The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state

Indexación: Web of ScienceWith about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients.http://www.nature.com/articles/srep2590

Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

Background:Trypanosoma cruzi ribosomal P proteins, P2β and P0, induce high levels of antibodies in patients with chronic Chagas' disease Cardiomyopathy (CCC). It is well known that these antibodies alter the beating rate of cardiomyocytes and provoke apoptosis by their interaction with β1-adrenergic and M2-muscarinic cardiac receptors. Based on these findings, we decided to study the cellular immune response to these proteins in CCC patients compared to non-infected individuals.Methodology/Principal findings:We evaluated proliferation, presence of surface activation markers and cytokine production in peripheral blood mononuclear cells (PBMC) stimulated with P2β, the C-terminal portion of P0 (CP0) proteins and T. cruzi lysate from CCC patients predominantly infected with TcVI lineage. PBMC from CCC patients cultured with P2β or CP0 proteins, failed to proliferate and express CD25 and HLA-DR on T cell populations. However, multiplex cytokine assays showed that these antigens triggered higher secretion of IL-10, TNF-α and GM-CSF by PBMC as well as both CD4+ and CD8+ T cells subsets of CCC subjects. Upon T. cruzi lysate stimulation, PBMC from CCC patients not only proliferated but also became activated within the context of Th1 response. Interestingly, T. cruzi lysate was also able to induce the secretion of GM-CSF by CD4+ or CD8+ T cells.Conclusions/Significance:Our results showed that although the lack of PBMC proliferation in CCC patients in response to ribosomal P proteins, the detection of IL-10, TNF-α and GM-CSF suggests that specific T cells could have both immunoregulatory and pro-inflammatory potential, which might modulate the immune response in Chagas' disease. Furthermore, it was possible to demonstrate for the first time that GM-CSF was produced by PBMC of CCC patients in response not only to recombinant ribosomal P proteins but also to parasite lysate, suggesting the value of this cytokine to evaluate T cells responses in T. cruzi infection.Fil: Longhi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Atienza, Augusto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Perez Prados, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Juan A. Fernández"; ArgentinaFil: Buying, Alcinette. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Santos, Radleigh. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Tasso, Laura Mónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bonato, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Chiale, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Pinilla, Clemencia. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Judkowski, Valeria A.. Torrey Pines Institute for Molecular Studies; Estados UnidosFil: Gomez, Karina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Abnormal behavior in mice mutant for the Disc1 binding partner, Dixdc1

Disrupted-in-Schizophrenia-1 (DISC1) is a genetic susceptibility locus for major mental illness, including schizophrenia and depression. The Disc1 protein was recently shown to interact with the Wnt signaling protein, DIX domain containing 1 (Dixdc1). Both proteins participate in neural progenitor proliferation dependent on Wnt signaling, and in neural migration independently of Wnt signaling. Interestingly, their effect on neural progenitor proliferation is additive. By analogy to Disc1, mutations in Dixdc1 may lead to abnormal behavior in mice, and to schizophrenia or depression in humans. To explore this hypothesis further, we generated mice mutant at the Dixdc1 locus and analyzed their behavior. Dixdc1−/− mice had normal prepulse inhibition, but displayed decreased spontaneous locomotor activity, abnormal behavior in the elevated plus maze and deficits in startle reactivity. Our results suggest that Dixdc1−/− mice will be a useful tool to elucidate molecular pathophysiology involving Disc1 in major mental illnesses

Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique

A new approach for the determination of the amyloid fibril – thioflavin T (ThT) binding parameters (the number of binding modes, stoichiometry, and binding constants of each mode) is proposed. This approach is based on the absorption spectroscopy determination of the concentration of free and bound to fibril dye in solutions, which are prepared by equilibrium microdialysis. Furthermore, the proposed approach allowed us, for the first time, to determine the absorption spectrum, molar extinction coefficient, and fluorescence quantum yield of the ThT bound to fibril by each binding modes. This approach is universal and can be used for determining the binding parameters of any dye interaction with a receptor, such as ANS binding to proteins in the molten globule state or to protein amorphous aggregates

Rationalising the role of Keratin 9 as a biomarker for Alzheimer’s disease

Keratin 9 was recently identified as an important component of a biomarker panel which demonstrated a high diagnostic accuracy (87%) for Alzheimer’s disease (AD). Understanding how a protein which is predominantly expressed in palmoplantar epidermis is implicated in AD may shed new light on the mechanisms underlying the disease. Here we use immunoassays to examine blood plasma expression patterns of Keratin 9 and its relationship to other AD-associated proteins. We correlate this with the use of an in silico analysis tool VisANT to elucidate possible pathways through which the involvement of Keratin 9 may take place. We identify possible links with Dickkopf-1, a negative regulator of the wnt pathway, and propose that the abnormal expression of Keratin 9 in AD blood and cerebrospinal fluid may be a result of blood brain barrier dysregulation and disruption of the ubiquitin proteasome system. Our findings suggest that dysregulated Keratin 9 expression is a consequence of AD pathology but, as it interacts with a broad range of proteins, it may have other, as yet uncharacterized, downstream effects which could contribute to AD onset and progression

Magnetic resonance imaging after most common form of concussion

<p>Abstract</p> <p>Background</p> <p>Until now there is a lack of carefully controlled studies with conventional MR imaging performed exclusively in concussion with short lasting loss of consciousness (LOC).</p> <p>Methods</p> <p>A MR investigation was performed within 24 hours and after 3 months in 20 patients who had suffered a concussion with a verified loss of consciousness of maximally 5 minutes. As a control group, 20 age- and gender matched patients with minor orthopaedic injuries had a MR investigation using the same protocol.</p> <p>Results</p> <p>In a concussion population with an average LOC duration of 1. 4 minutes no case with unequivocal intracranial traumatic pathology was detected.</p> <p>Conclusion</p> <p>An ordinary concussion with short lasting LOC does not or only seldom result in a degree of diffuse axonal injury (DAI) that is visualized by conventional MR with field strength of 1.0 Tesla (T). Analysis of earlier MR studies in concussion using field strength of 1.5 T as well as of studies with diffusion tensor MR imaging (MR DTI) reveal methodological shortcomings, in particular use of inadequate control groups. There is, therefore, a need for carefully controlled studies using MR of higher field strength and/or studies with MR DTI exclusively in common concussion with LOC of maximally 5 minutes.</p

The potential role for prolactin-inducible protein (PIP) as a marker of human breast cancer micrometastasis

The prolactin-inducible protein (PIP/GCPD15) is believed to originate from a limited set of tissues, including breast and salivary glands, and has been applied as a clinical marker for the diagnosis of metastatic tumours of unknown origin. We have investigated the potential role of PIP mRNA as a marker of human breast cancer metastasis. Using reverse transcription polymerase chain reaction and Southern or dot blot analysis, PIP mRNA was detected in 4/6 breast cell lines, independent of oestrogen receptor (ER) status. In breast primary tumours (n = 97), analysed from histologically characterized sections, PIP mRNA was detected in most cases. Higher PIP mRNA levels correlated with ER+ (P = 0.0004), progesterone receptor positive (PR+) (P = 0.0167), low-grade (P = 0.0195) tumours, and also PIP protein levels assessed by immunohistochemistry (n = 19, P = 0.0319). PIP mRNA expression was also detectable in 11/16 (69%) of axillary node metastases. PIP mRNA expression, however, was also detected in normal breast duct epithelium, skin, salivary gland and peripheral blood leucocyte samples from normal individuals. We conclude that PIP mRNA is frequently expressed in both primary human breast tumours and nodal metastases. However, the presence of PIP expression in skin creates a potential source of contamination in venepuncture samples that should be considered in its application as a marker for breast tumour micrometastases. © 1999 Cancer Research Campaig

Survivin expression in oral squamous cell carcinoma

A series of 110 cases of oral squamous cell carcinoma (SCC) together with six lymph node and one distant metastatic lesions was analysed for expression of survivin, a recent apoptosis inhibitor, by immunohistochemistry and Western blotting. In total, 91 cases (82.7%) of carcinoma and all metastasis (seven cases, 100%) were positive for survivin expression, with weighted survivin scores ranging from 1 to 4. In contrast, normal oral epithelium did not express survivin. There was no significant correlation between survivin expression and age, sex, tumour size, the presence of lymph node and distant metastases. Survivin expression was increased in poorly differentiated tumours, even if differences were not statistically significant. In contrast, when analysed for prognostic significance, patients with low survivin expression had statistically significant better survival rates than the group with high survivin expression (P < 0.05). These data suggest that survivin expression may identify cases of oral SCC with more aggressive and invasive phenotype

An in vivo cis-Regulatory Screen at the Type 2 Diabetes Associated TCF7L2 Locus Identifies Multiple Tissue-Specific Enhancers

Genome-wide association studies (GWAS) have repeatedly shown an association between non-coding variants in the TCF7L2 locus and risk for type 2 diabetes (T2D), implicating a role for cis-regulatory variation within this locus in disease etiology. Supporting this hypothesis, we previously localized complex regulatory activity to the TCF7L2 T2D-associated interval using an in vivo bacterial artificial chromosome (BAC) enhancer-trapping reporter strategy. To follow-up on this broad initial survey of the TCF7L2 regulatory landscape, we performed a fine-mapping enhancer scan using in vivo mouse transgenic reporter assays. We functionally interrogated approximately 50% of the sequences within the T2D-associated interval, utilizing sequence conservation within this 92-kb interval to determine the regulatory potential of all evolutionary conserved sequences that exhibited conservation to the non-eutherian mammal opossum. Included in this study was a detailed functional interrogation of sequences spanning both protective and risk alleles of single nucleotide polymorphism (SNP) rs7903146, which has exhibited allele-specific enhancer function in pancreatic beta cells. Using these assays, we identified nine segments regulating various aspects of the TCF7L2 expression profile and that constitute nearly 70% of the sequences tested. These results highlight the regulatory complexity of this interval and support the notion that a TCF7L2 cis-regulatory disruption leads to T2D predisposition

Mouse Acetylcholinesterase Enhances Neurite Outgrowth of Rat R28 Cells Through Interaction With Laminin-1

The enzyme acetylcholinesterase (AChE) terminates synaptic transmission at cholinergic synapses by hydrolyzing the neurotransmitter acetylcholine, but can also exert ‘non-classical’, morpho-regulatory effects on developing neurons such as stimulation of neurite outgrowth. Here, we investigated the role of AChE binding to laminin-1 on the regulation of neurite outgrowth by using cell culture, immunocytochemistry, and molecular biological approaches. To explore the role of AChE, we examined fiber growth of cells overexpressing different forms of AChE, and/or during their growth on laminin-1. A significant increase of neuritic growth as compared with controls was observed for neurons over-expressing AChE. Accordingly, addition of globular AChE to the medium increased total length of neurites. Co-transfection with PRIMA, a membrane anchor of AChE, led to an increase in fiber length similar to AChE overexpressing cells. Transfection with an AChE mutant that leads to the retention of AChE within cells had no stimulatory effect on neurite length. Noticeably, the longest neurites were produced by neurons overexpressing AChE and growing on laminin-1, suggesting that the AChE/laminin interaction is involved in regulating neurite outgrowth. Our findings demonstrate that binding of AChE to laminin-1 alters AChE activity and leads to increased neurite growth in culture. A possible mechanism of the AChE effect on neurite outgrowth is proposed due to the interaction of AChE with laminin-1