Synthesis and biological evaluation of caracasine acid derivatives.

A series of caracasine acid (1) derivatives were synthesized and evaluated for their in vitro cytotoxicity on human cancer-derived cell lines MCF-7 and PC-3, as well as for other activities such as antibacterial, antileishmanial and antitrypanosomal activity. Compound 1 was more effective than any of its derivatives against tested human cancer cell lines. PC-3 cells were more sensitive than MCF-7 to all compounds, particularly the methyl ester (2), the amide (9) and the epoxide (10). The evaluation of antiparasitic activity revealed that ester derivatives (2-8) and the amide derivative (9) were the most effective antileishmanial and antitrypanosomal compounds, even though their effect on Trypanosoma cruzi was modest. Finally, compound 1 and the derivatives evidenced a broad spectrum of antibacterial activity, as assayed against Gram-positive and Gram-negative bacteria.

Synthesis and biological evaluation of ( - )-13,14-dihydroxy-8,11,13-podocarpatrien-7-one and derivatives from (+)-manool.

13,14-Dihydroxy-8,11,13-podocarpatrien-7-one (1) and a series of ring C aromatic diterpene derivatives were synthesised from (+)-manool (4) and evaluated for their cytotoxic, leishmanicidal and trypanocidal activities. Our results indicated that compound 1 and other podocarpane-type intermediates are cytotoxic. Cleavage of C6-C7 bond of compound 7 improved cytotoxic activity, indicating that, in particular, the 6,7-seco-podocarpane-type compound 20 might serve as a lead compound for further development.

The marine sponge toxin agelasine B increases the intracellular Ca(2+) concentration and induces apoptosis in human breast cancer cells (MCF-7).

PURPOSE: In search for new drugs derived from natural products for the possible treatment of cancer, we studied the action of agelasine B, a compound purified from a marine sponge Agelas clathrodes. METHODS: Agelasine B was purified from a marine sponge Agelas clathrodes and assayed for cytotoxicity by MTT on two human breast cancer cells (MCF-7 and SKBr3), on a prostate cancer cells (PC-3) and on human fibroblasts. Changes in the intracellular Ca(2+) concentrations were assessed with FURA 2 and by confocal microscopy. Determination of Ca(2+)-ATPase activity was followed by Pi measurements. Changes in the mitochondria electrochemical potential was followed with Rhodamine 123. Apoptosis and DNA fragmentation were determined by TUNEL experiments. RESULTS: Upon agelasine B treatment, cell viability of both human breast cancer cell lines was one order of magnitude lower as compared with fibroblasts (IC(50) for MCF-7 = 2.99 µM; SKBr3: IC(50) = 3.22 µM vs. fibroblasts: IC(50) = 32.91 µM), while the IC(50) for PC-3 IC(50) = 6.86 µM. Agelasine B induced a large increase in the intracellular Ca(2+) concentration in MCF-7, SKBr3, and PC-3 cells. By the use of confocal microscopy coupled to a perfusion system, we could observe that this toxin releases Ca(2+) from the endoplasmic reticulum (ER). We also demonstrated that agelasine B produces a potent inhibition of the ER Ca(2+)-ATPase (SERCA), and that this compound induced the fragmentation of DNA. Accordingly, agelasine B reduced the expression of the anti-apoptotic protein Bcl-2 and was able to activate caspase 8, without affecting the activity of caspase 7. CONCLUSIONS: Agelasine B in MCF-7 cells induce the activation of apoptosis in response to a sustained increase in the [Ca(2+)]( i ) after blocking the SERCA activity. The reproduction of the effects of agelasine B on cell viability and on the [Ca(2+)]( I ) obtained on SKBr3 and PC-3 cancer cells strongly suggests the generality of the mechanism of action of this toxin.

The natural diterpene ent-16ß-17α-dihydroxykaurane down-regulates Bcl-2 by disruption of the Ap-2α/Rb transcription activating complex and induces E2F1 up-regulation in MCF-7 cells.

ent-Kauranes are diterpene-type compounds commonly found in most plant species, especially from the Euphorbiaceae family. These compounds have been studied due to their anti-inflammatory and anti-tumor properties. Regulation of apoptosis, or programmed cell death, is commonly bypassed by tumoral cells, giving rise to uncontrolled proliferating cells, which eventually become carcinogenic. In a previous work, we showed that both mRNA and protein expression levels of the antiapoptotic gene Bcl-2 are reduced in MCF-7 cancer cells by the effect of the natural diterpene ent-16ß-17α-dihydroxykaurane (DHK). This effect was not directly associated with the inactivation of NF-κB, as has been shown with other diterpenes compounds. Herein, we report that DHK is dissociating the Ap2α-Rb activating complex, affecting its binding ability for the Bcl-2 gene promoter. These events down-regulate Bcl-2 and is temporally accompanied by the induction of E2F1 and its target pro-apoptotic gene Puma. Disruption of the Rb-Ap2α activation complex was corroborated by chromatin immunoprecipitation and protein immunolocalization, which also revealed that Ap2α sorts out from the nucleus and relocalizes in the cell periphery. Taken together, our study confirms the regulation of Bcl-2 gene transcription by the Ap2α-Rb complex and describes a singular protein relocalization for Ap2α induced by DHK, implicating a new potential therapeutic target to differentially onset apoptosis in tumor cells.

Trypanosoma cruzi calmodulin: cloning, expression and characterization.

We have cloned and expressed calmodulin (CaM) from Trypanosoma cruzi, for the first time, to obtain large amounts of protein. CaM is a very well conserved protein throughout evolution, sharing 100% amino acid sequence identity between different vertebrates and 99% between trypanosomatids. However, there is 89% amino acid sequence identity between T. cruzi and vertebrate CaMs. The results demonstrate significant differences between calmodulin from T. cruzi and mammals. First, a polyclonal antibody developed in an egg-yolk system to the T. cruzi CaM recognizes the autologous CaM but not the CaM from rat. Second, it undergoes a larger increase in the alpha-helix content upon binding with Ca(2+), when compared to CaM from vertebrates. Finally, two classic CaM antagonists, calmidazolium and trifluoperazine, capable of inhibiting the action of CaM in mammals when assayed on the plasma membrane Ca(2+) pump, showed a significant loss of activity when assayed upon stimulation with the T. cruzi CaM.

Cytotoxic and apoptosis-inducing effect of ent-15-oxo-kaur-16-en-19-oic acid, a derivative of grandiflorolic acid from Espeletia schultzii.

ent-Kaurenic acid and many natural derivatives of this diterpene are known to have interesting biological properties. ent-15-Oxo-kaur-16-en-19-oic acid can be easily obtained from grandiflorolic acid which was first isolated from Espeletia grandiflora. The present work describes the proapoptotic effect of ent-15-oxo-kaur-16-en-19-oic acid on the human prostate carcinoma epithelial cell line PC-3 as evidenced by the changes in the expression level of proteins associated with the execution and regulation of apoptosis. Cell viability was affected upon exposure to the compound, the IC(50) were determined as 3.7 microg/ml, which is 4 times lower than that corresponding to a primary cell culture of fibroblasts (14.8 microg/mL). Through Western blot analysis, active forms of caspace-3 associated with the specific proteolysis of Poly(ADP-ribose) polymerase (PARP) were detected. Reduced levels of the antiapoptotic protein Bcl-2, as well as the appearance of internucleosomal DNA fragmentation, were also demonstrated. Thus, ent-15-oxo-kaur-16-en-19-oic acid may be a promising lead compound for new chemopreventive strategies, alone or in combination with traditional chemotherapy agents to overcome drug resistance in tumoral cells.

La muerte celular programada en las plantas: ¿es semejante a la Apoptosis en animales? / Programmed cell death in plants: it is similar to apoptosis in animals

La muerte celular programada (MCP) es esencial para mantener la homeostasis tisular en muchas formas de vida. En las plantas, de igual forma que en los animales, la MCP es el mecanismo por el cual se regulan una serie de procesos fisiológicos tales como la germinación, diferenciación, crecimiento, reproducción y desarrollo de semillas. La MCP también juega un papel importante en otros procesos como la resistencia a condiciones ambientales desfavorables. A diferencia de los modelos animales, la MCP en plantas está poco descrita al nivel molecular, lo que ha dado lugar a debates sobre el paralelismo entre este tipo de muerte programada y el proceso conocido en animales como apoptosis. Sin embargo, en los últimos años han surgido evidencias importantes que permiten concluir que en las plantas existe tal proceso. En esta revisión se analizan hallazgos recientes del reino vegetal en esta área, que comienzan a vislumbrar elementos claves sobre la modulación de la respuesta de la planta ante muy diversos factores de estrés, tanto bióticos como abióticos, abriendo además la posibilidad de utilizarlos en programas de mejoramiento genético de rubros agrícolas estratégicos.

Polycyclic aromatic hydrocarbon degradation by biosurfactant-producing Pseudomonas sp. IR1.

We characterized a newly isolated bacterium, designated as IR1, with respect to its ability to degrade polycyclic aromatic hydrocarbons (PAHs) and to produce biosurfactants. Isolated IR1 was identified as Pseudomonas putida by analysis of 16S rRNA sequences (99.6% homology). It was capable of utilizing two-, three- and four-ring PAHs but not hexadecane and octadecane as a sole carbon and energy source. PCR and DNA hybridization studies showed that enzymes involved in PAH metabolism were related to the naphthalene dioxygenase pathway. Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by IR1 during growth on both water miscible and immiscible substrates. The biosurfactants lowered the surface tension of medium from 54.9 dN cm(-1) to 35.4 dN cm(-1) and formed a stable and compact emulsion with an emulsifying activity of 74% with diesel oil, when grown on dextrose. These findings indicate that this isolate may be useful for bioremediation of sites contaminated with aromatic hydrocarbons.

A system for the detection and typing of human papillomavirus of the lower genital tract: in situ hybridization screening and polymerase chain reaction confirmation.

We developed a simplified and non-isotopic in situ hybridization procedure for the detection of Human Papillomavirus (HPV) in routine Papanicolaou cervical smears. The assay involves one oligonucleotide (malignant probe) which recognizes high risk HPV 16 and 18, and another which detects HPV 6 and 11 (benign probe). We adapted the system to fulfill the requirements of gynecologists and cytologists, assimilating their protocols and simplifying the in situ hybridization assay. When we compared the detection levels reached by the in situ hybridization versus a ladder PCR assay in 156 clinical samples, original designed for this work, the kappa coefficient between both methods is 0.945, indicating a strong agreement between the ISH and the PCR assays.

A system for the detection and typing of human papillomavirus of the lower genital tract: in situ hybridization screening and polymerase chain reaction confirmation

Se desarrollo un procedimiento de Hibridación in situ no-isotópico y simplificado para la detección de papilomavirus humano (HPV) en citologías de rutina de Papanicolaou. El ensayo involucra un oligonucleotido (sonda maligna) el cual reconoce a los VPH de alto riesgo: 16 y 18, y otra que detecta VPH 6 y 11 (sonda benigna). Se adaptó completamente el sistema a los requerimientos de ginecólogos y citotecnólogos, asimilando sus protocolos y simplificando el ensayo de hibridación in situ. Se compararon 156 muestras clínicas, los niveles de detección tanto en hibridación in situ como en el ensayo de PCR (diseño original para este trabajo), se obtuvieron valores de co-positividad y co-negatividad cercanos a la unidad (0,98)

The role of biotechnology in art preservation.

Biotechnology has played a key role in medicine, agriculture and industry for over 30 years and has advanced our understanding of the biological sciences. Furthermore, the tools of biotechnology have a great and largely untapped potential for the preservation and restoration of our cultural heritage. It is possible that these tools are not often applied in this context because of the inherent separation of the worlds of art and science; however, it is encouraging to see that during the past six years important biotechnological applications to artwork preservation have emerged and advances in biotechnology predict further innovation. In this article we describe and reflect upon a unique example of a group of scientists and art restoration technicians working together to study and treat of a piece of colonial art, and review some of the new applications in biotechnology for the preservation of mankind's cultural heritage. We predict an expansion in this field and the further development of biotechnological techniques, which will open up new opportunities to both biologists and artwork preservers.

Cytotoxic and proapoptotic activity of ent-16beta-17alpha-dihydroxykaurane on human mammary carcinoma cell line MCF-7.

Here we describe the cytotoxic and proapoptotic effect of an ent-kaurane (ent-16beta-17alpha-dihydroxykaurane), compound isolated from Croton malambo barks, on malignant cell growth. When MCF-7 mammary carcinoma cells were treated with increasing concentrations of the ent-kauranoid, its cytotoxic activity showed an IC50 of 12.5microg/ml, dose that is 2.66-fold lower than the corresponding value for non-malignant cells. At this growth inhibitory dose, both mRNA and protein levels for Bcl-2 as well as mRNA for hTERT were significantly reduced. The observed preapoptotic activity seemed to be triggered by a mechanism that is not directly affecting NF-(kappa)B binding ability. The potential use of this plant-derived compound as a cancer chemotherapy agent is discussed.