Quinolinotriazole antiplasmodials via click chemistry: synthesis and in vitro studies of 7-Chloroquinoline-based compounds

Malaria is nowadays one of the most serious health concerns in a global scale and, although there is an evident increase in research studies in this area, pointed by the vast number of hits and leads, it still appears as a recurrent topic every year due to the drug resistance shown by the parasite exposing the urgent need to develop new antimalarial medications. In this work, 38 molecules were synthesized via copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) or "click" chemistry, following different routes to produce 2 different organic azides, obtained from a 4,7 dicholoquinoline, reacted with 19 different commercially available terminal alkynes. All those new compounds were evaluated for their in vitro activity against the chloroquine resistant malaria parasite Plasmodium falciparum (W2). The cytotoxicity evaluation was accomplished using Hep G2 cells and SI index was calculated for every molecule. Some of the quinoline derivatives have shown high antimalarial activity, with IC50 values in the range of 1.72-8.66 µM, low cytotoxicity, with CC50>1000 µM and selectivity index (SI) in the range of 20-100, with some compounds showing SI>800. Therefore, the quinolinotriazole hybrids could be considered a very important step on the development of new antimalarial drugs

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)

Plants of the Araceae family for malaria and related diseases: a review / Plantas da família Araceae para a malária e doenças relacionadas: uma revisão

ABSTRACTIn the current work we performed a review of the Araceae family species traditionally used to treat malaria and its symptoms. The aim is to reveal the large number of antimalarial Araceae species used worldwide and their great unexplored potential as sources of antimalarial natural products. The SciFinder Scholar, Scielo, PubMed, ScienceDirect and Google books search engines were consulted. Forty-three records of 36 species and 23 genera of Araceae used for malaria and symptoms treatment were found. The neotropical genera Philodendron Schott and Anthurium Schott were the best represented for the use in the treatment of malaria, fevers, liver problems and headaches. Leaves and tubers were the most used parts and decoction was the most common preparation method. The extracts of Araceae species inhibit the in vitro growth of the human malaria parasite, the Plasmodium falciparum Welch, and significant median inhibitory concentrations (IC50) for extracts of guaimbê-sulcado (Rhaphidophora decursiva (Roxb.) Schott), aninga (Montrichardia linifera (Arruda) Schott), Culcasia lancifolia N.E. Br. and forest anchomanes (Anchomanes difformis (Blume) Engl.) have been reported demonstrating the antimalarial and cytotoxicity potential of the extracts and sub-fractions. In the only report about the antimalarial components of this family, the neolignan polysyphorin and the benzoperoxide rhaphidecurperoxin presented strong in vitro inhibition of the D6 and W2 strains of Plasmodiumfalciparum (IC50 = 368-540 ng/mL). No live study about antimalarial activity in animal models has been conducted on a species of Araceae. More bioguided chemical composition studies about the in vitro and also thein vivo antimalarial activity of the Araceae are needed in order to enhance the knowledge about the antimalarial potential of this family.

RESUMONo presente trabalho realizamos uma revisão das espécies da família Araceae usadas para tratar malária e seus sintomas. O objetivo foi revelar o grande número de espécies da família usadas no mundo, assim como seu potencial como fontes de produtos naturais antimaláricos. Foram consultadas as plataformas de busca SciFinder Scholar, Scielo, PubMed, ScienceDirect e Google books. Encontramos quarenta e três registros de 36 espécies e 23 generos de Aráceas usadas para tratar malária e seus sintomas. Os generos neotropicais Philodendron Schott e Anthurium Schott foram os melhor representados, úteis para o tratamento da malária, febres, problemas hepáticos e dores de cabeça. Folhas e tubérculos foram as partes mais utilizadas, enquanto a decocção foi o método de preparo mais comum. Os extratos de espécies de Araceae inibem o crescimento in vitro do parasito da malária humana, Plasmodium falciparum Welch, e concentrações inibitórias medianas (CI50) significativas foram relatadas para extratos de guaimbê-sulcado (Rhaphidophora decursiva (Roxb.) Schott), aninga (Montrichardia linifera (Arruda) Schott), Culcasia lancifoliaN.E. Br. e anchomanes do mato (Anchomanes difformis (Blume) Engl.), demonstrando o potencial antimalárico e citotóxico de extratos e subfrações. No único relato sobre os componentes antimaláricos dessa família, a neolignana polisiforina e o benzoperóxido rafidecurperoxina apresentaram forte inibição das cepas D6 e W2 de Plasmodiumfalciparum in vitro (CI50 = 368-540 ng/mL). Nenhum estudo sobre a atividade antimalárica in vivo em modelo animal foi realizado com espécies da família Araceae. Mais trabalhos biomonitorados pela composição química sobre a atividade antimalárica in vitro, assim como estudos in vivo, são necessários para aprofundar os conhecimentos sobre potencial antimalárico da familia.

Partial characterization of Plasmodium falciparum trophozoite proteome under treatment with quinine, mefloquine and the natural antiplasmodial diosgenone / Caracterización parcial del proteoma del trofozoíto de Plasmodium falciparum bajo tratamiento con quinina, mefloquina y el compuesto natural diosgenona

Introduction: Despite efforts to control malaria, around 10% of the world population is at risk of acquiring this disease. Plasmodium falciparum accounts for the majority of severe cases and deaths. Malaria control programs have failed due to the therapeutic failure of first-line antimalarials and to parasite resistance. Thus, new and better therapeutic alternatives are required. Proteomic analysis allows determination of protein expression levels under drug pressure, leading to the identification of new therapeutic drug targets and their mechanisms of action. Objective: The aim of this study was to analyze qualitatively the expression of P.falciparum trophozoite proteins (strain ITG2), after exposure to antimalarial drugs, through a proteomic approach. Materials and methods: In vitro cultured synchronized parasites were treated with quinine, mefloquine and the natural antiplasmodial diosgenone. Protein extracts were prepared and analyzed by two-dimensional electrophoresis. The differentially expressed proteins were selected and identified by MALDI-TOF mass spectrometry. Results: The following proteins were identified among those differentially expressed in the parasite in the presence of the drugs tested: enolase (PF10_0155), calcium-binding protein (PF11_0098), chaperonin (PFL0740c), the host cell invasion protein (PF10_0268) and proteins related to redox processes (MAL8P1.17). These findings are consistent with results of previous studies where the parasite was submitted to pressure with other antimalarial drugs. Conclusion: The observed changes in the P. falciparum trophozoite protein profile induced by antimalarial drugs involved proteins mainly related to the general stress response.

Introducción. A pesar de los esfuerzos para controlar la malaria, esta sigue siendo un problema de salud pública. Plasmodium falciparum es responsable de la mayoría de los casos graves y de las muertes. Los programas de control de la malaria han sido cuestionados debido al fracaso del tratamiento y a la resistencia del parásito a los antipalúdicos de primera línea, por lo que se requieren nuevas y mejores alternativas. El análisis proteómico permite identificar y determinar los niveles de expresión de las proteínas bajo la presión de los medicamentos, lo que posibilita la identificación de nuevos blancos terapéuticos y mecanismos de acción. Objetivo. Analizar cualitativamente la expresión diferencial de proteínas del citosol del trofozoíto de P. falciparum bajo tratamiento con quinina, mefloquina y el compuesto natural diosgenona mediante una aproximación proteómica. Materiales y métodos. Se trataron trofozoítos sincronizados y cultivados in vitro de P. falciparum (cepa ITG2) con quinina, mefloquina y el compuesto natural diosgenona. Los extractos proteicos se prepararon y analizaron por electroforesis bidimensional. Las proteínas con aparente expresión diferencial se seleccionaron e identificaron mediante espectrometría de masas MALDI-TOF. Resultados. Se encontraron las siguientes proteínas diferencialmente expresadas en el trofozoíto: la enolasa (PF10_0155), la proteína de unión a calcio (PF11_0098), la chaperonina (PFL0740c), la proteína de invasión a la célula del huésped (PF10_0268) y la proteína relacionada con procesos de reducción y oxidación (redox) (MAL8P1.17). Estos hallazgos son congruentes con resultados previos de estudios en los que el parásito fue presionado con otros medicamentos antipalúdicos. Conclusión. Los cambios observados en el perfil de proteínas del trofozoíto de P. falciparum tratado con antipalúdicos involucraron preferencialmente proteínas relacionadas con la respuesta al estrés general.

Identification of Anopheles fauna in a hyperendemic falciparum area of Orissa State, India.

BACKGROUND & OBJECTIVE: Keonjhar district of Orissa State has been hyperendemic for falciparum malaria since many years with alarming deaths due to cerebral malaria. Therefore an entomological investigation to know more about the relative prevalence of Anopheles species was done. METHODS: Daytime indoor resting and outdoor resting, light trap and double bed net collections were made. Surveys were also made to collect Anopheles immature in streams and paddy fields. The Anopheles mosquitoes obtained by different catching methods were identified and the known vector species were subjected to gut and salivary gland dissection for vector incrimination. The infected specimens of An. fluviatilis and An. minimus were subjected to polymerase chain reaction assay for identification of sibling species. RESULTS: Of the anophelines collected, the most abundant was An. splendidus (18.2%) and An. fluviatilis (17.7%), followed by An. maculatus (14.0%) and An. minimus (9.0%). The sporozoite rate of An. fluviatilis and An. minimus was 0.9 and 1.4 respectively. The infected specimens have been identified as sibling species S of the An. fluviatilis complex and A of the An. minimus complex. INTERPRETATION & CONCLUSION: An. fluviatilis and An. minimus are the major two species in the transmission of malaria in Keonjhar district in Orissa.

Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum

In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.

Oral and parenteral immunization with synthetic retro-inverso peptides induce antibodies that cross-react with native peptides and parent antigens.

The objective of this study was to determine whether certain retro-inverso peptides have the potential to act as synthetic vaccines in mice, when immunized by injection or orally. Immunization of mice parenterally with conjugates of three such retro-inverso peptides and orally with the unconjugated peptides elicited generally high titres of anti-peptide antibodies. Antibodies against the same three peptides cross-reacted by binding strongly in ELISA to the native peptides and vice versa, regardless of the mode of immunization. Antibodies against a retro-inverso diphtheria peptide also reacted strongly with diphtheria toxin. Seven of 8 mice, immunized by injection of the conjugate of a retro-inverso derivative of robustoxin [a lethal spider (Atrax robustus) venom toxin] were protected from challenge involving injection with twice the minimum lethal dose of A. robustus venom containing the toxin.

Three-dimensional structure of heat shock protein 90 from Plasmodium falciparum: molecular modelling approach to rational drug design against malaria.

We have recently implicated heat shock protein 90 from Plasmodium falciparum (PfHsp90) as a potential drug target against malaria. Using inhibitors specific to the nucleotide binding domain of Hsp90, we have shown potent growth inhibitory effects on development of malarial parasite in human erythrocytes. To gain better understanding of the vital role played by PfHsp90 in parasite growth,we have modeled its three dimensional structure using recently described full length structure of yeast Hsp90.S equence similarity found between PfHsp90 and yeast Hsp90 allowed us to model the core structure with high confidence. The superimposition of the predicted structure with that of the template yeast Hsp90 structure reveals an RMSD of 3.31 Angstrom. The N-terminal and middle domains showed the least RMSD (1.76 Angstrom) while the more divergent C-terminus showed a greater RMSD (2.84 Angstrom) with respect to the template. The structure shows overall conservation of domains involved in nucleotide binding, ATPase activity, co-chaperone binding as well as inter-subunit interactions. Important co-chaperones known to modulate Hsp90 function in other eukaryotes are conserved in malarial parasite as well. An acidic stretch of amino acids found in the linker region, which is uniquely extended in PfHsp90 could not be modeled in this structure suggesting a flexible conformation. Our results provide a basis to compare the overall structure and functional pathways dependent on PfHsp90 in malarial parasite. Further analysis of differences found between human and parasite Hsp90 may make it possible to design inhibitors targeted specifically against malaria.

Efficacy of arteether in chloroquine resistant falciparum malaria in eastern India.

BACKGROUND & OBJECTIVES: Morbidity and mortality due to falciparum malaria are increasing in many tropical areas. The situation is further complicated by drug resistant malaria. The present study was undertaken to evaluate the efficacy of arteether on acute chloroquine resistant Plasmodium falciparum malaria in eastern coalfield area of Asansol. METHODS: A total of 30 patients with chloroquine resistant falciparum malaria smear and histidine-rich protein II (HRPII) antigen positive were given arteether intramuscularly in a single daily dose of 150 mg (3 mg/kg body weight in case of children) for three consecutive days. They were followed up to 28 days of arteether therapy. Each patient was assessed in terms of fever clearance time, parasite clearance time and parasite reappearance rate. RESULTS: The cure rate was found to be 100% with fever clearance time between 1-3 days (mean +/- SD 48.2 +/- 10.6 h) and mean parasite clearance time of 1.2 +/- 0.3 days. Parasite reappearance rate was found to be 0%. No adverse effect due to arteether therapy was observed following the treatment. INTERPRETATION & CONCLUSION: The results indicated that arteether was effective in patients with acute chloroquine resistant, complicated as well as uncomplicated, falciparum malaria and might be considered as a suitable alternative to quinine.

Goat serum: an alternative to fetal bovine serum in biomedical research.

Serum is frequently added to the defined basal medium as a source of certain nutritional and macromolecular growth factors essential for cell growth. Although a number of synthetic media have been prepared serum continues to be used in cell culture by many investigators. The best supplementation to a basal medium is fetal bovine serum (FBS) that is most frequently used for all types of cell cultures. During last four decades National Institute of Virology, Pune, has been working on isolation and identification of viruses from clinical specimens, employing tissue culture. Initially FBS was used for this purpose. However, due to its prohibitive cost and uncertain supply an alternative was sought. Commercially available sera from newborn calf, sheep, horse, human and serum obtained from goat blood (available from local abattoir) were tried. Goat serum (GS) was found to be suitable for most of the cell lines and primary cultures. Primary cultures from guinea pig embryo, monkey kidney, chick embryo, mouse peritoneal macrophages, and established cell lines were prepared and grown in growth media supplemented with GS. These cultures were studied for their morphology and growth in comparison with cultures grown in FBS containing media, and were used for mass cultivation of cells, quantitation and susceptibility of various virus strains, studies on effects of different nutrients and natural substances on cellular metabolism and virus replication, epitope analysis of various strains of Japanese encephalitis (JE) virus, strain differentiation studies, studies on antibody dependent plaque enhancement, assay of murine migration inhibition factor. Monoclonal antibodies against JE virus adapted to GS were characterised for their retention of functionalities. The results were comparable to those of cell cultures grown in FBS containing media. Similar results on chromosome studies were obtained from patient's whole blood cultures prepared in GS and FBS containing growth media. Organ cultures from mammalian, reptile and avian hosts; successfully grown in GS supplemented growth media, were used for different virological studies. Growth media supplemented with GS were used for in vitro cultivation of malarial parasites. Thus since the last three decades many scientists are using GS in place of FBS, in various fields of biomedical research. The present article reviews an account of the same.

Expression & immunogenicity of malaria merozoite peptides displayed on the small coat protein of chimaeric cowpea mosaic virus.

BACKGROUND & OBJECTIVES: Foreign peptide sequences can be inserted into the betaB-betaC loop of the cowpea mosaic virus (CPMV) small coat protein (SCP) to yield functional chimaeric viruses. Immunisation with chimaeric CPMV elicits immune responses that protect against human immunodeficiency and mink enteritis viruses. The present study was undertaken to investigate the expression of a B cell epitope from the merozoite surface antigen-1 of the malaria parasite Plasmodium falciparum (PfMSP1) in CPMV for an epitope based vaccine. METHODS: DNA encoding a 19 aa sequence (VTHESYQEL VKKLEALEDA, termed P109), the N-terminus of the mature PfMSP1, was cloned into SCP gene yielding a chimaeric virus CPMV-P109. CPMV-P109 was propagated in cowpea plants. The immunogenicity of purified recombinant virus in rabbits was investigated. RESULTS: CPMV-P109 developed a systemically spreading infection in cowpea, with normal viral morphology. The P109 epitope was detected on CPMV-P109 by ELISA with an antiserum produced against homopolymeric P109. Immunisation of rabbits with CPMV-P109 yielded antibodies that, although were predominantly directed against virus-specific epitopes, also recognized the P109 peptide on the recombinant virus and free P109 peptide. These antibodies however, did not react with the native antigen on merozoite by immunofluorescence. INTERPRETATION & CONCLUSION: The results indicate that selecting immunodominant peptide epitopes and presenting them in a near native conformation are important for generating biologically relevant antibodies in the CPMV expression system. Further, the findings draw attention to the importance of measuring immune responses to the viral vector antigens, a preponderance of which can result in undesirable effects such as autoimmunity and hypersensitivity in immunized hosts.

Possible role of Granulocyte Macrophage Colony Stimulating Factor receptor (GM-CSF R) in malaria.

Malaria has been reportedly increasing in incidence on the globe. Evidence from clinical studies supports a role for cytokines in the pathogenesis of cerebral malaria. Given the stimulatory effect of the ligand GM-CSF on the synthesis and release of the pyrogenic cytokine TNF alpha, the present study has been undertaken to investigate a possible role of GMCSF receptor in the pathogenesis of both Plasmodium vivax and Plasmodium falciparum malaria. An enzyme immunoassay developed by us at our laboratory for the quantitation of GM-CSF receptor has been used. No changes in the concentration of the receptor have been indicated either at the time of diagnosis or after treatment. In addition, an intercomparison of the receptor concentration between the P. vivax and P. falciparum groups does not show any significant difference. The results suggest that GM-CSF receptor has no significant role in the pathogenesis of either type of malaria.

The intracellular trafficking of proteins in Plasmodium falciparum-infected erythrocytes - A review

Durante a invasäo de merozoitos nos eritrócitos, proteínas do Plasmodium säo secretadas pelo parasita induzindo várias alterações nas células infectadas do hospedeiro. Entre essas alterações está a habilidade dessas células de aderir ao endotélio vascular, e assim escapar da destruiçäo no baço. O tráfego de proteínas no Plasmodium é semelhante ao de células eucarióticas em diversos aspectos, todavia, à presença de um complexo de Golgi em P. falciparum era controverso até muito recentemente. Proteínas do parasita säo secretadas através da membrama do Retículo Endoplasmático (RE), ou inseridas nele co- ou pós-traduçäo. O parasita pode ainda secretar polipeptídeos por vias independentes da via clássica RE-complexo de Golgi e possuir duas diferentes vias, representadas pelo RE e por sERA, para o destino fina do polipeptídeo. Embora bastante progresso tenha sido feito nos últimos anos, os mecanismos envolvidos na secreçäo de proteínas pelo parasita, através de diferentes membranas para atingir a superfície do eritrócito ainda näo estäo completamente elucidados.

A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro

The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes.

Aislamiento y caracterización de proteínas de unión a calmodulina en el Plasmodium falciparum

Se detectaron en el citoplasma de P. falciparum, por Cromatografía de afinidad a calmodulina-agarosa en presencia de 0.2 mM calcio, nueve proteínas de unión a calmodulina de 135.0, 81.5, 66.0, 58.6, 45.0, 41.0, 36.5, 30.0 y 24.5 kDa que fueron eluidas con 1mM EGTA. Las mismas proteínas fueron identificadas independientemente por prueba de immunocoprecipitación de un extracto del parásito marcado con superíndice 35S-Met, usando calmodulina bovina y un anticuerpo monoclonal contra ésta. Cuando las nueve proteínas fueron sometidas a SDS-PAGE y transferidas a membrana de PVDF, ocho de ellas mantuvieron la habilidad de unirse a calmodulina biotinilada. Las proteínas fueron aisladas por Cromatografía de afindad, seguida por electroforesis preparativa e inoculadas en ratones Balb-C para producir anticuerpos monoclonales con los que se estudió su localización por Inmunofluorescencia e Inmunomicroscopia electrónica en los diferentes estadios del parásito. Igualmente se detectaron por inmunocoprecipitación dos bandas con peso mol superior a 205.0 kDa con capacidad de unión a un anticuerpo anti-espectrina. Una de las proteínas de unión a CaM (36.500) fue aislada y sometida a ruptura enzimática con tripsina, diseñandose a partir del extremo N-terminal de los fragmentos obtenidos, oligonucleótidos degenerados con los que se hizo amplificación del genoma del parásito por PCR, lográndose aislar un fragmento de 554 pb, que presentó una alta homología con la glutamato sintasa, proteína altamente relacionada con cloroplastos de algas y plantas, planteándose la probable importancia que esta proteína tiene tanto en la historia evolutiva de los aplicomplexas, como en el diseño de fármacos que permitan nuevas alternativas para elcontrol de las enfermedades ocasionadas por éste grupo de organismos

Glycosyl-phosphatidylinositols in protozoa structure, biosynthesis and intracellular localisation.

We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.

Caracterización parcial de la calmodulina de Plasmodium falciparum / Partial characterization of Plasmodium falciparum calmoduline

Se describe una alternativa para la purificación parcial de Calmodulina (CaM) a partir de Plasmodium falciparum, que de acuerdo con anticuerpos monoclanes altamente específicos contra CaM, permite separar por lo menos dos formas de la proteína, que aunque difieren en sus pesos moleculares (12.600 y entre 36.000 y 50.000 dalton), son capaces de estimular a la ATPasa de calcio del eritrocito por separado. La forma de menor peso molecular, que es la mejor representada, a diferencia de la CaM purificada de otras fuentes muestra un comportamiento electroforético independiente de la presencia de calcio: lo que conjuntamente con evidencias experimentales que sugieren una disminución en la carga negativa total a pH neutral y en la hidrofobicidad en su estado asociado al calcio, permite plantear la posibilidad de una modificación estructural de la CaM de Plasmodium falciparum, que de todas maneras no interfiere con su función como activador de la ATPasa de calcio. Los estudios cinéticos en presencia de dos inhibidores específicos de CaM (Calmidazolium (CdZ) y W-7) confirman la identidad de la proteína purificada del parásito como CaM y por otro lado revelan que aparentemente la CaM de Plasmodium falciparum es inhibible en menor grado que la CaM de eritrocito en su función estimuladora de la ATPasa de calcio del eritrocito, lo que hace pensar en una mayor afinidad de la proteína del parásito por ésta enzima o en modificación de la zona regulatoria a la que se unen los inhibidores. Se plantea la posibilidad de que la CaM de Plasmodium falciparum no constituya un blanco ideal para el desarrollo de agentes quimioterapeúticos sintéticos contra la malaria, así como de que los inhibidores de CaM, Calmidazolium (CdZ) y W-7, no sean útiles como drogas para tratar la enfermedad

Serum alpha-1 antichymotrypsin is a possible growth inhibitor of Plasmodium falciparum.

Serum protease inhibitors were determined in paired sera from 7 patients with cerebral malaria and 2 patients with acute malaria showing high and low growth inhibition activity in the initial and follow-up sera respectively. Alpha-1 antichymotrypsin and alpha-1 antitrypsin but not alpha-2 macroglobulin showed direct correlation with the growth inhibition activity. When alpha-1 antitrypsin was deliberately added to the malarial culture no growth inhibition occurred indicating that the alpha-1 antichymotrypsin was the most likely factor responsible for inhibition of growth of malarial parasites in vitro.