Establishment and characterisation of a new cell line derived from Culex quinquefasciatus (Diptera: Culicidae)

Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28ºC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6) was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep / Protocolos para obtenção e isolamento de células tronco mesenquimais de duas fontes em ovinos

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

OBJETIVO: Testar diferentes protocolos para o isolamento de células tronco a partir de sangue de cordão umbilical e tecido adiposo de ovinos. MÉTODOS: Foram utilizadas cinco amostras de sangue de cordão umbilical e cinco amostras de tecido adiposo perirrenal de 10 fêmeas de ovelha. A coleta das amostras foi realizada através de procedimento cirúrgico para coleta do material de forma mais asséptica possível. Foram realizados três protocolos de isolamento e cultivo das células-tronco do cordão umbilical e quatro protocolos para o isolamento e cultivo das células-tronco de gordura de ovinos RESULTADOS: Somente um dos protocolos utilizados para o isolamento das células-tronco de cordão umbilical foi efetivo. Dos quatro protocolos utilizados para isolamento das células-tronco de gordura, da mesma forma, apenas um obteve sucesso. Foi realizado o ensaio de unidades formadoras de colônias destas células, sendo contadas 58 colônias ao final de sete dias. Na citometria de fluxo essas células mostraram-se positivas para CD44 e negativas para CD38, CD45, CD41/61. Estas células apresentaram curva de crescimento com fases de LOG, LAG e PLATEAU bem definidas, características das curvas de crescimento das células-tronco de origem mesenquimal. CONCLUSÕES: O isolamento e cultivo das células-tronco mesenquimais do cordão umbilical de ovinos é de difícil realização, exigindo maiores ensaios e estudos profundos. Células tronco do tecido adiposo de ovelhas demonstraram características mesenquimais, de acordo com a curva de crescimento, habilidade de formação de colônias, células com morfologia fibroblastóide e reação positiva ao anticorpo CD44.

Development and characterization of two new cell lines from common carp, Cyprinus carpio (Linn)

Two new cell lines (CCF and CCH) were established from fin and heart tissues of common carp, Cyprinus carpio. The cells were optimally maintained in Leibovitz-15 medium supplemented with 10 percent fetal bovine serum (FBS) and 10 ng/ml of basic fibroblastic growth factor (bFGF). The effects of temperature, concentration of FBS and bFGF on the growth of CCF and CCH cells were examined. The temperature ranged from 24 to 32 °C for good growth of the cells. The growth rate of cells was higher in medium containing 10 percent FBS and the addition of bFGF to the medium significantly increased the growth rate. The CCF cells were found to be epithelial, while the CCH cells were fibroblastic in nature. The cytogenetic analysis of the cell lines revealed a diploid number of 100 chromosomes in C. carpio. The viability of CCF and CCH cell lines were 70 and 72 percent, respectively, after six months of storage in liquid nitrogen (-196 ° C). Molecular characterization of the cell lines using 16S rRNA and Cytochrome Oxidase Subunit I (COI) revealed the origin of the cell lines. These new cell lines will be useful for isolation of fish viruses and other in vitro biotechnological studies.

A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20 percent fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10 percent fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma

Caracterización morfológica de una linea celular ß de páncreas (P1) en cultivo continuo mediante técnicas de microscopía óptica, de fluorescencia y electrónica / Morphological characterization of a ß -pancreas (P1) cell line in continuous culture by light, fluorescent and electron microscopy techniques

La línea celular P1 fue obtenida por cultivo continuo a partir de explantes de páncreas endocrino de ratas macho Fisher 344, inoculadas con células MIT productoras de tumores localizados. Mediante radioinmunoensayo (RIA), se ha comprobado que presenta respuesta a glucosa y secreta insulina. Se realiza caracterización morfológica para complementar los estudios bioquímicos y fisiológicos existentes, mediante técnicas de microscopía, de fluorescencia y electrónica. Las preparaciones se trataron con aldehído-tionina para microscopía óptica; ácido glioxílico, glutaraldehído y paraformaldehído para microscopía de fluorescencia; osmio, uranilo y citrato de plomo para microscopía electrónica. Microscopio de luz de fluorescencia Nikon, y electrónico de transmisión Zeiss EM-109, fueron utilizados para las observaciones. Los resultados obtenidos muestran gránulos intracitoplasmáticos en color azul a microscopía óptica, amarillo-verdoso a microscopía de fluorescencia y que ultraestructuralmente corresponden a vesículas con centro electrodenso. Los métodos utilizados permiten la tipificación de la línea celular P1 y concluir, consecuentemente, que las células ß mantienen sus propiedades endocrinas en condiciones definidas de cultivo

Citotoxicidad inducida por el tabaco / Cytotoxic and proliferative effect of tobacco

El condensado de humo (CH) a una concentración de 0.40 mg/ml y el uretano a dosis de 5.0 mg/ml disminuyeron la adherencia y la proliferación celulares a porcentajes menores del 30 y 15 por ciento respectivamente, en comparación con las células controles que tuvieron 100 por ciento de adherencia y proliferación. El extracto de tabaco (ET) a una concentración de 16 mg/ml provocó 65 por ciento de inhibición de la proliferación; sin embargo, con 0.79 mg/ml, o dosis menores, los resultados fueron similares a los cultivos celulares testigos y con 0.1 mg/ml se incrementó la proliferación hasta 65 por ciento por arriba de las células testigo. El CH (0.5 mg/ml) dió valores semejantes en la adherencia y proliferación celulares con respecto a los controles. Las células MDCK tratadas con etilen-metanosulfonato y que después fueron expandidas en condiciones habituales, se incubaron con diferentes concentraciones del CH, ET y el uretano, observándose un comportamiento similar al de las celulas MDCK controles. Los cultivos celulares que se incubaron tres días posteriores a su enfrentamiento con el CH (0.05 mg/ml), o con el uretano (2 mg/ml), tuvieron el 50 por ciento de inhibición de la proliferación al compararlos con los cultivos controles. Los datos obtenidos en este trabajo indican que las dosis altas de los productos del tabaco son citotóxicas, reflejándose este hecho en la disminución de la adherencia y proliferación de las células y que las dosis bajas de los PDCT estimulan estos fenómenos celulare

Regulation of gamma T-cell antigen receptor expression by intracellular calcium in acute lymphoblastic leukemia cell line DND41

The calcium inophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4-CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibitis the increased intracellular calcium promoted by ionomycin but not the increase promoted by antiCD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4-CD8- cells, and this is due to cytosolic calcium mobilization

Effect of growth factors on the expression of proto-oncogenes c-fos and c-myc in FRTL-5 cell line

This study was performed to prove the hypothesis that oncogene expressions would have the same patterns with those of cellular growth to growth factors in FRTL-5 cells. Ribonucleic acids of FRTL-5 were extracted at 15', 30', 60' and 120' after administration of growth factors to quiescent FRTL-5, and blotted to the nitrocellulose membrane. They were hybridized with radiolabelled c-fos, c-myc and beta-actin probes. Hybridized dot blots were autoradiographed and the amount of radioactivity was measured by densitometry. Densitometric readings were used as the indices of oncogene expressions. Expressions of c-fos and c-myc were more prominent in combined administrations of TSH (10 mU/ml) and IGF-I (100 ng/ml) or IgG of Graves' disease (Graves' IgG; 1 mg/ml) and IGF-I than in combined administration of TSH and Graves' IgG. IgG of primary myxedema suppressed oncogene expressions by TSH or Graves' IgG, but not by IGF-I. From the above results, it was suggested that expressions of c-fos and c-myc to growth factors would have similar patterns with those of cell growth to growth factors in FRTL-5, and the actions of TSH and Graves' IgG would be manifested through same signal transduction system, but IGF-I would be manifested by its own.

Características de uma nova linhagem de levedura "Killer" / Study of some characteristics of newly isolated killer yeas

Foi isolada uma nova linhagem de Saccharomyces cerevisuiae 337, de usina de cana de açucar, que apresentou ativudade "Killer" semelhante ao fenótipo K.A eletroforese em gel de agarose mostrou que a linhagem S.cerevisiae 337 possui dois ds-RNAs. A atividade "killer" da linhagem foi comparada com a levedura padräo"Killer" K

Efecto de varios factores durante la criopreservación sobre la morfología y la viabilidad de algunas líneas celulares poikilotérmicas / Effect of several factors during cryopreservation on the morphology and viability of some poikilothermic cell lines

Se estudió el efecto de varios factores durante la criopreservación sobre 3 líneas celulares poikilotérmicas utilizando como indicadores la morfología y la viabilidad. Se comprobó que evitar la toxicidad en el medio de suspensión es de vital importancia en el éxito de la congelación. Además, uno de los sistemas estudiados y de gran aplicación en Virología no soporta largas hibernaciones. Finalmente se discute el método más adecuado de congelación para este tipo de células