The inhibitory effect of MK886 on the inflammatory process caused by Paracoccidioidomycosis brasiliensis infection in genetically selected mice

Leukotrienes are classic inflammatory response mediators considered chemotactic agents and microbicidal activity regulators in cells of the innate immune system, playing a protective role against different infectious agents. In this study, we investigated the involvement of leukotrienes in the course of murine paracoccidioidomycosis based on the following immunologic parameters: cell influx, mieloperoxydase activity, NO production, cytokine production, and fungal recovery in lungs of mice selected according to the intensity of their low (AIRmin) and high (AIRmax) acute inflammatory response. Infection by P. brasiliensis induced considerable production of IL-6, IL-10, IFN-gamma and TNF-alpha cytokines, and led to cell recruitment, as well as NO production in lungs at different study periods. In animals treated with MK886, a leukotriene biosynthesis inhibitor, IFN-gamma, IL-6 and TNF-alpha production was lower, while neutrophil influx and NO production decreased. These results may explain the higher fungal load in lungs of animals in which leukotriene synthesis was inhibited, suggesting that leukotrienes have a possible protective role in experimental paracoccidioidomycosis. AIRmax animals had lower fungal load in comparison with AIRmin ones, which can be related to the AIR phenotype regarding neutrophil migration, besides lower production of NO and pro-inflammatory cytokines. Thus, mice presenting AIRmax background are more resistant to infection by P. brasiliensis.

Efeito de um inibidor de leucotrienos no processo inflamatório decorrente da infecção por Paracoccidioides brasiliensis em camundongos geneticamnte selecionados

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

5-Lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases

5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor α, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 μM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.

Arachidonic acid release from mammalian cells transfected with human groups IIA and X secreted phospholipase A(2) occurs predominantly during the secretory process and with the involvement of cytosolic phospholipase A(2)-alpha

Stable expression of human groups IIA and X secreted phospholipases A(2) (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum- and interleukin-1beta-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan- or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A(2) inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cell-impermeable, secreted phospholipase A(2) trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1beta-dependent release of arachidonate is promoted by secreted phospholipase A(2) expression and is completely dependent on cytosolic (group IVA) phospholipase A(2). These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIA-transfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.

Leukotriene A4 hydrolase: protection from mechanism-based inactivation by mutation of tyrosine-378.

Leukotriene A4 (LTA4) hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7, 9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme that catalyzes the final step in the biosynthesis of the potent chemotactic agent leukotriene B4 (LTB4). LTA4 hydrolase/aminopeptidase is suicide inactivated during catalysis via an apparently mechanism-based irreversible binding of LTA4 to the protein in a 1:1 stoichiometry. Previously, we have identified a henicosapeptide, encompassing residues Leu-365 to Lys-385 in human LTA4 hydrolase, which contains a site involved in the covalent binding of LTA4 to the native enzyme. To investigate the role of Tyr-378, a potential candidate for this binding site, we exchanged Tyr for Phe or Gln in two separate mutants. In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, were exchanged for Ala. The mutated enzymes were expressed as (His)6-tagged fusion proteins in Escherichia coli, purified to apparent homogeneity, and characterized. Enzyme activity determinations and differential peptide mapping, before and after repeated exposure to LTA4, revealed that wild-type enzyme and the mutants [S379A] and [S380A]LTA4hydrolase were equally susceptible to suicide inactivation whereas the mutants in position 378 were no longer inactivated or covalently modified by LTA4. Furthermore, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single-point mutation in LTA4 hydrolase, catalysis and covalent modification/inactivation have been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.

Dynamic compression improves biosynthesis of human zonal chondrocytes from osteoarthritis patients

Objective: We hypothesize that chondrocytes from distinct zones of articular cartilage respond differently to compressive loading, and that zonal chondrocytes from osteoarthritis (OA) patients can benefit from optimized compressive stimulation. Therefore, we aimed to determine the transcriptional response of superficial (S) and middle/deep (MD) zone chondrocytes to varying dynamic compressive strain and loading duration. To confirm effects of compressive stimulation on overall matrix production, we subjected zonal chondrocytes to compression for 2 weeks. Design: Human S and MD chondrocytes from osteoarthritic joints were encapsulated in 2% alginate, pre-cultured, and subjected to compression with varying dynamic strain (5, 15, 50% at 1 Hz) and loading duration (1, 3, 12 h). Temporal changes in cartilage-specific, zonal, and dedifferentiation genes following compression were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The benefits of long-term compression (50% strain, 3 h/day, for 2 weeks) were assessed by measuring construct glycosaminoglycan (GAG) content and compressive moduli, as well as immunostaining. Results: Compressive stimulation significantly induced aggrecan (ACAN), COL2A1, COL1A1, proteoglycan 4 (PRG4), and COL10A1 gene expression after 2 h of unloading, in a zone-dependent manner (P < 0.05). ACAN and PRG4 mRNA levels depended on strain and load duration, with 50% and 3 h loading resulting in highest levels (P < 0.05). Long-term compression increased collagen type II and ACAN immunostaining and total GAG (P < 0.05), but only S constructs showed more PRG4 stain, retained more GAG (P < 0.01), and developed higher compressive moduli than non-loaded controls. Conclusions: The biosynthetic activity of zonal chondrocytes from osteoarthritis joints can be enhanced with selected compression regimes, indicating the potential for cartilage tissue engineering applications. © 2012 Osteoarthritis Research Society International.

Bacterial kinetics-controlled shape-directed biosynthesis of silver nanoplates using Morganella psychrotolerans

We show for the first time that by controlling the growth kinetics of Morganella psychrotolerans, a silver-resistant psychrophilic bacterium, the shape anisotropy of silver nanoparticles can be achieved. This is particularly important considering that there has been no report that demonstrates a control over shape of Ag nanoparticles by controlling the growth kinetics of bacteria during biological synthesis. Additionally, we have for the first time performed electrochemistry experiments on bacterial cells after exposing them to Ag(+) ions, which provide significant new insights about mechanistic aspects of Ag reduction by bacteria. The possibility to achieve nanoparticle shape control by using a "green" biosynthesis approach is expected to open up new exciting avenues for eco-friendly, large-scale, and economically viable shape-controlled synthesis of nanomaterials.

Transcriptional regulation of flavonoid biosynthesis in nectarine (Prunus persica) by a set of R2R3 MYB transcription factors

Background Flavonoids such as anthocyanins, flavonols and proanthocyanidins, play a central role in fruit colour, flavour and health attributes. In peach and nectarine (Prunus persica) these compounds vary during fruit growth and ripening. Flavonoids are produced by a well studied pathway which is transcriptionally regulated by members of the MYB and bHLH transcription factor families. We have isolated nectarine flavonoid regulating genes and examined their expression patterns, which suggests a critical role in the regulation of flavonoid biosynthesis. Results In nectarine, expression of the genes encoding enzymes of the flavonoid pathway correlated with the concentration of proanthocyanidins, which strongly increases at mid-development. In contrast, the only gene which showed a similar pattern to anthocyanin concentration was UDP-glucose-flavonoid-3-O-glucosyltransferase (UFGT), which was high at the beginning and end of fruit growth, remaining low during the other developmental stages. Expression of flavonol synthase (FLS1) correlated with flavonol levels, both temporally and in a tissue specific manner. The pattern of UFGT gene expression may be explained by the involvement of different transcription factors, which up-regulate flavonoid biosynthesis (MYB10, MYB123, and bHLH3), or repress (MYB111 and MYB16) the transcription of the biosynthetic genes. The expression of a potential proanthocyanidin-regulating transcription factor, MYBPA1, corresponded with proanthocyanidin levels. Functional assays of these transcription factors were used to test the specificity for flavonoid regulation. Conclusions MYB10 positively regulates the promoters of UFGT and dihydroflavonol 4-reductase (DFR) but not leucoanthocyanidin reductase (LAR). In contrast, MYBPA1 trans-activates the promoters of DFR and LAR, but not UFGT. This suggests exclusive roles of anthocyanin regulation by MYB10 and proanthocyanidin regulation by MYBPA1. Further, these transcription factors appeared to be responsive to both developmental and environmental stimuli.

The role of enoyl reductase genes in phloridzin biosynthesis in apple

Phloridzin is the predominant polyphenol in apple (Malus× domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple.

Identification and characterisation of F3GT1 and F3GGT1, two glycosyltransferases responsible for anthocyanin biosynthesis in red-fleshed kiwifruit (Actinidia chinensis)

Much of the diversity of anthocyanins is due to the action of glycosyltransferases, which add sugar moieties to anthocyanidins. We identified two glycosyltransferases, F3GT1 and F3GGT1, from red-fleshed kiwifruit (Actinidia chinensis) that perform sequential glycosylation steps. Red-fleshed genotypes of kiwifruit accumulate anthocyanins mainly in the form of cyanidin 3-O-xylo-galactoside. Genes in the anthocyanin and flavonoid biosynthetic pathway were identified and shown to be expressed in fruit tissue. However, only the expression of the glycosyltransferase F3GT1 was correlated with anthocyanin accumulation in red tissues. Recombinant enzyme assays in vitro and in vivo RNA interference (RNAi) demonstrated the role of F3GT1 in the production of cyanidin 3-O-galactoside. F3GGT1 was shown to further glycosylate the sugar moiety of the anthocyanins. This second glycosylation can affect the solubility and stability of the pigments and modify their colour. We show that recombinant F3GGT1 can catalyse the addition of UDP-xylose to cyanidin 3-galactoside. While F3GGT1 is responsible for the end-product of the pathway, F3GT1 is likely to be the key enzyme regulating the accumulation of anthocyanin in red-fleshed kiwifruit varieties.

Transcriptional analysis of apple fruit proanthocyanidin biosynthesis

Proanthocyanidins (PAs) are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. Many flavonoids have antioxidant properties and may have beneficial effects for human health. PAs are found in the seeds and fruits of many plants. In apple fruit (Malus × domestica Borkh.), the flavonoid biosynthetic pathway is most active in the skin, with the flavan-3-ols, catechin, and epicatechin acting as the initiating units for the synthesis of PA polymers. This study examined the genes involved in the production of PAs in three apple cultivars: two heritage apple cultivars, Hetlina and Devonshire Quarrenden, and a commercial cultivar, Royal Gala. HPLC analysis shows that tree-ripe fruit from Hetlina and Devonshire Quarrenden had a higher phenolic content than Royal Gala. Epicatechin and catechin biosynthesis is under the control of the biosynthetic enzymes anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR1), respectively. Counter-intuitively, real-time quantitative PCR analysis showed that the expression levels of Royal Gala LAR1 and ANR were significantly higher than those of both Devonshire Quarrenden and Hetlina. This suggests that a compensatory feedback mechanism may be active, whereby low concentrations of PAs may induce higher expression of gene transcripts. Further investigation is required into the regulation of these key enzymes in apple.

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.

Overexpression of the kiwifruit SVP3 gene affects reproductive development and suppresses anthocyanin biosynthesis in petals, but has no effect on vegetative growth, dormancy, or flowering time

SVP-like MADS domain transcription factors have been shown to regulate flowering time and both inflorescence and flower development in annual plants, while having effects on growth cessation and terminal bud formation in perennial species. Previously, four SVP genes were described in woody perennial vine kiwifruit (Actinidia spp.), with possible distinct roles in bud dormancy and flowering. Kiwifruit SVP3 transcript was confined to vegetative tissues and acted as a repressor of flowering as it was able to rescue the Arabidopsis svp41 mutant. To characterize kiwifruit SVP3 further, ectopic expression in kiwifruit species was performed. Ectopic expression of SVP3 in A. deliciosa did not affect general plant growth or the duration of endodormancy. Ectopic expression of SVP3 in A. eriantha also resulted in plants with normal vegetative growth, bud break, and flowering time. However, significantly prolonged and abnormal flower, fruit, and seed development were observed, arising from SVP3 interactions with kiwifruit floral homeotic MADS-domain proteins. Petal pigmentation was reduced as a result of SVP3-mediated interference with transcription of the kiwifruit flower tissue-specific R2R3 MYB regulator, MYB110a, and the gene encoding the key anthocyanin biosynthetic step, F3GT1. Constitutive expression of SVP3 had a similar impact on reproductive development in transgenic tobacco. The flowering time was not affected in day-neutral and photoperiod-responsive Nicotiana tabacum cultivars, but anthesis and seed germination were significantly delayed. The accumulation of anthocyanin in petals was reduced and the same underlying mechanism of R2R3 MYB NtAN2 transcript reduction was demonstrated.

Regulation of carotenoid biosynthesis in banana fruit

This thesis investigated the basis for massive differences in provitamin A carotenoid content in banana fruits. Rather than gene expression levels, carotenoid storage capacity and product degradation explained much of the differences. Such information should provide important insights for future developments in the biofortification of banana. A high carotenoid-containing cultivar, 'Asupina' and a popular commercial but low carotenoid-containing cultivar, 'Cavendish' were used in the investigations.

Variation in the complex carbohydrate biosynthesis loci of Acinetobacter baumannii genomes

Extracellular polysaccharides are major immunogenic components of the bacterial cell envelope. However, little is known about their biosynthesis in the genus Acinetobacter, which includes A. baumannii, an important nosocomial pathogen. Whether Acinetobacter sp. produce a capsule or a lipopolysaccharide carrying an O antigen or both is not resolved. To explore these issues, genes involved in the synthesis of complex polysaccharides were located in 10 complete A. baumannii genome sequences, and the function of each of their products was predicted via comparison to enzymes with a known function. The absence of a gene encoding a WaaL ligase, required to link the carbohydrate polymer to the lipid A-core oligosaccharide (lipooligosaccharide) forming lipopolysaccharide, suggests that only a capsule is produced. Nine distinct arrangements of a large capsule biosynthesis locus, designated KL1 to KL9, were found in the genomes. Three forms of a second, smaller variable locus, likely to be required for synthesis of the outer core of the lipid A-core moiety, were designated OCL1 to OCL3 and also annotated. Each K locus includes genes for capsule export as well as genes for synthesis of activated sugar precursors, and for glycosyltransfer, glycan modification and oligosaccharide repeat-unit processing. The K loci all include the export genes at one end and genes for synthesis of common sugar precursors at the other, with a highly variable region that includes the remaining genes in between. Five different capsule loci, KL2, KL6, KL7, KL8 and KL9 were detected in multiply antibiotic resistant isolates belonging to global clone 2, and two other loci, KL1 and KL4, in global clone 1. This indicates that this region is being substituted repeatedly in multiply antibiotic resistant isolates from these clones.