Experimental carcinogenesis with 7, 12-dimethylbenz(a)anthrazene (DMBA) and its inhibition with isothi-ocyanates / Carcinogénesis experimental con 7, 12-dimetilbenzantraceno (DMBA) y su inhibición con isitiocianatos

Introduction: DMBA is a chemical carcinogen that induces carcinomas within a few weeks of its application. We developed an experimental model of carcinogenesis induced by DMBA dissolved in 0,5% paraffin oil (DMBA-PO), verifying the inhibitory effect of the carcinogenicity of phenyl isothiocyanate (PhITC), phenethyl (PhnITC) and benzyl isothiocyanate (BITC). Material and Methods: For this, 88 hamsters were distributed into three groups: one exposed to DMBA-PO (Group 1, n=12), three subgroups (n=12) exposed to PhITC, PhnITC, BITC and DMBA-PO (Group 2, n=36) and four control subgroups (n=10) that were not exposed to the carcinogen in which PO (paraffin oil) and isothiocyanates were applied (Group 3, n=40). Results: The experiment had a duration of 20 weeks, at the end of which the inhibitory effect was established by comparing the lesions developed in the groups that received isothiocyanates with the group that was only treated with DMBA-PO. The carcinogenic effect of DMBA-PO is 100% (35 carcinomas) and the inhibitory effect was 0, whereas in the presence of isothiocyanates the carcinogenic effect decreases, with an inhibitory effect of 86% for BITC (5 carcinomas) and 74% for PhITC (9 carcinomas). Conclusion: The inhibitory effect for PhnITC is 80% in relation to invasive OSCC (1 carcinoma).

Introducción: El DMBA es un carcinógeno químico que induce carcinomas a las pocas semanas de su aplicación. Desarrollamos un modelo experimental de carcinogénesis inducida por DMBA disuelto en aceite de parafina al 0,5% (DMBA-Ap) comprobando el efecto inhibidor de la carcinogénesis de los isotiocianatos fenil (PhITC), fenetil (PhnITC) y bencil isotiocianato (BITC). Material y Métodos: Para ello, se distribuyeron 88 hámsteres en 3 grupos: uno expuesto al DMBA-Ap (Grupo 1, n=12), tres subgrupos (n=12) expuestos a PhITC, PhnITC, BITC y DMBA-Ap (Grupo 2, n=36) y cuatro subgrupos controles (n=10), no expuestos al carcinógeno en el que se aplicaron Ap e isotiocianatos (Grupo 3, n=40). Resultados:El experimento tuvo una duración de 20 semanas, al final de la cual se establece de forma comparativa el efecto inhibidor comparando las lesiones desarrolladas en los grupos que recibieron isotiocianatos con respecto al grupo tratado sólo con DMBA-Ap. El efecto carcinógeno del DMBA-Ap es del 100% (35 carcinomas) y el efecto inhibidor 0, mientras que en presencia de isotiocianatos el efecto carcinógeno disminuye, con un efecto inhibidor del 86% para BITC (5 carcinomas) y del 74% para el PhITC (9 carcinomas). Conclusión:El efecto inhibidor del PhnITC es del 80% en relación con el COCE invasivo (1 carcinoma).

Application of lectins for detection of glycomic differences in the epididymal duct in water buffalo (Bubalus bubalis) and dromedary camel (Camelus dromedarius) / Aplicación de lectinas para la detección de diferencias glucómicas en el conducto epididimario en búfalos de agua (Bubalus bubalis) y dromedarios (Camelus dromedarius)

SUMMARY: The present study was conducted to detect the differences in glycohistochemical features in the epididymal duct of the dromedary camel and the water buffalo. Epididymal sections (caput, corpus and cauda) from both species were stained with fluorescein isothiocyanate (FITC) conjugated lectins. Binding sites for five lectins (DBA, GSA-1, HPA, PNA and WGA) have been found in both species. The binding sites of different lectins showed significant variations in the pattern of distribution in both a species. This included both species-specific and region-specific order. Additionally, only three (GSA-1, PNA and WGA) out the five lectins studied exhibited binding sites in all epididymal regions in both species. The other two lectins (DBA and HPA) followed the same order recorded for the other three (GSA-1, PNA and WGA) in buffalo, but failed to show any binding sites in cauda epididymis in camel. In conclusion, the variable regional and species-specific distribution features of lectins revealed that both species have diverse glycomic characteristics that may be related to their different reproductive patterns. However, the glycome-associated functional capacities remain obscured and need further profound investigations.

RESUMEN: El presente estudio se realizó para detectar las diferencias en las características glicohistoquímicas del conducto epididimal del dromedario y el búfalo de agua. Las secciones del epidídimo (cabeza, cuerpo y cola) de ambas especies se tiñeron con lectinas conjugadas con isotiocianato de fluoresceína (FITC). Se encontraron sitios de unión para cinco lectinas (DBA, GSA-1, HPA, PNA y WGA) en ambas especies. Los sitios de unión de diferentes lectinas mostraron variaciones significativas en el patrón de distribución en ambas especies. Esto incluía tanto el orden específico de la especie como el específico de la región. Además, solo tres (GSA-1, PNA y WGA) de las cinco lectinas estudiadas exhibieron sitios de unión en todas las regiones del epidídimo en ambas especies. Las otras dos lectinas (DBA y HPA) siguieron el mismo orden registrado para las tres restantes (GSA-1, PNA y WGA) en búfalos, pero no mostraron ningún sitio de union en la cola del epidídimo en camellos. En conclusión, las características de distribución regionales y específicas de especies variables de las lectinas revelaron que ambas especies tienen características glucómicas diversas que pueden estar relacionadas con sus diferentes patrones reproductivos. Sin embargo, las capacidades funcionales asociadas con el glicoma permanecen desconocidas y requieren mayor investigación.

Benzyl isothiocyanate inhibits invasion and induces apoptosis via reducing S100A4 expression and increases PUMA expression in oral squamous cell carcinoma cells

Benzyl isothiocyanate (BITC) has been shown to inhibit invasion and induce apoptosis of various types of cancer. However, its role on human oral squamous cell carcinoma (OSCC) cells is still not well elucidated. In the present study, we investigated the effect of BITC on apoptosis and invasion of SCC9 cells, and its underlying mechanisms in vitro and in vivo. SCC9 cells were exposed to BITC (5 and 25 μM) for 24 and 48 h. Cell growth, apoptosis, invasion, and migration were detected in vitro by MTT, FITC-conjugated annexin V/propidium iodide staining followed by flow cytometry, Matrigel-coated semi-permeable modified Boyden, and wound-healing assay. S100A4, PUMA, and MMP-9 expressions were detected to investigate its mechanisms. Xenotransplantation experiments were used to investigate the role of BITC on tumor growth and lung metastasis. BITC inhibited cell viability and induced cell apoptosis in a dose- and time-dependent manner through upregulation of PUMA signals. BITC inhibited cell invasion and migration by downregulation of S100A4 dependent MMP-9 signals. The ip administration of BITC reduced tumor growth but not lung metastasis of SCC9 cells subcutaneously implanted in nude mice. BITC treatment activated pro-apoptotic PUMA and inhibited S100A4-dependent MMP-9 signals, resulting in the inhibition of cell growth and invasion in cultured and xenografted SCC9 cells. Thereby, BITC is a potential therapeutic approach for OSCC.

Analysis of a suppressive subtractive hybridization library of Alternaria alternata resistant to 2-propenyl isothiocyanate

Background Isothiocyanates (ITCs) are natural products obtained from plants of the Brassicas family. They represent an environmentally friendly alternative for the control of phytopathogenic fungi. However, as it has been observed with synthetic fungicides, the possibility of inducing ITC-resistant strains is a major concern. It is, therefore, essential to understanding the molecular mechanisms of fungal resistance to ITCs. We analyzed a subtractive library containing 180 clones of an Alternaria alternata strain resistant to 2-propenyl ITC (2-pITC). After their sequencing, 141 expressed sequence tags (ESTs) were identified using the BlastX algorithm. The sequence assembly was carried out using CAP3 software; the functional annotation and metabolic pathways identification were performed using the Blast2GO program. Results The bioinformatics analysis revealed 124 reads with similarities to proteins involved in transcriptional control, defense and stress pathways, cell wall integrity maintenance, detoxification, organization and cytoskeleton destabilization; exocytosis, transport, DNA damage control, ribosome maintenance, and RNA processing. In addition, transcripts corresponding to enzymes as oxidoreductases, transferases, hydrolases, lyases, and ligases, were detected. Degradation pathways for styrene, aminobenzoate, and toluene were induced, as well as the biosynthesis of phenylpropanoid and several types of N-glycan. Conclusions The fungal response showed that natural compounds could induce tolerance/resistance mechanisms in organisms in the same manner as synthetic chemical products. The response of A. alternata to the toxicity of 2-pITC is a sophisticated phenomenon including the induction of signaling cascades targeting a broad set of cellular processes. Whole-transcriptome approaches are needed to elucidate completely the fungal response to 2-pITC.

Studies on statistical optimization of sulforaphane production from broccoli seed

Background: Natural sulforaphane (SF) has been of increasing interest for nutraceutical and pharmaceutical industries due to its anti-cancer effect. The main objective of the present work was to optimize the production of SF from broccoli seed using response surface methodology. Results: Three major factors (hydrolysis time, water volume and ethyl acetate volume) were screened out through Plackett-Burman (PB) factorial design. The methods of steepest ascent combined with central composite design (CCD) were employed for optimization of the SF production process. The optimal extraction conditions for SF production were a hydrolysis time of 13 min, a hydrolysis volume/weight ratio of 2.9:1 (v/g) and an extraction volume/weight ratio of 17.5:1 (v/g). The maximum SF yield was 14.8 ± 0.1 mg/g, a value that was in perfect agreement with the actual experimental value (14.8 mg/g). Conclusions: These results suggested that PB design combined with CCD were proved effective in screening and optimization of the parameters of SF production.

El aceite canola y sus efectos en la salud / Canola oil and health effects

El término canola (Canadian Oil Low Acid), designa a una variedad de semillas desarrolladas durante la década de los setenta por métodos tradicionales de fitomejoramiento de la colza. La canola se ubica en el segundo lugar como planta oleaginosa cultivada en el mundo, y no sólo se utiliza en la fabricación de aceite para ensaladas y frituras, sino también en la elaboración de margarinas, mantecas y otros productos alimenticios. El presente trabajo tiene como objetivo realizar una revisión de la información científica disponible sobre los antecedentes que motivaron el surgimiento de las semillas variedad canola, así como la seguridad y efectividad del consumo de su aceite sobre algunos de los factores de riesgo de enfermedad cardiovascular (ECV). La búsqueda y localización de la información, incluyó una revisión de artículos científicos, para lo cual se utilizaron los descriptores: Canola, enfermedad cardiovascular, aterosclerosis, y antioxidantes, fundamentalmente. La mayoría de los artículos seleccionados estaban relacionados con el efecto de la composición de ácidos grasos de aceites comestibles en la ECV, así como estudios de análisis de antioxidantes en este aceite. Además, se localizaron artículos de organismos reguladores sobre la seguridad del consumo del aceite de canola(AU)

The term canola (Canadian Oil Low Acid) refers to a variety of seeds developed in the seventies by traditional breeding methods of rapeseed. Canola is located in the second most cultivated oilseed plant in the world, and not only used in the manufacture of salad oil and frying, but also in the development of margarines, shortenings and other food products. This paper aims to conduct a review of the available scientific information on the background that led to the emergence of canola seed variety and the safety and effectiveness of its oil consumption on some of the risk factors for cardiovascular disease (CVD). Finding and locating information, including a review of scientific articles. The descriptors were used: Canola, cardiovascular disease, atherosclerosis, and antioxidants, mainly. Most selected articles were related to the effect of fatty acid composition of edible oils in VCE and analysis studies of antioxidants in this oil. In addition, articles were located regulators about the safety of canola oil consumption(AU)

Increased presevation of sliced mozzarella cheese by antimibrobial sachet incorporated with allyl isothiocyanate

There is an increasing tendency to add natural antimicrobials of plant origin into food. The objective of this work was to develop a microbial sachet incorporated with allyl isothiocyanate (AIT), a volatile compound of plant origin, and to test its efficiency against growth of yeasts and molds, Staphylococcus sp. and psychrotrophic bacteria on sliced mozzarella cheese. Another objective was to quantify the concentration of AIT in the headspace of cheese packaging. A reduction of 3.6 log cycles was observed in yeasts and molds counts in the mozzarella packed with the antimicrobial sachet over 15-day storage time. The sachet also showed an antibacterial effect on Staphylococcus sp., reducing 2.4 log cycles after 12-day storage. Psychrotrophic bacteria species were the most resistant to the antimicrobial action. The highest concentration of AIT (0.08µg.mL-1) inside the active packaging system was observed at the 6-day of storage at 12 ºC ± 2 ºC. At the end of the storage time, AIT concentration decreased to only 10 percent of the initial concentration. Active packaging containing antimicrobial sachet has a potential use for sliced mozzarella, with molds and yeasts being the most sensitive to the antimicrobial effects.

Conjugação e validação de controle isotípico IgG1-FITC para uso em citometria de fluxo / Conjugation and validation of IgG1-FITC isotype control to be used in flow cytometry

Em meados da década de 50 iniciou-se o desenvolvimento da citometria de fluxo, tecnologia que permite verificar características físico-químicas de células ou partículas suspensas em meio fluido. Esta tecnologia utiliza anticorpos monoclonais marcados com fluorocromos como ferramenta de investigação em diversas análises e necessita de controles isotípicos para definição da região negativa (background). Estes controles são constituídos por imunoglobulinas de mesmo isotipo e fluorocromo dos anticorpos testes, sendo o isotiocianato de fluoresceína (FITC) o marcador fluorescente mais utilizado na conjugação de anticorpos. Os controles isotípicos têm como função definir a fluorescência inespecífica (células negativas) e as regiões fluorescentes (células positivas). No presente estudo foi selecionado anticorpo monoclonal murino (AcMm) dirigido contra antígeno eritrocitário canino, produzido no Laboratório de Anticorpos Monoclonais do Hemocentro de Botucatu, o qual reage positivamente com hemácias de cães, mas nunca com leucócitos humanos, tendo, portanto, potencial utilidade como controle negativo em citometria de fluxo. A purificação do AcMm da subclasse IgG1 foi feita por cromatografia de afinidade em Proteína-A Sepharose, e o controle da purificação realizado por eletroforese em géis de ágarose e poliacrilamida (SDS-PAGE). A imunoglobulina purificada foi conjugada ao FITC e filtrado em coluna de Sephadex G-25 para separação das proteínas marcadas e não-marcadas. O AcMm conjugado foi testado contra hemácias de cães, e o êxito da conjugação comprovado por testes de fluorescência, sendo a mediana de positividade de 94,70. Frente a leucócitos humanos a mediana de positividade foi 0,03 contra 0,50 dos reagentes comerciais. Os testes estatísticos não-paramétricos de Wilcoxon e correlação de Spearman comprovaram a eficiência e validam o controle isotípico produzido em comparação aos reagentes comerciais testados.

It was during the 1950's that the development of flow cytometry started, technology that allow to measure physiochemical characteristics of cells or suspended particles in fluid. This technology uses monoclonal antibodies labeled by fluorochromes as investigation tool in several analysis and needs isotype controls to define the negative region (background). These controls are constituted by immunoglobulins of the same isotype and fluorochrome from test antibodies, being fluorescein isothiocyanate (FITC) the most fluorescent marker used in antibody conjugations. The isotype controls have the function to define the unspecific fluorescence (negative cells) and the fluorescent regions (positive cells). In this study was selected monoclonal antibody (mAb) against canine erythrocyte antigen, produced in the Monoclonal Antibodies Laboratory - Blood Center of Botucatu, which reacts positively with dog red blood cells, but never with human leukocytes, having therefore, utility potential as negative control in flow cytometry. The purification mAb of IgG1 subclass was made by affinity chromatography in Sepharose Protein-A and the purification control was performed by electrophoresis in ágarose and polyacrylamide gels (SDS-PAGE). The purified immunoglobulin was conjugated to FITC and after was filtered in Sephadex G25 column to separation of labeled and unlabeled proteins. The conjugated mAb was tested against dog red blood cells and the conjugation success was verified by fluorescence tests, being the median positivity of 94.70. To the human leucocytes the positivity median was 0.03 against 0.50 of the commercial reagents. The nonparametric statistical tests of Wilcoxon and the correlation Spearman showed the efficiency and validate the isotype control produced in relation to the tested commercial reagents.

Cuantificación de sulforafano procedente de glucorafanina en retoños de brassica oleracea var. italica por cromatografía líquida de alta resolución / Quantification of sulforaphane from glucoraphanin in shoots of brassica oleracea var. italica by high performance liquid chromatography

Se determino la presencia de 4.76 mg/g (0.476 % P/P) de Sulforafano de la muestra de retoños de brócoli que se cultivó bajo un sistema controlado (Tabla No. 3), temperatura 18-22 °C, pH 6.5, fotoperíodo de 11-13 horas de luz, humedad relativa intermedia a baja, además el brócoli fue cortado al 5º. día de retoño (Anexo No. 1, Fotografías No. 1-13), para la obtención de óptimos resultados. Bajo este proceso se obtuvieron 16 retoños de brócoli (ver anexo No. 1, fotografía No. 12), con lo que se procedió a la fase de extracción. En el proceso de extracción se realizó una hidrólisis con una solución de ácido clorhídrico a pH 3 con lo que la glucorafanina se transforma a Sulforafano. Posteriormente se realizaron extracciones líquido-líquido usando como solvente diclorometano quedando un gel de color café (Anexo No. 1, Fotografías No. 14-17). Se separó el Sulforafano presente de los otros compuestos contaminantes usando cromatografía por exclusión de tamaño (Sephadex) y una placa cromatográfica de sílica gel preparativa, identificando y cuantificando de esta manera el Sulforafano presente en el extracto por Cromatografía líquida de Alta Resolución con detector de luz ultravioleta - visible (UV-Vis).

Alimentos funcionais: [revisão] / Functional foods: [review]

Faz-se uma revisão sumária sobre alimentos funcionais com destaque em seus grupos químicos, sua ação fisiológica e suas fontes na dieta.

A brief survey of functional foods is presented with special emphasis on chemical groups, physiological actions, and dietary sources.

Comparison of four extraction methods to detect hepatitis A virus RNA in serum and stool samples

The efficiency of extraction methods for hepatitis A virus (HAV) RNA in clinical samples is of great importance for molecular diagnosis, especially in regions endemic for HAV, such as Brazil. We compared the efficiency of four different extraction techniques in serum and stool samples for the detection of hepatitis A virus by reverse transcription PCR (RT-PCR). We used PCR to analyse serum and stool samples of 12 patients who were referred to the Brazilian Reference Center for Viral Hepatitis (BRCVH) in Rio de Janeiro. The methods tested were Proteinase K, Silica, TRIzol and Guanidine isothiocyanate. Proteinase K extraction was the best method for serum samples; it detected the HAV-RNA in 11 of the 12 samples. The guanidine isothiocyanate method was the most effective for stool samples, detecting HAV-RNAs in 9 of the samples. The TRIzol« method worked best with serum samples, and the silica method was unsatisfactory with both serum and stool samples. The RNA extraction method affected the outcome. The use of appropriate RNA extraction methods is a critical step for successful and valid PCR studies on clinical samples. We recommend that RNA extraction techniques be carefully selected for their efficiency with each type of specimen

Separación de aminoácidos mediante cromatografía líquida de alta resolución / Amino acid analysis by high-performance liquid chromatography

Se presenta una compilación de la aplicación de la cromatografía líquida de alta resolución en el análisis de aminoácidos. Se analizan las formas de derivatización, se comparan los distintos derivados según sus alcances y limitaciones, la forma de detección y los distintos modos cromatográficos: cromatografía de intercambio iónico, cromatografía en fase normal, cromatografía en fase reversa y cromatografía en fase quiral. También se presentan los métodos automatizados comerciales más recientes para este tipo de análisis. Se dan ejemplos de aplicación de interés en el campo clínico-bioquímico, con el análisis de muestras de líquido cafalorraquídeo, de orina y de plasma/sangre o manchas de sangre seca. El análisis de los aminoácidos de hidrolizados de proteínas y de péptidos resulta más complejo y las condiciones de hidrólisis juegan un rol muy importante en los resultados, que son tratados en esta compilación. Se incluyen, además, aspectos referidos al análisis de los aminoácidos lábiles en proteínas, la preparación de las muestras, las recomendaciones prácticas al hacer HPLC y la influencia de los buffers