Antônio José Rocha

Differential expression of cysteine peptidase genes in the inner integument and endosperm of developing seeds of Jatropha curcas L. (Euphorbiaceae)

In several plant tissues, programmed cell death (PCD) is mediated by the combined action of cysteine peptidases, namely KDEL-tailed cysteine peptidases (KDEL-CysEP) and vacuolar processing enzymes (VPE). Here, we performed a search of the draft genome of Jatropha curcas L. (Euphorbiaceae) and identified 2 genes for KDEL-CysEP (Jc-CysEP1 and Jc-CysEP2) and 3 genes for VPE (Jc-βVPE, Jc-γVPE and Jc-δVPE) and determined the expression patterns of these genes by RT-qPCR in integument and cellular endosperm of seeds collected at seven different developmental stages. We were able to demonstrate that the expression of Jc-CysEP1, Jc-CysEP2, Jc-βVPE and Jc-γVPE proceeded rapidly from Stage IV, with Jc-CysEP2 displaying the highest relative expression; expression of Jc-δVPE could not be detected in any of the tissues/developmental stages analyzed. Additionally, we showed that the expression pattern of these peptidases correlates with anatomical changes in integument and cellular endosperm, thus suggesting a role for both classes of peptidases in PCD and in protein processing, both of which occur simultaneously in each of these tissues.

Bioethical conflicts of gene therapy: a brief critical review

Methods and techniques employed in gene therapy are reviewed in parallel with pertinent ethical conflicts. Clinical interventions based on gene therapy techniques preferentially use vectors for the transportation of therapeutic genes, however little is known about the potential risks and damages to the patient. Thus, attending carefully to the clinical complications arising as well as to security is essential. Despite the scientific and technological advances, there are still many uncertainties about the side effects of gene therapy. Moreover, there is a need, above all, to understand the principles of bioethics as both science and ethics, in accordance with its socioecological responsibility, in order to prioritize the health and welfare of man and nature, using properly natural resources and technology. Therefore, it is hard to determine objective results and to which extent the insertion of genes can affect the organism, as well as the ethical implication.

Identification of Suitable Reference Genes for Gene Expression Normalization in Jatropha curcas L During Development and Under Stress Conditions Using Real Time Quantitative PCR

Jatropha curcas L represent a potential source of raw material for the production of biodiesel. The aim this study was to find potential candidate reference genes in J. curcas tissues. Three softwares were utilized to verify which would be the most stable reference genes in qPCR assay: GeNorm, NormFinder and BestKeeper. The most stable reference genes in developing J. curcas seeds suggested by GeNorm were GAPDH, UCP, actin. However, the best combinations of stable genes in each tissue were identified separately under stress conditions: EF1-α, PP2A2 and GAPDH in total stress, however, in SA stress, four genes were required for normalization: PP2A2, EF1-α, GAPDH and PUB. In PEG stress, four genes also were required: PP2A2, EF1-α, GAPDH and PUB, while in NaCl stress, five genes were necessary: PP2A2, GAPDH, EF1-α, PUB and Tβ2. These results are in accordance with two other programs used in this study (NormFinder, BestKeeper). In addition, the transcript levels of Jc-SRG-2 seem to be more correlated with stress responses than changes in transcript levels of Jc-SRG-1, mainly of leaves in exposure to 3-12 h on PEG and NaCl stress. Taken together, GAPDH and PP2A2 were regarded as being the best reference to provide guidelines for the selection of potential references genes under these study conditions.

Trombose na endoprótese do aneurisma da aorta: avaliação por TC multidetector

OBJECTIVE: Evaluate the imaging findings of thrombosis in a series of patients submitted to endovascular repair of aortic abdominal aneurysm. METHODS: MDCT images of 30 patients submitted to endovascular repair of aortic abdominal aneurysm were obtained by a 64 slice scanner, 5 to 29 months after the endovascular treatment. RESULTS: Thrombosis was diagnosed in 10 patients (33.3%), and in three patients thrombosis was total in an iliac branch. CONCLUSION: MDCT allowed diagnosis of different types of endoluminal thrombosis in patients submitted to endovascular repair of aortic abdominal aneurysm. Utilization of this minimally invasive diagnostic technique should be encouraged in clinical practice.

Cloning of cDNA sequences encoding cowpea (Vigna unguiculata) vicilins: Computational simulations suggest a binding mode of cowpea vicilins to chitin oligomers

Vicilins are 7S globulins which constitute the major seed storage proteins in leguminous species. Variant vicilins showing differential binding affinities for chitin have been implicated in the resistance and susceptibility of cowpea to the bruchid Callosobruchus maculatus. These proteins are members of the cupin superfamily, which includes a wide variety of enzymes and non-catalytic seed storage proteins. The cupin fold does not share similarity with any known chitin-biding domain. Therefore, it is poorly understood how these storage proteins bind to chitin. In this work, partial cDNA sequences encoding β-vignin, the major component of cowpea vicilins, were obtained from developing seeds. Three-dimensional molecular models of β-vignin showed the characteristic cupin fold and computational simulations revealed that each vicilin trimer contained 3 chitin-binding sites. Interaction models showed that chito-oligosaccharides bound to β-vignin were stabilized mainly by hydrogen bonds, a common structural feature of typical carbohydrate-binding proteins. Furthermore, many of the residues involved in the chitin-binding sites of β-vignin are conserved in other 7S globulins. These results support previous experimental evidences on the ability of vicilin-like proteins from cowpea and other leguminous species to bind in vitro to chitin as well as in vivo to chitinous structures of larval C. maculatus midgut.

Guidelines for Successful Quantitative Gene Expression in Real- Time qPCR Assays

This chapter was developed to provide some important guidelines for studies with quantitative PCR (qPCR) using either dyes or probes, citing several essential compo‐ nents necessary for a good PCR assay. The efficiency and specificity of quantitative PCR (qPCR) depend on several parameters related to mRNA quantification that must be controlled to avoid mistakes in data interpretation. Avoiding contamination with proteins, carbohydrate and phenolic compounds during RNA extraction and purifica‐ tion processes will improve RNA quality and provide reliable results. Specific primers and sensible probes are also crucial to intensify efficiency, specificity and fluorescence. Other parameters such as the optimization of primer concentrations and efficiency primer curves must be done. During gene‐expression profile quantification, qPCR assays using reference genes are required to normalize the target gene expression data. These reference genes are checked for stability to identify the most stable genes among a group of candidate genes that will be used to normalize the qPCR data, using programs such as geNorm, BestKeeper and NormFinder. Additionally, the choice of appropriate reference genes for a specific experimental condition is fundamental. The main aim of this chapter is to provide guidelines and highlight precautions to obtain a successful qPCR assays.

Stent thrombosis in aortic aneurysm: evaluation by multidetector CT

OBJECTIVE Evaluate the imaging findings of thrombosis in a series of patients submitted to endovascular repair of aortic abdominal aneurysm. METHODS MDCT images of 30 patients submitted to endovascular repair of aortic abdominal aneurysm were obtained by a 64 slice scanner, 5 to 29 months after the endovascular treatment. RESULTS Thrombosis was diagnosed in 10 patients (33.3%), and in three patients thrombosis was total in an iliac branch. CONCLUSION MDCT allowed diagnosis of different types of endoluminal thrombosis in patients submitted to endovascular repair of aortic abdominal aneurysm. Utilization of this minimally invasive diagnostic technique should be encouraged in clinical practice.