Expression of alpha- and beta-tubulin genes during growth of Trypanosoma cruzi epimastigotes.

The expression of tubulin genes was studied during the growth of epimastigotes of Trypanosoma cruzi. Northern blot analysis showed that there was a decrease in the levels of alpha- and beta-tubulin mRNAs as epimastigotes changed from the logarithmic to the stationary phase. The changes were associated with a similar decrease in the rates of transcription for both of these genes as measured by run-on assays using permeabilized parasites. In contrast to these results, ubiquitin transcription increased slightly. The levels of alpha-tubulin protein per parasite also decreased in stationary compared with logarithmic phase epimastigotes, in close agreement with the decrease in transcription. However, beta-tubulin protein levels did not change significantly. Our results thus indicated that during the growth of epimastigotes, the expression of alpha-tubulin is controlled partially at the transcriptional level. On the other hand, the experiments also suggested that beta-tubulin expression is controlled at a post-transcriptional level.

Evaluation of recombinant antigens for serodiagnosis of Chagas' disease in South and Central America.

The commercially available diagnostic tests for Chagas' disease employ whole extracts or semipurified fractions of Trypanosoma cruzi epimastigotes. Considerable variation in the reproducibility and reliability of these tests has been reported by different research laboratories, mainly due to cross-reactivity with other pathogens and standardization of the reagents. The use of recombinant antigens for the serodiagnosis of Chagas' disease is recommended to increase the sensitivity and specificity of serological tests. Expressed in Escherichia coli, as fusion products with glutathione S-transferase, six T. cruzi recombinant antigens (H49, JL7, A13, B13, JL8, and 1F8) were evaluated in an enzyme-linked immunosorbent assay for Chagas' disease. The study was carried out with a panel of 541 serum samples of chagasic and nonchagasic patients from nine countries of Latin America (Argentina, Bolivia, Brazil, Chile, Colombia, El Salvador, Guatemala, Honduras, and Venezuela). The optimal concentration of each recombinant antigen for coating of plates was determined with the help of 125I-labelled recombinant proteins. While the specificity of the epimastigote antigen was 84% because of false positives from leishmaniasis cases, for the recombinant antigens it varied from 96.2 to 99.6%. Recombinant antigens reacted with 79 to 100% of serum samples from chronic chagasic patients. In this way, it is proposed that a mixture of a few T. cruzi recombinant antigens should be employed in a diagnostic kit to minimize individual variation and promote high sensitivity in the diagnosis of Chagas' disease.

Cloning and sequence analysis of a Trypanosoma cruzi alpha-tubulin cDNA.

A cDNA clone derived from the Trypanosoma cruzi alpha-tubulin gene was isolated and sequenced (Tc alpha tub; L37345). Tc alpha tub revealed an 87.79% and an 85.36% identity with the DNA sequence of T. brucei and Leishmania, respectively. This clone was used to study, by Northern blots, alpha-tubulin gene expression in epimastigotes, cell-cultured derived trypomastigotes and extracellular amastigotes. alpha-tubulin MRNA levels were the same in epimastigotes and trypomastigotes, however, there was a drastic decrease in amastigotes. This clone could be useful to elucidate the regulatory mechanisms of alpha-tubulin gene expression during the differentiation of T. cruzi.

A combined polymerase chain reaction-colour development hybridization assay in a microtitre format for the detection of Clostridium spp.

We have developed a rapid and sensitive assay for the detection of clostridial cells or spores in liquid food samples. The method recognizes Clostridium 16S (small ribosomal subunit RNA) rDNA (ribosomal DNA) sequences by a polymerase chain reaction-digoxigenin-labelling protocol, coupled to a capture oligonucleotide immobilized on a microtitre plate. The positive results are revealed by means of a colour reaction. In 6 h of non-intensive labour, we can detect as few as two to five clostridial cells or spores in experimentally contaminated soft drinks.

A double sandwich monoclonal enzyme immunoassay for detection of hepatitis B surface antigen.

An enzyme immunoassay for detection of hepatitis B surface antigen based on the use of 3 monoclonal antibodies (mAbs) was developed: an IgM as capture and 2 IgG1 for detection. The system biotin-streptavidin was compared with direct conjugation of mAbs to peroxidase and was preferred because of its higher signal to noise ratio. The possibility of simultaneous addition of human serum and biotin-mAb was discarded because of an evident prozone effect with some sera containing high HBsAg levels. The conjugation of biotin to IgG1 mAbs through a spacer arm (amidocaproyl) and the use of a highly sensitive substrate (tetramethylbenzIdine) improved the assay detection limit by about 10 times.

cAMP receptor protein from Trypanosoma cruzi: purification and cloning of a short sequence of the corresponding cDNA.

cAMP is involved in the differentiation of Trypanosoma cruzi, the causative agent of Chagas' disease. cAMP levels are elevated in the infective, non-dividing metacyclic trypomastigote stage, with respect to the non-infective, proliferative, epimastigote stage. In both stages three is a cAMP receptor protein (CARPT), with unique properties that differentiate it from the regulatory subunits of the cAMP-dependent protein kinase (RI and RII). The CARPT from T. cruzi epimastigotes was purified using ion-exchange chromatography, affinity chromatography and gel filtration. After the final step of purification, two protein bands were obtained, p89 and p70, corresponding to the intact CARPT and its proteolytic product. These two CARPT polypeptides were utilized to prepare polyclonal antibodies in rabbits. Previous results from our laboratory showed that CARPT cross-reacts with polyclonal antibodies prepared against the regulatory subunit (RII) of the cAMP-dependent protein kinase (PKA). As expected from these results, the anti-CARPT antibody recognized purified RII protein in an ELISA assay. The anti-CARPT antibodies were used for immunoblot analyses of epimastigote lysates. The two bands corresponding to the CARPT (p89 and p70), as well as a p40 band, were recognized. Immunoscreening of a T. cruzi lambda ZAP cDNA library with these anti-CARPT polyclonal antibodies yielded one positive clone (pBSCARPT) which contained a 540 bp insert. Northern analyses using the pBSCARPT clone as a probe, showed a 5.2 kb mRNA band in epimastigotes, which were grown in culture from 2 to 10 days in LIT medium. Sequence analyses of the 540 bp insert have failed to show homology to other gene sequences in the database.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential turn-over of beta-tubulin during the cell differentiation of Trypanosoma cruzi.

We investigated the expression of beta-tubulin during the differentiation of non-infective epimastigotes to infective metacyclics of Trypanosoma cruzi to underlay some of the regulatory mechanisms of the gene expression in this pathogenic parasite. Given the strong evolutionary conservation of tubulin, it was possible to study its translational and transcriptional products with heterologous probes. Quantitative Western blotting with specific monoclonal antibodies against beta-tubulin revealed an increase in the relative amounts of this protein in metacyclics with respect to epimastigotes. Pulse-chase experiments with radioactive methionine followed by immunoprecipitation and polyacrylamide gel electrophoresis showed that beta-tubulin has a slower degradation in metacyclics, which may contribute to its relative higher abundance in these parasite forms. In contrast with these results, both in vitro translation of poly (A+) mRNA in a wheat germ system and Northern blots of total and poly (A+) mRNA with a heterologous DNA probe from Leishmania enriettii, revealed a significant decrease (5 fold) in the specific transcripts of beta-tubulin in the metacyclics with respect to epimastigotes. It thus appeared that after differentiation of T. cruzi the translational machinery for a key protein such as beta-tubulin is shut off by a decrease in its specific message. The protein levels of this protein are maintained, however, by a compensatory mechanism that involves a slower turn-over of the synthesized protein.

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop.

A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.

Cyclic AMP in the Malpighian tubule fluid and in the urine of Rhodnius prolixus.

We demonstrate the presence of cyclic adenosine monophosphate (cAMP) in the fluid secreted by isolated Malpighian tubules of Rhodnius prolixus. In addition, we show that fifth-instar R. prolixus excrete cAMP in the urine after a meal of human blood. Nonstimulated isolated Malpighian tubules secrete small amounts of cAMP that increase about 10-fold after the addition of 5-hydroxytryptamine (5-HT). 5-HT is known to mimic R. prolixus diuretic hormone. The present findings demonstrate that 5-HT also acts via cAMP. The presence of cAMP in the rectal sac of the insect could be of importance in the differentiation of Trypanosoma cruzi and in the cycle of Chagas disease.

Intracellular signaling transduction in the differentiation of Trypanosoma cruzi: role of cAMP.

We have studied the cell differentiation of Trypanosoma cruzi in an vitro system that allows the transformation of epimastigotes into metacyclic trypomastigotes. Intracellular cAMP levels of epimastigotes increased 3 fold prior to their differentiation into metacyclics where cAMP remained elevated 3.7 fold with respect to epimastigotes. We also observed a 3 fold increase in the specific activity of cAMP-binding of metacyclics crude homogenates. This activity resided in a cAMP-binding receptor protein (CARPT) which was different from the typical cAMP-binding subunits (RI and RII) of cAMP-dependent protein kinases, as shown by the use of polyclonal antibodies prepared against these two types of proteins. Anti-RI antibodies did not react with CARPT, and anti-RII antibodies gave a cross reaction with CARPT which was at least 1,000 fold less sensitive than the one shown by the homologous antigen. On Western blots CARPT displayed a major band with Mr = 87,000 instead of Mr = 56,000 for RII. These studies implicate that cAMP may act as a mediator of the cell differentiation of T. cruzi by a mechanism involving a novel type of cAMP-binding receptor.

Cyclic AMP as an inducer of the cell differentiation of Trypanosoma cruzi.

The addition to epimastigotes cultures of T. cruzi, of either cAMP, monobutyryl-cAMP, dibutyryl-cAMP, 8-Br-cAMP (at 2 mM each), or the cAMP-phosphodiesterase inhibitor, papaverine (0.2 mM), promoted the in vitro differentiation of these parasite forms into metacyclics. This effect of cAMP may also be exerted in vivo in the insect vector, since cAMP was detected in the urine and in the Malpighi secretion fluids of Rodnius prolixus.

[Immunoenzyme assay using micro Dot on nitrocellulose (Dot-ELISA) in the diagnosis of Chagas' disease. I. Comparative study of 2 antigenic preparations of Trypanosoma cruzi]. / El ensayo inmunoenzimático en microgotas sobre nitrocelulosa (Dot-ELISA) en el diagnóstico de la enfermedad de Chagas. I. Estudio comparativo de dos preparaciones antigénicas de Trypanosoma cruzi.

Using the Dot-ELISA technique, two antigenic preparations of Trypanosoma cruzi epimastigote forms have been compared for the diagnosis of Chagas' disease: (1) The cytoplasmic fraction (cytoplasmic antigen) and (2) whole formalin fixed epimastigotes (integral antigen). There was been used sera from 95 chagasic patients with chronic cardiomyopathy, positive conventional serology and either positive or negative xenodiagnosis; 74 subjects with negative conventional serology, and either clinically normal or presenting cardiomyopathy; 74 patients with different diseases including syphilis, toxoplasmosis, leishmaniasis or autoantibodies such as rheumatoid factor and antinuclear antibodies. By defining the diagnostic titers (cut off): 1:512 for cytoplasmic antigen and 1:128 for the integral antigen, a sensitivity of 100% has been obtained with both antigenic preparations, being the specificity of 96% for the former and 100% for the latter when leishmaniasis sera were not included. A comparative study with conventional serology was carried out using 147 sera from a Laboratory of Chagas' diagnosis; Dot-ELISA with cytoplasmic antigen showed co-positivity index of 1.0, co-negativity 0.989 and efficiency of 0.993, and Dot-ELISA with integral antigen 1.0, 0.979 and 0.986 respectively. According to this evaluation, Dot-ELISA using whole formalin fixed epimastigotes might be a practical alternative for the serological diagnosis of Chagas' disease.

Possible role of cAMP in the differentiation of Trypanosoma cruzi.

To assess the possible action of cAMP on the cell differentiation of Trypanosoma cruzi, we determined both cAMP levels and cAMP-binding activities of epimastigotes and trypomastigotes of this parasite. Trypomastigotes showed a 4-fold higher cAMP content and a 2.5-fold increase in the specific activity of a cAMP-binding protein with identical properties to that of epimastigotes. The high levels of cAMP present in trypomastigotes strongly suggest a role of this cyclic nucleotide on the differentiation of T. cruzi.

Trypanosoma cruzi: polypeptide markers of epimastigotes and trypomastigotes.

We compared the major polypeptides of epimastigotes and trypomastigotes of T. cruzi, by submitting total parasite lysates to electrophoresis in polyacrylamide gels (SDS-PAGE), protein staining with Coomassie brilliant blue, laser densitometry, or immunoblotting with sera derived from infected individuals (Chagas' disease). Epimastigotes and trypomastigotes displayed extensive homology, the differences being quantitative, except for a trypomastigote-specific band of Mr 75,000 which reacted with chagasic sera. Immunoblotting with chagasic sera confirmed the electrophoretic homology of epimastigotes and trypomastigotes. Upon antigenic dilution, a cluster of antigenic bands in the range of Mr 150,000 to 75,000 prevailed in the trypomastigotes, whereas the epimastigotes displayed more abundance of antigenic bands in the range of Mr 72,000 to 36,000.

A unique type of cyclic AMP-binding protein of Trypanosoma cruzi.

On centrifugation on sucrose density gradients, the cyclic AMP-receptor protein of Trypanosoma cruzi was clearly resolved from the type II regulatory subunit of protein kinase from bovine heart (S20,W = 8.25 and 4.1, respectively). The binding of cyclic [3H]AMP to these two proteins was affected to different extents by several cyclic AMP analogues. Such differences between the cyclic AMP-receptor protein of T. cruzi and cyclic AMP-binding proteins of other eukaryotes might be exploitable by chemotherapy.