Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Lectin histochemistry for detecting cadmium-induced changes in the glycosylation pattern of rat placenta

Cadmium (Cd) is an industrial and environmental pollutant that produces toxic effects on gametogenesis, pre- and post-implantation embryos, and the placenta. Because the effects of acute Cd intoxication on the placenta are not well understood, we investigated changes in its glycosylated components in Cd treated dams at days 4, 7, 10 and 15 of gestation using lectin histochemistry. CdCl₂ was administered to pregnant rats; control animals received sterile normal saline. Placentas were processed for DBA, Con A, SBA, PNA, UEA-I, RCA-I and WGA lectin histochemistry to evaluate changes in the carbohydrate pattern of the placenta that might modify cell interactions and contribute to embryonic alterations. Lectin binding was analyzed in the yolk sac; trophoblast giant cells; trophoblast I, II and III; spongiotrophoblast cells and endovascular trophoblast cells in the chorioallantoic placenta. Our lectin binding patterns showed that Cd caused alteration of SBA and DBA labeling of trophoblast-derived cells, which suggested increased expressions of α and β GalNAc. Cd also caused decreased UEA-1 binding affinity, which indicated fewer α-L-Fuc residues in placentas of Cd treated dams. The nonreactivity in trophoblast I of the control placentas incubated with Con-A contrasted with the labeling in placentas of experimental dams, which indicated increased expression of terminal α-D-Man, and α-D-Glc residues. We found that Cd altered the reactivity of placenta to several lectins, which indicated modification of the glycotype presented by the fetal component of the placenta. We report that Cd exerts a deleterious effect on the glycosylation pattern of the placenta.     

Suppression of citrus canker disease mediated by flagellin perception

Citrus cancer, caused by strains of Xanthomonas citri (Xc) and Xanthomonas aurantifolii (Xa), is one of the most economically important citrus diseases. Although our understanding of the molecular mechanisms underlying citrus canker development has advanced remarkably in recent years, exactly how citrus plants fight against these pathogens remains largely unclear. Using a Xa pathotype C strain that infects Mexican lime only and sweet oranges as a pathosystem to study the immune response triggered by this bacterium in these hosts, we herein report that the Xa flagellin C protein (XaFliC) acts as a potent defence elicitor in sweet oranges. Just as Xa blocked canker formation when coinfiltrated with Xc in sweet orange leaves, two polymorphic XaFliC peptides designated flgIII-20 and flgIII-27, not related to flg22 or flgII-28 but found in many Xanthomonas species, were sufficient to protect sweet orange plants from Xc infection. Accordingly, ectopic expression of XaFliC in a Xc FliC-defective mutant completely abolished the ability of this mutant to grow and cause canker in sweet orange but not Mexican lime plants. Because XaFliC and flgIII-27 also specifically induced the expression of several defence-related genes, our data suggest that XaFliC acts as a main immune response determinant in sweet orange plants.     

Abiotic and Biotic Degradation of Oxo-Biodegradable Plastic Bags by Pleurotus ostreatus

In this study, we evaluated the growth of Pleurotus ostreatus PLO6 using oxo-biodegradable plastics as a carbon and energy source. Oxo-biodegradable polymers contain pro-oxidants that accelerate their physical and biological degradation. These polymers were developed to decrease the accumulation of plastic waste in landfills. To study the degradation of the plastic polymers, oxo-biodegradable plastic bags were exposed to sunlight for up to 120 days, and fragments of these bags were used as substrates for P. ostreatus. We observed that physical treatment alone was not sufficient to initiate degradation. Instead, mechanical modifications and reduced titanium oxide (TiO₂) concentrations caused by sunlight exposure triggered microbial degradation. The low specificity of lignocellulolytic enzymes and presence of endomycotic nitrogen-fixing microorganisms were also contributing factors in this process.     

Challenges and Solutions in the Development of Genomic Biomarker Panels: A Systematic Phased Approach

In the post-genome era, high throughput gene expression profiling has been successfully used to develop genomic biomarker panels (GBP) that can be integrated into clinical decision making. The development of GBPs in the context of personalized medicine is a scientifically challenging and resource-intense process. It needs to be accomplished in a systematic phased approach to address biological variation related to a clinical phenotype (e.g. disease etiology, gender, etc.) and minimize technical variation (noise). Here we present the methodological aspects of GBP development based on the experience of the Cardiac Allograft Rejection Gene Expression Observation (CARGO) study, a study that lead to the development of a molecular classifier for rejection screening in heart transplant patients.     

Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs’ protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.     

Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells

Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor κB (NF-κB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-κB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-κB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.     

Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Supports Mitochondrial Metabolism and Host-Cell Invasion of Trypanosoma cruzi

Proline is crucial for energizing critical events throughout the life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease. The proline breakdown pathway consists of two oxidation steps, both of which produce reducing equivalents as follows: the conversion of proline to Δ¹-pyrroline-5-carboxylate (P5C), and the subsequent conversion of P5C to glutamate. We have identified and characterized the Δ¹-pyrroline-5-carboxylate dehydrogenase from T. cruzi (TcP5CDH) and report here on how this enzyme contributes to a central metabolic pathway in this parasite. Size-exclusion chromatography, two-dimensional gel electrophoresis, and small angle x-ray scattering analysis of TcP5CDH revealed an oligomeric state composed of two subunits of six protomers. TcP5CDH was found to complement a yeast strain deficient in PUT2 activity, confirming the enzyme''s functional role; and the biochemical parameters (K_m, k_cat, and k_cat/K_m) of the recombinant TcP5CDH were determined, exhibiting values comparable with those from T. cruzi lysates. In addition, TcP5CDH exhibited mitochondrial staining during the main stages of the T. cruzi life cycle. mRNA and enzymatic activity levels indicated the up-regulation (6-fold change) of TcP5CDH during the infective stages of the parasite. The participation of P5C as an energy source was also demonstrated. Overall, we propose that this enzymatic step is crucial for the viability of both replicative and infective forms of T. cruzi.