bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphism and Ultrastructure of a Colpodean Ciliate of the Genus Platyophryides Foissner, 1987
Authors: DE PUYTORAC PIERRE , KATTAR M. RAGEL , GROLIERE C. ALAIN , NETO INACIO DA SILVA
Published: May 2007

ABSTRACT. A ciliate isolated from a pond in Brazil, transformed to a giant form when its food was shifted from a bacterial prey to a ciliate prey. This polymorphism is immediately reversible when the prey ciliates, either Tetrahymena or Colpidium, disappear from the culture medium. By its life cycle, morphology, and ultrastructure, this ciliate belongs to the Class Colpodea. it could belong to the genus Platyophryides Foissner, 1987, except that its micronucleus is not enveloped by the macronuclear membrane. The systematic position of the genus Platyophryides, the validity of the three species in this genus, and the characteristics of the Cyrtolophosidida are discussed.
A Temperature‐Sensitive Mutation Affecting Synthesis of Outer Arm Dyneins in Tetrahymena thermophila
Authors: ATTWELL GWILYM J. , BRICKER CONNIE S. , SCHWANDT ANITA , GOROVSKY MARTIN A. , PENNOCK DAVID G.
Published: May 2007

ABSTRACT. We have characterized a novel, temperature‐sensitive mutation affecting motility in Tetrahymena thermophila. Mutants grew and divided normally at the restrictive temperature (38°C), but became nonmotile. Scanning electron microscopic analysis indicated that nonmotile mutants contained the normal number of cilia and that the cilia were of normal length. Transmission electron microscopic analysis indicated that axonemes isolated from nonmotile mutants lacked outer dynein arms, so the mutation was named oad I (outer arm defficient). Motile mutants shifted to 38° C under conditions that prevent cell growth and division (starvation) remained motile suggesting that once assembled into axonemes at the permissive temperature (28° C) the outer arm dyneins remain functional at 38° C. Starved, deciliated mutants regenerated a full complement of functional cilia at 38° C, indicating that the mechanism that incorporates the outer arm dynein into developing axonemes is not affected by the oad I mutation. Starved, nonmotile mutants regained motility when shifted back to 28° C, but not when incubated with cycloheximide. We interpret these results to rule out the hypothesis that the oad I mutation affects the site on the microtubules to which the outer arm dyneins bind. Axonemes isolated from mutants grown for one generation at 38° C had a mean of 6.0 outer arm dyneins, and axonemes isolated from mutants grown for two generations at 38° C had a mean of 3.2 outer arm dyneins. Taken together, these results indicate that the oad I mutation affects the synthesis of outer arm dyneins in Tetrahymena.
Chemosensory Response in Blepharisma. I. Accumulation of Cells in Products of Bacterial Metabolism
Authors: MATSUOKA TATSUOMI , TAKAHASHI MAKOTO , WADA KEISUKE , TANEDA KOJI
Published: May 2007

ABSTRACT. Blepharisma cells were attracted by a pellet of live bacteria (Enterobacter) which was separated from the Blepharisma suspension by a cellulose membrane (fractionation: M.W. 14,000). The cells, however, were not attracted by killed bacteria. Crude and heat‐treated supernatants obtained from bacterial suspension also induced chemoaccumulation of cells. These results suggest that the cells of Blepharisma detect certain small molecules, produced by live bacteria, that can pass through the cellulose membrane and are stable to heat. From the live bacteria supernatant, several ninhydrin‐positive substances were isolated by means of two‐dimensional thin‐layer chromatography. Several of the spots contained substances that attracted the cells, indicating that certain ninhydrin‐positive components, such as peptides or free amino acids (probably products of bacterial metabolism), may serve as a signal for food.
Effect of Shaking on the Growth of Diluted Cultures of Tetrahymena
Authors: HELLUNG‐LARSEN PER , LYHNE INGER
Published: May 2007

ABSTRACT. Cultures of Tetrahymena pyriformis, T. thermophila and T. pigmentosa have been studied with regard to growth rates in shaken and unshaken flasks. In the standard medium, a minimum doubling time of 170 min was obtained for T. pyriformis at 28° C in the unshaken cultures. If the depth of the medium was less than 1 cm, the gyratoric shaking increased the doubling time to 340 min. The effect of shaking could be reduced by the addition of dextrane. Cells subjected to shaking were observed in different media and at different growth temperatures. If cultures were inoculated with 104 cell·ml−1 or more, the effect of shaking was absent. However, with inoculates of 103 or 102 cell·ml−1, the doubling times for T. pyriformis increased to 240 and 275 min, respectively. Periods of 2 min shaking followed by rest for 60 min could not induce an effect.
Sustained Food Vacuole Formation by Axenic Paramecium tetraurelia and the Inhibition of Membrane Recycling by Alcian Blue
Authors: KANESHIRO EDNA S. , REUTER STEVEN F. , QUATTRONE FRANK J. , MORRIS RANDAL E.
Published: May 2007

It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue‐grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue‐grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue‐treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.
Surface Domains in the Pathogenic Protozoan Tritrichomonas foetus
Authors: BENCHIMOL MARLENE , KACHAR BECHARA , SOUZA WANDERLEY DE
Published: May 2007

ABSTRACT The quick‐freezing and freeze‐etching techniques were used to analyze surface domains of Tritrichomonas foetus. The surface of the protozoan body was not smooth, presenting surface projections, except on the flagellar surface. Images of the actual surface of the anterior flagella revealed the presence of intramembranous particles that form rosettes, as observed on the protoplasmic fracture face. They may represent integral transmembrane proteins exposed at the cell surface. Surface specializations were also observed at the flagella base and where the recurrent flagellum attaches to the cell body.
An Isoenzyme Analysis of the Genus Phytomonas: Genetic, Taxonomic and Epidemiologic Significance
Authors: GUERRINI FRANCOISE , SEGUR CENDRINE , GARGANI DIDIER , TIBAYRENC MICHEL , DOLLET MICHEL
Published: May 2007

ABSTRACT We studied 31 Phytomonas stocks isolated from various hosts and a broad geographical range by isoenzyme electrophoresis (14 loci) and population genetics analysis. The total variability is considerable since many stocks share no allele. Population genetic analysis strongly suggests that Phytomonas zymodemes behave as natural clones, as already proposed by us for several other protozoan species. These clones should be considered as actual taxa in all applied studies. Latex plants and phloemic plants (coconut and palm trees) harbor distinct sets of clones; hence, latex plants studied in this article are probably not a reservoir for parasites of the coconut and palm tree.
Ciliate Protozoa in the Rumen of Kafue Lechwe, Kobus leche kafuensis, in Zambia, with the Description of Four New Species
Authors: IMAI SOICHI , TSUTSUMI YOSHIATSU , YUMURA SHOJIRO , MULENGA ALBERT
Published: May 2007

ABSTRACT The composition of the rumen ciliate fauna in 76 Kafue lechwe inhabiting a limited area in Zambia was surveyed and five genera containing 24 species with 16 formae belonging to the family Ophryoscolecidae were identified. Four new species belonging to Diplodiniinae were recognized and described as Diplodinium lochinvarense n. sp., Diplodinium leche n. sp., Diplodinium zambiense n. sp., and Metadinium ossiculi n. sp. In addition, Ostracodinium gracile form fissilaminatum Dogiel, 1932 was found for the second time and described as Metadinium fissilaminatum n. comb. The species composition was fairly unusual. Seven of the species have been found only in African wild antelopes and these species were found more frequently than cosmopolitan species. There was no evidence of isotrichid species. The average density of ciliates per 1 ml of the rumen fluid was 25.7 times 104, and the number of ciliate species per head of host was 10.8.
Production and Characterization of Three Polyclonal Antibodies Raised Against Cyst Wall Proteins of a Hypotrichous Ciliate
Authors: RIOS ROSA M. , MARTIN JESUS , TORRES ANTONIO , FEDRIANI CONCEPCION
Published: May 2007

ABSTRACT Three polyclonal antibodies raised against Paraurostyla sp. cyst wall polypeptides of molecular weight 110,000 (p110), 66,000 (p66) and 52,000 (p52) have been obtained. The specificity of the antisera was tested by immunoblotting. Anti‐p110 antibody detected five bands of 300, 170, 135, 110 and 40 kDa, respectively. Antiserum obtained against p66 recognized only this protein. Anti‐p52 antiserum showed reaction for two different bands of 52 and 44 kDa, respectively. The precise localization of these proteins in the cyst wall was assessed by light microscope immunocytochemistry. Anti‐p110 antiserum produced a strong positive reaction in both the ectocyst and endocyst. Both anti‐p66 and anti‐p52 antibodies recognized the ectocyst.
Trypanosoma brucei brucei: In Vitro Production of Metacyclic Forms
Authors: HIRUMI H. , HIRUMI K. , MOLOO S. K. , SHAW M. K.
Published: May 2007

ABSTRACT An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast‐like cells (feeder cell layers); 2) maintained in glucose‐free Eagle's minimum essential medium supplemented with 10 mM L‐proline, 2 mM L‐glutamine and 20% (v/v) fetal bovine serum at 27° C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27° C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long‐term cultures also appeared in diethylaminoethyl cellulose‐isolated populations along with metacyclic forms.
Ultrastructural Study of the Development of Vairimorpha necatrix (Kramer, 1965) (Protozoa, Microsporida) in Larvae of the Corn Earworm, Heliothis zea (Boddie) (Lepidoptera, Noctuidae) with Emphasis on Sporogony
Authors: MITCHELL MAUREEN J. , CALI ANN
Published: May 2007

The ultrastructure of the life cycle of Vairimorpha necatrix (Kramer) was studied in Heliothis (Helicoverpa) zea larvae raised at temperatures ranging from 15° C to 30° C. In contrast to the description of the type species, both patterns of development (Nosema‐like and Thelohania‐like) that characterize the genus Vairimorpha were present at all temperatures examined. No temperature‐related differences in the ultrastructure of Vairimorpha necatrix were observed; however, the effects of temperature variation were apparent in 1) the time between inoculation with V. necatrix and the initiation of the parasite life cycle and 2) the duration (or length) of the parasite life cycle. An additional form of the parasite, that of a diplokaryon in a sporophorous vesicle, was present during early sporogony. The nuclei of the diplokaryon separated followed by cytokinesis, forming two monokaryotic parasites, and eventually two plasmodia, within the sporophorous vesicle. This pattern of development is characteristic neither of Nosema nor Thelohania, nor has it been described for Vairimorpha.
Production and Characterization of Monoclonal Antibodies Against a Highly Immunogenic Fraction of Entamoeba histolytica (NIH:200) and Their Application in the Detection of Current Amoebic Infection
Authors: SENGUPTA KRISANU , DAS PRADEEP , JOHNSON TONY M. , CHAUDHURI PARTHA P. , DAS DEBASISH , NAIR G. BALAKRISH
Published: May 2007

Six monoclonal antibodies (MAbs) were produced against a highly immunogenic fraction derived by the chromatographic separation of the soluble preparation of axenic Entamoeba histolytica (strain NIH:200) trophozoites. Isotype characterization of the six MAbs revealed that four belonged to the IgM class and one each to the IgG1 and the IgG2a subclasses. The immunoreactivity patterns and the specificity of the MAbs with homologous and heterologous antigens were analyzed by the enzyme‐linked immunotransfer blot technique and by the enzyme‐linked immunosorbent assay. The MAbs reacted intensely with isolates of E. histolytica (strain NIH:200 as well as a local isolate MX1) but showed no reactivity with Entamoeba coli, Iodamoeba butschlii, Endolimax nana, Entamoeba hartmanni, free‐living amoeba (Acanthamoeba harticolus) and other enteric parasites. Using the IgG1 MAb as a detecting antibody, a polyclonal‐monoclonal antibody‐based enzyme‐linked immunosorbent assay was developed for the detection of E. histolytica antigens in stool samples of infected patients. The detection limit of the assay was 8 ng of amoebic antigen. This test was found to be specific and sensitive and yielded 100% positive results in cases with amoebiasis but did not react with controls included in the evaluation. The MAb‐based enzyme‐linked immunosorbent assay developed in this study will be an important test for the diagnosis of E. histolytica in the feces of infected humans; however, the limitation of the test is the inability to discriminate the pathogenic status of the amoeba detected in the stool.
Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes
Authors: ALAM SHARIFUL , BANNO YOSHIKO , NOZAWA YOSHINORI
Published: May 2007

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X‐100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q‐Sepharose, hydroxyapatite HCA‐100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS‐polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent Km value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca2+ but not Mg2+ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (∼ 9%) and phosphatidylethanolamine (∼ 2%).
Pharmacological Characterization of an Opioid Receptor in the Ciliate Tetrahymena
Authors: CHIESA RICARDO , SILVA WALTER I. , RENAUD FERNANDO L.
Published: May 2007

A pharmacological characterization has been performed of the opioid receptor involved in modulation of phagocytosis in the protozoan ciliate Tetrahymena. Studies on inhibition of phagocytosis by mammalian prototypic opioid agonists revealed that morphine and β‐endorphin have the highest intrinsic activity, whereas all the other opioids tested can only be considered partial agonists. However, morphine (a mu‐receptor agonist) is twice as potent as β‐endorphin (a delta‐receptor agonist). Furthermore, the sensitivity for the opioid antagonist naloxone, determined in the presence of morphine and β‐endorphin, is very similar to the sensitivity exhibited by mammalian tissues rich in mu‐opioid receptors. We suggest that the opioid receptor coupled to phagocytosis in Tetrahymena is mulike in some of its pharmacological characteristics and may serve as a model system for studies on opioid receptor function and evolution.
In Vitro Susceptibility of Acanthamoeba Culbertsoni to Inhibitors of Folate Biosynthesis1
Authors: MEHLOTRA RAJEEV K. , SHUKLA ONKAR P.
Published: May 2007

ABSTRACT. The effects of different sulphonamides, dihydrofolate reductase inhibitors and other inhibitors of folate metabolism on growth of Acanthamoeba culbertsoni in a chemically defined medium are reported. Among the sulphonamides, sulphamethoxazole and sulphadiazine were most effective followed by sulphanilamide and sulphaguanidine. Inhibition by each sulphonamide was reversed by p‐aminobenzoic acid as well as folic acid. 7‐Methylguanosine, a pteridine synthesis‐inhibitor, did not inhibit multiplication of A. culbertsoni. Among the dihydrofolate reductase inhibitors, pyrimethamine blocked the amoebic growth at 100 μg/ml, while trimethoprim and cycloguanil palmoate failed to cause significant inhibition of growth even at 250 μg/ml. Metoprine inhibited amoebic growth completely at 50 μg/ml. Methotrexate and a thymidylate synthetase inhibitor 5‐fluorouracil inhibited growth strongly, with IC50 values (the concentration of the drug which causes 50% inhibition of the growth at 72 h) of 1.97 and 2.45 μg/ml, respectively. Inhibition by methotrexate, metoprine or 5‐fluorouracil could not be reversed by folic acid, folinic acid, thymidine, or folinic acid plus thymidine. the results indicate unusual features in A. culbertsoni folate metabolism.
The Invasive Nature of an Infectious Bacterial Symbiont
Authors: SOLDO ANTHONY T. , MUSIL GEORGE , BRICKSON SYLVIA A.
Published: May 2007

ABSTRACT. Xenosomes are infectious bacterial symbionts that exist exclusively in the cytoplasm of the small philasterine marine ciliate Parauronema acutum. We have used this host‐symbiont system as a model to study infection. In the past we postulated that infection took place by a process in which the symbionts escaped digestion and entered into the host's cytoplasm through the food vacuole during phagocytosis. This is clearly not the case. We now present evidence based on electron microscopic observations that the symbionts infect in a manner involving direct penetration of the protozoan's cell membranes. We have obtained additional data that suggest that, following entrance of the symbionts into the cytoplasm, only a single xenosome is required to establish an infection.
Spatial and Temporal Aspects of Mixotrophy In Chesapeake Bay Dinoflagellates
Authors: BOCKSTAHLER KATRIN R. , COATS D. WAYNE
Published: May 2007

ABSTRACT. Gymnodinium sanguineum, Gyrodinium uncatenum, and Ceratium furca are large phototrophic dinoflagellates that commonly form red tides in the mesohaline portion of Chesapeake Bay during the summer. Examination of protargol‐stained specimens revealed that these dinoflagellates also feed heterotrophically as indicated by the presence of food vacuoles containing partially digested prey. Ingested prey were generally identified as nanociliates (≥20 μm) belonging to the oligotrich genera Strobilidium and Strombidium; occasionally other small ciliates (e.g. Balanion sp. and Mesodinium sp.), dinoflagellates, and diatoms were observed in early stages of digestion. the percentage of these mixotrophs that had ingested prey was usually less than 20%, but approached 30% in some samples. Occurrence of food vacuoles in Gymno. sanguineum was positively correlated with ≤20 μm oligotrichous ciliate density; limited data for Gyro. uncatenum suggests a similar relationship, but C. furca feeding was not related to nanociliate densities.
Reclinomonas americana N. G., N. Sp., a New Freshwater Heterotrophic Flagellate
Authors: FLAVIN MARTIN , NERAD THOMAS A.
Published: May 2007

ABSTRACT. A new heterotrophic flagellate has been discovered from sites in Maryland, Michigan and Wyoming. The flagellate resides within a lorica constructed of a meshwork of intertwined fibrils with the outer surface invested with nail‐shaped spines. The organism “reclines” within the lorica with its ventral aspect directed upward, and has two heterodynamic flagella, neither of which bears mastigonemes. One flagellum is directed upward and the other is arched over the ventral aspect of the body. Ingestion of bacteria takes place at the left posterior half of the cell. The organism is anchored to the lorica on the right posterior side by a series of regularly spaced cytoplasmic bridges and at the left anterior of the cell by a cytoplasmic appendage similar to the “languette cytoplasmique” found in some bicosoecids. The right side of the cell is raised into a flattened lip with the outer margin reinforced by a ribbon of microtubules. The new flagellate has mitochondria with tubular cristae and lacks a Golgi. A new genus is created to accommodate both the new flagellate described herein and Histiona campanula Penard. A new family is proposed to include the new genus and Histiona.
Absence of Transitory [Ca2+]I Flux During Early In Vitro Metacyclogenesis of Trypanosoma cruzi1
Authors: KRASSNER STUART M. , CHANG JOHNNY , PAK SUNG , LUC KIM‐OANH , GRANGER BARBARA
Published: May 2007

ABSTRACT. The phorbol ester TPA (phorbol 12‐myristate 13‐acetate) substitutes for CO2 as an agonist for transforming Trypanosoma cruzi epimastigotes to the metacyclic trypomastigote stage in a starvation medium consisting of phosphate buffered saline + 10 mM proline, 10 mM sodium acetate and 0.035% NaHCO3. Since TPA is thought to stimulate protein kinase C by mimicking the activity of the secondary messenger diacylglycerol, the above result suggested that T. cruzi metacyclogenesis could be activated by a Ca2+‐dependent protein kinase C signal induction pathway. Accordingly, cytosolic calcium flux ([Ca2+]i) in epimastigotes, activated with 5% CO2 or TPA (10‐7 M), was measured with the Ca2+ molecular probe, fluo‐3AM. In addition, [Ca2+]i was measured in cells incubated with putative metacyclogenic agonists (e.g. proline, glutamate, bioamines, ionophores and catecholamines). None of the compounds studied, except for EGTA, affected cytosolic Ca2+ levels. Control assays with 11 μM thapsigargin, which mobilizes noncytoplasmic Ca2+ stores by inhibiting endoplasmic reticulum Ca2+‐ATPase. validated our fluorometric assay procedure. Although thapsigargin significantly increases cytoplasmic Ca2+ fluorescence, it has no effect on transformation. The protein kinase C inhibitors staurosporine, H‐7 and HA 1004 were tested for their effect on T. cruzi metacyclogenesis. Low concentrations of staurosporine and HA 1004 significantly elevated Pent strain transformation while H‐7 had no effect on Peru strain metacyclogenesis. Inhibitor H‐7 did significantly depress CL transformation. the results indicate that induction of T. cruzi metacyclic trypomastigote formation by CO2 and TPA is not accompanied by changes in cytosolic Ca2+ and do not provide supporting evidence for participation of a protein kinase C‐mediated phosphoinositide cascade in metacyclogenesis.
Antagonistic Action of the Bacterium Bacillus Licheniformis M‐4 Toward the Amoeba Naegleria Fowleri
Authors: CORDOVILLA PILAR , VALDIVIA EVA , GONZALEZ‐SEGURA ALICIA , GALVEZ ANTONIO , MARTINEZ‐BUENO MANUEL , MAQUEDA MERCEDES
Published: May 2007

ABSTRACT. Free‐living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis). In a broad bacterial screening from soil and water we have isolated three strains (M‐4, D‐13 and A‐12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp. and also against different Gram‐positive and Gram‐negative bacteria. Physical‐chemical characteristics, partial purification and biological activities of a substance produced by the M‐4 strain have been investigated. This substance (m‐4) is stable at high temperature (up to 100°C) and extremes of pH (2.5‐9.5) and also at ‐20°C for months. Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium. Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m‐4.
Gametocytogenesis of Leucocytozoon Smithi
Authors: STEELE EDNA JOSEF , NOBLET GAYLE PITTMAN
Published: May 2007

ABSTRACT. Development of young gamelocytes of Leucocytozoon smithi into morphologically mature forms was studied using electron microscopy. Gametocytogenesis began on day seven post inoculation when merozoites, released from ruptured hepatic schizonts, developed into gametocytes within mononuclear phagocytes or leukocytes (monocytes or lymphocytes). No gametocytes were observed in any erythrocytes or polymorphonuclear leukocytes. Two gametocyte forms, round and elongate, were observed. Immature round gametocytes occurred on days 7‐10 post inoculation in the deep vasculature of liver, lung and spleen. Mature elongate gametocytes were observed beginning on day 12 post inoculation in both the deep tissue vasculature and peripheral circulation of the turkey host. Growth and elongation of the gametocyte resulted in distortion of the host cell and its nucleus. the host cell nucleus initially was elongated and displaced to one side or indented by the growing parasite. Eventually, the nucleus was laterally compressed or split into two or three fragments. the compressed host cell cytoplasm was displaced longitudinally and stretched over the parasite to form hornlike cytoplasmic extensions from each end. the potential role of microtubules in the elongation of the gametocyte and its host cell, and possibly in the indentation and splitting of the host cell nucleus, is discussed.
The Nuclear Apparatus of the Ditransversal Ciliate Homalozoon Vermiculare (Ciliophora, Rhabdophora) During Interphase and Division Ii. the Micronuclei and Some Observations On Conjugation
Authors: LEIPE DETLEF D. , HAUSMANN KLAUS
Published: May 2007

ABSTRACT. The nuclear apparatus of H. vermiculare consists of a single moniliform macronucleus and about 25 micronuclei. the micronuclei are about 3 μm in diameter and characterized by a meshwork of thick condensed chromatin. Mitosis is intranuclear and acentric as in all other ciliates. In metaphase, interpolar and chromosomal microtubules are abundant and the length of the micronuclei increases to about 5 μm. In late anaphase, interzonal microtubules become prominent and the spindle elongates to about 50 μ. In meta‐ and anaphase, the microtubules of the spindle are attached to the polar vesicles, and in anaphase, chromosomes become attached to it. In contrast to most other eukaryotes, micronuclear mitosis is not strictly bound to cell division in H. vermiculare. While most of the micronuclei divide prior to cytokinesis, others retain their interphasic shape or degenerate. In addition, some micronuclei divide in the interdivision period, i.e. between two successive divisions of the cell and macronucleus. Mating cells of H. vermiculare become joined to each other in the cilia‐free region covering the cytostome. In the course of conjugation, the cell membranes and the underlying oral filamentous sheaths of both cells fuse, thus uniting the endoplasm of both cells in the mouth region. Synaptonemal complexes in the meiotic chromosomes are more distinct in H. vermiculare than in most other dilates. the micrographs presented here depict dearly the central filament, transverse elements, and other substructures.
Genetic Diversity of Pneumocystis carinii Derived from Infected Rats, Mice, Ferrets, and Cell Cultures
Authors: WEINBERG GEOFFREY A. , DURANT PAMELA J.
Published: May 2007

ABSTRACT. The degree of strain and/or species diversity among Pneumocystis carinii isolates is unknown. As a first approach to the study of P. carinii genetic relatedness, we compared the pulsed field gel electrophoretic karyotypes of P. carinii derived from lung homogenates of three immunosuppressed host animals: rats transtracheally inoculated with P. carinii‐infected rat lung; mice transtracheally inoculated with P. carinii‐infected mouse lung; and ferrets which developed reactivated latent P. carinii pneumonia. Rat P. carinii propagated on HEL299 cells was also examined. Karyotypes of P. carinii DNA from both rat lung homogenate and cell culture were identical (14 bands, 315–680 kb). In contrast, mouse and ferret P. carinii DNA karyotypes were each distinctly different from the rat P. carinii samples (mouse P. carinii 15 bands, 315–610 kb; ferret P. carinii nine bands, 410–760 kb). Three distinct rat P. carinii gene probes reacted with both Southern‐transferred rat and mouse P. carinii DNA but not with ferret P. carinii DNA. Thus, P. carinii from rat, mouse, and ferret are genetically diverse. The results are consistent with recently reported antigenic and nucleic acid sequence differences among P. carinii isolates recovered from different hosts.
Trypanosoma brucei brucei and T. b. gambiense: Stumpy Bloodstream Forms Express More CB1 Epitope in Endosomes and Lysosomes than Slender Forms
Authors: BRICKMAN MARLA J. , BALBER ANDREW E.
Published: May 2007

CB1‐glycoprotein is a component of flagellar pocket, endosome, and lysosome membranes of long, slender bloodstream forms of the Trypanosoma brucei subgroup of African trypanosomes. We have used immunoblotting, immunofluorescence, and cryoimmunoelectron microscopy to study CB1‐glycoprotein expression as long, slender bloodstream forms of pleomorphic T. b. brucei and T. b. gambiense transform through intermediate stages into short, stumpy forms. Intermediate and stumpy forms express more CB1‐glycoprotein than long, slender forms. These results, coupled with previous work showing that procyclic forms do not express CB1‐glycoprotein, show that the expression of lysosomal membrane glycoproteins is regulated coordinately with other aspects of lysosome and endosome function as these trypanosomes go through their life cycle.
Artificial Infections of Pneumocystis carinii in the SCID Mouse and their Use in the In Vivo Evaluation of Antipneumocystis Drugs
Authors: COMLEY JOHN C. W. , STERLING ALETHEA M.
Published: May 2007

A model for the in vivo evaluation of antipneumocystis drugs has been developed in SCID mice infected intratracheally with cryopreserved mouse‐derived Pneumocystis carinii. The development of a highly reproducible fatal P. carinii pneumonia occured within 10 weeks (mean survival time ± SEM = 72.2 ± 1.2 days). Continuous administration of dexamethasone (2 mg/liter in the drinking water) exacerbated the rate of onset of severe P. carinii pneumonia (mean survival time ± SEM = 63 ± 1.3 days) in SCID mice. The number of cysts per g of lung homogenate (homogenate counts) were maximal with an inoculum of 20,000 cysts at 6 weeks post infection. Homogenate counts correlated with infection scores (graded assessments of immunofluorescent cysts on lung impression smears) suggesting that infection scoring accurately and rapidly reflects the severity of P. carinii pneumonia in SCID mice. These studies led to the development of a drug screening protocol in which Pneumocystis‐free female SCID mice (20–25 g) were started on dexamethasone 7 days prior to IT inoculation with a single dose of 20,000 cysts. Drugs were evaluated either for: a) prophylaxis (continuously from day 1 post infection) or b) treatment (from day 21 post infection) until day 42 post infection, when all mice were killed and infection scores determined. Co‐trimoxazole (at 250 mg sulfamethoxazole + 50 mg trimethoprim/kg/day) given in the drinking water was found to be highly effective in both the prophylaxis and treatment of mouse P. carinii pneumonia. Co‐trimoxazole remained very effective in the prophylaxis P. carinii pneumonia in the SCID mouse at 125 mg sulfamethoxazole + 25 mg trimethoprim/kg/day p.o. and showed some enhancement of efficacy over sulfamethoxazole alone at 125 mg/kg/day p.o., suggesting limited synergy between sulfamethoxazole and trimethoprim. The results presented provide confirmation of the usefulness and predictability of the model.
Molecular Characterization of a Cytoplasmic Dynein from Dictyostelium
Authors: KOONCE MICHAEL P. , GRISSOM PAULA M. , LYON MARY , POPE THERESA , McINTOSH J. RICHARD
Published: May 2007

Cytoplasmic dynein is a high molecular weight, microtubule‐based mechanochemical ATPase that is believed to provide motive force for a number of intracellular motilities, including transport of membrane‐bound organelles. Cytoplasmic dynein also localizes to the mitotic spindles of some organisms and to the kinetochore regions of some condensed chromosomes, where it may play an active role in spindle assembly, spindle position, and/or chromosome movement during cell division. Despite active research efforts from a number of laboratories, little detail is yet available about dynein‐based cellular activities. This paper describes our efforts to characterize cytoplasmic dynein from Dictyostelium and to use this protist as a molecular genetic factory to probe structure‐function relationships of this molecule.
Spontaneous All‐or‐Nothing Action Potentials in the Ciliate Bursaridium difficile
Authors: BERG TROND OLAV , SAND OLAV
Published: May 2007

The electrical membrane properties and the swimming behaviour of the freshwater ciliate Bursaridium difficile were studied by current clamp recordings and video analysis. The resting membrane potential was –45 ± 6 mV (mean ± SD, n = 80), and the input resistance and membrane capacitance were 109 ± 42 megaohms (MΩ) (n = 63) and 457 ± 150 picofarads (pF) (n = 42), respectively. Based on an estimated surface area of 6.8 × 10‐4 cm2, the corresponding specific membrane resistance and capacitance are 7.4 × 104Ω× cm2 and 0.7 μF/cm2. Bursaridium difficile generates spontaneous, all‐or‐nothing action potentials with a well‐defined threshold in normal medium. The spontaneous firing frequency was 0.22 ± 0.06 Hz (n = 80). The maximum rate of rise of the action potentials was less than 1 V/s, and they displayed a prolonged plateau phase (0.5–1 s). The action potentials were abolished in nominal Ca2+‐free solution and are thus Ca2+‐spikes. The swimming pattern of Bursaridium in homogeneous surroundings is composed of forward swimming periods interrupted by regular, short periods of backward swimming followed by a change in the forward swimming direction. The turning frequency corresponded to the spontaneous firing frequency, and only forward swimming was observed in nominal Ca2+‐free solution. The periods of backward swimming activity are thus linked to the spontaneous action potentials.
kDNA and rDNA Sequences Reveal a Phylogenetic Cluster of Species Originally Placed in Different Genera of Trypanosomatids
Authors: NUNES LUIZ R. , TEIXEIRA MARTA M. G. , CAMARGO ERNEY P. , AFFONSO HELOIZA T.
Published: May 2007

ABSTRACT Hybridization using kDNA and rDNA sequences as probes was performed to study phylogenetic relatedness of different species of trypanosomatids. Using this approach, we identified five organisms which had been classified as Phytomonas and Herpetomonas that were more closely correlated to each other phylogenetically than to any other species or isolates from either genera. These findings raise doubts about the validity of the current classification of Trypanosomatidae. Finally, we demonstrated the usefulness of kDNA sequences as an alternative to genomic sequences in obtaining phylogenetic information on trypanosomatids.
Monoclonal Antibody to a Bacterial Endonuclear Symbiont Holospora Cross Reacts with Proteins of Contractile Vacuole Radial Canals of Paramecium Species
Authors: DOHRA HIDEO , FUJISHIMA MASAHIRO , FOK AGNES K. , ALLEN RICHARD D.
Published: May 2007

ABSTRACT A monoclonal antibody (mAb) IR‐2‐1 was raised against a 67‐kDa protein purified from the macronucleus‐specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67‐kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR‐2‐1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa‐free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air‐dried P. caudatum. These results suggest that the 67‐kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule‐specific mAb, DS‐1, the antigens for IR‐2‐1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium. Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.
Effect and Localization of Trifluralin in Plasmodium falciparum Gametocytes: An Electron Microscopic Study1
Authors: KAIDOH TOSHIYUKI , NATH JAYASREE , FUJIOKA HISASHI , OKOYE VINCENT , AIKAWA MASAMICHI
Published: May 2007

Trifluralin, a herbicide which is known to bind to plant and algal tubulin, induced ultrastructural changes in the microtubules of the mature Plasmodium falciparum gametocytes in vitro. Trifluralin treatment led to disassembly of the well ordered subpellicular microtubules, whereas it had no effect on microtubules of human platelets or of rat neuronal cells in vitro. The disassembled microtubules showed fragmented large tubular structures, which frequently were associated with the pellicular membranes. Electron microscopic autoradiography showed radioactive trifluralin associated with the microtubule fragments. These results provide evidence that trifluralin selectively binds to microtubules in malaria parasites and causes disruption of their structure.
Molecular Evolutionary Analyses of Nuclear‐Encoded Small Subunit Ribosomal RNA Identify an Independent Rhizopod Lineage Containing the Euglyphina and the Chlorarachniophyta
Authors: BHATTACHARYA DEBASHISH , HELMCHEN THOMAS , MELKONIAN MICHAEL
Published: May 2007

The Rhizopoda comprise a diverse assemblage of protists which depend on lobose or filose pseudopodia for locomotion. The biochemical and morphological diversity of rhizopods has led to an uncertain taxonomy. Ribosomal RNA sequence comparisons offer a measure of evolutionary relatedness that is independent of morphology and has been used to demonstrate a polyphyletic origin of the Lobosea. We sequenced complete small subunit ribosomal RNA coding regions from the filose amoebae, Euglypha rotunda and Paulinella chromatophora (Euglyphina) to position these taxa in the eukaryote phylogeny. The neighbor‐joining analyses show that E. rotunda and P. chromatophora share a monophyletic origin and are not closely related to any lobose amoebae in our analyses. Instead, the Euglyphina form a robust sister group to the Chlorarachniophyta. These results provide further evidence for the polyphyly of the Rhizopoda and support the creation of a new amoeboid lineage which includes the Euglyphina and the chlorarachniophyte algae; taxa with tubular mitochondrial cristae and filose or reticulate pseudopodia.
Phylogeny of Adeleid Blood Parasites with a Partial Systematic Revision of the Haemogregarine Complex
Authors: SIDDALL MARK E.
Published: May 2007

ABSTRACT. The phylogenetic relationships of taxa representative of blood parasitic adeleids were investigated in a cladistic analysis. Two phylogenetic analyses were performed. Monophyly of species of Haemogregarina (sensu lato) of some marine fishes with species of Haemogregarina (sensu stricto) was not supported in either analysis. A new genus, Desseria n. g., was created to accommodate these species. The historical burden placed on the genus Haemogregarina Danilewsky, 1885 as a repository for poorly known and inadequately described species is partially relieved through taxonomic revisions involving the genera Haemogregarina, and Desseria n. g.
YI‐S, a Casein‐free Medium for Axenic Cultivation of Entamoeba histolytica, Related Entamoeba, Giardia intestinalis and Trichomonas vaginalis
Authors: DIAMOND LOUIS S. , CLARK C. GRAHAM , CUNNICK CAROL C.
Published: May 2007

ABSTRACT. Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen‐dwelling parasitic protozoa, especially Entamoeba, Giardia, and trichomonads. The digest used almost exclusively in the development of these media, Medo‐Peptone (Trypticase® BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo‐Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica. In response to this problem we have developed a casein‐free medium, YI‐S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein‐dependent medium TYI‐S‐33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen‐dwellers.
In Vitro Characteristics of the Microsporidian: Enterocytozoon salmonis
Authors: WONGTAVATCHAI JANENUJ , CONRAD PATRICIA A. , HEDRICK RONALD P.
Published: May 2007

ABSTRACT. Enterocytozoon salmonis, an intranuclear microsporidian of salmonid fish, was propagated in vitro using chinook salmon mononuclear leukocytes. Characteristic morphology and infectivity of the cultured parasites were evaluated to determine the effect of in vitro maintenance and passage on the parasites. Cultured parasites developed through several stages from meronts to infectious spores. Parasites obtained from in vitro passages tested up to the 17th subculture, retained their morphological characteristics and pathogenicity for chinook salmon (Oncorhynchus tshawytscha). The disease induced by experimental infections with parasites from in vitro cultures was ideniical to that observed in naturally infected chinook salmon. An examination of supernatants obtained from the infected cultures revealed evidence of soluble factor(s) produced by E. salmonis‐infected cells that stimulated uninfected target cells in vitro. This observation may explain in part the proliferative disease of hematopoietic tissues which characterizes the disease in infected chinook salmon.
Cloning and Analysis of a Cryptosporidium parvum Gene Encoding a Protein with Homology to Cytoplasmic Form Hsp70
Authors: KHRAMTSOV NIKOLAI V. , TILLEY MICHAEL , BLUNT DENNIS S. , MONTELONE BETH A. , UPTON STEVE J.
Published: May 2007

ABSTRACT. An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.
Phylogeny of the Large Extrachromosomal DNA of Organisms in the Phylum Apicomplexa
Authors: EGEA NANCY , LANG‐UNNASCH NAOMI
Published: May 2007

ABSTRACT. Organisms in the phylum Apicomplexa appear to have a large extrachromosomal DNA which is unrelated to the mitochondrial DNA. Based on the apparent gene content of the large (35 kb) extrachromosomal DNA of Plasmodium falciparum, it has been suggested that it is a plastid‐like DNA, which may be related to the plastid DNA of rhodophytes. However, phylogenetic analyses have been inconclusive. It has been suggested that this is due to the unusually high A + T content of the Plasmodium falciparum large extrachromosomal DNA. To further investigate the evolution of the apicomplexan large extrachromosomal DNA, the DNA sequence of the organellar ribosomal RNA gene from Toxoplasma gondii, was determined. The Toxoplasma gondii rDNA sequence was most similar to the large extrachromosomal rDNA of Plasmodium falciparum, but was much less A + T rich. Phylogenetic analyses were carried out using the LogDet transformation to minimize the impact of nucleotide bias. These studies support the evolutionary relatedness of the Toxoplasma gondii rDNA with the large extrachromosomal rDNA of Plasmodium falciparum and with the organellar rDNA of another parasite in the phylum Apicomplexa, Babesia bovis. These analyses also suggest that the apicomplexan large extra‐chromosomal DNA may be more closely related to the plastid DNA of euglenoids than to those of rhodophytes.