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1.  De Novo Assembly and Transcriptome Analysis of the Rubber Tree (Hevea brasiliensis) and SNP Markers Development for Rubber Biosynthesis Pathways 
PLoS ONE  2014;9(7):e102665.
Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.
PMCID: PMC4105465  PMID: 25048025
Currently multiresistant Staphylococcus aureus is one common cause of infections with high rates of morbidity and mortality worldwide, which directs scientific endeavors in search for novel antimicrobials. In this study, nine extracts from Bidens pilosa (root, stem, flower and leaves) and Annona crassiflora (rind fruit, stem, leaves, seed and pulp) were obtained with ethanol: water (7:3, v/v) and their in vitro antibacterial activity evaluated through both the agar diffusion and broth microdilution methods against 60 Oxacillin Resistant S. aureus (ORSA) strains and against S. aureus ATCC6538. The extracts from B. pilosa and A. crassiflora inhibited the growth of the ORSA isolates in both methods. Leaves of B. pilosa presented mean of the inhibition zone diameters significantly higher than chlorexidine 0.12% against ORSA, and the extracts were more active against S. aureus ATCC (p < 0.05). Parallel, toxicity testing by using MTT method and phytochemical screening were assessed, and three extracts (B. pilosa, root and leaf, and A. crassiflora, seed) did not evidence toxicity. On the other hand, the cytotoxic concentrations (CC50 and CC90) for other extracts ranged from 2.06 to 10.77 mg/mL. The presence of variable alkaloids, flavonoids, tannins and saponins was observed, even though there was a total absence of anthraquinones. Thus, the extracts from the leaves of B. pilosa revealed good anti-ORSA activity and did not exhibit toxicity.
PMCID: PMC4131820  PMID: 25076435
Plant extracts; Products with antimicrobial action; Staphylococcus aureus; ORSA; Toxicity tests; Microbial sensitivity tests
3.  Role of Chitin and Chitinase/Chitinase-Like Proteins in Inflammation, Tissue Remodeling, and Injury 
Annual review of physiology  2011;73:10.1146/annurev-physiol-012110-142250.
The 18 glycosyl hydrolase family of chitinases is an ancient gene family that is widely expressed from prokaryotes to eukaryotes. In mammals, despite the absence of endogenous chitin, a number of chitinases and chitinase-like proteins (C/CLPs) have been identified. However, their roles have only recently begun to be elucidated. Acidic mammalian chitinase (AMCase) inhibits chitin-induced innate inflammation; augments chitin-free, allergen-induced Th2 inflammation; and mediates effector functions of IL-13. The CLPs BRP-39/YKL-40 (also termed chitinase 3-like 1) inhibit oxidant-induced lung injury, augments adaptive Th2 immunity, regulates apoptosis, stimulates alternative macrophage activation, and contributes to fibrosis and wound healing. In accord with these findings, levels of YKL-40 in the lung and serum are increased in asthma and other inflammatory and remodeling disorders and often correlate with disease severity. Our understanding of the roles of C/CLPs in inflammation, tissue remodeling, and tissue injury in health and disease is reviewed below.
PMCID: PMC3864643  PMID: 21054166
asthma; fibrosis; BRP-39/YKL-40; AMCase; chitotriosidase
4.  Use of sodC versus ctrA for real-time polymerase chain reaction-based detection of Neisseria meningitidis in sterile body fluids 
Memórias do Instituto Oswaldo Cruz  2013;108(2):246-247.
We evaluated the use of a newly described sodC-based real-time-polymerase chain reaction (RT-PCR) assay for detecting Neisseria meningitidis in normally sterile sites, such as cerebrospinal fluid and serum. The sodC-based RT-PCR assay has an advantage over ctrA for detecting nongroupable N. meningitidis isolates, which are commonly present in asymptomatic pharyngeal carriage. However, in our study, sodC-based RT-PCR was 7.5% less sensitive than ctrA. Given the public health impact of possible false-negative results due to the use of the sodC target gene alone, sodC-based RT-PCR for the diagnosis of meningococcal meningitis should be used with caution.
PMCID: PMC3970655  PMID: 23579808
sodC-based RT-PCR; N. meningitidis diagnostics; meningitis
5.  Chitin Particles Are Multifaceted Immune Adjuvants 
Rationale: Chitin is a ubiquitous polysaccharide in fungi, insects, allergens, and parasites that is released at sites of infection. Its role in the generation of tissue inflammation, however, is not fully understood.
Objectives: We hypothesized that chitin is an important adjuvant for adaptive immunity.
Methods: Mice were injected with a solution of ovalbumin and chitin.
Measurements and Main Results: We used in vivo and ex vivo/in vitro approaches to characterize the ability of chitin fragments to foster adaptive immune responses against ovalbumin and compared these responses to those induced by aluminum hydroxide (alum). In vivo, ovalbumin challenge caused an eosinophil-rich pulmonary inflammatory response, Th2 cytokine elaboration, IgE induction, and mucus metaplasia in mice that had been sensitized with ovalbumin plus chitin or ovalbumin plus alum. Toll-like receptor-2, MyD88, and IL-17A played critical roles in the chitin-induced responses, and MyD88 and IL-17A played critical roles in the alum-induced responses. In vitro, CD4+ T cells from mice sensitized with ovalbumin plus chitin were incubated with ovalbumin-stimulated bone marrow–derived dendritic cells. In these experiments, CD4+ T-cell proliferation, IL-5, IL-13, IFN-γ, and IL-17A production were appreciated. Toll-like receptor-2, MyD88, and IL-17A played critical roles in these in vitro adjuvant properties of chitin. TLR-2 was required for cell proliferation, whereas IL-17 and TLR-2 were required for cytokine elaboration. IL-17A also inhibited the generation of adaptive Th1 responses.
Conclusions: These studies demonstrate that chitin is a potent multifaceted adjuvant that induces adaptive Th2, Th1, and Th17 immune responses. They also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves and is regulated by TLR-2, MyD88, and IL-17A.
PMCID: PMC3029935  PMID: 20656945
chitin; adjuvant; ovalbumin; aluminum hydroxide; alum
7.  Chitin Regulation of Immune Responses: An Old Molecule With New Roles 
Current opinion in immunology  2008;20(6):684-689.
Chitin, the second most abundant polysaccharide in nature, is commonly found in lower organisms such as fungi, crustaceans and insects, but not in mammals. Although the non-specific anti-viral and anti-tumor activities of chitin/chitin derivatives were described two decades ago, the immunological effects of chitin have been only recently been addressed. Recent studies demonstrated that chitin has complex and size-dependent effects on innate and adaptive immune responses including the ability to recruit and activate innate immune cells and induce cytokine and chemokine production via a variety of cell surface receptors including macrophage mannose receptor, toll-like receptor 2 (TLR-2), and Dectin-1. They also demonstrated adjuvant effects of chitin in allergen-induced Type 1 or Type 2 inflammation and provided insights into the important roles of chitinases and chitinase-like proteins (C/CLP) in pulmonary inflammation. The status of the field and areas of controversy are highlighted.
PMCID: PMC2605627  PMID: 18938241
chitin; chitinases; chitinase-like protein; innate and adaptive immunity
8.  Role of breast regression protein 39 (BRP-39)/chitinase 3-like-1 in Th2 and IL-13–induced tissue responses and apoptosis 
The Journal of Experimental Medicine  2009;206(5):1149-1166.
Mouse breast regression protein 39 (BRP-39; Chi3l1) and its human homologue YKL-40 are chitinase-like proteins that lack chitinase activity. Although YKL-40 is expressed in exaggerated quantities and correlates with disease activity in asthma and many other disorders, the biological properties of BRP-39/YKL-40 have only been rudimentarily defined. We describe the generation and characterization of BRP-39−/− mice, YKL-40 transgenic mice, and mice that lack BRP-39 and produce YKL-40 only in their pulmonary epithelium. Studies of these mice demonstrated that BRP-39−/− animals have markedly diminished antigen-induced Th2 responses and that epithelial YKL-40 rescues the Th2 responses in these animals. The ability of interleukin13 to induce tissue inflammation and fibrosis was also markedly diminished in the absence of BRP-39. Mechanistic investigations demonstrated that BRP-39 and YKL-40 play an essential role in antigen sensitization and immunoglobulin E induction, stimulate dendritic cell accumulation and activation, and induce alternative macrophage activation. These proteins also inhibit inflammatory cell apoptosis/cell death while inhibiting Fas expression, activating protein kinase B/AKT, and inducing Faim 3. These studies establish novel regulatory roles for BRP-39/YKL-40 in the initiation and effector phases of Th2 inflammation and remodeling and suggest that these proteins are therapeutic targets in Th2- and macrophage-mediated disorders.
PMCID: PMC2715037  PMID: 19414556
9.  Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica 
Genetics and Molecular Biology  2009;32(4):802-810.
In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.
PMCID: PMC3036905  PMID: 21637458
coffee; cup quality; differential expression; naturally decaffeinated
Chitin, is a ubiquitous polysaccharide in fungi, insects and parasites. To test the hypothesis that chitin is an important immune modulator, we characterized the ability of chitin fragments to regulate murine macrophage cytokine production in vitro and induce acute inflammation in vivo. Here we show that chitin is a size-dependent stimulator of macrophage interleukin (IL)-17A production and IL-17A receptor (R) expression and demonstrate that these responses are Toll-like Receptor (TLR)-2 and MyD88-dependent. We further demonstrate that IL-17A pathway activation is an essential event in the stimulation of some but not all chitin-stimulated cytokines and that chitin utilizes a TLR-2, MyD88- and IL-17A-dependent mechanism(s) to induce acute inflammation. These studies demonstrate that chitin is a size-dependent pathogen-associated molecular pattern (PAMP) that activates TLR-2 and MyD88 in a novel IL-17A / IL-17AR-based innate immunity pathway.
PMCID: PMC2577310  PMID: 18768886
Monocytes/Macrophages; Cytokines; Inflammation; Lung; Rodent
11.  Neurospora crassa mat A-2 and mat A-3 proteins weakly interact in the yeast two-hybrid system and affect yeast growth 
Genetics and Molecular Biology  2009;32(2):354-361.
Mating-type genes control the entry into the sexual cycle, mating identity and sexual development in fungi. The mat A-2 and mat A-3 genes, present in the mat A idiomorph of the filamentous fungus Neurospora crassa, are required for post-fertilization functions but are not essential for mating identity. Their putative roles as transcription factors are based on the similarity of mat A-2 with the Podospora anserina SMR1 gene and an HMG motif present in the mat A-3 gene. In this work the yeast two-hybrid system was used to identify transcriptional activity and protein-protein interaction of N. crassamat A-2 and mat A-3 genes. We observed that the mat A-3 protein alone is capable of weakly activating transcription of yeast reporter genes; it also binds with low specificity to the GAL1 promoter sequence, possibly due to its HMG domain. Our results also indicate that mat A-3 is capable to form homodimers, and interact with mat A-2. Interference on yeast growth was observed on some transformants suggesting a toxic action of the mat A-2 protein. Our data on pattern of interactions of mat proteins contributes towards understanding the control of vegetative and sexual cycles in filamentous fungi.
PMCID: PMC3036917  PMID: 21637691
Neurospora crassa; mating-type; yeast two-hybrid system
12.  Marked stem cell factor expression in the airways of lung transplant recipients 
Respiratory Research  2006;7(1):90.
Airways repair is critical to lung function following transplantation. We hypothesised that the stem cell factor (SCF) could play a role in this setting.
We studied 9 lung transplant recipients (LTx recipients) during their first year postgraft, and evaluated SCF mRNA expression in bronchial biopsy specimens using on-line fluorescent PCR and SCF protein levels in bronchoalveolar lavage (BAL) and serum using ELISA. The expression of SCF receptor Kit was assessed using immunostaining of paraffin-embedded bronchial sections.
SCF mRNA was highly expressed during the early postgraft period [Month (M)1-M3] (300% increase vs controls: 356 vs 1.2 pg SCF/μg GAPDH cDNA, p < 0.001) and decreased thereafter (M4-M12: 187 pg/μg), although remaining at all times 10–100 times higher than in controls. While SCF protein levels in BAL were similar in LTx recipients and in controls, the SCF serum levels were at all times higher in LTx recipients than in controls (p < 0.05), with no relationship between these levels and the acute complications of the graft. Finally, Kit was strongly expressed by the mast cells as well as by the bronchial epithelium of LTx recipients.
SCF and Kit are expressed in bronchial biopsies from lung transplant recipients irrespective of the clinical status of the graft. A role for these factors in tissue repair following lung transplantation is hypothesised.
PMCID: PMC1513216  PMID: 16780589
13.  Paradoxical early glucocorticoid induction of stem cell factor (SCF) expression in inflammatory conditions 
British Journal of Pharmacology  2003;141(1):75-84.
Stem cell factor (SCF) is a major growth factor for mast cells, promoting their differentiation and chemotaxis. Its expression is regulated by glucocorticoids in inflammatory conditions, showing an early increased protein expression, before the expected anti-inflammatory decrease (Da Silva et al., Br. J. Pharmacol. 2002:135,1634).We here evaluated the early kinetic of SCF expression regulated by interleukin (IL)-1β, budesonide and the combination of both in human lung fibroblasts in culture.Budesonide potentiated the IL-1β-enhanced expression of SCF mRNA (+103%) and protein (+98%) very shortly after treatment (at 30 min and 1 h, respectively). A gentle downregulation followed. This potentiating effect of budesonide was related to increased SCF mRNA stability and SCF gene transcription.Deletion of a κB-like site that we identified in the first intron of the SCF gene, in a luciferase reporter system, abolished the potentiation by budesonide, as well as the effect of IL-1β alone, as compared to the wild-type construction activity.All budesonide-induced effects were glucocorticoid-receptor dependent, since they were reproduced by dexamethasone and blocked by RU486.IL-1β+budesonide did not affect the relative expression of the soluble and membrane-bound forms of SCF.In conclusion, our results clearly show that glucocorticoids act very early to adversely increase the expression of SCF mRNA and protein in the inflammatory conditions created by IL-1β, and that this effect involves increased mRNA stability and increased gene expression through activation of the NF-κB-like responsive element.
PMCID: PMC1574180  PMID: 14662725
SCF; IL-1β; glucocorticoid; NF-κB; inflammation; fibroblast; mast cell; lung; asthma

Results 1-13 (13)