bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribosomal RNA Sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: Phylogenetic Construction and Structural Correspondence
Authors: ZHU XIAOLONG , WITTNER MURRAY , TANOWITZ HERBERT B. , CALI ANN , WEISS LOUIS M.
Published: May 2007

ABSTRACT. The microsporidian species Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis were compared by using sequence data of their rRNA gene segments, which were amplified by polymerized chain reaction and directly sequenced. The forward primer 530f (5′‐GTGCCATCCAGCCGCGG‐3′) was in the small subunit rRNA (SSU‐rRNA) and the reverse primer 580r (5′‐GGTCCGTGTTTCAAGACGG‐3′) was in the large subunit rRNA (LSU‐rRNA). We have utilized these sequence data, the published data on Encephalitozoon cuniculi and Encephalitozoon hellem and our cloned SSU‐rRNA genes from E. bieneusi and S. intestinalis to develop a phylogenetic tree for the microsporidia involved in human infection. The higher sequence similarities demonstrated between S. intestinalis and E. cuniculi support the placement of S. intestinalis in the family Encephalitozoonidae. This method of polymerized chain reaction rRNA phylogeny allows the establishment of phylogenetic relationships on limiting material where culture and electron microscopy are difficult or impossible and can be applied to archival material to expand the molecular phylogenetic analysis of the phylum Microspora. In addition, the highly variable region (E. coli numbering 590–650) and intergenic spacer regions in the microsporidia were noted to have structural correspondence, suggesting the possibility that they are coevolving.
Ultrastructure and Sporogony of Eimeria (syn. Epieimeria) anguillae (Apicomplexa) in the Eel (Anguilla anguilla)
Authors: BENAJIBA MOHAMED HASSAN , MARQUES ADAM , LOM JIŘÍ , BOUIX GEORGES
Published: May 2007

ABSTRACT. Ultrastructural studies on Eimeria (syn. Epieimeria) anguillae (Apicomplexa), parasite of the digestive tract of the eel, have shown that the development of this parasite takes place completely within the host cell. Merogony and gamogony are intracellular but in the epicytoplasmic position. Sporogony is also located within the epithelial cells, which agrees with assignment of this coccidian in the family Eimeriidae. However, depending on the intensity of infection and the physiopathological reaction of the host, the gamont may behave in two ways. 1) In massive infections, gamogony stages cause a genuine destruction of intestinal epithelium. Large numbers of gamonts form nodules and parts of the seriously destroyed epithelium peel off and are released into the lumen of the gut and quickly discharged into the outer environment. This discharged epithelium envelops cells containing immature oocysts that then sporulate outside the host. 2) In light infections, the host cells, which are necrotic due to the presence of a zygote, are pushed between the surrounding intact cells towards the base of the epithelium. Closely above its basal lamella, the oocyst then undergoes sporulation. These results show no taxonomically important biological features (e.g. special mode of implantation to the host cell or active movement of the zygote). Because the morphological characteristics of Epieimeria do not differ significantly from Eimeria, we propose to suppress the genus Epieimeria Dyková and Lom, 1981, and relegate its species into the genus Eimeria.
Divisional Morphogenesis in Amphisiellides illuvialis N. Sp., Paramphisiella caudata (Hemberger) and Hemiamphisiella terricola Foissner, and Redefinition of the Amphisiellidae (Ciliophora, Hypotrichida)
Authors: EIGNER PETER , FOISSNER WILHELM
Published: May 2007

ABSTRACT. Classification of hypotrich ciliates is bewildering, possibly due to many unrecognized convergencies and the lack of detailed ontogenetic data in most species. A puzzling case are hypotrichs which have an obliquely extending “median cirral row” on the ventral surface between the right and left marginal cirral row. Such species are often assigned to the poorly defined family Amphisiellidae. However, we show by a comparative analysis of the divisional morphogenesis of three amphisiellid morphotypes and by a reevaluation of literature data that a median cirral row can be formed by at least four non‐homologous processes. These data are used to define the “amphisiellid median cirral row/’(a row containing all or most cirri from at least two rightmost anlagen, arranged one behind the other during cytokinesis), to redefine the Amphisiellidae (Euhypotrichina with an amphisiellid median cirral row of which the anterior segment is formed by cirri of the rightmost ventral anlage and the posterior segment by cirri of the second ventral anlage from right; a middle segment may be formed by neighboring anlagen), and to improve the diagnoses of the seven genera assigned by us to this family. Attempts to reconstruct the evolution within the Amphisiellidae failed, in spite of the detailed morphological and ontogenetic data available. We thus conclude that such data, although highly valuable, are insufficient to light up the supergeneric phylogeny of hypotrich ciliates. For this, molecular markers and investigations of the morphogenetic processes at electron microscopic level appear indispensable.
Purification and Characterization of Isocitrate Lyase from Ethanol‐grown Euglena gracilis
Authors: ONO KOUKI , OKIHASHI MASAHIRO , INUI HIROSHI , MIYATAKE KAZUTAKA , KITAOKA SHOZABURO , NAKANO YOSHIHISA
Published: May 2007

Isocitrate lyase was purified to homogeneity from ethanol‐grown Euglena gracilis. The specific activity was 0.26 μmol/min/mg protein. The molecular mass of the enzyme was calculated to be 380 kDa by gel filtration on a Superose 6 column. The subunit molecular mass of the enzyme was 116 kDa as determined by SDS‐polyacrylamide gel electrophoresis. These results showed that the native form of this enzyme was a trimer composed of three identical subunits. The pH optimum for cleavage and condensation reactions was 6.5 and 7.0, respectively. The Km values for isocitrate, glyoxylate and succinate were 3.8, 1.3 and 7.7 mM, respectively. Isocitrate lyase absolutely required Mg for enzymatic activity. This is the first report of the purification of isocitrate lyase to homogeneity from Euglena gracilis.
The Salient Features of Five Marine Ciliates in the Class Spirotrichea (Oligotrichia), with Notes on their Culturing and Behaviour
Authors: MONTAGNES DAVID J. S. , TAYLOR F. J. R.
Published: May 2007

Descriptive information is provided for five ciliates collected from marine coastal British Columbian surface waters. In this paper we identify three new species, Strobilidium neptuni n. sp., Strobilidium veniliae n. sp., and Strombidinopsis multiauris n. sp.; provide a new name and description for one previously described species, Strombidium siculum nom. nov. and provide more taxonomic data for Strombidinopsis cheshiri Snyder and Ohman, 1991.
Chilodonella spp. at Four Fish Farms in Northern Finland
Authors: RINTAMÄKI PÄIVI , TORPSTRÖM HEIKKI , BLOIGU AINI
Published: May 2007

A total of 4.1% infestation with Chilodonella spp. was found among fish studied in 144 tanks in 1987–1989, representing 14.0% of the tanks in which fish are reared at four salmonid farms in northern Finland. Two species were found, C. hexasticha and C. piscicola, and both occurred on salmon (Salmo salar L.), sea trout [S. trutta m. trutta (L.)] and brown trout [S. t. m. lacustris (L.)]. Variability was observed in the length and width of the C. piscicola specimens and the number of ciliary rows or kineties. Large specimens which had more kinetics than average for C. piscicola were found mainly on the skin of salmon aged 1–2 years. The number of kinetics in the right ciliary band was found in stepwise logistic regression analysis to be of importance when typing C. piscicola specimens. Fingerlings were found to be more susceptible to Chilodonella infestation than older fish, and mortality varied in the range 2–10% in the course of the epizootics in the three fish species. Most mortality cases were caused by C. hexasticha, occurring mainly on the gills of the fish. Chilodonella piscicola was most often found in salmon and occurred at lower water temperatures than C. hexasticha (mean water temperature when found for the first time being 13° C and 16° C, respectively).
The Journey of Malaria Sporozoites in the Mosquito Salivary Gland
Authors: PIMENTA PAULO F. , TOURAY MUSA , MILLER LOUIS
Published: May 2007

The life cycle of malaria parasites in the mosquito vector is completed when the sporozoites infect the salivary gland and are ready to be injected into the vertebrate host. This paper describes the fine structure of the invasive process of mosquito salivary glands by malaria parasites. Plasmodium gallinaceum sporozoites start the invasion process by attaching to and crossing the basal lamina and then penetrating the host plasma membrane of the salivary cells. The penetration process appears to involve the formation of membrane junctions. Once inside the host cells, the sporozoites are seen within vacuoles attached by their anterior end to the vacuolar membrane. Mitochondria surround, and are closely associated with, the invading sporozoites. After the disruption of the membrane vacuole, the parasites traverse the cytoplasm, attach to, and invade the secretory cavity through the apical plasma membrane of the cells. Inside the secretory cavity, sporozoites are seen again inside vacuoles. Upon escaping from these vacuoles, sporozoites are positioned in parallel arrays forming large bundles attached by multilammelar membrane junctions. Several sporozoites are seen around and inside the secretory duct. Except for the penetration of the chitinous salivary duct, our observations have morphologically characterized the entire process of sporozoite passage through the salivary gland.
Immunoelectron Microscopy of Giardia lamblia Cytoskeleton Using Antibody to Acetylated α‐Tubulin
Authors: SOLTYS BOHDAN J. , GUPTA RADHEY S.
Published: May 2007

Giardia lamblia trophozoites contain acetylated α‐tubulin but lack detectable levels of tyrosinolated α‐tubulin, as demonstrated in immunoblots with monoclonal antibodies specific for these tubulin forms. By immunofluorescence microscopy, acetylated α‐tubulin is localized in axonemes, median bodies and in the adhesive disk. Post‐embeddment immunogold labeling of thin sections of cells was used to evaluate acetylation at the level of individual microtubules by electron microscopy. Cells were fixed with glutaraldehyde and embedded in the acrylic resin LR Gold. Results indicate all microtubules in adhesive disk, axonemes, basal bodies, funis and the median bodies contain acetylated α‐tubulin. Unlike immunofluorescence labeling, all microtubules of the adhesive disk and the funis could be gold labeled. No nonspecific labeling of the cytoplasm or of structures other than microtubules was observed. Acetylated microtubules in G. lamblia do not appear to be a subset of microtubules and acetylation appears uniform along the entire length of individual microtubules. Acetylation and the tyrosinolation state of microtubules in Giardia are discussed in the context of microtubule stability and crosslinked features of the cytoskeleton.
Incorporation of Fatty Acids and Amino Acids by Cultured Pneumocystis carinii
Authors: PAULSRUD JOHN R. , QUEENER SHERRY F.
Published: May 2007

Cultured P. carinii rapidly took up a variety of fatty acids. The relative rates of uptake for four fatty acids were 18:1 >> 16:0 ≅ 18:0 ≅ 18:2. Fatty acids were primarily incorporated into phospholipids and the uptake process was specifically inhibited by 2.2 and 22 μM primaquine, a concentration having no effect on host cells. Amino acids were also taken up by cultured P. carinii in a primaquine sensitive process. Radiolabeled leucine was incorporated into the major surface glycoprotein of P. carinii. The formation of radioactive P. carinii‐specific proteins indicated that the cultured organisms carried out transcription and translation and that the incorporation of amino acids was dependent upon P. carinii rather than rare HEL human embryonic lung cells. The spinner flask culture system provides convenient access to P. carinii for metabolic studies in defined medium for a period of 5–14 days after inoculation.
F‐actin Distribution of Dictyostelium Myosin I Double Mutants
Authors: PETERSON MICHELLE D. , TITUS MARGARET A.
Published: May 2007

The roles of the myosin I class of mechanoenzymes have been investigated by single and double gene knockout studies in the amoeba Dictyostelium discoideum. Cells lacking different myosin I pairs (myoA‐/myoB‐, myoB‐/myoC‐, and myoA‐/myoC‐) were examined with respect to their cytoskeletal organization. F‐actin localization by rhodamine‐phalloidin staining of cells indicates that the myoA‐/myoB‐, myoB‐/myoC‐, and myoA‐/myoC‐ cells appear to redistribute their F‐actin more slowly than wild type cells upon adhesion to a substrate. These studies suggest that Dictyostelium myoA, myoB, and myoC may have overlapping roles in maintaining the integrity or organization of the cortical membrane cytoskeleton.
A Comparative Study of Lipid Compositions of Cryptosporidium parvum (Apicomplexa) and Madin‐Darby Bovine Kidney Cells
Authors: MITSCHLER RALENE R. , WELTI RUTH , UPTON STEVE J.
Published: May 2007

Membrane lipid compositions of Cryptosporidium parvum and Madin‐Darby bovine kidney cells, an epithelial‐like cell line commonly used to study coccidia in vitro, were analyzed using both thin‐layer chromatography and gas‐liquid chromatography. Phosphatidylcholine was the predominant lipid in both C. parvum and Madin‐Darby bovine kidney cells, comprising 65% and 41% of the total phospholipids, respectively. Phospholipids of C. parvum contained twice the level of 16:0 and twenty‐fold more 18:2 than the Madin‐Darby bovine kidney cell line. We suggest that the parasite may be capable of sequestering specific complex membrane lipids at concentrations greater than those in the host cells. This study constitutes the first report of the lipid composition of C. parvum.
Evidence for the Presence of “Metabolic Sterols” in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols
Authors: KANESHIRO EDNA S. , ELLIS JAYNE E. , JAYASIMHULU KOKA , BEACH DAVID H.
Published: May 2007

Mixed life cycle stages of rat‐derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas‐liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii, respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas‐liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost‐5‐en‐3‐ol), cholest‐5‐en‐3‐one, and β‐sitosterol (stigmast‐5‐en‐3‐ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C28 and C29 sterols with one or two double bonds, and a hydroxyl group at C‐3 (ergost‐5‐en‐3‐ol, ergost‐7‐en‐3‐ol, ergosta‐dien‐3‐ol, stigmast‐5‐en‐3‐ol, stigmast‐7‐en‐3‐ol and stigmasta‐dien‐3‐ol). Steryl esters of P. carinii, probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3‐hydroxy‐3‐methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own “metabolic sterols”
The Micronuclear Gene Encoding β‐Telomere Binding Protein in Oxytricha nova
Authors: MITCHAM JENNIFER L. , PRESCOTT DAVID M. , MILLER MICHAEL K.
Published: May 2007

ABSTRACT The micronuclear version of the gene encoding β‐telomere binding protein (β‐TBP) in Oxytricha nova has been sequenced and compared to the macronuclear β‐TBP gene, previously described. The micronuclear gene contains three AT‐rich internal eliminated sequences (IES) of 37, 40, and 43 bp and four macronuclear destined sequences (MDS). The IES interrupt the gene once near the 5′ end of the coding region and twice in the 3′ trailer downstream from the TGA stop codon. The sequences of the micronuclear and macronuclear genes are colinear. Thus, the micronuclear β‐TBP gene is not scrambled, which contrasts with the highly scrambled state among the 14 MDS in the micronuclear α;‐TBP gene.
“Fenestrin” and Conjugation in Tetrahymena thermophila
Authors: NELSEN E. MARLO , WILLIAMS NORMAN E. , YI HONG , KNAAK JENNIFER , FRANKEL JOSEPH
Published: May 2007

ABSTRACT Certain monoclonal antibodies interact with proteins of Tetrahymena thermophila found in the conjugation junction as well as around the gametic nuclei (pronuclei) of conjugating cells; they also react with the oral primordium and fission zone of vegetative cells and with the cytoproct and contractile vacuole pores of all cells. One of these (FXIX‐3A7) was investigated in detail. Immunogold labelling suggests that the material labelled by the 3A7 monoclonal antibody, which we call “fenestrin,” is located beneath the epiplasm (membrane skeleton). Immunoblots reveal that the major and perhaps sole antigen is a 64 kDa polypeptide, found in two isoelectric variants. Developmental studies implicate fenestrin in two processes involved in conjugation. The first is “tip transformation.” During preliminary starvation (“initiation”), labelling of fenestrin first appeared as a spot at the anterior end of starved mature cells, then after mixing of different mating types (“costimulation”) it extended posteriorly along the anterior suture. After pairing, this region spread to form a widened plate. The second process is pronuclear transfer. Fenestrations representing channels between the conjugating cells began to appear 0.5 to 1 h after the conjugants united, and eventually merged to form a small number of temporary large holes during exchange of the transfer pronuclei. A fenestrin envelope also enclosed both the transfer and resident pronuclei; a strand of fenestrin connected the two. Shortly after pronuclear transfer, both transfer and resident pronuclei were released from fenestrin caps and fused to produce a zygotic nucleus (synkaryon) not associated with fenestrin. Fenestrin thus appears to be intimately involved in the process of pronuclear exchange.
A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity
Authors: BHAUD YVONNE , BARBIER MICHÈLE , SOYER‐GOBILLARD MARIE‐ODILE
Published: May 2007

ABSTRACT By adding the protein synthesis inhibitor, emetine (10‐4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2‐Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non‐dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non‐dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.
Polymorphism of the Thymidylate Synthase Gene of Pneumocystis carinii from Different Host Species
Authors: MAZARS EDITH , ÖDBERG‐FERRAGUT CARMEN , DEI‐CAS EDUARDO , FOURMAUX MARIE‐NOELLE , ALIOUAT EL MOUKHTAR , BRUN‐PASCAUD MONIQUE , MOUGEOT GENEVIEVE , CAMUS DANIEL
Published: May 2007

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400‐bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat‐derived P. carinii was hybridized with the amplified products from rat‐ and mouse‐derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host‐species specific. A specific probe which recognized all human P. carinii isolates was defined.
Isotricha jalaludinii N. Sp. Found from the Rumen of Lesser Mouse Deer, Tragulus javanicus, in Malaysia
Authors: IMAI SOICHI , KUDO HIROSHI , FUKUTA KATSUHIRO , ABUDULLAH NORHAMI , HO YIN‐WAN , ONODERA RYOJI
Published: May 2007

Isotricha jalaludinii n. sp. found in the rumen of lesser mouse deer, Tragulus javanicus, in Malaysia was described and illustrated. This new species is characterized by the location and direction of the vestibulum, shape of the macronucleus, and absence of a dent at the vestibular opening. The presence of single peculiar isotrichid species in the rumen of mouse deer, which is recognized as one of the most primitive ruminants, suggests that the isotrichid ciliates similar to I. jalaludinii and Isotricha intestinalis were established at a fairly early period during the evolution of ruminants.
The Regulation of Dictyostelium Development by Transmembrane Signalling
Authors: GINSBURG GAIL T. , GOLLOP RACHEL , YU YIMIN , LOUIS JOHN M. , SAXE CHARLES L. , KIMMEL ALAN R.
Published: May 2007

ABSTRACT. Dictyostelium discoideum has a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughout Dictyostelium development to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type‐specific gene expression.
Plasmodium falciparum Rhoptry Proteins of 140/130/110 kd (Rhop‐H) Are Located in an Electron Lucent Compartment in the Neck of the Rhoptries
Authors: SAM‐YELLOWE TOBILI Y. , FUJIOKA HISHASHI , AIKAWA MASAMICHI , MESSINEO DANIELA G.
Published: May 2007

ABSTRACT. To investigate in more detail the structure of the high molecular weight rhoptry protein complex of Plasmodium falciparum, Rhop‐H (140/130/110 kd), the complex was affinity purified from parasite extracts using rhoptry protein specific antisera prepared against Rhop‐H proteins bound to and eluted from Balb/c mouse erythrocytes, using 0.5 M NaCl. The individual proteins (140 kd/Rhop‐1, 130 kd/Rhop‐2, and 110 kd/Rhop‐3) were separated, electroeluted, and monospecific polyclonal antisera prepared against the individual proteins, and against the affinity purified complex. Immunofluorescence assays and immunoelectron microscopic studies were performed to verify the subcellular localization of the Rhop‐H epitopes. Immunoblotting and immunoprecipitation assays were also performed. We report novel findings regarding the localization of the rhoptry proteins to an electron lucent compartment in the neck of the rhoptries. Analysis of the amino acid composition of the individually purified Rhop‐H proteins demonstrated a predominance of negatively charged (E, D) as well as hydrophobic residues (L, A, P, S) in the three proteins. The percentage of negatively charged residues was high for all three proteins. Similarities in amino acid composition for the three proteins supports the previous data demonstrating shared properties such as erythrocyte and liposome binding, for the three proteins. Results of antibody characterizations using rhoptry protein specific antisera demonstrate the immunodominance of the Rhop‐H complex.
Kyaroikeus cetarius N. G., N. Sp.: A Parasitic Ciliate from the Respiratory Tract of Odonticete Cetacea
Authors: SNIEZEK JAMES H. , COATS D. WAYNE , SMALL EUGENE B.
Published: May 2007

ABSTRACT. Examination of mucus discharged from the blowholes of live odonticete Cetacea and material collected from nasal orifices of dead hosts routinely revealed infestations of a large spindle‐shaped ciliate. Kyaroikeus cetarius n. g., n. sp. These ciliates had a prominent posterior podite and were holotrichously ciliated except for a conspicuous bipolar stripe extending along the left margin of the cell. Most specimens were free‐swimming and moved slowly through freshly collected mucus, but some individuals were attached to host cells or cellular debris by a transparent thread secreted from the podite. The oral architecture of protargol‐impregnated K. cetarius consisted of one preoral and two circumoral kinetics set within a deep oral cavity continuous with an extensive, posteriorly directed cytopharynx. Somatic kineties were composed entirely of monokinetids and formed an expansive right ciliary field covering most of the cell surface, a reduced left ciliary field adjacent to the oral cavity, and a group of four kinetal fragments located mid‐ventrally within a shallow pocket. Subkinetal microtubules were associated with the kineties of the right and left fields, and the non‐ciliated stripe was underlain by a series of longitudinal fibers. The ciliate's large, heteromeric macronucleus was centrally positioned and clearly evident in living or stained specimens, while the ellipsoid micronucleus was located adjacent to the cytopharynx and often inconspicuous. K. cetarius has several morphological attributes typical of phyllopharyngian ciliates (e.g., adhesive organelle, heteromeric macronucleus, somatic kineties of monokinetids, subkinetal microtubules), and, except for its nearly holotrichous ciliation, most closely resembles dysteriine ciliates.
Ultrastructure and Ecology of Perispira ovum (Ciliophora: Litostomatea): An Aerobic, Planktonic Ciliate that Sequesters the Chloroplasts, Mitochondria, and Paramylon of Euglena proxima in a Micro‐oxic Habitat
Authors: JOHNSON PAUL W. , DONAGHAY PERCY L. , SMALL EUGENE B. , SIEBURTH JOHN MCN.
Published: May 2007

ABSTRACT. High resolution sampling of the stratified water column in a fjord‐like ecosystem revealed a green‐pigmented planktonic ciliate that was found to be a ravenous predator of Euglena proxima. The vertical distributions of both predator and prey were coincident, and maximum populations occurred across the transition from oxic to anoxic water. This ciliate was identified as Perispira ovum (family Spathidiidae; Order Haptorida). P. ovum was observed by transmission electron microscopy to retain not only the chloroplasts, but also the mitochondria and paramylon reserve of its algal prey. A mechanism of sequestration of algal organelles is demonstrated for the first time. This mechanism includes: recognition, capture, and ingestion of prey; rupture and release of algal cell contents; and enrobing of individual organelles and paramylon by the host vacuolar membrane. The structural integrity, peripheral location, and association with host endoplasmic reticulum suggests the sequestered organelles may be functional within P. ovum. The occurrence and high biomass of this aerobic ciliate in an oxygen‐limited environment also suggests that the sequestered chloroplasts are photosynthetically active and may provide additional substrates (such as oxygen) and metabolic capabilities that are crucial for its survival.
Alterations Induced by the Antifungal Compounds Ketoconazole and Terbinafine in Leishmania
Authors: VANNIER‐SANTOS MARCOS A. , URBINA JULIO A. , MARTINY ANDREA , NEVES ANDREA , DE SOUZA WANDERLEY
Published: May 2007

ABSTRACT. The antiproliferative effects and ultrastructural alterations induced in vitro by two antifungal compounds, the azole ketoconazole and the allylamine terbinafine on Leishmania amazonensis are reported. Promastigotes treatment with ketoconazole and terbinafine induced growth arrest and cell lysis in 72 hours. Combination of the two agents produced additive effects on promastigote axenic growth and synergistic effects on intracellular amastigote proliferation. The amastigotes, either axenically grown or infecting murine macrophages, were about 100‐fold more sensitive to the drugs. These compounds induced the appearance of large multivesicular bodies, especially after ketoconazole treatment, increased amount of lipid inclusions as well as numerous, polymorphic volutin granules, particularly in terbinafine‐treated cells. Multivesicular bodies were observed in close apposition with organelles such as mitochondria, which also showed alterations in the distribution and appearance of cristae, and the formation of paracrystalline arrays within the matrix. Some cells presented large portions of cytoplasm wrapped by endoplasmic reticulum and many parasites also presented myelin‐like endoplasmic reticulum profiles. Such alterations together with the strong acid phosphatase activity observed in the multivesicular bodies and volutin granules may indicate the existence of an unusual autophagic process in cells treated with ergosterol biosynthesis inhibitors.
Characterization of Glycogen and Amino Acid Pool of Entamoeba histolytica by 13C‐NMR Spectroscopy
Authors: BAKKER‐GRUNWALD TILLY , MARTIN JEAN‐BAPTISTE , KLEIN GERARD
Published: May 2007

ABSTRACT. Trophozoite extracts of axenic Entamoeba histolytica were investigated by natural‐abundance 13C‐NMR spectroscopy. The extracts were found to contain a high level of glycogen (30 mM glucose equivalents), which had a compact structure as suggested by α (1 → 6) branch points every 5–6 glucose residues. As other major metabolites, we identified putrescine (9.5 mM) and the following free amino acids: tyrosine and phenylalanine (1 mM), glycine, lysine and methionine (2 mM), isoleucine (5 mM), proline and valine (6–7 mM), leucine (11 mM) and glutamate (22 mM). Glutamate and proline may serve, together with putrescine, as intracellular osmolytes.
Encephalitozoon cuniculi Isolated from the Urine of an AIDS Patient, which Differs from Canine and Murine Isolates
Authors: HOLLISTER WAFAA S. , CANNING ELIZABETH U. , COLBOURN NICOLA I. , AARONS EMMA J.
Published: May 2007

ABSTRACT. A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymoprhic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi. However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi, with a band at 42–45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.
Use of a Tetrazolium‐based Cell Proliferation Assay to Measure Effects of In Vitro Conditions on Perkinsus marinus (Apicomplexa) Proliferation
Authors: DUNGAN CHRISTOPHER F. , HAMILTON ROSALEE M.
Published: May 2007

ABSTRACT. Because the in vitro cell cycle of the apicomplexan oyster pathogen Perkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for determining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium‐based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus. at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected culture system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populations propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F‐12. Temperatures of 10–40° C permitted growth, which was maximized at 35° C. pH 6.0–8.5 permitted growth, which was maximized at 7.0–7.5. Osmolalities of 340–1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1–10% (v/v) did not enhance log phase growth, but enhanced stationary phase metabolic activity in proportion to concentration. Our isolate (ATCC 50439) has a 13 h log phase doubling time when propagated under optimized conditions: 28° C, 800 mOsm, pH 7.0, 1:1 DME/Ham's F‐12 medium, 5% (v/v) FBS. It is tolerant of antibacterial agents at concentrations commonly used in vertebrate tissue culture, but is inhibited by several antimycotics at similar concentrations.
Monoclonal Antibodies Identify a Subset of Dense Granules in Cryptosporidium parvum Zoites and Gamonts
Authors: BONNIN ALAIN , GUT JIRI , DUBREMETZ JEAN FRANCOIS , NELSON RICHARD G. , CAMERLYNCK PATRICK
Published: May 2007

ABSTRACT. Two monoclonal antibodies raised against purified oocysts and excysted sporozoites of Cryptosporidium parvum identified antigens located in the anterior half of sporozoites by indirect immunofluorescence microscopic assay. The monoclonal antibodies also reacted with Triton X‐100‐insoluble antigens of asexual and sexual stage parasites developing in epithelial cells in vitro and identified a 110 kilodalton antigen on immunoblots of sodium dodecyl sulfate‐extracted oocysts. Immunoblotting reactivity was abolished by prior treatment of blotted antigen with periodic acid suggesting that the monoclonal antibodies recognize a carbohydrate or carbohydrate‐dependent epitope(s). By immunoelectron microscopy, the antibodies reacted with a family of small, electron‐dense granules located predominantly in the central region of merozoites and also with a population of cytoplasmic inclusions in macrogamonts. In addition, the monoclonal antibodies prominently labeled the parasitophorous vacuole membrane of all intracellular stages examined suggesting that the corresponding antigen(s) may be exocytosed from the granules to become associated with Triton X‐100‐insoluble components of the vacuolar membrane or cytoskeleton.
Phylogeny of Trichomonads Inferred from Small‐Subunit rRNA Sequences
Authors: GUNDERSON JOHN , HINKLE GREGORY , LEIPE DETLEF , MORRISON HILARY G. , STICKEL SHAWN K. , ODELSON DAVID A. , BREZNAK JOHN A. , NERAD THOMAS A. , MÜLLER MIKLOS , SOGIN MITCHELL L.
Published: May 2007

ABSTRACT. Small subunit (16S‐like) ribosomal RNA sequences were obtained from representatives of all four families constituting the order Trichomonadida. Comparative sequence analysis revealed that the Trichomonadida are a monophyletic lineage and a deep branch of the eukaryotic tree. Relative to other early divergent eukaryotic assemblages the branching pattern within the Trichomonadida is very shallow. This pattern suggests the Trichomonadida radiated recently, perhaps in conjunction with their animal hosts. From a morphological perspective the Devescovinidae and Calonymphidae are considered more derived than the Monocercomonadidae and Trichomonadidae. Molecular trees inferred by distance, parsimony and likelihood techniques consistently show the Devescovinidae and Calonymphidae are the earliest diverging lineages within the Trichomonadida, however bootstrap values do not strongly support a particular branching order. In an analysis of all known 16S‐like ribosomal RNA sequences, the Trichomonadida share most recent common ancestry with unidentified protists from the hindgut of the termite Reticulitermes flavipes. The position of two putative free‐living trichomonads in the tree is indicative of derivation from symbionts rather than direct descent from some free‐living ancestral trichomonad.
Uptake and Metabolism of L‐Serine by Pneumocystis carinii carinii
Authors: FLORIN‐CHRISTENSEN MONICA , FLORIN‐CHRISTENSEN JORGE , KANESHIRO EDNA S.
Published: May 2007

ABSTRACT. Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid‐precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin‐layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 11% in long chain bases and other compounds. Four long chain bases were detected by thin‐layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were tentatively identified by their migrations on thin‐layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidylethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L‐Cycloserine and β‐CI‐D‐alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids.
Acanthamoeba pearcei N. Sp. (Protozoa: Amoebida) from Sewage Contaminated Sediments
Authors: NERAD THOMAS A. , SAWYER THOMAS K. , LEWIS EARL J. , MCLAUGHLIN SHAWN M.
Published: May 2007

ABSTRACT. Seabottom sediments from a discontinued Philadelphia‐Camden 40‐Mile ocean sewage disposal site were cultured for cyst‐forming free‐living amoebae. Barge delivered wastes were discharged at the site from 1973 until 1980 when the site was closed. One station at the southeast margin of the site was sampled at a depth of approximately 50 m, twice in 1978 and once in 1982, 1983 and 1984. Sediment from the 1978 collection yielded Acanthamoeba polyphaga, Vahlkampfia sp., and an unknown amoeba with stellate endocysts similar to those of A. astronyxis. Trophozoites and cysts of the isolate were typical of those described for the genus Acanthamoeba. Biochemical tests employing enzyme electrophoresis and morphological studies on live and stained specimens showed that the isolate was distinct from other well‐described species within the family Acanthamoebidae Sawyer & Griffin, 1975.
Evolution of Cell Adhesion Systems: Evidence for Arg‐Gly‐Asp‐Mediated Adhesion in the Protozoan Neoparamoeba aestuarina
Authors: CUSTODIO MARCIO R. , IMSIECKE GEORG , BOROJEVIC RADOVAN , RINKEVICH BARUCH , ROGERSON ANDREW , MÜLLER WERNER E. G.
Published: May 2007

ABSTRACT. Developmental processes in multicellular organisms require structural elements, such as adhesion molecules, to stabilize cells at functional positions. In vertebrates, a series of extracellular matrix proteins, e.g. fibronectin and laminin are involved in cell adhesion. These proteins contain Arg‐Gly‐Asp [RGD] at their binding sites. Here we show that at concentrations above 2 mM the peptide GRGDSPK, comprising the tripeptide RGD (Arg‐Gly‐Asp), prevents the adhesiveness of cells of the marine amoeba Neopar‐amoeba aestuarina. In addition, elevated levels of GRGDSPK cause cells to alter their shapes from those with digitiform subpseudopodia to rounded cells with small lobed pseudopodia. These cells detach from the substratum. These results are specific for the RGD sequence, because incubation in GRGESPK solution at the same concentrations had no effect on cell attachment or structure. From these data we suggest that the structural adhesion molecules identified in vertebrates shows amino acid homologies with those found in unicellular protozoa.
Spironucleus vortensN. Sp. from the Freshwater AngelfishPterophyllum scalare:Morphology and Culture
Authors: POYNTON SARAH L. , FRASER WILLIAM , FRANCIS‐FLOYD RUTH , RUTLEDGE PHILLIP , REED PEGGY , NERAD THOMAS A.
Published: May 2007

ABSTRACT.A new diplomonad flagellate,Spironucleus vortensn. sp., is described from the intestinal lumen of the freshwater angelfish, (Pterophyllum scalare), bred in Florida. Live organisms are pyriform, and measure 12.5–20.5 μm long by 5.0–11.2 üm wide. Scanning electron microscopy shows that the trophozoite bears two compound lateral longitudinal ridges, each originating posterior to three emerging anterior flagella, and continuing posteriorly to the emergence of the posterior flagellum. Each ridge comprises a broad central part, surrounded by a peripheral ridge. At the opening of the flagellar pocket, the broader right peripheral ridge crosses to the other side of the body, and then back again. The posterior end of the body bears two papillae. Transmission electron microscopy shows that the compound lateral ridges are supported by microtubules, and bear microfibrillar structures in discrete longitudinal plaques. The serendipitous growth ofS. vortensin a culture system with lip tumor tissue, facilitated axenic cultivation in a modified TYM medium (trypticase, yeast extract, maltose). The flagellate is now routinely maintained in an axenic TYI‐S‐33 medium (trypticase, yeast extract, iron serum), and is stabilized in the cryopreserved state.Spironucleus vortensis an aerotolerant anaerobe that can be cultured at 25° C, 28° C and 30° C.
Deciliation Induces Phosphorylation of a 90‐kDa Cortical Protein in Tetrahymena thermophila
Authors: GITZ DARRELL L. , PENNOCK DAVID G.
Published: May 2007

ABSTRACT. We have used the anti‐phosphoprotein antibody MPM‐2 to examine changes in phosphorytation of cortical proteins during cilia regeneration in Tetrahymena thermophila. Although numerous cortical proteins are phosphorylated in both nondeciliated and deciliated cells, deciliation induces a dramatic increase in the phosphorylation of a 90‐kDa cortical protein. The 90‐kDa protein remained phosphorylated during cilia regeneration and then gradually became dephosphorylated. The 90‐kDa protein was phosphorylated and dephosphorylated normally in Tetrahymena mutants that assemble short cilia, suggesting that achievement of full length is not the signal that triggers dephosphorylation of the 90‐kDa protein. When initiation of cilia assembly is blocked, the 90‐kDa protein becomes phosphorylated and remains phosphorylated for an extended period of time, suggesting that initiation of cilia elongation triggers eventual dephosphorylation of the 90‐kDa protein, regardless of how long the cilia actually become.
Morphology and Ecology of Siroloxophyllum utriculariae (Penard, 1922) N. G., N. Comb. (Ciliophora, Pleurostomatida) and an Improved Classification of Pleurostomatid Ciliates
Authors: FOISSNER WILHELM , LEIPE DETLEV
Published: May 2007

ABSTRACT. The morphology and infraciliature of Siroloxophyllum utriculariae (Penard, 1922) n. g., n. comb. were studied in live cells, with the scanning and transmission electron microscope, as well as in specimens impregnated with protargol and silver carbonate. The new genus, Siroloxophyllum, belongs to the Loxophyllidae and has a specific combination of characters, viz. an oral bulge surrounding almost the entire cell, three perioral kineties, a single brush kinety, and a single right dorsolateral kinety. The ecology and faunistics of S. utriculariae are reviewed. It is a rare and infrequent predator preferring clean freshwaters. The somatic monokinetid of S. utriculariae has typical haptorid ultrastructure, including two transverse microtubular ribbons. The oral bulge is patterned string‐like with riffles containing the transverse microtubular ribbons originating from the oral kinetids. Perioral kineties 1 and 2 consist of dikinetids having one basal body each ciliated; the nonciliated basal body is associated with a nematodesmal and a transverse microtubular ribbon. Perioral kinety 3 consists of ciliated monokinetids having a fine structure similar to the somatic kinetids; they form triads with the dikinetids from perioral kinety 2. The classification of pleurostomatid ciliates is reviewed. Two suborders (Amphileptina, Litonotina) and three families (Amphileptidae, Litonotidae, Loxophyllidae n. fam.) are recognized and defined.
Short‐Term In Vitro Culture and Molecular Analysis of the Microsporidian, Enterocytozoon bieneusi
Authors: VISVESVARA GOVINDA S. , LEITCH GORDON J. , PIENIAZEK NORMAN J. , SILVA ALEXANDRE J. DA , WALLACE SARA , SLEMENDA SUSAN B. , WEBER RAINER , SCHWARTZ DAVID A. , GORELKIN LEO , WILCOX C. MEL , BRYAN RALPH T.
Published: May 2007

ABSTRACT. The microsporidium, Enterocytozoon bieneusi, causes a severe, debilitating, chronic diarrhea in patients with the acquired immunodeficiency syndrome. Specific diagnosis of intestinal microsporidiosis, especially due to Enterocytozoon, is difficult and there is no known therapy that can completely eradicate this parasite. Preliminary studies indicate that a short term (about 6 months) in vitro culture of this parasite yielding low numbers of spores, may be established by inoculating human lung fibroblasts and/or monkey kidney cell cultures with duodenal aspirates and or biopsy from infected patients. The cultures may subsequently be used for the isolation and molecular analysis of parasite DNA.