bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemosensory Adaptation to Lysozyme and GTP Involves Independently Regulated Receptors in Tetrahymena thermophila
Authors: KURUVILLA HEATHER G. , KIM MARK Y. , HENNESSEY TODD M.
Published: May 2007

ABSTRACT. Chemosensory adaptation is seen in Tetrahymena thermophila following prolonged exposure (ten minutes) to micromolar concentrations of the chemorepellents lysozyme or guanosine triphosphate (GTP). Since these cells initially show repeated backward swimming episodes (avoidance reactions) in these repellents, behavioral adaptation is seen as a decrease in this repellent‐induced behavior. The time course of this behavioral adaptation is paralleled by decreases in the extents of surface binding of either [32P]GTP or [3H]lysozyme in vivo. Scatchard plot analyses of repellent binding in adapted cells suggests the behavioral adaptation is due to a dramatic decrease in the number of surface binding sites, as represented by decreased Bmax values. The estimated KD values for nonadapted cells are 6.6 μM and 8.4 μM for lysozyme and GTP binding, respectively. Behavioral adaptation and decreased surface receptor binding are specific for each repellent. The GTP adapted cells (20 μM for ten minutes) still respond behaviorally to 50 μM lysozyme and bind [3H]lysozyme normally. Lysozyme adapted cells (50 μM for ten minutes) still bind [32P]GTP and respond behaviorally to GTP. All the behavioral and binding changes seen are also reversible (deadaptation). Neomycin was shown to be a competitive inhibitor of [3H]lysozyme binding and lysozyme‐induced avoidance reactions, but it had no effect on either [32P]GTP binding or GTP‐induced or avoidance reactions. These results are consistent with the hypothesis that there are two separate repellent receptors, one for GTP and the other for lysozyme, that are independently downregulated during adaptation to cause specific receptor desensitization and consequent behavioral adaptation.
Evidence for Symbiont‐induced Alteration of a Host's Gene Expression: Irreversible Loss of SAM Synthetase fromAmoeba proteus
Authors: CHOI JI YOUNG , LEE TAE WON , JEON KWANG W. , AHN TAE IN
Published: May 2007

ABSTRACT.Symbiont‐bearing xD amoebae no longer produce a 45‐kDa cytoplasmic protein that functions as S‐adenosylmethionine synthetase in symbiont‐free D amoebae. The absence of the protein in xD amoebae is attributable to xD amoeba's failure to transcribe the corresponding gene as a result of harboring bacterial symbionts. However, xD amoebae have about half the level of enzyme activity found in D amoebae, indicating that they use an alternative source for the enzyme. xD amoebae originated from D amoebae by bacterial infection and now depend on their symbionts for survival. xD amoebae exhibit irreversible nucleolar abnormalities when their symbionts are removed, suggesting that X‐bacteria supply the needed enzyme. A monoclonal antibody against the 45‐kDa protein was produced and used as a probe in cloning its corresponding cDNA. The product of the cDNA was found to have S‐adenosylmethionine synthetase activity. These results show how symbiotic X‐bacteria may become essential cellular components of amoebae by supplementing a genetic defect for an amoeba's house‐keeping gene that is brought about by an action of X‐bacteria themselves. This is the first reported example in which symbionts alter the host's gene expression to block the production of an essential protein.
The Fine Structure of the Scopula‐Stalk Region of Vorticella convallaria
Authors: WIBEL RON , VACCHIANO EMANUEL J. , MACIEJEWSKI JOHN J. , BUHSE HOWARD E. , CLAMP JOHN
Published: May 2007

ABSTRACT. We examined by SEM and TEM the stalk‐scopular junction, the stalk, and stalk formation in Vorticella convallaria Linnaeus, 1767. The stalk sheath is anchored to the walls of the scopular lip and to the scopular cilia by thin fibrils. Experimental extraction of these fibrils weakens this junction enough to separate the stalk from the cell body. Telotrochs escape from the stalk by means of violent contractions of the cell body, accelerated beating of the trochal band cilia, and twisting of the cell body against the stalk. The edges of the scopular lip spread over the scopular cilia after escape and, in some cases, fuse to enclose the entire, aboral scopular surface in a cupola‐like structure. The sessile cells contain fewer and smaller scopular granules than telotrochs. The presence of disintegrating scopular granules in the stalk matrix of some sessile cells suggests that they contain material which is secreted over a period of time to form the stalk. Eruptive formation of the initial adhesive pad and quick elongation of the distal part of the stalk suggests a rapid exocytosis of the larger, more numerous granules of the telotroch. The stalk sheath is formed of fibrils making up complete and incomplete compartments peripherally arranged along the major stalk axis.
Detection of Penicillin‐binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei
Authors: MOTTA MARIA CRISTINA M. , LEAL LUIS HENRIQUE M. , SOUZA WANDERLEY DE , DE ALMEIDA DARCY F. , FERREIRA LUIS CARLOS S.
Published: May 2007

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the β‐lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with β‐lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of β‐lactam antibiotic were two membrane‐bound penicillin‐binding proteins (PBPs) detected in the Sarkosyl‐soluble fraction of purified symbionts labeled with [3H]‐benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [3H]‐benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than [3H]‐benzylpenicillin and indicated that PBP1 is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.
Inhibition of Early Endosome Fusion by Trypansom cruzi‐Infected Macrophage Cytosol
Authors: OCHATT CLAUDIA M. , MAYORGA LUIS S. , ISOLA ELVIRA L. D. , WILKOWSKY SILVINA , TORRES HÉCTOR N. , TÉLLEZ‐INÒN MARÍA T.
Published: May 2007

ABSTRACT. Trypanosoma cruzi trypomastigotes survive inside macrophages by promoting fusion between the parasitophorous vacuole and mature host lysosomes upon internalization. Since trypomastigotes can evade the lytic pathway, the earliest steps of endocytosis, such as early endosome fusion, may be affected. To test this hypothesis, we used an in vitro early endosome fusion assay. Our results show that trypomastigote‐infected macrophage cytosols cannot promote fusion between early endosomes, compared to mock‐infected cytosols (heat‐killed trypomastigotes were used in the parasite‐macrophage interaction assay). GTPγS addition potentiates the fusogenic activity driven by trypomastigote‐infected macrophage cytosol‐mediated assays, unlike the biphasic fusogenic effect obtained with GTPγS treatment of macrophage cytosol controls. Calcium‐stimulated early endosome fusogenic processes are not affected in the assays mediated by infected macrophage cytosol. We conclude that GTP‐regulated factors, and not calcium‐regulated elements, are involved in the inhibition of the early endosome fusogenic process by the trypomastigote‐infected macrophage cytosol. This primary impediment to the progress of a normal endocytosis may be a relevant step required for the lysosomal recruitment‐fusion of the host lysosmes upon trypomastigote infection and further survival of the parasite within its host.
Comparative Aspects of Energy Metabolism in Plant Trypanosomatids
Authors: FERNÁNDEZ‐BECERRA CARMEN , SÁNCHEZ‐MORENO MANUEL , OSUNA ANTONIO , OPPERDOES FRED R.
Published: May 2007

ABSTRACT. The energy metabolism was compared among four different representatives of the genus Phytomonas isolated from different plants and localities: the sieve tubes of the hartrot‐infected coconut palm in French Guyana, the latex fluid of Euphorbia hyssopifolia in French Guyana and the fruits of tomato and cherimoya in Spain. All four isolates produced acetate, ethanol, glycerol and glycine as metabolic end‐products. In addition, small amount of succinate and pyruvate were excreted. Only minor quantitative differences were observed in the four isolates. Glycosomes, harboring the glycolytic enzymes, were present in all isolates. No evidence was found for an active involvement of the mitochondrion in metabolism. Respiration was insensitive to the classical inhibitors of the respiratory chain, such as antimycin and potassium cyanide, but inhibited by salicylhydroxamic acid. No evidence was found for the functioning of a citric‐acid cycle. It is concluded that representative of this genus share the same highly active carbohydrate metabolism combined with a complete suppression of mitochondrial activity.
The Striated Bands of Paramecium Are Immunologically Distinct from the Centrin‐Specific Infraciliary Lattice and Cytostomal Cord
Authors: Allen Richard D. , Aihara Marilynn S. , Fok Agnes K.
Published: May 2007

The pellicle of Paramecium has three two‐dimensionally arrayed systems that occupy separate but closely paralleling planes. All three systems are now distinguishable by their differing immunological properties. This study focused on the two deeper systems. The infraciliary lattice lies innermost and labels with centrin‐specific antibodies. The middle system, the striated bands, is specifically labeled with a monoclonal antibody that we have raised to a 110 kDa conical antigen in P. multimicronucleatum. This antibody labels a similar geometric cortical pattern in at least two species, P. multimicronucleatum and P. tetraurelia. Centrin‐specific structures appear to be net‐like in the above two species but show a more interrupted pattern in P. caudatum. The cytostomal cord is an essentially unbranched extension of the net‐like infraciliary lattice and, like it, is centrin‐specific. The cord has a unique association with the alveolar sacs which suggest these calcium‐storing compartments contribute to the calcium fluxes required for contraction of the cord. A structural rather than a contractile function is favored for the striated bands, based solely on their morphology.
Development of Tick Gut Forms of Babesia bigemina In Vitro
Authors: Golgh Joanne M. , Jorgensen Wayne K. , Kemp David H.
Published: May 2007

Development of a laboratory cultured tick‐transmissible strain of Babesia bigemina was followed in vitro after addition of gut material from engorged female Boophilus microplus ticks and incubation at 37° C. Sequential development of stages, from intraerythrocytic strahlenkörper through multiplication to the fusion of what is assumed to be two gametes, is described. A change in physical environment (temperature, gas composition) experienced during passage of Babesia stages into the in vitro culture tubes possibly mimics the changes experienced in passage from host blood to the midgut of the tick vector. The effect in vitro was to induce the erythrocytic parasites to remain inactive at a trophozoite‐like stage. Addition of factor(s) within midgut initiated further development of strahlenkörper. Two populations of strahlenkorper were recognized; an elongated form which did not appear to develop further, and a polymorphic population which underwent further multiplication initiated while the parasites were still within the erythrocyte, and continuing after they had emerged. These strahlenkorper increased in size as multiple division of nuclei occurred, with cell division being completed more slowly. Large aggregations of multinucleated strahlenkorper formed, but once division was complete, single‐nucleated strahlenkorper emerged from the aggregates. Two individuals of post‐aggregation strahlenkorper, assumed to be gametes, fused together. The morphology and ultrastructure of all stages of development are described and compared with forms already described from the tick midgut.
Cell Wall Antigens of Pneumocystis carinii Trophozoites and Cysts: Purification and Carbohydrate Analysis of these Glycoproteins
Authors: Stefano Jamie A. De , Myers John D. , Pont David Du , Foy Jillana M. , Theus Sue A. , Walzer Peter D.
Published: May 2007

We have purified and biochemically analyzed individual cell wall glycoproteins of Pneumocystis carinii. Our results show that corresponding core glycoproteins constitute the cell wall antigens in both trophozoites and cysts, and glycosylation of these glycoproteins does not appear to be significantly altered during development. Cysts and trophozoites in rat‐derived organism preparations were separated from each other by counterflow centrifugal elutriation, then treated with Zymolyase to obtain the cell wall fractions. Gel electrophoresis patterns of these fractions from both life‐cycle stages were qualitatively similar. Ten major antigenic glycoproteins in these fractions were purified by preparative continuous elution gel electrophoresis. All ten glycoproteins from cysts and trophozoites contained mannose, glucose, galactose. and N‐acetylglucosamine, and some contained traces of fucose. The glycoproteins of cysts had more mannose than their trophozoite counterparts. The trophozoite glycoproteins differed from those of the cyst by the presence of xylose. To examine the species‐specificity of glycoprotein glycosylation, preparations of human‐derived P. carinii (comprised of mixed life‐cycle stages) were also examined and found to contain the same sugars as those found in rat‐derived organisms. Most of the purified rat‐derived glycoproteins bound Concanavalin A, which was abolished by treatment with N‐glycanase. This suggested that the majority of the oligosaccharides were N‐linked to the proteins, but attempts to identify carbohydrate linkage sites by amino acid sequencing were hampered by apparent modifications of residues. The peptides derived by cyanogen bromide cleavage revealed distinct size patterns for each glycoprotein, suggesting that they were distinct proteins. Most of the glycoproteins reacted with monoclonal antibodies which recognize a highly conserved epitope on rat P. carinii. Four of the individually purified glycoprotein preparations elicited in vitro cellular immune responses, implicating their involvement in the recognition of P. carinii by host T cells. The identification and characterization of P. carinii cell wall proteins will be helpful in analyzing the relationship of the organism to its mammalian host. Supplementary key words. Biochemical analysis, developmental stages, opportunistic pathogen, structure.
Efficacy of Novel Antimicrobials Against Clinical Isolates of Opportunistic Amebas
Authors: Schuster Frederick L. , Visvesvara Govinda S.
Published: May 2007

ABSTRACT We examined the effects of the macrolide antimicrobial agent azithromycin and phenothiazine compounds against clinical isolates of Acanthamoeba spp. and Balamuthia mandrillaris, opportunistic pathogens of human beings and other animals. Acanthamoeba growth was inhibited in vitro at 1,5, and 10 μg/ml of azithromycin, but not the macrolides, erythromycin, and clarithromycin. In experiments attempting to simulate in vivo conditions, azithromycin protected monolayers of rat glioma cells from destruction by Acanthamoeba at a concentration of 0.1 μg/ml, and delayed destruction at concentrations of 0.001 and 0.01 μg/ml. We concluded that the minimal inhibitory concentration of azithromycin was 0.1 μg/ml. Our results, however, suggested that the drug was amebastatic but not amebicidal, since ameba growth eventually resumed after drug removal. The phenothiazines (chlorpromazine, chlorprothixene, and triflupromazine) inhibited Acanthamoeba growth by 70‐90% at 5 and 10 μg/ml, but some of these compounds were toxic for rat glioma cells at 10 μg/ml. Azithromycin was not very effective against B. mandrillaris in an in vitro setting, but was amebastatic in tissue culture monolayers at concentrations of 0.1 μg/ml and higher. Balamuthia amebas showed in vitro sensitivity to phenothiazines. Ameba growth was inhibited 30‐45% at 5 μg/ml in vitro, but completely at 5 μg/ml in the rat glioma model. In spite of their potential as antiamebic drugs in Balamuthia infections, toxicity of phenothiazines limits their use in clinical settings.
Cloning of Two Putative Giardia lamblia Glucosamine 6‐Phosphate Isomerase Genes Only One of Which is Transcriptionally Activated During Encystment
Authors: Keulen Harry Van , Steimle Paul A. , Bulik Dorota A. , Borowiak R. Kevin , Jarroll Edward L.
Published: May 2007

ABSTRACT The biosynthesis of the carbohydrate component of the cyst wall of the protozoan parasite Giardia lamblia, a polymer of N‐acetylgalactosamine (GalNac). is by a pathway that is initiated with the conversion of fructose 6‐phosphate to glucosamine 6‐phosphate by an aminating isomerase. glucose 6‐phosphate isomerase. This enzyme appears only after Giardia trophozoites an: induced to start the production of cyst wall components after bile is added. To investigate whether induction of glucosamine 6‐phosphate isomerase is by protein modification or by transcription activation, its gene was cloned and sequenced. Two genes, gpi1 and gpi2, encoding putative glucosamine 6‐phosphate isomerases were identified but only one, gpi1 was expressed. The transcript for gpi1 appeared not earlier than 6 h after cells were induced with bile salts. These results show that the first enzyme in the pathway leading to GalNac synthesis in encysting Giardia cyst wall biosynthesis is under transcriptional control.
Phylogenetic Diversity of Parabasalian Symbionts from Termites, Including the Phylogenetic Position of Pseudotrypanosoma and Trichonympha
Authors: Keeling Patrick J. , Poulsen Nicole , Mcfadden Geoffrey I.
Published: May 2007

ABSTRACT The phylogenetic diversity of parabasalian flagellates from termite hindguts has been examined by small subunit ribosomal RNA (rRNA) amplification and sequencing. Two species of particular interest, the giant trichomonad Pseudotrypanosoma giganteum and the hypermastigote Trichonympha magna, were isolated from the gut of Porotermes adamsoni by micropipetting. and the rRNA genes from these small populations amplified and sequenced. rRNA genes representing Hypermastigida and the Trichomonadida families Devescovinidae and Trichomonadidae. were also recovered by amplification from whole hindguts of three termites, P. adamsoni, Cryptotermes brevis, and Cryptotermes dudleyi. The parabasalian rRNA genes from C. brevis were found to comprise a unique and extremely heterogeneous lineage with no clear affinities to any known parabasalian rRNAs. In addition, one of the sequences isolated from P. Adamsoni was found to be similar to another uncharacterised rRNA gene from Reticulitermes flavipes. The phylogeny of all known parabasalian small subunit rRNAs was examined with these new sequences. We find many taxonomic groups to be supported by rRNA, but not all. We have found the root of parabasalia to be very difficult to discern accurately, but have nevertheless identified several possible positions.
Encephalitozoon hellem in Two Eclectus Parrots (Eclectus roratus): Identification from Archival Tissues
Authors: Pllparampil Nicole , Graham David , Phalen David , Snowden Karen
Published: May 2007

ABSTRACT Members of the phylum Microspora are obligate, intracellular, single‐celled parasites identified in a wide range of vertebrate and invertebrate hosts. Only a few cases of microsporidial infections have been documented in psittacine birds including peach‐faced, masked, and Fischer's lovebirds (Agapornis roseicollis, A. personata, and A. fischeri. respectively), budgerigars (Melopsittacus undulatus), and a double yellow‐headed Amazon parrot (Amazona ochrocephala). Parasite identification has typically been limited to phylum or genus, and no avian species of microsporidia has clearly been described. In this report, microsporidia were identified in the kidney and intestine of a new host, the eclectus parrot (Eclectus roratus). Parasites were identified as Encephalitozoon hellem using morphologic, ultrastructural, and small subunit ribosomal RNA gene sequence data obtained from archived tissues. This parasite species was first identified in immunocompromised humans and may be a potential zoonotic pathogen. The epidemiology and prevalence of this parasite in humans and birds should be further explored.
The Development of Trypanosoma cruzi (Trypanosomatidae) in the Reduviid Bug Triatoma infestans (Insecta): Influence of Starvation
Authors: Kollien Astrid H. , Schaub Günter A.
Published: May 2007

ABSTRACT Fifth instars of Triatoma infestans with established Trypanosoma cruzi infections were dissected after different periods of starvation to determine the population density and the percentage of different developmental stages of T. cruzi in the small intestine and rectum of the bugs. After a short starvation period of 20 days, the population density in the small intestine was 20% (about 60,000) of the rectal population. The population in the small intestine was strongly reduced after an additional ten days of starvation, and no flagellates could be found there 60, 90 and 120 days after the last feeding. In the rectum, this reduction went down to 1% of the initial population, but a total elimination never occurred. Usually the remaining population contained more live than dead flagellates. Starvation also resulted in an increase in the rectum in the number and percentage of drop‐like forms, intermediates between sphere‐ and epi‐ or trypomastigotes, from 1% initially to about 10% after 90 days of starvation. The percentage of spheromastigotes increased from 2% at 20 days after the last feeding to about 20% after an additional 40 and 70 days. Therefore, the spheromastigotes of T. cruzi seem to be induced by stress conditions.
The Effects of Cooling Conditions on the Behavior of Oxytricha bifaria (Ciliophora Hypotrichida)
Authors: Ricci Nicola , Barbanera Fillppo , Erra Fabrozio
Published: May 2007

The new analytic approach to the behavior of ciliates represented by the ethogram was used to study the locomotion ofOxytricha bifariaat different temperatures, isotropically applied according to a new protocol. It was shown that under these experimental conditions as the temperature dropped in stages from 24°C to 19°C to 14°C to 9°C, the general mobility of experimental populations decreased, as indicated by 1) the decreasing percentage of mobile organisms, 2) their decreasing velocity, 3) their prolonged backward creeping, and 4) the increasing length of their immobilization periods. The ethogram more particularly revealed that decreasing temperatures induced 1) the appearance of rightward arcs (several of them being travelled by specimens sliding on the substrate). 2) the reduction of both the radius and the velocity along the normal leftward arcs (A‐) and the segments (S), and 3) the symmetric increase of both the central angle and the duration of the A‐ and S. These changes were reversibly induced: they disappeared when the temperature returned to 24°C. Moreover. a new behavioral pattern, the prolonged Side Stepping Reaction, was found. Helicoidal swimming, occurring only at 14°C, was analyzed. Among all of the behavioral parameters, nine were shown to change dramatically between 14°C and 9°C, demonstrating that the linear cooling of the populations induced clear non‐linear effects.
Stage‐Specific Substrate Phosphorylation by a Ca2+/Calmodulin‐Dependent Protein Kinase in Trypanosoma cruzi
Authors: Ogueta Sandra B. , Macintosh Gustavo C. , Téllez‐No̊n Maria T.
Published: May 2007

The presence of (Ca2/calmodulin (Ca2/CaM)‐dependent protein kinase (TcCaM K) and some stage‐specific substrates that appeared during morphogenesis of the parasite Trypanosoma cruzi were identified. Western blot analysis using a polyclonal antibody against rat brain CaM K type II recognized the same subunit composition (52, 59/62 kDa) observed for the mammalian enzyme, as well as the previously characterized TcCaM K found in epimastigote forms. Differential protein phosphorylation profiles were observed after enzyme activation in the stages of T. cruzi. Co‐immunoprecipitation of stage‐specific substrates with the TcCaM K suggested that the enzyme might be involved in the phosphorylation of a different set of proteins through the life cycle. Three phosphoproteins, pp105 and pp87 from epimastigotes and pp23 from trypomastigotes were identified as potential substrates for TcCaM K. The characterization of these endogenous stage markers might be a useful tool to understand the developmental cycles of these pathogenic protozoa.
Relative Potencies of Different Cytochalasins for the Inhibition of Phagocytosis in Ciliates
Authors: Zackroff Robert V. , Hufnagel Linda A.
Published: May 2007

Ciliate actins have been reported to exhibit an unusual degree of sequence divergence, within the phylum Ciliophora, and when compared to actins from other organisms. To determine whether these primary structural differences are correlated with pharmacological differences, we investigated the effects of seven cytochalasins on phagocytosis, which has been shown to be actin‐dependent in eukaryotic cells. The relative potencies of cytochalasin inhibition of phagocytosis in Spirostomun ambiguum and Paramecium multimicronucleatum were similar. Dihydrocytochalasin B and cytochalasin A were the most potent of the seven cytochalasins in both ciliates, and strongly inhibited phagocytosis at 20‐40 μM. Dihydrocytochalasin B was considerably more potent than either cytochalasins B or D, a result unexpected on the basis of reports utilizing other organisms. However, even at concentrations up to 100 μM, dihydrocytochalasin B did not inhibit the rate of Spirostomum defecation. After long‐term treatment of Spirostomum with 50 μM dihydrocytochalasin B, the sensitivity of phagocytosis to the drug was reduced. These results indicate that the relative cytochalasin sensitivities of actin‐dependent functions in ciliates differ from those of other organisms, and are consistent with the hypothesis that sequence differences might give rise to substantial differences in the pharmacological properties of ciliate actins.
A Gene Encoding the Plant‐Like Alternative Oxidase is Present in Phytomonas but Absent in Leishmania spp.
Authors: Hellemond Japp J. Van , Simons Bert , Millenaar Frank F. , Tielens Aloysius G.M.
Published: May 2007

The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome‐containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron‐transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant‐like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei, trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant‐like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant‐like alternative oxidase.
The Interaction of Acanthamoeba spp. with Activated Macrophages and with Macrophage Cell Lines
Authors: Marciano‐Cabral Francine , Toney Denise M.
Published: May 2007

Acanthamoeba spp. are free‐living amebae associated with amebic keratitis and chronic granulomatous amebic encephalitis. The present studies were undertaken to compare the pathogenicity of three species of Acanthamoeba in B6C3F1 mice after intranasal challenge with Acanthamoeba‐induced cytopathogenicity for different macrophage populations. The ability of murine macrophage cell lines and activated murine peritoneal macrophages to lyse Acanthamoeba has been assessed by coincubating macrophages with 3H‐uridine labeled amebae. Conversely, destruction of macrophages by Acanthamoeba was determined by measuring the release of chro‐mium‐51 from radiolabeled macrophages. Acanthamoeba culbensoni, which is highly pathogenic for mice, destroys macrophage cultures in vitro. Activated primary peritoneal macrophages were more resistant to Acanthamoeba‐mediated destruction than macrophage cell lines activated in vitro. Activated macrophages were capable of limited destruction of Acanthamoeba polyphaga and Acanthamoeba castellanii. Acanthamoeba‐specific antibodies increased the amebicidal activity of activated macrophages. Macrophage‐mediated destruction was by contact‐dependent cytolysis and by ingestion of amebae. Conditioned medium obtained from macrophage cultures after treatment with lipopolysaccharide and interferon gamma was neither cytolytic nor cytostatic for Acanthamoeba spp. Purified recombinant cytokines including tumor necrosis factor α. interleukin 1α, and interleukin 1β, alone or in combination, were not cytolytic for Acanthamoeba trophozoites.
Impact of a Novel, Feminising Microsporidium on its Crustacean Host
Authors: Terry Rebecca S. , Smith Judith E. , Dunn Alison M.
Published: May 2007

We describe the transmission and pathogenic effects of a novel, feminising microsporidium, probably a Nasema species, on its crustacean host Gammarus duebeni. The parasite prevalence in the field was high (46% of females were infected) and the parasite was transovarially transmitted to 91% of embryos of infected females. The impact of the parasite on the host was assessed by means of a host breeding experiment. The parasite feminised 66% of infected host young and was transovarially transmitted by these individuals to the next host generation. The parasite differed from other feminising microsporidia in G. duebeni in that early embryos had a high parasite burden (288 parasites per embryo) and the infection was pathogenic, causing a reduction in both the growth rate of young hosts and in adult size. This study suggests that feminising microsporidia are a diverse group in which a variety of host/pathogen relationships have evolved.
Molecular Phylogeny of Microsporidians with Particular Reference to Species that Infect the Muscles of Fish
Authors: Nilsen Frank , Endresen Curt , Hordvik Ivar
Published: May 2007

Ribosomal DNA from eight species of microsporidians infecting fish have been sequenced. Seven of these species infect the skeletal muscle of fish (Pleistophora spp.) and one species infects migratory mesenchyma cells (Glugea anomala). These sequences, in addition to other available microsporidian rDNA sequences from a broad range of host taxa, have been used in phylogenetic analysis. This analysis revealed that muscle‐infecting microsporidians from fish are a polyphyletic group, indicating that characters supposed to be important in the classification of the genus Pleistophora have to be re‐evaluated. One character that probably has a polyphyletic origin is the amorphous coat, which has been extensively used in the definition of this genus. Furthermore, our results showed that the insect parasitizing Pleistophora spp. are not related to the true pleistophorans parasitic in skeletal muscle of fish. Phylogenetic analysis of small subunit rDNA sequences revealed disagreements between the molecular phylogeny and classifications based upon ultrastruclure. Many of the morphological characters claimed to be important in microsporidian classifications appeared to have arisen several times during evolution: for example, the diplokaryon and sporophorous vesicles.
A Method for the Synchronous Induction of Large Numbers of Telotrochs in Vorticella convallaria by Monocalcium Phosphate at Low pH
Authors: BAUFER PAUL J. DE , AMIN AMR A. , PAK SAMANTHA C. , BUHSE HOWARD E.
Published: May 2007

ABSTRACT We have devised a two step method for the synchronous induction of telotrochs in the peritrich ciliate, Vorticella convallaria. The method is easy, reliable, and allows us to study the earliest events of telotroch formation at the ultrastructural, biochemical, and molecular levels. The steps involved are: (1) excising the cell body from the stalk in a large population (7.4 times 104 cells) of EDTA‐treated, attached cells by the application of monocalcium phosphate monohydrate solution at pH 3.2, (2) rinsing and suspending the isolated cell bodies in inorganic medium. Within 90 min, 80% of the population forms telotrochs. Analysis of factors that are important for maximum stalk excision and transformation shows that the population must not be older than 2 d and the most effective concentration of monocalcium phosphate is 4.8 mM for a 20 min exposure. The most effective monocalcium phosphate is in the monohydrated form. A pH value of 3.2, produced by the addition of hydrochloric acid in the presence or absence of calcium is not sufficient to initiate stalk excision and telotroch formation. This observation leads us to conclude that stalk excision is dependent on monocalcium phosphate or its hydrolysis products.
Assembly of the Paraflagellar Rod and the Flagellum Attachment Zone Complex During the Trypanosoma brucei Cell Cycle
Authors: KOHL LINDA , SHERWIN TREVOR , GULL KEITH
Published: May 2007

ABSTRACT Trypanosomes possess a single flagellum that is attached to their cell body via the flagellum attachment zone (FAZ). The FAZ is composed of two structures: a cytoplasmic filament complex and four microtubules situated next to it. There is a complex transmembrane crosslinking of this FAZ to the paraflagellar rod (PFR) and axoneme within the flagellum. We have partially purified the FAZ complex and have produced monoclonal antibodies both against the FAZ and the paraflagellar rod. The two antibodies against the FAZ (L3B2 and L6B3) recognise the cytoplasmic filament in immunofluorescence and in immunoelectron microscopy. On Western blot, they detect a doublet of high molecular weight (M, 200,000). Two anti‐PFR antibodies (L13D6 and L8C4) recognise the paraflagellar rod in immunofluorescence, but show a difference on Western blot: L13D6 recognises both major PFR proteins, whereas L8C4 is specific for only one of them. Using these new antibodies we have shown that although the growth of both cytoplasmic FAZ filament and external PFR are related, their growth initiates at different time points during the cell cycle and the two structures elongate at distinct rates.
Critical Analysis of Eukaryotic Phylogeny: A Case Study Based on the HSP70 Family
Authors: GERMOT AGNÈS , PHILIPPE HERVÉ
Published: May 2007

ABSTRACT Trichomonads, together with diplomonads and microsporidia, emerge at the base of the eukaryotic tree, on the basis of the small subunit rRNA phylogeny. However, phylogenies based on protein sequences such as tubulin are markedly different with these protists emerging much later. We have investigated 70 kDa heat‐shock protein (HSP70), which could be a reliable phylogenetic marker. In eukaryotes, HSP70s are found in cytosol, endoplasmic reticulum, and organelles (mitochondria and chloroplasts). In Trichomonas vaginalis we identified nine different HSP70‐encoding genes and sequenced three nearly complete cDNAs corresponding to cytosolic, endoplasmic reticulum, and mitochondrial‐type HSP70. Phylogenies of eukaryotes were reconstructed using the classical methods while varying the number of species and characters considered. Almost all the undoubtedly monophyletic groups, defined by ultrastructural characters, were recovered. However, due to the long branch attraction phenomenon, the evolutionary rates were the main factor determining the position of species, even with the use of a close outgroup, which is an important advantage of HSP70 with respect to many other markers. Numerous variable sites are peculiar to Trichomonas and probably generated the artefactual placement of this species at the base of the eukaryotes or as the sister group of fast‐evolving species. The inter‐phyla relationships were not well supported and were sensitive to the reconstruction method, the number of species, and the quantity of information used. This lack of resolution could be explained by the very rapid diversification of eukaryotes. likely after the mitochondrial endosymbiosis.
Cloning and Expression of a cDNA Encoding a Vorticella convallaria Spasmin: an EF‐Hand Calcium‐Binding Protein
Authors: MACIEJEWSKI JOHN J. , VACCHIANO EMANUEL J. , MCCUTCHEON SUZANNE M. , BUHSE HOWARD E.
Published: May 2007

ABSTRACT The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium‐binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20‐kD calcium‐binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20‐kD protein with calcium‐binding helix‐loop‐helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly‐produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel‐shift assays and was recognized by the anti‐spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF‐hand superfamily of calcium‐binding proteins.
Morphology and Morphogenesis of Metopus hasei Sondheim, 1929 and M. inversus (Jankowski, 1964) nov. comb. (Ciliophora, Metopida)
Authors: FOISSNER WILHELM , AGATHA SABINE
Published: May 2007

ABSTRACT The morphology and morphogenesis of Metopus hasei Sondheim, 1929 and M. inversus (Jankowski, 1964) n. comb, were investigated using live observation, silver impregnation, and scanning electron microscopy. Metopus has a spiral body organization and the ventral margin of the preoral dome bears five specialized ciliary rows, that form the so‐called perizonal stripe. Division is homothetogenic, occurs in freely motile (i. e. non‐encysted) condition, and includes a partial reorganization of the parental oral apparatus. During division, the complicated cell shape becomes ellipsoidal and all ciliary rows arrange meridionally. Stomatogenesis is entirely somatic (≅ pleurotelokinetal) and commences with the formation of kinetofragments in some dorsolateral kineties. The fragments become the opisthe's adoral membranelles, while the paroral membrane is generated by the left two perizonal ciliary rows, which proliferate kinetids intrakinetally. The perizonal stripe of the opisthe is generated by the three right parental perizonal kineties, which divide, and by two dorsolateral ciliary rows, which are added. The morphogenetic processes, especially the unique mode of formation of the paroral membrane, are used to define the order Metopida Jankowski, 1980 n. stat. more properly. The ontogenetic, ultrastructural, and sequence data available give no clear indication about metopid phylogeny, but definitely exclude metopids from the classical heterotrichs, with which they were classified for more than 100 years. Accordingly, we place the Metopida as incertae sedis in the subphylum Intramac‐ronucleata Lynn, 1996.
A Comparative Genomics Approach to the Evolution of Eukaryotes and their Mitochondria1
Authors: LANG B. FRANZ , SEIF ELIAS , GRAY MICHAEL W. , O'KELLY CHARLES J. , BURGER GERTRAUD
Published: May 2007

The Organelle Genome Megasequencing Program (OGMP) investigates mitochondrial genome diversity and evolution by systematically determining the complete mitochondrial DNA (mtDNA) sequences of a phylogenetically broad selection of protists. The mtDNAs of lower fungi and choanoflagellates are being analyzed by the Fungal Mitochondrial Genome Project (FMGP), a sister project to the OGMP. Some of the most interesting protists include the jakobid flagellates Reclinomonas americana, Malawimonas jakobiformis, and Jakoba libera, which share ultrastructural similarities with amitochondriate retortamonads, and harbor mitochondrial genes not seen before in mtDNAs of other organisms. In R. americana and J. libera, gene clusters are found that resemble, to an unprecedented degree, the contiguous ribosomal protein operons str, S10, spc, and alpha of eubacteria. In addition, their mtDNAs code for an RNase P RNA that displays all the elements of a bacterial minimum consensus structure. This structure has been instrumental in detecting the rnpB gene in additional protists. Gene repertoire and gene order comparisons as well as multiple‐gene phylogenies support the view of a single endosymbiotic origin of mitochondria, whose closest extant relatives are Rickettsia‐type α‐Proteobacteria.
Investigating Deep Phylogenetic Relationships among Cyanobacteria and Plastids by Small Subunit rRNA Sequence Analysis1
Authors: TURNER SEÁN , PRYER KATHLEEN M. , MIAO VIVIAN P. W. , PALMER JEFFREY D.
Published: May 2007

Small subunit rRNA sequence data were generated for 27 strains of cyanobacteria and incorporated into a phylogenetic analysis of 1,377 aligned sequence positions from a diverse sampling of 53 cyanobacteria and 10 photosynthetic plastids. Tree inference was carried out using a maximum likelihood method with correction for site‐to‐site variation in evolutionary rate. Confidence in the inferred phylogenetic relationships was determined by construction of a majority‐rule consensus tree based on alternative topologies not considered to be statistically significantly different from the optimal tree. The results are in agreement with earlier studies in the assignment of individual taxa to specific sequence groups. Several relationships not previously noted among sequence groups are indicated, whereas other relationships previously supported are contradicted. All plastids cluster as a strongly supported monophyletic group arising near the root of the cyanobacterial line of descent.
Phagotrophic Mechanisms and Prey Selection in Free‐living Dinoflagellates1
Authors: HANSEN PER JUEL , CALADO ANTÓNIO J.
Published: May 2007

Three types of feeding mechanisms are known in dinoflagellates: pallium feeding, tube feeding, and direct engulfment. Pallium feeding has only been described for heterotrophic thecate species (Protoperidinium, Diplopsalis group). Tube feeding is commonly found among both naked and thecate species of mixotrophic and heterotrophic dinoflagellates (e.g. Amphidinium, Dinophysis, Gyrodinium, Peridiniopsis). Direct engulfment is mainly found among naked species (e.g. Gymnodinium, Gyrodinium, Noctiluca): recently, however, some thecate species have been shown to use this feeding mechanism as well. Feeding behavior in dinoflagellates involves several steps prior to actual ingestion, including precapture, capture, and prey manipulation. As feeding mechanisms allow the ingestion of relatively large prey or parts thereof, dinoflagellates are regarded as raptorial feeders. While prey size plays an important role in the ability of dinoflagellates to ingest food, this alone cannot explain observed prey preferences. Some dinoflagellate species can be very selective in their choice of prey, while others show a remarkable versatility.
Mixotrophy among Dinoflagellates1
Authors: STOECKER DIANE K.
Published: May 2007

Mixotrophy, used herein for the combination of phototrophy and phagotrophy, is widespread among dinoflagellates. It occurs among most, perhaps all, of the extant orders, including the Prorocentrales, Dinophysiales. Gymnodiniales, Noctilucales, Gonyaulacales, Peridiniales, Blastodiniales. Phytodiniales, and Dinamoebales. Many cases of mixotrophy among dinoflagellates are probably undocumented. Primarily photosynthetic dinoflagellates with their “own” plastids can often supplement their nutrition by preying on other cells. Some primarily phagotrophic species are photosynthetic due to the presence of kleptochloroplasts or algal endosymbionts. Some parasitic dinoflagellates have plastids and are probably mixotrophic. For most mixotrophic dinoflagellates, the relative importance of photosynthesis, uptake of dissolved inorganic nutrients, and feeding are unknown. However, it is apparent that mixotrophy has different functions in different physiological types of dinoflagellates. Data on the simultaneous regulation of photosynthesis, assimilation of dissolved inorganic and organic nutrients, and phagotophy by environmental parameters (irradiance. availablity of dissolved nutrients, availability of prey) and by life history events are needed in order to understand the diverse roles of mixotrophy in dinoflagellates.
Molecular Phylogeny of Labyrinthulids and Thraustochytrids Based On the Sequencing of 18s Ribosomal Rna Gene
Authors: HONDA DAISKE , YOKOCHI TOSHIHIRO , NAKAHARA TORO , RAGHUKUMAR SESHAGIRI , NAKAGIRI AKIRA , SCHAUMANN KARSTEN , HIGASHIHARA TAKANORI
Published: May 2007

ABSTRACT Labyrinthulids and thraustochytrids are unicellular heterotrophs, formerly considered as fungi, but presently are recognized as members in the stramenopiles of the kingdom Protista sensu lato. We determined the 18S ribpsomal RNA gene sequences of 14 strains from different species of the six genera and analyzed the molecular phylogenetic relationships. the results conflict with the current classification based on morphology, at the genus and species levels. These organisms are separated, based on signature sequences and unique inserted sequences, into two major groups, which were named the labyrinthulid phylogenetic group and the thraustochytrid phylogenetic group. Although these groupings are in disagreement with many conventional taxonomic characters, they correlated better with the sugar composition of the cell wall. Thus, the currently used taxonomic criteria need serious reconsideration.
Analysis of Lysine‐Rich Histones From the Unicellular Green Alga Chlamydomonas Reinhardtii
Authors: MORRIS RODNEY L. , SALINGER ANDREW P. , RIZZO PETER J.
Published: May 2007

ABSTRACT The H1 histones of the unicellular green alga Chlamydomonas reinhardtii were extracted from isolated nuclei, fractionated by high performance liquid chromatography, and analyzed by two‐dimensional electrophoresis. peptide mapping, and N‐terminal sequencing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 5% perchloric acid extracts of isolated C. reinhardtii nuclei revealed two H1 proteins (H1A and H1B). Two‐dimensional gel analysis did not reveal heterogeneity of either algal H1 protein, but did detect differences in the hydrophobic amino acid content of the C. reinhardtii H1A and H1B. Digestion of H1A and H1B with V8 protease revealed two distinctly different peptide maps. C. reinhardtii H1 peptide maps were not at all similar to those of Pisum H1, but algal and pea H2B peptide maps did show some peptides in common. Seventeen amino acid residues were obtained from C. reinhardtii H1A amino terminal sequencing, while the H1B N‐terminus was blocked. A search of protein data bases revealed no sequence homology of the H1A N‐terminus with any known protein. Chlamydomonas histones fractionated by high performance liquid chromatography revealed minor components (histone variants) for H2A and H2B. the amino acid composition of Chlamydomonas lysinerich histones was compared to those of various other unicellular algae.