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.

Identification of Staphylococcus aureus Carrying the mecA Gene in Ready-to-Eat Food Products Sold in Brazil

Since Staphylococcus aureus can cause several types of diseases, the development of antibiotic resistance poses an even greater threat to public health. S. aureus is known to possess the adaptive capability to promptly respond to antibiotics, making it resistant and increasingly difficult to treat; methicillin-resistant strains of S. aureus are a major concern with regard to this species. Previous studies reported the identification of methicillin-resistant S. aureus in food, demonstrating that this can represent a source of S. aureus which may carry the mecA gene. Fifty-seven S. aureus isolates, previously obtained from different types of food, were screened by polymerase chain reaction with specific primers for the mecA gene, which mediates methicillin resistance. Five (9%) isolates showed the presence of mecA gene, demonstrating that food may contain microorganisms possessing resistance genes. This study emphasizes the need to include food as a possible source of S. aureus carrying mecA gene and the need to monitor these products. Moreover, this is the first report of the presence of mecA genes in S. aureus isolated from ready-to-eat food in Brazil and Latin America.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. one hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p < 0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p < 0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p = 0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4 +/- 12.4%) and apolipoprotein B (apoB) (17.0 +/- 31.3%) when compared with the higher quartile (>0.085: LDL-c = 40.3 +/- 14.3%; apoB = 32.5 +/- 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism. and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin. (C) 2008 Elsevier Inc. All rights reserved.

EIN3-like gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Grande naine)

Ethylene signal transduction initiates with ethylene binding at receptor proteins and terminates in a transcription cascade involving the EIN3/EIL transcription factors. Here, we have isolated four cDNAs homologs of the Arabidopsis EIN3/EIN3-like gene, MA-EILs (Musa acuminata ethylene insensitive 3-like) from banana fruit. Sequence comparison with other banana EIL gene already registered in the database led us to conclude that, at this day, at least five different genes namely MA-EIL1, MA-EIL2/AB266318, MA-EIL3/AB266319, MA-EIL4/AB266320 and AB266321 exist in banana. Phylogenetic analyses included all banana EIL genes within a same cluster consisting of rice OsEILs, a monocotyledonous plant as banana. However, MA-EIL1, MA-EIL2/AB266318, MA-EIL4/AB266320 and AB266321 on one side, and MA-EIL3/AB266319 on the other side, belong to two distant subclusters. MA-EIL mRNAs were detected in all examined banana tissues but at lower level in peel than in pulp. According to tissues, MA-EIL genes were differentially regulated by ripening and ethylene in mature green fruit and wounding in old and young leaves. MA-EIL2/AB266318 was the unique ripening- and ethylene-induced gene; MA-EIL1, MA-EIL4/Ab266320 and AB266321 genes were downregulated, while MA-EIL3/AB266319 presented an unusual pattern of expression. Interestingly, a marked change was observed mainly in MA-EIL1 and MA-EIL3/Ab266319 mRNA accumulation concomitantly with changes in ethylene responsiveness of fruit. Upon wounding, the main effect was observed in MA-EIL4/AB266320 and AB266321 mRNA levels, which presented a markedly increase in both young and old leaves, respectively. Data presented in this study suggest the importance of a transcriptionally step control in the regulation of EIL genes during banana fruit ripening.

Silencing of mitochondrial alternative oxidase gene of Aspergillus fumigatus enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 mu g/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox`s lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell

Paracoccidioides brasiliensis infectious process relies on the initial expression of virulence faactors that are assumed to be controlled by molecular mechanisms through which the conidia and/or mycelial fragments convert to yeast cells. In order to analyze the profile of the thermally-induced dimorphic gene expression, 48 h C-L transition cultures which had been incubated at 36 degrees C were studied. By this time approximately 50% of the conidial population had already reverted to yeast form cells. At this transition time, an EST-Orestes library was constructed and characterized. As a result, 79 sequences were obtained, of which 39 (49.4%) had not been described previously in other libraries of this fungus and which could represent novel exclusive C-Y transition genes. Two of these sequences are, among others, cholestanol delta-isomerase, and electron transfer flavoprotein-ubiquinoneoxidoreductase (ETF-QO). The other 40 (50.6%) sequences were shared with Mycelia (M), Yeast (Y) or Mycelia to yest transition (M-Y) libraries. An important component of this group of sequences is a putative response regulator receiver SKN7, a protein of high importance in stress adaptation and a regulator of virulence in some bacteria and fungi. This is the first report identifying genes expressed during the C-Y transition process, the initial step required to understand the natural history of P brasiliensis conidia induced infection.

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5` extremity of each mRNA, supplying the 5`-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the Virulence of the parasite in in vivo assays. The results presented here extend those findings to Vionnia subgenus. Leishmania (Vionnia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Transcription regulation of the PbGP43 gene by nitrogen in the human pathogen Paracoccidioides brasiliensis

We show indirect evidences for the possible involvement of NIT-2-like binding motifs in transcription modulation of the PbGP43 gene, which codes for an important antigen from the human fungal pathogen Paracoccidioides brasiliensis. This investigation was motivated by the finding of 23 NIT2-like sites within the proximal -2047 nucleotides of the PbGP43 5` intergenic region from the Pb339 isolate. They compose four clusters, two of them identical. We found four NIT2-containing probes that were positive in electrophoretic mobility shift assays and further analyzed them. PbGP43 could be modulated by nitrogen primary sources in Pb339, Pb3 and Pb18 isolates, as observed by reverse transcription (RT) real time-PCR. Gene reporter assays conducted in Aspergillus nidulans suggested that the minimal fragment responsible for nitrogen modulation lies within -480 bp of the PbGP43 gene. This is the first report on PbGP43 transcription modulation in response to nitrogen primary sources, which might help understand its regulation during infection. (C) 2008 Elsevier Inc. All rights reserved.

The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats

Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet`s effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet`s effects on genomic stability and DNA methylation. (C) 2011 Elsevier ay. All rights reserved.

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2 crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for similar to 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (similar to 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed. (C) 2010 Elsevier B.V. All rights reserved.

Paracoccin from Paracoccidioides brasiliensis; purification through affinity with chitin and identification of N-acetyl-beta-D-glucosaminidase activity

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most frequent systemic mycosis in Latin America. Our group has been working with paracoccin, a P. brasiliensis lectin with MM 70 kDa. which is purified by affinity, with immobilized N-acetylglucosamine (GlcNAc). Paracoccin has been described to play a role in fungal adhesion to extracellular matrix components and to induce high and persistent levels or TNF alpha. and nitric oxide production by macrophages. In the cell wall, paracoccin colocalizes with the beta-1,4-homopolymer of GlcNAc into the budding sites of the P. brasiliensis yeast cell. In this paper we present a protocol for the chitin-affinity purification or paracoccin. This procedure provided higher yields than those achieved by means of the technique based oil the affinity of this lectin with GlcNAc and had an impact on downstream assays. SDS-PAGE and Western blot analysis revealed similarities between the N-acetylglucosamine- and chitin-bound fractions, confirmed by MALDI-TOF-MS of trypsinic peptides. Western blot of two-dimensional gel electrophoresis of the yeast extract showed a major spot with M(r) 70000 and pl approximately 5.63. Moreover, an N-acetyl-beta-D-glucosaminidase activity was reported for paracoccin, thereby providing new insights into the mechanisms that lead to cell wall remodelling and opening new perspectives for its structural characterization. Copyright (C) 2009 John Wiley & Sons. Ltd.

Crotalus durissus collilineatus venom gland transcriptome: Analysis of gene expression profile

Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A(2) (PLA(2)) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA(2) inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF. (C) 2009 Published by Elsevier Masson SAS.

Downregulation of ultraspiracle gene expression delays pupal development in honeybees

Ecdysteroids regulate many aspects of insect physiology after binding to a heterodimer composed of the nuclear hormone receptor proteins ecdysone receptor (EcR) and ultraspiracle (Use). Several lines of evidence have suggested that the latter also plays important roles in mediating the action of juvenile hormone (JH) and, thus, integrates signaling by the two morphogenetic hormones. By using an RNAi approach, we show here that Us p participates in the mechanism that regulates the progression of pupal development in Apis mellifera, as indicated by the observed pupal developmental delay in usp knocked-down bees. Knock-down experiments also suggest that the expression of regulatory genes such as ftz transcription factor 1 (ftz-f1) and juvenile hormone esterase (jhe) depend on Usp. Vitellogenin (vg), the gene coding the main yolk protein in honeybees, does not seem to be under Usp regulation, thus suggesting that the previously observed induction of vg expression by JH during the last stages of pupal development is mediated by yet unknown transcription factor complexes. (C) 2008 Elsevier Ltd. All rights reserved.

A honeybee storage protein gene, hex 70a, expressed in developing gonads and nutritionally regulated in adult fat body

In preparing for metamorphosis, insect larvae store a huge amount of proteins in hemolymph, mainly hexamerins. Out of the four hexamerins present in the honeybee larvae, one, HEX 70a, exhibited a distinct developmental pattern, especially since it is also present in adults. Here, we report sequence data and experimental evidence suggesting alternative functions for HEX 70a, besides its well-known role as an amino acid resource during metamorphosis. The hex 70a gene consists of 6 exons and encodes a 684 amino acid chain containing the conserved hemocyanin N, M, and C domains. HEX 70a classifies as an arylphorin since it contains more than 15% of aromatic amino acids. In the fat body of adult workers, hex 70a expression turned out to be a nutrient-limited process. However, the fat body is not the only site for hex 70a expression. Both, transcript and protein subunits were also detected in developing gonads from workers, queens and drones, suggesting a role in ovary differentiation and testes maturation and functioning. In its putative reproductive role, HEX 70a however differs from the yolk protein, vitellogenin, since it was not detected in eggs or embryos. (C) 2008 Elsevier Ltd. All rights reserved.