bluuebunny/crossref_metadata_2025 huggingface dataset:
Ecology of the Red‐Tide Dinoflagellate Ceratium furca: Distribution, Mixotrophy, and Grazing Impact on Ciliate Populations of Chesapeake Bay
Authors: SMALLEY GABRIELA W. , COATS D. WAYNE
Published: July 2005

ABSTRACT. Ceratium furca is a primarily photosynthetic dinoflagellate also capable of ingesting other protists. During 1995 and 1996, we documented the abundance of C. furca in Chesapeake Bay and determined grazing rates on prey labeled with fluorescent microspheres. Abundance usually remained below 20 cells ml−1, although the species was capable of localized late‐summer blooms (≤ 478 cells ml−1) in the more saline lower to mid‐Bay region. Feeding rates ranged from 0 to 0.11 prey dinoflagellate−1 h−1 or from 0 to 37 pg C dinoflagellate−1 h−1 and were highest at lower salinities. Clearance rates averaged 2.5 ± 0.35 μl dinoflagellate−1 h−1. Impact of C. furca feeding on prey populations was higher in the lower Bay, averaging 67% of Strobilidium spp. removed d−1. Ingeslion rates were positively correlated with prey abundance and dissolved inorganic nitrogen, but negatively with salinity, depth, dissolved inorganic phosphorus, and inorganic P:N ratio. Daily consumption of prey biomass by C. furca averaged 4.6% of body carbon, 6.5% of body nitrogen, and 4.0% of body phosphorus, with maximal values of 36, 51, and 32%, respectively. Thus, the ability to exploit an organic nutrient source when inorganic nutrients are limiting may give C. furca a competitive advantage over purely photosynthetic species.
Microtubules Mediate Germ‐nuclear Behavior after Meiosis in Conjugation of Paramecium caudatum
Authors: NAKAJIMA YUKA , ISHIDA MASAKI , MIKAMI KAZUYUKI
Published: July 2005

ABSTRACT. Microtubule dynamics in Paramecium caudatum were investigated with an anti‐α‐tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus‐specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so‐called first assembly of microtubules, AM‐I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti‐α‐tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so‐called second assembly of microtubules, AM‐II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone.
Arginine Kinase: A Common Feature for Management of Energy Reserves in African and American Flagellated Trypanosomatids
Authors: PEREIRA CLAUDIO A. , ALONSO GUILLERMO D. , TORRES HÉCTOR N. , FLAWIÁ MIRTHA M.
Published: July 2005

ABSTRACT. This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 μmol min−1mg−1. Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356‐amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.
A Structurally Deviant Member of the Euplotes raikovi Pheromone Family: Er‐23
Authors: DI GIUSEPPE GRAZIANO , MICELI CRISTINA , ZAHN RALPH , DAMBERGER FRED , WÜTHRICH KURT , LUPORINI PIERANGELO
Published: July 2005

ABSTRACT. Pheromones of Euplotes raikovi form a homologous family of proteins with 37‐ to 40‐amino acid residues, including six cysteines that form three strictly conserved disulfide bridges. The determination of the primary structure of the pheromone Er‐23, which was isolated from cells derived from natural populations of E. raikovi that secrete the other known pheromones, has now revealed a novel structure type. The polypeptide chain of this pheromone contains 51 residues, 10 of which are cysteines presumably involved in the formation of five disulfide bridges, and lacks a carboxyl‐terminal tail following the last cysteine of the sequence. The elongation of the Er‐23 molecule is presumed to result from multiple events of gene duplication starting from an ancestral motif Xxx2–4‐Cys‐Xxx5–7‐Cys.
Fine Structure of the Myxosporean, Henneguya curimata n. sp., Parasite of the Amazonian Fish, Curimata inormata (Teleostei, Curimatidae)
Authors: AZEVEDO CARLOS , MATOS EDILSON
Published: July 2005

ABSTRACT. Henneguya curimata n. sp. (Myxozoa, Myxobolidae) is described from the kidney of the teleost Curimata inormata collected in an estuarine region of the Amazon River, near Belem. Brazil. This myxosporean produces large cysts (0.6–1.2 mm in diam.) that represent plasmodia containing all life cycle stages, including spores. The spore body is ellipsoidal (∼ 16.6 μn in length and ∼ 6.2 μm in width), and each valve presents a tapering tail (∼ 19.1 μm in length). These valves surround the binucleate sporoplasm cell and two ellipsoidal polar capsules located side‐by‐side at the same level, measuring 6.5 × 1.2 μm each and containing 10–11 coils of the polar filament. On the basis of its host specificity and on data collected by light and electron microscopy, the organism, H. curimata n. sp. is distinguished as a new species. The taxonomic affinities and morphological comparisons with other similar species of the same genus are discussed.
In Vitro Effect of Nitazoxanide Against Entamoeba histolytica, Giardia intestinalis and Trichomonas vaginalis Trophozoites
Authors: CEDILLO‐RIVERA ROBERTO , CHÁVEZ BIBIANA , GONZÁLEZ‐ROBLES ARTURO , TAPIA AMPARO , YÉPEZ‐MULIA LILIÁN
Published: July 2005

ABSTRACT. Nitazoxanide, a 5‐nitrothiazolyl derivative, is effective in the treatment of a broad range of parasitic infections. In vitro, it is active against several protozoa, including Cryptosporidiwn parvum, Blastocystis hominis, and Giardia intestinalis. The objective of this study was to determine the in vitro effect of nitazoxanide on the growth and morphology of three anaerobic protozoa (Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis) and to compare these effects with those of metronidazole and albendazole. A subculture method was used to determine the concentrations required to inhibit growth by 50% or 90% (IC50 and IC90). Nitazoxanide exhibited IC50 and IC90 values of 0.017 and 0.776 μg/ml respectively, against E. histolytica, 0.004 and 0.067 μg/ml against G. intestinalis, and 0.034 and 2.04 6 μg/ml against T. vaginalis. Based on the IC90 values, nitazoxanide was more toxic than metronidazole and albendazole against E. histolytica; albendazole and nitazoxanide were more toxic than metronidazole against G. intestinalis; and metronidazole was the most toxic drug against T. vaginalis. The effects of nitazoxanide on trophozoite ultrastructure of all three parasites included cell swelling and distorted cell shape, a redistribution of vacuoles, plasma membrane damage, and the formation of extensive empty areas in the cytoplasm of the protozoa.
The Fine Structure of the Gamont of Pterospora floridiensis (Apicomplexa: Eugregarinida)
Authors: LANDERS STEPHEN C.
Published: July 2005

ABSTRACT. Transmission electron microscopy of the gamont stage of Pterospora floridiensis has revealed a number of features. The gamont's surface varies from smooth to crenulate, with numerous pockets and folds. The pellicle is composed of an outer membrane, a middle lucent region, and an inner dense layer comprised of two tightly appressed membranes. Short ridges on the pellicle are 200–300+ nm long, 75–100 nm wide, and have a height of ∼ 50 nm. The thickness of the pellicle is 100 nm when measured from the inner membrane to the top of a ridge. The ridges are formed by the plasma membrane and an underlying structure that is circular in cross‐section. The surface folds and the pellicular ridges are distributed over the soma and the cell's unusual branching arms, though both are reduced near the junction between two gamonts in syzygy, and are absent at the central area of the junctional site. The cell has numerous active Golgi complexes associated with vesicles, as well as scattered dense mitochondria, lipid droplets, and paraglycogen granules. The nucleus has a large (13 μm) endosome, eccentrically located, and peripheral chromatin along the inner nuclear membrane.
How Oxymonads Lost Their Groove: An Ultrastructural Comparison of Monocercomonoides and Excavate Taxa
Authors: SIMPSON ALASTAIR G. B. , RADEK RENATE , DACKS JOEL B. , O'KELLY CHARLES J.
Published: July 2005

ABSTRACT. Despite being amongst the more familiar groups of heterotrophic flagellates, the evolutionary affinities of oxymonads remain poorly understood. A re‐interpretation of the cytoskeleton of the oxymonad Monocercomonoides hausmanni suggests that this organism has a similar Ultrastructural organisation to members of the informal assemblage ‘excavate taxa’. The preaxostyle, ‘Rl’ root, and ‘R2’ root of M. hausmanni are proposed to be homologous to the right, left, and anterior roots respectively of excavate taxa. The ‘paracrystalline’ portion of the preaxostyle, previously treated as unique to oxymonads, is proposed to be homologous to the I fibre of excavate taxa. Other non‐microtubular fibres are identified that have both positional and substructurel similarity to the distinctive B and C fibres of excavate taxa. A homologue to the ‘singlet root’, otherwise distinctive for excavate taxa, is also proposed. The preaxostyle and C fibre homologue in Monocercomonoides are most similar to the homologous structures in Trimastix, suggesting a particularly close relationship. This supports and extends recent molecular phylogenetic findings that Trimastix and oxymonads form a clade. We conclude that oxymonads have an excavate ancestry, and that the‘excavate taxa’ sensu stricto form a paraphyletic assemblage.
Antigenic Diversity of Encephalitozoon hellem Demonstrated by Subspecies‐Specific Monoclonal Antibodies
Authors: MO LAN , DRANCOURT MICHEL
Published: July 2005

ABSTRACT. Encephalitozoon hellem is a unicellular, obligate intracellular microsporidian species detected and isolated in HIV‐infected patients presenting with keratoconjunctivitis, sinusitis, tracheobronchitis, nephritis, cystitis, and disseminated infection. A total of 24 monoclonal antibodies were produced against E. hellem and characterized. The monoclonal antibodies were of the immunoglobulin (Ig) G and Ig M subclasses, and, when incorporated into indirect immunofluorescence and immunoblotting assays, reacted against 13 isolates of E. hellem originating from three geographic regions. These monoclonal antibodies did not react with one strain each of either Encephalitozoon intestinalis or Encephalitozoon cuniculi, demonstrating their specificity. Two monoclonal antibodies reacted with all karyotype B‐E. hellem isolates but did not react with karyotype A‐isolates from North America and the Netherlands, thus demonstrating antigenic diversity among E. hellem isolates. These results add to the increasing evidence for diversity among E. hellem, which therefore may be reclassified into subspecies.
α‐Proteobacterial Relationship of Apicomplexan Lactate and Malate Dehydrogenases
Authors: ZHU GUAN , KEITHLY JANET S.
Published: July 2005

ABSTRACT. We have cloned and sequenced a lactate dehydrogenase (LDH) gene from Cryptosporidium parvum (CpLDHl). With this addition, and that of four recently deposited α‐proteobacterial malate dehydrogenase (MDH) genes, the phylogenetic relationships among apicomplexan LDH and bacterial MDH were re‐examined. Consistent with previous studies, our maximum likelihood (ML) analysis using the quartet‐puzzling method divided 105 LDH/MDH enzymes into five clades, and confirmed that mitochondrial MDH is a sister clade to those of γ‐proteobacteria, rather than to α‐proteobacteria. In addition, a Cryptosporidium parvum MDH (CpMDHl) was identified from the ongoing Cryptosporidium genome project that appears to belong to a distinct clade (III) comprised of 22 sequences from one archaebacterium, numerous eubacteria, and several apicomplexans. Using the ML puzzling test and bootstrapping analysis with protein distance and parsimony methods, the resulting trees not only robustly confirmed the α‐proteobacterial relationship of apicomplexan LDH/MDH, but also supported a monophyletic relationship of CpLDHl with CpMDHl. These data suggest that, unlike most other eukaryotes, the Apicomplexa may be one of the few lineages retaining an α‐proteobacterial‐type MDH that could have been acquired from an ancestral α‐proteobacterium through primary endosymbiosis giving rise to the mitochondria, or through an unknown lateral gene transfer (LGT) event.
Plateins: A Novel Family of Signal Peptide‐Containing Articulins in Euplotid Ciliates1
Authors: KLOETZEL JOHN A. , BAROIN‐TOURANCHEAU ANNE , MICELI CRISTINA , BARCHETTA SABRINA , FARMAR JAMES , BANERJEE DEBEN , FLEURY‐AUBUSSON ANNE
Published: July 2005

ABSTRACT. In euplotid ciliates, the cortex is reinforced by alveolar plates—proteinaceous scales located within the membranous alveolar sacs, forming a monolayer just below the plasma membrane. This system appears to play a cytoskeletal role analogous to that provided by the fibrous epiplasm found beneath the cortical alveoli in other ciliates. In Euplotes aediculatus, the major alveolar plate proteins (termed α‐,β‐, and γ‐plateins) have been identified. Using anti‐platein antibodies, an expression library of Euplotes genes was screened, and a platein gene identified, cloned, and completely sequenced. Comparison of its derived amino acid sequence with micro‐sequences obtained directly from purified plateins identified this gene as encoding one of the closely related β‐or μ‐plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (pI = 4.88). Microsequences from authentic ot‐platein were then used to design oligonucleotide primers, which yielded, via a PCR‐based approach, the sequences of two α‐platein genes from E. aediculatus. Even more acidic proteins, the derived al‐ and a2‐plateins contain 536 and 501 residues, respectively. Analyses of their amino acid sequences revealed the plateins to be members of the articulin superfamily of cytoskeletal proteins, first described in Euglena and now identified in the ciliate Pseudomicrothorax and in Plasmodium. The hallmark articulin repetitive motifs (based on degenerate valine‐and proline‐rich 12‐mers) are present in all three plateins. In β/γ‐platein this primary motif domain (27 repeats) is central in the molecule, whereas the primary repeats in the α‐plateins lie near their C‐termini. A cluster of proline‐rich pentameric secondary repeats is found in the C‐terminus of pAy‐platein, but near the N‐terminus of a‐plateins. All three plateins contain canonical N‐terminal signal sequences, unique among known cytoskeletal proteins. The presence of start‐transfer sequences correlates well with the final intra‐alveolar location of these proteins. This feature, and significant differences from known articulins in amino acid usage and arrangement within the repeat domains, lead us to propose that the plateins comprise a new family of articulin‐related proteins. Efforts to follow microscopically the assembly of plateins into new alveolar plates during pre‐fission morphogenesis are underway.
Light Microscopical Observation of Infraciliary Bands of Eodinium posterovesiculatum in Comparison with Entodinium bursa and Diplodinium dentatum
Authors: ITO AKIRA , IMAI SOICHI
Published: July 2005

ABSTRACT. Morphological features of the four morphotypes of the rumen ciliate, Eodinium posterovesiculatum, are described from pyridinated. silver carbonate‐impregnated specimens. Infraciliary bands are compared with those of Entodinium bursa and Diplodinium dentatum. In Entodinium bursa, the adoral polybrachykinety is “C” shaped and the vestibular polybrachykinety extends from the dorsal extremity of the adoral polybrachykinety as in other Entodinium species. In Diplodinium dentatum, the adoral polybrachykinety encircles most of the circumference of the vestibular opening and the vestibular polybrachykinety extends from the inner side of the adoral polybrachykinety as in other Diplodinium species. Infraciliary “bands in Eodinium posterovesiculatum, encircle the whole circumference of the vestibular opening and are, therefore, distinct from Entodinium, Diplodinium and other ciliates in the family Ophryoscolecidae. In Eodinium posterovesiculatum, the adoral polybrachykinety and the anterior part of the vestibular polybrachykinety encircle the vestibular opening. The wide and long vestibular polybrachykinety extends along the right wall of the tubular vestibulum and is bordered by a kinety. Kinetids in the central part of the vestibular polybrachykinety are randomly disposed. The genus Eodinium is valid because of this characteristic polybrachykinety arrangement in Eodinium posterovesiculatum.
Trichomonas vaginalis: In Vitro Attachment and Internalization of HIV‐1 and HIV‐1‐Infected Lymphocytes
Authors: RENDÓN‐MALDONADO JOSÉ , ESPINOSA‐CANTELLANO MARTHA , SOLER CARMEN , TORRES JOSÉ V. , MARTÍNEZ‐PALOMO ADOLFO
Published: July 2005

ABSTRACT. Sexually transmitted diseases (STDs) caused by bacteria and protozoa play an important role in the epidemiology of human immunodeficiency virus (HIV‐1) infection. Human trichomoniasis, produced by the protozoan parasite Trichomonas vaginalis, is one of the most common STDs, and is a cause of mucosal lesions in the urogenital tract, which may increase the risk for HIV infection. However, there are no reports concerning the outcome of in vitro interactions between HIV particles and trichomonads. Therefore, we incubated T. vaginalis with three subtypes of HIV‐1 (A, B, and D), as well as with HIV‐1‐infected lymphocytes, and analyzed the interactions with immunofluorescence microscopy and transmission electron microscopy. Our results demonstrated that HIV‐1 particles attach and are incorporated into T. vaginalis through endocytic vesicles and are degraded within cytoplasmic vacuoles in approximately 48 h. There was no ultrastructural evidence of HIV‐1 replication in trichomonads. These results demonstrated that trichomonads may internalize and harbor HIV‐1 particles for short periods of time. In addition, under in vitro conditions, T. vaginalis ingests and digests HIV‐1‐infected lymphocytes.
Platyamoeba nucleolilateralis n. sp. from the Chesapeake Bay Region
Authors: ANDERSON O. ROGER , NERAD THOMAS A. , COLE JEFFREY C.
Published: July 2005

ABSTRACT. Members of the genus Platyamoeba are among the most common of the free‐living brackish and marine amoebae; yet, to date only twelve species have been documented in the literature and only a limited number of habitats have been sampled globally. During the course of a systematic survey of salt‐marsh amoebae along the east coast of the United States, a new species of Platyamoeba was discovered in sediment samples obtained from a salt marsh at Assateague Island, VA. The species can be distinguished from all other described species within the genus by the presence of a nucleus with a single parietal nucleolus and a floating form with long tapering pseudopods. Its shape varies from flabellate to spatulate as described for species of Platyamoeba and Vannella. The fine structure of the glycocalyx, however, is characteristic of Platyamoeba.
Characterization of sams Genes of Amoeba proteus and the Endosymbiotic X‐Bacteria
Authors: JEON TAECK J. , JEON KWANG W.
Published: July 2005

ABSTRACT. As a result of harboring obligatory bacterial endosymbionts, the xD strain of Amoeba proteus no longer produces its own S‐adenosylmethionine synthetase (SAMS). When symbiont‐free D amoebae are infected with symbionts (X‐bacteria), the amount of amoeba SAMS decreases to a negligible level within four weeks, but about 47% of the SAMS activity, which apparently comes from another source, is still detected. Complete nucleotide sequences of sams genes of D and xD amoebae are presented and show that there are no differences between the two. Long‐established xD amoebae contain an intact sams gene and thus the loss of xD amoeba's SAMS is not due to the loss of the gene itself. The open reading frame of the amoeba's sams gene has 1,28] nucleotides, encoding SAMS of 426 ammo acids with a mass of 48 kDa and pi of 6.5. The amino acid sequence of amoeba SAMS is longer than the SAMS of other organisms by having an extra internal stretch of 28 amino acids. The 5′‐flanking region of amoeba sams contains consensus‐binding sites for several transcription factors that are related to the regulation of sams genes in E. coli and yeast. The complete nucleotide sequence of the symbiont's sams gene is also presented. The open reading frame of X‐bacteria sams is 1,146 nucleotides long, encoding SAMS of 381 amino acids with a mass of 41 kDa and pi of 6.0. The X‐bacteria SAMS has 45% sequence identity with that of A. proteus.
Analysis of Chlamydomonas reinhardtii Genome Structure Using Large‐Scale Sequencing of Regions on Linkage Groups I and III
Authors: LI JIN BILLY , LIN SHAOPING , JIA HONGGUI , WU HONGMIN , ROE BRUCE A. , KULP DAVID , STORMO GARY D. , DUTCHER SUSAN K.
Published: July 2005

ABSTRACT. Chlamydomonas reinhardtii is a unicellular green alga that has been used as a model organism for the study of flageila and basal bodies as well as photosynthesis. This report analyzes finished genomic DNA sequence for 0.5% of the nuclear genome. We have used three gene prediction programs as well as EST and protein homology data to estimate the total number of genes in Chlamydomonas to be between 12,000 and 16,400. Chlamydomonas appears to have many more genes than any other unicellular organism sequenced to date. Twenty‐seven percent of the predicted genes have significant identity to both ESTs and to known proteins in other organisms, 32% of the predicted genes have significant identity to ESTs alone, and 14% have significant similarity to known proteins in other organisms. For gene prediction in Chlamydomonas, GreenGenie appeared to have the highest sensitivity and specificity at the exon level, scoring 71% and 82%, respectively. Two new alternative splicing events were predicted by aligning Chlamydomonas ESTs to the genomic sequence. Finally recombination differs between the two sequenced contigs. The 350‐Kb of the Linkage group III contig is devoid of recombination, while the Linkage group I contig is 30 map units long over 33‐kb.
Analysis of Four Human Microsporidian Isolates by MALDI‐TOF Mass Spectrometry
Authors: MOURA HERCULES , OSPINA MARIA , WOOLFITT ADRIAN R. , BARR JOHN R. , VISVESVARA GOVINDA S.
Published: July 2005

ABSTRACT. Spores of four species of microsporidia isolated from humans were analyzed by matrix‐assisted laser desorption ioni‐zation time of flight mass spectrometry (MALDI‐TOF MS) and specific biomarkers were found for each. The microsporidia analyzed included three species, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis and the fourth organism is the recently described Brachiola algerae. Whole spores, spore shells, and soluble fractions were applied directly to the MALDI target without further purification steps. MALDI‐TOF MS analysis of both whole spores and soluble fractions of the four isolates revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 2,000‐8,000 Da. Statistical analysis of the averaged centroided masses uncovered two distinct sets of unique peptides or biomarkers, one originated from whole spores and the other from soluble fractions, that can differentiate the four microsporidian species studied. MALDI‐TOF MS analysis of whole organisms is a rapid, sensitive, and specific option to characterize microsporidian isolates and has the potential for several applications in parasi‐tology.
Identification of the Inositol Isomers Present inTetrahymena
Authors: KERSTING MICHAEL C. , BOYETTE MICHELE , MASSEY JOSEPH H. , RYALS PHILLIP E.
Published: July 2005

ABSTRACT.The inositol isomer composition of phosphoinositides, polyphosphoinositols, phosphatidylinositol‐linked glycans, and glycosyl phosphatidylinositol‐anchored proteins of logarithmic phaseTetrahymena voraxwas determined by GC‐MS analysis of tri‐methylsilylimadaz.ole derivatives. The most abundant inositol found was the myoisomer; however, appreciable percentages ofscyllo‐inositol were present in the free inositol pool, phosphatidylinositol‐linked glycan fraction, and glycosyl phosphatidylinositol‐anchored protein fraction. Trace quantities ofchiro‐ andneo‐inositols also were present.
Molecular Characterization of Encephalitozoon intestinalis (Microspora) Replication Kinetics in a Murine Intestinal Cell Line
Authors: WASSON KATHERINE , BARRY PETER A.
Published: July 2005

ABSTRACT. Microsporidia are obligate intracellular pathogens of invertebrate and vertebrate animals. Most human infections are caused by Enterocytozoon bieneusi or Encephalitozoon intestinalis, and result in chronic diarrhea. In order to determine the signals involved in microsporidial spore activation and invasion, kinetics of in vitro E. intestinalis replication were defined using real‐time quantitative PCR. Segments of small subunit ribosomal RNA and polar tube protein 2 genes of E. intestinal is were used to quantify parasite gene copy number following infection in murine colon carcinoma cells. Parasite DNA was detectable in small but significant amounts within host cells as early as 4 h postinoculation, genome replication was completed by 36 h, and parasite progeny were released into the supernatant beginning 72 h postinoculation. Heat‐treating spores did not prevent transfer of parasite DNA into cells, but did inhibit parasite replication. Treating cell cultures with albendazole suppressed but did not completely inhibit parasite replication. These results confirm observations that E. intestinalis completes its life cycle within the turnover time of its target host cells; invasion into susceptible host cells occurs independently of spore viability; and real‐time quantitative PCR is a sensitive and reproducible method with which to monitor microsporidial infection under varying treatments or conditions.
Molecular Phylogeny of Three Oxymonad Genera: Pyrsonympha, Dinenympha and Oxymonas
Authors: MORIYA SHIGEHARU , DACKS JOEL B. , TAKAGI AIKA , NODA SATOKO , OHKUMA MORIYA , DOOLITTLE W. FORD , KUDO TOSHIAKI
Published: July 2005

ABSTRACT. Oxymonads are a morphologically well‐characterized and highly diverse lineage of protists. They are, however, under sampled at a molecular level. It has recently been demonstrated that a genus of oxymonads, Pyrsonympha, is phylogenetically related to the excavate taxon Trimastix. Here, we addressed issues of internal oxymonad evolution. Pyrsonympha and Dinenympha are shown, by fluorescent in situ hybridization and phylogenetic evidence, to be separate genera and not morphotypes of the same organism. We demonstrated that three genera of oxymonads, Dinenympha, Pyrsonympha, and Oxymonas are each monophyletic and together form a clade which excludes other known eukaryotes. We have presented a taxonomic scheme of oxymonads taking into account their sisterhood with Trimastix and speculated on morphological evolution of oxymonads, particularly of their attachment apparatuses. Our biogeographical analysis with Japanese and Canadian Pyrsonympha and Dinenympha suggests that these genera diverged before the separation of termites that inhabit Eastern Asia and Western North America.
Use of Random Amplified Polymorphic DNA (RAPD) Analysis for the Identification of Giardia intestinalis Subtypes and Phylogenetic Tree Construction
Authors: ŠEDINOVÁ JITKA , FLEGR JAROSLAV , EY PETER L. , KULDA JAROSLAV
Published: July 2005

ABSTRACT. A comparison of random amplified polymorphic DNA (RAPD) was used to investigate genetic polymorphisms among 25 isolates of Giardia intestinalis and to assess the utility of RAPD for subtype detection and genealogical analysis. Using data obtained for six human and 19 animal‐derived isolates in polymerase chain reactions using 13 different primers, phylogenetic trees were constructed and bootstrap values computed by the program FreeTree. Three major clades were distinguished, corresponding to previously defined genetic assemblages A, B, and E. The purported specificity of assemblage E genotypes for artiodactyl hosts was supported. Assemblages A and B showed wide host spectra, including human and animal hosts. No correlation was found between the genotype of analyzed isolates and the presence or absence of the double‐stranded RNA Giardiavirus. The results indicate that RAPD data provide reliable genetic information that can be used for both “fingerprinting” and genealogical purposes.
Metacystis borrorin. sp. (Ciliophora: Metacystidae) on the SeagrassThalassia testudinum
Authors: ALADRO‐LUBEL MA. ANTONIETA , MARTINEZ‐MURILLO MA. ESTHER
Published: July 2005

ABSTRACT.A new epibiontic ciliate of the genus Metacystis is described on the seagrassThalassia testudinumof the coral reef lagoons of Veracruz, Mexico. The ciliate was studied in living and stained specimens and under the scanning electron microscope. The cell body (10–35 × 10–18 μm in vivo) is transversely annulated (4–6 rings). The somatic ciliature consists of 22–30 longitudinal kineties, and patterned as 5–7 transverse kinetics. The circumoral kinety is composed of kinetosomes closely spaced. The macronucleus diameter measures about 3–7 μm. The lorica (18–61 × 11–26 μm) has the posterior end round to conical or irregular with mucoid filaments. This prostomatid colonizes both natural and artificial substrates placed in an aquarium.Metacystis borrorin. sp. is a species that forms part of the ciliate community onThalassia testudinumwith a temperature range of 21–26°C and a salinity of 32–40‰.
Influence of Diet on the Rumen Protozoal Fauna of Indigenous African Wild Ruminants
Authors: DEHORITY BURK A. , ODENYO AGNES A.
Published: July 2005

ABSTRACT. A study was carried out to determine if the protozoal fauna of indigenous African wild ruminants was different from that found in their domestic counterparts and if the animal's diet influenced the number and types of protozoa. Samples of rumen contents were collected in 1997 and 2001 from various indigenous African wild ruminants in Kenya. All three ruminant feeding types were sampled: browsers or concentrate selectors (giraffe and Guenther's dik‐dik); intermediate or adaptable mixed feeders (impala, Thomson's gazelle, Grant's gazelle and eland); grass or roughage eaters (hartebeest and wildebeest). Total concentration of ciliate protozoa and percentage generic distribution were determined. In general, protozoal concentrations were higher in concentrate selectors, followed by the intermediate or opportunistic mixed feeders and lowest in the grass and roughage eaters. Both Thomson's and Grant's gazelle were protozoa‐free in the 2001 samples. Entodinium percentages were considerably higher in concentrate selectors and intermediate mixed feeders, compared to roughage eaters. Two genera of protozoa previously found in only a few African ruminants, Epiplastron and Opisthotrichum, were observed in several additional animal species and represent new host records. A difference was noted in the protozoal species composition of the indigenous wild ruminants from that previously observed in African domestic ruminants.
Two New Small‐Subunit Ribosomal RNA Gene Lineages within the Subclass Gymnamoebia
Authors: PEGLAR MICHAEL T. , ZETTLER LINDA A. AMARAL , ANDERSON O. ROGER , NERAD THOMAS A. , GILLEVET PATRICK M. , MULLEN THOMAS E. , FRASCA SALVATORE , SILBERMAN JEFFREY D. , O'KELLY CHARLES J. , SOGIN MITCHELL L.
Published: July 2005

ABSTRACT. Phylogenetic analysis of small‐subunit ribosomal RNA gene sequences for gymnamoebae of the families Vexilliferidae, Paramoebidae, and Vannellidae identified two distinct lineages that are supported by gross morphological characters. This analysis indicates that paramoebids and vexilliferids are part of one lineage and that vannellids belong to another. A shared morphological character unique to the paramoebid/vexilliferid lineage members is the presence of dactylopodiate subpseudopodia. However, cell surface structures, normally used for taxonomic discrimination, range from simple hair‐like filaments without any apparent organization (Neo‐paramoeba), to hexagonal glycostyles (Vexillifera) or more elaborate surface scales (Korotnevella). Taxa within the vannellid lineage all lack subpseudopodia and appear flabellate, spatulate or linguif'orm while in locomotion. Cell surface structures of taxa within the vannellid lineage range from filaments organized into hexagonal arrays (Lingulamoeba, Platyamoeba) to pentagonal glycostyles (Cly‐donella, Vannella). Vannellid lineage members of the genera Clydonella and Lingulamoeba were studied at the level of electron microscopy. Unique cell surface features validate these as genera distinct from Vannella and Platyamoeba. Genetic and ultrastructural data are used to discuss the phylogenetic interrelationships for the taxa studied.
Structure of N‐glycosidic Carbohydrates of Secretory Proteins of Tetrahymena thermophila
Authors: BECKER BURKHARD , RÜSING MATTHIAS
Published: July 2005

ABSTRACT. Glycoproteins secreted by Tetrahymena into the culture medium were isolated and the N‐glycosidic oligosaccharides analyzed using lectin blots and fluorophore‐assisted carbohydrate gel electrophoresis (FACE). Lectin blots showed that the glycoproteins secreted by Tetrahymena contain only N‐glycosidic structures of the high mannose type. Further analysis using the FACE technology revealed the presence of four different N‐glycosidic structures differing only in the number of mannose residues attached to the core chitobiose unit.
Ultrastructure of Besnoitia besnoiti Tissue Cysts and Bradyzoites
Authors: DUBEY J. P. , SHKAP V. , PIPANO E. , FISH L. , FRITZ D. L.
Published: July 2005

ABSTRACT. Besnoitia besnoiti is an economically important tissue cyst‐forming apicomplexan of cattle in Africa and Israel. Tissue cysts and bradyzoites of B. besnoiti from the skin of a naturally infected bull were studied by transmission electron microscopy. Tissue cysts enclosed host cell and bradyzoites. Bradyzoites were 6–7. 5 × 1. 9–2. 3 μm in size and contained organelles found in coccidian merozoites including numerous micronemes, rhoptries, amylopectin granules, and a posteriorly located nucleus. Enigmatic bodies, characteristically found in Besnoitia sp. bradyzoites, were not observed. Therefore, enigmatic bodies should be removed as a generic character of the bradyzoites of Besnoitia species.
Profilin Functions in Cytokinesis, Nuclear Positioning, and Stomatogenesis in Tetrahymena thermophila
Authors: WILKES DAVID E. , OTTO JOANN J.
Published: July 2005

ABSTRACT. Expression of the actin‐binding protein profilin was disrupted in the ciliate Tetrahymena thermophila by an antisense ribosome method. In cells with the antisense disruption no profilin protein was detected. Cultures of cells with the antisense disruption could be maintained, indicating that profilin was not essential for cytokinesis or vegetative growth. Disruption of the expression of profilin resulted in many cells that were large and abnormally shaped. Formation of multiple micronuclei, which divide mitotically, was observed in cells with a single macronucleus, indicating a defect in early cytokinesis. Some cells with the antisense disruption contained multiple macronuclei, which in Tetrahymena may indicate a function late in cytokinesis. The lack of profilin also affected cytokinesis in the cells that could divide. Normal‐sized and normal‐shaped cells with the antisense disruption took significantly longer to divide than control cell types. The profilin disruption revealed two new processes in which profilin functions. In cells lacking profilin, micronuclei were not positioned at their normal site on the surface of the macronucleus and phagocytosis was defective. The defect in phagocytosis appeared to be due to disruption of the formation of oral apparatuses (stomatogenesis) and a possible failure in the internalization of phagocytic vacuoles.
Detection of Naegleria sp. in a Thermal, Acidic Stream in Yellowstone National Park
Authors: SBEEBAN KATHY B. , FERRIS MICHAEL J. , BENSON JOAN M.
Published: July 2005

ABSTRACT. An initial survey of sequences of PCR‐amplified portions of the 18S rRNA genes from a community DNA clone library, prepared from an algal mat in a thermal, acidic stream in Yellowstone National Park, WY, USA, revealed among other sequences, several that matched Vahlkampfia. This finding prompted further investigation using primers specific for Naegleria. Sequences from a subsequent DNA clone library, prepared from the 5.8S rRNA gene and the adjacent internal transcribed spacer (ITS) regions of the rRNA, closely matched Naegleria and formed an independent lineage within a clade containing Naegleria sturti and Naegleria niuginiensis. The sequences may represent a new Naegleria species.
Resolution of a Taxonomic Conundrum: an Ultrastructural and Molecular Description of the Life Cycle of Pleistophora mulleri (Pfeiffer 1895; Georgevitch 1929)
Authors: TERRY REBECCA S. , MACNEIL CALUM , DICK JAIMIE T. A. , SMITH JUDITH E. , DUNN ALISON M.
Published: July 2005

ABSTRACT. The classification of a microsporidian parasite observed in the abdominal muscles of amphipod hosts has been repeatedly revised but still remains inconclusive. This parasite has variable spore numbers within a sporophorous vesicle and has been assigned to the genera Glugea, Pleistophora, Stempellia, and Thelohania. We used electron microscopy and molecular evidence to resolve the previous taxonomic confusion and confirm its identification as Pleistophora mulleri. The life cycle of P. mulleri is described from the freshwater amphipod host Gammarus duebeni celticus. Infection appeared as white tubular masses within the abdominal muscle of the host. Light and transmission electron microscope examination revealed the presence of an active microsporidian infection that was diffuse within the muscle block with no evidence of xenoma formation. Paucinucleate merogonial plasmodia were surrounded by an amorphous coat immediately external to the plasmalemma. The amorphous coat developed into a merontogenetic sporophorous vesicle that was present throughout sporulation. Sporogony was polysporous resulting in uninucleate spores, with a bipartite polaroplast, an anisofilar polar filament and a large posterior vacuole. SSU rDNA analysis supported the ultrastructural evidence clearly placing this parasite within the genus Pleistophora. This paper indicates that Pleistophora species are not restricted to vertebrate hosts.
The Molecular Evolution and Structural Organization of Self‐Splicing Group I Introns at Position 516 in Nuclear SSU rDNA of Myxomycetes
Authors: HAUGEN PEIK , COUCHERON DAG H. , RØNNING SISSEL B. , HAUGLI KARI , JOHANSEN STEINAR
Published: July 2005

ABSTRACT.Group I introns are relatively common within nuclear ribosomal DNA of eukaryotic microorganisms, especially in myxomycetes. Introns at position S516 in the small subunit ribosomal RNA gene are particularly common, but have a sporadic occur rence in myxomycetes.Fuligo septica, Badhamia gracilis, andPhysarum flavicomum, all members of the family Physaraceae, contain related group IC1 introns at this site. TheF. septicaintron was studied at the molecular level and found to self‐splice as naked RNA and to generate full‐length intron RNA circles during incubation. Group I introns at position S516 appear to have a particularly widespread distribution among protists and fungi. Secondary structural analysis of more than 140 S516 group I introns available in the database revealed five different types of organization, including IC1 introns with and without His‐Cys homing endonuclease genes, complex twin‐ribozyme introns, IE introns, and degenerate group I‐like introns. Both intron structural and phylogenetic analyses indicate a multiple origin of the S516 introns during evolution. The myxomycete introns are related to S516 introns in the more distantly related brown algae andAcanthamoebaspecies. Possible mechanisms of intron transfer both at the RNA‐ and DNA‐levels are discussed in order to explain the observed widespread, but scattered, phylogenetic distribution.
The Endosymbiotie Bacterium Holospora obtusa Enhances Heat‐Shock Gene Expression of the Host Paramecium caudatum
Authors: HORI MANABU , FUJISHIMA MASAHIRO
Published: July 2005

ABSTRACT. The bacterium Holospora oblusa is a macronuclear‐specific symbiont of the ciliate Paramecium caudatum. H. obtusa‐bearing paramecia could survive even after the cells were quickly heated from 25°C to 35°C. To determine whether infection with H. obtusa confers heat shock resistance on its host, we isolated genes homologous to the heat shock protein genes hsp60 and hsp70 from P. caudatum. The deduced amino acid sequences of both cDNAs were highly homologous to hsp family sequences from other eukaryotes. Competitive PCR showed that H. obtusa‐free paramecia expressed only trace amounts of hsp60 and hspVO mRNA at 25°C, but that expression of hsp70 was enhanced immediately after the cells were transferred to 35°C. H. obtusa‐bearing paramecia expressed high levels of hsp70 mRNA even at 25°C and the level was further enhanced when the cells were incubated at 35°C. In contrast, the expression pattern of hsp60 mRNA was the same in H. obtusa‐bearing as in H. obtusa‐freeparamecia. These results indicate that infection with its endosymbiont can confer a heat‐shock resistant nature on its host cells.
Catalase is the Bacteria‐Derived Detoxifying Substance against Paramecia‐ Killing Toxin in Wheat Grass Powder Infusion
Authors: MIZOBUCHI NAOMI , YOKOIGAWA KUMIO , HARUMOTO TERUE , FUJISAWA HIROMI , TAKAGP YOSHIOMI
Published: July 2005

ABSTRACT. Paramecium cells are usually cultured in a wheat grass powder infusion inoculated with Klebsiella pneumoniae. However, non‐bacterized wheat grass powder infusion is toxic to paramecia, and bacteria‐derived substance detoxifies the toxic substance. Here, the detoxifying substance from K. pneumoniae, which was found to be proteinaceous, was purified to homogeneity. The protein had an apparent molecular mass of about 200 kDa by gel filtration and 92 kDa by SDS‐polyacrylamide gel electrophoresis. Although the amino acid sequence of the amino terminal region did not show a high sequence homology with any reported proteins, amino acid sequences of internal regions of the protein were nearly identical to catalase HPII from Escherichia coll. When the wheat grass powder infusion was treated at 25°C for 1 h with commercially available catalase from bovine liver, the toxicity of the infusion against paramecia was completely abolished. The initial concentration of hydrogen peroxide in the wheat grass powder infusion was about 30 uM and was completely decomposed by the catalase treatment. Therefore, the toxic substance in the wheat grass powder infusion and the detoxifying substance from K. pneumoniae are considered as hydrogen peroxide and catalase, respectively.
The Accidental Protozoologist—My Journey Through the World of Ciliates1,2
Authors: BLEYMAN LEA K.
Published: July 2005

ABSTRACT. The Past‐President's Address has been the opportunity for the speaker to reminisce about the road traveled to get to this time in life. In this paper, I continue in that tradition. During my journey to the present day, I visited different laboratories, studying the genetics of mating and mating types in Paramecium, Tetrahymena, Blepharisma and Euplotes. I have met and worked with many distinguished scientists, including other Past‐Presidents of the Society. I also became an active participant in the Society of Protozool‐ogists. I hope the recounting of my trip will be both entertaining and enlightening.
Lack of Arginine Decarboxylase in Trypanosoma cruzi Epimastigotes
Authors: CARRILLO CAROLINA , CEJAS SILVINA , HUBER ALEJANDRA , GONZÁLEZ NÉLIDA S. , ALGRANATI ISRAEL D.
Published: July 2005

ABSTRACT. The presence of arginine decarboxylase (ADC) enzymatic activity in Trypanosoma cruzi epimastigotes is still a matter of controversy due to conflicting results published during the last few years. We have investigated whether arginine might indeed be a precursor of putrescine via agmatine in these parasites. We have shown that wild‐type T. cruzi epimastigotes cultivated in a medium almost free of polyamines stopped their growth after several repeated passages of cultures in the same medium, and that neither arginine nor ornithine were able to support or reinitiate parasite multiplication. In contrast, normal growth was quickly resumed after adding exogenous putrescine or spermidine. The in vivo labelling of parasites with radioactive arginine showed no conversion of this amino acid into agmatine, and attempts to detect ADC activity measured by the release of CO2 under different conditions in T. cruzi extracts gave negligible values for all strains assayed. The described data clearly indicate that wild‐type T. cruzi epimastigotes lack ADC enzymatic activity.
Isolation and Characterization of Glutathione Reductase from Physarum polycephalum and Stage‐Specific Expression of the Enzyme in Life‐Cycle Stages with Different Oxidation‐Reduction Levels
Authors: MINAMI YOSHIKO , KOHAMA TAKESHI , SEKIMOTO YU‐JI , AKASAKA KENICHI , MATSUBARA HIROSHI
Published: July 2005

ABSTRACT. Physarum polycephalum has a life cycle with several distinct phases that have different oxidation‐reduction requirements. To investigate the relationship between the life cycle and the oxidation‐reduction state, we isolated glutathione reductase (GR; EC 1.6.4.2) from Physarum microplasmodia. The enzyme was found to be a homodimer with a subunit Mr of 49,000, and Km values for oxidized glutathione and NADPH of 40 and 28.6 μM, respectively. We then constructed a cDNA library from microplasmodium mRNA and cloned GR cDNA from the library. The isolated cDNA consisted of 1,475 bp encoding a polypeptide of 452 amino acids. The amino acid sequence similarity was about 50% with GRs of other organisms, and several conserved sequence motifs thought to be necessary for activity are evident in the Physarum. enzyme. Escherichia coli transformed with an expression vector containing the cDNA synthesized the active GR. Genomic Southern blot analysis indicated that the GR gene is present as a single copy in the Physarum genome. Immunoblot analysis and RT‐PCR analysis detected GR mRNA expression in the microplasmodium, plasmodium, and scle‐rotium, but not in the spore or flagellate. GR activity was low in the spore and flagellate. These results suggest that the glutathione oxidation‐reduction system relates to the Physarum life cycle.