[Microcultures of Entamoeba]. / Microcultivos de Entamoeba.

The purpose of this study was the development of a microculture system for Entamoeba using ultrathin culture chambers, characterized by constant culture conditions, where the growing amebas can constantly be observed under the microscope, and which allows rapid variations in the characteristics of the culture medium, so as to determine the optimal conditions for growth and encystment of the ameba. After a lag phase the number of amebas increased following exponential kinetics. The E. invadens distribution in the culture chamber continued up to a total surface of (444 mm2) with in 7 days. On the whole E. invadens showed a tendency of not forming aggregates and growing until a confluent monolayer was reached. On the other hand, E. histolytica grew faster, covering the internal surface of the chamber in 5 days, and its growth went beyond a monolayer, since it covered all the internal walls, forming multiple aggregates. Encystment was induced in E. invadens by changing the feeding medium in the chamber for encystment medium. The encystment we observed was lower than that previously reported.

Characterization of a calcium-dependent ATPase in Entamoeba invadens.

A high-affinity calcium-dependent ATPase (Ca2+-ATPase) was identified in a crude plasma membrane fraction from Entamoeba invadens (IP-1 strain). The Ca2+-ATPase activity was solubilized from the membrane by utilizing the non-ionic detergent octylglucoside. The activity had an apparent half maximal saturation constant of 0.4 +/- 0.05 microM for free calcium. The calcium activation of ATPase activity followed a cooperative mechanism (Hill number of 2.3 +/- 0.13) which suggests that two interacting sites were involved. The high-affinity Ca2+-ATPase appeared to be magnesium-independent, since by lowering contaminant free magnesium with trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid did not modify the activity observed with Ca2+. The apparent Km of the enzyme for ATP was 31 microM. The observed activity had an optimum pH of 8.8. The enzyme was insensitive to various agents such as Na+, K+, ouabain, dicyclohexylcarbodiimide, KCN, NaN3, mersalyl, quercetin, ruthenium red and vanadate. Only lanthanum (0.5 mM) inhibited 100% the enzymatic activity. Calmodulin and trifluoperazine at the concentrations tested did not modify the Ca2+-ATPase activity.

Induction of encystation of Entamoeba invadens by removal of glucose from the culture medium.

A procedure has been developed for the effective induction of axenic encystation of Entamoeba invadens strain IP-1. Low concentrations of glucose in Diamond's axenic growth medium cause stimulation of the differentiative process. The time course of encystation depended on the density of inoculum. While a culture with an inoculum of 5 X 10(4) cells/ml required about 3 days to initiate encystment, a culture with an inoculum of 1 X 10(6) cells/ml required only 8 hr. Cyst yield was optimal (70%) when a density of 5 X 10(5) cells/ml was employed for the inoculum. Under above conditions it was found that serum is absolutely required while vitamins are not. It was found that encystation could be triggered without change in the osmolarity of the medium and that oxygen does not influence this process.

Location on chitin in the cyst wall of Entamoeba invadens with colloidal gold tracers.

Chitin was located in the cyst wall of Entamoeba invadens with colloidal gold-linked wheat germ agglutinin. Cysts stained differentially from trophozoites when encysting cultures were treated with the gold tracer; cysts acquired a wine-red coloration while, in general trophozoites remained unstained. Observation of cells with the electron microscope revealed that the tracer particles were bound specifically to the walls of the surface of the cyst when cells were exposed in suspension, and to the cyst wall cross-section, when cells were exposed to the tracer in thin section, indicating that chitin fibers were distributed on the surface as well as throughout the matrix of the cyst wall.

Alteracion del enquistamiento de Entamoeba invadens por calcofluor M2R. / Changes in insheathing of Entamoeba invadens by calcofluor M2R

Se estudio el efecto del colorante calcofluor M2R sobre el enquistamiento de Entamoeba invadens. Los resultados observados indican que el calcofluor interfiere con el enquistamiento de la amiba.En presencia de calcofluor las amibas no enquistaron normalmente, solo se formaron paredes de aspecto anormal y la sintesis de quintina se inhibio 72 por ciento

Actividad quitinolitica de Entamoeba invadens. / Chitin activity of Entamoeba invadens

Se investigo la presencia de actividad quitinolitica en extractos de Entamoeba invadens.Para medir esta actividad enzimatica se utilizo como sustrato quitina radioactiva. Los productos de la reaccion se analizaron mediante cromatografia en placa de DEAE-celulosa y papel. Se encontro actividad quitimolitica principalmente en extractos de celulas en proceso de enquistamiento. Los principales productos de hidrolisis de quitina detectados fueron: N-acetilglucosamina y N,N-diacetilquitobiosa. Mediante la determinacion de estos productos de hidrolisis de quitina, fue posible detectar la presencia de actividad de quitina sintetasa en extractos de la amiba

Identification of the structural component in the cyst wall of Entamoeba invadens.

Cyst walls of Entamoeba invadens were isolated and purified. Both whole cysts and purified walls appeared intensely fluorescent when stained with Calcofluor white M2R. Examination of positive replicas of purified cyst walls with the electron microscope revealed the presence of a microfibrillar structure. The main sugars detected in acid hydrolysates from the walls were hexosamines. X-ray diffraction analysis of purified cyst walls demonstrated that the crystalline polymer present was chitin.

[Staining of cysts of Entamoeba invadens, Entamoeba histolytica and Entamoeba coli with wheat germ agglutinin labelled with colloidal gold]. / Tinción de quistes de Entamoeba invadens, Entamoeba histolytica y Entamoeba coli con aglutinina de germen de trigo marcada con partículas de oro coloidal.

Cysts of Entamoeba invadens obtained under axenic conditions were stained with wheat germ agglutinin labelled with colloidal gold. Microscopic observation of encysting cultures revealed that upon staining, cysts acquired a red coloration, while trophozoites remained unstained. E. histolytica and E. coli cysts obtained from asymptomatic patients were also stained red by this technique. Electronmicroscopic examination of stained cells showed a layer of colloidal gold granules attached to the surface of cysts, while trophozoites remained free of gold granules. The results of the staining procedure developed, agreed with the known facts that wheat germ agglutinin is a lectin that binds specifically to chitin and its oligomers, and that chitin is present in the cyst wall of E. invadens. The results suggested that chitin is also present in the wall of cysts of E. histolytica and E. coli.

[Origin of sac-shaped walls in Entamoeba invadens cultures during the encystment process]. / Origen de las paredes en forma de saco observadas en cultivos de Entamoeba invadens en proceso de enquistamiento.

Changes in the population of trophozoites, cysts and bag-shaped walls in an encysting culture of Entamoeba invadens were determined. The results indicated that bag-shaped walls do not play a precursor role in the encystation of the organism. Samples of cells from encysting cultures were stained in vivo with Calcofluor M2R. Microscopic observations indicated also that bag-shaped walls do not play a precursor role in the formation of the cyst. It was suggested that bag-shaped walls arise from an abnormal response of some trophozoites when the culture is transferred to the encystation medium.

[Formation of the cell wall during the formation of Entamoeba invadens cysts]. / Formación de la pared celular durante el enquistamiento de Entamoeba invadens.

The presence of chitin in the walls of E. invadens cysts has provided a new way to study the process of encystation, since chitin stains intensely in the presence of Calcofluor white M2R, a fluroescent dye. With this technique we have observed various stages in the differentiation of trophozoite membrane, then the cyst is formed inside the trophozoite and finally the cyst leaves the trophozoite after partial degradation of trophozoite body.

[Composition of the cell wall of Entamoeba invadens cysts]. / Composición de la pared celular de quistes de Entamoeba invadens.

Cyst walls isolated from Entamoeba invadens have a microfibrillar structure. The main sugars detected in acid hydrolysates from walls were hexosamines. X-ray diffraction analysis of the cyst walls demonstrated that the only crystalline polymer present was chitin.

In vitro formation of an active multienzyme complex in the tryptophan pathway of Neurospora crassa.

A multienzyme complex that catalyzes the anthranilate synthetase, phosphoribosylanthranilate (PRA) isomerase, and indoleglycerolphosphate (InGP) synthetase reactions was produced in vitro when extracts from a tryp-1 mutant and a tryp-2 mutant of Neurospora crassa were mixed. The sedimentation values and the molecular weights for the interacting components obtained from the mutants were estimated by sucrose gradients and by gel filtration on Sephadex columns. The component coded for by the tryp-2 gene (a-component) which is present in the tryp-1-17 mutant has a sedimentation coefficient of 4.5S and molecular weight of 70,000. The component coded for by the tryp-1 gene (i-component) which is present in the tryp-2-6 mutant has a sedimentation coefficient 7.5S and a molecular weight of 170,000. The complex formed in vitro had similar properties to those previously reported for the wild-type complex. A Q(10) value of 3 and an apparent activation energy of 18,000 cal/mole were calculated for the formation of the complex from the two components.