Inhibitor tolerance of a recombinant flocculating industrial Saccharomyces cerevisiae strain during glucose and xylose co-fermentation

ABSTRACT Lignocellulose-derived inhibitors have negative effects on the ethanol fermentation capacity of Saccharomyces cerevisiae. In this study, the effects of eight typical inhibitors, including weak acids, furans, and phenols, on glucose and xylose co-fermentation of the recombinant xylose-fermenting flocculating industrial S. cerevisiae strain NAPX37 were evaluated by batch fermentation. Inhibition on glucose fermentation, not that on xylose fermentation, correlated with delayed cell growth. The weak acids and the phenols showed additive effects. The effect of inhibitors on glucose fermentation was as follows (from strongest to weakest): vanillin > phenol > syringaldehyde > 5-HMF > furfural > levulinic acid > acetic acid > formic acid. The effect of inhibitors on xylose fermentation was as follows (from strongest to weakest): phenol > vanillin > syringaldehyde > furfural > 5-HMF > formic acid > levulinic acid > acetic acid. The NAPX37 strain showed substantial tolerance to typical inhibitors and showed good fermentation characteristics, when a medium with inhibitor cocktail or rape straw hydrolysate was used. This research provides important clues for inhibitors tolerance of recombinant industrial xylose-fermenting S. cerevisiae.

Squamocin, an annonaceous acetogenin, enhances naphthalene degradation mediated by Bacillus atrophaeus CN4 / Squamocin, una acetogenina annonácea, estimula la degradación de naftaleno mediada por Bacillus atrophaeus CN4

Squamocin belongs to a group of compounds called annonaceous acetogenins. They are secondary products of Annonaceae metabolism and can be isolated from Annona cherimolia seeds. This paper deals with the stimulation of biofilm formation of Bacillus atrophaeus CN4 by employing low squamocin concentrations to increase naphthalene degradation. Bacillus atrophaeus CN4, isolated from contaminated soil, has the ability to degrade naphthalene as the only source of carbon and energy. In the absence of additional carbon sources, the strain removed 69% of the initial concentration of naphthalene (approx. 0.2 mmol/l) in the first 12 h of incubation. The addition of squamocin in LB medium stimulated Bacillus atrophaeus CN4 biofilm formation and enhanced naphthalene removal. Squamocin (2.5 pg/ml) does not affect planktonic growth and therefore, the observed increases are solely due to the stimulation of biofilm formation.

Squamocin pertenece a un grupo de compuestos llamados acetogeninas annonáceas (ACG). Las ACG son productos secundarios del metabolismo de plantas de la familia Annonaceae y se pueden aislar a partir de semillas de Annona cherimola. Este artículo trata de la estimulación de la formación de biofilm de Bacillus atrophaeus CN4 mediante el empleo de bajas concentraciones de squamocin para optimizar la degradación de naftaleno. B. atrophaeus CN4, aislado de suelo contaminado, tiene la capacidad de emplear naftaleno como única fuente de carbono y energía. En ausencia de fuentes de carbono adicionales, la cepa degradó el 69% de la concentración inicial de naftaleno (aprox. 0,2 mmol/l) en las primeras 12h de incubación. La adición de squamocin en medio LB estimula la formación de biofilm y la remoción naftaleno de B. atrophaeus CN4. Squamocin (2,5 µg/ml) no afecta al crecimiento planctónico y, por lo tanto, los incrementos observados se deben únicamente a la estimulación de la formación de biofilm.

Furan type lignans with antimycobacterial activity / Lignanos del tipo furano con actividad antimicobacterial

Compounds such as triclosan, diclofenac and trimetropin posses antibacterial activity, including mycobacterial; their structures are based on two aromatic rings joined by a methylene or a heteroatom. Since a similar structural system is found in natural diarylfuran- based lignans, we studied plants known with this type of lignans, as potential active against Mycobacterium tuberculosis. Fractions of the active extracts were tested for anti-TB activity and their chemical constituents analyzed by NMR spectroscopy. Several extracts and chromatographical fractions exhibited > 90 percent inhibition of M. tuberculosis at 128 ug/mL. Methylpluviatilol, a pure compound isolated from Virola sebifera, was active at this concentration.. These findings suggest that plant species of the families here studied may yield novel lead compounds for the development of antimycobacterial agents.

Compuestos tales como triclosan, diclofenac y trimetoprim poseen actividad antibacterial, incluyendo la antimicobacterial; sus estructuras están basadas en dos anillos aromáticos unidos por un metileno o un heteroátomo. Debido a que en la naturaleza se encuentra un sistema estructural similar del tipo diarilfurano en los lignanos, así como otros subtipos, nosotros estudiamos plantas contra Mycobacterium tuberculosis, de las que se sabe contienen lignanos Las fracciones cromatográficas de los extractos activos fueron ensayadas para actividad anti.Tb y sus constituyentes químicos se analizaron por espectroscopía de RMN. Varios extractos y fracciones cromatográficas exhibieron una inhibición superior al 90 por ciento a 128 ug/mL; el compuesto metilpluviatilol, aislado de mostró una inhibición del 99 por ciento a esa concentración. Esos hechos sugieren que las especies de plantas de las familias aquí estudiadas podrían suministrar nuevos compuestos líderes para el desarrollo de agentes antimicobacteriales.

Introduction of soft drinks and processed juice in the diet of infants attending public day care centers / Introdução de refrigerantes e sucos industrializados na dieta de lactentes que frequentam creches públicas

OBJECTIVE: Identifying at what age infants enrolled in public day care centers are introduced to soft drinks and industrialized juice, as well as comparing the nutritional composition of these goods with natural fruit juice. METHODS: A cross-sectional study with the mothers of 636 children (aged 0 to 36 months) from nurseries of day care centers, who were asked questions about the age of feeding introduction. This study evaluated the proximate composition of soft drinks and artificial juice, comparing them with those of natural fruit juice regarding energy, sugar, fiber, vitamin C, and sodium values. The chemical composition of fruit juice was obtained by consulting the Table of Food Composition and, for industrialized drinks, the average nutritional information on the labels of the five most consumed product brands. RESULTS: The artificial drinks were consumed before the first year of life by more than half of the children studied, however, approximately 10% consumed them before the age of 6 months. With regard to the comparison among the drinks, artificial fruit juice beverages and soft drinks proved to contain from nine to 13 times higher amounts of sodium, and 15 times less vitamin C than natural juices. CONCLUSIONS: The introduction of soft drinks and industrialized juice in the diet of infants was inopportune and premature.. When compared to natural fruit juice, these have inferior nutritional composition, which suggests the urgent need for measures based on strategies for food and nutrition education in order to promote awareness and the maintenance of healthy eating habits. .

OBJETIVO: Identificar a idade de introdução do refrigerante e de sucos industrializados na dieta de lactentes matriculados em berçários de creches públicas e comparar as composições nutricionais dessas bebidas com as do suco de fruta natural. MÉTODOS: Estudo transversal com 636 crianças (de zero a 36 meses) de berçários de creches, cujas mães foram entrevistadas sobre idade de introdução dos alimentos. Avaliaram-se as composições centesimais do refrigerante e sucos industrializados, comparando-as com as do suco de laranja natural para valor energético, açúcar, fibra, vitamina C e sódio. A composição centesimal do suco de laranja foi obtida por meio de consulta à Tabela de Composição de Alimentos e, para as bebidas industrializadas, utilizaram-se as médias das informações nutricionais contidas nos rótulos de cinco marcas mais consumidas dos produtos. RESULTADOS: O refrigerante e suco industrializado foram consumidos antes do primeiro ano de vida por mais da metade das crianças estudadas, sendo que cerca de 10% o consumiram antes dos seis meses. Quando comparadas à composição do suco de laranja natural, bebidas forneceram quantidades de 9 a 13 vezes superiores de sódio e 15 vezes inferiores de vitamina C. CONCLUSÕES: A introdução de refrigerantes e sucos industrializados na dieta dos lactentes foi inoportuna e precoce. Comparados ao suco de fruta natural, tais bebidas possuem composição nutricional inferior, sugerindo a necessidade de medidas fundamentadas em estratégias de educação alimentar e nutricional como forma de promover a formação e manutenção de hábitos alimentares saudáveis. .

beta-TrCP1 degradation is a novel action mechanism of PI3K/mTOR inhibitors in triple-negative breast cancer cells

An F-box protein, beta-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulation by beta-TrCP has been widely studied, the regulation of beta-TrCP itself is not well understood yet. In this study, we found that the level of beta-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of beta-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of beta-TrCP1 prior to its degradation. In addition, knockdown of beta-TrCP1 inhibited the proliferation of TNBC cells. We further identified that pharmacological inhibition of mTORC2 was sufficient to reduce the beta-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of beta-TrCP1 in TNBC cells and targeting beta-TrCP1 is a potential approach to treat human TNBC.

Arctiin blocks hydrogen peroxide-induced senescence and cell death though microRNA expression changes in human dermal papilla cells

BACKGROUND: Accumulating evidence indicates that reactive oxygen species (ROS) are an important etiological factor for the induction of dermal papilla cell senescence and hair loss, which is also known alopecia. Arctiin is an active lignin isolated from Arctium lappa and has anti-inflammation, anti-microbial, and anti-carcinogenic effects. In the present study, we found that arctiin exerts anti-oxidative effects on human hair dermal papilla cells (HHDPCs). RESULTS: To better understand the mechanism, we analyzed the level of hydrogen peroxide (H2O2)-induced cytotoxicity, cell death, ROS production and senescence after arctiin pretreatment of HHDPCs. The results showed that arctiin pretreatment significantly inhibited the H2O2-induced reduction in cell viability. Moreover, H2O2-induced sub-G1 phase accumulation and G2 cell cycle arrest were also downregulated by arctiin pretreatment. Interestingly, the increase in intracellular ROS mediated by H2O2 was drastically decreased in HHDPCs cultured in the presence of arctiin. This effect was confirmed by senescence associated-beta galactosidase (SA-ß-gal) assay results; we found that arctiin pretreatment impaired H2O2-induced senescence in HHDPCs. Using microRNA (miRNA) microarray and bioinformatic analysis, we showed that this anti-oxidative effect of arctiin in HHDPCs was related with mitogen-activated protein kinase (MAPK) and Wnt signaling pathways. CONCLUSIONS: Taken together, our data suggest that arctiin has a protective effect on ROS-induced cell dysfunction in HHDPCs and may therefore be useful for alopecia prevention and treatment strategies.

Interacciones genotóxicas de mutágenos en mezclas binarias mediante ensayo cometa alcalino en linfocitos humanos / Interaction of mutagens in binary mixtures using the alkaline comet assay in human lymphocytes

Introducción. Los mutágenos contenidos en mezclas complejas presentan interacciones de sinergismo, aditivas o antagónicas. Se han desarrollado enfoques experimentales que permitan dilucidar el responsable de las interacciones en la mezcla. Objetivo. Desarrollar un diseño experimental para comprender los procesos que se llevan a cabo entre los compuestos presentes en las mezclas complejas. Materiales y métodos. Se expusieron linfocitos humanos a mezclas binarias de mutágenos B[a]P, DMBA, Trp-P-1 y MX durante una hora, con activación metabólica y sin ella. La viabilidad se evaluó con azul de tripano y, la genotoxicidad, con cometa alcalino. Resultados. Ningún hidrocarburo tuvo efecto con furanona. Con S9 y sin él, se observó que se presentaban interacciones tóxicas entre hidrocarburos. Se observó sinergismo sin S9 entre B[a]P y Trp-P-1 y, con actividad metabólica, entre DMBA y Trp-P-1. Sin S9 se observó interacción antagónica entre Trp-P-1 y DMBA y, con S9, entre Trp-P-1 y MX y entre MX y DMBA. Se observó un incremento dependiente de la dosis en la longitud de la cola. Hubo daño genotóxico medio y aumento de las células dañadas. Para todas las mezclas se pudo determinar la concentración mínima en la que se observaban efectos adversos y solo para algunas se determinó la concentración máxima en la cual no se observaron efectos adversos. Conclusión. Se hace un aporte para comprender los procesos que ocurren cuando en una mezcla hay presentes, al menos, dos mutágenos y se valida un modelo de análisis que permite dilucidar el compuesto que tiene efecto sobre otro. También, se demostró que según el tipo de compuestos en la mezcla, se tendrá o no un umbral de riesgo.

Introduction. Mutagens contained in complex mixtures can present synergistic interactions, either additive or antagonistic. Therefore, development of experimental approaches is necessary to elucidate which is the responsible agent for the effect in the mixtures. Objective. An experimental design was developed that allowed an understanding of the processes between the compounds of complex mixtures. Materials and methods. Human lymphocytes were exposed to binary mixtures of the mutagens B[a]P, DMBA, Trp-P-1 and MX for 1 hour with or without S9. Viability was assessed with trypan blue dye and the genotoxicity by the comet assay. Results. All of the hydrocarbon showed an effect with furanone. With and without S9, the most toxic interactions were observed between hydrocarbons. Synergistic interaction was observed without S9 between B [a] P and Trp-P-1 and between DMBA and Trp-P-1 with metabolic activity. Without S9 antagonistic interaction was observed only between Trp-P-1+DMBA, and with S9 between Trp-P-1+MX and MX+DMBA. It observed an increase dose dependent in tail length. Half the cultures showed genotoxic damage and increased cell damage. For each mixture, minimum concentrations were determined at which adverse effects are observed; for some only the maximum concentration was determined at which no adverse effects are observed. Conclusion. The processes between mutagens present in a mixture have become better understood, and the results validated an analytical model that determined which component had an effect on another. The results also showed that the type of compounds in the mixture determined whether or not a risk threshold was present.

Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS-/- mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor NG-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca2+ levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca2+ levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol-treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca2+ chelator, calmodulin antagonist, and CaMKKbeta siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca2+-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca2+/CaMKKbeta-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization.

p53 and DNA-dependent protein kinase catalytic subunit independently function in regulating actin damage-induced tetraploid G1 arrest

We previously reported that the p53 tumor suppressor protein plays an essential role in the induction of tetraploid G1 arrest in response to perturbation of the actin cytoskeleton, termed actin damage. In this study, we investigated the role of p53, ataxia telangiectasia mutated protein (ATM), and catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in tetraploid G1 arrest induced by actin damage. Treatment with actin-damaging agents including pectenotoxin-2 (PTX-2) increases phosphorylation of Ser-15 and Ser-37 residues of p53, but not Ser-20 residue. Knockdown of ATM and DNA-PKcs do not affect p53 phosphorylation induced by actin damage. However, while ATM knockdown does not affect tetraploid G1 arrest, knockdown of DNA-PKcs not only perturbs tetraploid G1 arrest, but also results in formation of polyploidy and induction of apoptosis. These results indicate that DNA-PKcs is essential for the maintenance of actin damage induced-tetraploid G1 arrest in a p53-independent manner. Furthermore, actin damage-induced p53 expression is not observed in cells synchronized at G1/S of the cell cycle, implying that p53 induction is due to actin damage-induced tetraploidy rather than perturbation of actin cytoskeleton. Therefore, these results suggest that p53 and DNA-PKcs independently function for tetraploid G1 arrest and preventing polyploidy formation.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil / Atividade leishmanicida e citotoxicidade de constituintes químicos de duas espécies de Annonaceae cultivadas no nordeste do Brasil

INTRODUCTION: Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. METHODS: Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10 percent phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. RESULTS: O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. CONCLUSIONS: These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

INTRODUÇÃO: A leishmaniose visceral é uma enfermidade endêmica em 88 países, com um total de 12 milhões de pessoas infectadas e 350 milhões em risco. Na procura de novos agentes com ação leishmanicida, alcalóides e acetogeninas isoladas de Annona squamosa e Annona muricata, foram testados contra as formas promastigotas e amastigotas de Leishmania chagasi. MÉTODOS: Foram preparados extratos com metanol: água (80: 20) das folhas de A. squamosa e sementes de A. muricata que foram extraídos com solução de ácido fosfórico 10 por cento e solventes orgânicos, para obter extratos ricos em alcalóides e acetogeninas. Estes extratos foram cromatografados em coluna de sílica gel sendo eluídos com solventes de diferentes polaridades para o isolamento dos constituintes, e feita a determinação estrutural por análise espectroscópica. Os constituintes isolados foram testados contra Leishmania chagasi, responsável pela leishmaniose visceral, utilizando o teste MTT. Testes de toxicidade foram realizados em todos os compostos isolados, sendo utilizadas células RAW 264.7. RESULTADOS: Um alcalóide benzilisoquinolínico, O-metilarmepavina, e uma C37-triidróxi-acetogenina com anel bistetrahidrofurânico adjacente foram isolados de A. squamosa e duas acetogeninas annonacinona e corossolona da A. muricata. O alcalóide mostrou um índice de inibição médio (IC50) de 23,3µg/mL e as acetogeninas apresentaram IC50 variando entre 25,9 a 37,6µg/mL contra promastigotas, e no ensaio de amastigotas, o IC50 valores variaram entre 13,5 a 28,7 µg/mL. A toxicidade mostrou resultados que variaram entre 43,5 a 79,9µg/mL. CONCLUSÕES: Estes resultados caracterizam A. squamosa e A. muricata como fontes potenciais de agentes leishmanicidas.

In vitro anti-proliferation/cytotoxic activity of epingaione and its derivatives on the human SH-SY5Y neuroblastoma and TE-671 sarcoma cells / Actividad citotóxica antiproliferativa in vitro de la epingaiona y sus derivados, sobre el neuroblastoma humano sh-sy5y y las células del sarcoma te-671

Epingaione (4-Methyl-1-(5-methyl-2, 3,4,5-tetrahydro-[2,3']bifuranyl-5-yl)-pentan-2-one) was isolated as one of the major lipophilic secondary metabolites from the leaves and stems of Bontia daphnoides L. The compound gave 79.24and 50.83anti-proliferation/cytotoxic activity on the human SH-SY5Y neuroblastoma and TE-671 sarcoma cells in vitro at 50 pg/mL, respectively. Epingaione was transformed into eleven derivatives under laboratory conditions using ethanol, some gave greater anti-proliferation/cytotoxic activity on the cancer cell lines tested. One of the derivatives (compound 2) with enhanced cytotoxic activity was elucidated as 5'-Ethoxy-5-methyl-5-(4-methyl-2-oxo-pentyl)-2,3,4,5-tetrahydro-5'H-[2,3']bifuranyl-2'-one. Both epingaione and compound 2 caused an accumulation of arrested or dead SH-SY5Y neuroblastoma in the m-phase of the cell cycle as revealed by the m-phase specific marker KE 67.

La epingaiona (4-Metil-1-(5-metil-2,3,4,5-tetrahidro-[2,3']bifuranil-5-il)-pentan-2-uno) fue aislada como uno de los principales metabolitos lipofilicos secundarios de las hojas y tallos de Bontia daphnoides L. El compuesto produjo 79.24 % y 50.83 % de actividad citotóxica/anti-proliferación sobre el neuroblastoma humano SH-SY5Y y las células del sarcoma TE-671 in vitro a 50 µg/mL, respectivamente. La epingaiona fue transformada en once derivados en condiciones de laboratorio, utilizando etanol. Algunos produjeron mayor actividad citotóxica y antiproliferativa sobre las líneas celulares cancerosas sometidas a ensayo. Uno de los derivados (compuesto 2) de elevada actividad citotóxica fue identificado como 5'-Etoxi-5-metil-5-(4-metil-2-oxo-pentil)-2,3,4,5-tetrahidro-5'H- [2,3']bifuranil-2'-uno. Tanto la epingaiona como el compuesto 22 causaron una acumulación de neuroblastomas SH-SY5Y muertos o detenidos en la fase m del ciclo celular, según lo revela el marcador KE 67 específico de la fase m.

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 æM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60 percent at 20 æg/ml (59 and 90 æM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 æg/ml (0.6-1.5 mM).

Correlation of phospholipid loss in goat whole blood with solvochromic properties of antiamebics like emetine, metronidazole and diloxanide furoate.

Phospholipid content of whole blood lipid decreases significantly when goat blood is incubated for different length of time with different amebicidal agents (e.g., emetine, metronidazole and diloxanide furoate). The plots of relative per cent phosphate loss against incubation period show biphasic nature and suggest that the rates of phospholipid loss bears some relation with the drug's lipophilicity (log P in 1 octanol/water system). The absolute phospholipid loss seems to be governed by the drug's aquasolubility. Implication of these finding were discussed in terms of their clinical profiles assuming that the loss of phospholipid is due to drug's binding with the phospholipid layer in amebic cyst-coat, being the first step which may trigger a chain of events leading to the onset of drug action.