p38 MAPK regulates IL-1β induced IL-6 expression through mRNA stability in osteoblasts

Osteoblast-derived IL-6 functions in coupled bone turnover by supporting osteoclastogenesis favoring bone resorption instead of bone deposition. Gene regulation of IL-6 is complex occurring both at transcription and post-transcription levels. The focus of this paper is at the level of mRNA stability, which is important in IL-6 gene regulation. Using the MC3T3-E1 as an osteoblastic model, IL-6 secretion was dose dependently decreased by SB203580, a p38 MAPK inhibitor. Steady state IL-6 mRNA was decreased with SB203580 (2 μM) ca. 85% when stimulated by IL-1β (1-5 ng/ ml). These effects require de novo protein synthesis as they were inhibited by cycloheximide. p38 MAPK had minor effects on proximal IL-6 promoter activity in reporter gene assays. A more significant effect on IL-6 mRNA stability was observed in the presence of SB203580. Western blot analysis confirmed that SB203580 inhibited p38 MAP kinase, in response to IL-1β in a dose dependent manner in MC3T3-E1 cells. Stably transfected MC3T3-E1 reporter cell lines (MC6) containing green fluorescent protein (GFP) with the 3′untranslated region of IL-6 were constructed. Results indicated that IL-1β, TNFα, LPS but not parathyroid hormone (PTH) could increase GFP expression of these reporter cell lines. Endogenous IL-6 and reporter gene eGFP-IL-6 3′UTR mRNA was regulated by p38 in MC6 cells. In addition, transient transfection of IL-6 3′UTR reporter cells with immediate upstream MAP kinase kinase-3 and -6 increased GFP expression compared to mock transfected controls. These results indicate that p38 MAPK regulates IL-1β-stimulated IL-6 at a post transcriptional mechanism and one of the primary targets of IL-6 gene regulation is the 3′UTR of IL-6.

Analysis of the E1-ubiquitin activating enzyme, Uba1, in cell death and tissue growth in Drosophila

Ubiquitination is an essential process involved in basic biological processes such as the cell cycle and cell death. Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Subsequently, ubiquitin is transferred to target proteins via ubiquitin ligases (E3). Defects in ubiquitin conjugation have been implicated in several forms of malignancy, the pathogenesis of several genetic diseases, immune surveillance/viral pathogenesis, and the pathology of muscle wasting. However, the consequences of partial or complete loss of ubiquitin conjugation in multi-cellular organisms are not well understood. Here, we report the characterization of nba1, the sole E1 in Drosophila. We have determined that weak and strong nba1 alleluias behave genetically different and sometimes in opposing phenotypes. For example, weak uba1 alleluias protect cells from cell death whereas cells containing strong loss-of-function alleluias are highly apoptotic. These opposing phenotypes are due to differing sensitivities of cell death pathway components to ubiquitination level alterations. In addition, strong uba1 alleluias induce cell cycle arrest due to defects in the protein degradation of Cyclins. Surprisingly, clones of strong uba1 mutant alleluias stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner resulting in severe overgrowth phenotypes in the mosaic fly. I have determined that the observed overgrowth phenotypes were due to a failure to downregulate the Notch signaling pathway in nba1 mutant cells. Aberrant Notch signaling results in the secretion of a local cytokine and activation of JAK/STAT pathway in neighboring cells. In addition, we elucidated a model describing the regulation of the caspase Dronc in surviving cells. Binding of Dronc by its inhibitor Diap1 is necessary but not sufficient to inhibit Dronc function. Ubiquitin conjugation and Uba1 function is necessary for the negative regulation of Dronc. ^

Quantifying uncertainty in parameter estimates for stochastic models of collective cell spreading using approximate Bayesian computation

Wound healing and tumour growth involve collective cell spreading, which is driven by individual motility and proliferation events within a population of cells. Mathematical models are often used to interpret experimental data and to estimate the parameters so that predictions can be made. Existing methods for parameter estimation typically assume that these parameters are constants and often ignore any uncertainty in the estimated values. We use approximate Bayesian computation (ABC) to estimate the cell diffusivity, D, and the cell proliferation rate, λ, from a discrete model of collective cell spreading, and we quantify the uncertainty associated with these estimates using Bayesian inference. We use a detailed experimental data set describing the collective cell spreading of 3T3 fibroblast cells. The ABC analysis is conducted for different combinations of initial cell densities and experimental times in two separate scenarios: (i) where collective cell spreading is driven by cell motility alone, and (ii) where collective cell spreading is driven by combined cell motility and cell proliferation. We find that D can be estimated precisely, with a small coefficient of variation (CV) of 2–6%. Our results indicate that D appears to depend on the experimental time, which is a feature that has been previously overlooked. Assuming that the values of D are the same in both experimental scenarios, we use the information about D from the first experimental scenario to obtain reasonably precise estimates of λ, with a CV between 4 and 12%. Our estimates of D and λ are consistent with previously reported values; however, our method is based on a straightforward measurement of the position of the leading edge whereas previous approaches have involved expensive cell counting techniques. Additional insights gained using a fully Bayesian approach justify the computational cost, especially since it allows us to accommodate information from different experiments in a principled way.

Fator de crescimento derivado de plaquetas humanas obtido por uma metodologia rápida e de baixo custo

Human platelet-derived growth factor (PDGF) was purified from lysates of clinically outdated human platelets by ionic exchange chromatography in CM-Sepharose. The eluated fraction was submitted to the Immunoblot/Slot Blot assay using anti-PDGF-AA and anti-PDGF-BB polyclonal antibodies and was evaluated as to its biological activity through the test of [H 3]-thymidine incorporation in NIH/3T3 cell line fibroblasts in culture. The Immunoblot/Slot Blot assay using anti-PDGF-AA and anti-PDGF-BB antibodies proved the presence of the PDGF in chromatographic cationic fraction. The comparison of biological activities between fiblobrast stimulation assay using recombinant PDGF-AB and partially purified PDGF was demonstrated in 165.796 and 157.567 cpm, respectively. This result, proved the potent mitogenic effect of partially purified PDGF and consequently their evidence about the wound healing activity.

Mixing-cell boundary conditions and apparent mass balance errors for advective-dispersive solute transport

In many field or laboratory situations, well-mixed reservoirs like, for instance, injection or detection wells and gas distribution or sampling chambers define boundaries of transport domains. Exchange of solutes or gases across such boundaries can occur through advective or diffusive processes. First we analyzed situations, where the inlet region consists of a well-mixed reservoir, in a systematic way by interpreting them in terms of injection type. Second, we discussed the mass balance errors that seem to appear in case of resident injections. Mixing cells (MC) can be coupled mathematically in different ways to a domain where advective-dispersive transport occurs: by assuming a continuous solute flux at the interface (flux injection, MC-FI), or by assuming a continuous resident concentration (resident injection). In the latter case, the flux leaving the mixing cell can be defined in two ways: either as the value when the interface is approached from the mixing-cell side (MC-RT -), or as the value when it is approached from the column side (MC-RT +). Solutions of these injection types with constant or-in one case-distance-dependent transport parameters were compared to each other as well as to a solution of a two-layer system, where the first layer was characterized by a large dispersion coefficient. These solutions differ mainly at small Peclet numbers. For most real situations, the model for resident injection MC-RI + is considered to be relevant. This type of injection was modeled with a constant or with an exponentially varying dispersion coefficient within the porous medium. A constant dispersion coefficient will be appropriate for gases because of the Eulerian nature of the usually dominating gaseous diffusion coefficient, whereas the asymptotically growing dispersion coefficient will be more appropriate for solutes due to the Lagrangian nature of mechanical dispersion, which evolves only with the fluid flow. Assuming a continuous resident concentration at the interface between a mixing cell and a column, as in case of the MC-RI + model, entails a flux discontinuity. This flux discontinuity arises inherently from the definition of a mixing cell: the mixing process is included in the balance equation, but does not appear in the description of the flux through the mixing cell. There, only convection appears because of the homogeneous concentration within the mixing cell. Thus, the solute flux through a mixing cell in close contact with a transport domain is generally underestimated. This leads to (apparent) mass balance errors, which are often reported for similar situations and erroneously used to judge the validity of such models. Finally, the mixing cell model MC-RI + defines a universal basis regarding the type of solute injection at a boundary. Depending on the mixing cell parameters, it represents, in its limits, flux as well as resident injections. (C) 1998 Elsevier Science B.V. All rights reserved.

Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.

Gemcitabine reactivates epigenetically silenced genes and functions as a DNA methyltransferase inhibitor

Gemcitabine is indicated in combination with cisplatin as first-line therapy for solid tumours including non-small cell lung cancer (NSCLC), bladder cancer and mesothelioma. Gemcitabine is an analogue of pyrimidine cytosine and functions as an anti-metabolite. Structurally, however, gemcitabine has similarities to 5-aza-2-deoxycytidine (decitabine/Dacogen®), a DNA methyltransferase inhibitor (DNMTi). NSCLC, mesothelioma and prostate cancer cell lines were treated with decitabine and gemcitabine. Reactivation of epigenetically silenced genes was examined by RT-PCR/qPCR. DNA methyltransferase activity in nuclear extracts and recombinant proteins was measured using a DNA methyltransferase assay, and alterations in DNA methylation status were examined using methylation-specific PCR (MS-PCR) and pyrosequencing. We observe a reactivation of several epigenetically silenced genes including GSTP1, IGFBP3 and RASSF1A. Gemcitabine functionally inhibited DNA methyltransferase activity in both nuclear extracts and recombinant proteins. Gemcitabine dramatically destabilised DNMT1 protein. However, DNA CpG methylation was for the most part unaffected by gemcitabine. In conclusion, gemcitabine both inhibits and destabilises DNA methyltransferases and reactivates epigenetically silenced genes having activity equivalent to decitabine at concentrations significantly lower than those achieved in the treatment of patients with solid tumours. This property may contribute to the anticancer activity of gemcitabine.

Comparative genomics of koala, cattle and sheep strains of Chlamydia pecorum

Background Chlamydia pecorum is an important pathogen of domesticated livestock including sheep, cattle and pigs. This pathogen is also a key factor in the decline of the koala in Australia. We sequenced the genomes of three koala C. pecorum strains, isolated from the urogenital tracts and conjunctiva of diseased koalas. The genome of the C. pecorum VR629 (IPA) strain, isolated from a sheep with polyarthritis, was also sequenced. Results Comparisons of the draft C. pecorum genomes against the complete genomes of livestock C. pecorum isolates revealed that these strains have a conserved gene content and order, sharing a nucleotide sequence similarity > 98%. Single nucleotide polymorphisms (SNPs) appear to be key factors in understanding the adaptive process. Two regions of the chromosome were found to be accumulating a large number of SNPs within the koala strains. These regions include the Chlamydia plasticity zone, which contains two cytotoxin genes (toxA and toxB), and a 77 kbp region that codes for putative type III effector proteins. In one koala strain (MC/MarsBar), the toxB gene was truncated by a premature stop codon but is full-length in IPTaLE and DBDeUG. Another five pseudogenes were also identified, two unique to the urogenital strains C. pecorum MC/MarsBar and C. pecorum DBDeUG, respectively, while three were unique to the koala C. pecorum conjunctival isolate IPTaLE. An examination of the distribution of these pseudogenes in C. pecorum strains from a variety of koala populations, alongside a number of sheep and cattle C. pecorum positive samples from Australian livestock, confirmed the presence of four predicted pseudogenes in koala C. pecorum clinical samples. Consistent with our genomics analyses, none of these pseudogenes were observed in the livestock C. pecorum samples examined. Interestingly, three SNPs resulting in pseudogenes identified in the IPTaLE isolate were not found in any other C. pecorum strain analysed, raising questions over the origin of these point mutations. Conclusions The genomic data revealed that variation between C. pecorum strains were mainly due to the accumulation of SNPs, some of which cause gene inactivation. The identification of these genetic differences will provide the basis for further studies to understand the biology and evolution of this important animal pathogen. Keywords: Chlamydia pecorum; Single nucleotide polymorphism; Pseudogene; Cytotoxin

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

Preparation of rare earth ion induced conformation-specific anti-calmodulin monoclonal antibody

Monoclonal antibody technique was employed to detect the conformational change of calmodulin induced by metal ions. Bovine calmodulin was firstly modified by 2,4-dinitrofluorobenzene to improve its immunogenicity, then, the derived protein was saturated with trivalent europium ions and injected to Balb/c mice as antigen. After four times of immunization, a corresponding antibody was detected and its titer in serum was determined as 1 : 12 000. By fusing of the spleen cells with hybridoma cells, a europium induced conformation-specific anti-calmodulin monoclonal antibody cell strain named as 2C3 was produced successfully. The molecular recognition ability of antibody to apocalmodulin and holocalmodulin showed a significant difference, indicating that this antibody could be applied to the studies of different effects of metal ions on the conformational change of calmodulin and its interaction with target molecules.

Développement de procédés efficaces pour la construction et la production de vecteurs adénoviraux

L’adénovirus possède plusieurs caractéristiques faisant de ce virus un candidat de choix pour la construction de vecteurs utiles dans les études de génomique fonctionnelle. Dans la majorité de ces applications, on a recours à un vecteur adénoviral de première génération délété de sa région E1. L’utilisation de vecteurs adénoviraux comprend deux maillons faibles : la construction du vecteur et la production subséquente de ce dernier. Le développement de méthodes alternatives est donc nécessaire pour renforcer ces deux maillons, permettant ainsi une utilisation étendue de ces vecteurs. Ce développement va s’articuler sur deux axes : l’ingénierie du vecteur de transfert pour la construction de l’adénovirus recombinant et l’ingénierie d’une lignée cellulaire pour la production du vecteur. En utilisant un vecteur de transfert adénoviral co-exprimant, à partir d’un promoteur régulable à la tétracycline, la protéase de l’adénovirus et une protéine de fluorescence verte (GFP) par l’intermédiaire d’un site d’entrée ribosomal interne (IRES), notre groupe a établi que la sélection positive, via l’expression ectopique de la protéase, est un processus efficace pour la création de librairie d’adénovirus recombinants. Par contre, la diversité atteinte dans ce premier système est relativement faible, environ 1 adénovirus recombinant par 1 000 cellules. Le travail effectué dans le cadre de cette thèse vise à construire un nouveau transfert de vecteur dans lequel l’expression de la protéase sera indépendante de celle du transgène permettant ainsi d’optimiser l’expression de la protéase. Ce travail d’optimisation a permis de réduire le phénomène de transcomplémentation du virus parental ce qui a fait grimper la diversité à 1 virus recombinant par 75 cellules. Ce système a été mis à l’épreuve en générerant une librairie adénovirale antisens dirigée contre la GFP. La diversité de cette librairie a été suffisante pour sélectionner un antisens réduisant de 75% l’expression de la GFP. L’amplification de ce vecteur adénoviral de première génération doit se faire dans une lignée cellulaire exprimant la région E1 telle que les cellules 293. Par contre, un adénovirus de première génération se répliquant dans les cellules 293 peut échanger, par recombinaison homologue, son transgène avec la région E1 de la cellule créant ainsi un adénovirus recombinant réplicatif (RCA), compromettant ainsi la pureté des stocks. Notre groupe a déjà breveté une lignée cellulaire A549 (BMAdE1) exprimant la région E1, mais qui ne peut pas recombiner avec le transgène du virus. Par contre, le niveau de réplication de l’adénovirus dans les BMAdE1 est sous-optimal, à peine 15-30% du niveau obtenu dans les cellules 293. Le travail fait dans le cadre de cette thèse a permis de mettre en évidence qu’une expression insuffisante d’E1B-55K était responsable de la mauvaise réplication du virus dans les BMAdE1. Nous avons produit de nouveaux clones à partir de la lignée parentale via une transduction avec un vecteur lentiviral exprimant E1B-55K. Nous avons confirmé que certains clones exprimaient une plus grande quantité d’E1B-55K et que ces clones amplifiaient de manière plus efficace un vecteur adénoviral de première génération. Ce clone a par la suite été adapté à la culture en suspension sans sérum.

Anti-genotoxic effect of aqueous extracts of sun mushroom (Agaricus blazei Murill lineage 99/26) in mammalian cells in vitro

The sun mushroom is the popular name for the Agaricus blazei Murill fungus, a mushroom native to south-eastern Brazil, which has been frequently used in popular medicine mainly in the form of tea to treat various ailments (stress, diabetes, etc.). In the present study, the genotoxic and/or anti-genotoxic effects ofA. blazei on mammalian cells in culture was assessed by checking the increase or reduction of micronucleus (MN) frequency and comets. The sun mushroom (lineage 99/26) was used as aqueous extracts prepared (2.5%) at three different temperatures (60, 25 and 4°C). The in vitro micronucleus (MN) test in binucleated cells and comet assay were used in V79 cells cultivated in HAM-F10+DMEM medium (1:1), supplemented with 10% of fetal bovine serum. The experiments were divided into four treatment types: 1. Negative control; 2. Positive control with MMS; 3. Treatments with the three forms of extracts (60, 25 and 4°C); and 4. Treatments with the extracts in different associations (simultaneous, pre-treatment, post-treatment and simultaneous after pre-incubation for 1 h) with MMS. None of the A. blazei extracts show genotoxic activity. In the comet assay no protecting effect was found. The results obtained in the MN test showed that the three forms of extracts used had protective activity, suggesting that the compound or active ingredients of A. blazei are always present in these extracts. The greater protective efficiency of the simultaneous treatment and simultaneous treatment with pre-incubation mixture with MMS suggests that the extracts have an antimutagenic action of the desmutagenic type. © 2002 Elsevier Science Ltd. All rights reserved.

Catecholamine effects on human melanoma cells evoked by α1-adrenoceptors

The biological effects of catecholamines in mammalian pigment cells are poorly understood. Our previous results showed the presence of α1-adrenoceptors in SK-Mel 23 human melanoma cells. The aims of this work were to (1) characterize catecholamine effects on proliferation, tyrosinase activity and expression, (2) identify the α1- adrenoceptor subtypes, and (3) verify whether chronic norepinephrine (NE) treatment modified the types and/or pharmacological characteristics of adrenoceptors present in SK-Mel 23 human melanoma cells. Cells treated with the aradrenergic agonist, phenylephrine (PHE, 10-5 or 10-4 M), for 24-72 h, exhibited decreased cell proliferation and enhanced tyrosinase activity, but unaltered tyrosinase expression as compared with the control. The proliferation and tyrosinase activity responses were inhibited by the α1-adrenergic antagonist prazosin, suggesting they were evoked by α1-adrenoceptors. The presence of actinomycin D, a transcription inhibitor, did not diminish PHE-induced effects. RT-PCR assays, followed by cloning and sequencing, demonstrated the presence of α1A- and α1B-adrenoceptor subtypes. NE-treated cells (24 or 72 h) were used in competition assays, and showed no significant change in the competition curves of α1-adrenoceptors as compared with control curves. Other adrenoceptor subtypes were not identified in these cells, and NE pretreatment did not induce their expression. In conclusion, the activation of SK-Mel 23 human melanoma α1- radrenoceptors elicit biological effects, such as proliferation decrease and tyrosinase activity increase. Desensitization or expression of other adrenoceptor subtypes after chronic NE treatment were not observed.

Genotoxic and antigenotoxic effects of organic extracts of mushroom Agaricus blazei Murrill on V79 cells

Agaricus blazei Murrill, popularly known as the sun mushroom, is a native mushroom in SP, Brazil, that has been widely used in the treatment of cancer and many other pathologies in different parts of the world. A water-soluble protein-polysaccharide complex (1 → 6)β-D-glucan has been isolated from its fruiting body that showed immune-modulation activity. From organic extracts, linoleic acid has been isolated and determined to be the main substance with antimutagenic activity. Using both the micronucleus (MN) and comet (single cell microgel electrophoresis) assays, this study determined the genotoxic and antigenotoxic potential of A. blazei (AB) obtained from commercial sources or the following strains: a) strains AB 97/29 (young and sporulated phases); b) a mixture taken from AB 96/07, AB 96/09 and AB 97/ 11 strains; and c) commercial mushrooms from Londrina, PR and Piedade, SP, designated as AB PR and AB SP, respectively. The extracts from these mushrooms were isolated in chloroform:methanol (3:1) and used in vitro at three different concentrations. V79 cells (Chinese hamster lung cells) were exposed to the extracts under pre-, simultaneous and post-treatment conditions, combined with methyl methanesulfonate (MMS). Under the circumstances of this study, these organic extracts did not show any genotoxic or mutagenic effects, but did protect cells against the induction of micronuclei by MMS. Copyright by the Brazilian Society of Genetics.

On the application of nanostructured electrodes prepared by Ti/TiO2/WO3 template : A case study of removing toxicity of indigo using visible irradiation

New assays with HepG2 cells indicate that Indigo Carmine (IC), a dye that is widely used as additive in many food and pharmaceutical industries exhibited cytotoxic effects. This work describes the development of a bicomponent nanostructured Ti/TiO2/WO3 electrode prepared by template method and investigates its efficiency in a photoelectrocatalytic method by using visible light irradiation and applied potential of 1V. After 2h of treatment there are reduction of 97% discoloration, 62% of mineralization and formation of three byproducts assigned as: 2-amine-5-sulfo-benzoic acid, 2,3-dioxo-14-indole-5-sulfonic acid, and 2-amino-α-oxo-5-sulfo-benzeneacetic acid were identified by HPLC-MS/MS. But, cytotoxicity was completely removed after 120min of treatment. © 2013 Elsevier Ltd.