Molecular evidence for the expression of nicotinic acetylcholine receptor alpha-chain in mouse thymus.

The presence and structure of nicotinic acetylcholine receptor (nAChR) in the thymus has been a subject of interest for many years because of its possible role in the pathogenesis of the autoimmune disease myasthenia gravis. Using the polymerase chain reaction with primers specific for the alpha-chain of nAChR (nAChR-alpha), an 880-bp homologous band was found after amplification of cDNA prepared from mouse thymus, thymic medullary and cortical epithelial cell lines, but not from thymocytes or kidney. Sequencing of the polymerase chain reaction product from the thymus and thymic medullary and cortical epithelial lines showed identity with skeletal muscle nAChR-alpha over the region examined. This region includes the domains of the molecule on which B cell and T cell autoantigenic targets have been described. No evidence was found in mouse tissue for the exon 3A, which has been described in human muscle and the human rhabdomyosarcoma cell line TE671. Our results provide evidence at the RNA level for the expression of the nAChR-alpha on stromal cells but not on thymocytes in normal murine thymus and are consistent with a role for intrathymic autoantigen expression in the pathogenesis of myasthenia gravis.     

Fc gamma receptor-mediated interplatelet activation by a monoclonal antibody against beta 2 microglobulin.

Three different mAb directed against beta 2 microglobulin (two IgG1 and one IgG2a) were tested for their ability to activate human platelets. Although all three antibodies bound to platelets, only one of them, B2.62.2, of the IgG1 subclass, induced platelet activation. This activation is similar to the activation by SYB-1, a CD9 antibody of the same subclass previously described as activating platelets through platelet Fc gamma R. These similarities include serotonin secretion, a lag time preceding aggregation and the induction of a strong intracellular calcium mobilization from storage pools. As with CD9 antibodies, the F(ab')2 fragments of B2.62.2 did not induce activation but blocked the activation by the native antibody, by preventing the binding to beta 2 microglobulin. Also, this activation was inhibited by pretreating the platelet with IV-3, a mAb that blocks the Fc binding site of the FcR. Inasmuch as the same antibody does not prevent the binding of B2.62.2 on platelets, we conclude that the activation by B2.62.2 is mediated by the FcR. Nevertheless, there were differences with the activation by SYB-1. B2.62.2 activation was more dependent on thromboxane A2 formation and no cytoplasmic alkalinization was detected. Finally, contrary to SYB-1, B2.62.2 activation proved to be sensitive to platelet count, suggesting that it involves the formation of immune complexes consisting of antibodies and platelets, that activate nearby platelets.     

Bartonella spp. Bacteremia in Blood Donors from Campinas,Brazil

Bartonella species are blood-borne, re-emerging organisms, capable of causing prolonged infection with diverse disease manifestations, from asymptomatic bacteremia to chronic debilitating disease and death. This pathogen can survive for over a month in stored blood. However, its prevalence among blood donors is unknown, and screening of blood supplies for this pathogen is not routinely performed. We investigated Bartonella spp. prevalence in 500 blood donors from Campinas, Brazil, based on a cross-sectional design. Blood samples were inoculated into an enrichment liquid growth medium and sub-inoculated onto blood agar. Liquid culture samples and Gram-negative isolates were tested using a genus specific ITS PCR with amplicons sequenced for species identification. Bartonella henselae and Bartonella quintana antibodies were assayed by indirect immunofluorescence. B. henselae was isolated from six donors (1.2%). Sixteen donors (3.2%) were Bartonella-PCR positive after culture in liquid or on solid media, with 15 donors infected with B. henselae and one donor infected with Bartonella clarridgeiae. Antibodies against B. henselae or B. quintana were found in 16% and 32% of 500 blood donors, respectively. Serology was not associated with infection, with only three of 16 Bartonella-infected subjects seropositive for B. henselae or B. quintana. Bartonella DNA was present in the bloodstream of approximately one out of 30 donors from a major blood bank in South America. Negative serology does not rule out Bartonella spp. infection in healthy subjects. Using a combination of liquid and solid cultures, PCR, and DNA sequencing, this study documents for the first time that Bartonella spp. bacteremia occurs in asymptomatic blood donors. Our findings support further evaluation of Bartonella spp. transmission which can occur through blood transfusions.     

Proteomic profiling of cerebrospinal fluid in Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare fatal neurodegenerative disease belonging to the group of transmissible spongiform encephalopathies or prion diseases. The agent responsible for the disease is the prion protein in an altered conformational form. Although there have been countless studies performed on the prion protein, the mechanisms that induce the structural change of the normal protein, and the harmful action the altered protein has on nervous cells, are still not fully understood. Furthermore, the final diagnosis for CJD can only occur with a postmortem histopathological analysis of the brain; the antemortem diagnosis is only possible for some specific CJD forms. Finally, there is no current treatment able to stop or delay the progression of the disease. Studies directed at resolving these issues are, therefore, extremely relevant. The proteomic approach is a very good strategy to be applied in such contexts because it allows easy identification of proteins and peptides possibly involved in the disease processes. In this article, the existing data regarding prion infection, biomarkers for CJD diagnosis and the use of several modern proteomic technologies for the identification of new cerebrospinal fluid polypeptides involved in CJD are reviewed.     

Analysis of human skeletal muscle after 48 h immobilization reveals alterations in mRNA and protein for extracellular matrix components.

We examined the effects of 48 h of knee immobilization on alterations in mRNA and protein in human skeletal muscle. We hypothesized that 48 h of immobilization would increase gene expression and respective protein products for ubiquitin-proteasome pathway (UPP) components. Also, we used microarray analysis to identify novel pathways. Biopsies were taken from the vastus muscle of five men (20.4 +/- 0.5 yr) before and after 48-h immobilization. Global changes in gene expression were analyzed by use of Affymetrix GeneChips. Candidate genes were confirmed via quantitative RT-PCR. Western blotting (WB) was used to quantify protein products of candidate genes and to assess Akt pathway activation. Immunohistochemistry was used to localize proteins found to be altered when assessed via WB. The greatest percentage of genes showing altered expression with the GeneChip included genes involved in the UPP, metallothionein function, and extracellular matrix (ECM) integrity. Quantitative RT-PCR analysis confirmed increases in mRNA for UPP components [USP-6, small ubiquitin-related modifier (SUMO-1)] and the metallothioneins (MT2A, MT1F, MT1H, MT1X) and decreases in mRNA content for matrix metalloproteinases (MMP-28, TIMP-1) and ECM structural components [collagen III (COLIII) and IV (COLIV)]. Only phosphorylated Akt (Ser473, Thr308), COLIII and COLIV protein levels were significantly different postimmobilization (25, 10, 88, and 28% decrease, respectively). Immunohistochemistry confirmed WB showing decreased staining for collagens postimmobilization. Our results suggest that 48 h of immobilization increases mRNA content for components of the UPP and metallothionein function while decreasing mRNA and protein for ECM components as well as decreased phosphorylation of Akt.     

Adenosine A3 receptor stimulation reduces muscle injury following physical trauma and is associated with alterations in the MMP/TIMP response

We have previously demonstrated that in response to traumatic injury in skeletal muscle, there is a dysregulation of the matrix metalloproteases (MMPs) and their inhibitors (TIMPs), a response hypothesized to interfere with proper skeletal muscle regeneration. Moreover, we have shown that pharmacological activation of the adenosine A(3) receptor by Cl-IBMECA in skeletal muscle can protect against ischemia-reperfusion and eccentric exercise injury. However, the mechanism by which Cl-IBMECA protects muscle tissue is poorly defined. This study evaluated the effects of Cl-IBMECA on MMP/TIMP expression in skeletal muscle and tested the hypothesis that adenosine A(3) receptor-stimulated protection of skeletal muscle following traumatic injury is associated with a blunting of MMPs involved in inflammatory processes and collagen degradation, and an increase in MMPs associated with extracellular matrix remodeling. Sixty C57BL/6J male mice were injected with Cl-IBMECA (n = 30) or a vehicle (n = 30), and Evans blue dye. Injury was induced by applying a cold steel probe (-79°C) to the tibialis anterior (TA) muscle for 10 s. TA muscles from uninjured and injured legs were collected 3, 10, and 24 h postinjury for analysis of muscle injury and MMP/TIMP mRNA and protein levels. Twenty-four hours postinjury, 56.8% of the fibers were damaged in vehicle-treated mice vs. 35.4% in Cl-IBMECA-treated mice (P = 0.02). Cl-IBMECA treatment reduced membrane type 1 (MT1)-MMP, MMP-3, MMP-9, and TIMP-1 mRNA expression 2- to 20-fold compared with vehicle-treated mice (P < 0.05). Cl-IBMECA decreased protein levels of latent/shed MT1-MMP 23-2,000%, respectively, 3-10 h postinjury. In Cl-IBMECA-treated mice, latent MMP-2 was decreased 20% 3 h postinjury, active MMP-3 was decreased 64% 3 h postinjury, and latent/active MMP-9 was decreased 417,631% 3 h postinjury and 20% 10 h postinjury. Protein levels of active MMP-2 and latent MMP-3 were increased 25% and 74% 3 h postinjury, respectively. The present study elucidates a new protective role of adenosine A(3) receptor stimulation in posttraumatic skeletal muscle injury.     

Heparin Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides: STRUCTURAL FEATURES AND BIOLOGICAL PROPERTIES

The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (A_NAc,6S-GlcUA-A_NS,3,6S-I_2S-A_NS,6S). By using the highly chemoselective depolymerization to prepare new ultra low molecular weight heparin and coupling it with the original separation techniques, it was possible to isolate a polysaccharide with a biosynthetically unexpected structure and excellent antithrombotic properties. It consisted of a dodecasaccharide containing an unsaturated uronate unit at the nonreducing end and two contiguous AT-binding sequences separated by a nonsulfated iduronate residue. This novel oligosaccharide was characterized by NMR spectroscopy, and its binding with AT was determined by fluorescence titration, NMR, and LC-MS. The dodecasaccharide displayed a significantly increased anti-FXa activity compared with those of the pentasaccharide, fondaparinux, and low molecular weight heparin enoxaparin.     

miRTissue: a web application for the analysis of miRNA-target interactions in human tissues

Background

microRNAs act as regulators of gene expression interacting with their gene targets. Current bioinformatics services, such as databases of validated miRNA-target interactions and prediction tools, usually provide interactions without any information about what tissue that interaction is more likely to appear nor information about the type of interactions, causing mRNA degradation or translation inhibition respectively.

Results

In this work, we introduce miRTissue, a web application that combines validated miRNA-target interactions with statistical correlation among expression profiles of miRNAs, genes and proteins in 15 different human tissues. Validated interactions are taken from the miRTarBase database, while expression profiles are downloaded from The Cancer Genome Atlas repository. As a result, the service provides a tissue-specific characterisation of each couple of miRNA and gene together with its statistical significance (p-value). The inclusion of protein data also allows providing the type of interaction. Moreover, miRTissue offers several views for analysing interactions, focusing for example on the comparison between different cancer types or different tissue conditions. All the results are freely downloadable in the most common formats.

Conclusions

miRTissue fills a gap concerning current bioinformatics services related to miRNA-target interactions because it provides a tissue-specific context to each validated interaction and the type of interaction itself. miRTissue is easily browsable allowing the user to select miRNAs, genes, cancer types and tissue conditions. The results can be sorted according to p-values to immediately identify those interactions that are more likely to occur in a given tissue. miRTissue is available at http://tblab.pa.icar.cnr.it/mirtissue.html.