Evaluación de la proteólisis en células aisladas vivas de Plasmodium falciparum aisladas durante los estadios asexuales sanguíneos / Proteolysis assessment in isolated Plasmodium falciparum living cells at the asexual blood stages

It has been demonstrated that proteases play crucial roles in Plasmodium falciparum infection and therefore have been considered as targets for the development of new therapeutic drugs. The aim of this study was to describe the specific proteolytic activity profile in all blood stages of P. falciparum isolated parasites in order to explore new antimalarial options. For this purpose, we used the fluorogenic substrate Z-Phe-Arg-MCA (Z: carbobenzoxy, MCA: 7-amino-4-methyl coumarine) and classic inhibitors for the different classes of proteolytic enzymes, such as phenylmethylsulfonyl fluoride (PMSF), 1.10-phenantroline, pepstatin A and E64 to study the inhibition profiles. As expected, due to the high metabolic activity in mature stages, the substrate was mostly degraded in the trophozoite and schizont, with specific activities ~ 20 times higher than in early stages (merozoite/rings). The major actors in substrate hydrolysis were cysteine proteases, as confirmed by the complete hydrolysis inhibition with E64 addition. Proteolytic activity was also inhibited in the presence of PMSF in all but the schizont stage. However, PMSF inhibition was the result of unspecific interaction with cysteine proteases as demonstrated by reversion of inhibition by dithiotreitol (DTT), indicating that serine protease activity is very low or null. To our knowledge, this is the first report aiming to describe the proteolytic profile of P. falciparum isolated parasites at all the erythrocytic cycle stages. The results and protocol described herein can be useful in the elucidation of stage specific action of proteolysis-inhibiting drugs and aid in the development of antimalarial compounds with protease inhibitory activity(AU)

e ha demostrado que las proteasas desempeñan funciones vitales en la infección por Plasmodium falciparum, y por lo tanto se consideran dianas en la elaboración de nuevos medicamentos terapéuticos. El objetivo del estudio era describir el perfil de actividad proteolítica específica de todas las etapas sanguíneas de parásitos aislados de P. falciparum con vistas a explorar nuevas opciones antimaláricas. Con ese propósito, utilizamos el sustrato fluorogénico Z-Phe-Arg-AMC (Z: carbobenzoxi, AMC: 7-amino-4-metilcumarina) e inhibidores clásicos para las diferentes clases de enzimas proteolíticas, tales como el fluoruro de fenilmetilsulfonilo (PMSF), 1,10-fenantrolina, pepstatina A y E64 para estudiar los perfiles de inhibición. Como se esperaba, debido a la elevada actividad metabólica de las etapas de madurez, el sustrato fue degradado mayormente en el trofozoíto y el esquizonte, con actividad específica ~ 20 veces superior a la de las etapas tempranas (merozoíto/ anillos). Los principales actores en la hidrólisis del sustrato fueron las cisteínas proteasas, lo que fue confirmado por la inhibición completa de la hidrólisis con la adición de E64. La actividad proteolítica también fue inhibida en presencia de PMSF en todas las etapas excepto el esquizonte. Sin embargo, la inhibición del PMSF fue resultado de una interacción inespecífica con las cisteínas proteasas, según lo demuestra la reversión de la inhibición con el ditiotreitol (DTT), lo que indica que la actividad de la serina proteasa es muy baja o inexistente. Que sepamos, este es el primer informe dirigido a describir el perfil proteolítico de parásitos aislados de P. falciparum en todas las etapas del ciclo eritrocítico. Los resultados y el protocolo que aquí se describen pueden ser útiles para dilucidar la acción específica de los medicamentos inhibidores de proteólisis en cada etapa, así como contribuir al desarrollo de compuestos antimaláricos con actividad inhibidora de la proteasa(AU)

Cell Death and Its Different Modes: History of Understanding and Current Trends

Discussions about what is life continue to struggle; there are pros and cons for whether a virus is alive. However, an opposite thing –cell death –appears to be tantamount important and equally not-easygoing to define. Nevertheless, our current knowledgeabout eukaryotic cell death has made a long way and resulted in a fruitful outcome: starting from three types of cell death (type I, II and III which are mainly applicable to eukaryotic cells of organisms from the biological kingdom animalia) in 1970s, Nomenclature Committee on Cell Death has named already twelve cell death forms in 2018, including the above mentioned apoptosis, autophagy and necrosis among them. How the scientific attitude towards cellular demise evolved and various aspects of different cell death modes are reviewed in this article.

Effect of procyanidin on expression of Caspase -3 in SD rats with type 2 diabetes mellitus and focal cerebral ischemia / 中国基层医药

Objective To investigated the effect of procyanidin (PC) on the expression of cysteine proteinase -3 (Caspase -3) in type 2 diabetes mellitus SD rats with focal cerebral ischemia. Methods Following the random principle, 40 healthy Sprague - Dawley (SD) rats were numbered sequentially and randomly divided to normal rats with focal cerebral ischemia group,type 2 diabetes mellitus SD rats with focal cerebral ischemia group,PC low/ middle/ high -dose groups,with 8 rats in each group. The type 2 diabetes mellitus - MCAO model was set up. The doses of PC for low,middle and high - dose groups were 50 mg/ kg,100 mg/ kg,200 mg/ kg. Immunohistochemistry method was used to measure the activity of Caspase - 3. Results Compared with that in the normal rats with focal cerebral ischemia group[(11. 42 ±2. 52)],the expression of Caspase -3 increased in the type 2 diabetes with ischemia group[(15. 00 ± 2. 38)](t = 2. 17,P < 0. 01). Compared with that in the type 2 diabetes with ischemia group,the expression of Caspase - 3 decreased in the PC groups[(9. 38 ± 2. 00),(7. 71 ± 1. 55),(6. 96 ± 1. 57)](t = 2. 86,3. 13,3. 36,all P < 0. 01),whereby the middle and high - dose groups showed more significant decrease (t = 1. 92,2. 03,all P <0. 01) and with no statistically significant difference between the two groups(t = 1. 13,P > 0. 05). Conclusion PC can decrease the expression of Caspase - 3 protein in type 2 diabetes mellitus SD rats with focal cerebral ischemia, finally may inhibit the apoptosis.

Aplicação de planejamento baseado na estrutura do receptor na busca de inibidores de cisteíno-proteases parasitárias (cruzaína (T. cruzi) e PCB (Leishmanioses) / Structure-based virtual screening in the search of parasitic cysteine-proteases inhibitors

Doenças causadas por agentes infecciosos e parasitários são chamadas negligenciadas por não despertarem interesse das indústrias farmacêuticas para o desenvolvimento de novas alternativas terapêuticas. Essas doenças são responsáveis por levar milhões de pessoas à morte todos os anos e afetam principalmente os países pobres e em desenvolvimento. Dentre estas, a doença de Chagas e as leishmanioses, parasitoses causadas por parasitas flagelados pertencentes à família Trypanosomatidae, T. cruzi e Leishmaina sp., respectivamente, se apresentam como um sério problema de saúde pública mundial. Endêmicas em vários países e causando milhões de mortes anualmente, ainda hoje não existem fármacos eficientes e seguros para o tratamento dessas doenças. Este panorama torna eminente a necessidade de pesquisa e desenvolvimento de novos fármacos para essas parasitoses. A busca por agentes quimioterápicos envolve a seleção de vias metabólicas essenciais à sobrevivência dos parasitas. Dentre estas, destacamse cisteíno-proteases presentes nesses tripanossomatídeos, deste modo a cruzaína no T. cruzi, e a CPB2.8 na Leishmania mexicana, se mostram como alvos bioquímicos promissores. A disponibilidade de estruturas cristalográficas da cruzaína e do sequenciamento genômico da CPB2.8, nos permite utilizar estratégias de planejamento de fármacos baseado no receptor (SBDD) na identificação de candidatos a fármacos para essas doenças. Entre as técnicas modernas de SBDD utilizadas, a triagem virtual possibilita identificar promissores candidatos a novos fármacos. Assim neste trabalho, obteve-se por meio da técnica de modelagem comparativa o modelo da enzima CPB2.8 de L. mexicana, visto a indisponibilidade da estrutura cristalográfica no Protein Data Bank (PDB). De modo a refinar o modelo construído realizou-se a simulação por dinâmica molecular de 100ns, apresentando estabilização a partir de 80ns. A simulação por dinâmica molecular foi validada por meio do gráfico de Ramachandran, gráfico de raio de giro, RMSD, gráfico de superfície hidrofóbica. Foram calculados os mapas de interação molecular no programa GRID das seguintes proteínas: cruzaína, CPB2.8, catepsina B e catepsina L, e, posteriormente, foi construído um modelo farmacofórico baseado no sítio ativo das enzimas cruzaína e CPB2.8. O modelo farmacofórico da cruzaína foi validado por curva ROC apresentando valor de AUC 61%. A triagem virtual foi realizada para ambas as proteínas e foram obtidos 369 compostos para a cuzaína e 225 compostos para a CPB2.8. Foi realizado o ancoramento molecular desses compostos obtidos pela triagem virtual a fim de diminuir a quantidade de compostos a serem avaliados experimentalmente

Neglected diseases are caused by parasites and infectious agents and affect mainly people in poor areas being prevalent in 149 countries and causing 534,000 deaths per year. Among neglected diseases we can highlight Chagas Disease and Leishmaniasis, both have a high rate of morbidity and mortality and both are addressed in this project in the search of new drugs against a NTD. Nowadays, the search for new drugs involves the selection of biological pathways essential for parasite survival, in this class of parasites we can suggest the cysteine proteases, a proteases family present in Trypanosoma cruzi and and Leishmania ssp. In order to obtain a new agent against Neglected Disease in this work was obtained the model of the enzyme CPB2.8 of L. mexicana using the comparative modeling technique, due to the unavailability of the crystallographic structure in the Protein Data Bank (PDB). In order to refine the constructed model was performed the molecular dynamics simulation of 100ns, stabilization was achieved from 80ns. Molecular dynamics simulation was validated using the Ramachandran graph, radius of rotation graph, RMSD, hydrophobic surface area graph. The molecular interaction fields were calculated in the GRID program to cruzain, CPB2.8, cathepsin B and cathepsin L. Based on molecular interaction fields generated pharmacophoric models were constructed using information about the active site of the enzymes cruzain and CPB2.8. The pharmacophoric model of cruzain was validated by ROC curve presenting AUC value of 61%. Virtual screening was performed for both proteins and 369 compounds were obtained for cuzain and 225 compounds for CPB2.8. Docking studies of these compounds was performed in order to decrease the amount of compounds to be evaluated experimentally

Observation of the effect of clopidogrel combined with aspirin in the treatment of ischemic cerebrovascular disease and its influence on serum HIF-1 alpha and caspase-3 / 中国基层医药

Objective To compare the effect of clopidogrel combined with aspirin and dipyridamole combined with aspirin in the treatment of ischemic cerebrovascular disease,and to analyze its influence on the levels of serum hypoxia-inducible factor 1 alpha (HIF-1 alpha),cysteine proteinase 3 (caspase-3).Methods 96 patients with ischemic cerebrovascular disease were selected,and they were randomly divided into A group (n =48) and B group(n =48) according to the digital table.A group was treated with clopidogrel and aspirin,B group was treated with dipyridamole and aspirin.The clinical efficacy,adverse reactions and serum HIF-1 alpha,caspase-3 expression before and after treatment were compared between the two groups.Results The total effective rate of A group was 93.8％,which was significantly higher than 83.3％ of B group (x2 =12.760,P ＜ 0.05).Before treatment,the MESSS score had no statistically significant difference between the two groups(t =0.174,P ＞ 0.05).After 4 weeks of treatment,the MESSS score of A group was (5.07 ± 1.26) points,which of B group was (6.12 ± 1.48) points,the MESSS scores of the two groups were decreased significantly than those before treatment,and the MESSS score of A group after treatment was significantly lower than that of B group (t =7.306,P ＜ 0.05).Before treatment,the levels of HIF-1 alpha,caspase-3 had no statistically significant differences between the two groups (t =0.205,0.337,all P ＞ 0.05).After 4 weeks of treatment,the levels of HIF-1 and caspase-3 in A group were (248.55 ± 12.44) ng/mL,(7.34 ± 1.77) ng/mL,respectively,which in B group were (301.63 ± 14.86) ng/mL,(8.85 ± 1.94) ng/mL,respectively.The levels of HIF-1 alpha and caspase-3 levels in A group were significantly lower than those in B group (t =8.778,9.156,all P ＜ 0.05).The incidence rate of adverse reaction of A group was 4.2％,which was significantly lower than 10.4％ of B group (x2 =8.461,P ＜ 0.05).Conclusion Compared with dipyridamole combined with aspirin,clopidogrel combined with aspirin in the treatment of ischemic cerebrovascular disease can effectively improve the patients' neurological impairment,inhibit the expression of serum HIF-1,caspase-3,with less adverse reaction,and it is worthy of application and promotion.

Análisis de anticuerpos antifosfolipídicos y cistatina C en pacientes con esclerosis múltiple / Analysis of antiphospholipid antibodies and cystatin C in multiple sclerosis patients / Teste de anticorpos antifosfolípídes e cistatina C em pacientes com esclerose múltipla

La cistatina C es considerada el inhibidor fisiológico más importante de las proteasas de cisteína endógenas. Se cree que el papel de la cistatina C es el de modular la actividad de las proteasas secretadas o liberadas de células dañadas o en proceso de necrosis, siendo por tanto las cistatinas fundamentales para los procesos de regulación y prevención del potencial daño proteolítico local. Los anticuerpos antifosfolípidos se usan para esclarecer el diagnóstico de esclerosis múltiple (EM) ya que existen patologías que pueden cursar con sintomatología o hallazgos paraclínicos semejantes. El objetivo de este trabajo fue analizar la concentración de cistatina C y la presencia o ausencia de anticuerpos antifosfolipídicos en pacientes diagnosticados de esclerosis múltiple remitente recurrente (EMRR) como marcadores de desmielinización. Este trabajo se llevó a cabo conjuntamente por el laboratorio de Riesgo Vascular, el laboratorio de Autoinmunidad y la Unidad de Esclerosis Múltiple del Hospital Universitario Virgen Macarena de Sevilla, España, con una duración de un año. Se seleccionaron dos tipos de poblaciones: grupo 1, n=30 pacientes con EMRR y un segundo grupo, denominado grupo control, n=30. Se determinó cistatina C y anticuerpos antifosfolípidos IgG e IgM, anticuerpos anticardiolipina IgG e IgM y anticuerpos f>2 glicoproteína IgG e IgM. Los pacientes diagnosticados de EMRR presentan títulos negativos de anticuerpos antifosfolípidos IgG e IgM, anticardiolipina IgG e IgM y f>2 glicoproteína IgG e IgM. La concentración de cistatina C es menor en el grupo de pacientes diagnosticados de EM, lo que podría producir un déficit en la modulación de las proteasas de cisteína endógenas. Dicha desmielini-zación agudizaría el progreso de la EM.

Cystatin C is considered the most important physiological inhibitor of endogenous cysteine proteases; the role of cystatin C is believed to be to modulate the activity of proteases secreted or released from damaged cells or in the process of necrosis, therefore cystatins being fundamental regulatory processes and a potential prevention of local proteolytic damage. Antiphospholipid antibodies are used to clarify the diagnosis of diseases like multiple sclerosis (MS) and other pathologies could present similai symptoms or paraclinical findings. The objective of the present work is to analyze the concentration of cystatin C and the presence or absence of antiphospholipid antibodies in patients diagnosed with relapsing remitting multiple sclerosis (RRMS) as markers of demyelization. This work was carried out jointly by the Vascular Risk Laboratory, the Laboratory of Autoimmunity and Multiple Sclerosis Unit, Hospital Universitario Virgen Macarena in Seville in one year. Two types of people were selected: Group 1 (n = 30) RRMS group and a control group, n = 30. Cystatin C and antiphospholipid antibodies IgG and IgM, IgG and IgM anticardiolipin, $2 glycoprotein IgG and IgM were determined. Patients showed negative titers of antiphospholipid antibodies IgG and IgM, IgG and IgM anticardiolipin, $2 glycoprotein IgG and IgM. Cystatin C concentration is lower in the group of patients diagnosed with MS, which could give rise to a decrease in the modulation of endogenous cysteine proteases. This would exacerbate the progress of demyelization in MS.

A cistatina C é considerada o inibidor fisiológico das proteases de cisteína endógenas mais importante. Acredita-se que o papel da cistatina C é o de modular a atividade de proteases secretadas ou liberadas a partir de células danificadas ou em processo de necrose, sendo por isso as cistatinas fundamentais para os processos de regulagao e prevengao do potencial dano proteolítico local. Anticorpos antifosfolípides sao usados para esclarecer o diagnóstico de EM, visto que existem patologias que podem apresentar sintomas ou achados paraclínicos semelhantes. O objetivo deste trabalho foi o de analisar a concentra-gao de cistatina C e a presenga ou ausencia de anticorpos antifosfolípides em pacientes diagnosticados com esclerose múltipla recidivante - remitente (EMRR) como marcadores de desmielinizagao. Este trabalho foi realizado em conjunto pelo laboratório de Risco Vascular, o laboratório de Autoimunidade e a Unidade de Esclerose Múltipla do Hospital Universitario Virgen Macarena, de Sevilha, Espanha, com uma duragao de um ano. Foram selecionados dois tipos de populagdes-. Grupo 1 (n = 30) pacientes com EMRR e um segundo grupo, chamado de grupo controle, n = 30. Determinou-se cistatina C e anticorpos antifosfolípides IgG e IgM, anticorpos anticardiolipina IgG e IgM, e anticorpos $2 glicoproteína IgG e IgM. Pacientes diagnosticados com EMRR apresentam títulos negativos de anticorpos antifosfolípides IgG e IgM, anticardiolipina IgG e IgM e $2 glicoproteína IgG e IgM. A concentragao de cistatina C é menor no grupo de pacientes diagnosticados com EM, o que poderia produzir um déficit na modulagao das proteases de cisteína endógenas. Tal desmielinizagao agravaría o progresso da EM.

Obtención de anticuerpos monoclonales de ratón contra proteasa de cisteína 5 recombinante de Entamoeba histolytica / Production of monoclonal antibodies against cysteine protease 5 of Entamoeba histolytica

Objetivo. Obtener anticuerpos monoclonales de ratón contra la proteasas de cisteína 5 (EhCP5) de Entamoeba histolytica. Materiales y métodos. Se inmunizaron ratones BALB/c por vía intraperitoneal con adyuvante de Freund completo e incompleto con la proteína recombinante EhCP5 obtenida a partir del cultivo de E.coli DH5α trasfectada con el vector recombinante pJC45 que expresa dicha proteína. Se seleccionó el animal con mejor respuesta de anticuerpos. Al cual se le extrajo su bazo como fuente de linfocitos B, los cuales se fusionaron utilizando PEG con células de mieloma de ratón SP2-0/Ag14. Se procedió a selección de los hibridomas y a la evaluación de los sobrenadantes de las colonias que crecieron a los 7 días mediante ELISA. Los hibridomas con valores más altos de anticuerpos específicos contra la proteína EhCP5r se seleccionaron, y los clones obtenidos por diluciones limitantes fueron expandidos. Resultados. A partir de un clon secretor estable se purifico el anticuerpo monoclonal anti EhCP5r del isotipo IgG1 por cromatografía de afinidad con proteína G. Los clones fueron expandidos in vivo ein vitro. Con el anticuerpo purificado se diseñaron tres sistemas de captura para evaluar la aplicabilidad del anticuerpo monoclonal anti EhCP5r como método inmunodiagnóstico. Conclusiones. Se logro la producción de un anticuerpo monoclonal específico contra EhCP5r que permite diferenciar Entamoeba histolytica de Entamoeba dispar.

Objective. Obtain mouse monoclonal antibodies against cysteine proteases 5 (EhCP5) of Entamoeba histolytica. Materials and methods. BALB/c mice were immunized intraperitoneally with complete and incomplete Freund adjuvant EhCP5 with the recombinant EhCP5 protein obtained from E.coli DH5α culture transfected with the recombinant vector pJC45 that expresses said protein. The animal with the best antibody response was selected. Its spleen was extracted as a source of B-lymphocytes, which were merged using PEG miceSP2-0/Ag14 myeloma cells. The team proceeded to undergo the selection of the hybridomas and the evaluation of the supernatants of the colonies that grew after 7 days by ELISA. The hybridomas with higher values of specific antibodies against the protein EhCP5r were selected, and clones obtained by limiting dilution were expanded Results. With the use of a stable secreting clone the monoclonal antibody anti EhCP5r IgG1 isotype was purified by affinity chromatography with protein G. The clones were expanded in vivo and in vitro. Three capture systems were designed with the purified antibody to assess the applicability of the monoclonal antibody anti EhCP5r as an immunodiagnostic method. Conclusions. The production of a specific monoclonal antibody against EhCP5r was achieved to differentiate Entamoeba histolytica from Entamoeba dispar.

Cysteinyl Cathepsins: Multifunctional Enzymes in Cardiovascular Disease / 전남의대학술지

Until recently, the role of lysosomal cysteine protease cathepsins in intracellular protein degradation was believed to be mainly restricted to scavenging. However, recent studies have revealed nontraditional roles for cysteine protease cathepsins in the extracellular space during the development and progression of cardiovascular disease. Although the precise mechanisms are unknown, data from animal studies suggest that members of the cathepsin family, like other extracellular proteases, contribute to extracellular matrix protein remodeling and interstitial matrix degradation, as well as to cell signaling and cell apoptosis in heart disease. Inflammatory cytokines and hormones regulate the expression and secretion of cathepsins in cultured cardiovascular cells and macrophages. Serum levels of cathepsins L, S, and K and their endogenous inhibitor cystatin C may be useful predictive biomarkers in patients with coronary artery disease and cardiac disease. Furthermore, in vivo pharmacological intervention with a synthetic cathepsin inhibitor and cardiovascular drugs (including statins and angiotensin II type 1 receptor antagonists) has the potential for pharmacologic targeting of cathepsins in cardiovascular disease. This review focuses on cathepsin biology (structure, synthesis, processing, activation, secretion, activity regulation, and function) and the involvement of cysteinyl cathepsins in the pathogenesis of several heart and vessel diseases, especially with respect to their potential application as diagnostic and prognostic markers and drug targets to prevent inappropriate proteolysis in cardiovascular disease.

Cysteinyl Cathepsins: Multifunctional Enzymes in Cardiovascular Disease / 전남의대학술지

Until recently, the role of lysosomal cysteine protease cathepsins in intracellular protein degradation was believed to be mainly restricted to scavenging. However, recent studies have revealed nontraditional roles for cysteine protease cathepsins in the extracellular space during the development and progression of cardiovascular disease. Although the precise mechanisms are unknown, data from animal studies suggest that members of the cathepsin family, like other extracellular proteases, contribute to extracellular matrix protein remodeling and interstitial matrix degradation, as well as to cell signaling and cell apoptosis in heart disease. Inflammatory cytokines and hormones regulate the expression and secretion of cathepsins in cultured cardiovascular cells and macrophages. Serum levels of cathepsins L, S, and K and their endogenous inhibitor cystatin C may be useful predictive biomarkers in patients with coronary artery disease and cardiac disease. Furthermore, in vivo pharmacological intervention with a synthetic cathepsin inhibitor and cardiovascular drugs (including statins and angiotensin II type 1 receptor antagonists) has the potential for pharmacologic targeting of cathepsins in cardiovascular disease. This review focuses on cathepsin biology (structure, synthesis, processing, activation, secretion, activity regulation, and function) and the involvement of cysteinyl cathepsins in the pathogenesis of several heart and vessel diseases, especially with respect to their potential application as diagnostic and prognostic markers and drug targets to prevent inappropriate proteolysis in cardiovascular disease.

Caracterización cinética de un preparado semipurificado de bromelina para uso antitumoral / Kinetic characterization of a semipurified preparation from bromelain for antitumor use

INTRODUCCIÓN: la mayor parte de la producción mundial de enzimas está destinada a la obtención de proteasas. Las aplicaciones de estas biomoléculas con fines terapéuticos son muy variadas. Algunas se conocen como remedios naturales y otras son fármacos modificadores de la respuesta biológica. Las plantas de piña contienen varias cisteíno-proteasas, el componente mayoritario aislado del tallo es la bromelina de tallo (EC 3.4.22.32). En el campo de la salud se le atribuyen varias acciones entre las que se destaca la de inducir la diferenciación de células tumorales y atenuar el crecimiento del tumor. OBJETIVOS: caracterizar cinéticamente un preparado semipurificado de bromelina obtenida por un procedimiento desarrollado en Cuba para ser evaluado en el campo de la salud. MÉTODOS: el extracto crudo de bromelina, obtenido a partir de tallos de plantas adultas de Ananas comosus (L.) Merr cv Española roja (piña) se purificó por cromatografía de intercambio iónico en carboximetilcelulosa-52 en un sistema semibach. RESULTADOS: la caracterización cinética del extracto purificado permitió comprobar la existencia de un preparado activo y estable con una actividad específica de 0,94 U/mg de proteínas (2,34 veces superior a la del extracto crudo original), con una fracción proteica mayoritaria de masa molar 24 500 Da y un punto isoeléctrico, localizado en la zona de pH básico (9,55). Tiene pH óptimo 6,8 al utilizar hemoglobina 2 por ciento como sustrato a 37 ºC y una estabilidad en función del pH y de la temperatura aceptable. Si se conserva liofilizado a - 20 ºC mantiene 80 por ciento de la actividad durante 1 año. CONCLUSIONES: la cromatografía de intercambio iónico en carboximetilcelulosa-52 posibilitó la obtención de un preparado semipurificado de bromelina, con una actividad específica superior a la del extracto original que puede ser utilizado en la medicina como antitumoral

INTRODUCTION: most of the world production of enzymes is devoted to proteases obtaining. Applications of these biomolecules for therapeutical objectives are very varied. Some are known as natural remedies and other are drugs modifying the biological response. Pineapple plants contain some cysteine-proteases, the majority component isolated from stem is the stem Bromelain (EC 3.4.22.32). In the health field it has attributed some actions including the differentiation induction of tumor cells and attenuation of tumor growth. OBJECTIVES: to characterize kinetically a semipurified preparation from Bromelain obtained by a procedure developed in Cuba to be assessed in the health field. METHODS: the crude extract from Bromelain obtained from the adult plant stems of Ananas comosus (L.) Merr Cv Spanish red (pineapple) was purified by ion-exchange chromatography in carboxylmethylcelulose-52 in semibatch system. RESULTS: kinetic characterization of purified extract allowed us to verify the existence of a stable and active preparation with a specific activity of 0,94 U/mg of proteins (2,34 times superior to that of original crude extract) with a protein fraction in the main of molar mass 24 500 Da and a isoelectric point, located in the basic pH zone (9,55). It has a 6,8optimal pH using the 2 percent hemoglobin as substrate at 37ºC and stability depending on pH and on acceptable temperature. If it is preserved lyophilized at 20 ºC it maintains the 80 percent of activity for a year. CONCLUSIONS: the ion-exchange chromatography in carboxyl methylcellulose-52 allowed the achievement of semipurified preparation from Bromelain with a specific activity higher to that of original extract that may to be used in medicine as antitumor agent

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na+-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na+-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na+-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na+ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na+-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A2 (cPLA2). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA2 specific inhibitor AACOCF3. ATP depletion-induced alterations in Na+-dependent phosphate uptake and cell viability were prevented by AACOCF3. Inhibition of Na+-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA2 activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

Mycophenolic Acid Induced Apoptotic Signal Transduction in Molt-4 T-cells

PURPOSE: Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH), is the active metabolite of the immunosuppressive drug, mycophenolate mofetil (MMF). MMF is used to prevent an immune- mediate rejection response following organ transplantation via the inhibition of the IMPDH and GTP biosynthesis pathway. This study was designed to elucidate the mechanism by which MPA exerts its cytotoxic effect on human T lymphocytic and monocytic cell lines. METHODS: MOLT-4 and U937 cell lines were treated with MPA. Cell viability, expression of Bcl2 family proteins and Fas/Fas-L, effects of antioxidants and intracellular Ca2+ regulating agents and apoptosis were measured using a variety of microscopic and biochemical techniques. RESULTS: MPA induced the death of U937 and MOLT-4 cells in dose and time dependent manners, which was revealed an apoptosis with a characteristic ladder pattern of DNA fragmentation. In addition, BAPTA/AM, an intracellular Ca2+ chelator protected MOLT-4 cells from MPA treated apoptosis, although it did not have an additive with thapsigargin, and increases cytosolic Ca2+ stores. However, antioxidants including reduced glutathione (GSH) and N-acetyl-L-cysteine (NAC) did not inhibit the apoptosis of cells by MPA. Furthermore, guanosine suppressed MPA induced apoptosis of MOLT-4 lymphocytes, although adenosine did not. MPA also increased the catalytic activity of caspase family cysteine proteases including caspase-8, 9 and 3 proteases in MOLT-4 cells. Sequential activation indicated that the cleavage of caspase-8 and 9 precedes those of caspase-3. CONCLUSION: The results suggest that MPA induces the apoptotic death of MOLT-4 lymphocytes via the activations of caspase family proteases and the depletion of GTP.