Immunological responses and cytokine gene expression analysis to Cooperia punctata infections in resistant and susceptible Nelore cattle

Cellular and humoral immune response, as well as cytokine gene expression, was assessed in Nelore cattle with different degrees of resistance to Cooperia punctata natural infection. One hundred cattle (male, weaned, 11-12 months old), kept together on pasture, were evaluated. Faecal and blood samples were collected for parasitological and immunological assays. Based on nematode faecal egg counts (FEC) and worm burden, the seven most resistant and the eight most susceptible animals were selected. Tissue samples of the small intestine were collected for histological quantification of inflammatory cells and analysis of cytokine gene expression (IL-2, IL-4, IL-8, IL-1 2p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, and MUC- 1) using real-time RT-PCR. Mucus samples were also collected for IgA levels determination. Serum IgG1 mean levels against C. punctata antigens were higher in the resistant group, but significant differences between groups were only observed 14 days after the beginning of the experiment against infective larvae (1-3) and 14 and 84 days against adult antigens. The resistant group also presented higher IgA levels against C. punctata (L3 and adult) antigens with significant difference 14 days after the beginning of the trial (P < 0.05). In the small-intestine mucosa, levels of IgA anti-L3 and anti-adult C. punctata were higher in the resistant group, compared with the susceptible group (P < 0.05). Gene expression of both T(H)2 cytokines (IL-4 and IL-13) in the resistant group and T(H)1 cytokines (IL-2, IL-1 2p35, IFN-gamma and MCP-1) in the susceptible group was up-regulated. Such results suggested that immune response to C. punctata was probably mediated by TH2 cytokines in the resistant group and by T(H)1 cytokines in the susceptible group. (C) 2008 Elsevier B.V. All rights reserved.

mRNA profile of Nellore calves after primary infection with Haemonchus placei

Haemonchus parasites are responsible for many losses in animal production. However, few studies are available, especially of zebu cattle. In this study, we investigated mRNA differences of immune response genes in naive Nellore calves infected with Haemonchus placei, relating these differences to patterns of cellular infiltrate. Calves were infected with 15,000 H. placei 13 larvae and after 7 days lymph node and abomasum tissues were collected. IL-2, IL-4, IL-8, IL-12, IL-13, IFN-gamma, MCP-1, lysozyme, pepsinogen and TNF-alpha genes were evaluated by qPCR. Mast cells, eosinophils and globular leukocytes were counted by abomasum histology. In the infected group, IL-4, IL-13 and TNF-alpha were up-regulated in the abomasal lymph node. In the abomasum, IL-13 increased and TNF-alpha was down-regulated (p < 0.05). No differences were detected for mast cells and eosinophil counts in abomasal tissue (p > 0.05). We conclude that for this infection time, there was Th2 polarization but that cellular infiltrate in abomasal tissue takes longer to develop. (C) 2010 Elsevier B.V. All rights reserved.

Spatial patterns of soil attributes and components in a mangrove system in Southeast Brazil (Sao Paulo)

Purpose Among environmental factors governing innumerous processes that are active in estuarine environments, those of edaphic character have received special attention in recent studies. With the objectives of determining the spatial patterns of soil attributes and components across different mangrove forest landscapes and obtaining additional information on the cause-effect relationships between these variables and position within the estuary, we analyzed several soil attributes in 31 mangrove soil profiles from the state of So Paulo (Guaruja, Brazil). Materials and methods Soil samples were collected at low tide along two transects within the CrumahA(0) mangrove forest. Samples were analyzed to determine pH, Eh, salinity, and the percentages of sand, silt, clay, total organic carbon (TOC), and total S. Mineralogy of the clay fraction (< 2 mm) was also studied by X-ray diffraction analysis, and partitioning of solid-phase Fe was performed by sequential extraction. Results and discussion The results obtained indicate important differences in soil composition at different depths and landscape positions, causing variations in physicochemical parameters, clay mineralogy, TOC contents, and iron geochemistry. The results also indicate that physicochemical conditions may vary in terms of different local microtopographies. Soil salinity was determined by relative position in relation to flood tide and transition areas with highlands. The proportions of TOC and total S are conditioned by the sedimentation of organic matter derived from vegetation and by the prevailing redox conditions, which clearly favored intense sulfate reduction in the soils (similar to 80% of the total Fe is Fe-pyrite). Particle-size distribution is conditioned by erosive/deposition processes (present and past) and probably by the positioning of ancient and reworked sandy ridges. The existing physicochemical conditions appear to contribute to the synthesis (smectite) and transformation (kaolinite) of clay minerals. Conclusions The results demonstrate that the position of soils in the estuary greatly affects soil attributes. Differences occur even at small scales (meters), indicating that both edaphic (soil classification, soil mineralogy, and soil genesis) and environmental (contamination and carbon stock) studies should take such variability into account.

Out of the Andes: patterns of diversification in clearwing butterflies

Global biodiversity peaks in the tropical forests of the Andes, a striking geological feature that has likely been instrumental in generating biodiversity by providing opportunities for both vicariant and ecological speciation. However, the role of these mountains in the diversification of insects, which dominate biodiversity, has been poorly explored using phylogenetic methods. Here we study the role of the Andes in the evolution of a diverse Neotropical insect group, the clearwing butterflies. We used dated species-level phylogenies to investigate the time course of speciation and to infer ancestral elevation ranges for two diverse genera. We show that both genera likely originated at middle elevations in the Andes in the Middle Miocene, contrasting with most published results in vertebrates that point to a lowland origin. Although we detected a signature of vicariance caused by the uplift of the Andes at the Miocene-Pliocene boundary, most sister species were parapatric without any obvious vicariant barrier. Combined with an overall decelerating speciation rate, these results suggest an important role for ecological speciation and adaptive radiation, rather than simple vicariance.

Increased expression of fructan 1-exohydrolase in rhizophores of Vernonia herbacea during sprouting and exposure to low temperature

Rhizophores of Vernonia herbacea, an Asteraceae found in the Brazilian Cerrado, store high amounts of fructans that vary in composition over the phenological cycle. Fructan 1-exohydrolase (1-FEH) activity is detectable during the sprouting phase, mainly in the proximal regions of rhizophores, of plants induced to sprout by defoliation and/or cold storage. We found an increase in 1-FEH gene expression during natural and induced sprouting and further enhancement through low-temperature treatment. Furthermore, a comparative analysis of 1-FEH gene expression in different regions of the rhizophores during the transition from dormancy to sprouting is presented. Transcripts were detected mainly in the proximal region, coinciding with high 1-FEH activity and a high concentration of free fructose. Low temperature promoted the accumulation of fructans of a low degree of polymerization (DP) and enhanced 1-FEH activity and gene expression. It is hypothesized that a set of 1-FEH proteins acts in two different ways during fructan mobilization: (1) by hydrolyzing fructo-oligosaccharides and -polysaccharides in sprouting plants (naturally or induced) for carbon supply and (2) by hydrolyzing preferably fructo-polysaccharides under low temperature to maintain the oligosaccharide pool for plant cold acclimation. (C) 2010 Elsevier GmbH. All rights reserved.

A phospholipase A(2) from Bothrops asper snake venom activates neutrophils in culture: Expression of cyclooxygenase-2 and PGE(2) biosynthesis

In this study, the production of prostaglandin E(2) (PGE(2)) and up-regulation in cyclooxygenase (COX) pathway induced by a phospholipase A(2) (PLA(2)), myotoxin-III (MT-III), purified from Bothrops asper snake venom, in isolated neutrophils were investigated. The arachidonic acid (AA) production and the participation of intracellular PLA(2)s (cytosolic PLA(2) and Ca(2+)-independent PLA(2)) in these events were also evaluated. MT-III induced COX-2, but not COX-1 gene and protein expression in neutrophils and increased PGE(2) levels. Pretreatment of neutrophils with COX-2 and COX-1 inhibitors reduced PGE(2) production induced by MT-III. Arachidonyl trifluoromethyl ketone (AACOCF(3)), an intracellular PLA(2) inhibitor, but not bromoenol lactone (BEL), an iPLA(2) inhibitor, suppressed the MT-III-induced AA and PGE(2) release. In conclusion, MT-III directly stimulates neutrophils inducing COX-2 mRNA and protein expression followed by production of PGE(2). COX-2 isoform is preeminent over COX-1 for production of PGE(2) stimulated by MT-III. PGE(2) and AA release by MT-III probably is related to cPLA(2) activation. (c) 2010 Elsevier Ltd. All rights reserved.

Virulence features of atypical enteropathogenic Escherichia coli identified by the eae(+) EAF-negative stx(-) genetic profile

This study characterized 76 atypical enteropathogenic Escherichia coli (aEPEC) strains, previously classified by the eae(+) EAF-negative stx(-) genotype, isolated from children with diarrhea in Brazil. Presence of bfpA and bfpA/perA was detected in 2 and 6 strains, respectively. The expression of bundle-forming pilus (BFP), however, was observed by immunofluorescence in 1 bfpA and 3 bfpA/perA strains, classifying them as typical EPEC (tEPEC). The remaining 72 aEPEC strains were characterized by serotyping, intimin typing, adherence patterns to HEp-2 cells, capacity to induce actin aggregation (fluorescent actin staining test), and antimicrobial resistance. Our results show that aEPEC comprise a very heterogeneous group that does not present any prevalence or association regarding the studied characteristics. It also suggest that tEPEC and aEPEC must not be classified only by the reactivity with the EAF probe, and that the search of other markers present in pEAF, as well as the BFP expression, must be considered for this matter. (C) 2009 Elsevier Inc. All rights reserved.

The expression of efflux and uptake transporters are regulated by statins in Caco-2 and HepG2 cells

Aim: Statin disposition and response are greatly determined by the activities of drug metabolizing enzymes and efflux/uptake transporters. there is little information on the regulation of these proteins in human cells after statin therapy. In this study, the effects of atorvastatin and simvastatin on mRNA expression of efflux (ABCB1, ABCG2 and ABCC2) and uptake (SLCO1B1, SLCO2B1 and SLC22A1) drug transporters in Caco-2 and HepG2 cells were investigated. Methods: Quantitative real-time PCR was used to measure mRNA levels after exposure of HepG2 and Caco-2 cells to statins. Results: Differences in mRnA basal levels of the transporters were as follows: ABCC2>ABCG2>ABCB1>SLCO1B1>>>SLC22A1>SLC O2B1 for HepG2 cells, and SLCO2B1>>ABCC2>ABCB1>ABCG2>>>SLC22A1 for Caco-2 cells. While for HepG2 cells, ABCC2, ABCG2 and SLCO2B1 mRnA levels were significantly up-regulated at 1, 10 and 20 mu mol/L after 12 or 24 h treatment, in Caco-2 cells, only the efflux transporter ABCB1 was significantly down-regulated by two-fold following a 12 h treatment with atorvastatin. Interestingly, whereas treatment with simvastatin had no effect on mRNA levels of the transporters in HepG2 cells, in Caco-2 cells the statin significantly down-regulated ABCB1, ABCC2, SLC22A1, and SLCO2B1 mRnA levels after 12 or 24 h treatment. Conclusion: These findings reveal that statins exhibits differential effects on mRNA expression of drug transporters, and this effect depends on the cell type. Furthermore, alterations in the expression levels of drug transporters in the liver and/or intestine may contribute to the variability in oral disposition of statins.

Decreased ABCB1 mRNA expression induced by atorvastatin results from enhanced mRNA degradation in HepG2 cells

The mechanisms underlying atorvastatin supression of ABCB1 gene expression, at transcriptional and post-transcriptional levels of ABCB1 gene in HepG2 (human hepatocellular carcinoma) cells were investigated. Quantitative real-time PCR was used to measure mRNA levels, as well as to estimate the half-life of ABCB1 mRNA. Western blotting analysis was performed in order to measure protein levels of ABCB1. Electrophoretic mobility shift assay (EMSA) was used to evaluate interactions between protein(s) and ABCB1 promoter region. Exposure to atorvastatin for 24 h resulted in a dose-dependent decrease of ABCB1 mRNA and protein levels, which was not abolished by addition of farnesyl or geranylgeranyl pyrophosphate. After removing fetal bovine serum from the media, however, ABCB1 expression was decreased by 2-fold in either HepG2 cells treated and non-treated with atorvastatin. Addition of cholesterol to serum free media abolished this latter effect on ABCB1 mRNA levels. In EMSA using a 5`-end-labeled 241 bp ABCB1 promoter DNA fragment (-198 to +43) as probe, the binding of the proteins to the probe was reduced by NF-Y, but not changed by NF kappa B, AP-1, and SP1. However, the NF-Y binding activity was similar in control and atorvastatin-treated cells. mRNA stability studies revealed that ABCB1 mRNA degradation was increased in 1, 10 and 20 mu M atorvastatin-treated versus control cells (half-lives of 2 h versus 7 h). Therefore, evidence is provided that decreased mRNA stability by atorvastatin treatment may explain the decrease in ABCB1 transcript levels. (C) 2009 Elsevier B.V. All rights reserved.

Statin regulation of CYP3A4 and CYP3A5 expression

CYP3A4 and CYP3A5 are cytochrome P450 enzymes that are highly expressed in the liver and gut and metabolize endogenous compounds and xenobiotics. Statins are cholesterol-lowering drugs that are extensively metabolized by CYP3A4 and CYP3A5. The bioavailability of statins is affected by CYP3A4 and CYP3A5 and glucuronidases metabolism as well as uptake and efflux transporters that affect drug disposition. CYP3A4 and CYP3A5 variants have been demonstrated to influence the pharmacokinetics, efficacy and safety of statins. Inducers and inhibitors of CYP3A4 and CYP3A5 play an important role in reducing statin efficacy and increase the risk of adverse effects, respectively. Statins have been demonstrated to increase CYP3A expression in vitro, most likely because they are ligands to nuclear receptors (pregnane X receptor and constitutive androsterone receptor) that form heterodimers with retinoid X receptors and bind to responsive elements in the CYP3A4 and CYP3A5 promoter regions. This special report outlines the earlier studies on variability of response to statins owing to CYP3A variants and highlights findings on the induction of CYP3A4 and CYP3A5 expression by statins.

ABCA1 expression and statins: inhibitory effect in peripheral blood mononuclear cells

The ATP-binding cassette transporter A1 (ABCA1) has an essential role in the formation of nascent high-density lipoprotein particles and also participates in the cholesterol efflux from macrophages in the artery wall. Several substances, such as statins, or even gene variants are able to modulate ABCA1 expression. There is strong evidence that statin treatment downregulates the ABCA1 expression in nonloaded macrophages. Interestingly, in cholesterol-loaded macrophages, which are more relevant to atherogenesis, this effect is lost. We observed an inhibitory effect of atorvastatin in peripheral blood mononuclear cells of hypercholesterolemic individuals. Moreover, in these individuals, the ABCA1 -14C > T polymorphism was associated with high baseline gene-expression levels. Other studies are needed to evaluate how relevant these findings are to the formation of arterial foam cells in vivo.

Atorvastatin effects on SREBF1a and SCAP gene expression in mononuclear cells and its relation with lowering-lipids response

Background: The transcription factors SREBP1 and SCAP are involved in intracellular cholesterol homeostasis. Polymorphisms of these genes have been associated with variations on serum lipid levels and response to statins that are potent cholesterol-lowering drugs. We evaluated the effects of atorvastatin on SREBF1a and SCAP mRNA expression in peripheral blood mononuclear cells (PBMC) and a possible association with gene polymorphisms and lowering-cholesterol response. Methods: Fifty-nine hypercholesterolemic patients were treated with atorvastatin (10 mg/day for 4 weeks). Serum lipid profile and mRNA expression in PBMC were assessed before and after the treatment. Gene expression was quantified by real-time PCR using GAPD as endogenous reference and mRNA expression in HepG2 cells as calibrator. SREBF1 -36delG and SCAP A2386G polymorphisms were detected by PCR-RFLP. Results: Our results showed that transcription of SREBF1a and SCAP was coordinately regulated by atorvastatin (r=0.595, p<0.001), and that reduction in SCAP transcription was associated with the 2386AA genotype (p=0.019). Individuals who responded to atorvastatin with a downregulation of SCAP had also a lower triglyceride compared to those who responded to atorvastatin with an upregulation of SCAP. Conclusion: Atorvastatin has differential effects on SREBF1a and SCAP mRNA expression in PBMC that are associated with baseline transcription levels, triglycerides response to atorvastatin and SCAP A2386G polymorphism. (c) 2008 Elsevier B.V. All rights reserved.

Heterologous expression of a thermophilic esterase in Kluyveromyces yeasts

In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 A degrees C and 45 A degrees C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.

Nisin expression production from Lactococcus lactis in milk whey medium

BACKGROUND: Nisin is a commercially available bacteriocin produced by Lactococcus lactis ATCC 11454 and used as a natural agent in the biopreservation of food. In the current investigation, milk whey, a byproduct from dairy industries was used as a fermentation substrate for the production of nisin. Lactococcus lactis ATCC 11454 was developed in a rotary shaker (30 degrees C/36 h/100 rpm) using two different media with milk whey (i) without filtration, pH 6.8, adjusted with NaOH 2 mol L-1 and without pH adjustment, both autoclaved at 121 degrees C for 30 min, and (ii) filtrated (1.20 mu m and 0.22 mu m membrane filter). These cultures were transferred five times using 5 mL aliquots of broth culture for every new volume of the respective media. RESULTS: The results showed that culture media composed of milk whey without filtration supplied L. lactis its adaptation needs better than filtrated milk whey. Nisin titers, in milk whey without filtration (pH adjusted), was 11120.13 mg L-1 in the second transfer, and up to 1628-fold higher than the filtrated milk whey, 6.83 mg.L-1 obtained in the first(t) transfer. CONCLUSIONS: Biological processing of milk byproducts (milk whey) can be considered a profitable alternative, generating high-value bioproducts and contributing to decreasing river disposals by dairy industries. (C) 2008 Society of Chemical Industry.

Transcriptional responses of host peripheral blood cells to tuberculosis infection

Host responses following exposure to Mycobacterium tuberculosis (TB) are complex and can significantly affect clinical outcome. These responses, which are largely mediated by complex immune mechanisms involving peripheral blood cells (PBCs) such as T-lymphocytes, NK cells and monocyte-derived macrophages, have not been fully characterized. We hypothesize that different clinical outcome following TB exposure will be uniquely reflected in host gene expression profiles, and expression profiling of PBCs can be used to discriminate between different TB infectious outcomes. In this study, microarray analysis was performed on PBCs from three TB groups (BCG-vaccinated, latent TB infection, and active TB infection) and a control healthy group. Supervised learning algorithms were used to identify signature genomic responses that differentiate among group samples. Gene Set Enrichment Analysis was used to determine sets of genes that were co-regulated. Multivariate permutation analysis (p < 0.01) gave 645 genes differentially expressed among the four groups, with both distinct and common patterns of gene expression observed for each group. A 127-probeset, representing 77 known genes, capable of accurately classifying samples into their respective groups was identified. In addition, 13 insulin-sensitive genes were found to be differentially regulated in all three TB infected groups, underscoring the functional association between insulin signaling pathway and TB infection. Published by Elsevier Ltd.