bluuebunny/crossref_metadata_2025 huggingface dataset:
The Euplotes crassus Conjugation‐specific conN1 Gene Encodes a Transcription Elongation Factor TFIIS‐like Protein
Authors: GHOSH SUSMITA , BARRETT DAVID M. , KLOBUTCHER LAWRENCE A.
Published: July 2005

ABSTRACT. The Euplotes crassus macronuclear DNA molecule containing the conjugation‐specific conN1 gene has been sequenced, along with a cDNA clone. The results indicate that the conN1 gene encodes a protein similar to the transcription elongation factor TFIIS proteins identified in other eukaryotes.
Organization of Telomeres During the Cell and Life Cycles of Trypanosoma brucei
Authors: PÉREZ‐MORGA DAVID , AMIGUET‐VERCHER AMELIA , VERMIJLEN DAVID , PAYS ETIENNE
Published: July 2005

ABSTRACT. The genome of Trypanosoma brucei contains about 120 chromosomes, which do not visibly condense during mitosis. We have analyzed the organization and segregation of these chromosomes by in situ hybridization using fluorescent telomere probes. At the onset of mitosis, telomeres migrate from their nuclear peripheral location and congregate into a central zone. This dense group of telomeres then splits into two entities that migrate to opposite nuclear poles. Segregation continues until the double‐sized nucleus divides and, before cytokinesis occurs, the telomeres reorganize into the discrete foci observed at interphase. During migration, the telomeres are located at the free end of the mitotic spindle. Treatment with the microtubule polymerization inhibitor rhizoxin prevents telomere clustering and chromosomal segregation. In the insect‐specific procyclic form as well as in the non‐dividing bloodstream stumpy form, telomeres tend to cluster close to the nuclear periphery at interphase. In contrast, in the proliferative bloodstream slender form the telomeres preferentially locate in the central zone of the nucleus. Thus, telomeres are closer to the nuclear periphery during those life cycle stages where the telomeric expression sites for the variant surface glycoprotein are all inactive, suggesting that transcriptional inactivation of these sites is related to their subnuclear localization.
Pseudoloma neurophilia n. g., n. sp., a New Microsporidium from the Central Nervous System of the Zebrafish (Danio rerio)
Authors: MATTHEWS JENNIFER L. , BROWN AMANDA M. V. , LARISON KAREN , BISHOP‐STEWART JANELL K. , ROGERS PAUL , KENT MICHAEL L.
Published: July 2005

ABSTRACT. An unusual xenoma‐forming microsporidium was discovered in the central nervous system of moribund zebrafish from a laboratory colony in Eugene, Oregon. Infected fish were often emaciated and lethargic, and histological examination commonly revealed severe myelitis and myositis associated with the infection. Based on its structure, development, and small subunit ribosomal DNA sequence it is unique among fish microsporidia. Spores are uninucleate, ovoid to pyriform, with a prominent posterior vacuole. Spores average 5.4 × 2.7 μm with 13–16 coils of the polar filament. The microsporidium produces xenomas within the spinal cord and hindbrain of fish, and xenomas contained sporophorous vesicles with up to 16 spores. Sporoblasts and presporoblast stages (probably sporonts) are found occasionally in small aggregates dispersed randomly throughout xenomas. It clustered in the “Ichthyosporidium group” along with other fish microsporidian genera based on rDNA sequence analysis. The rDNA sequence of the zebrafish microsporidium was most similar to that of Ichthyosporidium, but showed only 12.1% similarity and therefore this microsporidium can be considered a distinct genus and species, which we have named Pseudoloma neurophilia n. g., n. sp.
Fine Oral Filaments in Paramecium: a Biochemical and Immunological Analysis
Authors: CLEROT JEAN‐CLAUDE , IFTODE FRANCINE , BUDIN KARINE , JEANMAIRE‐WOLF RACHEL , COFFE GÉRARD , FLEURY‐AUBUSSON ANNE
Published: July 2005

ABSTRACT. In Paramecium, several kinds of the oral networks of fine filaments are defined at the ultrastructural level. Using the sodium chloride‐treated oral apparatus of Paramecium as an antigen to produce monoclonal antibodies, we have begun to identify the proteins constituting these networks. Immunoblotting showed that all positive antibodies were directed against three bands (70–, 75‐and 83‐kD), which corresponded to quantitatively minor components of the antigen; there was no antibody specific for the quantitatively major components (58‐ and 62‐kD). Immunolocalization with four of these antibodies directed against one or several of these three bands showed that these proteins are components of the fine filaments supporting the oral area; a decoration of the basal bodies and the outer lattice was also observed on the cortex. Immunofluorescence on interphase cells suggested that the three proteins colocalized on the left side of the oral apparatus, whereas only the 70‐kD band was detected on the right side. During division, the antigens of the antibodies were detected at different stages after oral basal body assembly. The antibodies cross‐reacted with the tetrins, which are oral filament‐forming proteins in Tetrahymena, demonstrating that tetrin‐related proteins are quantitatively minor components of the oral and the somatic cytoskeleton of Paramecium.
Identification of Heterotrophic Nanoflagellates by Restriction Fragment Length Polymorphism Analysis of Small Subunit Ribosomal DNA
Authors: LIM EE LIN , DENNETT MARK R. , CARON DAVID A.
Published: July 2005

ABSTRACT. Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau 3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the ‘naked’ flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.
Phylogenetic Relationships Among Microsporidia Based on rDNA Sequence Data, With Particular Reference to Fish‐Infecting Microsporidium Balbiani 1884 Species.
Authors: BELL ANDREW S. , AOKI TAKASHI , YOKOYAMA HIROSHI
Published: July 2005

ABSTRACT. Recently, large discrepancies have been identified between microsporidian systematics based on molecular and traditional characteristics. In the current study the 530f–580r region of the rRNA gene of eight microsporidian species was cloned and sequenced. Included were two unclassified species of Microsporidium Balbiani, 1884 and an unidentified microsporidian that infects the musculature of different sea bream species. Sequence identities in excess of 98% indicated that these three species almost certainly are members of the same genus. Phylogenetic analyses of all microsporidian sequence data available for this region of the gene (20 species) and for partial small subunit sequences (51 species of 21 genera) revealed these species to be distinct from the family Pleistophoridae Doflein, 1901 and closely related them to the genus Spraguea Weissenberg, 1976. This clade was found to comprise a sister taxon to that containing the vast majority of fish‐infecting species. Broad cladistic divisions were found between terrestrial insect‐infecting and fish‐infecting species, which together are distant from the aquatic insect‐infecting microsporidia. The rRNA gene of certain fish‐infecting genera was found to be more highly conserved than previously reported. This has implications for its utility in diagnostic assays and phylogenetic studies at, or close to, the species level.
Development in Electrofused Conjugants of Tetrahymena thermophila
Authors: COLE ERIC S. , VIRTUE MARK A. , STUART KATHLEEN R.
Published: July 2005

ABSTRACT. Electric shock can create parabiotic fusions of living Tetrahymena cells. In this study, cells were mated and successful pairs were electrofused with either vegetatively growing cells or other mating pairs. In particular, we electrofused pairs from normal [diploid × diploid] matings with vegetatively dividing cells in G‐ or M‐phase of the cell cycle. We also fused [diploid × diploid] conjugants with mating pairs involving an aneuploid partner [diploid ×“star”], which typically undergo an abortive conjugal pathway termed genomic exclusion. Using such parabiotic fusions we identified and characterized two developmentally critical landmarks: I) the “abort” signal, which is initiated in pairs with nuclear defects (this first becomes evident soon after the completion of Meiosis I or the beginning of Meiosis II); and 2) the “terminal commitment point”, a developmental stage in normal [diploid × diploid] pairs after which conjugation no longer responds to a parabiotically transmitted abort signal (this correlates with the onset of the second postzygotic nuclear division). Finally we demonstrate that a conjugal‐arrest‐activity varies with the vegetative cell cycle, reaching its highest level of activity during M‐phase and dropping just after cytokinesis.
The Nucleariid Amoebae: More Protists at the Animal‐Fungal Boundary
Authors: ZETTLER LINDA A. AMARAL , NERAD THOMAS A. , O'KELLY CHARLES J. , SOGIN MITCHELL L.
Published: July 2005

ABSTRACT. Nucleariid amoebae are naked amoebae, generally characterized by a spherical or sometimes flattened body with radiating filopodia. Most species preferentially consume algal prey or cyanobacteria. Phylogenetic analyses of the small‐subunit rRNA coding regions from four nucleariid amoebae place these species near the origin of the animal‐fungal divergence, together with the choanofiagellate‐Corallochytrium and the ichthyosporean clades. The species Nuclearia delicatula, N. moebiusi, and N. simplex form a monophyletic group, while ATCC 30864, tentatively but possibly incorrectly assigned to Nuclearia sp., represents a separate line of descent. These nucleariids are unrelated to the lineage containing the testate filose amoebae (Testaceafilosia). Our findings expand the morphological and phylogenetic diversity of protists at the animal‐fungal divergence.
Growth and Grazing Rates of the Heterotrophic Dinoflagellate Polykrikos kofoidii on Red‐Tide and Toxic Dinoflagellates
Authors: JEONG HAE JIN , KIM SOO KYEONG , KIM JAE SEONG , KIM SEONG TAEK , YOO YEONG DU , YOON JOO YIH
Published: July 2005

ABSTRACT. We investigated growth rates, grazing rates, and prey selection of Polykrikos kofoidii when feeding on several species of red‐tide and/or toxic dinoflagellates. Polykrikos kofoidii ingested all prey species used in this study, exhibiting positive growth on Lingulodinium polyedrum, Scrippsiella trochoidea, Ceratium furca, Gymnodinium catenatum, Gyrodinium impudicum, Prorocentrum micans, and the toxic dinoflagellate Amphidinium carterae, but not on P. minimum. Specific growth rates of P. kofoidii increased rapidly with increasing density of L. polyedrum, S. trochoidea, C. furca, and G. catenatum before saturating between 500–2,000 ng C ml 1. Specific growth rates increased continuously when P. kofoidii was fed the other prey species. Maximum specific growth rates of P. kofoidii on G. catenatum (1.12 d−1), S. trochoidea (0.97 d−1), and L. polyedrum (0.83 d−1) were higher than those on C. furca (0.35 d−1), A. carterae (0.10 d−1), P. micans (0.06 d−1), G. impudicum (0.06 d−1), and P. minimum (−0.03 d−1). Threshold prey concentrations (where net growth = 0) were 54–288 ng C ml−1. Maximum ingestion and clearance rates of P. kofoidii on these dinoflagellates were 5–24 ng C pseudocolony−1 d−1 and 1.0–5.9 μl pseudocolony−1 h−1, respectively. Polykrikos kofoidii strongly selected L. polyedrum over S. trochoidea in prey mixtures. Polykrikos kofoidii exhibited higher maximum growth, ingestion, and clearance rates than previously reported for the mixotrophic dinoflagellate Fragilidium cf. mexicanum or the heterotrophic dinoflageilates Protoperidinium cf. divergens and P. crassipes, when grown on the same prey species. Grazing coefficients calculated by combining field data on abundances of Polykrikos spp. and co‐occurring red‐tide dinoflagellate prey with laboratory data on ingestion rates obtained in the present study suggest that Polykrikos spp. sometimes have a considerable grazing impact on prey populations.
Lipid Class and Fatty Acid Composition of the Protozoan Parasite of Oysters, Perkinsus marinus Cultivated in Two Different Media
Authors: SOUDANT PHILIPPE , CHU FU‐LIN E.
Published: July 2005

ABSTRACT. The meront stage of the oyster protozoan parasite, Perkinsus marinus, cultivated in two media with different fatty acid profiles was analyzed for its fatty acid and lipid class composition. The composition of fatty acids in the prezoosporangium stage of the parasite as well as that of the host oyster were investigated. Although the lipid class composition of meronts was dominated by phospholipids and triacylglycerol, there was no triaclgycerol detected in either culture medium. Despite the difference in fatty acid composition of the two media, the fatty acid composition of meronts in each medium was dominated by 14:0, 16:0, 18:0, 18:1(n‐9), 20:1(n‐9), 18:2(n‐6) and 20:4(n‐6), a profile that differed from its host. The quantities of total lipids and fatty acids in meronts increased as the number of meronts increased and far exceeded the initial amounts in the media and in the initial cell inoculum. The meronts harvested 25 d post‐inoculation, had about 3 to 6 times higher total lipids and 4 to 13 times higher fatty acids than the amounts contained in the media. The fatty acid profiles of both prezoosporangia and oysters resembled each other and consisted primarily of 16:0, 20:4(n‐6), 20:5(n‐3), 22:2Δ7,15, and 22:6(n‐3). These results indicate that during meront proliferation, the parasite synthesizes certain fatty acids and lipid classes. For development from meront to prezoosporangium, the parasite may rely on its host for lipid resources.
The Interaction of Protozoa with their Potential Prey Bacteria in the Rhizosphere
Authors: JJEMBA PATRICK K.
Published: July 2005

ABSTRACT. Extensive colonization of the rhizosphere by beneficial bacteria is desirable but it is not yet clear whether the feeding patterns of protozoa exert any ecological impact on the competence of bacteria in the rhizosphere. To determine whether the successful colonization of the rhizosphere by bacteria can be explained by inherent abilities of the bacteria to resist predation, six bacteria categorized as poor, mediocre or good rhizosphere colonists were tested. The populations of protozoa in the rhizosphere consistently increased to a maximum density in two days but then declined to a stable level. Grazing by protozoa increased the doubling time of all of the test bacteria but did not significantly affect the final densities of bacteria in the rhizosphere. The differences in the colonization ability of the six bacteria tested were not attributable to inherent differences in their susceptibility to predation by protozoa.
Diplonemaspp. Possess Spliced Leader RNA Genes Similar to the Kinetoplastida
Authors: STURM NANCY R. , MASLOV DMITRI A. , GRISARD EDMUNDO C. , CAMPBELL DAVID A.
Published: July 2005

ABSTRACT.The phylogenetic placement of the genusDiplonemain relation to fellow phylum membersEuglenaandTrypanosomahas been uncertain. The spliced leader RNA gene, present in the euglenids and kinetoplastids in distinct forms, was a potential target for resolving this question. The first indication supporting a closer relationship to the kinetoplastids was the recognition of potential spliced leader RNA exon sequences in the genomic DNA of twoDiplonemaisolates. Examination of total cell RNA revealed transcripts in the anticipated size range at approximately 120 and 130 nt. Specific PCR amplification of a spliced leader RNA gene repeat was performed. The hallmark features of the kinetoplastid‐type spliced leader RNA, specifically the 39‐nt exon, splice‐donor site, Sm‐binding site and poly‐T tract and the potential to form the requisite stem‐loop structures, were found.Diplonemaspp. are different from the kinetoplastids by virtue of C residues at positions 4 and 18 in the exon. While the intergenic spacer regions varied in size, each contained the complete sequence or remnants of a 5S ribosomal RNA gene. Possession of a functional spliced leader RNA gene of the kinetoplastid variety inDiplonemasupports a closer evolutionary relationship with this group than with the euglenids.
The I‐antigens of Ichthyophthirius multifiliis are GPI‐Anchored Proteins
Authors: CLARK THEODORE G. , GAO YAN , GAERTIG JACEK , WANG XUTING , CHENG GEORGINA
Published: July 2005

ABSTRACT. The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i‐antigens) that when clustered, trigger rapid, premature exit from the host. Similar antigens are present in free‐living ciliates and are GPI‐anchored in both Paramecium and Tetrahymena. Although transmembrane signalling through GPI‐anchored proteins has been well‐documented in metazoan cells, comparable phenomena have yet to be described in protists. Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i‐antigens are GPI‐anchored in these cells as well. Based on their solubility properties in Triton X‐114, the i‐antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase. Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic. Concomitantly, they are recognized by antibodies against a cross‐reacting determinant exposed on virtually all GPI‐anchored proteins following cleavage with phospholipase C. Finally, when expressed in recombinant form in Tetrahymena thermophila, full‐length i‐antigens are restricted to the membrane, while those lacking hydrophobic C‐termini are secreted from the cell. Taken together, these observations argue strongly that the i‐antigens of Ichthyophthirius multifiliis are, in fact, GPI‐anchored proteins.
Morphology and Infraciliature of a Marine Scuticociliate with a Polymorphic Life Cycle: Urocryptum tortum n. gen., n. comb.
Authors: PÉREZ‐UZ BLANCA , GUINEA ALMUDENA
Published: July 2005

ABSTRACT. A new genus and species combination are proposed for Urocryptum tortum n. gen., n. comb., a scuticociliate with a polymorphic life cycle. This marine ciliate was isolated from a sample taken at Gokasho Bay in Mie Prefecture (Japan). Specimens from different phases of the growth cycle were examined in vivo and with two silver staining techniques. Three life‐history stages were observed: an exponential growth phase stage (trophont), a stationary phase stage (tomite), and finally a resting stage (cyst). The exponential growth form is laterally flattened and ovoid; it has 20–24 somatic kineties (SK) and a typical complement of scuticociliate oral structures. Polykinetid 1 (Pk1) has two longitudinal files of 6 kinetosomes (Ks); sometimes one or two additional kinetosomes are located anteriorly. Polykinetid 2 (Pk2) has two files of 6 or 7 Ks; a third file of three widely spaced kinetosomes is located on its right side, as well as a small curved row of 5 Ks positioned on the right hand side of the posterior end of Pk2. Polykinetid 3 (Pk3) has 3 rows of 4, 5, and 7 Ks's perpendicular to the haplokinety (Hk) or paroral membrane. The stationary growth phase cell is spindle‐shaped and has a similar number of SK that are much more closely spaced than in exponentially growing specimens. Oral infraciliary structures are reduced in size, having fewer kinetosomes and being positioned more anteriorly in the cell. Pk1 is composed of two files of 5–6 Ks, Pk2 has only two files of 6–7 Ks, and Pk3 has two to three rows of 3–4 Ks. The Hk is displaced anteriorly and becomes straight, losing the shape typical of exponential growth phase cells. Cysts are pyriform with a wide cyst wall; no infraciliary structures were visible.
Ultrastructural Characteristics of the In Vitro Cell Cycle of the Protozoan Pathogen of Oysters, Perkinsus marinus
Authors: SUNILA INKE , HAMILTON ROSALEE M. , DUNCAN CHRISTOPHER F.
Published: July 2005

ABSTRACT. Ultrastructural characteristics of vegetative and zoosporangial stages of cultured Perkinsus marinus, a pathogen of the eastern oyster, Crassostrea virginica, were examined by transmission electron microscopy. An axenic cell culture was propagated from infected Chesapeake Bay oyster hemolymph. Different stages of the in vitro cell cycle, including schizonts and different size trophonts, were examined. Trophonts had spherical nuclei with wide perinuclear spaces, mitochondria with tubular cristae, and vacuoles with vacuoplasts. There were micropores on the inside of cell walls. A tubular network in the cytoplasm connected lomasomes to vacuoles, and contained vacuoplast precursor material. Vacuoplasts and precursor material diminished when cell cultures were not fed, suggesting a function in metabolite storage. Cells divided by schizogony or binary fission. Daughter cells in a schizont were not alike, and may specialize for different functions. Some of the daughter cells in a schizont died. Some hypnospores, directly isolated from infected oyster hemolymph enlarged in Ray's fluid thioglycollate medium, and were induced to zoosporulate. Zoosporangia contained varicose, hypha‐like structures, whose apical tips gave rise to prezoospores. Ultrastructural characteristics of the vegetative and zoosporangial stages did not resemble any apicomplexan parasites other than members of the genus Perkinsus.
Molecular Identification of Algal Endosymbionts in Large Miliolid Foraminifera: 1. Chlorophytes
Authors: PAWLOWSKI JAN , HOLZMANN MARIA , FAHRNI JOSÉ F. , HALLOCK PAMELA
Published: July 2005

ABSTRACT. Large miliolid foraminifers bear various types of algal endosymbionts including chlorophytes, dinoflagellates, rhodophytes, and diatoms. Symbiosis plays a key role in the adaptation of large foraminifera to survival and growth in oligotrophic seas. The identity and diversity of foraminiferal symbionts, however, remain largely unknown. In the present work we use ribosomal DNA (rDNA) sequences to identify chlorophyte endosymbionts in large miliolid foraminifera of the superfamily Soritacea. Partial 18S and complete Internal Transcribed Spacer (ITS) rDNA sequences were obtained from symbionts of eight species representing all genera of extant chlorophyte‐bearing Soritacea. Phylogenetic analysis of the sequences confirms the previous fine structure‐based identification of these endosymbionts as belonging to the genus Chlamydomonas. All foraminiferal symbionts form a monophyletic group closely related to Chlamydomonas noctigama. The group is composed of seven types identified in this study, including one previously morphologically described species, Chlamydomonas hedleyi. Each of these types can be considered as a separate species, based on the comparison of genetic differences observed between other established Chlamydomonas species. Several foraminiferal species share the same symbiont type, but only one species, Archaias angulatus, was found to bear more than one type.
Molecular Identification of Algal Endosymbionts in Large Miliolid Foraminifera: 2. Dinoflagellates
Authors: PAWLOWSKI JAN , HOLZMANN MARIA , FAHRNI JOSÉ F. , POCHON XAVIER , LEE JOHN J.
Published: July 2005

ABSTRACT.Large miliolid foraminifers of the subfamily Soritinae bear symbiotic dinoflagellates morphologically similar to the species of the “Symbiodinium” complex, commonly found in corals and other marine invertebrates. Soritid foraminifers are abundant in coral reefs and it has been proposed that they share their symbionts with other dinoflagellate‐bearing reef dwellers. In order to test this hypothesis, we have analysed partial large subunit ribosomal DNA sequences from dinoflagellates symbionts obtained from 28 foraminiferal specimens, and compared them to the corresponding sequences ofSymbiodinium‐like endosymbionts from various groups of invertebrates. Phylogenetic analysis of our data shows that all soritid symbionts belong to the “Symbiodinium” species complex, within which they form seven different molecular types (Fr1–Fr7). Only one of these types (Fr1) branches within a group of invertebrate symbionts, previously described as type C. The remaining six types form sister groups to coral symbionts previously designed as types B, C, and D. Our data indicate a high genetic diversity and specificity ofSymbiodinium‐like symbionts in soritids. Except for type C, we have found no evidence for the transmission of symbionts between foraminifers and other symbiont‐bearing invertebrates from the same localities. However, exchanges must have occurred frequently between the different species of Soritinae, as suggested by the lack of host specificity and some biogeographical patterns observed in symbiont distribution. Our data suggest that members of the subfamily Soritinae acquired their symbionts at least three times during their history, each acquisition being followed by a rapid diversification and independent radiation of symbionts within the foraminiferal hosts.
Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi
Authors: BACCHI CYRUS J. , LANE SCHENNELLA , WEISS LOUIS M. , YARLETT NIGEL , TAKVORIAN PETER , WITTNER MURRAY
Published: July 2005

ABSTRACT. Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK‐13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051–1.059/ g/ml in a microgradient procedure, and 1.102–1.119/g/ml in a scaled‐up procedure were nearly homogenous, consisting of pre‐emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre‐emergent spores incubated with [3H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL‐α‐difluoromethylornithine (DFMO) but not by DL‐α‐difluoro‐methylarginine (DFMA). Cell‐free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S‐adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1‐acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine‐stimulated and inhibited by methylglyoxal‐bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre‐emergent spores have a eukaryotic‐type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre‐emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture super‐natants had little metabolic activity.
Searching for Excystment‐Regulated Genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): A mRNA Differential Display Analysis of Gene Expression in Excysting Cells
Authors: VILLALOBO EDUARDO , MOCH CLARA , PERASSO ROLAND , BAROIN‐TOURANCHEAU ANNE
Published: July 2005

ABSTRACT. In the absence of food, the oxytrichid Sterkiella histriomuscorum transforms like many ciliates into resting cysts. When transferred back into feeding medium, the cyst re‐transforms into a vegetative cell. The entry into and exit from the dormant cyst stage are complex developmental processes still poorly investigated at the molecular level. Assuming that these changes in state could involve changes in gene expression, we have used the technique of mRNA differential display to detect differentially expressed genes in cysts and two different stages of excysting cell. Variation in the temporal expression pattern of transcripts could be detected and, in using an inverse‐PCR strategy on circularized macronuclear DNA, we have sequenced the macronuclear genes of three of the isolated cDNAs, which correspond to 1) a nucleotide‐binding domain‐encoding gene, 2) a DHHC‐domain‐carrying gene, and 3) a phosphatase type 2C‐ encoding gene. For the first two genes, Northern blot analyses supported an excystment‐associated regulated gene expression. We discuss their possible role during excystment and we show that the combination of differential display and inverse PCR constitutes a powerful approach to isolate excystment‐regulated genes in hypotrichs.
Behind the Discovery of “Nissenbaum's Fixative”
Authors: NISSENBAUM GERALD
Published: July 2005

ABSTRACT. The author describes the serendipitous discovery, conception, development, and history of Nissenbaum's Fixative while an undergraduate biology major in the early 1950s. The subsequent uses, applications, and modifications over the past forty‐seven years are also described. Some of the modifications omitted from his short original paper are mentioned. Highlights of his subsequent career in the field of medicine are noted.
Encystation in Acanthamoeba castellanii: Development of Biocide Resistance1
Authors: LLOYD D. , TURNER N. A. , KHUNKITTI W. , HANN A. C. , FURR J. R. , RUSSELL A. D.
Published: July 2005

ABSTRACT. Since the early 1960s, axenic culture and the development of procedures for the induction of encystation have made Acanthamoeba spp. superb experimental systems for studies of cell biology and differentiation. More recently, since their roles as human pathogens causing keratitis and encephalitis have become widely recognized, it has become urgent to understand the parameters that determine differentiation, as cysts are much more resistant to biocides than are the trophozoites.

Viability of trophozoites of the soil amoeba Acanthamoeba castellanii (Neff), is conveniently measured by its ability to form plaques on a lawn of Escherichia coli. Use of confocal laser scanning microscopy with Calcofluor white, Congo Red or the anionic oxonol dye, DiBAC4(3) or flow cytometry with propidium iodide diacetate and fluorescein or oxonol provides more rapid assessment. For cysts, the plaque method is still the best, because dye exclusion does not necessarily indicate viability and therefore the plate count method has been used to study the sequence of development of biocide resistance during the differentiation process. After two hours, resistance to HC1 was apparent. Polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromo‐ propamine isethionate, and H2O2 and moist heat, all lost effectiveness at between 14 and 24 h after trophozoites were inoculated into encystation media. Chlorhexidine diacetate resistance was observed at between 24 and 36 h. The molecular biology and biochemistry of the modifications that underlie these changes are now being investigated.
A Role for a Galactose Lectin and Its Ligands During Encystment of Entamoeba1
Authors: EICHINGER DANIEL
Published: July 2005

ABSTRACT. In the life cycle of Entamoeba parasites alternate between the colon‐dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human‐infecting Entamoeba, parasites express a galactose/N‐acetylgalactosamine (gal/galNAc)‐binding lectin, which plays demonstrated roles in contact‐ dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal‐terminated ligands in the induction medium. Precise concentration ranges of several gal‐terminated ligands, such as asialofetuin, gal‐bovine serum albumin (gal‐BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up‐regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C‐terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment.
Regulation of Carbohydrate Metabolism During Giardia Encystment1
Authors: JARROLL EDWARD L. , MACECHKO P. TIMOTHY , STEIMLE PAUL A. , BULIK DOROTA , KARR CRAIG D. , van KEULEN HARRY , PAGET TIMOTHY A. , GERWIG GERRIT , KAMERLING JOHANNES , VLIEGENTHART JOHANNES , ERLANDSEN STANLEY
Published: July 2005

ABSTRACT. Giardia intestinalis trophozoites encyst when they are exposed to bile. During encystment, events related to the inducible synthesis of a novel N‐acetyl‐d‐galactosamine (GalNAc) homopolymer, occur. Within the first 6 h of encystment, mRNA for glucosamine 6‐P isomerase (GPI), the first inducible enzyme unique to this pathway appears, oxygen uptake rates double from non‐encysting levels, and metronidazole (MTZ) inhibits oxygen uptake. Within 12 h, GPI and its activity are detectable and OU decreases 50% from non‐encysting levels; glucose's stimulation and MTZ's inhibition of oxygen uptake cease. In contrast, aspartate uptake remained constant throughout the 40 h monitored. Two genes, gpi 1 and 2 encode for GPI, but only gpi l is expressed during encystment. Glucosamine 6‐P (GlcN6P), the synthetic product of GPI, activates UDP‐N‐acetylglucosamine (UDP‐GlcNAc) pyrophosphorylase, a downstream enzyme, 3 to 5‐fold in the direction of UDP‐GlcNAc synthesis. UDP‐GlcNAc is epimerized to UDP‐GalNAc and UDP‐GalNAc is polymerized by “cyst wall synthase” (β 1 → 3 GalNAc transferase) into a highly insoluble β 1,3‐linked homopolymer. This GalNAc polysaccharide, the major component of cyst wall filaments, forms, in conjunction with polypeptides, the outer cyst wall of Giardia.
Phosphatidylinositol‐Specific Phospholipase C (PI‐PLC) Cleavage of GPI‐Anchored Surface Molecules of Trypanosoma cruzi Triggers In Vitro Morphological Reorganization of Trypomastigotes
Authors: MORTARA RENATO A. , MINELLI LINDAMAR M. S. , VANDEKERCKHOVE FILIP , NUSSENZWEIG VICTOR , RAMALHO‐PINTO F. JUAREZ
Published: July 2005

ABSTRACT. Trypanosoma cruzi trypomastigotes treated with phosphatidylinositol‐specific phospholipase C (PI‐PLC) in vitro are rapidly induced to differentiate into round forms. Using confocal microscopy, we were able to show that trypomastigotes treated with PI‐PLC initiate the process of flagellum remodeling by 30 sec after contact with the enzyme and amastigote‐like forms are detected as early as 10 min after PI‐PLC treatment. Scanning and transmission electron microscopy indicate that trypomastigotes undergo a previously undescribed process of flagellum circularization and internalization. Analysis of the flagellar complex with monoclonal antibody 4D9 shows heterogeneous labeling among the parasites, suggesting a remodeling of these molecules. After PI‐PLC treatment, parasites rapidly lose the surface marker Ssp‐3 and 24 h post‐treatment they begin to exhibit a circular nucleus and a rod‐shaped kinetoplast. By flow cytometry analysis and confocal microscopy, the Ssp‐4 amastigote‐specific epitope can be detected on the parasite surface. This indicates that the release of trypomastigote GPI‐anchored molecules by exogenous PI‐PLC in vitro can trigger morphological changes.
Fine Structure of Clonally Propagated In Vitro Life Stages of a Perkinsus sp. Isolated from the Baltic Clam Macoma balthica
Authors: COSS CATHLEEN A. , ROBLEDO JOSÉ A. F. , VASTA GERARDO R.
Published: July 2005

ABSTRACT. We established monoclonal in vitro cultures of a Perkinsus sp. isolated from the baltic clam Macoma balthica and compared morphological features of various life stages by light and transmission electron microscopy to those of the currently accepted Perkinsus species: Perkinsus marinus, Perkinsus olseni, Perkinsus atlanticus, and Perkinsus qugwadi. Except that trophozoites were slightly larger than those of P. marinus, and that they underwent zoosporulation in culture, observation of our isolate under light microscopy did not reveal striking differences from any Perkinsus species. Perkinsus sp. from M. balthica shared fine structural characteristics with other Perkinsus species that clearly place it within this genus. Although zoospores of Perkinsus sp. from M. balthica were slightly smaller than those from other species, the ultrastructural arrangement and appearance of the apical complex and flagella seem to be identical to those of P. marinus and P. atlanticus. Our isolate also appeared, in some sections, to have cortical alveolar expansions of the plasmalemma at regions other than the anterior end and lobulated mitochondria that were reported as unique for P. qugwadi. Little consensus exists among authors in the assignment of taxonomic weight to any particular morphological feature to designate Perkinsus species. The present study of gross morphology and ultrastructure was complemented with molecular studies reported elsewhere, which propose that Perkinsus sp. from Macoma balthica is a distinct species.
Description of Perkinsus andrewsi n. sp. Isolated from the Baltic Clam (Macoma balthica) by Characterization of the Ribosomal RNA Locus, and Development of a Species‐Specific PCR‐Based Diagnostic Assay
Authors: COSS CATHLEEN A. , ROBLEDO JOSÉ A. F. , RUIZ GREGORY M. , VASTA GERARDO R.
Published: July 2005

ABSTRACT. A Perkinsus species was isolated from the baltic clam Macoma balthica and an in vitro culture established under conditions described for P. marinus. As reported previously, morphological features remarkable enough to clearly indicate that this isolate is a distinct Perkinsus species were lacking. In this study, regions of the rRNA locus (NTS, 18S. ITS1, 5.8S. and 1TS2) of this isolate were cloned, sequenccd, and compared by alignment with those available for other Perkinsus species and isolates. Sequence data from the rRNA locus and species‐specific PCR assays indicated not only that Perkinsus sp. from M. balthica was not P. marinus, but it was different from P. atlanlicus and P. olseni. The degree of difference was comparable to or greater than differences between accepted Perkinsus species. In particular, NTS sequence and length were dramatically different from that of P. marinus and P. atlanlicus. Therefore, we formally propose to designate the Perkinsus sp. from M. balthica as a separate species, P. andrewsi n. sp. Primers based on P. andrewsi NTS sequence were used to develop a PCR‐based diagnostic assay that was validated for species‐specificity and sensitivity. PCR‐based assays specific for cither P. andrewsi or P. marinus were used to test for their presence in bivalve species sympatric to M. balthica. Although isolated from M. balthica, P. andrewsi was also detected in the oyster Crassostrea virginica and clams Macoma mitchelli and Mercenaria mercenaria, and could coexist with P. marinus in all four bivalve species tested.
Morphological, Biochemical and Molecular Characterization of Herpetomonas samuelpessoai camargoi n. subsp., a Trypanosomatid Isolated from the Flower of the Squash Cucurbita moschata
Authors: FIORINI JOAO E. , TAKATA CARMEN S. A. , TEOFILO VIRGINIA M. , NASCIMENTO LUIZ C. , FARIA‐E‐SILVA PAULO M. , SOARES MAURILIO J. , TEIXEIRA MARTA M. G. , DE SOUZA WANDERLEY
Published: July 2005

ABSTRACT. We report the morphological, biochemical and molecular characteristics of a trypanosomatid isolated from the flower of Cucurbita moschata. Although the trypanosomatid was isolated from a plant, the lack of recognition of Phytomonas‐spccific molecular markers based on spliced‐leader and ribosomal genes as well as by monoclonal antibodies specific for Phytomonas argues against assigning it to this genus. Because the isolate displayed typical opisthomastigote forms in culture, it is assigned to the genus Herpetomonas. Analysis of randomly amplified polymorphic DNA (RAPD) patterns and characterization of ribosomal SSU and ITS markers suggest that it is more closely related to H. samuelpessoai than to any other species. However, the presence of spined flagellates in culture (displaying lateral expansions of the plasma membrane originating near the flagellar pocket) and isolate‐specific RAPD fingerprints argue strongly that the trypanosomatid belongs to a new subspecies, for which the name Herpetomonas samuelpessoai camargoi n. subsp. is proposed.
Divisional Morphogenesis in Uroleptus caudatus (Stokes, 1886), and the Relationship Between the Urostylidae and the Parakahliellidae, Oxytrichidae, and Orthoamphisiellidae on the Basis of Morphogenetic Processes (Ciliophora, Hypotrichida)
Authors: EIGNER PETER
Published: July 2005

ABSTRACT. Morphogenetic processes during division in Uroleptus caudatus (Stokes, 1886) arc described using protargol impregnation. As is typical for the family Urostylidae Bütschli, 1889, zigzag midventral cirri develop, but contrary to most species of this family the adoral membranelles of the proter are not renewed during division and two dorsomarginal kincties develop. These two atypical features occur in only two other species of the Urostylidae (Uroleptus musculus, Holosticha diademata), and in all species of the family Parakahliellidae Eigner, 1997, and in many of the Oxytrichidae Ehrenberg, 1838. Membranelles arc also not renewed in the Orthoamphisiellidae Eigner, 1997. This shows that these three atypical members of the family Urostylidae are those which are most closely related to the other three hypotrichous families named above. The highly distinct zigzag midventral cirri clearly separate the family Urostylidae from the other three hypotrichous families. These cirri are used together with new morphogenctic features for a revised family diagnosis. The analysis of the Urostylidae revealed that during division all species of the family Urostylidae develop their cirral patterns, including the two rightmost ventral anlagen, in separate areas for each proter and opisthe. Thus, “long primary primordia” are absent in the Urostylidae. This is similar to the morphogenetic pattern by which the family Parakahliellidae is defined (“neokinetal 1”) indicating that the Urostylidae are more closely related to the Parakahliellidae than to either the Oxytrichidae or to the Orthoamphisiellidae. All 68 detailed descriptions of divisional morphogenesis in species of the Hypotrichida are analyzed now in this and two former papers. As a result all these species can be assigned or at least recognized to be closely related to one of the following four families: to the Orthoamphisiellidae (“within‐row” anlagen), to the Oxytrichidae (“neokinetal 3” anlagen), to the Parakahliellidae (“neokinetal 1” anlagen), and to the distinctly different Urostylidae (“midventral” anlagen). Thus, all Hypotrichida can most probably be assigned after morphogenctic investigation to one of the four families.
Analysis of Micronuclear, Macronuclear and cDNA Sequences Encoding the Regulatory Subunit of cAMP‐Dependent Protein Kinase of Euplotes octocarinatus: Evidence for a Ribosomal Frameshift
Authors: TAN MING , HECKMANN KLAUS , BRÜNEN‐NIEWELER CLAUDIA
Published: July 2005

ABSTRACT. We have isolated and characterized the micronuclear gene encoding the regulatory subunit of cAMP‐dependent protein kinase of the ciliated protozoan Euplotes octocarinatus, as well as its macronuclear version and the corresponding cDNA. Analyses of the sequences revealed that the micronuclear gene contains one small 69‐bp internal eliminated sequence (IES) that is removed during macronuclear development. The IES is located in the 5′‐noncoding region of the micronuclear gene and is flanked by a pair of tetranucleotide 5′‐TACA‐3’direct repeats. The macronuclear DNA molecule carrying this gene is approximately 1400 bp long and is amplified to about 2000 copies per macronucleus. Sequence analysis suggests that the expression of this gene requires a + 1 ribosomal frameshift. The deduced protein shares 31% identity with the cAMP‐dependent protein kinase type I regulatory subunit of Homo sapiens, and 53% identity with the regulatory subunit R44 of one of the two cAMP‐dependent protein kinases of Paramecium. In addition, it contains two highly conserved cAMP binding sites in the C‐terminal domain. The putative autophosphorylation site ARTSV of the regulatory subunit of E. octocarinatus is similar to that of the regulatory subunit R44 of Paramecium but distinct from the consensus motif RRXSZ of other eukaryotic regulatory subunits of cAMP‐dependent protein kinases.
Trichites of Strombidium (Ciliophora, Oligotrichida) Are Extrusomes
Authors: MODEO LETIZIA , PETRONI GIULIO , BONALDI MICHELA , ROSATI GIOVANNA
Published: July 2005

ABSTRACT. The trichites of Strombidium and related genera have been considered either as a cytoskeletal armature or as extrusomes. To demonstrate their true nature, a study was undertaken on two marine Strombidium species by ultrastructurai and cytocheinical analysis as well as in vivo experiments. Trichites, extending from the cortex into the cell, are rod‐shaped, membrane‐bounded, and have a complex structure. The following elements of the trichites, are distinguishable: an electron‐transparent lumen, a laminated layer, and a compact layer. In trichites of one species, thin “rings” surround the lumen. Numerous short, curved tubules with a polysaccharide wall are present in the cytoplasm surrounding the trichites. At the cortical end, each trichite is enveloped by a “cap” of electron‐dense proteinaceous material. In some cases, the cortical alveoli appear interrupted, forming a “hole” for trichite ejection. Ejection of rod‐shaped structures, up to 5 times longer than resting trichites, was obtained by in vivo treatments with dextran and aminoethyldextran. Negative staining indicated that these structures were transformed trichites. As no other possible extrusive structures were observed in the cytoplasm of Strombidium, trichites were considered extrusomes.