bluuebunny/crossref_metadata_2025 huggingface dataset:
Giardia lamblia: Identification and Characterization of a Variant‐Specific Surface Protein Gene Family
Authors: NASH THEODORE E. , CONRAD JOHN T. , MOWATT MICHAEL R.
Published: May 2007

ABSTRACT. Giardia lamblia trophozoites undergo antigenic variation by modulating the expression of variant‐specific surface proteins (VSP), which are encoded by a number of small multigene families. We characterized the genomic copy of the VSP gene expressed by the cloned trophozoite line H7, derived from the isolate GS/M, in addition to a related, but nonexpressed, family member. The coding regions of the two genes encode closely related polypeptides (86% identity). However, differences in the coding region of these genes reside solely in an 873 bp segment. Only four differences were found between the 5’flanking sequences (465 bp). The proximal 205 base pairs downstream from the coding regions were identical, but thereafter the sequences diverged (37% identity over the next 391 bp). Mapping studies indicated that no other VSP gene was located within 4 kb pairs of the expressed H7 VSP gene, and transcripts from the nonexpressed gene were detected in neither GS/H7 nor heterogeneous trophozoites populations derived from this cloned line. Any mechanisms responsible for the differential expression of VSP genes must reconcile the near identity of DNA sequences that flank the coding regions of expressed and nonexpressed VSP genes.
Molecular Cloning and Characterization of a Leishmania donovaniα‐Tubulin Gene
Authors: JOSHI MANJU , DWYER DENNIS M. , NAKHASI HIRA L.
Published: May 2007

ABSTRACT. We have isolated a cDNA for an α‐tubulin mRNA from L. donovani promastigotes and determined its complete nucleotide sequence. Both nucleotide and deduced amino acid sequence analysis of this cDNA showed significant similarity with a previously reported, partial sequence of an L. enriettiiα‐tubulin and the complete sequence of human α‐tubulin. Further, the in vitro translated L. donovaniaα‐tubulin gene product was specifically immunoprecipitated with a monoclonal antibody against human α‐tubulin. Northern blot analysis revealed that there was little change in the expression of the L. donovaniα‐tubulin RNA during parasite differentiation from promastigote to the in vitro grown “amastigote” form. Southern blot analysis revealed a simple genomic organization for the L. donovaniα‐tubulin gene with more than one copy of the α‐tubulin gene in the parasite genome. To our knowledge, this is the first complete sequence of an α‐tubulin for Leishmania to be reported in the literature.
An Improved Method to Obtain High Molecular Weight DNA from Purified Micro‐ and Macronuclei of Tetrahymena thermophila
Authors: CHAU MIU‐FUN , ORIAS EDUARDO
Published: May 2007

An improved method to obtain high molecular weight DNA from purified macro‐ and micronuclei of Tetrahymena thermophila is described. Micro‐ and macronuclear DNA obtained using previously described protocols was degraded and not suitable for the cloning of large (> 100 kb) DNA fragments. Based on the data reported here, we propose that DNA degradation is mainly due to nuclease activity; some micronuclear DNA degradation is due to mechanical shearing as a result of extended periods of blending. We have made modifications to reduce nuclease degradation by minimizing cell lysis, by the early addition of EDTA and by increasing the EDTA concentration (23 mM). To reduce mechanical shearing, cell and nuclear suspensions were blended for shorter periods. High molecular weight micro‐ and macronuclear DNA was obtained using the new protocol.
In Situ Hybridization as a Method to Study the Regulation of Gene Expression in Paramecium
Authors: CURTENAZ SOPHIE , BEISSON JANINE
Published: May 2007

A method for whole cell in situ hybridization in Paramecium was developed, which allowed us to visualize both the cytoplasmic mRNA and the transcripts in the macronucleus, and to estimate their respective variation throughout the cell cycle. Nonisotopic DNA probes, labeled with digoxigenin (DIG), were prepared by PCR from a central part of two genes of P. tetraurelia, respectively coding for a β‐tubulin and the 51A surface antigen (51A‐sAg). The probes were detected with an FITC‐conjugated anti‐DIG antibody. While no fluorescence is observed in the micronuclei, mRNA are detected in the macronucleus, as discrete spots which disappear after RNase H treatment, in the cytoplasm where they are uniformly distributed, and in the post‐autogamous fragments of the old macronucleus. The intensity of the cytoplasmic labeling observed with the two probes shows a clear and different cell cycle‐dependent modulation. For the β‐tubulin mRNA, a peak is observed at the beginning of mitosis, while for the 51A‐sAg, the peak occurs later, during cytokinesis. The macronuclear labeling, which likely reflects the transcription rate, seems roughly constant for the 51A‐sAg. In contrast, for the β‐tubulin genes, the intensity of the macronuclear signal shows a peak that just precedes the cytoplasmic peak.
Gametocytogenesis of Leucocytozoon caulleryi in In Vitro Culture: Effect of Human Red Blood Cells on the Development of Second‐Generation Merozoites
Authors: OHNISHI YOSHIHIRO , ONO TADASUKE , MORII TSUTOMU
Published: May 2007

For investigating development of second‐generation merozoites of Leucocytozoon caulleryi into mature gametocytes, infected erythrocytes from chickens at 15 d after sporozoites inoculation were cultured in RPMI‐1640 modified medium supplemented with 10% horse serum and 0.5 ml of human erythrocytes (type O). When culture was carried out at 37°C in a humidified atmosphere of 5% CO2 for 7 d, the very small number of second‐generation merozoites developed into morphologically mature gametocytes. However, in the high carbon dioxide and low oxygen condition, mature gametocytes weren't observed in cluture. The role of human erythrocytes added has not been clarified yet.
Repetitive Micronuclear Divisions in the Absence of the Macronucleus During Conjugation of Paramecium caudatum
Authors: MIKAMI KAZUYUKI
Published: May 2007

ABSTRACT. The germinal micronucleus divides six times during conjugation of Paramecium caudatum: this includes two meiotic divisions and one mitosis of haploid nuclei during mating, and three mitoses of a fertilization nucleus (synkaryon). Microsurgical removal of the macronucleus showed that micronuclei were able to divide repeatedly in the absence of the macronucleus, after metaphase of meiosis I of the micronucleus and also after synkaryon formation. When the macronucleus was removed after the first division of synkaryon, in an extreme case the synkaryon divided five times and produced 32 nuclei, compared to three divisions and eight nuclei produced in the presence of the macronucleus. Treatment with actinomycin D (100 μ/ml) inhibited the morphological changes of the macronucleus during conjugation and induced a multimicronucleate state in exconjugants. However, in other cells, it induced production of a few giant micronuclei. We conclude that the micronucleus is able to undergo repeated divisions at any stage of conjugation in the absence of the macronucleus once the factor(s) for induction of the micronuclear division has been produced by the macronucleus. The macronucleus may also produce a regulatory factor required to stop micronucler division.
Release of Membrane‐Bound Trails by Trypanosoma cruzi Amastigotes onto Modified Surfaces and Mammalian Cells
Authors: BARROS HELENA C. , SILVA SOLANGE DA , VERBISCK NEWTON V. , ARAGUTH MARCIA F. , TEDESCO ROBERTO C. , PROCOPIO DANIELA O. , MORTARA RENATO A.
Published: May 2007

ABSTRACT. Upon incubation at 37° C onto glass coverslips coated with Concanavalin A, poly‐L‐lysine, or a monoclonal antibody (1D9) directed to the parasite major surface glycoprotein Ssp‐4, extracellular Trypanosoma cruzi amastigotes release trails of material barely visible by light microscopy. This release is not associated with parasite movements. Immunolabeling studies confirmed that the material is derived from the parasite's membrane since thin section through samples labeled with 1D9 revealed that the trails are membrane‐bound structures. Scanning electron microscopy showed that the ∼0.1‐μm thick trails of material emerging from the amastigotes can be uniform or beaded, indicating a tendency to vesiculation. The trails are preferentially released from the flagellar pocket region and/or at the opposite posterior end of the parasite body, and seem to be devoid of microtubules. The release is time and temperature‐dependent and fixed parasites do not form trails. All attempts to inhibit trail release using drugs (antimycin A, sodium azide, cytochalasin D, nocodazole, genistein, staurosporine, EGTA) failed. The observation of trails associated with intracellular parasites and amastigotes invading Vero cells suggests that this is probably a physiological process.
Detection of Genomic Polymorphism in Plasmodium falciparum Using an Arbitrarily Primed PCR Assay
Authors: ROJAS MARIA O. , DE‐CASTRO JOHANNA , MARINO GIOVANNA , WASSERMAN MOISES
Published: May 2007

ABSTRACT. Modifications of the arbitrarily primed polymerase chain reaction assay (i.e. a low annealing temperature and a very slow increase in the temperature during the elongation steps during the amplification cycles) allowed it to be used with the AT‐rich Plasmodium falciparum DNA. The analysis of the products by polyacrylamide—urea gels, after silver staining, resulted in high resolution and sensitivity. Eighteen single and six combined pairs of arbitrary primers were tested. Two produced polymorphic patterns complex enough to differentiate between close Colombian isolates in a single assay. This method may be useful in studying the distribution and migration of strains in endemic areas, and for identifying intralaboratory cross‐contamination of cultures.
Paramecium Has Two Regulatory Subunits of Cyclic AMP‐Dependent Protein Kinase, One Unique to Cilia
Authors: HOCHSTRASSER MARTIN , CARLSON GAIL L. , WALCZAK CLAIRE E. , NELSON DAVID L.
Published: May 2007

ABSTRACT. The subunit composition and intracellular location of the two forms of cAMP‐dependent protein kinase of Paramecium cilia were determined using antibodies against the 40‐kDa catalytic (C) and 44‐kDa regulatory (R44) subunits of the 70‐kDa cAMP‐dependent protein kinase purified from deciliated cell bodies. Both C and R44 were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48‐kDa protein (R48) weakly recognized by one of several monoclonal antibodies against R44, but not recognized by an anti‐R44 polyclonal serum. Gel‐filtration chromatography of a soluble ciliary extract resolved a 220‐kDa form containing C and R48 and a 70‐kDa form containing C and R44. In the large enzyme, R48 was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R44 The subunits of the large enzyme subsequently were purified to homogeneity by cAMP‐agarose chromatography. Both C and R48 were retained by the column and eluted with 1 M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP‐dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell‐body enzyme, contains R44, whereas R48 is the R subunit of the large enzyme.
Ecological Genetics of Tetrahymena thermophila: Mating Types, i‐Antigens, Multiple Alleles and Epistasis
Authors: DOERDER F. PAUL , ARSLANYOLU MUHITTIN , SAAD YASSER , KACZMAREK MICHELLE , MENDOZA MANUEL , MITA BARRY
Published: May 2007

Until recently, Tetrahymena thermophila has rarely been isolated from nature. With improved sampling procedures, T. thermophila has been found in ponds in many northeastern states. The availability of resident populations makes possible both population and ecological genetic studies. All seven known mating types have been recovered; no eighth mating type has been found. Crosses among whole‐genome homozygotes derived from Pennsylvania isolates reveal a spectrum genotypes with mating type alleles resembling traditional A (IV‐ and VII‐) and B(I‐) categories. The genotypes differ significantly with respect to mating type frequency, both among themselves and from previously described genotypes. One A‐category genotype appears to lack mating type II, while one A‐category and all B‐category genotypes have low frequencies of mating type III, thus accounting for the low frequency of III in the pond. The low frequency of III in all five B‐category genotypes examined suggests that the founding allele in this region was low for III. These and other differences are discussed both in terms of mating type frequencies in the pond and in terms of the possible molecular structure of mat alleles. By contrast, numerous variants of the cell surface immobilization antigen are found in addition to the previously described i‐antigens. Variants of the known SerH alleles include those with restriction fragment length polymorphisms and temperature sensitivity as well as alleles with new antigenic specificity. Multiple alleles are present in single ponds. Genes exhibiting serially dominant epistasis over SerH genes also are found. In two instances (K and C), families of antigenically similar polypeptides are expressed in place of H i‐antigen. Molecular weight differences suggest that these paralogous i‐antigen genes evolve by gene duplication and unequal crossing over within central repeats. The existence of complex patterns of epistasis together with seasonal changes in i‐ag frequencies suggest that i‐ag play an important, but as yet unknown, ecological role related to the occurrence of frequent conjugation.
Molecular Identification of Nanoplanktonic Protists Based on Small Subunit Ribosomal RNA Gene Sequences for Ecological Studies1
Authors: LIM EE LIN
Published: May 2007

Nanoplanktonic protists are comprised of a diverse assemblage of species which are responsible for a variety of trophic processes in marine and freshwater ecosystems. Current methods for identifying small protists by electron microscopy do not readily permit both identification and enumeration of nanoplanktonic protists in field samples. Thus, one major goal in the application of molecular approaches in protistan ecology has been the detection and quantification of individual species in natural water samples. Sequences of small subunit ribosomal RNA (SSU rRNA) genes have proven to be useful towards achieving this goal. Comparison of sequences from clone libraries of protistan SSU rRNA genes amplified from natural assemblages of protists by the polymerase chain reaction (PCR) can be used to examine protistan diversity. Furthermore, oligonucleotide probes complementary to short sequence regions unique to species of small protists can be designed by comparative analysis of rRNA gene sequences. These probes may be used to either detect the RNA of particular species of protists in total nucleic acid extracts immobilized on membranes, or the presence of target species in water samples via in situ hybridization of whole cells. Oligonucleotide probes may also serve as primers for the selective amplification of target sequences from total population DNA by PCR. Thus, molecular sequence information is becoming increasingly useful for identifying and enumerating protists, and for studying their spatial and temporal distribution in nature. Knowledge of protistan species composition, abundance and variability in an environment can ultimately be used to relate community structure to various aspects of community function and biogeochemical activity.
Oxidation of Alanine, Acetate, Glutamate, and Succinate by Digitonin‐Permeabilized Leishmania major Promastigotes
Authors: BLUM J. J.
Published: May 2007

Leishmania major promastigotes were treated with digitonin and the rates at which [1 ‐14C]acetate, [1,4‐14C]succinate, [1‐14C]glutamate, and [U‐14C]alanine are oxidized were measured in the presence of suitable cofactors. Acetate was oxidized at the lowest rate of the four substrates examined, even in the presence of added NAD, CoA, ADP and acetyl‐CoA synthase. Its rate of oxidation was negligible if the permeabilized cells were washed before the cofactors were added, indicating the requirement for an as yet unknown factor. Succinate was oxidized at a rate much higher than the very slow rate at which it is oxidized by intact cells. Its rate of oxidation was strongly inhibited by antimycin A, but that of glutamate was scarcely affected. Fumarate inhibited the rate of oxidation of acetate, glutamate, and succinate, but increased that of alanine, Ca++ inhibited the rates of oxidation of alanine and succinate, but not of acetate or glutamate. Increasing the osmolality by addition of mannitol partially inhibited the rate of oxidation of alanine but had little effect on that of glutamate. These results show that appreciable transaminase activity remains in the permeabilized cells and support earlier data indicating the presence of a branched NAD‐to‐cytochrome oxidase system. These results also provide preliminary information on the sensitivity of the two branches to Ca++, hyperosmolality, and Krebs cycle intermediates
Subgenus Systematics of Acanthamoeba: Four Nuclear 18S rDNA Sequence Types
Authors: GAST REBECCA J. , LEDEE DOLENA R. , FUERST PAUL A. , BYERS THOMAS J.
Published: May 2007

ABSTRACT Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes (Rns) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S‐7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes, or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%–4.3%, whereas differences among sequence types were 6%–12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.
Ultrastructure of the Trichomonad Flagellate Stephanonympha nelumbium
Authors: RÖSEL JOACHIM , RADEK RENATE , HAUSMANN KLAUS
Published: May 2007

ABSTRACT Stephanonympha nelumbium is a large trichomonad measuring 45–60 μm in length and 20–40 μm in width. It is a member of the multinucleate and multiflagellate family Calonymphidae. While the numerous flagella arise in groups of four at the anterior cell pole, the posterior body portion is covered with attached spirochetes and rod‐like bacteria. Generally, in the apical body portion of S. nelumbium, 50–100 nuclei are arranged in five to seven circular rows. Each nucleus is associated with a typical mastigont system, comprised of three anterior flagella, one recurrent flagellum being attached to the cell surface for a certain distance, and several typical root structures. Akaryomastigonts and costas do not occur. The fine structure of S. nelumbium corresponds with that of other calonymphids. The main difference to Calonympha is that the axostyle does not embrace the nucleus but passes it in form of a flattened rod.
A Nucleoside Diphosphate Kinase from Paramecium tetraurelia with Protein Kinase Activity
Authors: ANN KYOUNG‐SOOK , NELSON DAVID L.
Published: May 2007

Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound α‐labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid‐stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross‐reacted with 16‐ to 20‐kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase‐like awd protein of Drosophila and Nm23 protein of humans.
A Redescription of Gymnodinioides caridinae (Miyashita 1933) from Palaemonetes sinensis (Sollaud 1911) in the Songhua River
Authors: BRADBURY PHYLLIS C. , ZHANG LI‐MIN , SHI XIN‐BAI
Published: May 2007

ABSTRACT The freshwater exuviotrophic apostome, Gymnodinioides caridinae, was discovered in the Songhua River where it flows through Harbin, People's Republic of China. This apostome species thus has been found in Japan, China, and Belgium, but on different species of shrimp in each place. Protargol impregnations of Gymnodinioides caridinae on Palaemonetes sinensis (Sollaud) confirmed most of Miyashita's original description of the infraciliature and may explain the structure he interpreted as an accessory contractile vacuolar canal.
Phagosomal Proteins of Dictyostelium discoideum
Authors: REZABEK BETSY L. , RODRIGUEZ‐PARIS JUAN M. , CARDELLI JAMES A. , CHIA CATHERINE P.
Published: May 2007

ABSTRACT. In recognizing food particles, Dictyostelium cell‐surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradietns. Protein analyses of these vesicles showed that they contained glycoproteins and surface‐labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa‐actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H+‐ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis.
In Vitro Cultivation of the Vascular Phase of Sarcocystis singaporensis
Authors: JÄKEL THOMAS , HENKE MARION , WEINGARTEN BERND , KLIEMT DAMARIS , SEIDINGER SILVIA
Published: May 2007

To establish an in vitro culture system for the precystic phase of Sarcocystis singaporensis, we initially tested various excysting fluids for sporocysts. An excysting fluid containing 2.5% bovine taurocholate and 10% bile of the specific intermediate host, Rattus norvegicus, in RPMI medium was the most suitable resulting in excystation of 80% of the sporozoites. Subsequently, we identified brain endothelial cells and pneumonocytes of the rat to promote growth of sporozoites to schizonts. Hepatoma, fibroblastic, or myoblastic cells were not suitable for the parasite's development. First‐generation schizonts were seen at days 3‐10 postinoculation (PI); a distinct second peak of schizogonic development only occurred in endothelial cells at days 14‐18 PI. First‐generation schizonts were 26.0 (± 3.8) μm in diameter and contained 32‐50 merozoites, second‐generation schizonts measured 34.4 (± 10.6) μm and contained 54‐72 merozoites. Merozoite yield at large‐scale culture conditions (75 cm2 flasks) using pneumonocytes as host cells was relatively low. Ultrastructurally, sporozoites and merozoites were quite similar to corresponding stages of other Sarcocystis species. With regard to host cell specificity and developmental kinetics, in vitro cultivation showed close similarities to the situation in vivo.
Fluorescently‐labeled Oligonucleotide Probes Can Be Used To Identify Protistan Food Vacuole Contents
Authors: GUNDERSON JOHN H. , GOSS SUSAN H.
Published: May 2007

ABSTRACT. In situ hybridization using fluorescent oligonucleotide probes complementary to unique regions of 16S rRNA molecules provides a way of identifying the food vacuole contents of bactivorous protists. Laboratory experiments with Tetrahymena showed rRNAs in food vacuoles are degraded slowly enough to permit their use as hybridization targets for such probes. A probe specific for a hypervariable region of the small subunit rRNA of an unnamed proteobacterium abundant in a local lake was then synthesized. It was used to probe the food vacuoles of the ciliates present in fixed water samples collected from the same lake. The vacuoles of several filter‐feeding ciliates bound the probe, indicating that such probes can be used to identify the food vacuole contents of ciliates collected from natural samples.
Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei
Authors: GOLDBERG BURT , YARLETT NIGEL , RATTENDI DONNA , LLOYD DAVID , BACCHI CYRUS J.
Published: May 2007

ABSTRACT. The fate of the [methyl‐14C] group of S‐adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl‐14C]methionine, [U‐14C]methionine, S‐[methyl‐14C]AdoMet or [35S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl‐14C] and [U‐14C]methionine to [35S]methionine. After 4‐h incubation with [U‐14C]methionine, [methyl‐14C]methionine or [35S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U‐14C] or [methyl‐14C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight‐ and twofold more rapidly, respectively, than cells incubated with [35S]methionine and cycloheximide. [Methyl‐14C] and [U‐14C]methionine incorporation were > 85% decreased by co‐incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4‐h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U‐14C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U‐14C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U‐14C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post‐methylation products of AdoMet in African trypanosomes.
Effects of Carboxylemethylation and Polyamine Synthesis Inhibitors on Methylation of Trypanosoma brucei Cellular Proteins and Lipids
Authors: GOLDBERG BURT , RATTENDI DONNA , YARLETT NIGEL , LLOYD DAVID , BACCHI CYRUS J.
Published: May 2007

ABSTRACT. The fate of methionine in eukaryotic cells is divided between protein synthesis and the branched pathway encompassing polyamine synthesis, methylation of proteins and lipids, and transsulphuration reactions. Aside from protein synthesis, the first step to all other uses of methionine is conversion to S‐adenosylmethionine. Blockade of polyamine synthesis in African trypanosomes by the ornithine decarboxylase inhibitor DL‐α‐difluoromethylomithine (Ornidyl, DFMO) the AdoMet decarboxylase inhibitor 5′‐{[(Z)‐4‐amino‐2‐butenyl]‐methylamino}‐5′‐deoxyadenosine or the protein methylase inhibitor sinefungin induces dramatic increases in intracellular AdoMet. In a previous study, distribution and pool sizes of [15S] or [U‐14C]methionine were followed in bloodform trypanosomes as incorporation into the total TCA precipitable fraction. In the present study, the effects of pretreatment with DFMO (1 mM), MDL 73811 (1 μM) and sinefugin (2 nM) on [35S] and [U‐14C]methionine incorporation were studied in blood forms. DFMO or MDL 73811 pretreatment increased protein methylation 1.5‐fold through incorporation of [U14C]methionine, while sinefungin caused a 40% reduction of incorporation. The increases in incorporation of [U‐14C]methionine due to DFMO and MDL 73811 were reduced 40% to 70% by including cold AdoMet (1 mM) in the incubation medium, an indication of AdoMet transport by bloodform trypanosomes and the utilization of [U‐14C]methionine as AdoMet. Exogenous AdoMet had no effect on [35S]methionine incorporation. The agents studied are curative for African trypnosomiasis infections, either clinically (DFMO) or in model infections (MDL 73811, sinefungin) and thus highlight interference with AdoMet metabolism and methylation reactions as biochemical consequences of these agents.
Effects of Dichloroisoproterenol on Macromolecular Synthesis and Differentiation in Tetrahymena vorax
Authors: RYALS PHILLIP E. , YANG XIANGLI
Published: May 2007

ABSTRACT. The effect of dichloroisoproterenol on macrostomal cell formation in Tetrahymena vorax was examined and the drug was found to be 50% inhibitory at a concentration of 88 μM. Cellular uptake and incorporation of a variety of radiolabelled precursors was monitored in the presence of dichloroisoproterenol. The results demonstrate a strong, concentration‐dependent inhibitory effect on RNA and protein biosynthesis, with a lesser inhibition observed for lipid biosynthesis. These data indicate that dichloroisoproterenol's reported effects on vacuole formation and processing in Tetrahymena are nonspecific with regard to phagocytic processes, but result from a general suppression of macromolecular synthesis.
Effect of Osmotic Stress on the Rate of Release of Acid Phosphatase by Leishmania donovani Promastigotes
Authors: LAWRENCE FRANÇOISE , BLUM JOSEPH J. , ROBERT‐GERO MALKA
Published: May 2007

ABSTRACT. The release of soluble acid phosphatase from Leishmania donovani promastigotes in axenic culture medium is altered by osmolarity changes: Hypoosmotic conditions increase its release whereas hyperosmotic ones decrease the release of acid phosphatase.
The Ultrastructure of Zosterodasys agamalievi (Ciliophora: Synhymeniida)
Authors: KIVIMAKI K. LINDHOLM , RIORDAN GAVIN P. , LIPSCOMB DIANA
Published: May 2007

ABSTRACT. The cell surface of the synhymeniid ciliate, Zosterodasys agamalievi, consists of shallow kinetal grooves separated by low cortical ridges. Numerous electron‐opaque bodies are located in the cortical ridges, inside the kinetal grooves, and are distributed in parallel rows between adjacent kineties. Well‐developed alveoli are present beneath the cell surface membrane. Zosterodasys agamalievi has a single micronucleus and a homomerous macronucleus. The infraciliature of the somatic monokinetid consists of an anteriorly‐directed kinetodesmal fiber, a well‐developed divergent postciliary microtubular ribbon, radially‐oriented transverse microtubules, and a short striated rootlet, which extends anteriorly from the base of the kinetosome into the cell. Zosterodasys agamalievi has a perioral band of paired cilia, the synhymenium, that winds obliquely across the ventral surface of the body, just posterior to the cytostome. The infraciliature of the anterior kinetosome of the synhymenium consists of two postciliary microtubules; a well‐developed, divergent post‐ciliary ribbon of microtubules and a short kinetodesmal fiber are associated with the posterior kinetosome. The cytopharynx is supported by 14‐16 nematodesmata which are capped distally by a capitulum. The cytopharynx is bound proximally by a fibrous sheath and is lined by radially‐arranged microtubular ribbons. No obvious oral ciliature is present.
A CDC2‐Related Kinase is Associated with Macronuclear DNA Synthesis in Paramecium tetraurelia
Authors: TANG LIREN , ADL SINA M. , BERGER JAMES D.
Published: May 2007

ABSTRACT. Cell division in higher eukaryotes is mainly controlled by CDK (serine/threonine) protein kinases and by other components of these kinase complexes. Previously, we showed homologous kinases also occur in the ciate Paramecium tetraurelia. In this report, a polyclonal antibody was produced against a GST fusion protein which contained the N‐terminal third of the previously isolated CDC2FTA protein. It recognized a 36 kDa polypeptide on western blots and did not cross‐react with the related 34 kDa polypeptide. This 36 kDa polypeptide showed no affinity for yeast P13suc1 protein. The CDC2PTA level was invariant throughout the cell cycle and decreased slightly when cells entered stationary phase. Indirect immunofluorescence showed CDC2PTA localized in the macronucleus, but not in micronuclei. Native CDC2FTA was immunoprecipitated and the kinase activity was assayed using histone HI as substrate in elutriation synchronized samples. The kinase activity was high during the early stages of the cell cycle and reached a peak at 2.5 h after elutriation, which corresponds to the time of initiation of macronuclear DNA synthesis. This suggests CDC2PTA kinase may be associated with the regulation of macronuclear DNA synthesis. These results allow us to draw concrete comarisons for the first time between the role of CDK in ciliates and higher eukaryotes.
Leidyana canadensis N. Sp. (Apicomplexa: Eugregarinida) from Larval Eastern Hemlock Looper, Lambdina fiscellaria fiscellaria (Lepidoptera: Geometridae)
Authors: CLOPTON RICHARD E. , LUCAROTTI CHRISTOPHER J.
Published: May 2007

ABSTRACT. Leidyana canadensis n. sp. (Apicomplexa: Eugregarinida) is described from larvae of the eastern hemlock looper, Lambdina fiscellaria (Lepidoptera: Geometridae) collected near St. Stephen, Charlotte County, New Brunswick, Canada. Gamonts solitary, located between host ventricular peritrophic membrane and ventricular epithelium. Protomerite very broadly ellipsoid with transverse posterior margin; length 43.7 μm, width 42.7 μm; protomerite‐deutomerite septum strongly constricted. Deutomerite narrowly obovoid with anterior transverse margin; length 186.6 μm, width 58.7 μm; total length 227.1 μm. Nucleus spherical to broadly ellipsoid; length 26.3 μm, width 20.2 μm; placement abaxial in the posterior 2/3 of the deutomerite. Nucleus often obscured in late gamonts. Association late, caudofrontal, ephemeral and leading directly to syzygy. Gametocysts roughly spherical; diameter 216.7 μm; hyaline coat increasing diameter to 359.1 μm. Gametocysts mature and dehisce through six short spore tubes within 48 hours. Oocysts axially symmetric, dolioform in dorsal aspect, compressed in the plane perpendicular to the major axis, very uniform in size and shape; length along major axis 5.2 μm, terminal width 1.8 μm, medial width 3.8 μm; extruded in chains.
Identification of Synapomorphic Characteris in the GenusSarcocystisBased on 18S rDNA Sequence Comparison
Authors: JEFFRIES ALEX C. , SCHNITIZLER BEATE , HEYDORN A. OTTO , JOHNSON ALAN M. , TENTER ASTRID M.
Published: May 2007

ABSTRACT.In order to further investigate synapomorphic characters in the genusSarcocystis, the small subunit ribosomal RNA gene sequences ofSarcocystis capracanisandSarcocystis mouleiwere determined and used to infer the phylogenetic position of these two organisms within the cyst‐forming coccidia. Phylogenies derived using distance, maximum parsimony and maximum likelihood methods demonstrated thatS. capracanisgroups withSarcoystis tenellaandSarcocystis arieticanisas a clade that shares the characteristic of using canids as their definitive host.S. mouleiwas shown to group withSarcocystis giganteaandSarcocystis fusiformisas a clade that shares the characteristic of using felids as their definitive host.
Annual Abundances, Diversity and Growth Potential of Gymnamoebae in a Shallow Freshwater Pond
Authors: ANDERSON O. ROGER
Published: May 2007

ABSTRACT. Abundance and diversity of gymnamoebae were assessed at monthly intervals for one year in the sediment surface layer of a shallow, highly productive, freshwater pond located in northeastern United States using laboratory culture enumeration techniques. The range in total abundance was 81/ml in January to 1,568/ml in June (spring peak) and 1,813/ml in October (autumn peak). The latter is equivalent to over 1.8 million per liter, one of the highest values reported for gymnamoebae from a freshwater pond. Maximum diversity (H = 3.3) occurred in April, July, and August. The growth potential of the gymnamoebae was determined in laboratory cultures (20° C) containing only the pond water (control culture) and pond water emended with malt‐yeast extract and glucose (nutrient culture) using pond water collected in December. The total abundance of gymanmoebae increased from 363/ml in the initial pond water sample to approximately 1,800/ml in each of the culture conditions in the first week. Therafter, abundances reached 5,678/ml (control culture) and 8,016/ml (nutrient culture) by the third week indicating the vigorous growth potential from winter standing stock.
Cloning and Sequencing of a cDNA Encoding Glyceraldehyde‐3‐Phosphate Dehydrogenase from Tetrahymena thermophila: Growth‐associated Changes in its mRNA Expression
Authors: ZHAO YUTONG , NAKASHIMA SHIGERU , ANDOH MASATAKA , NOZAWA YOSHINORI
Published: May 2007

ABSTRACT. Glyceraldehyde‐3‐phosphate dehydrogenase is a key enzyme in the glycolytic pathway. Since its transcript levels do not vary in most experimental conditions, it has been often used as a control in northern blot or reverse transcriptase‐polymerase chain reaction analysis. We have cloned and sequenced a gene encoding glyceraldehyde‐3‐phosphate dehydrogenase (Tthgapdh) from Tetrahymena thermophila cDNA library and determined whether the Tthgapdh mRNA is a loading control in gene expression studies of T. thermophila cell. The open reading frame encooded a protein of 341 amino acid residues (36.8 kDa) containing a nicotinamide adenine dinucleotide‐binding domain and a catalytic domain, which was highly similar to those of other organisms. Its mRNA levels at different growth stages were examined by northern blot analysis. The fragment of the isolated cDNA was hybridized to a 1.3kb mRNA transcript. There was a marked increase in Tthgapdh mRNA level at the mid‐exponential phase, followed by a gradual decrease. Therefore, much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot analysis in Tetrahymena.
Wittmannia antarctica N. G., N. Sp. (Nosematidae), a New Hyperparasite in the Antarctic Dicyemid Mesozoan Kantharella antarctica
Authors: CZAKER RENATE
Published: May 2007

ABSTRACT. Collections of the dicyemid mesozoan Kantharella antarctica were made in the Weddell Sea during the Antarctic Expedition of the research vessel RV Polarstern in 1990 and 1991. A diplokaryotic microsporidian was found infecting all nematogens from all the samples taken in both years. The infected cells contained all developmental stages. Merogony initially was monokaryotic and spoorogony of diplokaryotic sporonts was by multiple fission. The stained ovoidal spores measured between 4.3‐6 μm X 1.7‐2.3 μm. The ultrastructural findings come from 11 specimens of Kantharella antarctica that were cut in serial sections. All developmental stages were noteworthy because of the myelinosomes situated adjacent to each diplokaryon. Similarly conspicuous were some organelles in the spore: a prominent, extraordinarily electron dense anterior portion of the polaroplast and the posterior vacuole. The isofilar polar filament with a diameter of about 115 nm showed 9‐11 coils. The great number of empty spore cases together with an extruded polar filament are indicative of an autoinfection. Though these characteristics resemble in part those of the genus Nosema from the family Nosematidae, the species in Kantharella antarctica differs from the former by its unusual development, life cycle and unusual host. Thus, this new species has been placed in a new genus and the name Wittmannia antarctica proposed.
Heterogeneous Distribution of Membrane Cholesterol at the Attachment Site of Cryptosporidium muris to Host Cells
Authors: YOSHIKAWA HISAO , KIMATA ISAO , ISEKI MOTOHIRO
Published: May 2007

ABSTRACT. Distribution of membrane cholesterol at the attachment site of Cryptosporidium muris was investigated by freeze‐fracture cytochemistry using a polyene antibiotic filipin. Since the host plasma membrane enveloped C. muris, the inner and outer membranes were continuous with the parasite plasma membrane at the annular ring and with host membrane at the dense band, respectively. Although many filipin‐cholesterol complexes were observed on the plasma membrane of host cells and parasites, a line showing no complexes was evident at the above two membrane junctures. These observations indicate that parasitic infection of C. muris altered the organization of membrane cholesterol.
Intraspecific Variation and Phylogenetic Relationships in the Genus Entamoeba as Revealed by Riboprinting
Authors: CLARK C. GRAHAM , DIAMOND LOUIS S.
Published: May 2007

ABSTRACT. Eighty‐seven isolates of amebae assigned to the genus Entamoeba have been studied by riboprinting (restriction enzyme polymorphism analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes). Twenty‐four distinct patterns were obtained, most of which corresponded to previously described species. In three species (Entamoeba coli, Entamoeba gingivalis and Entamoeba moshkovskii) intraspecific variation was detected that led to the grouping of isolates into ‘ribodemes’ (populations of amebae that share the same riboprint pattern). The riboprint data were used to estimate genetic distances among and within species for the construction of phylogenetic trees based on parsimony and distance analyses. The trees obtained with the two methods are largely congruent. In some cases the estimated distances between species were greater than the upper limit recommended for the fragment comigration method of analysis indicating unusually deep branches within this genus. However, it appears that those species producing cysts with eight nuclei, those producing cysts with one nucleus, and those producing cysts with four nuclei form morphologically based groups that are supported by the riboprint data. The oral parasite Entamoeba gingivalis, which does not encyst, clusters with the third group indicating secondary loss of this ability.
Control of Trypanosoma cruzi Epimastigote Motility through the Nitric Oxide Pathway
Authors: PEREIRA CLAUDIO , PAVETO CRISTINA , ESPINOSA JOAQUIN , ALONSO GUILLERMO , FLAWÁ MIRTHA M. , TORRES HECTOR N.
Published: May 2007

ABSTRACT. Trypanosoma cruzi epimastigote motility can be enhanced by addition of L‐arginine, to the culture. This effect is blocked by N‐methyl‐L‐arginine, a competitive inhibitor of the nitric oxide synthase. N‐methyl‐D‐aspartate and L‐glutamate, two agonists of the NMDALglutamate receptor, also enhanced motility. This stimulation is blocked by MK‐801 a noncompetitive antagonist of the NMDA receptor. In addition, sodium nitroprusside, a guanylyl cyclase stimulator and 8‐Br‐cyclic GMP, an analog of cyclic GMP, also stimulated epimastigote motility. It is suggested that an increase of intracellular cyclic GMP levels mediated by nitric oxide may be responsible for the increase in epimastigote motility.
Entamoeba dispar: Cultivation with Sterilized Crithidia fasciculata1
Authors: Kobayashi Seiki , Imai Eiko , Tachibana Hiroshi , Fujiwara Tatsushi , Takeuchi Tsutomu
Published: May 2007

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica‐specific monoclonal antibody (4G6) could be grown in either Diamond's BI‐S‐33 medium, newly developed BCSI‐S (Biosate cysteine starch iron‐serum) medium, or casein‐free YI‐S medium in the presence of Crithidia fasciculata (ReF‐1:PRR) sterilized by heating 56° C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4° C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica‐ and E. dispar‐specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.
Molecular Analysis of the Gal/GalNAc Adhesin of Entamoeba histolytica1
Authors: Mann Barbara J. , Lockhart Lauren Ashley
Published: May 2007

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactosc/N‐acetyl D‐galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E. coli, retained at least some of its native conformation.