MLO-Y4 osteocytic cell clones express distinct gene expression patterns characteristic of different stages of osteocyte differentiation / Clones de las células osteocíticas MLO-Y4 tienen diferentes patrones de expresión génica característicos de diferentes estadíos de diferenciación osteocítica

Osteocytes are the most abundant bone cell and are formed when osteoblasts become embedded in the bone matrix. Through changes in gene expression and paracrine effects, osteocytes regulate the number of osteoblasts, bone forming cells, and osteoclasts, bone resorbing cells, which are needed to maintain bone mass. MLO-Y4 is the better characterized osteocytic cell line; however, lacks expression of sclerostin, the product of the SOST gene, which is fundamental for osteocyte function and blocks bone formation. With the objective to isolate MLO-Y4 clones with different gene expression profiles, we performed cultures at very low density of MLO-Y4 cells stably transfected with nuclear green fluorescent protein (MLOnGFP). Cell morphology was visualized under a fluorescence microscope. Once the cells reached 80% confluency, RNA was extracted and quantitative real time PCR was performed. Clones exhibit different sizes and morphology, with some cells showing a spindle-like shape and others with abundant projections and a star-like shape. Gene expression also differed among clones. However, none of the clones examined expressed SOST. We conclude that the MLO-nGFP clones constitute a useful tool to study osteocyte differentiation and the role of osteocytes in the control of bone formation and resorption in vitro. (AU)

Los osteocitos son las células más abundantes del hueso y se forman cuando los osteoblastos se encuentran rodeados de matriz ósea. A través de cambios en la expresión génica y efectos paracrinos, los osteocitos controlan el número de osteoblastos que forman el hueso, y osteoclastos que resorben el hueso, células necesarias para mantener la masa ósea. Las células MLO-Y4 son la línea celular osteocítica más investigada; sin embargo, no expresan esclerostina, el pro esclerostina, el producto del gen SOST que bloquea la formación ósea y es indispensable para la función de los osteocitos. Con el objetivo de aislar clones de las células MLO-Y4 con diferentes perfiles de expresión génica, realizamos cultivos a muy baja densidad de las células transfectadas en forma estable con proteína verde fluorescente nuclear (MLO-nGFP). La morfología celular fue evaluada utilizando un microscopio de fluorescencia. Una vez que las células alcanzaron el 80% de confluencia, el ARN fue extraído y analizado por PCR cuantitativa en tiempo real. Las células de los diferentes clones tienen diferentes tamaños y morfología, algunas células son fusiformes y otras con proyecciones citoplasmáticas abundantes y en forma de estrella. La expresión de los genes también varió en los distintos clones. Sin embargo, ninguno de ellos expresó SOST. En conclusión, los clones de las células MLO-nGFP constituyen una herramienta útil para estudiar la diferenciación de los osteocitos y el rol de estas células en el control de la formación y resorción ósea in vitro. (AU)

A phage display combined with DNA affinity magnetic system can be applied to a screening of DNA binding proteins, such as transcription factors / Un fago pantalla combinado con un sistema de afinidad magnético al ADN se puede aplicar al exámen de las proteínas de unión al ADN, tales como los factores de transcripción

Here we introduce a new approach for the screening of DNA binding proteins, using a phage library based on a phage display technique. In principal, a complementary DNA (cDNA) library based on the recombinant bacteriophage T7 expressing target proteins on its capsid (phage display) is constructed. These phage particles are hybridized with a biotinylated target DNA fragment which is immobilized on the surface of streptavidin paramagnetic particle (SA-PMP). The phage particles are released from the target DNA fragment by a nuclease treatment and the recovered phages are used to the next round of hybridization. These processes are repeated three times to amplify the target phages in the population. This simple method is faster, and more systemic than other current methods (e.g. yeast one hybrid system). As a proof of this principle, we tried to isolate transcription factors which specifically bind to the promoter region of the Arabidopsis thaliana AtGST11 gene. Two obtained candidates, RING zinc finger protein and AtHB6, showed DNA binding activity to the AtGST11 promoter region. We could validate that our new application of phage display is a superior method for isolation of DNA binding proteins with a broad range of potential applications.

Genotipificación de cepas de enterobacterias procedentes de 4 centros de salud del área de Caracas / Genotyping of enterobacterias strains from 4 healthcare centers in Caracas

Las infecciones nosocomiales pueden ser producidas por microorganismos resistentes a la acción de los antimicrobianos que han sido seleccionados por la mal uso ó el uso indiscriminado de los antibióticos en el ámbito hospitalario. En los últimos años se han desarrollado nuevas técnicas moleculares de tipificación basada en la reacción en cadena de la polimerasa (PCR), que han representado un avance importante en el estudio de las enfermedades infecciosas, siendo muy útiles al permitir diferenciar serotipos estrechamente relacionados y grupos de cepas no relacionadas clonalmente, debido a su gran poder discriminatorio. En Venezuela, son pocos los estudios de eepidemiología molecular de las infecciones intrahospitalarias, y por esta razón nos propusimos genotipificar cepas de Escherichia coli y Klebsiella pneumoniae provenientes de aislados nosocomiales de cuatro centros de salud del área metropolitana (Hospital "Dr. José María Vargas", Hospital "Dr. Domingo Luciani", Centro Médico de Caracas y Policlínica Metropolitana) con la finalidad de determinar la relación clonal existente entre estas cepas. El uso de ERIC-PCR permitió relacionar parcialmente las especies de E. coli aisladas en los cuatro centros de salud, sin embargo la técnica de REP-PCR permitió discriminar entre los patrones de bandas similares, mostrando un poder de resolución mayor. El uso de ERIC-PCR y REP-PCR no permitió tipificar la mayoría de las cepas de K. pneumoniae aisladas en los cuatro centros de salud en estudio. En el Centro Médico de Caracas se identificaron dos aislados clonales provenientes de diferentes áreas del hospital, Unidad de Terapia de Adultos y Hospitalización. En la Policlínica Metropolitana se identificaron tres aislados clonales, dos en la unidad de Terapia y uno en Hospitalización. En el Hospital "Dr. Domingo Luciani" y en el Hospital "Dr. José María Vargas" no se identificaron clones. Estos resultados proporcionan un aporte a los programas de vigilancia...

Nosocomial infections can be produced by microorganisms resistand to antimicrobial agents, and they have been selected by the bad use or abuse of antibiotics in the hospital environment. Recently, new molecular typing techniques have been developed, based on polymerase chain reaction (PCR). These techniques represent an important advantage the study of infectious diseases; they are able to discriminate relate closed serovars and groups of nonrelated isolates due to its great power discriminatory. In Venezuela, there is a small number of molecular epidemiology researches. The goal of the present study is genotyping Escherichia coli and Klebsiella pneumoniae isolated of nosocomial infected patients from four healthcare centers in the metropolitan area (Hospital "Dr. José María Vargas", Hospital "Dr. Domingo Luciani", Centro Médico de Caracas, Policlínica Metropolitana) in order to investigate the clonal relationship between the isolates. ERIC-PCR allowed us to correlate E. coli isolates however REP-PCR shows greater greater resolution. The use of both ERIC-PCR and REP-PCR, did not permit us typing K. pneumoniae isolates. Two clonally related isolates from Centro Médico de Caracas were identified. Three clonally related isolates from the Policlínica Metropolitana ere identified. No clones were identified in samples from Hospital "Dr. Domingo Luciani" and the Hospital "José María Vargas". These results contribute to monitoring programs, to improve the control of the bacterial infections, helping to establish efficient procedures and reduce nosocomials infections.

Morphological variation of dinoflagellate Prorocentrum lima (Ehrenberg) Dodge with geographical difference.

Prorocentrum lima (Ehrenberg) Dodge, one of the cosmopolitan dinoflagellates, is basically benthic and is found on the surface of macroalgae and detritus. The identification of this species requires detailed morphological observation because of its close similarity to other benthic Prorocentrum species. The purpose of this study is to detect the morphological variability of P. lima using culture clones collected from several areas to find an adequate way of subdividing the species. In this study, 33 clones of P. lima were collected from Saipan, Tahiti, Indonesia, Japan and Bermuda, and their thecal valves and periflagellar area were observed by means of light microscopy with differential interference contrast optics and scanning electron microscopy. In general cells have two centrally located pyrenoids and a posterior nucleus. The surface of both valves has many valve pores except the center. Evenly spaced marginal pores are located along the edge of the valves. P. lima samples studied herein were subdivided into four major types (ellipsoidal, general, short, and elongate ovoid) according to their shapes, length-to-width ratio and number of valve pores. The length-to-width ratios of ellipsoidal, general, short, and elongate ovoid types were 1.32, 1.33-1.43, 1.20-1.27, and 1.53-1.60 microm respectively. Also there were slight differences in the number of valve pores. The number of valve pores examined in this study ranges from 40 to 97: ellipsoidal, general, and short ovoid types range from 40 to 91, while an elongate ovoid type ranges from 80 to 97. The combination of valve shape, number of valve pores and length-to-width ratio provides useful information on the morphological variation of P. lima.

Estudo da regulação da expressão do gene da proteína prion celular / PrPc protein gene expression regulation study

A conversão da proteína prion celular normal(PrPc), cuja função ainda está sob investigação, para a forma infecciosa (PrPsc) é a causa de algumas doenças neurodegenerativas em humanos e animais. Vários estudos têm sido realizados e mostram que PrPc pode participar de processos normais como memória, estresse oxidativo, neuritogênese e outros. Portanto, a elucidação dos processos de regulação de sua expressão é importante tanto para definir um estratégia para controlar a infeccção quanto para entender melhor a função fisiológica de PrPc. Este trabalho tem objetivo avaliar a expressão de gene de PrPc, a partir da regulação da atividade de seu promotor frente a drogas que foram eleitas de acordo com a composição dos elementos...

Morphological and morphometric variability of three clones of Trypanosoma cruzi and the strain of origen (Bolivia) / Morphological and morphometric variability of three clones of Trypanosoma cruzi and the strain of origin (Bolivia)

Knowing the great divesity of medical and biological properties of Trypanosoma cruzi, the causal agent of Chagas'disease, we quantified the morphological parameters that typify the different forms of three clones of T. cruzi and their original strain, Bolivia, in comparison among themselves and with strain Bolivia, attempting to provide additional data concerning the clonal biological behaviour of this parasite. Blood forms morphology was quantified using a computarized image analysis (Videoplan/Kontron) and statistical analysis was determined using ANOVA-1 Test. Large number of quantitative differences among slender, broad, and stout forms were found. The comparison of clones I, II and III with their mother strain, leads to the emergence of significant differences in at least 12 parameters out of the 16 we studied. When clones were compared among themselves, the differences decrased. Variations of the percentagens of the three kinds of clones were found along the acute infection. These data are the first step in correlating the morphological and pathogenic characteristics of the parasite.

Establecimiento y caracterización de la línea celular CaLo y del CLON Kalo, obtenidos a partir de un carcinoma de cervix, y efecto de IL-2, IL-3, GM-CSF, M-CSF, TNF-Ó E IFN-ç sobre su proliferación / Establishing and characterization of the CaLo cell line and KaLo clon, ontained from a cervix carcinoma and IL-2, IL-3, GM-CSF, M-CSF, TNF-Ó and IFN-ç on it's proliferation

En este trabajo se describe la caracterización morfológica, cromosómica y presencia de desmosomas de una línea celular (CaLo) derivada de una biopsia de cáncer cervicouterino. Nuestros resultados indican que CaLo posee una morfología típica de células epiteliales, una aneuploidia cromosómica con número modal de 58 y presencia de desmosomas de bida a la positividad en membrana para la presencia de desmogleína-1. A partir de CaLo también se aisló un clon llamado KaLo. esta clonación nos proporciona una evidencia del origen maligno de estas células. Ambos tipos celulares fueron cultivados en presencia de algunas citocinas recientemente empleadas en ciertos protocolos clínicos (TNF-Ó, IL-2. IL-3 GM-CSF, M-CSF e IFN-ç). Nuestros resultados demuestran un efecto inhibidor de la proliferación por parte de TNF-Ó, IL-3 y GM-CSF, mientras que con M-CSF e IFN-ç no se detectó efecto. Por otra parte, obresvamos un incremento en la proliferación celular en presencia de la IL-2. Estos resultados dan indicios de que el TNF-Ó, la IL-3 y el M-CSF pueden tener posibilidades de aplicación clínica por sus propiedades inhibidoras; mientras que ponen en evidencia el riesgo de utilizar in vivo la IL-2, pues activa la proliferación de células tumorales de cérvix, tal como se ha informado para algunos carcinomas de cabeza y cuello

Plant tissue culture techniques

Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus) or organized tissues or organs put in culture, under controlled sterile conditions.