largely elusive as well as the functional interplay between Condensin I and Condensin II.

<h4>Aim</h4>Hormone replacement therapy (HRT) may reduce the risk of cardiovascular events in healthy postmenopausal women. However recent studies suggest a 2-4 fold increased risk of idiopathic venous thromboembolism (VTE) among users of HRT. Our aim was to evaluate the overall effect of HRT on hemostatic variables probably related to increased VTE risk reported in epidemiological studies.<h4>Methods</h4>Therefore, 100 healthy postmenopausal women aged 45-60 years divided into 50 HRT non-users and 50 HRT users were examined. The authors assayed on the automated coagulometer ACL7000 (Instrumentation Laboratory, Milan) the procoagulant proteins: factor VIII (VIII:C) and factor VII (VII:C); the natural anticoagulant proteins: antithrombin (ATIII), protein C (PC), protein S (PS) and the resistance to anticoagulant action of activated protein C (APC-Resistance). The free tissue factor pathway inhibitor (TFPI) was measured with an ELISA method (Diagnostica Stagò; France, Roche). The in vivo coagulation and fibrinolysis activation was evaluated by the assays of prothrombin fragment 1+2 (F1+2) and plasmin- antiplasmin complexes (PAP) using ELISA techniques.<h4>Results</h4>Increased levels of FVIII:C and FVII:C were observed in HRT users and HRT non-users women compared to controls (FVIII:C= 126+/-58%, 120+/-59% vs 85+/-15% p=0.0001; FVII: C 113+/-23%, 103+/-19% vs 90+/-16% p=0.0001). The activation peptides were significantly different compared to those found in control subjects; higher values were observed in HRT users compared to HRT non-users (F1+2=1.11+/-0.44 nM, 077+/-0.31 nM vs 0.45+/-0.35 p=0.00001; P-AP= 606+/-406 ng/ml, 514+/-205 ng/ml vs 235+/-59 p=0.0001). The ATIII and the PC were similar among the 3 different groups of subjects, but reduced levels of PS were observed in HRT users (PS 93+/-23%, 105+/-22% vs 109+/-12 p=0.0001). The mean normalized APC sensitivity ratio (APC-SR) was lower in the two populations of women as compared with that of controls (nAPC-SR 1.02+/-0.7, 1.02+/-0.8 vs 1.1+/-25 p=0.02). The values of free TFPI were reduced in HRT users compared to HRT non-users (9.1+/-1.9 ng/ml, 10.1+/-2.3 ng/ml vs 4.6+/-1.5 ng/ml p<0.0001).<h4>Conclusion</h4>HRT appears to be associated to a shift in the procoagulant-anticoagulant balance towards a procoagulant state. The changes in hemostatic system could explain the increased risk of VTE in healthy postmenopausal women during HRT, nevertheless this risk could be higher in women known to have a congenital or acquired thrombophilic state.

Huntington's disease (HD) is a dominantly transmitted neurodegenerative disorder with wide variation in onset age but with an average age at onset of 40 years. Children of HD gene carriers have a 50% chance of inheriting the disease. The characteristic symptoms of HD are involuntary choreiform movements, cognitive impairment, mood disorders, and behavioral changes which are chronic and progressive over the course of the illness. HD is a "trinucleotide repeat" disorder, which is caused by an increase in the number of CAG repeats in the HD gene. Repeats of 40 or larger are associated with disease expression, whereas repeats of 26 and smaller are normal. Intermediate numbers of repeats, between 27 and 35, are not associated with disease expression but may expand in paternal transmission, resulting in the disease in descendents. Repeats of 36-39 are associated with reduced penetrance whereby some develop HD and others do not. The identification of the genetic defect in HD permits direct genetic testing for the presence of the gene alteration responsible for the disease. Tests may be performed in three circumstances: (1) confirmation of diagnosis, (2) predictive testing of persons at genetic risk for inheriting HD, and (3) prenatal testing. Testing is widely available and much experience has been gained with protocols that assist the individual in making an informed choice about test options, and minimize the occurrence of adverse emotional outcomes.

Fifty cases comprising 11 cases of disseminated intravascular coagulation (DIC), 16 cases of venous thromboses and 23 cases of hepatic diseases were studied for AT III levels using clotting assay. Twelve samples were subjected to ATIII estimation by the commercially available synthetic chromogenic assay. Twenty age and sex matched controls were also analysed to find out the reference value for the techniques. Low AT III levels, if present, were correlated with other markers of DIC, viz FDP and D-dimer assays. There was a decrease in the AT III levels in all the three disease categories with a significant difference between the AT III levels of the three disease categories. In DIC, lowest levels were observed which correlated well with FDP and D-dimer levels. There was no significant difference between the average AT III levels measured by both the clotting and synthetic chromogenic assay with the former procedure being relatively inexpensive.