In vitro ovicidal activity of Brongniartia montalvoana against small ruminant gastrointestinal nematodes.

Several plants of the Fabaceae family have been assessed regarding their high nutritional value and anthelmintic properties. The ovicidal effect of the hydroalcoholic extract (Bm-HAE) and subfractions from the aerial parts of Brongniartia montalvoana (Fabaceae) against a mixed strain of gastrointestinal nematodes (GIN) (Haemonchus spp., Trichostrongylus spp. and Oesophagostomum spp.) resistant to albendazole sulfoxide, ivermectin and levamisole was evaluated by the egg hatch test (EHT). The Bm-HAE was subjected to liquid-liquid chemical separation with ethyl acetate giving two fractions, an aqueous (Bm-Aq) and an organic (Bm-EtOAct). The purification of the bioactive fraction (Bm-EtOAct) through chromatographic separation resulted in four bioactive subfractions (BmR6, BmR7, BmR8 and BmR10). The treatments were designed as follows: Bm-HAE at 800, 1,500, 3,000 and 6,000 µg/mL, and Bm-Aq, Bm-EtOAct and subfractions (BmR6, BmR7, BmR8 and BmR10) at 100, 200, 400 and 800 µg/mL. Two properly negative controls (distilled water and 2% methanol) and thiabendazole (100 µg/mL) as a positive control were used for each bioassay. The chemical identification of the extract, fractions and subfractions was performed through chromatographic processes like open column chromatography, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC-PDA). Additionally, the GIN eggs exposed to the bioactive compounds were observed through confocal laser scanning microscopy (CLSM). The Bm-HAE showed 99.5% egg hatching inhibition (EHI) at 6,000 µg/mL with a lethal concentration (LC50) of 1110 µg/mL. The Bm-EtOAc fraction displayed 99.1% EHI at 800 µg/mL with LC50 = 180 µg/mL. The ovicidal activity of the four subfractions was similar at 800 µg/mL: BmR6 (92% EHI); BmR7 (100% EHI); BmR8 (97.8%); and BmR10 (99.1%). The HPLC-PDA analysis of the bioactive subfractions allowed identification of p-coumaric acid, ferulic acid and coumarin derivatives as major compounds. The CLSM analysis allowed observation of morphological alterations in unhatched larvae caused by bioactive compounds present in the Bm-EtOAc and BmR10. In addition, the flavonoids eriodyctiol, luteolin and cynaroside were described for the first time for B. montalvoana.

In vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts against Haemonchus contortus (Nematoda) and their anthelmintic effect in gerbils.

The in vitro nematicidal effect of Chenopodium ambrosioides and Castela tortuosa n-hexane extracts (E-Cham and E-Cato, respectively) on Haemonchus contortus infective larvae (L3) and the anthelmintic effect of these extracts against the pre-adult stage of the parasite in gerbils were evaluated using both individual and combined extracts. The in vitro confrontation between larvae and extracts was performed in 24-well micro-titration plates. The results were considered 24 and 72 h post confrontation. The in vivo nematicidal effect was examined using gerbils as a study model. The extracts from the two assessed plants were obtained through maceration using n-hexane as an organic agent. Gerbils artificially infected with H. contortus L3 were treated intraperitoneally with the corresponding extract either individually or in combination. The results showed that the highest individual lethal in vitro effect (96.3%) was obtained with the E-Cham extract at 72 h post confrontation at 40 mg/ml, followed by E-Cato (78.9%) at 20 mg/ml after 72 h. The highest combined effect (98.7%) was obtained after 72 h at 40 mg/ml. The in vivo assay showed that the individual administration of the E-Cato and E-Cham extracts reduced the parasitic burden in gerbils by 27.1% and 45.8%, respectively. Furthermore, the anthelmintic efficacy increased to 57.3% when both extracts were administered in combination. The results of the present study show an important combined nematicidal effect of the two plant extracts assessed against L3 in gerbils.

The TcTASV proteins are novel promising antigens to detect active Trypanosoma cruzi infection in dogs.

In regions where Chagas disease is endemic, canine Trypanosoma cruzi infection is highly correlated with the risk of transmission of the parasite to humans. Herein we evaluated the novel TcTASV protein family (subfamilies A, B, C), differentially expressed in bloodstream trypomastigotes, for the detection of naturally infected dogs. A gene of each TcTASV subfamily was cloned and expressed. Indirect enzyme-linked immunosorbent assays (ELISA) were developed using recombinant antigens individually or mixed together. Our results showed that dogs with active T. cruzi infection differentially reacted against the TcTASV-C subfamily. The use of both TcTASV-C plus TcTASV-A proteins (Mix A+C-ELISA) enhanced the reactivity of sera from dogs with active infection, detecting 94% of the evaluated samples. These findings agree with our previous observations, where the infected animals exhibited a quick anti-TcTASV-C antibody response, coincident with the beginning of parasitaemia, in a murine model of the disease. Results obtained in the present work prove that the Mix A+C-ELISA is a specific, simple and cheap technique to be applied in endemic areas in screening studies. The Mix A+C-ELISA could help to differentially detect canine hosts with active infection and therefore with high impact in the risk of transmission to humans.

Anthelmintic effects of Prosopis laevigata n-hexanic extract against Haemonchus contortus in artificially infected gerbils ( Meriones unguiculatus).

The anthelmintic effect of Prosopis laevigata (mezquite) n-hexanic extract was evaluated against Haemonchus contortus endoparasitic stages in artificially infected gerbils (Meriones unguiculatus). Prosopis laevigata leaves were collected from the Sierra de Huautla, Ecological Reserve of the Biosphere, in Morelos State, Mexico; dehydrated under shade and macerated with n-hexane for 3 days, followed by distillation for 8 h. This procedure was repeated three times and the final extract was kept at 4 degrees C. The in vivo effect of the plant extract was evaluated in gerbils artificially infected with H. contortus. Plant extract concentration was 40 mg/ml. Three groups of gerbils were as follows: group 1 (n = 7), P. laevigata extract at 100 microl intraperitoneally (IP); group 2 (n = 6), control--Tween 20 in water at a single dose of 100 microl IP; group 3 (n = 8) also served as a control, receiving water only, to determine the mortality due to causes other than the plant extract. An additional group of seven gerbils (group 4) was administered fenbendazole, as a positive control. Five days later the animals were euthanized and stomach and mucosa removed to quantify the nematodes. Data were analysed using the Student's t-test to compare the mean of nematodes obtained in groups 1, 2 and 3. The parasite population in the plant extract treated group 1 was reduced by 42.5% (P < 0.05) with respect to the control group 2; and when control group 3 was used for comparison the parasitic reduction was estimated as 53.11%. This study shows the in vivo anthelmintic effect of P. laevigata n-hexane extract for the first time, using gerbils as an in vivo model, with potential use in sheep.

Two simultaneous hepatitis B virus epidemics among injecting drug users and men who have sex with men in Buenos Aires, Argentina: characterization of the first D/A recombinant from the American continent.

Previous studies have revealed that hepatitis B virus (HBV)/D and HBV/F predominate among blood donors from Buenos Aires, Argentina. In the present study, blood samples from two high-risk groups were analysed: 160 corresponding to street- and hospital-recruited injecting drug users [81.2% showing the 'anti-hepatitis B core antigen (anti-HBc) only' serological pattern] and 20 to hepatitis B surface antigen (HBsAg)(+)/anti-HBc(+) men who have sex with men. HBV genotypes were assigned by polymerase chain reaction amplification followed by restriction fragment length polymorphism and confirmed by nucleotide sequencing of two different coding regions. HBV DNA was detected in 27 injecting drug users (16.9%, occult infection prevalence: 7.7%), and 14 men who have sex with men (70%). HBV/A prevailed among injecting drug users (81.8%) while HBV/F was predominant among men who have sex with men (57.1%). The high predominance of HBV/A among injecting drug users is in sharp contrast to its low prevalence among blood donors (P = 0.0006) and men who have sex with men (P = 0.0137). Interestingly, all HBV/A S gene sequences obtained from street-recruited injecting drug users encoded the rare serotype ayw1 and failed to cluster within any of the known A subgenotypes. Moreover, one of the HBV strains from a hospital-recruited injecting drug user was fully sequenced and found to be the first completely characterized D/A recombinant genome from the American continent. Data suggest that two simultaneous and independent HBV epidemics took place in Buenos Aires: one spreading among injecting drug users and another one sexually transmitted among the homosexual and heterosexual population.

In vitro nematicidal effects of medicinal plants from the Sierra de Huautla, Biosphere Reserve, Morelos, Mexico against Haemonchus contortus infective larvae.

Twenty extracts from plants from Sierra de Huautla Biosphere Reserve, Morelos, Mexico were evaluated against Haemonchus contortus infective larvae in an in vitro assay. The plant species evaluated were Bursera copallifera, B. grandifolia, Lippia graveolens, Passiflora mexicana, Prosopis laevigata, Randia echinocarpa and Urtica dioica. The plants were separated into their parts and macerated with different solvents (n-hexane, acetone, ethanol and methanol). An in vitro assay was used to evaluate the anthelmintic activity against unsheathed third stage H. contortus infective larvae. The experiment was carried out in 24-well cell culture plates at room temperature with three replicates per treatment and using a concentration of 20 mg ml- 1. Ten 5 microl aliquots were taken from the corresponding wells and deposited on a slide for microscopical observation at 24, 48, 72 and 96 h post-exposure. The evaluation criteria were based on the average numbers of live and/or dead larvae in the different treatments. Alive and dead larval numbers were statistically analysed through the ANOVA test (P>0.01). The Tukey test was used as a complementary tool to determine which treatment was different from the other treatments (P>0.05). The highest mortality was observed with P. laevigata hexanic extract from stem and leaves combined, which produced 51%, 81% and 86% larval mortality at 24, 48 and 72 h post-exposure, respectively. On the other hand, B. copallifera stem acetonic extract exhibited 18%, 59% and 66% nematicidal activity after 24, 48 and 72 h of exposure, respectively.

Dynamics of a hepatitis B virus e antigen minus population ascribed to genotype F during the course of a chronic infection despite the presence of anti-HBs antibodies.

The in vivo evolution of genotype F HBV variants was recorded in a chronically infected patient throughout a 3-year observation period. Fluctuating levels of HBs Ag and anti-HBs antibodies were recorded, both of them cocirculating in peripheral blood samples at given times. Fifty S gene derived clones were sequenced and phylogenetically analyzed. As expected, some amino acid replacements within the S ORF were also observed within the P ORF while others were silent for the former. Such change was statistically significant for both S and P overlapping genes, which clearly indicates the appearance of a positive selection pressure. Supporting this notion, amino acid replacements were documented at both B and T cell epitopes in samples from 1997 and 1998. Several mutations were documented within and outside the "a" determinant in the major hydrophilic region. Such substitutions might have resulted from the attempt of HBV to evade both humoral and/or cellular immune response. To the best of our knowledge this unusual profile of HBV variants in presence of usually "neutralizing" anti-HBs antibodies was examined in vivo for the first time.

Hepatitis B virus S gene mutants in a patient with chronic active hepatitis with circulating Anti-HBs antibodies.

An adult male farmer with chronic active hepatitis and cirrhosis despite previous circulating anti-HBs antibodies was studied. No markers of other hepatotropic viral infection were observed. HBV DNA was detected in serum by PCR and was characterized further by restriction fragment length polymorphism (RFLP) and sequencing of cloned PCR products derived from the S gene. The HBV DNA was ascribed to genotype F, and single-strand conformational polymorphism (SSCP) demonstrated the co-circulation of multiple quasispecies. Some of the variants exhibited changes located within the neutralizing "a" determinant, located between amino acids 124-147 of the S protein. Within this region, two clones showed either C124R or C124Y mutations. Other mutations were Q129R, C138R, C139R, and S140T (one clone each). Outside the "a" determinant several substitutions were documented. The high degree of the quasispecies variability was probably linked to the severity of the infection. Most members of the patient's family were infected with HBV, all with genotype F.

Gene discovery through genomic sequencing of Brucella abortus.

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.

A random sequencing approach for the analysis of the Trypanosoma cruzi genome: general structure, large gene and repetitive DNA families, and gene discovery.

A random sequence survey of the genome of Trypanosoma cruzi, the agent of Chagas disease, was performed and 11,459 genomic sequences were obtained, resulting in approximately 4.3 Mb of readable sequences or approximately 10% of the parasite haploid genome. The estimated total GC content was 50.9%, with a high representation of A and T di- and trinucleotide repeats. Out of the estimated 5000 parasite genes, 947 putative new genes were identified. Another 1723 sequences corresponded to genes detected previously in T. cruzi through expression sequence tag analysis. 7735 sequences had no matches in the database, but the presence of open reading frames that passed Fickett's test suggests that some might contain coding DNA. The survey was highly redundant, with approximately 35% of the sequences included in a few large sequence families. Some of them code for protein families present in dozens of copies, including proteins essential for parasite survival and retrotransposons. Other sequence families include repetitive DNA present in thousands of copies per haploid genome. Some families in the latter group are new, parasite-specific, repetitive DNAs. These results suggest that T. cruzi could constitute an interesting model to analyze gene and genome evolution due to its plasticity in terms of sequence amplification and divergence. Additional information can be found at http://www.iib.unsam.edu.ar/tcruzi.gss. html.

A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication.

As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment.

AU-rich elements in the 3'-untranslated region of a new mucin-type gene family of Trypanosoma cruzi confers mRNA instability and modulates translation efficiency.

Trypanosoma cruzi has a complex mucin gene family of 500 members with hypervariable regions expressed preferentially in vertebrate associated stages of the parasite. In this work, a novel mucin-type gene family is reported, composed of two groups of genes organized in independent tandems and having very short open reading frames. The structures of deduced proteins share the N and C termini but differ in central regions. One group has repeats with the consensus Lys-Asn-Thr(7)-Ser-Thr(3)-Ser(Ser/Lys)-Ala-Pro and the other a Thr-rich sequence of the type Asp-Gln-Thr(17-20)-Asn-Ala-Pro-Ala-Lys-Asp-Thr(5-7)-Asn-Ala-Pro-Ala-L ys. In both cases, expected mature core proteins are around 7 kDa. Both groups, named L and S, respectively, differ in the structure of genomic loci and mRNA, with differential blocks in the 3'-untranslated region. The highest mRNA level for S and L groups are in the epimastigote stage but they show distinct developmentally regulated patterns. Transcripts are short lived and their steady-state abundance is regulated post-transcriptionally with increased mRNA stability in insect stage epimastigote. AU-rich sequences, similar to ARE motives known to cause mRNA instability in higher eukaryotes, are present in the 3'-untranslated region of the transcripts. In transfection experiments this sequence is shown to be functional for the L group destabilizing its mRNA in a stage-specific manner. Furthermore, an effect of this AU-rich region on translation efficiency is shown. To our knowledge, this is the first time that a functional ARE sequence-dependent post-transcriptional regulation mechanism is reported in a lower eukaryote.

Genetic diversity of GBV-C/HGV strains among HIV infected-IVDU and blood donors from Buenos Aires, Argentina.

GBV-C/HGV RNA was investigated in serum samples from 70 HIV(+) intravenous drug users (IVDU), as well as from 200 blood donors from Buenos Aires, Argentina. Viral RNA was demonstrated in 21 IVDU by reverse transcription-nested PCR of the 5' UTR. c-DNA amplified products were analyzed and their sequences compared with those downloaded from GenBank. A phylogenetic tree based on 171 sequences demonstrated the presence of three major genogroups, including two subgroups, within local samples, i.e. group 1 (n=1), 2a (n=11), 2b (n=4) and 3 (n=5). These results agreed entirely with those obtained by a novel RFLP (J. Clin. Microbiol. 37, 1340-1347, 1999) of the same 5' UTR amplicons. As expected, GBV-C/HGV RNA prevalence was significantly higher among IVDU than among blood donors (P<0.0001), although within the latter group an unexpectedly high rate was also detected, since 11 of 200 sera (5.5%) proved positive. These viral isolates were ascribed either to subgroup 2a (n=5), subgroup 2b (n=5) or genogroup 3 (n=1). Briefly, this partial view of GBV-C/HGV molecular epidemiology in Argentina shows: (i) different rates of GBV-C/HGV infection within both IVDU and blood donors; (ii) a high prevalence of viral RNA among blood donors; and (iii) a predominant circulation of genogroup 2, with minor contribution of groups 3 and 1.

Enzymically inactive members of the trans-sialidase family from Trypanosoma cruzi display beta-galactose binding activity.

trans-sialidase is a unique sialidase in that, instead of hydrolizing sialic acid, it preferentially transfers the monosaccharide to a terminal beta-galactose in glycoproteins and glycolipids. This enzyme, originally identified in Trypanosoma cruzi, belongs to a large family of proteins. Some members of the family lack the enzymatic activity. No function has been yet assigned to them. In this work, the gene copy number and the possible function of inactive members of the trans -sialidase family was studied. It is shown that genes encoding inactive members are not a few, but rather, are present in the same copy number (60-80 per haploid genome) as those encoding active trans -sialidases. Recombinant inactive proteins were purified and assayed for sialic acid and galactose binding activity in agglutination tests. The enzymatically inactive trans -sialidases were found to agglutinate de-sialylated erythrocytes but not untreated red blood cells. Assays made with mouse and rabbit red blood cells suggest that inactive trans -sialidases bind to beta, rather than alpha, terminal galactoses, the same specificity required by active trans -sialidases. A recombinant molecule that was made enzymatically inactive through a mutation in a single amino acid also retained the galactose binding activity. The binding was competed by lactose and was dependent on conservation of the protein native conformation. Therefore, at least some molecules in the trans -sialidase family that have lost their enzymatic function still retain their Gal-binding properties and might have a function as lectins in the parasite-host interaction.

GB virus C/hepatitis G virus groups and subgroups: classification by a restriction fragment length polymorphism method based on phylogenetic analysis of the 5' untranslated region.

A phylogenetic tree based on 150 5' untranslated region sequences deposited in GenBank database allowed segregation of the sequences into three major groups, including two subgroups, i.e., 1, 2a, 2b, and 3, supported by bootstrap analysis. Restriction site analysis of these sequences predicted that HinfI and either AatII or AciI could be used for genomic typing with 99.4% accuracy. cDNA sequencing and subsequent alignment of 21 Argentine GB virus C/hepatitis G virus strains confirmed restriction fragment length polymorphism patterns theoretically predicted. This method may be useful for a rapid screening of samples when either epidemiological or transmission studies of this agent are carried out.

[Organization of the network for the study of the Trypanosoma cruzi genome]. / Organización de la red para el estudio del genoma de Trypanosoma cruzi.

Five years ago the Special Programme for Research and Training in Tropical Diseases (TDR) from the World Health Organization (WHO) launched the Parasite Genome Project. The aims were to obtain information on genome organization and gene discovery in five parasites, namely, Schistosoma, Filaria, Leishmania and Trypanosomas brucei and cruzi. Organization of research networks for each parasite under study, promotion of international collaboration and training of researchers in developing countries, were also main objectives of the programme. After five years, a large amount of information has been obtained, which is now available to researchers in the field.

Gene discovery through expressed sequence Tag sequencing in Trypanosoma cruzi.

Analysis of expressed sequence tags (ESTs) constitutes a useful approach for gene identification that, in the case of human pathogens, might result in the identification of new targets for chemotherapy and vaccine development. As part of the Trypanosoma cruzi genome project, we have partially sequenced the 5' ends of 1, 949 clones to generate ESTs. The clones were randomly selected from a normalized CL Brener epimastigote cDNA library. A total of 14.6% of the clones were homologous to previously identified T. cruzi genes, while 18.4% had significant matches to genes from other organisms in the database. A total of 67% of the ESTs had no matches in the database, and thus, some of them might be T. cruzi-specific genes. Functional groups of those sequences with matches in the database were constructed according to their putative biological functions. The two largest categories were protein synthesis (23.3%) and cell surface molecules (10.8%). The information reported in this paper should be useful for researchers in the field to analyze genes and proteins of their own interest.

The Trypanosoma cruzi mucin family is transcribed from hundreds of genes having hypervariable regions.

In previous works we have identified genes in the protozoan parasite Trypanosoma cruzi whose structure resemble those of mammalian mucin genes. Indirect evidence suggested that these genes might encode the core protein of parasite mucins, glycoproteins that were proposed to be involved in the interaction with, and invasion of, mammalian host cells. We now show that the mucin gene family from T. cruzi is much larger and diverse than expected. A minimal number of 484 mucin genes per haploid genome is calculated for a parasite clone. Most, if not all, genes are transcribed, as deduced from cDNA analysis. Comparison of the cDNA sequences showed evidences of a high mutation rate in localized regions of the genes. Sequence conservation among members of the family is much higher in the untranslated (UTR) regions than in the sequences encoding the mature mucin core protein. Transcription units can be classified into two main subfamilies according to the sequence homologies in the 5'-UTR, whereas the 3'-UTR is highly conserved in all clones analyzed. The common origin of members of this gene family as well as their relationships can be defined by sequence comparison of different domains in the transcription units. The regions encoding the N and C termini, supposed to correspond to the leader peptide and membrane-anchoring signal, respectively, (Di Noia, J. M., Sánchez, D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146-24149) are highly conserved. Conversely, the central regions are highly variable. These regions encode the target sites for O-glycosylation and are made of a variable number of repetitive units rich in Thr and Pro residues or are nonrepetitive but still rich in Thr/Ser and Pro residues. The region putatively coding for the N-terminal domain of the mature core protein is hypervariable, being different in most of the transcripts sequenced. Nonrepetitive central domains are unique to each gene. Gene-specific probes show that the relative abundance of different mRNAs varies greatly within the same parasite clone.

High diversity in mucin genes and mucin molecules in Trypanosoma cruzi.

Mucins are highly O-glycosylated molecules which in mammalian cells accomplish essential functions, like cytoprotection and cell-cell interactions. In the protozoan parasite Trypanosoma cruzi, mucin-related glycoproteins have been shown to play a relevant role in the interaction with and invasion of host cells. We have previously reported a family of mucin-like genes in T. cruzi whose overall structure resembled that of mammalian mucin genes. We have now analyzed the relationship between these genes and mucin proteins. A monoclonal antibody specific for a mucin sugar epitope and a polyclonal serum directed to peptide epitopes in a MUC gene-encoded recombinant protein, detected identical bands in three out of seven strains of T. cruzi. Immunoprecipitation experiments confirmed these results. When expressed in eukaryotic cells, the MUC gene product is post-translationally modified, most likely, through extensive O-glycosylation. Gene sequencing showed that the central domains encoding the repeated sequences with the consensus T8KP2, varies in number from 1 to 10, and the number of Thr residues in each repeat could be 7, 8, or 10. A run of 16 to 18 Thr residues was present in some, but not all, MUC gene-derived sequences. Direct compositional analysis of mucin core proteins showed that Thr residues are much more frequent than Ser residues. The same fact occurs in MUC gene-derived protein sequences. Molecular mass determinations of the 35-kDa glycoproteins further extend the heterogeneity of the family to the natural mucin molecules. Difficulties in assigning each of the several MUC genes identified to a mucin product arise from the high diversity and partial sequence conservation of the members of this family.

A putative pyruvate dehydrogenase alpha subunit gene from Trypanosoma cruzi.

A full-length DNA clone encoding a putative pyruvate dehydrogenase alpha subunit (E1 alpha) gene was isolated from a Trypanosoma cruzi (RA strain) DNA library. Sequencing of this clone revealed it to encode a 378 amino acid protein (M(r) 42774) with high sequence similarity to E1 alpha obtained from different sources. The highest score is obtained with human E1 alpha: 43,3% similarity. Southern blot analysis is consistent with the existence of a single copy of this putative T. cruzi E1 alpha gene per haploid genome in different parasite strains. Expression of this gene was demonstrated by Northern blot analysis and its trans-splicing acceptor site was identified by Polymerase Chain Reaction-mediated amplification of its cDNA.