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At least 19 recordsLinked to original sources
PubMed1989

The effect of short-wavelength ultraviolet light on antigens, lectin receptors and the ultrastructure of Crithidia fasciculata.

Crithidia fasciculata is an important trypanosomatid parasite commonly affecting insects and is used extensively as a model for the study of the biochemistry, ultrastructure and organization of the kDNA network of trypanosomatids. The present study describes the evolution of UV-induced morphological changes detectable by transmission electron microscopy in Crithidia fasciculata. Although only rare and minor changes in kinetoplast DNA were demonstrable 7 h after UV irradiation, alterations of this organelle were present in almost all flagellates observed 24 h and 48 h after irradiation. Other cell structures were apparently undamaged. Ultrastructural changes in kDNA did not correspond to changes in antigenicity of protein bands in western blotting against serum from Chagas' disease patients or in the presence of 3 different lectin receptors on the surface of the parasite.

Animals↗
PubMedDate not supplied

Growth-phase dependent substrate adhesion in Crithidia fasciculata.

Crithidia fasciculata is a trypanosomatid flagellate that parasitizes several species of mosquito. Within the alimentary tract of its host, C. fasciculata exists in two forms: one is a non-motile form, attached in clusters to the lining of the gut, the other a more elongated form swimming freely in the gut lumen. We have developed an in vitro culture system that reproduces the appearance of these two distinct morphological forms. Using two different cultivation methods, shaking and stationary incubations, we have demonstrated that adherence phenotypes are growth-phase dependent. Organisms in the logarithmic phase of growth possess the ability to adhere to substrates; this ability is lost when the organism enters a stationary growth phase. Parasite adherence was independent of cultivation method or substrate. Furthermore, adherent forms of Crithidia maintained their adhesive properties following their removal from substrates. Our data reveal a growth-phase-regulated process of cell attachment that may influence the transmission and dissemination of this parasitic flagellate.

Animals↗
PubMed1987

The sequence-directed bent structure in kinetoplast DNA is recognized by an enzyme from Crithidia fasciculata.

Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) interrupts a single phosphodiester bond in duplex DNA circles from various sources, only in their supercoiled form, but not following their relaxation by DNA topoisomerases. However, this requirement for DNA substrate supercoiling was not observed using the natural kinetoplast DNA as a substrate. Relaxed kinetoplast DNA minicircles, either free or topologically linked, were efficiently nicked by the enzyme. Furthermore, bacterial plasmids, containing a unit length kinetoplast DNA minicircle insert, were used as substrates for nicking in their relaxed form. This capacity to activate a relaxed DNA topoisomer as a substrate for nicking is an intrinsic property of the sequence-directed bend, naturally present in kinetoplast DNA. The 211-base pair fragment of the bent region from C. fasciculata kinetoplast DNA could support the nicking of a relaxed DNA substrate in a reaction dependent upon the DNA helix curvature.

Animals↗
PubMed1986

Introduction of plasmid DNA into the trypanosomatid protozoan Crithidia fasciculata.

Crithidia fasciculata cells were treated with a plasmid (pDK96) containing pBR322 sequences, a Leishmania tarentolae maxicircle autonomously replicating sequence, and the bacterial gene for aminoglycoside 3' phosphotransferase I inserted between the yeast alcohol dehydrogenase 1 promotor and terminator sequences. Resistant colonies were selected on agar plates containing paromomycin and screened for vector DNA by hybridization. Approximately 1% of the resistant colonies contained detectable vector DNA, which was present as extrachromosomal closed circular molecules ranging in copy number from 1 to 160 per cell. The plasmids could be recovered from Escherichia coli transformed to ampicillin resistance with Crithidia total cell DNA. Most of the recovered plasmids were a deleted product of pDK96, which lacked the maxicircle autonomously replicating sequence and contained a unique fragment of Crithidia nuclear DNA present at a low copy number in the wild-type genome. The plasmid DNA in resistant Crithidia was unstable even under selective conditions and was lost within 30 cell divisions.

Ampicillin↗
PubMed1988

Identification of C3 acceptors responsible for complement activation in Crithidia fasciculata.

Crithidia fasciculata, an insect trypanosomatid is readily lysed by normal human serum at concentrations as low as 3%. Lysis occurs in the presence of Mg+2-EGTA and is antibody independent, indicating that the alternative pathway of complement activation is involved. Analysis of [131I]C3 deposition on C. fasciculata cells using C8-deficient serum, revealed that about 4 x 10(5) C3 molecules bound to each cell. Most of the C3 was bound to cells as C3b, part of it forming high molecular weight complexes, which could be dissociated by methylamine treatment at alkaline pH. To characterize the C3 acceptors on C. fasciculata, surface-iodinated cells were incubated with C8D or heat-inactivated serum, extracted and immunoprecipitated with anti-C3 or anti-arabinogalactan antisera. Analysis of the immunoprecipitated material on SDS gels showed high-molecular weight components, which disappeared after methylamine treatment, giving rise to a component of 200 kDa molecular size. This 200-kDa component corresponded to a purified arabinogalactan complex, which was immunoprecipitated from labeled cell extracts, without incubation with C8D, using anti-arabinogalactan antibodies. These results suggest that the arabinogalactan glycoconjugate is a C3 acceptor in C. fasciculata during complement activation. Purified arabinogalactan complexes were able to inactivate C3 in vitro. Solubilization in KOH to cleave the peptide moiety rendered it unable to inactivate C3. Apparently, the aggregated state of the purified arabinogalactan component at the cell surface is important for C3 deposition and activation.

Animals↗
PubMed1985

Interaction of aminoglycosides and ionophores in the killing of Crithidia fasciculata.

Crithidia fasciculata was utilized as a prescreen to determine the antiprotozoal action of aminoglycoside antibiotics alone and in combination with surface-altering agents. Paromomycin was tested with the carrier ionophores nigericin and valinomycin, the channel ionophore gramicidin and the polyene antibiotics amphotericin B and nystatin. After exposure to the drugs in suspension, organisms were plated out to determine the survival of C. fasciculata. Killing was time dependent for both the antibiotic and the ionophore. Paromomycin action was found to be potentiated by all the surface altering agents. The aminoglycosides kanamycin, gentamycin and streptomycin were studied alone and in combination with nigericin. Synergistic effects were demonstrated both with kanamycin and gentamycin in combination with nigericin. Streptomycin was ineffective both alone and with surface-altering agents.

Aminoglycosides↗
PubMed1978

The ribonucleic acids of Crithidia fasciculata.

Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (X 10(6) daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.

Animals↗
PubMed1991

Cell surface charge and sugar residues of Crithidia fasciculata and Crithidia luciliae.

The surface charge of Crithidia fasciculata and Crithidia luciliae was analysed by measurement of the zeta-potential and labelling of the protozoan surface with cationized ferritin particles. Both trypanosomatids have a net negative surface charge, with a zeta-potential of -10.39 mV and -11.12 mV for C. luciliae and C. fasciculata, respectively. Enzyme treatment showed that phosphate groups, but not sialic acid, significantly contributed to the negative surface charge. Lectin-induced agglutination was used to analyse the presence of surface-exposed carbohydrates in C. fasciculata and C. luciliae. The cells did not agglutinate when incubated in the presence of lectins which recognized L-fucose, N-acetyl-D-glucosamine and sialic acid. However, lectins which bind to N-acetyl-D-galactosamine, D-galactose and D-mannose agglutinated both protozoa.

Agglutination Tests↗
PubMed1986

N-linked high mannose-type oligosaccharides in the protozoa Crithidia fasciculata and Crithidia harmosa contain galactofuranose residues.

Incubation of Crithidia fasciculata cells with [U-14C] glucose led to the synthesis of Man-P-dolichol but not of Glc-P-dolichol. The main and largest dolichol-P-P-linked oligosaccharide formed was Man7GlcNAc2 whether labeling was performed in 5 mM sodium pyruvate or 5.5 mM glucose. The protein-linked, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides isolated from mature glycoproteins were Man7GlcNAc and Gal1Man6GlcNAc, the latter being a mixture of two isomers. All the galactose residues were present in the furanose configuration, as judged by their extreme lability to acid hydrolysis, by the products obtained upon mild periodate oxidation, and by their sensitivity to beta-galactofuranosidase. Labeling cells for short times or at low temperature yielded a protein-bound, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide whose composition was Glc1Man7GlcNAc, of transient existence, and that was mainly labeled in the glucose residue. The latter oligosaccharide was detected on paper chromatography only as a smearing of Man7GlcNAc and Gal1Man6GlcNAc when cells were labeled with [2-3H] mannose, thus indicating that it was only present in minute amounts. Protein-bound endo beta-N-acetylglucosaminidase H-resistant oligosaccharides liberated, upon a mild acid treatment, galactose residues and an unidentified substituent. The treatment rendered the oligosaccharides sensitive to endo beta-N-acetylglucosaminidase H, which liberated Man7GlcNAc and two isomers of Man6GlcNAc. An almost similar mechanism of protein N-glycosylation, including the existence of galactofuranose residues in N-linked oligosaccharides, was found to occur in Crithidia harmosa.

Animals↗
PubMed2004

Mitochondrial histone-like DNA-binding proteins are essential for normal cell growth and mitochondrial function in Crithidia fasciculata.

The Crithidia fasciculata KAP2 and KAP3 proteins are closely related kinetoplast-specific histone-like DNA-binding proteins. The KAP2 and KAP3 genes are 46% identical and are arranged in tandem on the chromosomal DNA. Disruption of both alleles of either gene alone shows no detectable phenotype. However, replacement of both copies of the sequence encoding the entire KAP2 and KAP3 locus increases maxicircle mRNA levels two- to fourfold. These double-knockout cells are viable but grow extremely slowly, have reduced respiration and very abnormal cell morphologies, and accumulate numerous large vacuoles. The extreme phenotype of these mutant cells suggests an important role for the KAP2 and KAP3 proteins in mitochondrial metabolism and cell growth.

Animals↗
PubMed1982

Nucleotide sequence of an exceptionally long 5.8S ribosomal RNA from Crithidia fasciculata.

In Crithidia fasciculata, a trypanosomatid protozoan, the large ribosomal subunit contains five small RNA species (e, f, g, i, j) in addition to 5S rRNA [Gray, M.W. (1981) Mol. Cell. Biol. 1, 347-357]. The complete primary sequence of species i is shown here to be pAACGUGUmCGCGAUGGAUGACUUGGCUUCCUAUCUCGUUGA ... AGAmACGCAGUAAAGUGCGAUAAGUGGUApsiCAAUUGmCAGAAUCAUUCAAUUACCGAAUCUUUGAACGAAACGG ... CGCAUGGGAGAAGCUCUUUUGAGUCAUCCCCGUGCAUGCCAUAUUCUCCAmGUGUCGAA(C)OH. This sequence establishes that species i is a 5.8S rRNA, despite its exceptional length (171-172 nucleotides). The extra nucleotides in C. fasciculata 5.8S rRNA are located in a region whose primary sequence and length are highly variable among 5.8S rRNAs, but which is capable of forming a stable hairpin loop structure (the "G+C-rich hairpin"). The sequence of C. fasciculata 5.8S rRNA is no more closely related to that of another protozoan, Acanthamoeba castellanii, than it is to representative 5.8S rRNA sequences from the other eukaryotic kingdoms, emphasizing the deep phylogenetic divisions that seem to exist within the Kingdom Protista.

Animals↗
PubMed1987

Multiple spacer sequences in the nuclear large subunit ribosomal RNA gene of Crithidia fasciculata.

In Crithidia fasciculata, a trypanosomatid protozoan, the nuclear-encoded ;28S' rRNA is multiply fragmented, comprising two large (c and d) and four small (e, f, g and j) RNA species. We have determined that the coding sequences for these RNAs (and that of the 5.8S rRNA, species i) are separated from one another by spacer sequences ranging in size from 31 to 416 bp. Coding and spacer sequences are presumably co-transcribed, with excision of the latter during post-transcriptional processing generating a highly fragmented large subunit (LSU) rRNA. Secondary structure modelling indicates that the C. fasciculata LSU rRNA complex (seven segments, including 5.8S rRNA) is held together in part by long-range intermolecular base pairing interactions that are characteristic of intramolecular interactions in the covalently continuous LSU (23S) rRNA of Escherichia coli. At least one functionally critical region (encompassing the alpha-sarcin cleavage site) is contained in a small RNA species (f) rather than in one of the two large RNAs. Within a proposed secondary structure model of C. fasciculata LSU rRNA, discontinuities between the different segments (created by spacer excision) map to regions that are highly variable in structure in covalently continuous LSU rRNAs. We suggest that ;rRNA genes in pieces' and discontinuous rRNAs may represent an evolutionarily ancient pattern.

Journal Article↗
PubMed2006

Development of a pharmacodynamic screening model with Crithidia fasciculata.

The genus Crithidia is a member of the family Trypanosomatidae and is related to the genera Leishmania and Trypanosoma with which it shares a variety of biochemical mechanisms, such as polyamine synthesis and methionin salvage. In consequence, a screening system for antiparasitic candidate material has been developed with Crithidia fasciculata, a parasite naturally occurring in insects and amphibians, but devoid of pathogenicity for humans. Initially a variety of culture media were evaluated of which TPS was best suited for the maintenance of stock cultures, and E-medium - a newly developed formula - for sensitivity testing. Optimal growth of C. fasciculata was observed under microaerophilic conditions. A system for sensitivity testing was developed and applied to the investigation of extracts from higher tropical plants of the genera Stemona and Aglaia for anticrithidial activity. Extracts with significant anti-crithidial activity were scheduled for chromatographic fractionation and the subsequent isolation, purification and structural identification of individual compounds for further sensitivity testing. Encouraging results were obtained with extracts from Aglaia odorata leaves, A. elaeagnoidea stem bark and A. edulis leaves, with EC(90) values of 1213 ng/ml, 1606 ng/ml, and 1462 ng/ml, respectively.

Aglaia↗
PubMed2002

A new expression vector for Crithidia fasciculata and Leishmania.

Crithidia fasciculata is a monogenetic parasite of insects. It grows in fully defined media without requiring serum, which facilitates biochemical analysis. We have constructed a series of expression systems that allows expression of transfected genes in the kinetoplastid protozoa Crithidia and Leishmania. These cells can be readily transfected with plasmid DNA by electroporation and transformants selected with various antibiotic resistance markers. 5'-Trans-splicing signals and poorly defined regions within the 3'-untranslated regions of genes are required for optimal expression of genes in trypanosomatids. We, therefore, inserted the intergenic region of the C. fasciculata phosphoglycerate kinase (PGK) genes A and B, which allows polyadenylation of the target gene and spliced leader addition to the selectable marker gene. Part of the intergenic region of the PGK locus was added upstream of the target gene to permit its trans-splicing. A 3'-untranslated sequence from the Crithidia glutathionylspermidine synthetase (GSPS) was also added to allow the polyadenylation of the selectable marker gene. Genes can be readily inserted using a multiple cloning site and can be expressed as a fusion protein with a poly-histidine sequence at either the N or C-terminus or fused with green fluorescent protein. Biologically active proteins can be expressed in C. fasciculata or L. amazonensis promastigotes and purified by affinity chromatography using a metal chelating column.

Animals↗
PubMed1982

Isolation and characterization of drug resistant mutants of Crithidia fasciculata.

Mutants of Crithidia fasciculata, resistant to Actinomycin D, Tubercidin, Crystal Violet, 6-Azauridine, and 5-Fluorouracil were obtained and characterized. The mutants were stable when maintained under nonselective conditions, and could be grouped in several subclasses on the basis of cross-resistance to other drugs. Actinomycin D- and Crystal Violet-resistant mutants appeared spontaneously whereas the others appeared only after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Analysis with a cell sorter indicated no large scale meiotic changes in ploidy under the various culture conditions employed. These mutants may provide suitable, selectable, genetic markers for use in studying the possibility of a sexual mating process in Crithidia.

Animals↗
PubMed1983

Structural and biochemical characterisation of the paraflagellar rod of Crithidia fasciculata.

The trypanosomatid Crithidia fasciculata possesses an intraflagellar structure known as the paraflagellar or paraxial rod which runs from a point 1 to 2 micrometer distal to the basal body to the flagellar tip. In longitudinal section the paraflagellar rod was composed of three "sets" of parallel filaments arranged in a lattice. In cross section it consisted of two electron dense "plaques", one near the flagellar membrane, the other near the axoneme, separated by 6 to 7 fibrous elements. The position of the paraflagellar rod in relation to the axonemal central pair remained static along the length of the flagellum and was the same in all flagella examined. The paraflagellar rod was anchored to the axoneme by a regular array of 5 to 7 nm diameter links. These rod/axoneme links were sensitive to trypsin digestion enabling the rod to be separated from the axoneme. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the paraflagellar rod consisted mainly of two proteins, PFR1 (76 000 Daltons) and PFR2 (68 000 Daltons). The isoelectric points of these two proteins were remarkably similar. A PFR-enriched fraction was obtained by prolonged dialysis of demembranated flagella against a low concentration buffer. The paraflagellar rod and the central pair of singlet microtubules went into solution, leaving only the outer doublets intact. The relevance of these results to the study of the role of the paraflagellar rod in flagellar motility were discussed.

Animals↗
PubMed1976

Hydrolysis of triphosphoinositides by a soluble fraction of Crithidia fasciculata.

Homogenates of Crithidia fasciculata (a species of Trypanosomidae) were shown to contain a phosphatase (EC 3.1.3.36) and a phosphodiesterase (EC 3.1.4.11) which hydrolyse triphosphoinositides. Approximately 30% of the diesterase and most of the phosphatase are present in the soluble fraction. The triphosphoinositide phosphatase is specifically dependent upon Mg(2+) and is stable to storage with or without freezing. The triphosphoinositide phosphodiesterase requires Ca(2+) and is inactivated during storage. Both activities are maximal in the presence of cetyltrimethylammonium bromide and require protection or reactivation by GSH or dithiothreitol. Unlike similar mammalian enzymes the protozoal triphosphoinositide phosphatase does not hydrolyse diphosphoinositides. The two enzymes may be separated by (NH4)2SO4 fractionation and gel filtration on Sephadex G-200.

Animals↗
PubMed1977

Subcellular fractionation by differential and zonal centrifugation of the trypanosomatid Crithidia fasciculata.

Homogenates of Crithidia fasciculata were fractionated by differential centrifugation. Mitochondria were sedimented quantitatively at 10(4) g-min and accounted for approximately 10% of the total recovered protein. Catalase was found exclusively in the supernatant fraction whilst NADH:cytochrome c oxidoreductase and p-nitrophenylphosphatase were found in all the fractions. Zonal centrifugation confirmed that catalase was non-sedimentable. Clean separation of mitochondria was obtained in both high-speed and rate zonal experiments, but no NADH:cytochrome c oxidoreductase activity could be detected in these organelles. Separation of large lysosomal vacuoles which contained p-nitrophenylphosphatase activity was obtained and these were clearly resolved from mitochondria by both high-speed and rate zonal centrifugation.

Cell Fractionation↗