Objetive: Analyze the antiparasitic activity of marine invertebrate Ophiocomina nigra in Entamoeb... more Objetive: Analyze the antiparasitic activity of marine invertebrate Ophiocomina nigra in Entamoeba invadens. Methodology. In O. nigra, an analysis of the quantitative proximal chemical composition (moisture, ash, protein, lipids and nitrogen-free extract) was performed. In addition, the determination of the total protein pattern (SDS-PAGE 10%). On the other hand, the antiparasitic activity of E. invadens trophozoites was determined, which were grown in TYI medium at 28 ° C and exposed to the aqueous extract of O. nigra for 24 hours. Subsequently, metabolic activity (XTT assay) was determined and morphology was analyzed. Cytotoxicity tests were performed on human liver cells (Hep G2) exposed for 24 hours to O. nigra (XTT test) and the biomass was determined (violet crystal staining). Contribution. The antiparasitic activity of O. nigra in E. invadens and the cytotoxic effect in human liver cells was determined. There are few scientific studies of this marine invertebrate on its use i...
Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stab... more Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40-73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS-PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5'-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of L-ornithine and PLP yielding S(0.5) values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by alpha-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes.
Entamoeba invadens es una especie importante dentro del reino Protozoa porque permite un amplio e... more Entamoeba invadens es una especie importante dentro del reino Protozoa porque permite un amplio estudio y comparacion con los individuos del mismo genero que resultan ser patogenos para los seres humanos, como lo es E. histolytica. Dentro de estos estudios resaltan los relacionados con su ciclo de vida, especificamente enquistamiento y desenquistamiento. En el presente trabajo se evaluaron dos enzimas presentes durante el enquistamiento amibiano y que pudiesen participar en el desenquistamiento: la quitinasa, encargada de la degradacion de la pared gruesa del quiste en dimeros o monomeros de GlcNac y la N-acetil-β-D-glucosaminidasa, que se encarga de la degradacion de los dimeros de GlcNac.
El genero Acanthamoeba es un grupo de protozoarios de vida libre, ampliamente distribuidos en la ... more El genero Acanthamoeba es un grupo de protozoarios de vida libre, ampliamente distribuidos en la naturaleza. Actualmente existen millones de usuarios de lentes de contacto (LC), los cuales proveen una superficie donde los microorganismos pueden adherirse y causar queratitis. Debido a la resistencia del estadio quistico de Acanthamoeba, es dificil su completa eliminacion aun utilizando las Soluciones Multiproposito (SMP) elaboradas para desinfectar los LC. En el estudio de la medicina tradicional, la Annona muricata contienen metabolitos secundarios con actividad anticancerigena, antiparasitaria e insecticida. En este estudio, se analizo el efecto de extractos de A. muricata sobre A. castellanii. Los trofozoitos, cultivados a 30 °C en medio Chang conteniendo 10 % de suero fetal de bovino (SFB), fueron tratados con extractos acuosos y etanolicos de A. muricata en presencia o ausencia de SMP. Los resultados muestran una disminucion de la actividad metabolica amebiana independientemente...
Actualmente se cuenta con informacion acerca del proceso de cultivo y enquistamiento de trofozoit... more Actualmente se cuenta con informacion acerca del proceso de cultivo y enquistamiento de trofozoitos en los medios TYI-S-33 y LG, sin embargo no se ha comparado la eficiencia entre uno y otro cuando se induce la diferenciacion celular en medio LG-47. En el presente trabajo se comparo el efecto de preinocular trofozoitos de Entamoeba invadens IP-1 en ambos medios de cultivo (TYI-S-33 y LG) y su efecto en el proceso de enquistamiento inducido en medio LG-47, observandose que no existen diferencias significativas entre utilizar como medio de preinoculo a uno u otro medio de cultivo para la diferenciacion celular de Entamoeba invadens IP-1 en medio LG-47.
Los hongos del orden Ophiostomatales son organismos saprobios y, algunos de ellos parasitos, que ... more Los hongos del orden Ophiostomatales son organismos saprobios y, algunos de ellos parasitos, que ocasionan danos en arboles maderables como el pino ya sea por efecto de dano directo a la albura o por la coloracion (manchado azul) de la misma. Su estudio se ha abordado desde enfoques moleculares y bioquimicos. Un metodo alternativo de estudio y que al mismo tiempo sirve para separar proteinas, es el isoelectroenfoque y la determinacion del pI de las proteinas. En este trabajo se determino el pI de algunas hidrolasas acidas secretadas por Ophisotoma piceae en medio de cultivo minimo (medio de Mathur) adicionado de diferentes fuentes de carbono: xilana, glucosa o alga Nori. El patron de actividades recuperadas en el medio de cultivo libre de celulas fue muy complejo, presentandose pI principalmente en el rango de pH 3 a 5, y dependiente de la fuente de carbono empleada para su induccion. La xilana adicionada al medio minimo a una concentracion del 1%v p/v resulto una fuente de carbono ...
Biomedical Engineering: Applications, Basis and Communications, 2021
Yeast cultures of Saccharomyces cerevisiae were exposed to pulsed magnetic fields of low intensit... more Yeast cultures of Saccharomyces cerevisiae were exposed to pulsed magnetic fields of low intensity. Field intensities from 0.92 mT to 2.73[Formula: see text]mT and frequencies of 20, 77.5, 135, 192.5 and 250[Formula: see text]Hz were employed. Proliferation and cell viability measurements were made in exposed and control cultures. For the frequency of 20[Formula: see text]Hz, statistically significant differences were found ([Formula: see text]) in cell proliferation using field intensities of 1.81 and 2.71[Formula: see text]mT. No changes were observed at this frequency for lower field strengths. At 77.5[Formula: see text]Hz, only changes in proliferation were observed for 0.92[Formula: see text]mT ([Formula: see text]). No changes were observed to any other frequency or intensity of the magnetic field. The cell viability studies were performed at different times, from 4 to 12[Formula: see text]h of exposure. At 1.81[Formula: see text]mT and 20[Formula: see text]Hz, an increase in ...
Epidemiological studies have associated long-term exposure to environmental air pollution particu... more Epidemiological studies have associated long-term exposure to environmental air pollution particulate matter (PM) with the development of diverse health problems. They include infectious respiratory diseases related to the deregulation of some innate immune response mechanisms, such as the host defense peptides' expression. Herein, we evaluated in BALB/c mice the effect of long-standing exposure (60 days) to urban-PM from the south of Mexico City, with aerodynamic diameters below 2.5 µm (PM2.5) and 10 µm (PM10) on the lung's gene expression and production of three host defense peptides (HDPs); murine beta-defensin-3, -4 (mBD-3, mBD-4) and cathelin-related antimicrobial peptide (CRAMP). We also evaluated mRNA levels of Il1b and Il10, two cytokines related to the expression of host defense peptides. Exposure to PM2.5 and PM10 differentially induced lung inflammation, being PM2.5, which caused higher inflammation levels, probably associated with a differential deposition on the airways, that facilitate the interaction with alveolar macrophages. Inflammation levels were associated with an early upregulation of the three HDPs assessed and an increment in Il1b mRNA levels. Interestingly, after 28 days of exposure, Il10 mRNA upregulation was observed and was associated with the downregulation of HDPs and Il1b mRNA levels. The upregulation of Il10 mRNA and suppression of HDPs might facilitate microbial colonization and the development of diseases associated with long-term exposure to PM.
The development of mature biofilms is an aid in numerous aspects of the life cycle of fungi. It i... more The development of mature biofilms is an aid in numerous aspects of the life cycle of fungi. It is well known that Sporothrix schenckii complex causes a benign subcutaneous mycosis, but recent studies have suggestedthat biofilm formation may be one of the important factors involved in its virulence. Here we report the study of the biomass organization and a model of the stages of S. schenckii biofilm development: adsorption, active adhesion, microcolony formation, maturation, and dispersal of biofilm fragments. During the development, the biofilm is surrounded by extracellular matrix, which contains glycoprotein (mannose rich), carbohydrates, lipids, and nucleic acid. In addition, the extracellular DNA increases in extracellular matrix as a key component to structural integrity and antifungal resistance. The study of S. schenckii biofilm contributes to a better understanding of growth biofilm and physiology, adding new insights into the mechanisms of virulence and persistence of pat...
Aim: Sporothrix schenckii is the causative agent of sporotrichosis. A 70-kDa glycoprotein, Gp70, ... more Aim: Sporothrix schenckii is the causative agent of sporotrichosis. A 70-kDa glycoprotein, Gp70, is a candidate for the development of prophylactic alternatives to control the disease, and its gene (GP70) is predicted to encode for a protein of 43 kDa, contrasting with the molecular weight of the native protein. Materials & methods: The GP70 was expressed in bacteria, the recombinant protein purified, used in immunoassays and injected to Galleria mellonella. Results & conclusion: The recombinant protein was detected by anti-Gp70 antibodies, confirming that the Gp70 backbone is a 43-kDa peptide. This protein showed enzyme activity of cyclase and was recognized by sera of patients with sporotrichosis. Although it was not useful for serodiagnosis of sporotrichosis, it conferred protection to animals against experimental sporotrichosis.
Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a so... more Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a soluble fraction by anionic exchange, size exclusion and hydroxyapatite adsorption chromatographies. The enzymes, named here as A, B and B', showed molecular weights of 64, 33.4 and 33.4 kDa, respectively, as measured by gel filtration. Comparison of their levels of specific activity in partially purified samples revealed
Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of ... more Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of encystation in axenic conditions using 4-MU(Ch)3 as substrate. Encystment was paralleled by chitinase activity which showed a peak after about 72 h of cultivation where cysts accounted for 63% of cell population. Thereafter, activity fell off rapidly, whereas encystment continued, reaching 80% at the end of the experiment (96 h). Comparison of activity between cysts and the total cell population in 48- and 72-h-old encysting cultures suggested that chitinase may start to accumulate in the pre-cyst forms. About 70% of the enzyme was recovered in the supernatant following low-speed centrifugation of whole extracts. Most of this activity represented soluble chitinase since it was not sedimented by further centrifugation at 105,000 x g. A minor proportion of enzyme activity remained associated to the buffer-washed, high-speed sediment. In addition to 4-MU(Ch)3, chitinase activity was also measured following the hydrolysis of other substrates such as nascent, preformed or colloidal chitin. Like other chitinases, the cyst enzyme preferred nascent over preformed chitin as substrate. Digestion of the former yielded GlcNAc and minor amounts of (GlcNAc)2 as products. Allosamidin strongly inhibited hydrolysis of the fluorogenic substrate by the amebic chitinase in vitro with a Ki of 0.065 microM. IC50 values were 0.085 microM and 0.16 microM at 5 microM and 10 microM 4-MU(Ch)3, respectively. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation.
A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized.... more A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized. Mice spleen leucocytes were utilized as target cells. Interaction time was reduced to 1 h by pelleting interacting cells. To assess target-cell killing by amoebae, a nigrosine exclusion test was employed. Fixation with glutaraldehyde stabilized the percentage of stained target cells. Similar results were obtained when cytotoxicity of the E. histolytica HM1 strain was tested by the traditional and proposed methods. The new method allowed quantification of the contribution of cytolysis and cytophagocytosis to amoebic cytotoxicity. It was also demonstrated that uncloned E. histolytica HM1 strain is a heterogeneous population with respect to cytotoxicity expression.
Objetive: Analyze the antiparasitic activity of marine invertebrate Ophiocomina nigra in Entamoeb... more Objetive: Analyze the antiparasitic activity of marine invertebrate Ophiocomina nigra in Entamoeba invadens. Methodology. In O. nigra, an analysis of the quantitative proximal chemical composition (moisture, ash, protein, lipids and nitrogen-free extract) was performed. In addition, the determination of the total protein pattern (SDS-PAGE 10%). On the other hand, the antiparasitic activity of E. invadens trophozoites was determined, which were grown in TYI medium at 28 ° C and exposed to the aqueous extract of O. nigra for 24 hours. Subsequently, metabolic activity (XTT assay) was determined and morphology was analyzed. Cytotoxicity tests were performed on human liver cells (Hep G2) exposed for 24 hours to O. nigra (XTT test) and the biomass was determined (violet crystal staining). Contribution. The antiparasitic activity of O. nigra in E. invadens and the cytotoxic effect in human liver cells was determined. There are few scientific studies of this marine invertebrate on its use i...
Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stab... more Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40-73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS-PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5'-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of L-ornithine and PLP yielding S(0.5) values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by alpha-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes.
Entamoeba invadens es una especie importante dentro del reino Protozoa porque permite un amplio e... more Entamoeba invadens es una especie importante dentro del reino Protozoa porque permite un amplio estudio y comparacion con los individuos del mismo genero que resultan ser patogenos para los seres humanos, como lo es E. histolytica. Dentro de estos estudios resaltan los relacionados con su ciclo de vida, especificamente enquistamiento y desenquistamiento. En el presente trabajo se evaluaron dos enzimas presentes durante el enquistamiento amibiano y que pudiesen participar en el desenquistamiento: la quitinasa, encargada de la degradacion de la pared gruesa del quiste en dimeros o monomeros de GlcNac y la N-acetil-β-D-glucosaminidasa, que se encarga de la degradacion de los dimeros de GlcNac.
El genero Acanthamoeba es un grupo de protozoarios de vida libre, ampliamente distribuidos en la ... more El genero Acanthamoeba es un grupo de protozoarios de vida libre, ampliamente distribuidos en la naturaleza. Actualmente existen millones de usuarios de lentes de contacto (LC), los cuales proveen una superficie donde los microorganismos pueden adherirse y causar queratitis. Debido a la resistencia del estadio quistico de Acanthamoeba, es dificil su completa eliminacion aun utilizando las Soluciones Multiproposito (SMP) elaboradas para desinfectar los LC. En el estudio de la medicina tradicional, la Annona muricata contienen metabolitos secundarios con actividad anticancerigena, antiparasitaria e insecticida. En este estudio, se analizo el efecto de extractos de A. muricata sobre A. castellanii. Los trofozoitos, cultivados a 30 °C en medio Chang conteniendo 10 % de suero fetal de bovino (SFB), fueron tratados con extractos acuosos y etanolicos de A. muricata en presencia o ausencia de SMP. Los resultados muestran una disminucion de la actividad metabolica amebiana independientemente...
Actualmente se cuenta con informacion acerca del proceso de cultivo y enquistamiento de trofozoit... more Actualmente se cuenta con informacion acerca del proceso de cultivo y enquistamiento de trofozoitos en los medios TYI-S-33 y LG, sin embargo no se ha comparado la eficiencia entre uno y otro cuando se induce la diferenciacion celular en medio LG-47. En el presente trabajo se comparo el efecto de preinocular trofozoitos de Entamoeba invadens IP-1 en ambos medios de cultivo (TYI-S-33 y LG) y su efecto en el proceso de enquistamiento inducido en medio LG-47, observandose que no existen diferencias significativas entre utilizar como medio de preinoculo a uno u otro medio de cultivo para la diferenciacion celular de Entamoeba invadens IP-1 en medio LG-47.
Los hongos del orden Ophiostomatales son organismos saprobios y, algunos de ellos parasitos, que ... more Los hongos del orden Ophiostomatales son organismos saprobios y, algunos de ellos parasitos, que ocasionan danos en arboles maderables como el pino ya sea por efecto de dano directo a la albura o por la coloracion (manchado azul) de la misma. Su estudio se ha abordado desde enfoques moleculares y bioquimicos. Un metodo alternativo de estudio y que al mismo tiempo sirve para separar proteinas, es el isoelectroenfoque y la determinacion del pI de las proteinas. En este trabajo se determino el pI de algunas hidrolasas acidas secretadas por Ophisotoma piceae en medio de cultivo minimo (medio de Mathur) adicionado de diferentes fuentes de carbono: xilana, glucosa o alga Nori. El patron de actividades recuperadas en el medio de cultivo libre de celulas fue muy complejo, presentandose pI principalmente en el rango de pH 3 a 5, y dependiente de la fuente de carbono empleada para su induccion. La xilana adicionada al medio minimo a una concentracion del 1%v p/v resulto una fuente de carbono ...
Biomedical Engineering: Applications, Basis and Communications, 2021
Yeast cultures of Saccharomyces cerevisiae were exposed to pulsed magnetic fields of low intensit... more Yeast cultures of Saccharomyces cerevisiae were exposed to pulsed magnetic fields of low intensity. Field intensities from 0.92 mT to 2.73[Formula: see text]mT and frequencies of 20, 77.5, 135, 192.5 and 250[Formula: see text]Hz were employed. Proliferation and cell viability measurements were made in exposed and control cultures. For the frequency of 20[Formula: see text]Hz, statistically significant differences were found ([Formula: see text]) in cell proliferation using field intensities of 1.81 and 2.71[Formula: see text]mT. No changes were observed at this frequency for lower field strengths. At 77.5[Formula: see text]Hz, only changes in proliferation were observed for 0.92[Formula: see text]mT ([Formula: see text]). No changes were observed to any other frequency or intensity of the magnetic field. The cell viability studies were performed at different times, from 4 to 12[Formula: see text]h of exposure. At 1.81[Formula: see text]mT and 20[Formula: see text]Hz, an increase in ...
Epidemiological studies have associated long-term exposure to environmental air pollution particu... more Epidemiological studies have associated long-term exposure to environmental air pollution particulate matter (PM) with the development of diverse health problems. They include infectious respiratory diseases related to the deregulation of some innate immune response mechanisms, such as the host defense peptides' expression. Herein, we evaluated in BALB/c mice the effect of long-standing exposure (60 days) to urban-PM from the south of Mexico City, with aerodynamic diameters below 2.5 µm (PM2.5) and 10 µm (PM10) on the lung's gene expression and production of three host defense peptides (HDPs); murine beta-defensin-3, -4 (mBD-3, mBD-4) and cathelin-related antimicrobial peptide (CRAMP). We also evaluated mRNA levels of Il1b and Il10, two cytokines related to the expression of host defense peptides. Exposure to PM2.5 and PM10 differentially induced lung inflammation, being PM2.5, which caused higher inflammation levels, probably associated with a differential deposition on the airways, that facilitate the interaction with alveolar macrophages. Inflammation levels were associated with an early upregulation of the three HDPs assessed and an increment in Il1b mRNA levels. Interestingly, after 28 days of exposure, Il10 mRNA upregulation was observed and was associated with the downregulation of HDPs and Il1b mRNA levels. The upregulation of Il10 mRNA and suppression of HDPs might facilitate microbial colonization and the development of diseases associated with long-term exposure to PM.
The development of mature biofilms is an aid in numerous aspects of the life cycle of fungi. It i... more The development of mature biofilms is an aid in numerous aspects of the life cycle of fungi. It is well known that Sporothrix schenckii complex causes a benign subcutaneous mycosis, but recent studies have suggestedthat biofilm formation may be one of the important factors involved in its virulence. Here we report the study of the biomass organization and a model of the stages of S. schenckii biofilm development: adsorption, active adhesion, microcolony formation, maturation, and dispersal of biofilm fragments. During the development, the biofilm is surrounded by extracellular matrix, which contains glycoprotein (mannose rich), carbohydrates, lipids, and nucleic acid. In addition, the extracellular DNA increases in extracellular matrix as a key component to structural integrity and antifungal resistance. The study of S. schenckii biofilm contributes to a better understanding of growth biofilm and physiology, adding new insights into the mechanisms of virulence and persistence of pat...
Aim: Sporothrix schenckii is the causative agent of sporotrichosis. A 70-kDa glycoprotein, Gp70, ... more Aim: Sporothrix schenckii is the causative agent of sporotrichosis. A 70-kDa glycoprotein, Gp70, is a candidate for the development of prophylactic alternatives to control the disease, and its gene (GP70) is predicted to encode for a protein of 43 kDa, contrasting with the molecular weight of the native protein. Materials & methods: The GP70 was expressed in bacteria, the recombinant protein purified, used in immunoassays and injected to Galleria mellonella. Results & conclusion: The recombinant protein was detected by anti-Gp70 antibodies, confirming that the Gp70 backbone is a 43-kDa peptide. This protein showed enzyme activity of cyclase and was recognized by sera of patients with sporotrichosis. Although it was not useful for serodiagnosis of sporotrichosis, it conferred protection to animals against experimental sporotrichosis.
Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a so... more Three chitinase forms were identified in Entamoeba invadens cysts following fractionation of a soluble fraction by anionic exchange, size exclusion and hydroxyapatite adsorption chromatographies. The enzymes, named here as A, B and B', showed molecular weights of 64, 33.4 and 33.4 kDa, respectively, as measured by gel filtration. Comparison of their levels of specific activity in partially purified samples revealed
Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of ... more Chitinase activity was measured in extracts of Entamoeba invadens cells as a function of time of encystation in axenic conditions using 4-MU(Ch)3 as substrate. Encystment was paralleled by chitinase activity which showed a peak after about 72 h of cultivation where cysts accounted for 63% of cell population. Thereafter, activity fell off rapidly, whereas encystment continued, reaching 80% at the end of the experiment (96 h). Comparison of activity between cysts and the total cell population in 48- and 72-h-old encysting cultures suggested that chitinase may start to accumulate in the pre-cyst forms. About 70% of the enzyme was recovered in the supernatant following low-speed centrifugation of whole extracts. Most of this activity represented soluble chitinase since it was not sedimented by further centrifugation at 105,000 x g. A minor proportion of enzyme activity remained associated to the buffer-washed, high-speed sediment. In addition to 4-MU(Ch)3, chitinase activity was also measured following the hydrolysis of other substrates such as nascent, preformed or colloidal chitin. Like other chitinases, the cyst enzyme preferred nascent over preformed chitin as substrate. Digestion of the former yielded GlcNAc and minor amounts of (GlcNAc)2 as products. Allosamidin strongly inhibited hydrolysis of the fluorogenic substrate by the amebic chitinase in vitro with a Ki of 0.065 microM. IC50 values were 0.085 microM and 0.16 microM at 5 microM and 10 microM 4-MU(Ch)3, respectively. When added to the axenic medium, the drug markedly retarded encystment though it was partially recovered after longer periods of incubation.
A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized.... more A simplified and reliable method to quantify Entamoeba histolytica cytotoxicity was standardized. Mice spleen leucocytes were utilized as target cells. Interaction time was reduced to 1 h by pelleting interacting cells. To assess target-cell killing by amoebae, a nigrosine exclusion test was employed. Fixation with glutaraldehyde stabilized the percentage of stained target cells. Similar results were obtained when cytotoxicity of the E. histolytica HM1 strain was tested by the traditional and proposed methods. The new method allowed quantification of the contribution of cytolysis and cytophagocytosis to amoebic cytotoxicity. It was also demonstrated that uncloned E. histolytica HM1 strain is a heterogeneous population with respect to cytotoxicity expression.
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