Production and purification of bacterial membrane vesicles for biotechnology applications: Challenges and opportunities.

Bacterial membrane vesicles (BMVs) are bi-layered nanostructures derived from Gram-negative and Gram-positive bacteria. Among other pathophysiological roles, BMVs are critical messengers in intercellular communication. As a result, BMVs are emerging as a promising technology for the development of numerous therapeutic applications. Despite the remarkable progress in unveiling BMV biology and functions in recent years, their successful isolation and purification have been limited. Several challenges related to vesicle purity, yield, and scalability severely hamper the further development of BMVs for biotechnology and clinical applications. This review focuses on the current technologies and methodologies used in BMV production and purification, such as ultracentrifugation, density-gradient centrifugation, size-exclusion chromatography, ultrafiltration, and precipitation. We also discuss the current challenges related to BMV isolation, large-scale production, storage, and stability that limit their application. More importantly, the present work explains the most recent strategies proposed for overcoming those challenges. Finally, we summarize the ongoing applications of BMVs in the biotechnological field.

Analysis of Tumor-Derived Exosomes by Nanoscale Flow Cytometry.

The study of tumor exosomes has gained relevance in the last decades due to their potential use for therapeutic and diagnostic application. Although there is extensive knowledge of exosome biology, some biological samples like tumor-derived exosomes have been difficult to characterize due to their complexity and heterogeneity. This distinctive feature makes difficult the identification of specific exosome subpopulations with a shared molecular signature that could allow for targeting of exosomes with therapeutic and diagnostic potential use in cancer patients. Nanoscale flow cytometry has lately emerged as an alternative tool that can be adapted to the study of nanoparticles, such as exosomes. However, the physicochemical properties of these particles are an important issue to consider as nanoparticles need the application of specific settings which differ from those used in conventional flow cytometry of cells. Therefore, in the last few years, one of the main aims has been the optimization of technical and experimental protocols to improve exosome analysis. In this chapter, we discuss several aspects of cytometric systems with a special emphasis in technical considerations of samples and equipment.

Analysis of RNA yield in extracellular vesicles isolated by membrane affinity column and differential ultracentrifugation.

Extracellular vesicles (EV) have attracted much attention as potential biomarkers due to their protein, RNA and other nucleic acid content. The most common method used for EV isolation is differential ultracentrifugation (DU), however given the DU technical difficulties, other more practical methods have surged, such as membrane-affinity column commercial kits. Here, we assessed one commercial kit in terms of EV recovery and EV-derived RNA yield and compared it with a DU protocol. Our data shows that the commercial kit preparation results in a lower count of EV-like structures and a reduced expression of EV markers when compared to DU samples. Thus, apparently suggesting that the commercial kit had a lower EV yield. However, these findings did not reflect on RNA yield, which was greater with the commercial kit, even after an enzymatic treatment with proteinase K and RNAse A. We conclude that the kit has a higher EV-derived RNA yield in comparison to our DU protocol, suggesting that it may be the method of choice for RNA sequencing purposes.

Production of Lentivirus for the Establishment of CAR-T Cells.

One of the most versatile gene transfer methods involves the use of recombinant lentiviral vectors since they can transduce both dividing and nondividing cells, are considered to be safe and provide long-term transgene expression since the integrated viral genome, the provirus, is passed on to daughter cells. These characteristics are highly desirable when a modified cell must continue to express the transgene even after multiple cell divisions. Lentiviral vectors are often used to introduce protein encoding cDNAs, such as reporter genes, or for noncoding sequences, such as mediators of RNA interference or genome editing, including shRNA or gRNA, respectively. In the gene therapy setting, lentiviral vectors have been used successfully for the modification of hematopoietic stem cells, resulting in restored immune function or correction of defects in hemoglobin, to name but a few examples. The success of chimeric antigen receptor (CAR) T cells for the treatment of B cell leukemias and lymphomas has been particularly striking and this approach has relied heavily on lentivirus-mediated gene transfer. Here we present a typical protocol for the production of lentivirus, concentration by ultracentrifugation and determination of virus titer. The resulting virus can then be used in laboratory assays of gene transfer, including the establishment of CAR T cells.

Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells.

Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1ΔCT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1ΔCT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1ΔCT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1ΔCT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.

Sample Preparation of Trypanosoma cruzi Surface, Extracellular, and Nuclear Subproteomes.

The preparation of subproteome fractions prior to proteome analysis provides both the enrichment of proteins sub-represented in global proteome analysis and information on the cellular localization of the identified proteins. Here we describe protocols for the preparation of Trypanosoma cruzi surface and extracellular and nuclear fractions for further subproteome analysis.

Isolation and Characterization of Extracellular Vesicles Derived from Trypanosoma cruzi.

Extracellular vesicles (EVs) are heterogeneous membrane-surrounded structures that participate in cellular communications, which comprise exosomes and microvesicles. These vesicles have different biogenesis, and their physiological and pathological roles in chronic and infectious diseases are under constant investigation. In Chagas disease, Trypanosoma cruzi EVs have been described using different approaches. The isolation of T. cruzi-derived EVs has been done mainly using the differential centrifugation technique, and different strategies have been employed for characterization of them. Here, we describe the method to isolate EVs by differential centrifugation and a detection protocol for EVs in T. cruzi-host cell interaction to allow further investigations about this parasite.

Physicochemical and Biological Characterization of RTXM83, a New Rituximab Biosimilar.

BACKGROUND: RTXM83 is a rituximab biosimilar with proven clinical safety and efficacy. It is the first rituximab biosimilar developed and approved in South America and is currently marketed in several Latin American, Middle Eastern and African countries. OBJECTIVE: The aim of this study was to present the physicochemical and biological characterization studies utilized to demonstrate the similarity between RTXM83 and its reference product. METHODS: Primary and higher order protein structures were analysed using peptide mapping with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), fluorescence spectroscopy and circular dichroism, and micro-differential scanning calorimetry, among other techniques. Charge variants were determined by cation-exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Glycosylation and glycoforms distribution were analysed using MS, normal phase high-performance liquid chromatography (NP-HPLC) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Size variants were evaluated by size-exclusion chromatography (SEC), sedimentation velocity analytical ultracentrifugation (SV-AUC), dynamic light scattering (DLS), and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS). Biological characterization included binding assays for complement C1q, CD20, and several Fc receptors (FcRs), as well as potency determination for in vitro apoptosis induction, complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). RESULTS: RTXM83 and the reference product showed identical primary sequences and disulfide bridge patterns, and similarity at higher order protein structures, post-translational modification profiles (amino acid modifications, charge variants, and glycosylation) and levels of purity and process-related impurities. Functional studies demonstrated that RTXM83 is similar to the reference product regarding the three known mechanisms of action of rituximab: CDC, ADCC, and apoptosis induction. Binding affinities to CD20, complement component C1q, and different FcRs were also equivalent. CONCLUSION: RTXM83 is similar to its reference product in all critical quality attributes.

Metodología para la obtención y evaluación de un extracto proteínico de una cepa autóctona de Helicobacter pylori / Methodology to obtain and evaluate a protein extract of an autochthonous Helicobacter pylori strain

Helicobacter pylori es una bacteria gram negativa que posee numerosos antígenos que juegan un importante papel en la patogénesis de las enfermedades gastroduodenales. Debido a la necesidad de métodos de diagnóstico estandarizados con antígenos locales u autóctonos nos propusimos el diseño de una estrategia para la obtención de extractos de antígenos con reactividad frente a sueros de pacientes infectados por H. pylori. Dos cepas de H. pylori, una autóctona (IPK196A) y una de referencia ATCC 43504, se cultivaron en un medio líquido modificado. Se sometieron a los protocolos de ruptura por ultrasonido aplicándose tres variantes de precipitación y al fraccionamiento celular mediante ultracentrifugación diferencial. Los extractos proteínicos se visualizaron mediante electroforesis en gel de poliacrilamida y se transfirieron para la detección de antígenos inmunorreactivos a sueros de pacientes con infección por H. pylori e individuos sanos. La variante de ultrasonido y precipitación con Coomasie fue la más efectiva para concentrar las muestras. El método de ultracentrifugación mejoró la resolución de las proteínas reactivas y permitió separarlas según su localización subcelular. El sistema de transferencia húmedo fue ideal para la inmunodetección de los antígenos obtenidos por ultrasonido mientras que el sistema semiseco permitió detectar las proteínas de membrana obtenidas por ultracentrifugación diferencial. La introducción de una metodología en el laboratorio para la obtención y evaluación de extractos proteínicos antigénicos a partir de cepas autóctonas de H. pylori, constituye la antesala para el diseño de futuros diagnosticadores y candidatos vacunales(AU)

Helicobacter pylori is a gram-negative spiral-shaped bacterium, which has many antigens that play an important role in the pathogenesis of gastroduodenal diseases. Due to the lack of standardized methods from native or autochthonous antigens, we proposed in this study, the design of a strategy for extracting and obtaining immunoreactive antigens against H. pylori infected-patient sera. Two H. pylori strains, one autochthonous (IPK196A) and one reference ATCC 43504, were cultured in a modified liquid medium. Both strains were subjected to the ultrasound rupture protocols applying three precipitation variants and cell fractionation by differential ultracentrifugation. Protein extracts were visualized by polyacrylamide gel electrophoresis and transferred for the detection of immunoreactive antigens to sera from patients with H. pylori infection and healthy individuals. The precipitation with Coomasie was the most effective variant. The ultracentrifugation extraction method optimized the resolution of the proteins, which could be separated according to their subcellular location. The wet transfer system was ideal for the immunodetection of the antigens obtained by ultrasound, while the semi-dry system allowed detecting the membrane proteins by differential ultracentrifugation. The introduction of a methodology in the laboratory for obtaining and evaluating antigenic antibodies from autochthonous strains of H. pylori, is the prelude to the design for future diagnostics and vaccine candidates(AU)

Spectrophotometry and Ultracentrifugation for Measurement of Plasma Lipids in Dogs with Diabetes Mellitus.

BACKGROUND: There are conflicting reports of plasma lipoprotein lipid content in dogs with diabetes mellitus (DM). OBJECTIVES: To determine lipoprotein lipid content of plasma of dogs with DM by spectrophotometry and ultracentrifugation; to compare lipoprotein lipid content in diabetic and healthy dogs; and to quantify apolipoprotein B-100 (ApoB) in dogs with DM. ANIMALS: 22 dogs with DM and 9 healthy dogs. METHODS: Cross-sectional study. Triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) concentrations were measured by spectrophotometry. Very low-density lipoprotein cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) concentrations were calculated after ultracentrifugation. Non-HDL-C cholesterol was calculated by subtracting HDL-C from TC. ApoB was quantified by ELISA. The Mann-Whitney test was used for comparison of median lipoprotein concentrations, and Spearman's correlation was used to assess associations between ApoB and lipoprotein fractions. RESULTS: All values are reported in mg/dL. Median TG (122), TC (343.5), HDL-C, (200), VLDL-C, (27) LDL-C (68), non-HDL-C (114), and ApoB (320) were significantly higher in dogs with DM, compared to healthy dogs (57, 197, 168, 12, 16, 31, and 258, respectively, P-values 0.0079, <0.001, 0.029, 0.011, <0.001, <0.001, 0.025, respectively). A significant association was found between ApoB and LDL-C (Spearman's rho = 0.41, P = 0.022) and between ApoB and non-HDL-C (Spearman's rho = 0.40, P = 0.027). CONCLUSIONS AND CLINICAL IMPORTANCE: Dyslipidemia of dogs with DM is characterized by pronounced increases in LDL-C and non-HDL-C concentrations, although all lipoprotein fractions are significantly increased. Knowledge of specific lipoprotein fraction alterations in dogs with DM can enhance treatment options for diabetic dyslipidemia in dogs.

Characterization of a cyclic olefin polymer microcentrifuge tube.

Here, we describe the properties of a prototype microcentrifuge tube made from the plastic cyclic olefin polymer (COP). This material has been used in the manufacture of primary containers including syringes and vials for the storage, shipment, and delivery of biotherapeutics, vaccines, and cell therapy products. Its low level of extractable substances and metals along with its glass-like clarity make COP an attractive material for the fabrication of microcentrifuge tubes and other consumable laboratory plasticware where contamination is an important consideration, such as in the storage and analysis of labile proteins, nucleic acids, and metabolites. We compare the performance of microcentrifuge tubes made of COP with that of several brands made of polypropylene (PP), the plastic most widely used in the manufacture of microcentrifuge tubes. Our results show COP microcentrifuge tubes perform as well as tubes made of PP, with reduced levels of compounds capable of leaching into solvents typically used in the laboratory.

Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of the ultracentrifugation and adsorption-elution methods for virus concentration.

Group A rotaviruses (RV-A) are the most common agents of viral gastroenteritis in children worldwide. The goal of this study was to compare two different methods to concentrate RV-A from sewage samples and to improve the detection and quantification of RV-A using a multiplex quantitative PCR assay with an internal control. Both RV-A and the internal control virus, bacteriophage PP7, were seeded into wastewater and then concentrated using either an ultrafiltration-based adsorption-elution protocol or an ultracentrifugation-based protocol. Real time multiplex quantitative PCR was used to quantify the purified RV-A and PP7, and the results of the multiplex assay were compared with the results of the monoplex assays. The ultracentrifugation-based method had a mean recovery rate of 47% (range: 34-60%), while the ultrafiltration-based adsorption-elution method had a mean recovery rate of 3.5% (range: 1.5-5.5%). These results demonstrate that ultracentrifugation is a more appropriate method for recovering RV-A from wastewater. This method together with the multiplex qPCR assay may be suitable for routine laboratory use.

Separación de poblaciones de glóbulos rojos senescentes por gradientes preformados de Percoll / Separation of populations of senescent red blood cells by Percoll performed gradients

Los mecanismos que determinan la senescencia de los eritrocitos han sido extensamente estudiados, sin embargo, no se han logrado conclusiones definitivas debido a la ausencia de una técnica que permita el aislamiento de grupos etáreos bien definidos. Los métodos más comúnmente empleados se basan en el aumento de densidad de los eritrocitos durante el envejecimiento. En este trabajo desarrollamos una técnica para la separación de glóbulos rojos de distintas edades empleando gradientes preformados de Percoll, un polímero sintético con propiedades fisicoquímicas adecuadas para trabajar con células vivas. En las suspensiones eritrocitarias obtenidas se realizaron determinaciones hematológicas, actividades de enzimas antioxidantes y el ensayo de eritrofagocitosis. Los valores de los parámetros hematológicos evaluados fueron significativamente mayores en las suspensiones de glóbulos rojos jóvenes. Las actividades enzimáticas mostraron una disminución de la capacidad antioxidante en las poblaciones de eritrocitos senescentes. Este proceso favorecería la interacción de los hematíes envejecidos con las células fagocíticas, demostrada median­te el ensayo de eritrofagocitosis. Los resultados obtenidos indican que el método de gradientes de Percoll permite una adecuada separación de las suspensiones eritrocitarias de distintas edades, con una eficiencia comparable a la observada en la técnica de centrifugación diferencial considerada de referencia.

The mechanisms that determine the senescence of the erythrocytes have been extensively studied; however. definitive conclusions have not been achieved mainly because of the lack of a technique that allows the isolation of well-defined etarian groups. The methods most commonly used for separating erythrocytes from different ages are based on the increase in density that these cells present during their aging. In the present work we have developed a technique for obtaining red blood cells from different ages using Percoll preformed gradients, a synthetic polymer with adequate physic-chemic properties to work with lives cells. In the erythrocytes suspensions we have made hematological determinations. activities of antioxidants enzymes and the essay of erythrophagocytosis. The values of the hematological parameters were significantly higher in the suspensions of young red blood cells. In the measurements of the enzymatic activity we observed a decrease of the antioxidant capacity in the populations of senescent erythrocytes. This process would promote the interaction between the old erythrocytes and the phagocyte cells, demonstrated by the erythrophagocytosis essay. The results obtained indicate that the method Percoll density gradients allows an appropriate separation of the erythrocytes suspensions of different ages with a comparable efficiency to that observed in the technique differential centrifugation, considered as reference.

Climbing the steps of viral atomic force microscopy: visualization of Dengue virus particles.

In recent years, the application of atomic force microscopy (AFM) to biological systems has highlighted the potential of this technology. AFM provides insights into studies of biological structures and interactions and can also identify and characterize a large panel of pathogens, including viruses. The Flaviviridae family contains a number of viruses that are important human and animal pathogens. Among them, Dengue virus causes epidemics with fatal outcomes mainly in the tropics. In this study, Dengue virus is visualized for the first time using the in air AFM technique. Images were obtained from a potassium-tartrate gradient-purified virus. This study enhances the application of AFM as a novel tool for the visualization and characterization of virus particles. Because flavivirus members are closely related, studies of the morphologic structure of the Dengue virus can reveal strategies that may be useful to identify and study other important viruses in the family, including the West Nile virus.

Transition on the entropic elasticity of DNA induced by intercalating molecules.

We use optical tweezers to perform stretching experiments on DNA molecules when interacting with the drugs daunomycin and ethidium bromide, which intercalate the DNA molecule. Our results show that the persistence length of the DNA-drug complexes increases strongly as the drug concentration increases up to some critical value. Above this critical value, the persistence length decreases abruptly and remains approximately constant for larger drug concentrations, at least in the concentration range used in our experiments. Measured intercalators critical concentrations for the persistence length transition coincide with the reported values for the helix-coil transition of DNA-drug complexes obtained from sedimentation experiments. The contour length of the molecules increases monotonically and saturates as the drug concentration increases. The neighbor exclusion model fits to our results for the total drug concentration as a function of the relative increase of the contour length.

Glicación in vitro de lipoproteínas de baja densidad y aterogenicidad en cultivos celulares / In vitro glycation of low-density lipoproteins and atherogenicity in cell culture

La hiperglucemia sostenida incrementa la glicación de proteínas. En particular, las modificaciones en las lipoproteínas de baja densidad (LDL) aumentan su potencial aterogénico. En este trabajo se comparan las modificaciones producidas por la glicación in vitro de LDL aisladas por dos métodos: precipitación selectiva (PS) y ultracentrifugación (UC). Para ello, se determinó el incremento de fructosamina, el consumo de los grupos e-amino de lisina, guanidinio de arginina y la disminución de residuos de triptofano. Para todos los analitos, los resultados cinéticos indicaron diferencias significativas con relación al basal (p<0,05), coincidentes para ambos métodos en el tiempo de aparición y en el porcentaje de variación. La aterogenicidad de las LDL glicadas separadas por PS fue estudiada en cultivos de macrófagos RAW 264.7 evaluando la formación de células espumosas y cuantificando la incorporación de LDL por tinción de los depósitos lipídicos con Oil Red. Los resultados indican que la captación de LDL modificadas aumentó con el tiempo de incubación, siendo mayor la aterogenicidad de las LDL glicadas respecto de las nativas (p<0,001, 1 h a 37 °C). El procedimiento de PS seleccionado, accesible al laboratorio bioquímico clínico, permite evaluar las modificaciones por glicación que sufren las LDL en pacientes diabéticos.

Long-term hyperglycemia increases protein glycation. In particular, modifications in the low-density lipoproteins (LDL) increase their atherogenic potential. In this study, the modifications caused by in vitro glycation of LDL separated by two methods: selective precipitation (SP) and ultracentrifugation (UC) were compared. Increase fructosamine level, decrease of e-amino group of lysine, guanidinio of arginine and triptophan fluorescence were determined. Results showed significant differences vs. basal (p<0.05) for all the tested parameters, with coincidence for the two separation methods both in time and grade of modifications. The atherogenicity of glycated LDL separated by SP was studied in macrophages RAW 264.7 in culture, through the formation of foam cells and the quantification of the dye taken up by the cellular storage lipids. Results show that the uptake of modified LDL by macrophages increased with the time of incubation, being the atherogenicity of glycated LDL greater than native LDL (p<0.001, 1 h at 37 °C). The selected SP procedure, within the facilities of routine biochemical laboratory, enables the evaluation of the modifications caused by glycation in the LDL of diabetic patients.

Biochemical and biophysical characterization of small heat shock proteins from sugarcane. Involvement of a specific region located at the N-terminus with substrate specificity.

When cells are submitted to an increase in temperature, heat shock proteins (Hsp) are synthesized to help heat stress resistance. Small Hsps, which are diverse and abundant in plants, have the major function of preventing irreversible protein aggregation. The diversity of small Hsps in plants is intriguing and characterization of their chaperone activity is important to understand plant tolerance to heat stress. A previous study showed that small Hsps, mainly represented by class I (cytosolic), correspond to about 5% of all sugarcane Expressed Sequencing Tags belonging to the molecular chaperone category. Here, we present biochemical and biophysical characterization of two sugarcane small Hsps from class I, which were named SsHsp17.2 and SsHsp17.9 according to their monomer molecular mass of 17.2 and 17.9 kDa, respectively. The recombinant proteins have identity of about 75% to each other and similar structural characteristics. However, their stability and their chaperone activity were not equivalent: SsHsp17.9 was more efficient in protecting citrate synthase and malate dehydrogenase from aggregation whereas SsHsp17.2 was more efficient in protecting luciferase from aggregation. There is only one region, which is located at the N-terminus, of low homology between these two proteins. Based on that and on previous works pointing to multiple sites, mainly at the N-terminus, involved with substrate specificity in small Hsps, we suggest that this specific region is one of these sites. In addition, this is the first report on the chaperone activity of sugarcane small Hsps.

Determination of the sedimentation constants for total suspended solids and the algal component in a full-scale primary facultative pond operating at high wind velocities under tropical conditions.

This study evaluated the amount, distribution and sedimentation constant of solids in a full-scale primary facultative pond operating mostly under high wind conditions and the contribution made by the algal biomass. Solids deposition rates were measured using sedimentation traps placed in the inlet and outlet zones of the pond. Most sludge accumulation occurred, not surprisingly, in the inlet zone A1 with a sludge volume of 9072.m3 accumulating over an operating time of approximately 3 years. However, sludge deposition within this zone was uneven and affected by wind action. Mean proportionality constant (K) values for solids sedimentation were 3.02 and 5.70 for depths of 50 cm and 100 cm respectively for A1. In contrast in zone A3, (the outlet zone), reduced K values of 1.38 and 3.22 were obtained for depths of 50 cm and 100 cm respectively. The algal sedimentation constant varied from 0.8 d(-1) in zone A1 to 0.02 d(-1) in A3. These data suggest that in this large facultative pond the wind, blowing predominantly from the direction of the outlets towards the pond inlets, had a greater influence on solids deposition than the bulk hydraulic flow and also kept the pond completely mixed for most of the time.

Isolation and purification of functional total RNA from different organs of cacao tree during its interaction with the pathogen Crinipellis perniciosa.

Witches' broom disease, caused by Crinipellis perniciosa, is one of the major fungal diseases causing severe losses to cacao tree (Theobroma cacao L.) plantations in South America. One of the challenges associated with the understanding of the cacao and Crinipellis interaction in genomic studies is the isolation of intact nucleic acids. In this report, we describe a new, successful, and reliable procedure for the isolation of RNA from tissues of cacao tree, both infected and uninfected by Crinipellis. This protocol overcomes the problems associated with the very high amount of polyphenols and polysaccharides present in cacao organs that are not easily removed by conventional extraction procedures. The protocol requires few reagents, uses ultracentrifugation and inexpensive consumables, and can be easily applied in any laboratory. This method produced high-quality RNA that was suitable for subsequent purposes, such as reverse transcription PCR and cDNA library construction. We also report the first evidence of RNA isolation from cacao organs infected by C. perniciosa such as meristems and fruits.

Current Triton X-100 treatments do not allow a complete plasminogen activator extraction from developing nervous tissue.

Determinations of plasminogen activator (PA) activity are usually performed in Triton X-100-treated tissue homogenates or crude membrane fractions. Such preparations usually involve a single Triton X-100 treatment. In the present paper we describe the pattern of variability of PA activity measured in different fractions obtained from the developing chick CNS by a repetitive procedure of Triton X-100 treatment and ultracentrifugation. To further characterize this PA activity we have also performed zymographic analyses during the embryonic development and the early postnatal life. Our results show that: a) a single Triton X-100 treatment does not completely extract the enzyme and this lead to an underestimation of the total PA activity; b) the PA activity is associated with the particulate component of the total tissue homogenate requiring its complete solubilization more drastic Triton X-100 treatments; c) better estimations of total and specific activities are obtained by using soluble fractions derived by ultracentrifugation from Triton X-100-treated membrane fractions; d) the developing chick optic lobe expresses only one kind of PA molecule along the entire development; e) the level of PA activity vary characteristically during the ontogeny and the early postnatal life indicating the existence of a developmentally regulated mechanism of PA expression.