Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Human urothelial cells grown on collagen adsorbed to surface-modified polymers.

OBJECTIVES: Tissue engineering methods can be applied to regenerate diseased, or congenitally missing, urinary tract tissues. Urinary tract tissue cell cultures must be established in vitro and adequate matrices, acting as cell carriers, must be developed. Although degradable and nondegradable polymer matrices offer adequate mechanical stability, they are not optimal for cell adherence and growth. To overcome this problem, extracellular matrix proteins, permitting cell adhesion and regulation of cell proliferation and differentiation, can be adsorbed to the surface-modified polymer. METHODS: In this study, nondegradable polymer films, poly(ethylene terephthalate), were used as an experimental model. Films were modified by graft polymerization of acrylic acid to subsequently allow collagen type I and III immobilization. The following adhesion, proliferation of human urothelial cells, and induction of their stratification were analyzed. RESULTS: Collagen adsorption on 0.2 microg/cm2 poly(acrylic acid)-grafted polymer films rendered the matrix apt for human urothelial cell adhesion and proliferation. Furthermore, stratification of urothelial cells was demonstrated on these surface-modified matrices. CONCLUSIONS: These results have shown that surface-modified polymer matrices can be used to act as cell carriers for cultured human urothelial cells. Such a cell-matrix construct could be applied in reparative surgery of the urinary tract.

Linear Aggregation In The Social Accounting Matrix Framework

In economic literature, information deficiencies and computational complexities have traditionally been solved through the aggregation of agents and institutions. In inputoutput modelling, researchers have been interested in the aggregation problem since the beginning of 1950s. Extending the conventional input-output aggregation approach to the social accounting matrix (SAM) models may help to identify the effects caused by the information problems and data deficiencies that usually appear in the SAM framework. This paper develops the theory of aggregation and applies it to the social accounting matrix model of multipliers. First, we define the concept of linear aggregation in a SAM database context. Second, we define the aggregated partitioned matrices of multipliers which are characteristic of the SAM approach. Third, we extend the analysis to other related concepts, such as aggregation bias and consistency in aggregation. Finally, we provide an illustrative example that shows the effects of aggregating a social accounting matrix model.

Preferential binding of the methyl-CpG binding domain protein 2 at methylated transcriptional start site regions.

Methyl-CpG Binding Domain (MBD) proteins are thought to be key molecules in the interpretation of DNA methylation signals leading to gene silencing through recruitment of chromatin remodeling complexes. In cancer, the MBD-family member, MBD2, may be primarily involved in the repression of genes exhibiting methylated CpG at their 5' end. Here we ask whether MBD2 randomly associates methylated sequences, producing chance effects on transcription, or exhibits a more specific recognition of some methylated regions. Using chromatin and DNA immunoprecipitation, we analyzed MBD2 and RNA polymerase II deposition and DNA methylation in HeLa cells on arrays representing 25,500 promoter regions. This first whole-genome mapping revealed the preferential localization of MBD2 near transcription start sites (TSSs), within the region analyzed, 7.5 kb upstream through 2.45 kb downstream of 5' transcription start sites. Probe by probe analysis correlated MBD2 deposition and DNA methylation. Motif analysis did not reveal specific sequence motifs; however, CCG and CGC sequences seem to be overrepresented. Nonrandom association (multiple correspondence analysis, p < 0.0001) between silent genes, DNA methylation and MBD2 binding was observed. The association between MBD2 binding and transcriptional repression weakened as the distance between binding site and TSS increased, suggesting that MBD2 represses transcriptional initiation. This hypothesis may represent a functional explanation for the preferential binding of MBD2 at methyl-CpG in TSS regions.

Cell Adhesion Peptide RGDSP and Laminin Support Proliferation and Migration of Postnatal Retinal Stem Cells in a 3D Hydrogel Culture System

Purpose: Retinal stem cells (RSCs) can be isolated from radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have great capacity to renew and generate neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, our published results showed poor integration and survival rate after cell grafting into the retina. The uncontrollable environment of retina seems to be the problem. To bypass this, we are trying to generate hemi-retinal tissue in vitro that can be used for transplantation. Methods: Expanded RSCs were seeded in a mixture of poly-ethylene-glycol (PEG)-polymer-based hydrogels crosslinked by peptides that also serve as substrates for matrix metalloproteinases. Different doses of crosslinker peptides were tested. Several growth factors were studied to stimulate cell proliferation and differentiation. Results: Cells were trapped in hydrogels and cultured in the presence of FGF2 and EGF. Spherical cell clusters indicating proliferation appeared within several days, but there was no cell migration within the gel. We then added cell adhesion molecules integrin ligand RGDSP, or laminin, or a combination of both, into the gel. Cells grown with laminin showed the best proliferation. Cells grown with RGDSP proliferated a few times and then started to spread out. Cells grown with the combination of RGDSP and laminin showed better proliferation than with RGDSP alone and larger spread-outs than with laminin alone. After stimulations with first FGF2 and EGF, and then only FGF2, some cells showed neuronal morphology after 2 weeks. The neuronal population was assessed by the presence of neuronal marker b-tubulin-III. Glial cells were also present. Further characterizations are undergoing. Conclusions: RSC can grow and migrate in 3D hydrogel with the addition of FGF2, EGF, RGDSP and laminin. Further developments are necessary to form a homogenous tissue containing retinal cells.

Octacalcium Phosphate (OCP) Crystals Induce Synovial Inflammation and Cartilage

Introduction: The presence of intra-articular basic calcium phosphate (BCP) crystals, including OCP, carbonated-apatite, hydroxyapatite and tricalcium phosphate crystals, is associated with severe osteoarthritis and destructive arthropathies such as Milwaukee shoulder. Although BCP crystals displayed, in vitro, mitogenic, anabolic and catabolic responses, their intra-articular effect was never assessed.Objective: To determine the effects of OCP crystals in joints in vivo.Methods: OCP crystals (200 ug in 20 ml PBS) were injected into the right knee joint (the contra-lateral knee joint injected with 20 ul of PBS serving as a control) of wild-type mice treated or not by the IL1R antagonist Anakinra or mice deficient for the inflammasome proteins ASC and NALP3. 4 days and 17 days after crystal injection, mice were sacrificed and knee joints dissected. Histological scoring for synovial inflammation and characterisation of macrophages, neutrophils and T cells were performed. Technetium (Tc) uptake was measured at 6h, 1 and 4 days after OCP injection. Cartilage degradation was evaluated by Safranin O staining and VDIPEN immunohistochemistry. Intra-articular localisation of injected OCP crystals was evidenced by Von Kossa staining.Results: The intra-articular localisation of injected OCP crystals was evidenced by Von Kossa staining performed on non-decalcified samples embedded in methyl-metacrylate. Injection of OCP crystals into knee joints led at day 4 to an inflammatory response with intense macrophage staining and also some neutrophil recruitment in the synovial membrane. This synovitis was not accompanied by increased Tc uptake into the knee joint, Tc uptake being similar in OCP crystal injected knee or control knee at all time points investigated (6h, 1 day, 4 days). The histological modifications persisted over 17 days, with an additional fibrosis evidenced at this later time-point. The OCP crystal-induced synovitis was totally IL-1a and IL-1 independent as shown by the absence of inhibitory effects of anakinra injected into wild-type mice. Accordingly, OCP crystal-induced synovitis was similar in ASC-/- and NALP3-/- mice as no alterations of inflammation were demonstrated between these mice groups. Concerning cartilage matrix degradation, OCP crystals induced a strong breakdown of proteoglycans 4 and 17 days after injection, as measured by loss of red staining from Safranin O-stained sections of cartilage surfaces. In addition, we also measured advanced cartilage matrix destruction mediated by MMPs, as evidenced by VDIPEN staining of cartilage. OCP-mediated cartilage degradation was similar in all experimental conditions tested (WT+Anakinra, or ASC or NALP3 deficient mice).Conclusion: These data indicate in vivo that the intra-articular presence of OCP crystals is associated with cartilage destruction along with synovial inflammation. This is an interesting and new model of destructive arthropathy related to BCP crystals which will allow to assess new therapies in this disease.

The nuclear matrix: a critical appraisal.

It is becoming increasingly clear that the cell nucleus is a highly structurized organelle. Because of its tight compartmentalization, it is generally believed that a framework must exist, responsible for maintaining such a spatial organization. Over the last twenty years many investigations have been devoted to identifying the nuclear framework. Structures isolated by different techniques have been obtained in vitro and are variously referred to as nuclear matrix, nucleoskeleton or nuclear scaffold. Many different functions, such as DNA replication and repair, mRNA transcription, processing and transport have been described to occur in close association with these structures. However, there is still much debate as to whether or not any of these preparations corresponds to a nuclear framework that exists in vivo. In this article we summarize the most commonly-used methods for obtaining preparations of nuclear frameworks and we also stress the possible artifacts that can be created in vitro during the isolation procedures. Emphasis is placed also on the protein composition of the frameworks as well as on some possible signalling functions that have been recently described to occur in tight association with the nuclear matrix.

Pathology of the spleen in hepatosplenic schistosomiasis. Morphometric evaluation and extracellular matrix changes

Histological, ultrastructural, morphometric and immunohistochemical data obtained from the study of spleens removed by splenectomy from 34 patients with advanced hepatosplenic schistosomiasis revealed that the main alterations were congestive dilatation of the venous sinuses and diffuse thickening of the splenic cords. Splenic cord thickening was due to an increase of its matrix components, especially type IV collagen and laminin, with the conspicuous absence of interstitial collagens, either of type I or type III. Deposition of interstitial collagens (types I and III) occurred in scattered, small focal areas of the red pulp, but in the outside of the walls of the venous sinuses, in lymph follicles, marginal zone, in the vicinity of fibrous trabeculae and in sidero-sclerotic nodules. However, fibrosis was not a prominent change in schistosomal splenomegaly and thus the designation "fibro-congestive splenomegaly" seems inadequate. Lymph follicles exhibited variable degrees of atrophy, hyperplasia and fibrous replacement, sometimes all of them seen in different follicles of the same spleen and even in the same examined section. Changes in white pulp did not seem to greatly contribute to increasing spleen size and weight, when compared to the much more significant red pulp enlargement.

Extracellular matrix and platelet function in patients with musculocontractural Ehlers-Danlos syndrome caused by mutations in the CHST14 gene.

We report on a consanguineous, Afghani family with two sisters affected with characteristic facial features, multiple contractures, progressive joint and skin laxity, hemorrhagic diathesis following minor trauma and multisystem fragility-related manifestations suggestive of a diagnosis of musculocontractural Ehlers-Danlos syndrome (EDS). This novel form of connective tissue disorder was recently reported in patients of Japanese, Turkish, and Indian descent who were formerly classified as having EDS type VIB and has now been recognized to be a part of spectrum including patients previously classified as having adducted thumb-clubfoot syndrome. We identified a previously unreported mutation in the CHST14 gene, which codes for the enzyme dermatan 4-O-sulfotransferase. We discuss the prenatal presentation, detailed clinical manifestations, and neurological findings in two sisters with this newly described musculocontractural EDS-CHST14 type. We demonstrate that fibroblasts from one of our patients produce more chondroitin sulfate than normal and show lower than normal deposition of collagens I and II and fibrillin 1-containing microfibrills. These findings suggest that the imbalance in the glycosaminoglycan content in developing tissues might interfere with normal deposition of other extracellular matrix components and ultimately contribute to the development of the phenotype observed in these patients. Furthermore, we ruled out the contribution of intrinsic platelet factors to the bleeding diathesis observed in some affected individuals. © 2012 Wiley Periodicals, Inc.

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

Quantitave and qualitative interferences of pentoxifillyne on hepatic Schistosoma mansoni granulomas: effects on extracellular matrix and eosinophil population

Mast cells and eosinophils actively participate in tissue repair and are prominent components of Schistosoma mansoni granulomas. Since pentoxifillyne (PTX) is an immunomodulatory and antifibrotic substance, we aimed to characterize, by morphological techniques, the effect of this drug on fibrosis developed inside murine hepatic schistosomal granulomatous reaction, beyond the quantification of eosinophil and mast cell populations. The drug (1 mg/100 g animal weight) was administrated from 35 to 90 days post-infection, when the animals were killed. The intragranulomatous interstitial collagen network was analyzed by confocal laser scanning microscopy, the number of eosinophils and mast cells was quantified and the results were validated by t-student test. Treatment did not interfere on the granuloma evolution but caused a significant decrease in the total and involutive number of hepatic granulomas (p = 0.01 and 0.001, respectivelly), and in the intragranulomatous accumulation of eosinophils (p = 0.0001). Otherwise, the number of mast cells was not significantly altered (p = 0.9); however, it was positively correlated with the number of granulomatous structures (r = 0.955). In conclusion, PTX does not affect development and collagen deposition in S. mansoni murine granuloma, but decreases the intragranulomatous eosinophil accumulation possibly due to its immunomodulatory capability, interfering in cellular recruitment and/or differentiation.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Derris (Lonchocarpus) urucu (Leguminosae) Extract Modifies the Peritrophic Matrix Structure of Aedes aegypti (Diptera:Culicidae)

Aqueous suspension of ethanol extracts of Derris (Lonchocarpus) urucu (Leguminosae), collected in the state of Amazonas, Brazil, were tested for larvicidal activity against the mosquito Aedes aegypti (Diptera:Culicidae). The aim of this study was to observe the alterations of peritrophic matrix in Ae. aegypti larvae treated with an aqueous suspension of D. urucu extract. Different concentrations of D. urucu root extract were tested against fourth instar larvae. One hundred percent mortality was observed at 150 µg/ml (LC50 17.6 µg/ml) 24 h following treatment. In response to D. urucu feeding, larvae excreted a large amount of amorphous feces, while control larvae did not produce feces during the assay period. Ultrastructural studies showed that larvae fed with 150 µg/ml of D. urucu extract for 4 h have an imperfect peritrophic matrix and extensive damage of the midgut epithelium. Data indicate a protective role for the peritrophic matrix. The structural modification of the peritrophic matrix is intrinsically associated with larval mortality.