bluuebunny/crossref_metadata_2025 huggingface dataset:
On Micromastigotes and the evolution of the hypermastigont condition in the Parabasalida
Authors: CAMERON STEPHEN L. , O'DONOGHUE PETER J.
Published: April 2005

This study redescribes the genus Micromastigotes, Hollande and Carruette‐Valentin, 1971, a parabasalid flagellate symbiotic in termites, on the basis of light and electron microscopy. The genus Micromastigotes is characterised by possessing flagella bands which spiral around the anterior portion of the cell, the location of the nucleus and Golgi bodies at the base of the anterior flagellated area, and an axostyle, which runs the length of the cell. Individual flagella are derived from the central axis of the cell exiting perpendicularly, each is offset from the preceding flagellum by 12–18° thus forming a spiral band. Ultrastructurally, the flagella bases form a structure resembling a spiral staircase. Peltoaxostylar and preaxostylar fibres arise from the anterior most kinetosomes and the striated lamina forms a weakly undulating sheet directed posteriorly from each flagellum. Dense lamina and parabasal fibres are absent. Micromastigotes was originally classified as part of the Spirotrichonymphina and there are some similarities to other genera in this group, but Micromastigotes lacks a flagella gutter, a U‐shaped band at the base of the flagella composed of the striated and dense lamina, which is diagnostic of the spirotrichonymphines. The spiral pattern in Micromastigotes is not consistent with previous schemes for the development of a polymastigont condition in spirotrichonymphines suggesting that Micromastigotes represents an independent derivation of a polymastigont condition from a trichomonad‐like ancestor.
Massive differential expansion of the Trichomonas vaginalis adaptin genomic complement
Authors: DACKS JOEL B. , CARLTON JANE , HIRT ROBERT P. , EMBLEY T. MARTIN
Published: April 2005

Membrane transport is crucial to eukaryotic cell function and its evolution is likely to be critical for eukaryogenesis. Tetrameric adaptin complexes form part of the transport machinery, selecting cargo for transport through the late secretory and endocytic systems. With a few protistan exceptions, the majority of adaptin studies have been performed on animal and yeast models. Trypanosomatids and yeast have a minimal complement of adaptin genes, while humans and Arabidopsis show a moderate expansion. This expansion is functional, with adaptin subunit isoforms substituting combinatorially into their complex to provide novel localization. To address questions of evolution, parasitology and comparative cell biology, we examined the adaptin complexes of the sexually transmitted parasite Trichomonas vaginalis. In coordination with the T. vaginalis genome project at The Institute for Genomic Research, we identified all adaptin genes in the current 3.3X genome coverage data (http://www.tigr.org/tdb/e2k1/tvg/). Surprisingly, T. vaginalis has massively and differentially expanded its adaptin arsenal, with six times as many genes as other unicellular organisms examined and three times as many multicellar organisms. Eight Trichomonas adaptins have been confirmed by cDNA, demonstrating concurrent expression of the same gene. Phylogenetic analysis shows that the duplications giving rise to the various adaptin subfamilies occurred prior to the last common ancestor of diverse protists. Our data provide important insight into the evolution of the adaptin complex and the complexity of the Trichomonas late secretory/endocytic system. The differential expansion may illustrate the relative functional importance of the adaptin subfamilies to Trichomonas and be a model in which to study the lesser expansion seen in other eukaryotes. Finally, the size of the expansion, and the concurrent expression of at least one gene, suggests that the combinatorial nature of the adaptin complex may be more important than previously expected.
Observations of asexual and sexual processes in Protoperidinium depressum (Dinophyceae)
Authors: GRIBBLE KRISTIN E. , ANDERSON DONALD M.
Published: April 2005

Life cycle stages of the heterotrophic dinoflagellate Protoperidinium depressum were determined for a culture isolated from the west coast of Ireland. Division, sexuality, DNA content, gamete morphology, and identity of morphologically distinct life‐history stages were revealed for the first time using a suite of morphological and molecular tools. Asexual division occurred by eleutheroschisis within a temporary, immotile cyst, yielding two daughter cells. Morphological features, including apical and antapical horns, were not fully formed upon emergence from the division cyst, but developed some time after cell division. Daughter cells were half to two‐thirds the size of parent cells and swam attached in an epitheca‐to‐hypotheca orientation before separating. Stages indicating a completed life cycle, including gametes, planozygotes, and nuclear cyclosis were observed. Small cells, believed to be gametes, formed in repeated clonal and non‐clonal re‐isolations of the culture. In addition to their small size (40–50 μm), blunt apical horn, small antapical horns, and lack of pink pigmentation, these putative gametes had a thecal plate morphology different from that described for P. depressum. The LSU rDNA sequence (D1–D3 region) of small cells was, however, the same as that of P. depressum. Gametes were never witnessed to feed, but did form fusing, isogamous pairs. When isolated to well plates, gamete pairs failed to complete fusion to form a planozygote. Thus, the morphology of the initial planozygote remains uncertain. Large cells believed to be planozygotes had “typical”P. depressum morphology, but possessed two longitudinal flagella and had 2C DNA based on DAPI. Nuclear cyclosis was documented in one such tri‐flagellated, motile cell. The cell remained motile after the completion of nuclear cyclosis, but did not progress to the next life stage. Dormant hypnozygotes were not observed.
Foraminiferal evolution as revealed by β‐tubulin data
Authors: HABURA ANDREA , WEGENER LAURA , TRAVIS JEFFREY L. , BOWSER SAMUEL S.
Published: April 2005

Foraminiferal taxonomy is traditionally based on morphological characteristics, particularly the structure and composition of the test (shell). Recently, a comprehensive SSU‐based molecular phylogeny confirmed some multi‐chambered foraminiferal groups while enhancing our understanding of the single‐chambered, or the so‐called “allogromiid”, taxa. However, some relationships, such as the origin of the foraminiferal order Miliolida, remain unresolved, suggesting an approach involving multiple data sources may improve understanding. Here, we discuss the evolutionary implications of β‐tubulin genes from several species of foraminifera. Foraminiferal β‐tubulin is highly divergent, possibly indicating a period of accelerated evolution of this gene at the base of the foraminiferal lineage, but it is well‐conserved within the group. We show its utility in helping to resolve the phylogenetic position of Miliammina fusca, a foraminiferan whose proper classification has been debated for 70 years. M. fusca is a morphological “chimera”, and we show that its placement in SSU trees is not particularly robust. However, β‐tubulin data assigns Miliammina to the Miliolida, some of whose members show a similar chamber coiling pattern. This placement agrees with an earlier observation that M. fusca's actin isoforms more closely resemble those of traditional milolids. This finding implies that test formation can revert from calcareous to agglutinated, suggesting a reexamination of some relationships inferred from the fossil record.

Supported by NSF OPP0003639.
Evolution and implications of genome rearrangements in ciliates
Authors: KATZ LAURA A.
Published: April 2005

We are exploring genome evolution in diverse ciliates to assess whether patterns of protein evolution correspond to the presence of genome duality, and to levels of genome rearrangements among ciliates. Ciliate genomes contain an estimated 25,000–35,000 genes, a number higher than that of other characterized microbial eukaryotes and on a similar scale to that of humans and Arabidopsis thaliana. We have previously hypothesized that the divergence among ciliate proteins, and the accumulation of paralogs within ciliate taxa, is due to differential selection on the functional macronucleus and the transcriptionally inactive micronucleus. To assess this hypothesis, we are expanding our analyses of protein‐coding gene sequences to compare the fate of proteins in ciliates to that of other eukaryotes, and to test whether the most divergent ciliate proteins are found in ciliates with extensively processed genomes. These analyses, combined with a phylogenetic perspective that reveals multiple origins of extensive fragmentation in ciliates, provide a possible explanation for the large genome size and diversity of proteins in ciliates.
Cloning of mitichondrial cytochrome b gene from Pseudourostyla cristata by heat shock
Authors: LIU XINYING , MAO YONGZHEN , DANIEL LEE KAM‐LEN , JIN LIPEI
Published: April 2005

The total RNA was extracted from Pseudourstyla cristata after 2 h of heat shock, and the cDNA was synthesized directly using Smart LD‐PCR. The amplified cDNA products showed a strong band around 1.5 kb. The products were purified, cloned and sequenced. The resulting full cDNA sequence had a total length of 1538 bp including 1068 bp of cytb gene and UTR at both sides and showed all of the expected features for a fully functional CYB. The predicted protein is 49% (30% identical) similar to Paramecium tetraurelia. The sequence predicts a UAA stop codon 207 nucleotides from the end of the transcript suggesting a 3′ UTR, unlike that of Paramecium tetraurelia. Prediction of transmembrane helices in the analysis of proteins indicates the expected eight transmembrane domains. The cytb gene expression profiles after heat shock and before heat shock were generated using quantitative real‐time PCR. After 2 h of heat shock, the cytb gene expression was significantly enhanced. This is the first report that shows complete cytb gene among hypotrichous ciliates. Currently, we are investigating whether the method of cloning cytb rapidly are fit for other hypotrichous ciliates so as to analyze their phylogenetic relationship.

This work was supported by a grant from NSFC (No: 30370210) to L. P. JIN.
Identification of mRNAs expressed differentially between micronucleates and amicronucleates of Pseudourostyla cristata during starvation
Authors: LIU XINYING , MAO YONGZHEN , DANIEL LEE KAM‐LEN , JIN LIPEI
Published: April 2005

The evident character of amicronucleates of Pseudourostyla cristata, as compared with the normal micronucleates, was that a substantial proportion of cells exhibited an abnormal AZM. Moreover, the percentage of abnormal AZMs in amicronucleates during starvation was much higher. To understand how the molecular events of the micronucleus govern this mode, we employed suppression subtractive hybridization (SSH) to identify candidate genes which express in micronucleate cells but not in amicronucleate cells during starvation. A total of 284 transcripts were screened by cDNA array and 17 clones were confirmed to be differentially expressed among micronucleate cell. All 17 ESTs were analyzed by blaster matching in GeneBank at deduced amino acid sequence: 10 clones, no homologous proteins; 4 clones, myoglobin protein; 1 clone, cytochrome P450 monooxygenase; 1 clone, cytosolic malate dehydrogenase; the last clone, homologs of hsp90, which is interesting. Traditionally, heat shock proteins are known for their function during cellular stress as molecular chaperones responsible for the folding, refolding and transport of newly synthesized protein. Why was it significantly enhanced in micronucleates but not in amicronucleates during starvation? Further research including RACE, immunoblotting and gel mobility shift will be carried out. This work was supported by a grant from NSFC (No: 30370210) to L. P. JIN.
Symbiotic cooperation between dinos and ciliates in “Sym City”
Authors: LOBBAN CHRISTOPHER S.
Published: April 2005

Two new ciliates—a heterotrich and a hypotrich—have recently been discovered in the shallow benthos of coral reefs on Guam. They are unusual for ciliates in having zooxanthellae, but they share this characteristic with corals and many other reef animals. The heterotrich was a new genus, named Maristentor dinoferus, the hypotrich a Euplotes sp., and the dinoflagellate partner a Symbiodinium sp. Apart from their interest as dual‐alveolate symbioses, the heterotrich has abundant, fluorescent pigment with intriguing possibilities for comparison with stentorin and blepharismin pigments from freshwater ciliates that serve in both phototropisms and chemical defense. Comparison with deep‐water corals also raises the possibility that the cortical pigment enhances photosynthesis of the zooxanthellae.
Phylogenetic position of the apostome ciliates tested using the small subunit rRNA gene sequence
Authors: LYNN DENIS H. , STRUEDER‐KYPKE MICHAELA , BRADBURY PHYLLIS C.
Published: April 2005

Apostome ciliates are exclusively symbionts, typically of crustaceans. Their life cycles are complex, but show remarkable similarities to ophyroglenines and to other symbiotic oligohymenophoreans, particularly those that undergo microstome–macrostome transformations. The highly modified structures around the cytostome—the rosette and x, y, and z kineties—have made the assessment of homologies to other groups of ciliates difficult. A description of the apostome somatic kinetids with well‐developed overlapping kinetodesmal fibrils suggested affinities with hymenostomes (Bradbury 1966. J. Protozool., 13:591). Further analysis of the ultrastructural features of the oral region of the tomite of Hyalophysa demonstrated homologies to the paroral and adoral ciliature of hymenostomes, confirming these affinities (Bradbury 1989. J. Protozool., 36:95). To test this hypothesis, we proposed to sequence the small subunit rRNA (SSrRNA) genes of apostomes. Specimens of Gymnodinioides sp. were collected from molts of the amphipod Marinogammarus obtusatus collected off the coast of Eastport, ME. Seawater was decanted from the substrate upon which tomonts had settled, and the dish was flooded with 70% ethanol. The ethanol‐fixed ciliates were collected by a micropipette, rinsed with distilled water, and DNA was extracted using the modified Chelex® protocol. The SSrRNA genes were amplified by PCR, and sequences obtained in both directions. Initial BLAST searches of the GenBank databases clearly demonstrated affinities with oligohymenophorean ciliates, particularly scuticociliates. In preliminary phylogenetic analyses which use likelihood, distance and parsimony methods, this relationship was confirmed. However, the apostome sequence diverged basal to the Subclasses Astomatia and Scuticociliatia consistent with the assignment of a Subclass Apostomatia within the Class Oligohymenophorea. We have made collections of Hyalophysa chattoni and anticipate that the SSrRNA gene sequences of this genus will confirm the relationship of apostomes to the oligohymenophoreans.

Funded by NSERC Canada Discovery Grant to D. H. L.
Analysis of centrin‐, actin‐, and tetrin‐containing filaments in the Tetrahymena vorax macrostomal oral apparatus and their relation to the microtubule cytoskeleton
Authors: MCLAUGHLIN NEIL B. , MCCUTCHEON SUZANNE M. , BUHSE HOWARD E.
Published: April 2005

Our laboratory studies how the contractile cytoskeleton contributes to the process of phagocytosis. Because of its larger size and ease of manipulation, we chose the macrostomal cell of Tetrahymena vorax as our model for analysis of the distribution of proteins of prominent filamentous structures within the large oral apparatus (OA). Previous work in our laboratory identified centrin as a component of the fine filamentous reticulum (FFR) and actin and tetrin as colocalizing components of the coarse filamentous reticulum (CFR) and cross‐connectives (CC) (J. Eukaryot. Microbiol., 51:253–257). Our new data also show that actin coimmunoprecipitates with tetrin proteins, confirming our actin–tetrin colocalization results. Because of its positioning around the cytostome, the actin‐containing CFR/CC is a logical candidate for involvement in phagosome “pinch‐off” following prey ingestion. We have analyzed this process by employing an assay that uses the addition of calcium to induce phagosome formation. We show that inhibitors of actin are able to block this event, indicating that actin is necessary for phagosome “pinch‐off”. The OA also contains precisely arranged arrays of microtubules. We have examined the spatial relationship of the microtubular arrays to the distribution patterns of centrin, actin and tetrin, and found that tubulin and centrin fluorescences overlap in the region of the undulating membrane (UM). Tubulin fluorescence overlaps with actin and tetrin labeling at the inner edge of the CFR, where the CFR and UM converge. In addition to ciliary and oral rib labeling, tubulin antibodies also recognize the outer microtubule bundle (OMB), which delineates the right and posterior boundary of the OA.
Two adjacent genes encoding copper‐induced‐type metallothioneins in Tetrahymena thermophila may have resulted from a relatively recent gene duplication
Authors: BAKSHANDEH ARTA , HAMILTON EILEEN P. , ORIAS EDUARDO
Published: April 2005

Tetrahymena thermophila is a valuable model organism for the study of metallothioneins, which play important roles in the metabolism of essential metal ions and in heavy‐metal detoxification. Starting from a T. thermophila expressed sequence tag (EST) originally annotated as a Zn–Cu–Cd metallothionein (GenBank Acc #BM394105), we determined the genomic sequence corresponding to this EST and its flaking region. The sequence contains two adjacent genes encoding metallothioneins of the copper‐induced type: the MTT2 gene (submitted to GenBank by Boldrin et al. as Cu‐inducible while this work was in progress) and a second, novel gene (MTT4), located ∼1.4 kb downstream from and with same orientation as MTT2. Our entire sequenced segment is contained in the T. thermophila whole‐genome sequence recently completed at TIGR. Both MTT4 and MTT2 are intronless and encode two nearly identical proteins (two differences out of 108 amino acids). The 5′ and 3′‐flanking regions of both the metallothionein genes show significant sequence similarity as well, suggesting recent gene duplication. All glutamines use the CAA codon, reminiscent of codon usage observed in highly expressed Tetrahymena genes. MTT4 and MTT2 map to a 1.5‐Mb macronuclear chromosome from the right arm of micronuclear chromosome 2.

Supported by NIH grant RR02931. Sequence data were obtained from The Institute for Genomic Research Tetrahymena website, http://www.tigr.org/tdb/e2k1/ttg/
Is it the tortoise or the hare? Ciliates grazing on dinoflagellates in boom–bust dynamics
Authors: MONTAGNES DAVID J.S.
Published: April 2005

This mini‐review examines the maximum growth rate and threshold levels (the food level where growth rate is zero) of marine planktonic ciliates and heterotrophic dinoflagellates. There are strong indications that ciliates will out‐compete dinoflagellates, as they have higher maximum growth rates, lower threshold levels and are able to rapidly exploit both harmful and non‐harmful dinoflagellates as prey. I suggest that ciliates are neither the tortoise (slow and steady) nor the hare (fast and foolish), but the best combination of both. To some extent, this argument is raised as a “straw‐man” to be knocked down by discussion; the session is intended to serve as a platform for evaluating the relative importance of ciliates and heterotrophic dinoflagellates in marine ecosystems.
The Tetrahymena thermophila genome sequence: 1. Genome‐wide uniformity of macronuclear chromosome copy number in spite of amitotic (random) copy distribution
Authors: ORIAS EDUARDO , SALZBERG STEVEN L. , DELCHER ARTHUR L.
Published: April 2005

In Ciliates, the diploid, mitotic, genetically silent micronucleus is the cell's germline, while the macronucleus is the exclusive site of gene expression. Pre‐genomic studies in Tetrahymena thermophila have shown that

The macronucleus contains 250–300 chromosomes, derived from the five germline chromosomes;

G2 copies of each chromosome are distributed amitotically (randomly) during macronuclear division.

In heterozygotes, this results in the segregation of vegetative descendants expressing only one of the two alleles (assortment). Phenotypic assortment rate measurements for a handful of loci suggested a uniform macronuclear chromosome G1 copy number (∼45 per macronucleus; rDNA minichromosome excepted). The NIGMS/NSF‐supported, whole‐genome‐shotgun sequencing of the T. thermophila macronuclear genome was recently completed at TIGR. (Sequence and scaffold databases, and related resources, are publicly available for blasting or downloading without restriction at http://www.tigr.org/tdb/e2k1/ttg/). Analysis of the depth of sequence coverage shows that (rDNA excepted) at least 95%—and possibly all—of the macronuclear genome is in chromosomes with uniform copy number. This implies the existence of regulatory mechanisms that independently correct the number of copies of nearly 300 different chromosomes after random distribution at every cell cycle.

Supported by NIH grant RR02931. Preliminary sequence data were obtained from The Institute for Genomic Research website.
Small subunit ribosomal RNA sequences of Phaeodarea challenge the monophyly of Haeckel's Radiolaria
Authors: POLET STEPHANE , BERNEY CÉDRIC , FAHRNI JOSÉ , PAWLOWSKI JAN
Published: April 2005

In his grand monography of Radiolaria, Ernst Haeckel originally placed Phaeodarea within the class Radiolaria, together with Acantharea and Polycystinea. Cytological and ultrastructural studies, however, questioned the monophyly of Radiolaria, suggesting the independent evolutionary origin of the three taxa. Some recent molecular analyses based on small subunit ribosomal RNA (SSU rRNA) sequences challenged this classification, supporting the sisterhood of Acantharea and Polycystinea. In order to further test the monophyly of all Haeckel's Radiolaria, and as no data on Phaeodarea were available, we sequenced the complete SSU rRNA gene of three Phaeodarea and three Polycystinea. Our analyses show that the monophyletic Phaeodarea clearly branch among Cercozoa, and confirm that Acantharea and Polycystinea share a common history. This result enhances the morphological variability within Cercozoa, a phylum already containing very heterogeneous groups of protists. Our study suggests that the ability to secrete SrSO4 and the organization of microtubules inside axopodia are better phylogenetic markers than the simple presence of a central capsule and axopodia.
Molecular phylogeny of phyllopharyngen ciliates and their novel Group I introns
Authors: SNOEYENBOS‐WEST OONA L.O. , COLE JEFFREY , CAMPBELL ANNA , COATS D. WAYNE , KATZ LAURA A.
Published: April 2005

We analyze small subunit ribosomal DNA (ssu‐rDNA) sequences to evaluate both the monophyly of the ciliate class Phyllopharyngea de Puytorac et al. (1974), and relationships among subclasses. Classifications based on morphology and ultrastructure divide the Phyllopharyngea into four subclasses: the Phyllopharyngia, Chonotrichia, Rhynchodia and Suctoria. Our analyses of ssu‐rDNA genealogies derived from sequence data collected from diverse members representing three of the four subclasses of Phyllopharyngea provide strong support for the monophyly of the Phyllopharyngea, and show that the Chonotrichia emerge from within the Phyllopharyngia. Based on this initial sampling, suctorian budding types are monophyletic, and exogenous budding appears to be basal to evaginative and endogenous budding. Further, we report the discovery of a group I intron at position 891 in the Suctoria Acineta sp. and Tokophrya lemnarum, and a second group I intron at position 1506 in T. lemnarum. These introns represent only the second examples of group I introns in a ciliate ribosomal gene, since the discovery of ribozymes in the LSU rRNA gene of Tetrahymena thermophila 20 years ago. Phylogenetic analyses of Group I introns suggest a complex evolutionary history involving either multiple loses or gains of introns within endogenously budding Suctoria.
Divergent β‐tubulins from foraminifera: Implications for microtubule assembly
Authors: WEGENER LAURA , TRAVIS JEFFREY L. , HABURA ANDREA , BOWSER SAMUEL S.
Published: April 2005

Foraminifera utilize an unusual and very rapid microtubule assembly/disassembly system: a state transition between microtubules and helical filaments. The helical filament is an assembly polymorph that forms when the microtubule lattice uncoils into a ribbon of tubulin dimers connected only by lateral subunit/subunit bonds. An unusual β‐tubulin sequence, which may be implicated in this process, has previously been reported from the foraminiferan Reticulomyxa filosa. In order to determine the taxonomic distribution and possible significance of this tubulin isoform, we sequenced and analyzed β‐tubulin genes from a broad taxonomic range of foraminifera. These genes contain a large number of substitutions, conserved within the group, which may alter the biochemical properties of β‐tubulin, especially in regions involved in subunit/subunit binding between α‐ and β‐tubulin in the microtubule lattice. In particular, the M‐loop, which is intimately involved in lateral subunit binding, shows only 30% homology to the consensus sequence for eukaryotic β‐tubulins. Multiple regions identified to be involved in longitudinal contacts with α‐tubulin are also highly substituted. In contrast, the foraminiferal α‐tubulin is well conserved with its homologs in other organisms. We suggest that these alterations to the consensus eukaryotic β‐tubulin sequence may help to explain the unusual assembly properties of foraminiferal microtubules.

This work was supported by NSF OPP0003639.
Genes for infectivity and endocellular lifestyle of symbiotic X‐bacteria (Candiatus “Legionella jeonii”) in Amoeba proteus
Authors: YANG GYONG S. , LEE KEONG M. , AHN TAE I.
Published: April 2005

Candiatus “Legionella jeonii” is an obligate endocellular symbiont in Amoeba proteus and cannot be cultivated in vitro. In order to detect genes of the bacteria related with intracellular survival and integration to the host, we performed comparative genomic analysis. A total of 486 non‐redundant clones from the genomic DNA library of 3×106 pfu were tagged by nucleotide sequencing. To date, we have tagged 1023 genes in 1.5 Mbp and identified 383 genes of assigned functions and 180 conserved hypothetical genes. The remaining 460 tags had low homology. The 69% of high‐and 28% of low‐homology tags had matches in Legionella pneumophila database. We predicted 26 genes to be related with infection and endocellular lifestyle of L. jeonii including stress proteins (dpsA, dnaK, dnaJ, mutT), virulence system (icmK, icmTS, dotA, lvrB, lvrC), infection and pathogenecity factors (rcp, sbpA, rhuM, rpfB), drug resistance proteins (norM, mdfA), transporter proteins (ceaA, prop, necA), type IV secretion system (virB3), a global response regulation sensor kinase (grrS), a GTP‐binding protein (rabB) and ppGpp‐regulated growth inhibitors (mazEF). The large number of putative insertion elements (26 genes) in the bacterial genome was unusual. To date, about a half genome of L. jeonii has been tagged in comparing with the genome of L. pneumophilla (3.4 Mbp). These results suggest that L. jeonii still contains most of those genes for infectivity and endocellular lifestyle of pathogenic bacteria. The symbiotic L. jeonii in A. proteus could be a model system for molecular pathogenicity and symbiosis.
The response of protist and metazoan communities in permeable pavement structures to high oil loadings
Authors: COUPE S. J. , SMITH H. G.
Published: April 2005

Permeable pavement structures (PPS) have been demonstrated to provide an efficient and sustainable method of controlling urban derived hydrocarbon contamination. Until recently, laboratory PPS mesocosm models have used crushed granite as the load bearing sub‐base material. However, the use of virgin stone may not be the optimal choice of substrate, as this is not necessarily sustainable or cost effective in the long term when compared to the use of recycled materials. However, recycled materials such as waste concrete may change the environmental conditions in PPS mesocosms, and the characteristics of the eukaryotic community may become different from those which have been previously described. In the current experiment, granite and recycled concrete sub‐base materials were compared for their ability to retain 900 g/m2 of clean mineral oil applied to the mesocosm surface. It was observed that, even at this very high oil loading, 99.95% of the applied oil was retained within granite and concrete‐based structures, but the effluent was two pH units more alkaline in concrete mesocosms than granite. The eukaryotic microfauna in the effluent from both mesocosm types showed a ten‐fold increase in protist abundance, and a doubling in the number of protist genera, compared with earlier work using only 18 g/m2 of applied oil. Five genera of testate amoebae not previously recorded in PPS were identified, these included Arcella, Assulina, Cryptodifflugia, Cyclopyxis and Difflugia in addition to the three genera observed previously using the lower oil application. Metazoan abundances increased from 1.5 × 101 organisms per ml using the lower oil loadings to 2.0 × 103/ml in the current experiment. Rotifers and nematodes were the most numerous, but tardigrades were also observed in both concrete and granite‐based mesocosms. Despite the differences in effluent pH, it was apparent that there were only marginal differences in the eukaryotic microbiology of the two mesocosm types. This was thought to be due to the layered structural arrangement of the pavement and the location of the highly oil‐retentive polypropylene geotextile and extensive biofilm layer positioned above the concrete sub‐base. Work is now underway to find oil loadings that will adversely affect the abundance and diversity of eukaryotic organisms in PPS mesocosms.
Identifying new vahlkampfiid amoebae by ITS and 5.8S ribosomal DNA sequencing
Authors: DE JONCKHEERE J. F. , BROWN S.
Published: April 2005

Small subunit ribosomal DNA (SSU rDNA) comparisons have revealed that not only species but even genus identification based on morphology is unreliable in the family Vahlkampfiidae, and SSU rDNA sequences have been used to redefine vahlkampfiid genera. However, it is time consuming to use SSU rDNA sequences for species identification. The ITS region (including the 5.8S rDNA) has the advantage that it is much shorter than the SSU rDNA and thus easier and quicker to obtain. Initially, 29 strains of 13 species belonging to seven vahlkampfiid genera were investigated. It was found that each species has a specific ITS sequence, which appears to be conserved within a species. The same had previously been found in the vahlkampfiid genus Naegleria. Consequently, we have demonstrated that the ITS region (including the 5.8S rDNA) can be used to identify vahlkampfiid amoebae. In the current investigation, the ITS sequences of 16 undescribed vahlkampfiid isolates were determined. Based on these sequences, seven isolates were identified as belonging to described species, while nine represent six new species. Four of the new species belong to the genus Tetramitus, and one each to the genera Vahlkampfia and Paravahlkampfia.
Prolyl oligopeptidase from Trypanosoma brucei hydrolyzes collagen and is involved in parasitic growth
Authors: BASTOS I. M. D. , SANTANA J. M. , GRELLIER P.
Published: April 2005

Proteases play important roles in several processes of the biology of parasites including interactions with their hosts. We have reported that prolyl oligopeptidase from Trypanosoma cruzi (POPTc80) is associated with the entry of trypomastigotes into mammalian host cells. In this study, the gene coding to prolyl oligopeptidase of Trypanosoma brucei (POPTb) was identified and characterized. It is represented by a single copy per haploid genome of the parasite and its deduced amino acid sequence shares 77% identity with POPTc80. Secondary structure predictions demonstrated that POPTb shows the highly preserved secondary structure composition and arrangement of prolyl oligopeptidases. Active recombinant POP Tb, produced in E. coli, displayed enzymatic activity on peptides containing Pro at P1 position and collagen at a slightly alkaline pH. Its enzymatic activity was highly sensitive to POPTc80 inhibitors. Furthermore, these inhibitors arrested growth of procyclic and bloodstream T. brucei forms in a dose‐dependent manner. These data suggest that POPTb activity is required for normal parasite development.
Effects of Ginkgo biloba extract and its constituents on chemotaxis and cGMP‐dependent protein kinase activity in Tetrahymena thermophila
Authors: CHEN F. , LEICK V.
Published: April 2005

Ginkgo biloba extract (GBE), which is one of the most sold herbal extracts in the world, is considered as a multifunctional material, which can promote radical scavenging activity and improve brain functioning. Although much research has carried out on its mechanisms against diseases, there is a need for further investigation for understanding molecular mechanisms, potential health benefits, and possible health risks. Recently, we have been developing an alternative screening method for studying phytochemical materials bioactivities with Tetrahymena thermophila as experimental organism. In this paper, the effects of GBE and its constituents were systematically investigated on chemoattraction and cGMP‐dependent protein kinase (PKG) in T. thermophila. GBE and its constituents exerted significant inhibitions of chemoattraction and PKG. The minimal concentrations to completely inhibit chemotaxis of T. thermophila were 2 mg/ml, 50, 25, 12, 100, 100, 400, 500, 300 and 400 μM for GBE, quercetin, kaempferol, isorhamnetin, myricetin, genistein, rutin, quercitrin, isoquercitrin and quercetin‐3‐d‐galactoside, respectively. The IC50 values for PKG were 0.15 mg/ml, 26, 22, 19, 160, 132, 81 and 186 μM for GBE, myricetin, kaempferol, quercetin, isorhamnetin, genistein, rutin and isoquercitrin, respectively. The results indicate that the inhibitions of GBE and its constituents of chemotaxis of T. thermophila and their effects on PKG are rather similar. This suggests that the ciliate of T. thermophila may be a potential experimental organism in screening such bioactive phytochemicals.
Body height, body mass index, waist‐hip ratio, fluctuating asymmetry and second to fourth digit ratio (2D : 4D) in subjects with latent toxoplasmosis
Authors: FLEGR J. , HRUSKOVÁ M. , HODNY Z. , HANUSOVÁ J.
Published: April 2005

Between 20% and 60% of the population of most countries are infected with the protozoan Toxoplasma gondii. Subjects with clinically asymptomatic life‐long latent toxoplasmosis differ from those who are Toxoplasma free in several behavioral parameters. Case–control studies cannot decide whether these differences already existed before infection or whether they were induced by the presence of Toxoplasma in the brain of infected hosts. Here, we searched for such morphological differences between Toxoxoplasma‐infected and Toxoplasma‐free subjects that could be induced by the parasite (body weight, body height, body mass index, waist–hip ratio), or could rather correlate with their natural resistance to parasitic infection (fluctuating asymmetry, 2D : 4D ratio). We found Toxoplasma‐infected men to be taller and Toxoplasma‐infected men and women to have lower 2D : 4D ratios previously reported to be associated with higher prenatal testosterone levels. The 2D : 4D ratio negatively correlated with the level of specific anti‐Toxoplasma antibodies in Toxoplasma‐free subjects. These results suggest that some of the observed differences between infected and noninfected subjects may have existed before infection and could be caused by the lower natural resistance to Toxoplasma infection in subjects with higher prenatal testosterone levels.
Modulation of murine cellular immune responses and cytokine production by sand fly saliva
Authors: ROHOUSOVA I. , LIPOLDOVA M. , VOLF P.
Published: April 2005

Sand flies (Diptera: Phlebotominae) are hematophagous arthropods and important vectors of Leishmania parasites. During blood feeding, both vector saliva and parasites are injected by an infected sand fly. The aim of this study was to characterize how sand fly saliva interferes with host immunity in order to clarify the mechanism underlying enhancing effect of sand fly saliva on Leishmania infection. Spleen cells from naive BALB/c mice were incubated in vitro with Phlebotomus papatasi, P. sergenti or Lutzomyia longipalpis salivary gland lysate (SGL). We studied the effect of saliva on cell proliferation and cytokine production in non‐stimulated and concanavalin A‐stimulated splenocytes. Both spontaneous and mitogen‐stimulated lymphocyte proliferation were significantly suppressed with SGLs of all three sand fly species and all SGLs doses tested (equivalent to 1/16, 1/4 and 1 gland per well). The proliferation index (a lysate‐treated/lysate‐untreated cells ratio) was significantly lower when cells were incubated with SGL and was ranging between 0.52 and 0.30 for non‐stimulated splenocytes and 0.69–0.32 for mitogen‐stimulated lymphocytes. This result indicates that saliva from different sand fly species is able to suppress host proliferative response even to the potent Leishmania‐unrelated mitogen. In parallel experiments, we analysed the effect of SGL (1 gland per well) on cytokine production. The levels of Th1 (IL‐2, IFN‐γ) and Th2 (IL‐4) cytokines were determined in cell culture supernatants by capture ELISA. Exposure to SGL alone did not affect levels of IL‐2 or IL‐4. The production of IFN‐γ was inhibited by P. papatasi SGL, whereas the SGLs of other two species were ineffective. In mitogen‐stimulated cells both Th1 cytokines were modulated; the level of IFN‐γ production was significantly suppressed and IL‐2 enhanced by SGL of all three sand fly species. The production of Th2 cytokine IL‐4 was inhibited only by L. longipalpis SGL; no changes in IL‐4 levels were noted after incubation with Phlebotomus saliva. This species‐specific immunomodulation is in agreement with our previous finding that the composition of sand fly saliva varies among species. Taken together, sand fly saliva modulate cell proliferation as well as cytokine production. These findings further increase our understanding of sand fly–host immune relationship.
Diagnostics of Neospora caninum antibodies of bovine milk by ELISA
Authors: HURKOVÁ L. , HARAGÁLOVÁ D. , MODRY D.
Published: April 2005

Neospora caninum (Apicomplexa: Sarcocystidae) is a coccidian parasite with a two‐host life‐cycle. Dogs and coyotes are definitive hosts. A wide spectrum of mammals serves as intermediate hosts (i.a. ruminants, horses, canids). The reproductive failures associated with abortion in cattle have an economic impact. A milk ELISA is a simple serological method for diagnostics of anti‐Neospora caninum antibodies in dairy herds. We used a commercial Neospora caninum iscom ELISA kit (Svanova, Sweden) designed to detect bovine Neospora‐specific antibodies in blood serum. We examined 332 dairy herds in the Czech Republic. Milk serum was examined at dilution 1:100 according to manufacturer's instructions. The number of milking cows in the examined herds varied, but never exceeded 200 per one bulk sample. Four out of 332 examined herds were significantly positive, the positive bulk milk samples were obtained from herds consisting of 7, 19, 35 and 98 cows. Hitherto, we have studied in detail the smallest herd. The milk and blood samples or only blood samples (in some animals, e.g. calves) were examined individually. We observed the correlation of positivity between milk and blood. Moreover, the mother‐descendant positivity/negativity was found. A dog living in the farm was examined, being coprologically negative but serologically positive (IFAT).

This is the next proof of the occurrence of N. caninum in cattle in the Czech Republic. In addition, our results refer to an importance of transplacental transmission in cattle.
Recent adaptation to symbiotic lifestyle: the Polynucleobacter–Euplotes system
Authors: VANNINI CLAUDIA , HAHN MARTIN , PETRONI GIULIO , VERNI FRANCO , LUCCHESI SAMUELE , ROSATI GIOVANNA
Published: April 2005

Bacteria belonging to the genus Polynucleobacter were previously described as endosymbionts inhabiting the cytoplasm of freshwater ciliates of the genus Euplotes having a 9 type I cirrus pattern. They were all found to be necessary for the division process of their hosts. Here, we report the finding of a Polynucleobacter bacterium as symbiont of E. harpa, a brackish species with ten frontoventral cirri. Identity of the symbionts was unambiguously determined by morphological description and by 16S rRNA gene sequence characterization. Furthermore, antibiotic treatments confirmed that these bacteria play the same functional role of the other Polynucleobacter symbionts; aposymbiotic E. harpa ceases to divide and eventually die. Phylogenetic analyses based on 18S rRNA gene show that Euplotes species harboring Polynucleobacter symbionts, included E. harpa, steadily cluster together. In order to clear whether this symbiosis was established as a single evolutionary event by a common ancestor of the ciliate hosts, or by multiple acquisition of different bacteria by the different Euplotes species, comparative phylogenetic analyses were performed within the so‐called Polynucleobacter‐cluster, comprising bacterial symbionts as well as strictly related free‐living bacteria. Due to a high degree of conservation of 16S rRNA gene sequences within this group, phylogenetic trees based on this gene did not resolve the matter. Surprisingly, even the characterization and the analysis of the 16‐23S ITS sequences, usually exhibiting faster evolutionary rates, revealed a high similarity (>98%), thus hindering a stable and clear phylogenetic reconstruction. Data obtained cannot at present definitively confirm or reject any hypothesis on the origin of this symbiosis, revealing, at the same time, that we are dealing with a relatively young symbiotic system. As shown by the close phylogenetic relationship between free‐living and symbiotic Polynucleobacter, this is an ideal model for studying development and adaptation to symbiotic lifestyle.
Molecular phylogeny of free‐living and symbiotic Verrucomicrobiales by using protein‐coding genes
Authors: BAUER ANDREAS , ROSATI GIOVANNA , LUDWIG WOLFGANG , SCHLEIFER KARL‐HEINZ , PETRONI GIULIO
Published: April 2005

Verrucomicrobiales are a newly proposed, deep branched division of bacteria from which only a handful of cultured isolates are available. Culture‐independent analysis of environmental samples shows that members of Verrucomicrobiales are widespread and can be divided into several subdivisions. Symbiotic species of Euplotidium (Ciliophora) and Nematodes, epixenosomes and Xiphinemobacter spp. respectively, are according to the 16S sequences also members of this group. Up to now, only minimal sequence data mainly focused on 16S rRNA gene sequences are available for this group of organisms. The phylogenetic relatives of Verrucomicrobiales are still unclear with Chlamydiales and Planctomycetes being the most probable candidates. The present study describes preliminary results on phylogenetic relationships of Verrucomicrobiales by using protein‐coding genes like Hsp70 and RpoC/B. The species which are used for this study are Prosthecobacter vanneervenii, P. debontii, Opitutus sp.VeGlc2, O. terrae and epixenosomes. The genes were amplified with designed consensus‐degenerated primers and results are available on the Hsp70. The sequences show several differences within the group. Verrucomicrobium and Prosthecobacter sequences cluster together, but in P. debontii, two sequences were found, and the second one appears quite divergent. Also the two Opitutus genes are quite atypical and cluster together in a deep position. Preliminary attempt was performed on Euplotidium‐epixenosome complex. However, amplified and cloned sequences revealed to be closely related to ciliate sequences. Further attempt will be performed applying microtiter‐plate‐subtractive‐hybridization to characterize epixenosome genes.
Euplotes daidaleos and its endocytobionts
Authors: FOKIN SERGEI I.
Published: April 2005

Among almost 200 endocytobiotic associations between freshwater ciliates and Chlorella‐like algae (A) one Euplotes species—E. daidaleos Diller, Kounaris, 1966 is existing. It was not so far under particular investigation. Comparative study of cell morphology and behavior of “green” and derived from it “white” stocks of the ciliate (C), collected in Italy and Russia were carried out, using light and electron microscopy. The green C usually maintain 30–80 units of A and some bacteria (B), belonging to the different types—Polynucleobacter‐like bacterium, β‐subgroup of Proteobacteria (size about 2−10 × 0.4−0.5 μm) and another eubacterium (size 1−2 × 0.6−0.8 μm). The number of the first B was much higher in the white C, but could vary between different host cells. According to data obtained with fluorescent microscopy it looks like these B can produce long chains, which consisted of quite short individuals with only one nucleoid. Association between Euplotes and Chlorella within the system is rather a close one: loss of the A after long time cultivation of C in darkness usually did not happen. The majority of the ciliates (96–100%) kept A, but this number often dropped down, apparently as a result of digestion by C of some of the A. Positive phototaxis is almost absent in green E. daidaleos in comparison with that of Paramecium bursaria–Chlorella association. The rate of division was not significantly deviated according to A presence. Moreover, cells of the green Euplotes did not like high level of illumination and in any cases needed some additional food. This situation is also quite opposite to the P. bursaria–Chlorella system. In between 2 and 3 months of cultivation, the main part of the white stocks have lost its viability partly because of disturbance in the cirral pattern. In about 90% of cells some frontal, ventral, transversal and caudal cirri disappeared in different combinations. It is the first indication on some connection between A‐symbionts and host morphogenesis. Large food vacuoles almost all time presented in such cells show some kind of problems with digestion as well. E. daidaleos could be considered as three‐lateral symbiotic system, promising for further investigations.
Oral hygiene: past history and future recommendations
Authors: Sgan‐Cohen Harold D
Published: April 2005

Review of the dental literature reveals a changing emphasis attached to maintenance of optimal oral hygiene among patients over time. Moreover, a different relationship to this topic has been evident in the USA, Britain and Scandinavia. These changes, differences, and the rationale behind them, are described. The age of initiating oral hygiene practice and the optimal frequency of dental cleaning is discussed. Finally, recommendations are offered concerning oral hygiene promotion, both at the individual and community levels.