bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipase D (PLD) is Present in Leishmania donovani and its Activity Increases in Response to Acute Osmotic Stress
Authors: BLUM J. JOSEPH , LEHMAN JASON A. , HORN JEFF M. , GOMEZ‐CAMBRONERO JULIAN
Published: July 2005

ABSTRACT. We report here that the signaling molecule phospholipase D (PLD) is present in the parasitic protozoan Leishmania donovani. In vitro enzymatic activity is dependent on Ca2+ and Mg2+ ions, its basal activity is stimulated by phosphatidyl‐inositol‐4.5‐bisphosphate (PIP2), and its pH optima are pH 8.0 and pH 6.0. PLD activity increases 3‐fold about 5 min after an abrupt decrease in osmolality from 317 mOsm (isosmotic) to 155 mOsm and increases 1.5‐fold in response to an abrupt increase in osmolality to 617 mOsM. Cells grown for > 24 h under the anisosmotic conditions showed only marginal changes in activity compared to the controls grown under isosmotic conditions, indicating an adaptation to long‐term exposure to hypo‐ or hyper‐osmolarity. Immunologically. two isofonns, PLD1 and PLD2, are present. An analysis of in vitro PLD activity in anti‐PLD immunocomplexes revealed that either hypotonic (cell swelling) or hypertonic stress (cell shrinking) causes an increase in PLD1 activation but a reduction in PLD2 activity. The interplay between these two isoforms results in a predominance for PLD1 in the observed increase when measuring total PLD activity. Finally, the increase in enzymatic activity in acute hyposmotic shock is accompanied by tyrosyl phosphorylation of the PLD1 isotorm. suggesting a role for protein tyrosine kinase in the control of PLD activity in response to osmotic stress.
Gametogenesis, Fertilization and Ookinete Differentiation of Leucocytozoon smithi
Authors: STEELE EDNA JOSEF , NOBLET GAYLE PITTMAN
Published: July 2005

ABSTRACT. Development of Leucocytozoon smithi during gametogenesis, fertilization, and ookinete differentiation was studied by light and electron microscopy. Gametogenesis occurred rapidly, within 1–2 min after gametocytes were ingested by black flies. Usually one axoneme, but not infrequently two, was observed in microgametes. The macrogamete nucleus was characteristically elongated and fragmented, with a convoluted nuclear envelope. Fertilization occurred within five min after ingestion of gametocytes by the vector. The entire axoneme and nucleus of the microgamete entered the cytoplasm of the macrogamete. Zygote differentiation resembled sporozoite formation in that a thickened inner membrane and subpellicular microtubules developed beneath the plasmalemma, followed by cytoplasmic protrusion or evagination to form the anterior end. Extension of the inner thickened membrane continued as the xygote elongated. Development of sausage‐shaped ookinetes was completed within 6–8 h after ingestion of a blood meal by a black fly. Mature ookinetes possessed a single nucleus, double‐layered pellicle, canopy, apical pore, polar ring complex, subpellicular microtubules, micronemes, crystalloids, abundant mitochondria, endoplasmic reticulum, and ribosomes. Comparison of development of L. smithi with species of Plasmodium and Haemoproteus revealed general similarities in both sexual and asexual development within the insect vector. A diagram summarizing life cycle events for L. smithi is included.
A Multilocus Genotypic Analysis of Cryptosporidium meleagridis
Authors: GLABERMAN SCOTT , SULAIMAN IRSHAD M. , BERN CARYN , LEVIOR JOSEF , PENG MICHAEL M. , MORGAN UNA , GILMAN ROBERT , LAL ALTAF A. , XIAO LIHUA
Published: July 2005

ABSTRACT. Cryptosporidium meleagridis is a common cause of cryptosporidiosis in birds. In addition, recent reports have described the parasite as an etiologic agent of cryptosporidiosis in both immunocompetent and immunocompromised humans. Therefore, it is important to genetically characterize isolates of C. meleagridis from different hosts and geographic areas, and to develop molecular tools to differentiate isolates from various hosts or areas. In this study, a total of 11 isolates of Cryptosporidium meleagridis from both human and avian hosts were examined at three genetic loci: the small‐subunit rRNA, 60‐kDa glycoprotein precursor, and 70‐kDa heat shock protein genes. Two genotypes of C. meleagridis were seen at the small‐subunit rRNA locus. These differed from each other by the presence or lack of a heterogeneous copy of the gene and an ATT repeat. The 60‐kDa glycoprotein precursor gene divided these eleven isolates of C. meleagridis into six genotypes with high sequence diversity between groups. The highest genetic heterogeneity, however, was seen at the 70‐kDa heat shock protein locus, and was primarily present at the 3′end of the gene. This heterogeneity separated eight isolates of C. meleagridis into six genotypes. These data could be useful in the development of molecular tools to promote understanding of the transmission of C. meleagridisiin humans.
Immunohistochemistry Based Assay to Determine the Effects of Treatments on Cryptosporidium parvunt Viability
Authors: PHELPS KALMIA K. , LINDSAY DAVID S. , SUMNER SUSAN S. , PAYER RON
Published: July 2005

Cell culture infectivity assays can provide an accurate means of detecting viable Cryptosporidium parvum oocysts from environmental samples or to test the effects of various treatments on oocyst infectivity. Cell culture assays can also be used to test candidate chemotherapeutic agents. The use of a human cell line provides a situation close to human infection. The present assay uses an anti‐Cryptospordium primary antibody, combined with a biotinylated secondary antibody, and an immunoperoxidase detection system. Cryptosporidium parvum oocysts excysted in vitro when placed on monolayers of HCT‐8 cells and developmental stages including schizonts and merozoites were visualized using light microscopy of the immunoperoxidase stained slides and by transmission electron microscopy of infected HCT‐8 cell cultures. Because the immunoperoxidase system used gives a permanent preparation, the cell cultures can be retained and examined later. Dose titration of oocysts indicated that as few as 50 inoculated oocysts could be detected. The activity of paromomycin was evaluated in this system and 500 μg/ml produced a 97.8% reduction in infection.
Inter‐Strain Variability of Insertion/Deletion Events in the Encephalitozoon cuniculi Genome: A Comparative KARD‐PFGE Analysis
Authors: BRUGERE JEAN‐FRANCOIS , CORNILLOT EMMANUEL , BOURBON THIERRY , METENIER GUY , VIVARÈS CHRISTIAN P.
Published: July 2005

ABSTRACT. We applied a two‐dimensional pulsed‐field gel electrophoresis procedure to the genomes of two karyotype variants assigned to two different strains of the microsporidian Encephalitozoon cuniculi, termed D (strain III) and F (strain II). Data obtained for BssHII and Mlul restriction fragment length polymorphisms in each chromosome are compiled and compared to the reference strain I variant A. Six Insertion/Deletion (InDels) are found in subterminal position, some of these being characteristic of either D or F. Like in strain I, the terminal fragments extending between each telomere and rDNA locus are conserved in length for each chromosome. They are however smaller than in reference variant. This size reduction is estimated to be 2.5 kbp for the strain III isolate and 3.5 kbp for the strain II isolate. We hypothesize that for the three E. cuniculi strains, all chromosome extremities are prone to a constant process of sequence homogenization through mitolic recombination between conserved regions.
The Identification of rRNA Maturation Sites in the Microsporidian Encephalitozoon cuniculi Argues Against the Full Excision of Presumed ITS1 Sequence
Authors: PEYRETAILLADE ERIC , PEYRET PIERRE , METENIER GUY , VTVARES CHRISTIAN P. , PRENSIER GERARD
Published: July 2005

ABSTRACT. In Encephalitozoon cuniculi like in other microsporidia, the primary transcript for SSU and LSU rRNAs includes only one internal transcribed spacer (ITS1) which separates SSU rRNA from the 5.8S region associated with LSU rRNA. The extraction of total RNA from E. cuniculi‐infected MRC5 cells using a hot phenol/chloroform procedure enabled us to perform primer extension and S1 nuclease protection experiments in the aim of identifying rRNA maturation sites. Our data support a simple processing (four cleavage sites) with elimination of only nine nucleotides between SSU and LSU rRNA regions. Most of the presumed ITS1 sequence characterized by strain‐dependent polymorphism therefore remains linked to SSU rRNA 3′ end. A new secondary structure for the sixth domain of E. cuniculi LSU rRNA is proposed following the identification of its 3′terminus.
Parallel Phylogenies of Pneumocystis Species and their Mammalian Hosts
Authors: GUILLOT JACQUES , DEMANCHE CHRISTINE , HUGOT JEAN PIERRE , BERTHELEMY MADELEINE , WAKEFIELD ANN E. , DEI‐CAS EDUARDO , CHERMETTE RENE
Published: July 2005

ABSTRACT. The single name Pneumocystis carinii consists of an heterogeneous group of specific fungal organisms that colonize a very wide range of mammalian hosts. In the present study, mitochondrial large subunit (mtLSU) and small subunit (mtSSU) rRNA sequences of P. carinii organisms from 24 different mammalian species were compared. The mammals were included in six major groups: Primates (12 species). Rodents (5 species). Carnivores (3 species). Bats (1 species), Lagomorphs (1 species), Marsupials (1 species) and Ungulates (1 species). Direct sequencing of PCR products demonstrated that specific mtSSU and mtLSU rRNA Pneumocystis sequence could be attributed to each mammalian species. No animal harbored P. carinii f. sp. hominis. Comparison of combined mtLSU and mtSSU aligned sequences confirmed cospeciation of P. carinii and corresponding mammalian hosts. P. carinii organisms isolated from mammals of the same zoological group systematically clustered together. Within each cluster, the genetic divergence between P. carinii organisms varied in terms of the phylogenetic divergence existing among the corresponding host species. However, the relative position of P. carinii groups (rodent, carnivore or primate‐derived P. carinii) could not be clearly determined. Further resolution will require the integration of additional sequence data.
Removal of Toxoplasma gondii Oocysts from Sea Water by Eastern Oysters (Crassostrea virginica)
Authors: LINDSAY DAVID S. , PHELPS KALMIA K. , SMITH STEPHEN A. , FLICK GEORGE , SUMNER SUSAN S. , DUBEY J. P.
Published: July 2005

SUMMARY. Toxoplasma gondii infections have been reported in a number of marine mammals. Presently it is not known how these animals acquire T. gondii from their aquatic environment. The eastern oyster, Crassostrea virginica, has been shown to remove Cryptosporidium oocysts from seawater and a similar phenomenon may be occurring with T. gondii oocysts and marine invertebrates. The present study was done to determine if eastern oysters could remove and retain T. gondii oocysts from seawater. Oocysts of the VEG strain of T. gondii (1 × 106 oocysts) were placed in seawater (32 ppt NaCl) containing live eastern oysters. The infected seawater was removed one day postinoculation (PI) and replaced with fresh seawater. Selected oysters were removed at 1, 3 and 6 days PL Hemolymph, gill washes, and oyster tissue were collected separately at each observation time. The oyster tissue was homogenized and all 3 samples fed separately to mice. Toxoplasma gondii positive mice were observed at each time period. The results indicate that T. gondii oocysts can be removed from seawater by eastern oysters and retain their infectivity. Contaminated raw oysters may serve as a source of T. gondii infection for marine mammals and humans.
Is Pneumocystis a Plant?
Authors: KANESHIRO EDNA S.
Published: July 2005

ABSTRACT. Pneumocystis, an AIDS‐associated opportunistic pathogen of the lung has some unusual features. This article focuses on work done by my group to understand the organism's distinct sterols. Although Pneumocystis is closely related to fungi, it lacks the major fungal sterol, ergosterol. Several Δ7 24‐alkysterols synthesized by P. carinii are the same as those reported in some basidiomycete rust fungi. The 24‐alkylsterols are synthesized by the action of S‐adenosyl‐l‐methionine:C‐24 sterol methyl transferase (SAM:SMT). Fungal SAM:SMT enzymes normally transfer only one methyl group to the C‐24 position of the sterol side chain and the cells accumulate C28 24‐alkylsterols. In contrast, the P. carinii SAM:SMT and those of some plants catalyze one or two methyl transfer reactions producing both C28 and C29 24‐alkylsterols. However, unlike most fungi, plants, and the kinetoplastid flagellates Leishmania and Trypanosoma cruzi, P. carinii does not appear to form double bonds at C‐5 of the sterol nucleus and C‐22 of the sterol side chain. Furthermore, the P. carinii SAM:SMT substrate preference for C30 lanosterol differs from that of homologous enzymes in any other organisms studied. C31 24‐Methylenelanosterol and C32 pneumocysterol, products of SAM:SMT activity on lanosterol, can accumulate in high amounts in some, but not all, human‐derived Pneumocystis jiroveci populations.
Isolation and Expression of Two Genes Encoding Eukaryotic Release Factor 1 from Paramecium tetraurelia
Authors: KERVESTIN STEPHANIE , GARNIER OLIVIER A. , KARAMYSHEV ANDREY L. , ITO KOICHI , NAKAMURA YOSHIKAZU , MEYER ERIC , JEAN‐JEAN OLIVIER
Published: July 2005

ABSTRACT. Paramecium tetraurelia, like some other ciliate species, uses an alternative nuclear genetic code where UAA and UAG are translated as glutamine and UGA is the only stop codon. It has been postulated that the use of stop codons as sense codons is dependent on the presence of specific tRNAs and on modification of eukaryotic release factor one (eRFl), a factor involved in stop codon recognition during translation termination. We describe here the isolation and characterisation of two genes, eRF1‐a and eRF1‐b, coding for eRFl in P. tetraurelia. The two genes are very similar, both in genomic organization and in sequence, and might result from a recent duplication event. The two coding sequences are 1,314 nucleotides long, and encode two putative proteins of 437 amino acids with 98.5% identity. Interestingly, when compared with the eRFl sequences either of ciliates having the same variant genetic code, or of other eukaryotes, the eRFl of P. tetraurelia exhibits significant differences in the N‐terminal region, which is thought to interact with stop codons. We discuss here the consequences of these changes in the light of recent models proposed to explain the mechanism of stop codon recognition in eukaryotes. Besides, analysis of the expression of the two genes by Northern blotting and primer extension reveals that these genes exhibit a differential expression during vegetative growth and autogamy.
Epidermal Growth Factor Binds to a Receptor on Trypanosoma cruziAmastigotes Inducing Signal Transduction Events and Cell Proliferation
Authors: GHANSAH TOMAR J. , AGER EDWARD C. , FREEMAN‐JUNIOR PHYLLIS , VILLALTA FERNANDO , LIMA MARIA F.
Published: July 2005

ABSTRACT
Host growth factors induce proliferation of Trypanosoma cruzi amastigotes by mechanisms that remain poorly defined. Here we examined human epidermal growth factor (EGF) for its ability to bind to the mammalian multiplicative forms of T. cruzi and to induce growth of the parasites. EGF stimulated incorporation of [3H] thymidine into DNA and growth of amastigotes both in a concentration‐dependent manner. Radiolabeled EGF was found to bind to amastigotes in a concentration‐dependent and saturable manner but it did not bind to trypomastigotes. Scatchard analysis showed a single class of receptors with a Kd of 0.8 nM and numbering 3.1 × 103 per amastigote. Results from internalization experiments provided evidence of receptor‐mediated endocytosis of EGF. Northern analysis showed a 3.0‐kb transcript for the putative EGF receptor (EGFR) homologue in amastigotes, but not trypomastigotes. Binding of EGF to amastigotes induced signal transduction events. EGF induced “in vitro” kinase activity as determined by γ‐[32P] ATP incorporation into amastigote proteins. EGF also increased protein kinase C activity in a concentration‐dependent manner and Mitogen Activated Protein (MAP) kinase activity in a time‐ and concentration‐dependent manner. A specific inhibitor (AG14782) of the EGFR and a MAP kinase inhibitor (PD98059) decreased EGF‐dependent T. cruzi MAP kinase activity. These results describe a novel mechanism used by amastigotes to regulate their proliferation mediated by an EGF‐dependent signal transduction pathway.
Laboratory Susceptibility of Wyeomyia smithii (Diptera: Culicidae) to Ascogregarina taiwanensis (Apicomplexa: Lecudinidae)
Authors: REEVES WILL K. , McCULLOUGH SHAUN D.
Published: July 2005

ABSTRACT
Gregarines in the genus Ascogregarina are not known to develop in sabethine mosquitoes, but we successfully infected larvae of Wyeomyia smithii with Ascogregarina taiwanensis in the laboratory. Ascogregarina taiwanensis is a natural parasite of the exotic Asian tiger mosquito, Aedes albopictus. Only 18% to 70% of the W. smithii larvae had visible trophozoites, with a range of 1–92 per larva. Trophozoites persisted in the midgut for more than 37 d, and one adult female W. smithii had gametocysts in its Malphigian tubules, which indicated that A. taiwanensis might fully develop in W. smithii. After 50 d, gregarines were not found in W. smithii larvae.
Sequence Survey of the Genome of the Opportunistic Microsporidian Pathogen, Vittaforma corneae
Authors: MITTLEIDER DEREK , GREEN LINDA C. , MANN VICTORIA H. , MICHAEL SCOTT F. , DIDIER ELIZABETH S. , BRINDLEY PAUL J.
Published: July 2005

ABSTRACT
The microsporidian Vittaforma corneae has been reported as a pathogen of the human stratum corneum, where it can cause keratitis, and is associated with systemic infections. In addition to this direct role as an infectious, etiologic agent of human disease, V. corneae has been used as a model organism for another microsporidian, Enterocytozoon bieneusi, a frequent and problematic pathogen of HIV‐infected patients that, unlike V. corneae, is difficult to maintain and to study in vitro. Unfortunately, few molecular sequences are available for V. corneae. In this study, seventy‐four genome survey sequences (GSS) were obtained from genomic DNA of spores of laboratory‐cultured V. corneae. Approximately, 41 discontinuous kilobases of V. corneae were cloned and sequenced to generate these GSS. Putative identities were assigned to 44 of the V. corneae GSS based on BLASTX searches, representing 21 discrete proteins. Of these 21 deduced V. corneae proteins, only two had been reported previously from other microsporidia (until the recent report of the Encephalitozoon cuniculi genome). Two of the V. corneae proteins were of particular interest, reverse transcriptase and topoisomerase IV (parC). Since the existence of transposable elements in microsporidia is controversial, the presence of reverse transcriptase in V. corneae will contribute to resolution of this debate. The presence of topoisomerase IV was remarkable because this enzyme, previously had been identified only from prokaryotes. The 74 c 26.7 kilobases of unique sequences from which two statistics were generated: GC content and codon usage. The GC content of the unique GSS was 42%, lower than that of another microsporidian, E. cuniculi (48% for protein‐encoding regions), and substantially higher than that predicted for a third microsporidian, Spraguea lophii (28%). A comparison using the Pearson correlation coefficient showed that codon usage in V. corneae was similar to that in the yeasts, Saccharomyces cerevisiae (r= 0.79) and Shizosaccharomyces pombe (r= 0.70), but was markedly dissimilar to E. cuniculi (r= 0.19).
Morphology and Infraciliature of a New Marine Ciliate Euplotidium smalli n. sp. with Description of a New Genus, Paraeuplotidium n. g. (Ciliophora, Euplotida)
Authors: LEI YANLI , CHOI JOONG KI , XU KUIDONG
Published: July 2005

ABSTRACT
The morphology and infraciliature of a new hypotrichous ciliate, Euplotidium smalli n. sp., isolated from eutrophic coastal water in Korea, were observed in living cells and investigated using the protargol impregnation technique. This new ciliate bears 13–14 frontoventral cirri, 7 transverse cirri, and 5–6 dorsal kinetics. Neither left marginal cirrus nor caudal cirrus is present. The new species differs from the related species, Euplotidium agitatumNoland, 1937 in the different number of frontoventral and transverse cirri and different body shape. With the exception of Euplotidium agitatum, the known species of the genus EuplotidiumNoland, 1937 with the presence of left marginal cirrus are assigned to a new genus, Paraeuplotidium n. g. Diagnosis of Paraeuplotidium is: Gastrocirrhidae with funnel‐shaped buccal cavity; with frontoventral and transverse cirri; left marginal cirrus present. Paraeuplotidium itoi (Ito, 1958) n. comb. is designated here the type species. Four additional species are included: Paraeuplotidium psammophilus (Vacelet, 1961) n. comb., Paraeuplotidium arenarium (Magagnini & Nobili, 1964) n. comb., Paraeuplotidium helgae (Hartwig, 1980) n. comb., and Paraeuplotidium prosaltans (Tuffrau, 1985) n. comb. An improved generic diagnosis of Euplotidium is suggested based on morphology and infraciliature characters: marine hypotrichs with a funnel‐shaped buccal cavity; with frontoventral and transverse cirri; neither left marginal cirrus nor caudal cirri present.
Significant Differences Between Procyclic and Bloodstream Forms of Trypanosoma brucei in the Maintenance of their Plasma Membrane Potential
Authors: VAN DER HEYDEN NICOLE , DOCAMPO ROBERTO
Published: July 2005

ABSTRACT
The plasma membrane potential (ΔΨ) of procyclic and bloodstream trypomastigotes of Trypanosoma brucei was studied using the potentiometric fluorescent dye bisoxonol. Our results suggest that a proton pump plays a significant role in the regulation of AΨ in procyclic and bloodstream forms, as evidenced by depolarization of the plasma membrane by H+‐ATPase inhibitors (e.g. dicyclohexylcarbo‐diimide, N‐ethylmaleimide, diethylstilbestrol, and bafilomycin A1). In bloodstream stages the plasma membrane was significantly depolarized by ouabain only when the cells were incubated in sodium‐rich buffers indicating that a sodium pump was being inhibited. In contrast, ouabain had no effect on the AΨ of the procyclic stages in a sodium‐rich buffer. However, it induced an additional significant depolarization in these stages when their plasma membrane was already partially depolarized by the H+‐ATPase inhibitor dicyclohexylcarbo‐diimide, indicating the presence of an ouabain‐sensitive sodium pump whose activity is masked by the H+‐ATPase. Unlike procyclic forms, the AΨ of bloodstream‐stage trypomastigotes was markedly sensitive to extracellular Na+ and K+concentrations. Thus, there are significant differences between procyclic and blooodstream forms in the maintenance of the AΨ and in their permeability to cations.
Development of an In Vitro Clonal Culture and Characterization of the rRNA Gene Cluster of Perkinsus atlanticus, a Protistan Parasite of the Clam Tapes decussatus
Authors: ROBLEDO JOSé A. F. , NUNES PATRÍCIA A. , CANCELA M. LEONOR , VASTA GERARDO R.
Published: July 2005

ABSTRACT
Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR‐based species‐specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5‐kb in length including 5S, IGS, SSU, ITS1, 5.8S, IS2, LSU, and an inter‐cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3–100% identical to P. atlanticus sequences previously obtained from clam tissue (non‐clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus“genus‐specific” PCR‐based assay complements the species‐specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.
Description of a New Marine Species of Askenasia Blochmann, 1895 (Ciliophora, Haptoria), With Notes on its Ecology
Authors: EARLAND KIMBERLEY A. , MONTAGNES DAVID J. S.
Published: July 2005

ABSTRACT
A marine planktonic ciliate, belonging to the genus Askenasia Blochmann, 1895, is described with notes on its distribution and ecology. Specimens of this new species were collected from four coastal sites across the northern hemisphere. Samples were collected between March and December, from depths of 1–20 m. Relatively low (< 1 ml−1) densities were recorded from Atlantic and Pacific Oceans and North Sea coastal sites, whereas samples from the English Channel suggested that this species has the ability to form blooms. Specimens were protargol‐stained, revealing the key features of the genus, including the diagnostic sub‐equatorial kinety belt consisting of three unciliated monokinetids. However, the species is larger than other Askenasia with a single distinctive, rope‐like, scalloped macronucleus. This species also appears to have an extra somatic kinety belt, which may be present but has not been observed in other Askenasia species. Based on size, kinety structure, kinety number, and macronuclear shape a new species has been established.
Phytate as the Sole Phosphate Source for Growth of Tetrahymena
Authors: ZIEMKIEWICZ HOPE T. , JOHNSON MARGARET D. , SMITH‐SOMERVILLE HARRIETT E.
Published: July 2005

ABSTRACT
Phytate, the storage form of phosphate in seeds and grains, is a major form of environmental phosphate loading from fertilizer inputs and agricultural runoff. We have investigated the ability of Tetrahymena populations to grow on phytate as their sole phosphate source. Populations grew equally well in chemically defined medium with phosphate and medium in which the phosphate was replaced with phytate in comparable concentrations between 0.5 mM and 6 mM. Intracellular phytate concentrations of cells grown in phytate showed a 4–6‐fold increase over those grown in phosphate when measured during the late stage of exponential growth. These results demonstrate that phytate can provide a source of adequate phosphate for sustained growth in phytate‐rich environments.
Cryptosporidium hominis n. sp. (Apicomplexa: Cryptosporidiidae) from Homo sapiens
Authors: MORGAN‐RYAN UNA M. , FALL ABBIE , WARD LUCY A. , HIJJAWI NAWAL , SULAIMAN IRSHAD , PAYER RONALD , THOMPSON R. C. ANDREW , OLSON M. , LAL ALTAF , XIAO LIHUA
Published: July 2005

ABSTRACT. The structure and infectivity of the oocysts of a new species of Cryptosporidium from the feces of humans are described. Oocysts are structurally indistinguishable from those of Cryptosporidium parvum. Oocysts of the new species are passed fully sporulated, lack sporocysts, and measure 4.4–5.4 μm (mean = 4.86) × 4.4–5.9 μm (mean = 5.2μm) with a length to width ratio 1.0–1.09 (mean 1.07) (n = 100). Oocysts were not infectious for ARC Swiss mice, nude mice, Wistar rat pups, puppies, kittens or calves, but were infectious to neonatal gnotobiotic pigs. Pathogenicity studies in the gnotobiotic pig model revealed significant differences in parasite‐associated lesion distribution (P = 0.005 to P = 0.02) and intensity of infection (P = 0.04) between C. parvitm and this newly described species from humans. In vitro cultivation studies have also revealed growth differences between the two species. Multi‐locus analysis of numerous unlinked loci, including a preliminary sequence scan of the entire genome demonstrated this species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further support for its species status. Based on biological and molecular data, this Cryptosporidium infecting the intestine of humans is proposed to be a new species Ciyptosporidium hominis n. sp.
Simple, Filter‐based PCR Detection of Thelohania solenopsae (Microspora) in Fire Ants (Solenopsis invicta)
Authors: SNOWDEN KAREN F. , LOGAN KATHLEEN S. , VINSON S. BRADLEIGH
Published: July 2005

ABSTRACT. Thelohania solenopsae is a microsporidian parasite that may serve as a biological control agent for the red imported fire ant, Solenopsis invicta. A rapid, filter‐based PCR amplification method detecting a portion of the small‐subunit ribosomal RNA gene was developed to facilitate field studies detecting the parasite in fire ants. Processing ant homogenates with a commercially available membrane‐based system, FTA Classic Card technology, compared favorably with traditional DNA extraction and PCR amplification methods. As few as 100 spores were detected. The FTA membrane system is a simple, extraction‐free method for detecting T. solenopsae in fire ants, and allows for easy archival storage of DNA samples.
Density and Diversity of Protozoa in Some Arid Australian Soils
Authors: ROBINSON BRET S. , BAMFORTH STUART S. , DOBSON PHILIP J.
Published: July 2005

ABSTRACT. This is the first extensive study of soil protozoa of arid lands. Twenty‐six samples from litters, soils, termitaria, and a cyanobacterial crust, collected from central and south Australian arid lands, were analyzed for numbers and species of gymnamoebae, ciliates, and testacea. Amoebae ranged from 1,000–5,000/g of material, and were two orders of magnitude more abundant than ciliates. Both groups increased in abundance and species richness from bare soils through spinifex to mulga to chenopod vegetations. Testacea ranged 900–5,000/g with similar species richness throughout vegetations, but reached 11,900/g with a doubling of species in a refugium in Kings Canyon. The most prevalent species of amoebae, ciliates, and testacea were taxa associated with ephemeral and disturbed habitats (r‐selection). The cyanobacterial crust might be considered a micro‐refugium because it contained a number of non‐encysting protozoa, including Thecamoeba sp. and Nassula picta, feeding on cyanobacterial filaments. The numbers and species richness of protozoa under shrubs were greater than in bare soils, supporting the resource island hypothesis that desert plants create soil heterogeneity by localizing soil fertility under their canopies.
An Extracellular monoADP‐ribosyl Transferase Activity in Entamoeba histolytica Trophozoites
Authors: DELGADO‐CORONA PATRICIA , MARTÍNEZ‐CADENA GUADALUPE , ALVAREZ ANGEL H. , TORRES‐CALZADA HORACIO E. , AVILA EVA E.
Published: July 2005

ABSTRACT. Due to the important role of monoADP‐ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP‐ribosyl transferase activity. Reactions were initiated using amebafree medium as the source of both enzyme and ADP‐ribosylation substrate(s) and [32P]NAD+ as source of ADP‐ribose. Proteins were analyzed by electrophoresis, and [32P]‐labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37,000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A‐treated trophozoites, indicating that the extracellular monoADP‐ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37‐kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to [32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP‐ribosylation since [14C‐carbonyl]NAD+ was not incorporated into ameba substrates and a 75‐fold molar excess of ADP‐ribose caused no detectable inhibition of the monoADP‐ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP‐ribosyl transferase of E. histolytica may be an arginine‐specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP‐ribosyl transferase, whose localization depends upon a secretion process.
Multiple Strains of the Parasitic Dinoflagellate Amoebophrya Exist in Chesapeake Bay
Authors: GUNDERSON JOHN H. , JOHN SHINU A. , CHANSON BOMAN W. , COATS D. WAYNE
Published: July 2005

ABSTRACT
Small subunit rRNA sequences were amplified from Amoebophrya strains infecting Karlodinium micrum, Gymnodinium instriatum and an unidentified Scrippsiella species in Chesapeake Bay. The alignable parts of the sequences differed from each other and from the previously reported rRNA sequence of the Amoebophrya strain infecting Akashiwo sanguinea in Chesapeake Bay by 4 to 10%. This is a greater degree of difference than sometimes found between sequences from separate genera of free‐living dinoflagellates. These sequence differences indicate that the Amoebophrya strains parasitizing dinoflagellates in Chesapeake Bay do not all belong to the same species. In spite of their relative dissimilarity, the sequences do group together into a single clade with high bootstrap support in phylogenetic trees constructed from the sequences.
Induction of Anti‐Actin Drug Resistance in Tetrahymena
Authors: ZACKROFF ROBERT V. , HUFNAGEL LINDA A.
Published: July 2005

ABSTRACT
Both cytochalasin D and latrunculin B reversibly inhibited Tetrahymena phagocytosis at concentrations similar to those effective in mammalian systems, even though ciliate actins are known to be highly divergent from mammalian actins. Overnight exposure to relatively low (0.25 μM) concentrations of latrunculin B induced resistance in Tetrahymena to the inhibitory effects of that drug, as well as cross‐resistance to cytochalasin D. However, much higher (>30 μM) concentrations of cytochalasin D were required for induction of cross‐resistance to latrunculin B. Anti‐actin drug resistance in Tetrahymena may involve a general multidrug resistance mechanism and/or specific feedback regulation of F‐actin assembly and stability.
The Phylogeny of Colpodellids (Alveolata) Using Small Subunit rRNA Gene Sequences Suggests They are the Free‐living Sister Group to Apicomplexans
Authors: KUVARDINA OLGA N. , LEANDER BRIAN S. , ALESHIN VLADIMIR V. , MYL'NIKOV ALEXANDER P. , KEELING PATRICK J. , SIMDYANOV TIMUR G.
Published: July 2005

ABSTRACT. In an attempt to reconstruct early alveolate evolution, we have examined the phylogenetic position of colpodellids by analyzing small subunit rDNA sequences from Colpodella pontica Myl'nikov 2000 and Colpodella sp. (American Type Culture Collection 50594). All phylogenetic analyses grouped the colpodellid sequences together with strong support and placed them strongly within the Alveolata. Most analyses showed colpodellids as the sister group to an apicomplexan clade, albeit with weak support. Sequences from two perkinsids, Perkinsus and Parvilucifera, clustered together and consistently branched as the sister group to dinoflagellates as shown previously. These data demonstrate that colpodellids and perkinsids are plesiomorphically similar in morphology and help provide a phylogenetic framework for inferring the combination of character states present in the last common ancestor of dinoflagellates and apicomplexans. We can infer that this ancestor was probably a myzocytotic predator with two heterodynamic flagella, micropores, trichocysts, rhoptries, micronemes, a polar ring, and a coiled open‐sided conoid. This ancestor also very likely contained a plastid, but it is presently not certain whether it was photosynthetic, and it is not clear whether extant perkinsids or colpodellids have retained the organelle.
The Phylogenetic Position of the Pelobiont Mastigamoeba balamuthi Based on Sequences of rDNA and Translation Elongation Factors EF‐1α and EF‐2
Authors: ARISUE NOBUKO , HASHIMOTO TETSUO , LEE JENNIFER A. , MOORE DOROTHY V. , GORDON PAUL , SENSEN CHRISTOPH W. , GAASTERLAND TERRY , HASEGAWA MASAMI , MÜLLER MIKLÓS
Published: July 2005

ABSTRACT. The taxonomic position and phylogenetic relationships of the Pelobionta, an amitochondriate amoeboflagellate group, are not yet completely settled. To provide more information, we obtained sequences for the large subunit rDNA gene, the gene for translation elongation factor la, and for a large part of the gene encoding translation elongation factor 2 from a representative of this group. Mastigamoeba balamuthi (formerly Phreatamoeba balamuthi). The gene for the large subunit rDNA was unusually large compared to those of other protists, a phenomenon that had previously been observed for the gene encoding the small subunit rDNA. Phylogenetic reconstruction using a maximum likelihood method was performed with these sequences, as well as the gene encoding the small subunit rDNA. When evaluated individually, the M. balamuthi genes for the small and large subunit rDNAs and elongation factor 1α had a most recent common ancestor with either the Mycetozoa (slime molds) or with Entamoeba histolytica. A clade formed by M. balamuthi, E. histolytica, and Mycetozoa was not rejected statistically for any of the sequences. A combined maximum likelihood analysis using 3,935 positions from all molecules suggested that these three taxonomic units form a robust clade. We were unable to resolve the closest group to this clade using the combined analysis. These findings support the notion, which had previously been proposed primarily on cytological evidence, that both M. balamuthi and E. histolytica are closely related to the Mycetozoa and that these three together represent a major eukaryotic lineage.
Serological Affinities of the Oyster Pathogen Perkinsus marinus (Apicomplexa) with Some Dinoflagellates (Dinophyceae)
Authors: BUSHEK DAVID , DUNGAN CHRISTOPHER F. , LEWITUS ALAN J.
Published: July 2005

ABSTRACT. The protozoan oyster pathogen Perkinsus marinus is classified in the phylum Apicomplexa, although molecular‐genetic and ultrastructural evidence increasingly concur on its closer phylogenetic relationship with the dinoflagellates. To test for evidence of serological epitopes common to P. marinus and dinoflagellates, we probed 19 free‐living and 8 parasitic dinoflagellate, or dinoflagellate‐like, species for cross‐reactivity with polyclonal antibodies to P. marinus. Three of 19 free‐living dinoflagellates (16%), and 7 of 8 parasitic dinoflagellates (88%) were labeled by anti‐P. marinus antibodies. In reciprocal immunoassays using polyclonal antibodies to the Hematodinium sp. dinoflagellate parasite of Norway lobsters, Nephrops norvegicus, P. marinus and the same 7 parasitic dinoflagellates labeled by anti‐P. marinus antibodies, were again labeled. The dinoflagellate‐like parasite of prawns Pandalus platyceros was not labeled by either antibody reagent. These reciprocal results confirm the presence of shared antibody‐binding epitopes on cells of P. marinus and several dinoflagellates. The apparent widespread serological affinity between P. marinus and the parasitic dinoflagellates suggests a closer phylogenetic link to the syndinean dinoflagellate lineage. The consistent failure of the dinoflagellate‐like prawn parasite to bind either antibody reagent shows that this parasite is serologically distinct from both P. marinus and Hematodinium‐species parasitic dinoflagellates.
Laboratory and Field‐based Studies of Abundances, Small‐scale Patchiness, and Diversity of Gymnamoebae in Soils of Varying Porosity and Organic Content: Evidence of Microbiocoenoses
Authors: ANDERSON O. ROGER
Published: July 2005

ABSTRACT. Soil samples (varying in granularity) from four natural sites were cultured in microcosms to determine small‐scale patchiness in abundance and diversity of gymnamoebae. Eighty grams of the same thoroughly mixed soil, either moistened with distilled water (– nutrients) or supplemented with an equivalent vol. of organically enriched water (+ nutrients), were placed in covered glass jars and incubated for 14 d (25°C). Abundances (number/gram soil) were assessed in each of 3 core samples (5–10 mm apart). Assay precision was estimated to be ± 4%. Abundances were similar in the 3 closely‐spaced samples, but occasional samples had higher abundances, probably representing localized enriched sites (“nutrient hot spots”). Diversity within the triplicate, closely spaced samples varied substantially. Mean abundance and diversity of amoebae were consistently higher in organically enriched soil and in soil of increasing granularity. Field samples collected directly from two of the sites showed similar patterns of abundance and diversity as found in the experimental studies, indicating substantial small‐scale compartmentalization of soil protist communities. These data provide evidence of soil eukaryotic microbiocoenoses and indicate that soil microfauna may encounter wide variations in resources and prey communities as they migrate within small distances of several millimeters or less.
Transplasma Membrane Electron Transport in Leishmania donovani Promastigotes
Authors: DATTA GAUTAM , BERA TANMOY
Published: July 2005

ABSTRACT. Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to –125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4‐fold less sensitive to chloroquine and 12‐fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.
Re‐examining Alveolate Evolution Using Multiple Protein Molecular Phylogenies
Authors: FAST NAOMI M. , XUE LINGRU , BINGHAM SCOTT , KEELING PATRICK J.
Published: July 2005

ABSTRACT. Alveolates are a diverse group of protists that includes three major lineages: ciliates, apicomplexa, and dinoflagellates. Among these three, it is thought that the apicomplexa and dinoflagellates are more closely related to one another than to ciliates. However, this conclusion is based almost entirely on results from ribosomal RNA phylogeny because very few morphological characters address this issue and scant molecular data are available from dinoflagellates. To better examine the relationships between the three major alveolate groups, we have sequenced six genes from the non‐photosynthetic dinoflagellate, Ciyptlzecodinium cohnii: actin, beta‐tubulin, hsp70, BiP, hsp90, and mitochondria1 hspl0. Beta‐tubulin, hsp70, BIP, and hsp90 were found to be useful for intra‐alveolate phylogeny, and trees were inferred from these genes individually and in combination. Trees inferred from individual genes generally supported the apicomplexa‐dinoflagellate grouping, as did a combined analysis of all four genes. However, it was also found that the outgroup had a significant effect on the topology within alveolates when using certain methods of phylogenetic reconstruction, and an alternative topology clustering dinoflagellates and ciliates could not be rejected by the combined data. Altogether, these results support the sisterhood of apicomplexa and dinoflagellates, but point out that the relationship is not as strong as is often assumed.
Establishment of a Spinated Type of Diplodinium rangiferi by Transfaunation of the Rumen Ciliates of Japanese Sika Deer (Cervus nippon centralist to the Rumen of Two Japanese Shorthorn Calves (Bos taurus taurus)
Authors: IMAI SOICHI , MATSUMOTO MITSUTO , WATANABE AKIRA , SATO HlROSHI
Published: July 2005

ABSTRACT. One liter of rumen fluid containing 4.7 × 104 ciliates/ml, representing four genera including nine species of ciliates from a Japanese sika deer was inoculated into two unfaunated Japanese shorthorn calves. Two weeks after inoculation, all species originally present in the inoculum were subsequently detected in the rumen fluid of one or both calves. Ciliate densities ranged from 105–106 cells/ml over the remainder of the 33‐wk experiment. The inoculum contained Diplodinium rangiferi. which lacks caudal appendages, as is characteristic for the species. However, three weeks later, the rumen fluid of both calves contained D. rangiferi, which possesses caudal appendages varying from a single spine to multiple spines with a complicated furcate appearance. The caudal spines of D. rangiferi did not disappear during the experiment, even when the diet of the calves was switched to the ration of sika deer from which the inoculum was obtained.
Phylogenetic Position of Blastocystis hominis and of Stramenopiles Inferred from Multiple Molecular Sequence Data
Authors: ARISUE NOBUKO , HASHIMOTO TETSUO , YOSHIKAWA HISAO , NAKAMURA YOSHIfflRO , NAKAMURA GEN , NAKAMURA FUMINORI , YANO TAKA‐AKI , HASEGAWA MASAMI
Published: July 2005

ABSTRACT. Blastocystis hominis, a parasite of the human intestine, has recently been positioned within Stramenopiles by the small subunit rRNA phylogeny. To further confirm its phylogenetic position using multiple molecular sequence data, we determined the nucleotide sequences putatively encoding small subunit ribosomal RNA, cytosolic‐type 70‐kDa heat shock protein, translation elongation factor 2, and the non‐catalytic ‘B’ subunit of vacuolar ATPase of B. hominis (HE87–1 strain). Moreover, we determined the translation elongation factor 2 sequence of an apicomplexan parasite, Plasmodium falciparum, that belongs to alveolates. The maximum likelihood analyses of small subunit rRNA and cytosolic‐type 70‐kDa heat shock protein clearly demonstrated that B. hominis (HE87–1 strain) is positioned within Stramenopiles, being congruent with the previous small subunit rRNA analysis, including the sequences of B. hominis (Nand strain) and a Blastocystis isolate from guinea pig. Although no clear resolution among major eukaryotic groups was obtained by the individual phytogenies based on the four molecules analyzed here, a combined analysis of various molecules, including these, clearly indicated that Blatocystis/stramenopiles are the closest relatives of alveolates.
Not‐Adaptive Behavior of Isotropically Heated, Inert Populations of Oxytricha bifaria (Ciliophora, Stichotrichia)
Authors: BARBANERA FILIPPO , ERRA FABRIZIO , BANCHETTI ROSALBA
Published: July 2005

ABSTRACT. The physiological effects on isotropically heated populations of Oxytricha bifaria cultured at 24°C were investigated. At 34.6°C ciliates became inert, and did not adaptively react to either cold or warm microgradients; they neither moved towards the favorable cold thermal source nor escaped from the unfavorable warm one. The inert oxytrichas were only able to perform the Side‐Stepping Reaction (SSR) on the same spot. However, mobile ciliates at 31.6°C reacted to the cold microgradient by immediately orienting themselves towards its source, without accelerating but reducing their SSR frequency. Moreover, in a warm microgradient such ciliates immediately increased their SSR frequency, then moved away from the thermal source. At 34.6°C the behavior of ciliates was not‐adaptive—not acting to guide the organisms to more favorable conditions—whereas at 31.6°C it was still clearly adaptive. Therefore, the locomotory inertness of the oxytrichas at 34.6°C was the result of thermal stress rather than their behavioral response to the environmental isotropy, in contrast to populations of the same species made inert at 9°C.