Cytogenetic bioassays as tools to distinguish between toxic and non-toxic varieties of Jatropha curcas (Euphorbiaceae)

Abstract The tropical and subtropical naturalized physic nut (Jatropha curcas L.), has been explored for biodiesel production in recent times. The oil is extracted from the seeds and, for the production to be feasible, utilization of the residual seed cake is crucial. Although the cake could be employed as a protein source in animal feed, it is rich in phorbol ester, which is toxic for animals. Therefore, breeding programs have been working to reduce or eliminate the phorbol ester content in physic nut. In this context, the present work aimed to evaluate the physic nut oil of toxic and non-toxic varieties (containing known or undetectable amounts of phorbol ester, respectively) with regards to phytotoxicity in a model experiment with Lactuca sativa L. For this, the percentage of germinated seeds was evaluated after 8, 16, 24, 36 and 48 hours of exposure to the treatments with toxic and non-toxic oil at concentrations of 22.5 %, 45 % and 67.5 % of emulsion (physic nut oil energetically mixed with distilled water). Root growth was determined after 48 hours of exposure and the germination speed index was obtained. The different stages of mitotic division as well as possible chromosomal and nuclear alterations were also recorded. The mitotic index was calculated as the number of dividing cells, as a fraction of the total number of cells, and the frequency of chromosome and nuclear alterations, expressed as the percentage of number of alterations divided by the total number of cells. Both varieties exhibited phytotoxicity, inducing significant reductions in percentage of germinated seeds (reduction of 98 %), germination speed index (reduction of 24.44) and root growth (reduction of 8.54 mm). In microscopic analysis, a mitodepressive effect was observed for both oils at the three concentrations used when compared to the negative control; however, it was possible to distinguish between the toxic and the non-toxic varieties based on the more expressive reduction of division promoted by the first, 2.19 %. Significant increments in the frequency of mitotic cells showing chromosome alterations as well, as the presence of condensed nuclei, were observed in the treated cells. However, these parameters were not significantly different from the control in the cells treated with both physic nut oils. In conclusion, the evaluation of root growth and cell division in the plant model L. sativa, can be proposed as an alternative to animal tests to distinguish the varieties with high and low phorbol ester concentration, thus contributing to the detection of toxicity in varieties used in breeding programs.

Resumen Jatropha curcas L., naturalizado tropical y subtropical, ha sido explorado para la producción de biodiesel. El aceite se extrae de las semillas y, para que la producción sea factible, la utilización de la torta de semillas residual es crucial. Aunque la torta se puede emplear como una fuente de proteína en la alimentación animal, es rica en éster de forbol, que es tóxico para los animales. Por lo tanto, los programas de mejoramiento han procurado reducir o eliminar el contenido de éster de forbol de J. curcas. En este contexto, el presente trabajo tuvo como objetivo evaluar el aceite de J. curcas de las variedades tóxicas y no tóxicas (con cantidades conocidas o indetectables de éster de forbol, respectivamente) con respecto a la fitotoxicidad en el modelo Lactuca sativa L. El porcentaje de semillas germinadas se evaluó después de 8, 16, 24, 36 y 48 horas de tratamiento. El crecimiento de la raíz se determinó después de 48 horas de exposición y se obtuvo el índice de velocidad de germinación. Se registraron las diferentes etapas de la división mitótica así como posibles alteraciones cromosómicas y nucleares. El índice mitótico se calculó como el número de células en división como una fracción del número total de células y la frecuencia de las alteraciones cromosómicas y nucleares, expresada como el porcentaje del número de alteraciones dividido entre el número total de células. Ambas variedades exhibieron fitotoxicidad, induciendo reducciones significativas en el porcentaje de semillas germinadas (Reducción del 98 %), índice de velocidad de germinación (Reducción de 24.44) y crecimiento de raíces (Reducción de 8.54 mm). En el análisis microscópico, se observó un efecto mitodepresivo para ambos aceites. Sin embargo, fue posible distinguir entre las variedades tóxicas y las no tóxicas basándose en la reducción más expresiva de la división promovida por la primera, 2.19 %. Se observaron incrementos significativos en la frecuencia de células mitóticas que mostraban alteraciones cromosómicas, así como la presencia de núcleos condensados en las células tratadas. Sin embargo, estos parámetros no fueron significativamente diferentes del control en las células tratadas con ambos aceites de J. curcas. En conclusión, la evaluación del crecimiento de las raíces y la división celular en el modelo L. sativa se puede proponer como una alternativa a las pruebas en animales para distinguir las variedades con concentraciones altas y bajas de éster de forbol, contribuyendo así a la detección de toxicidad en variedades utilizadas en programas de mejoramiento genético.

Euphorbia fischeriana extract reactivates latent HIV through nuclear factor-κB pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of Euphorbia fischeriana extract on latent HIV reactivation and the pathway involved in this process and discuss the value of Euphorbia fischeriana extract in eliminating HIV.</p><p><b>METHODS</b>Fresh tissues of Euphorbia fischeriana root were crushed into powder after quick freezing with liquid nitrogen and extracted with acetone followed by a three-day vacuum freeze-drying for dehydration of the extract. The extract (EFE) was separated using RP-C18 column with high-performance liquid chromatography (HPLC) and identified with mass spectrometry (MS). The activity of reactivated latent HIV was analyzed by fluorescence-activated cell sorting in a J-Lat 10.6 cell model treated with EFE (50 µg/mL) for 24 h, using TNF-α (10 ng/mL) as the positive control. The effect of a NF-κB pathway inhibitor (Bay 11-7082) on EFE activity was tested. The changes in P65 expression in the cell nuclei within 2 h and HIV protein p24 expression within 24 h were analyzed by Western blotting in cells treated with EFE.</p><p><b>RESULTS</b>EFE was obtained by one-step acetone extraction, and the concentration of prostratin in the extract was around 0.53 mmol/L. About 50% of the cells showed HIV reactivation after treatment with 50 µg/mL EFE for 24 h accompanied by a significantly increased p24 expression. The activity of EFE in reactivating latent HIV was inhibited by Bay 11-7082 in a concentration-dependent manner, and p65 accumulation was detected in the cell nuclei within 2 h.</p><p><b>CONCLUSION</b>EFE we obtained contains the active compounds of prostratin and its analogues and shows a strong capacity to reactivate latent HIV through classical NF-κB pathway.</p>

Effects of Euphorbia milii latex on mitogen-induced lymphocyte proliferation / Efeito do latex de Euphorbia milii sobre a proliferação de linfócitos induzida por mitogénio

The crude latex of "Crown-of-Thorns" (Euphorbia milii var hislopii, syn E.splendens) is a potent plant molluscicide. For this reason, toxicological studies have been performed to evaluate the health risks posed by its use in schistosomiasis control programs. The present study is part of a more comprehensive immunotoxicological evaluation of this molluscicide. Here, we investigated the effects of E. milii latex on the proliferation of human lymphocytes in vitro. Lyophilized latex of E. milii (0, 0.5, 5, 25 and 50 µg/ml) was incubated with whole blood in the presence of proliferation stimulators, i.e. lectins (phytohemagglutinin, concanavalin A and pokeweed mitogen), as well as with human monoclonal antibody against CD3 and tetanus toxoid. Cell proliferation was measured by ³H-thymidine incorporation, and the effects of latex on mitogen-induced cell proliferation were compared to the effects of 10 ng/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA). Results showed that mitogen-induced cell proliferation was markedly enhanced by E. milii latex. This synergistic effect of latex on mitogen-induced lymphocyte proliferation may be due to the presence of TPA-like phorbol esters and/or to mitogenic plant lectins.

O látexbrutoda "Coroa de Cristo" (Euphorbia miliivarhislopii, syn E.splendens) é um potente moluscicidavegetal. Neste sentido, são necessários estudos toxicológicosque visemavaliar possíveis riscos à saúdeassociados ao uso em larga escala desta espécie em áreas endêmicas para esquistossomose. O presente estudo é parte deuma avaliação mais abrangentesobre o potencial tóxico destemoluscicida. Foram investigados in vitro osefeitos dolátex da E.miliisobre a proliferação delinfócitoshumanos. O látexliofilizado (0; 0,5;5;25 e 50 µg/ml)foi incubado comsangue totalna presençade agentes mitogênicos, tais como lectinas(fitohemaglutinina, concanavalina Ae pokeweed), anticorpomonoclonalhumano anti-CD3etoxóide tetânico. A proliferação celularfoi quantificada atravésincorporaçãode ³H-timidina eos efeitos do látexnaproliferação celular induzida por agentes mitogênicosforam comparados comos efeitos de10 ng/mlde12-O-tetradecanoilforbol-13-acetato (TPA). Os resultados demonstram quea proliferação celular induzida poragentes mitogênicosfoimarcadamenteaumentada na presença do látex daE.milii.Oefeito sinérgico observado pode ser devidoà presença deésteres de forbol, como o TPA, e/oude lectinas com ação mitogênica presentes nesta espécie vegetal.

The inhibitory effect of As₂O₃ combined with phorbol ester on the proliferation of Kasumi-1 cells and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of arsenic trioxide (As₂O₃) combined with tetradecanoylphorbol acetate (TPA) on the proliferation of Kasumi-1 cell line and its mechanism.</p><p><b>METHODS</b>Kasumi-1 cells were treated with 200 nmol/L TPA, different concentrations of As₂O₃ alone and combined with 200 nmol/L TPA. The proliferative inhibition rates were determined with CCK-8. Annexin V was adopted to detect apoptosis. Colony formation assay was used to determine the cloning efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38.</p><p><b>RESULTS</b>The proliferation inhibition rates of Kasumi-1 cells by TPA combined with different concentrations of As₂O₃ (0.2, 2.0 and 20.0 mmol/L)for 48 h were (25.56 ± 7.29)%, (60.63 ± 6.64)%, and (73.37 ± 2.15)%, the apoptosis rates were (61.65 ± 2.62)%, (75.39 ± 1.04)%, and (89.95 ± 1.46)%, and the colony formation rates were (76.17 ± 2.06)%, (38.50 ± 1.87)%, and (18.53 ± 2.20)%, respectively, compared with the different concentrations of As₂O₃ alone groups, the difference was statistically significant (P<0.05). Cells treated with both TPA and As₂O₃ expressed more CD11b antigens compared with the cells exposed to As₂O₃ alone. TPA treated Kasumi-1 cells were arrested at G1 phase compared with the control group, while As₂O₃ increased the percentage of Kasumi-1 cells in the G2 phase. Combination treatment increased the expression of p-P38 of Kasumi-1 cells compared with the cells exposed to As₂O₃ alone.</p><p><b>CONCLUSION</b>TPA can enhance the effect of As₂O₃ on inducing apoptosis and regulating cell cycle, thereby enhancing its anti-leukemia effect.</p>

[Primary culture of human skin melanocyte and comparison of culture in the presence and absence of phorbol ester]

Primary culture takes place following the cell isolation from tissues. Isolation and culture of melanocytes based on their roll in the protection of body against hazardous sun rays, production of skin, cornea and hair color is really important. This study was done to set isolation, culture and proliferation of melanocytes from children foreskin and adult eyelashes, and also comparison of two types of melanocyte culture medium. Human foreskin and eyelash samples were used for melanocyte isolation and culture. After isolation of epidermis from dermis, epidermis cell suspensions were prepared by enzymatic digestion. The isolated cells were cultured in two melanocyte selective culture media. Immunocytochemistary and reverse transcription-polymerase chain reaction [RT-PCR] assays were used for confirmation of isolated and cultured melanocytes. Our results indicated that isolated melanocyte cultured in the selective medium without phorbol esters is better than the melanocytes cultured in selective medium containing phorbol esters not only morphologically but also physiologically and from the aspect of cell adhesion. In addition, the results showed that isolated melanocyte from adult eyelashes are more dendritic than melanocytes isolated from children foreskin. Conversely, our results indicated that the number of cell passages in melanocyte isolated from foreskin is more than melanocytes isolated from adult eyelashes. Melanocytes cultured in selective medium containing convenient growth factors in absence of phorbol esters show more native physiological and adhesive properties. In addition, melanocyte isolated from younger tissues such as foreskin have better proliferative and sub-culturing properties so we suggest isolation and culture of younger tissues.

Vulnerabilidades específicas de células malignas humanas dependentes de Ras oncogênico: FGF2 e PMA como supressores de tumor / Specific vulnerabilities of human malignant cells dependent on oncogenic Ras: FGF2 and PMA as tumor suppressors

Um passo limitante no desenvolvimento de fármacos para terapias do câncer está na descoberta de vulnerabilidades específicas de células tumorais que sirvam à identificação de alvos moleculares apropriados à intervenção farmacológica. Esta é a motivação central desta tese, cuja abordagem experimental focaliza a ação oncogênica das proteínas Ras. Amplificação ou mutação ativadora nos proto-oncogenes ras estão entre as alterações genéticas mais frequentes em cânceres. Essas lesões genéticas aparecem na origem etiológica de múltiplas formas de fenótipos malignos. Mas, essas lesões oncogênicas também conferem susceptibilidades letais às células malignamente transformadas frente a determinados agentes que não interferem significativamente nas funções vitais de células normais. Nos últimos anos nosso laboratório vem estudando os mecanismos moleculares da ação antiproliferativa do fator de crescimento FGF2 (Fibroblast Growth Factor2) e do éster de forbol PMA (Phorbol-12-Myristate-13-Acetate) em linhagens de células murinas malignas dependentes de ras oncogênico. Nesta tese investigamos quanto de nossas observações anteriores com células murinas são aplicáveis a células humanas. Nesse sentido focalizamos a linhagem HaCaT de queratinócitos humanos imortalizados e seus subclones malignizados por expressão ectópica de H-RasV12; além disso, numa triagem inicial também examinamos treze linhagens celulares humanas derivadas de tumores naturais portadores de mutação ativadora em H-Ras, N-Ras ou K-Ras. Nossos resultados mostram que os queratinócitos da linhagem parental HaCaT expressam receptores de FGFs e respondem mitogenicamente tanto a FGF2 como a PMA; portanto, ambos FGF2 e PMA são benéficos aos queratinócitos HaCaT. Por outro lado, o FGF2 mostrou-se citotóxico para subclones HaCaT que expressam H-RasV12 induzível, mas sublinhagens HaCaT com expressão constitutiva de H-RasV12 mostraram-se resistentes à ação citotóxica de FGF2. Diferentemente de FGF2, PMA bloqueou a proliferação de sublinhagens clonais HaCaT-H-RasV12 em ambos substrato sólido e suspensão de agarose e, também, reduziu a estratificação dos queratinócitos HaCaT-H-RasV12 em culturas organotípicas. PMA foi citotóxico e não citostático, pois induziu morte apoptótica sem causar arresto em nenhuma fase específica do ciclo celular. Em HaCaT parental, PMA induziu aumento transitório dos níveis intracelulares de espécies reativas de oxigênio (ROS), mas nos queratinócitos HaCaT-H-RasV12, PMA causou aumentos mais altos e persistentes de ROS, o que promove forte estresse oxidativo, provavelmente responsável pela toxidez deste ester de forbol. Entre as treze linhagens celulares humanas malignas com H-Ras, N-Ras ou K-Ras mutados, onze foram vulneráveis à ação citotóxica de PMA; mas apenas uma delas, a linhagem de tumor urotelial UM-UC-3, foi sensível ao efeito anti-proliferativo de FGF2. Em conclusão, células malignas humanas com Ras mutado parecem superar rapidamente uma possível toxidez de FGF2, mas não ultrapassam a toxidez causada por PMA

A challenge in drug development for cancer therapy is the discovering of molecular targets suitable for pharmacological interference. This challenge was the main motivation of the present thesis. Amplification or activating mutation in ras proto-oncogenes are among the most frequent genetic lesions in human cancer. Actually, mutated Ras onco-proteins are in the etiological roots of multiple malignant phenotypes; however these onco-proteins also cause specific lethal vulnerabilities even in robust malignant cells. Recently, our laboratory reported that malignant murine cell lines dependent on oncogenic Ras are prone to toxicity initiated by FGF2 (Fibroblast Growth Factor 2) and PMA (Phorbol-12-Myristate-13-Acetate), which are not harmful to normal cells. This cytotoxicity of FGF2 and PMA very likely follows different molecular mechanisms, which, however, are not yet completely understood. The aim of this thesis was to investigate whether these vulnerabilities found in murine malignant cells were also valid for human malignant cell lines dependent on oncogenic Ras. To this end the experimental approach was focused on the HaCaT cell line of immortalized human keratinocytes and its sublines transformed by H-RasV12 ectopic expression. In addition thirteen human cell lines derived from natural tumor carrying mutated H-Ras, N-Ras or K-Ras oncogenes were also screened. The results showed that HaCaT keratinocytes express FGF receptors and respond mitogenically to both FGF2 and PMA. On the other hand, FGF2 was cytotoxic to HaCaT subclones expressing inducible H-RasV12. But, HaCaT sublines constitutively expressing H-RasV12 were resistant to FGF2 toxicity. However, PMA was toxic to all HaCaT-H-RasV12 sublines, inhibiting proliferation in both solid substrate and agarose suspension cultures and, also reducing stratification in organotypic cultures. Furthermore, in HaCaT-H-RasV12 sublines, but not in the parental HaCaT line, PMA caused a persistently high increase in intracellular levels of reactive oxygen species (ROS) and concomitantly induced apoptosis. Moreover, eleven of the thirteen human tumor cell lines with mutated H-Ras, N-Ras or K-Ras, were growth inhibited by PMA, whereas only one of them was inhibited by FGF2, the urothelial tumor cell line UM-UC-3. In conclusion, human malignant cells driven by Ras oncogenes very likely rapidly overcome FGF2 toxicity, whereas they remain stably vulnerable to PMA cytotoxicity

Actividad antiinflamatoria de flores y hojas de Caesalpinia pulcherrima L. (Swartz) / Anti-inflammatory activity of flowers and leaves of Caesalpinia pulcherrima L. (Swartz)

Introducción: Partes aéreas de la planta Caesalpinia pulcherrima L. (Swartz) han sido usadas en medicina tradicional al sur del Departamento de Cundinamarca para el tratamiento de afecciones inflamatorias. Objetivo: Evaluar la actividad antiinflamatoria de flores, hojas y frutos verdes de Caesalpinia pulcherrima para cuantificar su actividad antiinflamatoria en modelos murinos de inflamación aguda y subcrónica. Metodología: Este estudio cuantificó la actividad antiinflamatoria de diferentes extractos de tejidos aéreos de esta especie encontrada en Colombia por dos modelos de inflamación aguda, el edema auricular inducido por TPA (acetato de 12-O-tetradecanoil-forbol) y el edema plantar inducido por carragenina; así como el modelo de inflamación sub-crónico de granuloma inducido por pellet de algodón. Resultados: Los extractos de flores mostraron la mayor actividad antiinflamatoria en el modelo del TPA, en tanto que las hojas fueron más efectivas en disminuir el granuloma, en el modelo del pellet de algodón. No se observó ninguna actividad antiinflamatoria de ningún extracto en el modelo de carragenina. Los frutos verdes no mostraron actividad en ningún modelo. Conclusión: Los resultados demostraron la efectividad que el uso etnobotánico le atribuye a esta planta. Los extractos activos obtenidos mostraron el potencial uso de esta planta en la fabricación fitoterapéuticos efectivos. Salud UIS 2011; 43 (3): 281-287.

Introduction: Aerial parts of Caesalpinia pulcherrima L. (Swartz) have been used in traditional medicine in southern Cundinamarca Department for the treatment of inflammatory diseases. Objective: Evaluate the antiinflammatory activity of flowers, leaves and green fruits of the plant to quantify inflammatory activity of acute and subchronic murine inflammation models. Methodology: This study quantified the anti-inflammatory activity of different extracts of aerial tissues of this species for two models of acute inflammation, the ear edema induced by TPA (12-O-tetradecanoyl-phorbol-13-acetate) and plantar edema induced by carrageenan, as well as the sub-chronic inflammation model of granuloma induced by cotton pellet. Results: The flower extracts proved to have the largest anti-inflammatory activity in the TPA model, while the leaves were more effective in reducing the granuloma in the cotton pellet model. There was no anti-inflammatory activity of any extract carrageenan model. The green fruits showed no activity in any model. Conclusion: The results demonstrated the effectiveness of the ethnobotanical use attributed to this plant. The active extracts obtained showed the potential use of this plant in making safe and effective phytomedicines. Salud UIS 2011; 43 (3): 281-287.

Hydrolysis technology optimization of phorbol esters by orthogonal experiment / 中国中药杂志

<p><b>OBJECTIVE</b>To establish the best hydrolytic conditions from phorbol esters.</p><p><b>METHOD</b>The orthogonal experiment was used to optimize 4 factors, which were reaction time, ratio of solid-to-liquid, hydrolytic times, and temperature. Diamonsil C18 column (4. 6 mm x 250 mm, 5 microm) was used and the mobile phase was consisted of acetonitrile and water for HPLC detection. The detection wavelength was set at 234 nm, the flow rate was 1 mL x min(-1), and the column temperature was 25 degrees C.</p><p><b>RESULT</b>The optimum conditions were 10 h of reaction time, 1:6 of solid-to-liquid (BaOH/MeOH) ratio, 25 degrees C of temperature, and one time of hydrolysis. There was a good linear relationship of phorbol in the range of 4.28-107 mg x L(-1) (r = 0.999 9), and the average recovery was 97.89%, with RSD 0.78%.</p><p><b>CONCLUSION</b>The method is steady, reliable and reproducible, and it provides a mean for future study.</p>

Role of transient receptor potential vanilloid 4 in the effect of osmotic pressure on myocardial contractility in rat / 生理学报

The aim of the present study was to investigate the influence of osmotic pressure on myocardial contractility and the possible mechanism. Electrical stimulation was used to excite papillary muscles of the left ventricle of Sprague-Dawley (SD) rats. The contractilities of myocardium in hyposmotic, isosmotic, and hyperosmotic perfusates were recorded. The influences of agonist and antagonist of the transient receptor potential vanilloid 4 (TRPV4) on the contractility of myocardium under hyposmotic, isosmotic and hyperosmotic conditions were observed. The results were as follows: (1) Compared with that under isosmotic condition (310 mOsm/L), the myocardial contractility was increased by 11.5%, 21.5% and 25.0% (P<0.05) under hyposmotic conditions when the osmotic pressure was at 290, 270 and 230 mOsm/L, respectively; and was decreased by 16.0%, 23.7% and 55.2% (P<0.05) under hyperosmotic conditions when the osmotic pressure was at 350, 370 and 390 mOsm/L, respectively. (2) When ruthenium red (RR), an antagonist of TRPV4, was added to the hyposmotic perfusate (270 mOsm/L), the positive inotropic effect of hyposmia was restrained by 36% (P<0.01); and when RR was added to the hyperosmotic perfusate (390 mOsm/L), the inhibitory effect of hyperosmia on myocardial contractility was increased by 56.1% (P<0.01). (3) When 4-α-phorbol-12,13-didecanoate (4α-PDD), an agonist of TRPV4, was added to the isosmotic perfusate (310 mOsm/L), the myocardial contractility did not change; and when 4α-PDD was added to the hyperosmotic perfusate (390 mOsm/L), the inhibition of myocardial contractility by hyperosmia was increased by 27.1% (P<0.01). These results obtained indicate that TRPV4 is possibly involved in the osmotic pressure-induced inotropic effect.

Expression of WT1 gene and its isomer ratio changes during phorbol ester induced differentiation of K562 cell line / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the changes in expression of WT1 gene and ration of its isomers during phorbol ester (TPA) induced differentiation of leukemia cell line K562 by fluorescence quantitative RT-PCR and analysis the relationship between different isomers and hematogenic cell differentiation.</p><p><b>METHODS</b>The degree of cellular maturation were verified by NBT reduction test and immunophenotyping. Expression of WT1 gene was determined by fluorescence quantitative RT-PCR during differentiation of K562 cell line. The relative ratio of the four splicing variants WT1 ( + / + ), WT1 ( + / - ), WT1 ( - / + ), WT1 ( - / - ) were calculated.</p><p><b>RESULTS</b>During the differentiation of K562 cell, the NBT reduction rate and the CD9 positive rate both increased significantly (P < 0. 05). The expression of WT1 gene decreased immediately to (1.67 +/- 0.45) x 10(-3) from (4.67 +/- 1.11) x 10(-3), and then increased again to (4.64 +/- 1.53) x 10(-3) at 96 hours. The ratio of WT1 ( + / + ) was decreased gradually, from 0 hour (39.65 +/- 19.46)% to 96 hour (15.25 +/- 7.27)%. While the ratio of WT1( - / - ) was increased, from 0 hour (15.38 +/- 11.34)%, to 96 hour (37.60 +/- 11.90)%. The other two isomers ratios did not change significantly.</p><p><b>CONCLUSION</b>During the TPA induced differentiation of K562 cell, there are two high expression levels of WT1 gene. Before differentiation, the majority is WT1 ( + / + ), and after differentiation, is WT1 ( - / - ). It indicates that WT1 gene may activate or inhibit cell differentiation by regulating the ratio of its four splicing variants.</p>

Protein kinase C isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from mouse pancreatic islets

Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assess the PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulin release (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 mM) enhanced PIR ~ 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by ~ 40-50 percent. Both effects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cch also evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronic TMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. In contrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-induced insulin secretion.

Effect of phorbol ester on K+ channel in an G292 osteoblast-like cell / 대한치과교정학회지

In order to investigate the action mechanism of protein kinase C on K+ channel in osteoblastic cell, effects of phorbol 12,13-dibutyrate on human osteoblast-like cells (G292) were studied by patch clamp technique with cell-attached configuration. In this experiment, 45pS ion channel was dominant in G292 cell line according to their approximate conductances in symmetrical 140mM KCl saline at holding potential of 60mV. In current-voltage relationship, reversal potential was 5.5mV at the condition of potassium enriched saline in the pipette and -27 mV at the condition of standard extracellular saline in the pipette. Phorbol 12, 13-dibutyrate 10 nM increased the open probability of 45 pS channel and staurosporine, an inhibitor of protein kinase C, suppressed this effect. Phorbol 12,13-dibutyrate moved the reversal potential of 45pS channel to more negative potential and increased the single channel current at the same membrame potential. In order to check the activation of protein kinase C in G292 cell by phorbol 12,13-dibutyrate, western blot of protein kinase C was performed. Phorbol 12,13-dibutyrate 0.1 micrometer translocated protein kinase C from cellular compartment to membrane compartment of the cell. These findings suggest that phorbol 12,13-dibutyrate, one of phorbol esters, activate 45pS channel in G292 cell and affect cell membrane potential, that regulate cellular function.

Papel dos proteoglicanos de heparam sulfato de matriz e superfície na divisäo celular / The role of proteoglycans of cell surface and extracellular matrx on the cell proliferation, using as a experiental model endothelial cells and melanoma

O presente trabalho visa o estudo do papel dos proteoglicanos (PGs) da superfície e matriz extracelular (MEC) na divisão celular empregando como modelo células endoteliais e de melanoma em cultura. O éster de forbol PMA, um ativador da PKC, apresentou um duplo efeito no crescimento de células endoteliais: ação mitogénica no início de G1 e ação antiproliferativa ao final de G1, bloqueando a passagem de G1 para a fase S. Por outro lado, PMA estimulou especificamente a síntese de PGHS de maneira dose-dependente em Gol Gl. Além do PMA, somente o ionóforo de Ca2+ foi capaz de induzir o aumento na síntese de proteoglicano de heparam sulfato (PGHS). De maneira semelhante ao PMA, a bradicinina (BK) estimulou a síntese de PGHS e também bloqueou o ciclo celular. O conjunto de dados sugere o envolvimento de diversas isoformas da PKC nestes dois processos, em especial as isoformas de PKC dependentes de cálcio. Utilizando n-butanol, um inibidor da PKC, observamos que a biossíntese de PGHS não foi totalmente abolida, indicando a existência de outra via para a biossíntese de PGs, independente da PKC. Esta outra via poderia ser representada por NO, pois dados da literatura mostram que BK interfere na produção deste composto. A importância dos PGs na proliferação celular foi estudada empregando-se xilosídeos ligados a diferentes agliconas, que atuam como moléculas aceptoras para a biossíntese dos glicosaminoglícanos (GAG). Dentre todos os compostos testados, o-nitrofenii-P-D-xilosídeo é o melhor aceptor para a síntese de HS sendo o único composto capaz de alterar o ciclo celular, por bloquear a entrada do ciclo na fase S. Este efeito é similar ao apresentado pelo tratamento de células endoteliais com PMA e BK. Estudando PGs de células de melanoma e expressão de glicosidases e proteases, foi possível demonstrar que as células com maior potencial invasivo, e portanto maior capacidade migratória, apresentaram aumento na excreção de cisteínoproteases (tipo tiol). Por outro lado, as células de menor potencial metastático, apresentam maior atividade das exoglicosidases...(au)

The Effect of Activation of Protein Kinase C on the Calcium-dependent K; Current in Rat Basilar Smooth Muscle Cells

OBJECTIVE: Activation of protein kinase C(PKC) may play a certain role in the development of cerebral vasospasm. However, this mechanism still elusive. This study was undertaken to investigate the action of protein kinase C on calcium-dependent K+ channels(KCa) and internal calcium concentration [Ca2+]i in freshly isolated smooth muscle cells. METHODS: Whole-cell patch clamp and calcium microfluorimetry technique were used for measuring the K Ca and internal calcium concentration. RESULTS: In patch-clamp studies, depolarization evoked an outward KCa current which is sensitive to caffeine and A23187, and shown to be blocked by TEA(tetraethylammonium) but not by glibenclamide. Activation of protein kinase C by phorbol 12-myristate 13-acetate(PMA:1-100nmol/1) and phorbol 12, 13-dibutyrate(PDB:1-100nmol/1) dose-dependently enhanced KCa current. Subsequent application of TEA(10-30mmol/1) but not glibenclamide(3-6nmol/1), in the presence of phorbol esters, reduced the potassium current activated by phorbol esters. Preincubation with 1-(5-isoquinoline sulphonyl)-2-methylpiperazine(H-7:10nmol/1), a protein kinase C inhibitor, prevented the effect of phorbol esters on KCa. In calcium microfluorimetric studies, PMA(100nmol/1) increased intracellular calcium concentration and this effect of PMA was prevented by pre-incubation of cells with H-7(10nmol/1). CONCLUSION: These results indicate that activation of PKC increases intracellular calcium concentration and elevation of internal calcium concentration activates KCa in cerebral vascular smooth muscle cells.

Calcium- and zinc-binding proteins in intracellular transport

The complex mechanism of intracellular transport is regulated by free calcium in different manners. Calcium binding proteins regulate several aspects of the vesicle fusion mechanism mediated by NSF (N-ethylmaleimide sensitive fusion factor). At least in some regulated exocytosis, calcium-binding proteins are the trigger for fusion downstream of NSF, Still, calcium-binding proteins, such as annexins, may be part of a different fusion mechanism mediating some specific transport steps or working in parallel to the NSF-dependent fusion process. Calcium is not the only ion necessary for the function of factors involved in vesicular transport. A zinc requirement has been also proposed. One of the zinc-dependent factors is probably a protein with a cysteine-rich region that coordinates zinc and binds phorbol esters. Although protein kinase C is the more prominent family of proteins carrying this domain, the factor necessary for transport does not appear to function as a kinase.

Pretreatment with phorbol ester and an LHRH asgonist reduces testosterone production and protein kinase C activity in rat Leydig cells challenged with PDBu and LHRH

We have investigated the role of protein kinase C (PK-C) in luteinizing hormone-releasing hormone (LHRH)-induced testosterone secretion from purified rat Leydig cells (70-80-day old Sprague-Dawley rats) by pretreating the cells in vitro with 200 mM phorbol 12,13-dibutyrate (PDBu) (a known procedure to down-modulate this enzyme in most cell types) and 1 muM [D-Ala6,Des-Glyl0]-LHRH ethylamide, an LHRH agonist (LHRH-A). Following pretreatment we measured PK-C activity and secretion of testosterone in response to subsequent challenges with the PK-C activator PDBu (20-2000 nM) and with LHRH (0.001-1.0 muM) and the Ca2+ mobilizing secretagogue A23187 (0.1-1OO muM) in the same cell preparation. PDBu and LHRH-A pretreatments caused a reduction in testosterone secretion in response to subsequent exposure to PDBu or LHRH. Both pretreatments decreased PK-C activity in crude and purified extracts of the same cells. The magnitude of reduction of the secretory response was greater than that of enzyme activity for both PDBu and LHRH-A pretreatment (68.9 per cent reduction of testosterone secretion vs 54.7 per cent reduction of PK-C activity in PDBu-pretreated cells and 78.6 per cent reduction of testosterone production vs 36.6 per cent reduction of PK-C activity in LHRH-A-pretreated cells). The effect of phorbol ester pretreatment on PDBu- or LHRH-stimulated testosterone secretion and PK-C activity was specific (no measurable effect with 4 alpha-PDBu, an inactive phorbol ester). While PDBu and LHRH-A pretreatment reduced Leydig cell responsiveness to PDBu or LHRH, the secretion of testosterone in response to the Ca2+ -mobilizing secretagogue A23187 was similar in PDBu- and LHRH-A-pretreated and in control (non-pretreated) cells. We conclude that down-modulation of protein kinase C by prolonged exposure of Leydig cells to phorbol esters or LHRH-A results in decreased PK-C activity and testosterone secretion. These results provide the first evidence that pretreatment with LHRH-A, which does not enter the cell, can affect the steroidogenesis and PK-C activity responses to PDBu (the intracellular ligand of PK-C).

Slow cluster formation of purified human or rhesus T cells requires protein kinasse C and LFA-1

Ab: Homotropic T cell adhesion, as generally studied, consists of a rapid, transient binding process that is measured over a 15-120 min. period. Here we report a slow type of adhesion process occurring with human or rhesus T cells, purified from peripheral blood, that manifests itself by the formation of rounded, multi-layer clusters which may contain hundreds of cells. The maximal number and size of the clusters peak 1-2 days after the addition of phorbol ester, an absolute requirement. The number of clusters formed is proportional to phorbol ester concentration up to 1.25 ng/mL. Phorbol esters such as phorbol myristate acetate (PMA), phorbol dibutyrate (PDB), and 7-octylindolactam (OIL) induced optimal cluster formation at 1-13 ng/mL, levels slightly higher than that required to induce mitogenesis of purified T cells. Phorbol itself and the alpha-form of the ester were inactive. Both cluster formation and mitogenesis (stimulated by Con A or anti-CD3) are completely inhibited by staurosporin at 12.5 ng/mL. Even at 2.5 ng/mL, 74 percent of cluster formation was inhibited, which strongly implies a crucial role for protein kinase C. In the presence of accessory cells, T cell clusters were suppressed. Monoclonal Ab such as anti-CD3, mouse anti-CD3 followed by anti-mouse IgG, anti-CD4, anti-CD4A, anti-CD2, anti-CD8, and anti-CD45 did not induce cluster formation. None were inhibitory or stimulatory in the presence of PMA, except for anti-CD3 which enhanced cluster formation by 26 percent. However, anti-LFA-1 beta-chain (mouse monoclonal) completely blocked cluster formation over the range studied (63-1000 ng/mL) for both human and rhesus cells; rat anti-LFA-1 only blocked human cell adhesion. Anti LFA-1 only partially inhibited T cell mitogenesis. These results show that slow cluster formation shares the LFA-1 and phorbol ester requirements of the rapid adhesion of T cells requiring LFA-1 and ICAM-1. However, cluster occurs at a very low phorbol ester concentration, appears more sensitive to staurosporin inhibition, and is not stimulated via the TCR receptor like the rapid adhesion process. We hypothesize that certain neuronal processes, induced by phorbol ester, and which also show a similar protein kinase C activation time course, may share mechanisms in common with cluster formation

Patterns of long-term steroidogenesis stimulation by ACTH and phorbol ester

Adrenocorticotropic hormone (ACTH) triggers well-defined responses in Y-1 cells. Among them is steroidogenesis stimulation. We have previously shown that phorbol 12-myristate 13-acetate (PMA), an activator of the calcium- and phospholipid-dependent protein kinase (PKC) is able to mimic all the responses triggered by ACTH in these cells, including steroidogenesis stimulation. Short (2 h) treatment with PMA leads to only 20-30 per cent of the maximal steroidogenesis stimulation obtained with ACTH. However, the steroid secretion in the 2 h that follows the short-term (2 h) PMA treatment reaches the same levels as observed with ACTH, i.e., a 12- to 15-fold increase. We also show that this effect is restricted to cells treated with PMA for up to 4 h, while treatment for longer periods of time causes a reduction of the steroid biosynthesis rate, an effect that is not observed in cells treated with ACTH or N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (dcAMP). These results suggest that activation of PKC can elicit the first phase of ACTH steroidogenesis stimulation, but not the second one, which strictly depends on activation of cAMP-dependent protein kinase.

Role of Oxygen Free Radical in the Expression of Interleukin-8 andInterleukin-1beta Gene in Mononuclear Phagocytic Cells / 결핵

BACKGROUND: Oxygen free radicals have generally been considered as cytotoxic agents. On the other hand, recent results suggest that small nontoxic amounts of these radicals may act a role in intracellular signal transduction pathway and many efforts to reveal the role of these radicals as secondary messengers have been made. It is evident that the oxygen radicals are released by various cell types in response to extracellular stimuli including LPS, TNF, IL-1 and phorbol esters, all of which translocate the transcription factor NFkappaB from cytoplasm to nucleus by releasing an inhibitory protein subunit, MB. Activation of NFkappaB is mimicked by exposure to mild oxidant stress, and inhibited by agents that remove oxygen radicals. It means the cytoplasmic form of the inducible tanscription factor NFkappaB might provide a physiologically important target for oxygen radicals. At the same time, it is well known that LPS induces the release of oxygen radicals in neutrophil with the activation of NFkappaB. From above facts, we can assume the expression of IL-8 and IL-1beta gene by LPS stimulation may occur through the activation of NFkappaB, which is mediated through the release of MB by increasing amounts of oxygen radicals. But definitive evidence is lacking about the role of oxygen free radicals in the expression of IL-8 and IL-1beta gene in mononuclear phagocytic cells. We conducted a study to determine whether oxygen radicals act a role in the expression of IL-8 and IL-1beta gene in mononuclear phagocytic cells. METHOD: Human peripheral blood monocytes were isolated from healthy volunteers. Time and dose relationship of H2O2-induced IL-8 and IL-1beta mRNA expression was observed by Northern blot analysis. To evaluate the role of oxygen radicals in the expression of IL-8 and IL-1beta mRNA by LPS stimulation, pretreatment of various antioxiants including PDTC, TMTU, NAC, ME, Desferrioxamine were done and Northern blot analysis for IL-8 and IL-1beta mRNA was performed. RESULTS: In PBMC, dose and time dependent expression of IL-8 and IL-1beta mRNA by exogenous H2O2 was not observed. But various antioxidants suppressed the expression of LPS-induced IL-8 and IL-1beta mRNA expression of PBMC and the suppressive activity was most prominant when the pretreatment was done with TMTU. CONCLUSION: Oxygen free radical may have some role in the expression of IL-8 and IL-1beta mRNA of PBMC but that radical might not be H2O2.

Phorbol Esters Attenuate The Action of Isoproterenol on Vascular Smooth Muscle

The effects of phorbol esters were studied in rabbit carotid artery to evaluate the action of protein kinase C on the regulation of vascular tone by isoproterenol. The vascular rings, 2 mm in width, were myographied isometrically in an isolated organ bath and the effects of phorbol 12,13-dibutyrate(PDBu) and phorbol 12-myristate 13-acetate(PMA) were determined. Isoproterenol, a beta adrenergic agonist, relaxed the vessel which was precontracted by phenylephrine, but not that by phorbol esters. The action of isoproterenol was attenuated by removal of endothelium or pretreatment with methylene blue or nitro-L-arginine. The pretreatment with phorbol esters at concentrations which did not induce contraction, decreased isoproterenol-induced relaxation of vascular rings with or without endothelium. The action of PDBu on isoproterenol-induced relaxation was less effective than that of PMA, unlike those observed in contractile response, but the contractile effect of the former was more potent than that of the latter. PMA did not affect relaxant effect of forskolin, an activator of adenyl cyclase. Staurosporine, a protein kinase C inhibitor, inhibited the action of these drugs on both isoproterenol-induced relaxation and the contractile response. These results suggest that the relaxation induced by isoproterenol was reducd by the activation of protein kinase C, which may be isozyme different from that involved in contractile response.