Estudos estruturais e funcionais de Tsa1 de Saccharomyces cerevisiae: um modelo biológico para o estudo de inibidores de crescimento celular para leucemia linfoide aguda / Functional and structural evaluation of Tsa1 from Saccharomyces cerevisiae: a biological model for study of cellular growth inhibitors for acute lymphocytic leukemia

As peroxirredoxinas (Prx) são enzimas antioxidantes que se destacam pela capacidade de decompor uma grande variedade de hidroperóxidos com elevada eficiência (106-108M-1s-1), mantendo essas moléculas em níveis adequados à homeostase celular. Entretanto, já foi demonstrado que em diversos tipos tumorais os níveis de Prx são extremamente aumentados e experimentos envolvendo sua inativação resultam na diferenciação ou apoptose de células tumorais. Recentemente, foi descoberto um diterpenóide denominado adenantina que seria o primeiro inibidor para as Prx1 e Prx2 de humanos e foi demonstrada que sua aplicação em células de leucemia mieloide aguda promoveu diferenciação ou apoptose dessas células. Nesse contexto, o presente trabalho apresenta duas vertentes: 1) A caracterização das alterações estruturais e funcionais promovidas pela ligação da adenantina ao sítio ativo das Prx utilizando Tsa1 de Saccharomyces cerevisiae como modelo biológico, em função da sua alta similaridade com Prx2 de humanos; 2) Avaliação da atividade antitumoral dose dependente de adenantina sobre as linhagens celulares REH e MOLT-4 de leucemia linfoide aguda. No que concerne à primeira linha de investigação, nossos resultados revelam que Tsa1 é suscetível à inibição por adenantina, uma vez que o tratamento reduziu em ~66 % a velocidade de decomposição de peróxido de hidrogênio. Adicionalmente, a mutação da Thr44 de Tsa1, pertencente à chamada tríade catalítica, por uma Ser resultou em uma proteína mais suscetível a alterações na estrutura secundária e à inibição da atividade peroxidásica em função da ligação com adenantina, apresentando uma diminuição de ~85% na velocidade de reação. Características semelhantes foram observadas para a proteoforma Tsa2 de S. cerevisiae, que carreia naturalmente a substituição da Thr44 pela Ser. Análises de sequências de Prx em bancos de dados revelaram que majoritariamente proteínas contendo Ser são encontradas em organismos procariotos, muitos deles patogênicos. Finalmente, demonstramos por meio de ensaios citotoxicidade que as bactérias Staphylococcus aureus e Staphylococcus epidermidis, que possuem uma Ser na tríade catalítica, têm seu crescimento inibido pelo tratamento com adenantina (IC50 de 460µM e 77µM, respectivamente), enquanto que para Escherichia coli, que possui Thr nessa posição, a toxicidade da adenantina foi bastante baixa (não foi possível determinar o IC50 nas condições utilizadas). Dessa forma, os dados apresentados neste trabalho demonstram o potencial da utilização da adenantina tanto como antibiótico quanto como antileucêmico

Peroxiredoxins (Prx) are antioxidant enzymes which stand out due the ability to decompose a wide variety of hydroperoxides with high efficiency (106-108M-1s-1) maintaining these molecules at suitable levels to cellular homeostasis and participating in several signaling events. However, it has been shown that, in many tumor types, Prx levels are extremely increased and experiments involving its inactivation have resulted in differentiation or apoptosis of tumor cells. It was recently found a diterpenoid, called adenanthin, that would be the first human Prx1 and Prx2 inhibitor and it was demonstrated that its application in acute myeloid leukemia cells was able to promote differentiation or apoptosis. In this context, this work presents two lines of research: 1) Characterization of structural and functional changes promoted by adenanthin binding to Prx active site using Tsa1 from Saccharomyces cerevisiae as biological model, due to its high similarity to human Prx2. 2) Evaluation of adenanthin dose-dependent antitumor activity over the acute lymphoid leukemia cell lines REH and MOLT-4. As regards the first line of research, our result reveal that Tsa1 is susceptible to inhibition by adenanthin, since the treatment with this binder reduced the hydrogen peroxide decomposition velocity in ~ 66%. In addition, the replacement of Thr44 from Tsa1, aminoacid belonging to the so-called catalytic triad, by a Ser resulted in a protein more susceptible to alterations in secondary structure and to peroxidase activity inhibition in function of adenanthin binding, presenting ~85% of decrease in reaction velocity. Similar characteristics were observed for Tsa2 proteoform from S. cerevisiae, which naturally carries the substitution of Thr44 by Ser. Prx sequences analyzes in databases revealed that mostly Ser-containing proteins are found in prokaryotic organisms, many of them pathogenic ones. Finally, we demonstrate through cytotoxicity assays that the bacteria Staphylococcus aureus and Staphylococcus epidermidis, which have a Ser in catalytic triad, have their growth inhibited by adenanthin treatment (IC50 of 460µM and 77µM, respectively), whereas for Escherichia Coli, which has Thr at that position, the tocyxicity of adenanthin was quite low (it was not possible to determine the IC50 under the used conditions). Regarding the second line of investigation, we found that adenanthin is able to induce the death of leukemic cell lines REH and MOLT-4, and for the last one, there was an unexpected proliferation of cells treated by the longest incubation period (72 hours), characterizing a possible indication of differentiation process. In this sense, the data presented here demonstrate the potential of adenanthin use in both antibiotic and antileukemic treatment

Effect of vaginally administered fumagillin on immunohistochemical distribution of leukemia inhibitory factor, interleukin 6, transforming growth factor beta and vascular endothelial growth factor in implantation stage endometrium of the rhesus monkey.

Intravaginal administration of an anti-angiogenic agent, fumagillin, during blastocyst implantation inhibits pregnancy establishment in a dose-related manner in the rhesus monkey. In the present study, mated female rhesus monkeys were vaginally inserted with tampons containing vehicle (group 1; n = 5) and test agent (fumagillin, 4 mg/animal; group 2; n = 6) on cycle day 20, and endometrial tissue samples were collected on cycle day 24 from all monkeys and processed for histological examination and immunohistochemical localization for LIF, IL-6, TGF-beta and VEGF. Concentrations of estradiol-17 beta, progesterone and chorionic gonadotrophin in peripheral circulation were determined. From the serum profiles of the hormones, 2 monkeys in group 1, and 1 monkey in group 2 appeared pregnant. However, endometrial morphology revealed histological evidence of pregnancy in 3 out of 6 fumagillin-treated animals. Histometric analysis of immunohistochemical staining in epithelial, stromal and vascular compartments revealed that per cent areas occupied by immunoprecipitate for the cytokines studies did not change in epithelial and stromal compartments, except that for TGF-beta which was higher (P < 0.05) in epithelial compartment in group 2. No change was observed in immunoprecipitation areas for IL-6 in epithelial, stromal and vascular compartments. On the other hand, changes (P < 0.05) for LIF, TGF-beta and VEGF were evident in the vascular compartment. It is possible that disparate responses observed in glandular, stromal and vascular compartments in implantation stage endometrium following fumagillin treatment actually caused from associated decline in progesterone concentration in peripheral circulation. It is also possible that fumagillin, an angiostatic agent, affects the synthesis and secretion of cytokines primarily in the vascular compartment of implantation stage endometrium, and thereby manifests differential responses in epithelial, stromal and vascular compartments.

Estudio comparativo "in vitro" de la inhibición del crecimiento bacteriano del cemento de conductos experimental Endobalsam y los selladores de conductos Endometasona, Grossman, Tubliseal y óxido de zinc-eugenol / An in vitro comparative study of the bacterial growth inhibition of Endobalsam experimental root canal sealer and of Endomethasone, Grossman, Tubliseal and zinc-eugenol oxide root canal sealers

Se comparó la acción inhibitoria de un cemento experimental de conductos a base de bálsamo del Perú con cuatro preparaciones con óxido de zinc-eugenol (Endometasona, Grossman, Tubliseal y óxido de zinc-eugenol) sobre el crecimiento de bacterias potencialmente patógenas de la pulpa y periápice (Staphylococcus aureus, estreptococos alfa-hemolíticos y Escherichia coli), usando mo modelo experimental el mótodo de sensibilidad antimicrobiana en placas Petri. Las cepas de S. aureus y E. coli fueron sembradas en placas Petri on agar Mueller Hinton y las cepas de estreptococos alfa-hemolíticos en agar soya tripticasa como base y sangre de carnero al 5 por ciento. A cada placa de agar inoculada con estos microorganismso se le realizó cinco pocillos. Mezclas frescas de los cementos fueron situadas dentro de cada pocillo. Todas las placas preparadas fueron incubadas a 37 grados C. Las lecturas se hicieron midiendo los diámetros de los halos de inhibición a las 24, 48 horas y 7 días. Para obtener validez estadística se repitió 9 veces todo el procedimiento. Todos los selladores estudiados inhibieron en algún grado el crecimiento de bacterias utilizadas. El cemento experimental Endobalsam demostró tener cierta actividad inhibitoria "in vitro" sobre el crecimiento bacteriano, requisito importante de un buen sellador de conductos

Actividad inmunoadyuvante del Staphylococcus aureus: efecto sobre la respuesta humoral y el sistema fagocítico mononuclear en ratas / Inmunoadyuvant activity of staphylococcus aureus:effect on humoral response and mononuclear phagocytic system in rats

Se estudió el efecto del S. aureus y de varios tipos celulares sobre: a) producción de anticuerpos (hemaglutininas) y b) estimulación del Sistema Fagocítico Mononuclear (S.F.M.). Ratas adultas, endocriadas,recibieron:I) células de exudado peritoneal (CEP); II) células del bazo (CB); III) células de bazo no-adherentes (CBN-A); IV) células de exudado peritoneal normal (CEPN); V) células de bazo normal (CBN); VI) células de bazo normal no-adherentes (CBN N-A); VII) S. aureas s.c. y VIII) (-). I, II y III proveían de ratas inoculadas 7 días antes con S. aureus y IV,V y VI de animales normales. Se sensibilizó con glóbulos rojos de carnero al 50 por ciento. Se determinó el título de hemaglutininas séricas totales y con 2 mercapto etanol en la Respuesta primaria y en la Respuesta secundaria, posterior al segundo inóculo. Se estimó peso y se practicó un estudio anatomopatológico del hígado y bazo de los animales tratados con S. aureus y testigos. Se hallaron diferencias significativas en el título de hemaglutininas en todos los días estudiados (Respuesta primaria y secundaria)-p<0,0l;p<0,05-entre los grupos I,II,III,VII vs. IV,V,VI y VIII. Se pesó el hígado y bazo (corregido/peso del animal) en ratas inoculadas con S. aureus (1) y sin tratamiento o control (2). Los valores fueron significativamente mayores en (1) (p<0,001). Hígado: a) 5,746 Ð 0,67; b) 3,609 Ð 0,456. Bazo: a)0,5698 Ð 0,067; b) 0,325 Ð 0,05. Las modificaciones anatomopatológicas en hígado y bazo se observaron con mayor frecuencia e intensidad en los animales tratados que en los testigos (p<0,01). Se confirma el efecto adyuvante del S. aureus, ya que produce aumento de peso e importantes modificaciones anatomopatológicas del órganos del S.F.M., eleva la respuesta humoral a un antígeno no relacionado y estimula la memoria inmunológica

The Azadirachtins: potent insect growth inhibitors

In the course of their coevolution with insects, plants have learnt to protect themselves by chemical means. Semiochemical act as antifeedants or deterrents, others by disrupting growth and development. By use of the Epilachna varivestis bioassay we isolated from Azadirachta indica seed a group of triterpenoids which interfee with larval growth and development in ppm range. Main components are the azadirachtins A and B with identical biological activity. Various other azadirachtins were obtained, either as minor seed components or by chemical modification of the naturally occuring compounds. Structure vs. activity relation studies enabled us to postulate a basic structural element that should still be biologically active and with much simpler chemical structure than natural compounds. What underlies the biological activity of these insect growth inhibitors? Their interference with the hormonal regulation of development and reproduction has been studied in Locusta migratoria and Rhodnius prolixus. In addition, tritiated dihydroazadirachtin A was used. With this approach, a precise correlation between administered dose, resulting effects, and retention of the compound was established. The azadirachtins either interrupt, delay, or deviate whole developmental programs. Results from these studies provide another chemical probe for studies in insect endocrinology and physiology.