bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemorepellents in Paramecium and Tetrahymena
Authors: FRANCIS JOSEPH T. , HENNESSEY TODD M.
Published: May 2007

Although Paramecium has been widely used as a model sensory cell to study the cellular responses to thermal, mechanical and chemoattractant stimuli, little is known about their responses to chemorepellents. We have used a convenient capillary tube repellent bioassay to describe 4 different compounds that are chemorepellents for Paramecium and compared their response with those of Tetrahymena. The classical Paramecium t‐maze chemokinesis test was also used to verify that this is a reliable chemorepellent assay. The first two compounds, GTP and the oxidant NBT, are known to be depolarizing chemorepellents in Paramecium but this is the first report of them as repellents in Tetrahymena. The second two compounds, the secretagogue alcian blue and the dye cibacron blue, have not previously been described as chemorepellents in either of these ciliates. Two other compounds, the secretagogue AED and the oxidant cytochrome c, were found to be repellents to Paramecium but not to Tetrahymena. The repellent nature of each of these compounds is not related to toxicity because cells are completely viable in all of them. More importantly, all of these repellents are effective at micromolar to nanomolar concentrations, providing an opportunity to use them as excitatory ligands in future works concerning their membrane receptors and possible receptor operated ion channels.
Induction of Antibiotic Resistance in Paramecium tetraurelia by the Bacterial Gene APH‐3'‐II
Authors: HAYNES W. JOHN , LING KIT‐YIN , SAIMI YOSHIRO , KUNG CHING
Published: May 2007

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'‐phosphotransferase‐II (APH‐3'‐II or neor) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non‐coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neor ORF was inserted, the plasmid was referred to as pPXV‐NEO. Delivery of approximately 10–20 picoliters of linearized PXV‐NEO at > 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neor‐specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G‐418, and the concentrations of neor‐specific DNA and neor‐specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon‐usage differences.
Inhibition of Pair Formation by Concanavalin A and Concanavalin A‐Binding Glycoconjugates in Euplotes octocarinatus
Authors: PLÜMPER EVELYN , FREIBURG MANFRED , HECKMANN KLAUS
Published: May 2007

ABSTRACT. Concanavalin A (≥ 50 μg/ml) inhibits pair formation in both of the two complementary mating types of Euplotes octocarinatus studied in this investigation. This effect can be reversed by methyl‐α‐d‐mannose. Concanavalin A is accessible for methyl‐α‐d‐mannose until pairs are formed. Methyl‐α‐d‐mannose as well as methyl‐α‐d‐glucose and 2‐acetamido‐2‐deoxy‐d‐glucose alone do not inhibit pair formation unless applied in concentrations ≥ 60 mM. The Concanavalin A‐sensitive phase of preconjugant interaction starts 2 h after cells are induced to conjugate. Based on these observations we suggest that Concanavalin A might exhibit its action by binding to carbohydrate moieties of preconjugation‐specific adhesion molecules and thereby might allosterically block interactions with their counterparts. To identify preconjugation‐specific alterations in number or localization of Concanavalin A‐binding glycoconjugates, we probed western blots of total cell proteins or fixed cells, respectively, with digoxigenin‐labeled Concanavalin A. On Concanavalin A blots 20 different Concanavalin A‐binding glycoconjugates were identified in mating‐competent cells. Localization of Concanavalin A‐binding sites on mating‐competent cells by light microscopy resulted in predominant labeling of a comma‐shaped structure near the paroral membranelle. During the preconjugation period no changes in number or localization of Con A‐binding glycoconjugates were detected. Possible reasons are discussed.
A New Method for Inoculation of Poor Germinator, Nosema sp. NIS M11 (Microsporida: Nosematidae), into an Insect Cell Culture
Authors: YASUNAGA CHISA , INOUE SHINO , FUNAKOSHI MASAKO , KAWARABATA TAKESHI , HAYASAKA SYOJI
Published: May 2007

ABSTRACT. Spores of Nosema sp. NIS M11, primed with 0.1 N KOH solution, were mixed with either physiological salt solutions or IPL‐41 medium, an insect cell culture medium, for germination. In the latter medium, only a few spores germinated, while high percentages of spore germination were obtained in physiological salt solutions, particularly in Rinaldini's solution. By using the salt solutions as inoculation media, KOH‐primed NIS M11 spores were inoculated into the Spodoptera frugiperda SF21AEII cell line. The initial infection levels were consistently higher than that obtained by using IPL‐41 medium. Among the salt solutions, Rinaldini's solution, containing KCl in place of NaCl, gave the highest percentage of initial cell infection. Increased osmolarity of salt solutions did not improve the efficiency of spore germination and infection of N. sp. NIS M11.
Effects of Temperature on Promastigotes of Several Species of Leishmania
Authors: LEON LEONOR L. , SOARES MAURILIO J. , TEMPORAL ROSANE M.
Published: May 2007

ABSTRACT. Six Leishmania species were studied comparatively, in order to determine the influence of temperature “in vitro” on differentiation, infectivily and protein synthesis. Differentiation ocurred in a heterogeneous manner, even in species that produce similar clinical manifestations. Thus, no association could be found between thermosensitivity and disease. The association between expression of proteins and increasing temperatures was analyzed at 34° C by polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS‐PAGE), using different incubation times, and employing a technique involving metabolic incorporation of [35S]‐methionine. Protein synthesis was very similar in all the New World species apart from L. amazonensis, which expressed a protein of approximately 80 kDa when incubated at 34° C for 2 hours. All the tested species had in common the expression of a 70 kDa protein. Differences, however, were observed in relation to the time interval for protein expression. in L. chagasi, synthesis was detected after 30 minutes of incubation at 34° C, while L. braziliensis required 1 hour at the same temperature. The “in vivo” and “in vitro” infectivity of the differentiated forms was also analyzed, but no significant differences were observed.
pH‐Dependent Effects of 2,4‐Dinitrophenol (DNP) on Proliferation, Endocytosis, Fine Structure and DNP Resistance in Tetrahymena
Authors: NILSSON JYTTE R.
Published: May 2007

ABSTRACT. In view of the importance of external pH on cytotoxic effects of ionizable agents, the pH‐dependent effects of 2,4‐dinitrophenol (DNP) were investigated. As uncoupler of oxidative phosphorylation. DNP interferes with the proton gradient across the inner mitochondrial membrane. DNP was added to proliferating Tetrahymena pyriformis in media of different initial pH. Effects studied were rates of cell proliferation and endocytosis, and fine structure. Findings correlated with the calculated concentration of undissociated DNP, taking into account that pH changes with time and cell density in Tetrahymena cultures. A linear relationship thus emerged between initial concentrations of undissociated DNP and lengths of the lag preceding cell proliferation. Once resumed, the rate of proliferation corresponded to that of control cells, even in different concentrations of undissociated DNP, presumably indicating an adaptation mechanism. Endocytosis was elevated throughout a wide range of undissociated DNP concentrations with a sharp transition towards inhibition at high DNP concentrations causing lethality with time. Changes in fine structure of DNP‐treated cells (mitochondria, peroxisomes, nucleoli) also depended on the concentration of undissociated DNP.
Adenyiyl Cyclase and G‐Proteins in Phytomonas
Authors: FARBER MARISA D. , MONTAGNA ANDREA E. , PAVETO CRISTINA , DOLLET MICHEL , SANCHEZ‐MORENO MANUEL , OSUNA ANTONIO , TORRES HECTOR N. , FLAWIA MIRTHA M.
Published: May 2007

ABSTRACT. Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A [35S]GTP‐γ‐S‐binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 32P]NAD+ led to incorporation of radioactivity into bands of about 40–44 kDa. Crude membranes were electrophoresed on SDS‐polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti‐αs antibody and the AS/7 antibody (anti‐α1, anti‐αi1, anti‐αi2). These procedures resulted in the identification of polypeptides of approximately 40–44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through Gαs, proteins.
Long‐Range Restriction Mapping of Megabase‐Sized Chromosomes that May Be Homologs in Trypanosoma brucei
Authors: BASTIAN L. SCOT , LODES MICHAEL J. , MYLER PETER J. , STUART KENNETH
Published: May 2007

ABSTRACT. Trypanosoma brucei is a blood‐borne pathogen that changes its variant surface glycoprotein coat, thus evading immune destruction. Restriction digestion, combined with probe hybridization studies, was used to construct long‐range restriction maps of the 1.4 (M4) and 1.5 megabase (M3) chromosomes from the IsTaR1 serodeme of T. b. brucei. Comparison of the two chromosomes suggests that they are a homologous pair. Hybridization with a repetitive sequence probe also identifies several copies on the M4 chromosome and a relative paucity of cross‐hybridizing repetitive sequence on the larger M3 chromosome.
A Sensitive and Specific DNA Probe for the Oyster Pathogen Haplosporidium nelsoni
Authors: STOKES NANCY A. , BURRESON EUGENE M.
Published: May 2007

ABSTRACT. Haplosporidium nelsoni is a pathogen of the eastern oyster, Crassostrea virginica, along the middle Atlantic coast of the U.S. Genomic DNA was extracted from H. nelsoni plasmodia and small subunit (SSU) rDNA was amplified by PCR, cloned and sequenced. The sequence of H. nelsoni SSU rDNA was aligned with that of another haplosporidian, Minchinia teredinis, and with SSU rDNA data of C. virginica and various protists in GenBank. A 21‐base oligonucleotide unique to H. nelsoni, designated MSX1347, was commercially synthesized and tested for sensitivity and specificity. In dot blot hybridizations the probe detected 100 pg of cloned H. nelsoni rDNA and the presence of H. nelsoni in 1 μg of genomic DNA from an infected oyster. It did not hybridize with 1 μg of genomic DNA from uninfected C. virginica or with cloned SSU rDNA of M. teredinis. The probe was further tested for specificity with in situ hybridizations on AFA‐fixed, paraffin‐embedded tissue sections. The probe hybridized well with H. nelsoni plasmodia and immature spores, but poorly with mature spores. The probe did not hybridize with oyster tissue, with other common oyster parasites such as P. marinus or Nematopsis sp., or with the haplosporidians Haplosporidium louisiana from mud crabs (Panopeus spp.), Haplosporidium costale from C. virginica or M. teredinis from shipworms (Teredo spp.).
Monoclonal Antibodies Reveal Complex Structure in the Membrane Skeleton of Tetrahymena
Authors: WILLIAMS NORMAN E. , HONTS JERRY E. , DRESS VIRGINIA M. , NELSEN E. MARLO , FRANKEL JOSEPH
Published: May 2007

ABSTRACT. Twelve monoclonal antibodies were raised that are specific for the membrane skeleton of Tetrahymena. Five were directed against T. pyrifomis and seven were directed against T. thermophila. Some cross‐reactivity between species was found. Each monoclonal antibody recognized one of the three major components of epiplasm, i.e. the bands A, B, and C identified in electrophoretic separations of epiplasmic proteins. It was found, using these antibodies, that the epiplasmic proteins A, B, and C have overlapping but independent distributions within the cell.
Preliminary Characterisation of Chlorarachniophyte Mitochondrial DNA
Authors: GILSON PAUL , WALLER ROSS , McFADDEN GEOFF
Published: May 2007

ABSTRACT. The division Chlorarachniophyta comprises amoeboflagellate protists with complex chloroplasts derived from the endosymbiosis of a eukaryotic alga. Analysis of chlorarachniophyte chromosomal DNAs by pulsed‐field gel electrophoresis revealed an apparently linear 36‐kb chromosome that could not be ascribed to either the host or endosymbiont nuclei. A single eubacterial‐like small subunit ribosomal RNA gene is encoded on this chromosome and phylogenetic analyses places this gene within a clade of mitochondrial genes from other eukaryotes. High resolution in situ hybridization demonstrates that transcripts of the small subunit ribosomal RNA gene encoded by the 36‐kb chromosome are exclusively located in the mitochondria. The 36‐kb chromosome thus likely represents a linear mitochondrial genome. Small amounts of an apparently dimeric (72 kb) form are also detectable in pulsed‐field gel electrophoresis.
Temporal and Spatial Abundance of Naked Amoebae (Gymnamoebae) in Marine Benthic Sediments of the Clyde Sea Area, Scotland
Authors: BUTLER HELEN , ROGERSON ANDREW
Published: May 2007

ABSTRACT. The abundances of benthic naked amoebae in the sediments of the Clyde Sea Area, Scotland, were studied throughout 1991. Four sites differing in sediment grain size and organic carbon content were chosen for study. Amoebae were enumerated by enrichment cultivation methods and found to be numerically important, attaining densities of up to 14,883 amoebae/cm3 on one occasion. They were most abundant, and temporally stable, in finer sediments where they averaged 2,224 cells/cm3 and lower and more variable in the sandy sediments, averaging 874 cells/cm3 throughout the year. In general, amoebae were most abundant in the surface sediment layers. Around 70 different morphotypes were recorded, and 61% of all amoebae counted were less than 10 μm in length. This is the first detailed quantitative study of benthic marine gymanamoebae and shows that naked amoebae and flagellates are numerically the dominant interstitial fauna in sediments of this area. Moreover, the gymnamoebae comprised the largest proportion of total protozoan biomass (excluding foraminiferans) and clearly need to be considered in future models of benthic carbon flow.
Ceratomyxa sparusaurati N. Sp. (Myxosporea: Bivalvulida), a New Parasite from Cultured Gilthead Seabream (Sparus aurata L.) (Teleostei: Sparidae): Light and Electron Microscopic Description
Authors: SITJÀ‐BOBADILLA ARIADNA , PALENZUELA OSWALDO , ÀLVAREZ‐PELLITERO PILAR
Published: May 2007

ABSTRACT. A new Myxosporea, Ceratomyxa sparusaurati n. sp., was found in the gall bladder and bile of cultured gilthead sea bream (Sparus aurata L.) from different Spanish fish farms. It is clearly different from all the previously reported Ceratomyxa from sparids, and it is distinguished from other members of the genus by the shape and size of the spores. Prevalence of infection was 2.15% in an Atlantic farm, 48.7% in a Mediterranean farm and 28.6% in the facilities of the Instituto de Acuicultura de Torre de la Sal. The cell‐in‐a‐cell pattern was found through all the sporogenesis and the general ultrastructure resembled other Myxosporea. Primary cells with two developing spores, harbored other secondary cells. Disporous sporoblasts contained numerous membrane‐bound inclusions, a few lipid droplets and polysaccharides as evidenced by cytochemistry. In mature spores, binucleate sporoplasmic cells contained abundant Thièry‐negative sporoplasmosomes.
Axenic Cultivation of Entamoeba dispar Brumpt 1925, Entamoeba insolita Geiman and Wichterman 1937 and Entamoeba ranarum Grassi 1879
Authors: CLARK C. GRAHAM
Published: May 2007

ABSTRACT. Three species of Entamoeba have been grown in axenic culture for the first time. In two cases, novel methods for adapting the organisms to growth without bacteria were employed. While E. ranarum was axenized by the classic technique of Diamond, from a monoxenic culture with Trypanosoma cruzi as the associate, both E. dispar and E. insolita were first grown in axenic culture medium supplemented with lethally irradiated bacteria. From there, E. insolita was axenized directly, but E. dispar initially required the presence of fixed bacteria. After prolonged culture under this technically axenic but unwieldy culture system, E. dispar was eventually adapted to growth in the absence of added bacteria.
Microsporidian Spore Wall: Ultrastructural Findings on Encephalitozoon hellem Exospore
Authors: BIGLIARDI ELISA , SELMI MARIA GLORIA , LUPETTI PIETRO , CORONA SILVIA , GATTI SIMONETTA , SCAGLIA MASSIMO , SACCHI LUCIANO
Published: May 2007

A study of the spore wall of Encephalitozoon hellem was performed on thin sections, freeze‐fracture, and deep‐etched samples to obtain information on spore wall organization and composition. Our observations demonstrate that the spore wall is formed by an inner 30–35 nm electron‐lucent endospore and an outer 25–30 nm electron‐dense exospore. The exospore is a complex of three layers: an outer spiny layer, an electron‐lucent intermediate lamina and an inner fibrous layer. Freeze‐fracture and deep‐etching techniques reveal that the intermediate lamina and the inner fibrous layer result from the different spatial disposition of the same 4‐nm thick fibrils. In thin sections the endospore reveals a scattered electron‐dense material that appears in the form of trabecular structures when analyzed in deep‐etched samples. The presence of chitin in the exospore is discussed.
Characterization of Encephalitozoon (Septata) intestinalis Isolates Cultured from Nasal Mucosa and Bronchoalveolar Lavage Fluids of Two AIDS Patients
Authors: DIDIER ELIZABETH S. , ROGERS LINDA B. , ORENSTEIN JAN M. , BAKER MICHAEL D. , VOSSBRINCK CHARLES R. , VAN GOOL TOM , HARTSKEERL RUDY , SOAVE ROSEMARY , BEAUDET LISA M.
Published: May 2007

ABSTRACT. Microsporidia are obligate intracellular protozoan parasites that can cause opportunistic infections in AIDS patients. Species from five genera of microsporidia are presently known to infect man. One species, Septata intestinalis originally was detected in stool specimens of individuals with chronic diarrhea and subsequently was found to disseminate to the kidneys, lungs, and nasal sinuses. This organism has since been reclassified as Encephalitozoon and in this study, we report the culture of Encephalitozoon intestinalis from a bronchoalveolar lavage specimen and a nasal mucus aspirate of two AIDS patients living in the USA. The bronchoalveolar and nasal microsporidian isolates grew in several continuous cell lines including RK‐13, MDCK, HT‐29, Caco‐2, Vero, and 1047. Transmission electron microscopy of the clinical and cell culture specimens revealed that the new isolates appeared to be E. intestinalis based on morphology and growth of organisms in septated membrane‐bound parasitophorous vacuoles. The new E. intestinalis isolates were characterized and compared with the first isolated E. intestinalis that was cultured from stool to confirm their identity and to determine if there existed any minor differences, as seen in the closely related Encephalitozoon cuniculi strains. By the methods of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis staining for proteins and carbohydrates, Western blot immunodetection, and polymerase chain reaction‐based methods with restriction endonuclease digestion, double‐stranded DNA heteroduplex mobility shift analysis, and DNA sequencing of the ribosomal DNA intergenic spacer region, the new isolates were identical to each other and to the reference isolate of E. intestinalis. In addition, with any of these methods, the E. intestinalis organisms could be distinguished from the three E. cuniculi strains, Encephalitozoon hellem, and Vittaforma corneae, which is important for diagnostics, therapeutic strategies, and epidemiology.
Molecular Cloning of a Gene Encoding Acid α‐Glucosidase from Tetrahymena pyriformis
Authors: ALAM MD. SHARIFUL , NAKASHIMA SHIGERU , DEYASHIKI VOSHIHIRO , BANNO YOSHIKO , KARA AKIRA , NOZAWA YOSHINORI
Published: May 2007

ABSTRACT. Lysosomal acid α‐glucosidase is essential for the degradation of glycogen to glucose in lysosomes. The ciliated protozoan Tetrahymena pyriformis secretes acid α‐glucosidase into its culture medium. We have earlier reported the purification and characterization of acid α‐glucosidase from T. pyriformis. The exact molecular mechanism of secretion of this enzyme has not yet been clarified. In the present study we have isolated a full length cDNA clone encoding acid α‐glucosidase from T. pyriformis. The isolated clone (3019 bp) contained an open reading frame encoding 923 amino acids, and has an estimated molecular mass of 104 kDa. Northern blot analysis revealed that the isolated cDNA hybridized to a 2.8‐kb mRNA transcript. N‐terminal amino acids after the first methionine fulfilled the requirement of a signal peptide. The deduced amino acid sequence contains the amino acid sequences determined of several peptides derived from the purified enzyme, and was found to have 34% identity and 45% similarity with that of human lysosomal enzyme, with 75% identity in the 16 amino acids at the proposed active site.
Molecular Characterization of the D Surface Protein Gene Subfamily in Paramecium primaurelia
Authors: BOURGAIN‐GUGLIELMETTI FLORENCE M. , CARON FRANCOIS M.
Published: May 2007

ABSTRACT. When Paramecium primaurelia expresses the D serotype, a major high molecular weight mRNA species is detected in the cytoplasm. Using the cDNA derived from this mRNA as a probe, three very similar genes, Dα, Dβ and Dγ, were cloned. Of these three genes, we show that only the Dα mRNA is present in the cytoplasm of cells expressing the D serotype and corresponds to the major mRNA species. The nucleotide sequence of the entire coding region of the Dα gene, as well as the upstream and downstream sequences, has been determined. The 7632‐nucleotide open reading frame encodes a putative protein that displays the characteristic cysteine residue periodicity of Paramecium surface antigens but does not contain central tandemly repeated sequences. Partial sequences of the two nonexpressed genes Dβ and Dγ indicate a high percentage of identity (90%–95%) with the Dα gene, suggesting that Dβ and Dγ genes are either very similar surface protein genes whose transcription is repressed trough mutual exclusion, or perhaps are pseudogenes. A region of variable DNA rearrangement was identified 1 kb upstream of the Dγ gene. This macronuclear region arises from the same micronuclear locus by alternative excision of internal eliminated sequences during macronuclear development.
Primary Structure and Phylogenetic Relationships of Glyceraldehyde‐3‐Phosphate Dehydrogenase Genes of Free‐Living and Parasitic Diplomonad Flagellates
Authors: ROZARIO CATHERINE , MORIN LOÏC , ROGER ANDREW J. , SMITH MICHAEL W. , MÜLLER MIKLÓS
Published: May 2007

ABSTRACT. Complete nucleotide sequences have been established for two genes (gap1 and gap2) coding for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, EC 1.2.1.12) homologs in the diplomonad Giardia lamblia. In addition, almost complete sequences of the GAPDH open reading frames were obtained from PCR products for two free‐living diplomonad species, Trepomonas agilis and Hexamita inflata, and a parasite of Atlantic salmon, an as yet unnamed species with morphological affinities to Spironucleus. Giardia lamblia gap 1 and the genes from the three other diplomonad species show high similarity to each other and to other glycolytic GAPDH genes. All amino‐acyl residues known to be highly conserved in this enzyme are also conserved in these sequences. Giardia lamblia gap2 gene is more divergent and its putative translation reveals the presence of a cysteine and serine‐rich insertion resembling a metal binding finger. This motif has not yet been noted in other GAPDH molecules. All sequences contain an S‐loop signature with characteristics close to those of eukaryotes. In phylogenetic reconstructions based on the derived amino acid sequences with neighborjoining, parsimony and maximum‐likelihood methods the four typical GAPDH sequences of diplomonads cluster into a single clade. Within this clade, G. lamblia gap1 shares a common ancestor with the rest of the genes. The latter are more closely related to each other, indicating an early separation of the lineage leading to the genus Giardia from the lineage encompassing the morphologically less differentiated genera, Trepomonas, Hexamita and that of the unnamed species. This result is discordant with the orthogonal evolution of diplomonads suggested on the basis of comparative morphology. In neighbor‐joining reconstructions G. lamblia gap2 occupies a variable position, due to its great divergence. In parsimony and maximum likelihood analysis however, it shares a most recent common ancestor with the typical G. lamblia gap1 gene, suggesting that it diverged after the separation of the Giardia lineage. The position of the diplomonad clade in broader phylogenetic reconstructions is firmly within the typical cytosolic glycolytic representatives of GAPDH of eukaryotes.
The 44‐kDa Regulatory Subunit of the Paramecium cAMP‐Dependent Protein Kinase Lacks a Dimerization Domain and May Have a Unique Autophosphorylation Site Sequence
Authors: CARLSON GAIL L. , NELSON DAVID L.
Published: May 2007

ABSTRACT. The 44‐kDa regulatory subunit (R44) of one form of cAMP‐dependent protein kinase of Paramecium was purified, and two partial internal amino acid sequences from it were used to clone the corresponding cDNA. This R44 cDNA clone was 1022‐bp long, including 978 bp of coding sequence and 7 bp and 37 bp of 5′ and 3′ untranslated sequences, respectively. A 1.1‐kb mRNA was labeled on a Northern blot. The deduced R44 amino acid sequence had 31%–38% positional identity to the sequences of other cloned cAMP‐dependent protein kinase regulatory subunits. R44 sequence showed equal sequence similarity to mammalian types I and II regulatory subunits. The N‐terminal sequence encoding the regulatory subunit dimerization domain found in most regulatory subunits is not present in the R44 clone, confirming the lack of regulatory subunit dimer formation previously reported for the Paramecium cAMP‐dependent protein kinase. The putative autophosphorylation site of R44 contains the amino acid sequence TRTS, distinct from the consensus sequence RRXS, where X is any residue, found in other autophosphorylated cAMP‐dependent protein kinase regulatory subunits and many cAMP‐dependent protein kinase substrates.
Ribosomal RNA‐Based Oligonucleotide Probes to Identify Marine Green Ultraphytoplankton1
Authors: KNAUBER DONNA C. , BERRY EUGENE S. , FAWLEY MARVIN W.
Published: May 2007

Oligonucleotide probes based on small subunit 18S rRNA variable region sequences, were designed and used to discriminate groups of marine green ultraphytoplankton of similar size and morphology. Hybridization of probes was visualized using a combination of biotinylated oligonucleotides and fluorescein labeled avidin. Increased fluorescent signal was generated by building a complex of biotinylated probe, fluorescein labeled avidin and biotinylated anti‐avidin. Cells of one phylogenetic group were easily differentiated from morphologically similar cells of a different phylogenetic group. Design of specific probes allowed identification of Mantoniella squamata, Micromonas pusilla, Pseudoscourfieldia marina and the micromonadophyte clone CCMP1194. This technique should be readily adaptable to open ocean samples and should greatly facilitate the description and quantification of ultraphytoplankton in the open ocean.
Ubiquity of Cysteine‐ and Metalloproteinase Activities in a Wide Range of Trypanosomatids
Authors: BRANQUINHA MARTA H. , VERMELHO ALANE B. , GOLDENBERG SAMUEL , BONALDO MYRNA C.
Published: May 2007

We have analysed the proteinase profiles of 11 species from 7 different genera of trypanosomatids by in situ detection of enzyme activities on SDS‐PAGE gels containing co‐polymerized gelatin as substrate, and the use of specific proteinase inhibitors. Our survey indicates that while cysteine‐ and metalloproteinases are distributed ubiquitously among trypanosomatids, there are marked differences between the enzyme profiles from the monogenetic (Crithidia, Herpetomonas, Leptomonas) and digenetic (Trypanosoma, Endotrypanum, Phytomonas, Leishmania) species. The detected metalloproteinase activities, ranging in size from 50–100 kDa, partitioned into the detergent‐phase after Triton X‐114 extraction, while most of cysteine proteinases, of three distinct molecular mass ranges (30–50 kDa, 80–100 kDa and 116–205 kDa), partitioned into the aqueous phase. Thus, within this group of organisms, the metalloproteinase activities seem to be predominantly membrane‐associated proteins. We also show that the plant parasites of the genus Phytomonas exhibit a distinctive cysteine proteinase profile that might be exploited further as a criterion for taxonomy of the genus.
Identifying and Distinguishing Sibling Species in the Tetrahymena pyriformis Complex (Ciliophora, Oligohymenophorea) Using PCR/RFLP Analysis of Nuclear Ribosomal DNA
Authors: JEROME CHERYL A. , LYNN DENIS H.
Published: May 2007

ABSTRACT We describe a riboprinting strategy for identifying and distinguishing among sibling species in the Tetrahymena pyriformis complex. It involves use of the polymerase chain reaction to amplify a large segment of the nuclear ribosomal DNA and internal transcribed spacers, and digestion of this DNA with restriction enzymes. Unique restriction fragment length patterns or haplotypes were then used to distinguish species into: (1) six taxa that were identifiable to the species level, (2) eight taxa that were separated into four pairs, and (3) a group of eight taxa that were identical to each other. The latter result indicates that a more variable molecule is needed to distinguish the most closely related species in the complex. There was no intraspecific variation between two strains from one species (Tetrahymena thermophila) nor among multiple isoiates from another species (Tetrahymena empidokyrea). This approach provides an alternative to traditional techniques for identifying T. pyriformis species that require living reference specimens and/or that reveal high levels of intraspecific variation.
Survival and Infectivity of Pneumocystis carinii Outside the Mammalian Host.
Authors: KANESHIRO EDNA S. , MAIORANO J. NICHOLAS
Published: May 2007

SUMMARY Air blower prefilters servicing HEPA‐filtered Biobubble housing Pneumocystis carinii‐infected rats were stored for up to five months at ‐80°C to room temperature. After storage, 76% of immunosuppressed rats exposed to these prefilters developed P. carinii infections. In contrast, only 4% of control immunosuppressed rats exposed to autoclaved filters had P. carinii infections. These observations indicate that the organism has a dormant form that remains infective for at least several months outside the mammalian host.
ITSs Typing of P. carinii Samples from Italy, The Netherlands and Tanzania.
Authors: ATZORI CHIARA , AGOSTONI FIORENZA , CARGNEL ANTONIETTA , PERENBOOM ROOS , BECKERS PETER
Published: May 2007

SUMMARY P. carinii molecular epidemiology appears a new interesting investigational field to understand distribution and incidence of isolates from different geographical locations. Recently a typing system, the Type Specific Oligoblotting (TSO) based on 6 different sequences of the Internal Transcribed Spacers (ITSs) of P. carinii rRNA has been developed [1]. By using P. carinii ITSs nested PCR followed by TSO hybridization we have typed 55 lung derived specimens collected in Italy, The Netherlands and sub‐Saharian Africa from pts with microwpically detected P. carinii pneumonia.
Development of a Rapid Detection Procedure for Ctyptosporidium, Using In Vitro Cell Culture Combined with PCR
Authors: ROCHELLE PAUL A. , FERGUSON DONNA M. , HANDOJO TROY J. , LEON RICARDO DE , STEWART MIC H. , WOLFE ROY L.
Published: May 2007

A detection, viability, and infectivity assay was developed for Cryptosporidiurn parvum. Oocysts or excysted sporozoites were inoculated onto monolayers of CaCo‐2 cells grown on chamber slides. C. parvum infection was monitored by three methods: a) application of a fluorescein‐labeled anti‐sporozoite antibody; b) PCR of a heat‐shock protein gene fragment; and c) detection of mRNA from the heat‐shock protein gene by RT‐PCR.
Gram‐Chromotrope: a New Technique that Enhances Detection of Microsporidial Spores in Clinical Samples
Authors: MOURA HERCULES , SILVA JORGE L. NUNES DA , SODRÉ FERNANDO C. , BRASIL PATRICIA , WALLMO KRISTY , WAHLQUIST SUSANNE , WALLACE SARA , CROPPO GIAN P. , VISVESVARA GOVINDA S.
Published: May 2007

We have developed a new staining procedure that combines the traditional Gram staining for bacteria and the Weber's chromotrope staining method, the standard technique for the detection of microsporidia spores in clinical Specimens. This “Gram‐chromotrope” staining technique enhances the staining characteristics of microsporidia spores and facilitates the easy detection and differentiation of spores from other microorganisms that are found in clinical specimens, especially stool samples. This new technique is fast, reliable, and simple to perform, and can be easily adapted for use in clinical laboratories.
Parasite Diversity in an Endemic Region for Avian Malaria and Identification of a Parasite Causing Penguin Mortality
Authors: MCCONKEY GLENN A. , LI JUN , ROGERS M. JOHN , SEELEY DOUGLAS C. , GRACZYK THADDEUS K. , CRANFIELD MICHAEL R. , MCCUTCHAN THOMAS F.
Published: May 2007

ABSTRACT Understanding the population structure of Plasmodium parasites is essential for malaria intervention. A survey of parasites in vectors and host infections was conducted in an area of intense mortality due to malaria in a captive penguin (Spheniscus demersus) colony, using a novel method for identification of Plasmodium species by amplification of ribosomal sequences in DNA or RNA. Three phylogenetically distinct groups of avian Plasmodium were detected in mosquitoes (Culex) collected at the study site (Baltimore Zoo, Baltimore, MD) during a period of high transmission. One of the three clades of Plasmodium was found to be prevalent in penguins monitored through the malaria transmission season and consistent with morphological identification as Plasmodium relictum. This parasite sequence was directly associated with the death of a penguin. Thus, a complete transmission cycle is defined at this site. Phylogenetic comparison of ribosomal sequences to an authenticated reference strain of Plasmodium relictum indicates that this is not the parasite causing death in the penguins, suggesting that different parasites may be morphologically indistinguishable.
Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM
Authors: ERLANDSEN STANLEY L. , MACECHKO PAUL T. , KEULEN HARRY VAN , JARROLL EDWARD L.
Published: May 2007

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14‐16 h. The intracellular phase lasted for 8‐10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4‐6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (‐15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.
Ribosomal RNA Analysis Indicates a Benthic Pennate Diatom Ancestry for the Endosymbionts of the Dinoflagellates Peridinium foliaceum and Peridinium balticum (Pyrrhophyta)
Authors: CHESNICK JOBY M. , KOOISTRA WIEBE H. C. F. , WELLBROCK URSULA , MEDLIN LINDA K.
Published: May 2007

ABSTRACT. The establishment of chloroplasts as cellular organelles in the dinoflagellate, heterokont (stramenopile), haptophyte, and cryptophyte algae is widely accepted to have been the result of secondary endosymbiotic events, that is, the uptake of a photosynthetic eukaryote by a phagotrophic eukaryote. However, the circumstances that promote such associations between two phylogenetically distinct organisms and result in the integration of their genomes to form a single functional photosynthetic cell is unclear. The dinoflagellates Peridinium foliaceum and Peridinium balticum are unusual in that each contains a membrance‐bound eukaryotic heterokont endosymbiont. These symbioses have been interpreted, through data derived from ultrastructural and biochemical investigations, to represent an intermediate stage of secondary endosymbiotic chloroplast acquistion. In this study we have examined the phylogenetic origin of the P. foliaceum and P. Balticum heterokont endosymbionts through analaysis of their nuclear small subunit ribosomal RNA genes. Our analyses clearly demonstrate both endosymbionts are pennate diatoms belonging to the family Bacillariaceae. Since members of the Bacillariaceae are usually benthic, living on shallow marine sediments, the manner in which establishment of a symbiosis between a planktonic flagellated dinoflagellate and a botton‐dwelling diatom is discussed. In particular, specific environmentally associated life strategy stages of the host and symbiont, coupled with diatom food preferences by the dinoflagellate, may have been vital to the formation of this association.
Distribution of Epitopes of Trypanosoma cruzi Amastigotes During the Intracellular Life Cycle within Mammalian Cells
Authors: BARROS HELENA C. , VERBISCK NEWTON V. , SILVA SOLANGE DA , ARAGUTH MARCIA F. , MORTARA RENATO A.
Published: May 2007

ABSTRACT. In this study we have examined the distribution of epitopes defined by monoclonal antibodies raised against Trypanosoma cruzi amastigotes during the intraceullar life cycle of the parasite. We have raised monoclonal antibodies towards amastigote forms and performed preliminary immunochemical characterization of their reactivities. MAB 1D9, 3G8, 2B7, 3B9, and 4B9, and 4B9 react with carbohydrate epitopes of the parasite major surface glycoprotein—Ssp‐4 defined by MAB 2C2 [5]: MAB 4B5 reacts with a noncarbohydrate epitope in all developmental stages of the parasite, and MAB 3B2 also detects a noncarbohydrate epitope preferentially in T. cruzi flagellared forms. Vero cells infected with tissue culture‐derived trypomastigotes of clone D11 (G strain) were fixed at different times during the intraceullular proliferation of parasites, and processed for immjno‐electron microscopy and confocal immunoflurescence with the different monoclonal antibodies. We observed that while the surface distribution of MAB 2C2 and 4B9 epitopes was uniform throughout the cycle, MAB 1D9, 3G8, and 2B7 reacted with cytoplasmic membrance‐bound compartments of the amastigotes. MAB 3B9 displayed a unique surface dentate pattern in some amastigotes. MAB 4B5 recognized a curved‐shaped structure at the flagellar pocket region in some intracellular amastigotes and localized to the membrane in dividing forms. In intracellular trypomastigotes, MAB 4B5 also displayed a punctate pattern near the flagellar pocket.
Conjugation‐Specific Genes in the Ciliate Euplotes crassus: Gene Expression from the Old Macronucleus
Authors: LING ZHONG , GHOSH SUSMITA , JACOBS MARY ELLEN , KLOBUTCHER LAWRENCE A.
Published: May 2007

ABSTRACT. Following mating or conjugation, the hypotrichous ciliate Euplotes crassus undergoes a massive genome reorganization process. While the nature of the rearrangement events has been well studied, little is known concerning proteins that carry out such processes. As a means of identifying such proteins, differential screening of a developmental cDNA library, as well as construction of a cDNA subtraction library, was used to isolate genes expressed only during sexual reproduction. Five different conjugation‐specific genes have been identified that are maximally expressed early in conjugation, during the period of micronuclear meiosis, which is just prior to macronuclear development and the DNA rearrangement process. All five genes are retained in the mature macronucleus. Micronuclear, macronuclear, and cDNA clones of one gene (conZ47) have been sequenced, and the results indicate that the gene encodes a putative DNA binding protein. In addition, the presence of an internal eliminated sequence in the micronuclear copy of the conZ47 gene indicates that this conjugation‐specific gene is transcribed from the old macronucleus.
Lipid Composition of the Marine Ciliates Pleuronema sp. and Fabrea salina: Shifts in Response to Changes in Diet1
Authors: HARVEY H. RODGER , EDERINGTON MELISSA C. , MCMANUS GEORGE B.
Published: May 2007

ABSTRACT. The composition and incorporation of lipids in two marine ciliates, Pleuronema sp. and Fabrea salina, was examined following growth on either an algal or bacterial diet. When allowed to feed on a natural bacterial community, Pleuronema sp. synthesized the triterpenoid alcohol gammaceran‐3β‐ol (tetrahymanol) and two hopanoids (hopan‐3β‐ol and one uncharacterized hopanoid). When fed the marine alga Isochrysis galbana, F. salina contained the major algal sterol 24‐methylcholesta‐5, 22‐dien‐3β‐ol and several long chain ketones specific to the alga. In both ciliates, fatty acids composition showed a general correspondence to that of the diet. Using a series of antibiotic treatments to alter the bacterial prey community, and thus fatty acid composition of the ciliate's diet, promoted changes in the fatty acid composition of Pleuronema sp. to resemble that of the bacterial prey. The addition of a mixture of algal sterols to a bacterized culture of another scuticociliate, Parauronema acutum, inhibited tetrahymanol synthesis and resulted in the incorporation of sterols into the ciliate.
Trophic Controls on Stage Transformations of a Toxic Ambush‐Predator Dinoflagellate1
Authors: BURKHOLDER JOANN M. , GLASGOW HOWARD B.
Published: May 2007

ABSTRACT. The toxic dinoflagellate, Pfiesteria piscicida, was recently implicated as the causative agent for about 50% of the major fish kills occurring over a three‐year period in the Albemarle‐Pamlico Estuarine System of the southeastern USA. Transformations between life‐history stages of this dinoflagellate are controlled by the availability of fresh fish secretions or fish tissues, and secondarily influenced by the availability of alternate prey including bacteria, algae, microfauna, and mammalian tissues. Toxic zoospores of P. piscicida subdue fish by excreting lethal neurotoxins that narcotize the prey, disrupt its osmoregulatory system, and attack its nervous system. While prey are dying, the zoospores feed upon bits of fish tissue and complete the sexual phase of the dinoflagellate life cycle. Other stages in the complex life cycle of P. piscidia include cryptic forms of filose, rhizopodial, and lobose amoebae that can form within minutes from toxic zoospores, gametes, or planozygotes. These cryptic amoebae feed upon fish carcasses and other prey and, thus far, have proven less vulnerable to microbial predators than flagellated life‐history stages. Lobose amoebae that develop from toxic zoospores and planozygotes during colder periods have also shown ambush behavior toward live fish. In the presence of abundant flagellated algal prey, amoeboid stages produce nontoxic zoospores that can become toxic and form gametes when they detect what is presumed to be a threshold level of a stimulatory substance(s) derived from live fish. The diverse amoeboid stages of this fish “ambush‐predator” and at least one other Pfiesteria‐like species are ubiquitous and abundant in brackish waters along the western Atlantic and Gulf Coasts, indicating a need to re‐evaluate the role of dinoflagellates in the microbial food webs of turbid nutrient‐enriched estuaries.
Small Subunit Ribosomal RNA Sequences Unite Alternate Actinosporean and Myxosporean Stages of Myxobolus cerebralis the Causative Agent of Whirling Disease in Salmonid Fish
Authors: ANDREE KARL B. , GRESOVIAC STEPHANE J. , HEDRICK RONALD P.
Published: May 2007

ABSTRACT. The alternating myxosporean and actinosporean stages of the myxozoan parasitc Myxobolus cerebralis (Hofer 1903) from its salmonid fish and aquatic oligochaete hosts, respectively, were compared for sequence homology of the small subunit (18S) ribosomal RNA genes. A 99.8% similarity between the sequences of these two stages was substantially greater than that of M. cerebralis compared to two other Myxobolus sp. from salmonid fish. Our results are the first molecular evidence confirming the alternating stages initially described by Wolf and Markiw [25] for the life cycle of M. cerebralis but found in two different taxonomic classes (Myxosporea and Actinosporea) are indeed forms of the same organism. Sequencing of rRNA genes of the actinosporean stage followed by development of specific primers for DNA amplification of the myxosporean stage, as in our study, should be applied to solve other myxozoan life cycles. Additionally, these approaches will in the future provide useful diagnostic reagents for the detection and study of this important group of fish pathogens.