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PubMed1982

Measurement of low avidity anti-dsDNA by the Crithidia luciliae test and the PEG assay.

With the immunofluorescence technique (IFT) using Crithidia luciliae as a substrate, 14,417 sera sent to our laboratory for routine anti-dsDNA determination, were screened for the presence of antibodies to dsDNA. The 1,260 sera that were found IFT positive were then assayed with the Farr radioimmunoassay, in which 3H-labelled PM2-DNA is used as antigen. Only 470 sera (37%) were found to be Farr positive. This discrepancy is, at least partially, caused by the fact that the Farr assay does not detect anti-DNA of low avidity, whereas the Crithidia-IFT does. Sixty-eight percent of the IFT-positive/Farr negative sera were found positive with the PEG assay, a radioimmunoassay that also employs double stranded PM2-DNA as antigen, and that also detects anti-dsDNA of low avidity. The IFT performed on IFT positive/Farr negative sera was found to be rather irreproducible. It was shown that this was due to local increases of the salt concentration resulting from the way the assay was performed. The problem could be overcome by careful control of the assay conditions, i.e. never letting Crithidia slides dry up after washing with PBS. In the PEG assay, these sera sometimes showed a DNA binding that decreased with time. It could be shown that this is caused by a parallel increase in pH during the incubation as a result of CO2 evaporation from the serum.

Antibodies, Antinuclear↗
PubMedDate not supplied

[Inhibition of oxidative phosphorylation in Crithidia fasciculata and Trypanosoma cruzi by lipophilic o-quinones and nifurtimox].

ATP and ADP levels were determined in Crithidia fasciculata and Trypanosoma cruzi. The nucleotide levels in crithidia or epimastigotes at the stationary phase of growth were, in nmol/10(8) cells, 15-40, and 3-7, for ATP and ADP, respectively. Incubation with the lipophilic o-naphthoquinones CG 8-935, CG 9-442 and CG 10-248 or the anti-chagasic nitrofuran nifurtimox, significantly decreased ATP level, with non-significant variations of the ADP level. The kinetics of ATP variation showed an initial 1-2 h lag and the diminution of the ATP level reached maximum value after 4-6 h incubation. Addition of L-glutamate or D-glucose as energy sources produced 2- or 3-fold increase of ATP level, after incubation the protozoa for 4-6 h with the corresponding substrates. Quinones and nifurtimox strongly depressed D-glucose or L-glutamate effects. Buthionine sulfoximine an inhibitor of glutathione biosynthesis, enhanced the effect of nifurtimox on ATP level in Crithidia fasciculata. It is concluded that by inhibiting ATP synthesis, either directly or-through oxygen radicals, the assayed drugs produced their cytotoxic action.

Adenosine Diphosphate↗
PubMed1975

Virulence of bacteria-associated, Crithidia-associated, and axenic Entamoeba histolytica: experimental hamster liver infections with strains from patients and carriers.

Experimental infections of the hamster liver were carried out with five strains from patients with clinical amoebiasis and ten strains from asymptomatic carriers. Inocula of comparatively small size (12000-36000 amoebae) were injected under the liver capsule. 1. The virulence of the patient strains varied from 21-96% (see article) and declined sharply within 7-15 weeks after elimination of the associated bacterial flora. The virulence of the carrier strains varied from 0-100%, probably fluctuating with changes in the concomitant bacterial flora (Table 1). 2. The interrelation between size of inoculum, period of bacteria-free growth, and virulence was demonstrated with a Crithidia-associated patient strains (Table 2). 3. A patient strain showed a faster decrease of virulence during axenic than in Crithidia-associated cultivation (Table 3). 4. Two successive passages through hamster liver resulted in a marked increase of virulence of two bacteria-free strains, lasting for several months (Table 4). 5. A significant enhancement of virulence of Crithidia-associated and axenic amoebae by reassociation with a mixed bacterial flora during two weeks, followed by elimination of the bacteria, was demonstrated with two strains. The restored virulence was lost again within a few weeks (Table 5). 6. The virulence of an attenuated patient strain did not become manifest by adding large numbers of dead amoebae to the inoculum (Table 6). 7. The pathology of the different lesions caused in the hamster liver by the amoebae is described, including one of a granulomatous type, frequently found after inoculation with bacteria-free amoebae. 8. In an attempt to explain the occurrence of strains differing in pathogenicity an hypothesis is put forward based on the idea of selection of virulence and avirulent amoebae.

Amebiasis↗
PubMed1970

Occurrence of Crithidia factors and folic acid in various bacteria.

Crithidia factors and folic acid were found to be widely distributed in culture fluids and in cells of 27 species of bacteria, when cultured under aerobic conditions into the stationary phase. Most bacteria excreted more Crithidia factors and folic acid than they retained in their cells. One Crithidia factor produced by Serratia indica and one produced by Bacillus cereus differed from biopterin in their chromatographic behavior. The factor excreted by S. indica appeared to be a 2-amino-4-hydroxy-6-substituted pteridine on the basis of KMnO(4) oxidation and ultraviolet absorption spectra. One of the folate compounds excreted by this organism was shown to be identical to 5,10-methylidynetetrahydrofolic acid by bioautography.

Bacteria↗
PubMed1969

Effects of some antitumor agents on growth and glycolytic enzymes of the flagellate Crithidia.

Some antitumor agents known to specifically inhibit certain tumor cell enzymes were examined for activity against glycolytic enzymes and growth of the insect trypanosomatid, Crithidia fasciculata. The cytoplasmic enzymes hexokinase, alpha-glycerophosphate dehydrogenase, malic dehydrogenase, and glucose-6-phosphate dehydrogenase were tested. Agaricic acid (2-hydroxy-1,2,3-nonadecane tricarboxylic acid) was highly inhibitory (50 to 100%) to malic and alpha-glycerophosphate dehydrogenases at approximately 3 x 10(-5)m; 2-(p-hydroxyphenyl)-2-phenylpropane (2 x 10(-4)m), and 5,6-dichloro-2-benzoxazolinone (5 x 10(-4)m) were less effective (50% inhibition) against them. The antiprotozoal agents primaquine (4 x 10(-4)m) and Melarsoprol (8 x 10(-4)m) were 30 to 40% inhibitory. Agaricic acid, 2-(p-hydroxyphenyl)-2-phenylpropane, and 5,6-dichloro-2-benzoxazolinone inhibited growth of Crithidia at less than 10(-4)m. Eight other test compounds from the Cancer Chemotherapy National Service Center (CCNSC) were not toxic to cell growth, although two (4-biphenylcarboxylic acid and 1-[p-chlorobenzyl]-2-ethyl-5-methyl-indole-3-acetic acid) inhibited Crithidia alpha-glycerophosphate dehydrogenase below 1 mm. All of the compounds used specifically inhibited cancer cell alpha-glycerophosphate dehydrogenase. The corresponding enzyme in pathogenic African trypanosomes is important in their terminal respiration. C. fasciculata may be useful in preliminary evaluation of chemotherapeutic agents as potential trypanocides.

Acetates↗
PubMed1985

The binding of antihistone antibodies to Crithidia luciliae kinetoplasts is growth cycle-dependent.

The Crithidia luciliae immunofluorescence (CLIF) assay is widely used to test for native DNA (nDNA) antibodies in the diagnosis and management of systemic lupus erythematosus. However, sera from patients with drug-induced lupus erythematosus or rheumatoid arthritis, which should not contain nDNA antibodies, occasionally react with the CL kinetoplast. We examined 36 sera from patients with systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, and drug-induced lupus erythematosus, who had positive CLIF tests. All 36 sera were also antinuclear antibody-positive with homogeneous and/or peripheral staining patterns on mouse kidney substrates. After hydrochloric acid extraction of the CL smears to remove histone and other nuclear protein antigens, 14 of the 36 sera no longer produced a positive result on the CLIF test. Ten of these 14 sera again gave a positive CLIF result after the hydrochloric acid-extracted Crithidia substrate had been reconstituted with purified histone. These studies demonstrated that kinetoplast binding was due to antihistone antibodies in at least 10 of 36 initially CLIF-positive sera. Antihistone antibodies were then purified with a histone-affinity column, and these purified antibodies were reactive with CL kinetoplasts. Thus, the CLIF test is not specific for nDNA antibodies. Additional studies using CL from different days of culture indicated that histone antigen expression in the CL kinetoplast was a function of the life cycle of this organism and is most readily detected 2 days after initiation of culture.

Animals↗
PubMed1996

Histone H1 and core histones in Leishmania and Crithidia: comparison with Trypanosoma.

The Trypanosomatidae family is characterized by flagellated protozoa presenting a kinetoplast. Several genera of this family contain species that are pathogenic to man and domestic animals. Their chromatin is not condensed into chromosomes during cell division. As a contribution to the understanding of basic aspects of their genome organization, we present a systematic characterization of the histones from three genera of the Trypanosomatidae family. Crithidia fasciculata and Leishmania mexicana show core nucleosomal histones with electrophoretic mobilities both similar to and different from those of Trypanosoma cruzi and higher eukaryotes. Another protein is extracted from the chromatin of these organisms by procedures designed to purify histone H1. This protein presents elution profiles by HPLC and amino acid composition of histone H1. Considering these data and the high mobility of this protein in Triton-acetic acid-urea-polyacrylamide gel electrophoresis, as well as its position relative to the nucleosomal core histones in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we postulate that Crithidia and Leishmania possess a histone H1 shorter than that of higher eukaryotes as we have previously shown to be the case for T. cruzi. The possible presence of a shorter histone H1 in these trypanosomatids may explain the absence of chromatin condensation during cell division in these flagellates.

Amino Acids↗
PubMed2001

Crithidia guilhermei: purification and partial characterization of a 62-kDa extracellular metalloproteinase.

An extracellular metalloproteinase from Crithidia guilhermei, a monoxenic trypanosomatid of insects, was purified 11-fold by ammonium sulfate precipitation, gel filtration on a Shinpack Diol-150 column, and anion-exchange chromatography in a MONO Q column, both using the HPLC system. The proteinase appeared as a single band with an apparent molecular mass of 62 kDa in SDS-PAGE, under reducing conditions, and was optimally active at 37 degrees C and pH 6.0. The enzyme showed 62% residual activity at 50 degrees C for 30 min. The proteinase was completely inhibited by 1, 10-phenanthroline, indicating that the enzyme belongs to the metalloproteinase class. This is the first report of the purification of an extracellular metalloproteinase from the Crithidia species. The possible role of this enzyme in the digestive tract of the insect host is discussed.

Animals↗
PubMed1986

Structure and evolution of the small subunit ribosomal RNA gene of Crithidia fasciculata.

We present the cloning and sequence analysis of the nuclear-encoded Crithidia fasciculata small subunit (SSU) rRNA gene, the longest (2,206 bp) such gene yet characterized by direct sequence analysis. Much of the sequence can be folded to fit a phylogenetically conserved secondary structure model, with the additional length of this gene being accommodated within discrete variable domains that are present in eukaryotic SSU rRNAs. On the basis of sequence comparisons, we conclude that Crithidia contains the most highly diverged SSU rRNA described to date among the eukaryotes, and therefore represents one of the earliest branchings within the eukaryotic primary kingdom.

Animals↗
PubMed1991

Class I and class II ribonuclease H activities in Crithidia fasciculata (Protozoa).

The protozoan Crithidia fasciculata contains two different ribonuclease H activities. These enzymes display similar physical and biochemical characteristics to their homologues in higher eukaryotes, for instance calf thymus class I and class II ribonuclease H. Class I ribonuclease H of lower and higher eukaryotes can be activated by Mg2(+)- and Mn2(+)-ions. However, the presence of Mn2(+)-ions is inhibitory for the Mg2(+)-dependent class II ribonuclease H activity of Crithidia fasciculata and calf thymus. The protozoan class I-homologue enzyme appears to be serologically related to the class I ribonuclease H of calf thymus.

Animals↗
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↗
PubMed1992

Identification of a surface metalloproteinase on 13 species of Leishmania isolated from humans, Crithidia fasciculata, and Herpetomonas samuelpessoai.

Promastigotes of thirteen species of Leishmania isolated from human patients, as well as L. enriettii, Crithidia fasciculata and Herpetomonas samuelpessoai, were examined for the expression of an amphiphilic, surface-oriented metalloproteinase by surface radioiodination of living cells, fractionation by Triton X-114 extraction and phase separation, and zymogram analysis by fibrinogen-SDS-PAGE. In all species of Leishmania, and the two monoxenous trypanosomatid parasites of insects, an ectoproteinase similar to the Promastigote Surface Protease, or PSP, was observed. In contrast, neither Phytomonas sp. nor 'Leishmania tarentolae' express a detectable surface metalloproteinase. The presence of the functionally conserved metalloproteinase at the surface of Crithidia and Herpetomonas suggest the enzyme may not be involved in the infection of the mammalian host by Leishmania, but rather contributes to the survival of the protozoan in the environment of the insect midgut.

Animals↗
PubMed1987

DNA sequence of Crithidia fasciculata kinetoplast minicircles.

Kinetoplast DNA (kDNA) networks of the insect trypanosomatid Crithidia fasciculata strain CF-C1 contain a nearly homogeneous population of kDNA minicircles as judged by restriction enzyme cleavage analysis. We have determined the entire nucleotide sequence of the major class of minicircles by analyzing M13 phage clones carrying half-length segments of the kDNA minicircle molecules. The 12 nucleotide sequence d(G-G-G-G-T-T-G-G-T-G-T-A) is the longest sequence common to kDNA minicircles from several trypanosome species examined to date. Two copies of this universal minicircle sequence were identified 180 degrees apart as direct repeats within the C. fasciculata kDNA minicircles. In addition, these universal minicircle sequences are contained within direct repeats with nearly identical sequences of 173 and 177 base pairs (bp) in length. These sequences are also conserved in the same arrangement in minor sequence classes of minicircles from this strain. Site-specific discontinuities on both strands of the minicircle, identified previously in minicircle replication intermediates, were localized within the 173 and 177 bp conserved sequences. These sequences were also found to have extensive homology with similar conserved sequences in kDNA minicircles from Leishmania tarentolae. We suggest that the two conserved sequences, each containing a single copy of the universal minicircle sequence, represent replication origins in the Crithidia minicircles.

Animals↗
PubMed1987

Inactivation of Crithidia fasciculata carbamoyl phosphate synthetase II by the antitumor drug acivicin.

In Crithidia fasciculata, carbamoyl phosphate synthetase II, which catalyses the first step of de novo pyrimidine biosynthesis, was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. The antitumor drug acivicin competitively inhibited the synthetase II activity with respect to L-glutamine, yielding an apparent Ki of 2 microM. In the absence of L-glutamine, acivicin resulted in a selective, time-dependent inactivation of L-glutamine-dependent activity of the enzyme, with an inactivation constant (Kinact) of 100 microM and a minimum inactivation half-time (T) of 0.2 min. L-Glutamine protected the enzyme from inactivation. These results are consistent with a postulate that acivicin is an active site-directed affinity analogue of L-glutamine, achieving irreversible inactivation. The inactivated enzyme retained ammonia-dependent activity. Acivicin stimulated the ammonia-dependent activity by increasing the Vmax value of the enzyme; apparent Km values for ammonia and MgATP were not affected. Differential action of acivicin on the Crithidia and mammalian synthetase II is discussed.

Ammonia↗
PubMed1993

Crithidia fasciculata contains a transcribed leishmanial surface proteinase (gp63) gene homologue.

We have cloned and analysed a gene from the insect parasite, Crithidia fasciculata, with homology to the gp63 metalloendoproteinase gene of Leishmania. The Crithidia gene homologue is arranged as a multicopy family comprised of approximately 7 genes. The mature transcript is 4.0 kb. The predicted amino acid sequence has significant homology with Leishmania gp63s, contains a zinc-binding motif and a potential site for addition of a glycosylphosphatidylinositol anchor. Demonstration of a gp63 homologue in C. fasciculata, a monogenetic parasite, suggests that the molecule may play a role in parasite survival within the insect gut.

Amino Acid Sequence↗
PubMed1987

Kinetic properties of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing) in the parasitic protozoan Crithidia fasciculata and separation of the enzyme from aspartate carbamoyltransferase.

A high specific activity of carbamoyl-phosphate synthetase II (glutamine-hydrolyzing; EC 6.3.5.5) was demonstrated in extract of the cultured Crithidia fasciculata. The enzyme was separated from aspartate carbamoyltransferase by ammonium sulfate fractionation. Apparent Km for the synthetase for L-glutamine, NH4+, MgATP or bicarbonate was 0.27, 26, 1.7 or 1.7 mM at 2.0% dimethyl sulfoxide plus 0.3% glycerol. 8.6% dimethyl sulfoxide plus 1.4% glycerol decreased Km for L-glutamine to 0.10 mM, while Km for MgATP was unaffected. The higher solvent concentrations made Vmax markedly reduced, yielding the inhibition of the activity. These properties are unique to the Crithidia synthetase, compared with the mammalian enzyme.

Ammonium Sulfate↗
PubMed2005

Identification and characterization of purine nucleoside transporters from Crithidia fasciculata.

To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by <2-fold. These data imply that this metabolic adaptation can mainly be ascribed to post-transcriptional events. Conversely, Southern analysis of CfNT1 suggests that it is a member of a highly homologous multi-copy gene family, indicating that adenosine transport by C. fasciculata is more complex than previously thought.

Adenosine↗
PubMed1998

The mitochondrial ND8 gene from Crithidia oncopelti is not pan-edited.

RNA editing in trypanosomatid mitochondria is a process involving the insertion and deletion of uridine residues within the coding region of maxicircle messenger RNA transcripts. Twelve of the 17 known genes need editing to produce functional molecules. We have analyzed the predicted editing sites for the Crithidia oncopelti mitochondrial NADH-ubiquinone oxidoreductase subunit 8 (ND8) gene based on known mRNAs from other trypanosomatid species. All studied ND8 mRNAs undergo editing throughout the coding (and 3' noncoding) sequences (pan-editing). The 5' part of the C. oncopelti ND8 gene undergoes editing (like in Leishmania tarentolae and Trypanosoma brucei) while the 3' part of the pre-edited gene corresponds to the 3' part of edited ND8 mRNAs from other organisms. The organization of the ND8 gene in C. oncopelti mitochondrial DNA differs from all organisms investigated so far -- this gene is not pan-edited. We have also localized the guide RNA for cytochrome b between 9S rRNA and the ND8 gene. This RNA shows high homology to the gCYb-II gene of L. tarentolae and the gCyb gene of Crithidia fasciculata. A hypothetical editing pattern for the cytochrome b gene in C. oncopelti maxicircles is proposed.

Amino Acid Sequence↗