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S Goldenberg

Publications and source records attributed to S Goldenberg.

At least 19 recordsLinked to original sources
PubMed2004

Immunization with cytoplasmic repetitive antigen and flagellar repetitive antigen of Trypanosoma cruzi stimulates a cellular immune response in mice.

In previous studies, we demonstrated that CRA and FRA recombinant proteins, used for diagnosis of Chagas' disease, elicited a humoral immune response in susceptible and resistant mice. To understand better the immune response to these proteins, we have evaluated, the cellular immune response in CRA- and in FRA-immunized BALB/c and C57BL/6 mice. A specific cellular lymphoproliferative response was observed in both strains of mice. Spleen cell cultures mainly from CRA-immunized C57BL/6 and FRA-immunized BALB/c mice produced high levels of IFN-y, indicating the induction of a Type 1 immune response. Regarding the T cell subsets, CD4+ T cells were the major source of IFN-y in CRA- and FRA-immunized mice. These results suggest that CRA and FRA are important immunogens in inducing a Type 1 immune response and that they may be considered as potential vaccine antigens.

Animals↗
PubMed2003

Chagas' disease diagnosis: comparative analysis of recombinant ELISA with conventional ELISA and the haemagglutination test.

BACKGROUND AND OBJECTIVES: Serological screening for Chagas' disease in the blood banks of South America is carried out by using two different assays that generally show a high number of inconclusive results. To establish a combination of two tests that can minimize the number of inconclusive results, we compared a recombinant enzyme-linked immunosorbent assay (ELISA) with two conventional tests. MATERIALS AND METHODS: Serum samples from chagasic patients (n = 112), from non-chagasic individuals (n = 143) and from patients with other diseases (n = 32) were tested using three assays: recombinant ELISA (Rec-ELISA); conventional ELISA (Con-ELISA); and the indirect haemagglutination (IHA) test. RESULTS: When we evaluated the data by matching the Rec-ELISA and the IHA test, 52 inconclusive results were obtained. When Rec-ELISA and Con-ELISA were matched, only four inconclusive results were observed. CONCLUSIONS: Our investigation indicates that the use of two ELISAs with different antigen preparations provides an effective test combination for blood bank screening of Chagas' disease.

Adult↗
PubMed2001

Crystal structure of the dimeric phosphoenolpyruvate carboxykinase (PEPCK) from Trypanosoma cruzi at 2 A resolution.

ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) (ATP: oxaloacetate carboxylyase (transphosphorylating), EC 4.1.1.49) is a key enzyme involved in the catabolism of glucose and amino acids in the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Due to the significant differences in the amino acid sequence and substrate specificity of the human enzyme (PEPCK (GTP-dependent), EC 4.1.1.32), the parasite enzyme has been considered a good target for the development of new anti-chagasic drugs. We have solved the crystal structure of the recombinant PEPCK of T. cruzi up to 2.0 A resolution, characterised the dimeric organisation of the enzyme by solution small angle X-ray scattering (SAXS) and compared the enzyme structure with the known crystal structure of the monomeric PEPCK from Escherichia coli. The dimeric structure possesses 2-fold symmetry, with each monomer sharing a high degree of structural similarity with the monomeric structure of the E. coli PEPCK. Each monomer folds into two complex mixed alpha/beta domains, with the active site located in a deep cleft between the domains. The two active sites in the dimer are far apart from each other, in an arrangement that seems to permit an independent access of the substrates to the two active sites. All residues of the E. coli PEPCK structure that had been found to interact with substrates and metal cofactors have been found conserved and in a substantially equivalent spatial disposition in the T. cruzi PEPCK structure. No substrate or metal ion was present in the crystal structure. A sulphate ion from the crystallisation medium has been found bound to the active site. Solution SAXS data suggest that, in solutions with lower sulphate concentration than that used for the crystallisation experiments, the actual enzyme conformation may be slightly different from its conformation in the crystal structure. This could be due to a conformational transition upon sulphate binding, similar to the ATP-induced transition observed in the E. coli PEPCK, or to crystal packing effects. The present structure of the T. cruzi PEPCK will provide a good basis for the modelling of new anti-chagasic drug leads.

Amino Acid Sequence↗
PubMed2001

A DnaJ-like protein homologous to the yeast co-chaperone Sis1 (TcJ6p) is involved in initiation of translation in Trypanosoma cruzi.

In eukaryotes, proteins homologous to the bacterial DnaJ protein are involved in regulation of the Hsp70 molecular chaperones, which are implicated in a variety of protein biogenesis pathways. We report herewith the molecular characterization of a T. cruzi DnaJ gene, termed TcJ6, encoding a protein that displays high sequence homology with the Saccharomyces cerevisiae Sis1 co-chaperone required for the initiation of translation. TcJ6 protein was expressed as a polypeptide of 36.5 kDa at a constant level during parasite differentiation and was associated to the cytoplasmic fraction. We showed that overexpression of TcJ6 complemented a temperature-sensitive yeast sis1 mutant. In addition, sucrose gradient sedimentation analysis of polysomes from T. cruzi and a yeast mutant overexpressing TcJ6p showed that the trypanosomal co-chaperone was closely associated with ribosomal subunits, 80 S monosomes and the smaller polysomes, as observed for Sis1p. Furthermore, in T. cruzi TcJ6p was also found to be preferentially concentrated around the nucleus, giving a speckled staining pattern. This suggests that TcJ6p is associated with the endoplasmic reticulum. Taken together, these data suggest that the trypanosomal DnaJ is involved in initiation of translation.

Amino Acid Sequence↗
PubMed2001

Trypanosoma cruzi: a gene family encoding chitin-binding-like proteins is posttranscriptionally regulated during metacyclogenesis.

The development of the representation of differential expression method has lead to the cloning of Trypanosoma cruzi stage-specific genes. We used this method to characterize a multicopy gene family differentially expressed during metacyclogenesis. The genomic and cDNA clones sequenced encoded three short cysteine-rich polypeptides, of two types, with predicted molecular masses of 7.1, 10.4, and 10.8 kDa. We searched GenBank for similar sequences and found that the sequences of these clones were similar to that encoding the wheat germ agglutinin protein. The region of similarity corresponds to the chitin-binding domain, with eight similarly positioned half-cysteines and conserved aromatic residues involved in chitin recognition. Multiple copies of the genes of this family are present on a high- molecular-mass chromosome. We studied the expression of genes of this family during metacyclogenesis by determining messenger RNA (mRNA) levels. The mRNAs for the members of this gene family were present in the total RNA fraction but were mobilized to the polysomal fraction of adhered (differentiating) epimastigotes during metacyclogenesis, with a peak of accumulation at 24 of differentiation. Polyclonal antisera were raised against a recombinant protein and a synthetic peptide. The specific sera obtained detected 7- and 11-kDa proteins in T. cruzi total protein extracts. The 11-kDa protein was present in similar amounts in the various cell populations, whereas the 7-kDa protein displayed differential synthesis during metacyclogenesis, with maximal levels in 24-h-adhered (differentiating) epimastigotes.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of the metacyclogenin gene, which is specifically expressed during Trypanosoma cruzi metacyclogenesis.

We isolated a gene that is differentially expressed during Trypanosoma cruzi metacyclogenesis by the representation of differential expression (RDE) method, using differentiating epimastigotes cultured in chemically defined medium. This gene, the metacyclogenin gene, encodes a 630-nucleotide mRNA that is specifically associated with the polysomes of epimastigotes allowed to differentiate for 24 h. We sequenced and characterized the metacyclogenin gene and found that there were at least three copies of the gene organized into tandem 2.8 kb repeats in the genome of T. cruzi Dm28c. We analyzed the repeats and found that they contained two other genes, one encoding tryparedoxin peroxidase and the other encoding a 0.6 kb mRNA (named associated gene or AG) with sequences showing no significant similarity to those in the GenBank database. Northern blot analysis of polysomal RNA extracted from replicating and differentiating epimastigotes showed that metacyclogenin and AG genes displayed similar patterns of expression. Their products were detected only in differentiating epimastigotes, whereas tryparedoxin peroxidase was detected only in the polysomal RNA fraction of replicating and differentiating epimastigotes. In Northern blots of total RNA from differentiating and replicating epimastigotes, the genes studied were detected in both cell populations. The differential expression of the metacyclogenin gene was confirmed by immunocytochemistry studies showing that the protein is detected only in differentiating (adhered) epimastigote. The results suggest that mRNA mobilization to polysomes is an important mechanism in the regulation of gene expression in T. cruzi.

Amino Acid Sequence↗
PubMed2001

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit.

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the referred kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.

Adolescent↗
PubMed2000

Decreased accumulation of [14C]carboplatin in human cisplatin-resistant cells results from reduced energy-dependent uptake.

We have isolated cisplatin-resistant human liver carcinoma (7404-CP20) cells with reduced accumulation of cisplatin and other drugs (methotrexate, arsenate, and arsenite) to which these cells are cross-resistant. To determine whether the reduction of drug accumulation in cisplatin-resistant cells results from impaired uptake or from active efflux, [(14)C]carboplatin was used for kinetic analysis of drug uptake and efflux. We demonstrate here that the uptake of [(14)C]carboplatin in 7404 parental cells is time, temperature, and energy dependent, and that the rate of uptake is reduced in 7404-CP20 cells. Efflux of [(14)C]carboplatin in cisplatin-resistant cells was comparable to efflux in the parental cisplatin-sensitive cells. There was little effect of temperature (between 37 degrees C and 4 degrees C) on efflux in cisplatin-resistant cells. Immunoblotting with specific antibodies directed to MRP1 and MRP2 (cMOAT) also showed that expression of these two ABC transporter genes was considerably reduced in 7404-CP20 cells and another cisplatin-resistant cell line KB-CP20, in contradistinction to previous studies suggesting that MRP might be responsible for cisplatin efflux. To rule out a generalized defect in uptake of small molecules, fluorescence-activated cell sorter (FACS) analysis of rhodamine 123 uptake showed that there was no difference between cisplatin-sensitive and -resistant cells. The presence of a pleiotropic defect in uptake of [(14)C]carboplatin, [(3)H]methotrexate, [(73)As]arsenate, and [(73)As]arsenite in cisplatin-resistant cells, in association with reduced expression of related cell surface proteins as demonstrated in our previous work, suggests a novel mechanism for acquisition of resistance to cisplatin associated with reduced activity of many different specific uptake systems.

Adenosine Triphosphate↗
PubMed2000

Gastrinaemia and G-cell density in the antral gastrocystoplasty: an experimental study in rats.

OBJECTIVE: To evaluate gastrinaemia and G-cell density in the antrum incorporated into the bladder of rats after antrocystoplasty. Materials and methods Thirty-two adult, female Wistar EPM-1 rats (body weight 200-220 g) were divided into four equal groups that underwent; group 1, no treatment (controls); group 2, a sham operation; group 3, antrectomy; and group 4, antrocystoplasty. The rats were assessed 2 months after treatment, and gastrinaemia and the G-cell density determined in the antrum mucosa incorporated into the bladder. RESULTS: Compared with group 1, serum gastrin was significantly lower in group 3 (P<0.05) and the G-cell density lower in group 4 (P<0.05), although there was no decrease in gastrinaemia in group 4. CONCLUSION: Antrocystoplasty in rats did not affect gastrinaemia but reduced the number of G cells in the antrum incorporated into the bladder.

Anastomosis, Surgical↗
PubMed2000

Comparative blood flow study of arteriovenous fistulae in canine femoral arteries: modified latero-lateral and end-lateral techniques.

The aim of this study was to evaluate the blood flow of an arteriovenous fistula comparing the modified latero-lateral (LLM) and end-lateral (TL) techniques in canine femoral arteries. Ten mongrel dogs were submitted to 2 arteriovenous fistulae each, with a LLM on one side and a TL procedure on the other side. Cranial and caudal average blood flow as well as average venous flow were measured by an electronic fluxometer 15, 20, and 25 min after surgery. Mann-Whitney, Friedman, and Wilcoxon nonparametric tests were used for data analysis (alpha </= 0,05). Femoral artery flow, cranial to the fistula, was increased 5(1/2) times in both groups. In the femoral artery caudal to the fistula, the flow in the majority of the animals was reversed 1.3 times using the TL technique and 2 times using the LLM technique. The average flow in the cranial vein was statistically different with an improved flow of 7.4 times versus 6.8 times for the LLM technique. These results suggest that LLM arteriovenous fistula in dogs leads to a larger venous flow than in the TL arteriovenous fistula and the reverse caudal arterial flow in the latero-lateral technique. The flow behavior (laminar or turbulent) in the 2 types of arteriovenous fistula was studied in terms of singular head loss.

Anastomosis, Surgical↗
PubMed2000

Video-endoscopic dissection of multiple pedicled arterial grafts for use in minimally invasive coronary artery bypass surgery.

BACKGROUND: We evaluated the possibility of video-endoscopic dissection of both internal thoracic arteries (rTAs) through the left thoracic approach and right gastroepiploic artery (GEA) via a small laparoscopic access, for use in minimally invasive coronary artery bypass surgery. METHODS: The procedure was performed on twenty-two mongrel dogs. Three 10 mm ports were inserted in the left hemithorax to enable the introduction of a rigid 0-degree videoscope and forceps which allowed access to the left and right ITAs. For dissection of the GEA, one 10 mm and two 5 mm ports were inserted in the para-umbilical region. RESULTS: The left and right ITAs and the GEA were easily visualized and dissection and complete mobilization was achieved without injury, which was checked by the presence of good blood flow. The ITAs and GEA were divided distally, exteriorized through a small left anterior thoracotomy and the length of these three grafts were able to reach all of the left coronary artery branches. CONCLUSIONS: We have demonstrated, in dogs, the feasibility of video-assisted dissection of both the left and right ITAs through the left thoracic approach, without sternotomy, avoiding the risks of sternal complications and expanding its use for all patients. Furthermore, a third arterial conduit (the RGA) can be used without requiring laparotomy.

Angioscopy↗
PubMed1999

Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein.

The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypanosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the addition of myristate and palmitate at its amino terminal segment and both modifications are required for calcium-modulated flagellar membrane association. FCaBP has four sequence motifs for potential calcium binding, and comparison to other calcium-acyl switch proteins, such as recoverin, suggested that only two of these sites are functional. Because it is not possible to predict with certainty the calcium binding affinity or selectivity based on motif analysis alone, we determined the quantitative calcium binding activity of FCaBP by direct ligand binding using the flow dialysis method. The results demonstrated the presence of two calcium binding sites in the full length FCaBP and in a mutant (FCaBPdelta12) lacking the amino terminal pair of sites. FCaBPdelta12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBPdelta34), did not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from other myristoyl switch proteins such as recoverin. The results add to a growing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the apparently novel mechanism by which FCaBP undergoes calcium modulated flagellar membrane association and its relation to calcium signal transduction.

Amino Acid Sequence↗
PubMed1999

Metabolic and ultrastructural effects of cyclosporin A on pancreatic islets.

We investigated the effect of different doses of cyclosporin A (CyA) on glucose and insulin levels, as well as its residual effects on pancreatic islets ultrastructure after discontinuation of the drug. We studied four groups of Wistar rats. One control- (n = 5) and three experimental groups, n = 10 each, were treated with different doses of CyA i.m. for 14 days: group I, 5 mg/Kg; group II, 15 mg/Kg; and group III, 25 mg/Kg. Five animals of each group were sacrificed after 14 days, and the remaining five after 21 days to assess residual CyA effects. On the day of sacrifice, the rats underwent maltose absorption test, and glucose and insulin levels were measured. Pancreatic biopsies were obtained on day 21 to evaluate islets ultrastructure by electron microscopy. As a result, statistically significant, dose dependent (P < 0.05) increases in glucose and insulin levels were observed in CyA-treated groups. Groups II and III showed insulin levels significantly higher after fasting (P < 0.05) on day 14 comparing to the controls, while in groups I and II values returned to normal after CyA discontinuation. Group III showed persistently increased insulin levels on day 21. Pancreatic ultrastructural changes were observed only in group III. We can conclude that CyA effects on glucose and insulin levels were temporary and reversible at low doses. Ultrastructural changes in the pancreatic islets may occur with high doses of CyA.

Animals↗
PubMed1999

Differential gene expression during Trypanosoma cruzi metacyclogenesis.

The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE). The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes) resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells), while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

Animals↗
PubMed1999

Tenorrhaphy and tendinous venous envelope (TVE). A morphofunctional study in 24 beagles dogs.

A morphofunctional study was undertaken in 24 Beagle dogs subjected to a a tenorrhaphy, in order to evaluate the results obtained with 3 different surgical procedures (16 hands each)--"traditional" (TRAD); the "tendinous venous envelope" (TVE), and the "simulated procedure" (SHAM). The surgical tissue (n = 48) was the extensor digitorum communis muscle of the hand (forepaw) bilaterally. For TRAD procedure, the Bunnell suture technique was applied. For the TVE procedure, the tenorrhaphy site was wrapped in a segment of autologous femoral vein and for the SHAM procedure, the tendon was exposed over 3 cm without sectioning it and the wound was closed. In each case, the limb was immobilised for 21 days in a cast. Functional data (288 X-rays) was obtained on angle variations of the radio-carpal joint in 2 different positions--passive (physiological), and forced (with an additional weight of 150 g). For each position, the angle variation obtained for each one of the surgical procedures were compared at 3 different times: day zero (surgical intervention), day 21 (cast removal), and day 35 (surgical tissue removal). Morphological data (144 sections) was obtained by analyzing the surgical tissue (tendinous fibrosis; fusion of the tendon clusters with the epimysium and the neighboring tissues; synovitis and the virtual space between the tendon and the neosheath). Tests of variance analysis by posts (critical H = 5.99), showed that the TVE procedure produced better results as compared to TRAD (SHAM approximately equal to TVE > TRAD) on day 21 (H = 22.58) as well as on day 35 (H = 8.08).

Analysis of Variance↗
PubMed1998

TcDJ1, a putative mitochondrial DnaJ protein in Trypanosoma cruzi.

A full length cDNA encoding a novel Trypanosoma cruzi DnaJ protein was cloned and characterized. The 324 amino acid protein encoded by the cDNA (TcDJ1) displays a characteristics J-domain, but lacks the Gly-Phe and zinc finger regions present in some other DnaJ proteins. Relative to four other T. cruzi DnaJ proteins, TcDJ1 has an amino terminal extension containing basic and hydroxylated resides characteristic of mitochondrial import peptides. A T. cruzi transfectant expressing epitope-tagged TcDJ1 was generated and subcellular fractions were produced. Western blot analysis revealed that the protein has a molecular mass of 29 kDa and is found in the mitochondrial fraction. The expression of TcDJ1 is developmentally regulated since the levels of both mRNA and protein are much higher in epimastigotes (replicative form) than in metacyclic trypomastigotes (infective form). Thus it may participate in mitochondrial biosynthetic processes in this organism.

Amino Acid Sequence↗
PubMed1998

Expression and cellular localization of Trypanosoma cruzi type II DNA topoisomerase.

Topoisomerases are enzymes that participate in many cellular functions involving topological manipulation of DNA strands. There are two types of topoisomerases in the cell: (a) type I topoisomerases; and (b) type II topoisomerases (topo II). Previously we have cloned and sequenced the gene encoding Trypanosoma cruzi topo II (TcTOP2). This study group has raised an antiserum against recombinant type II DNA topoisomerase (TctopoII) to study the expression of this gene during T. cruzi differentiation and to determine the cellular location of the enzyme. Western blot analysis showed that T. cruzi TctopoII is expressed in the replicative epimastigotes but not in the infective and non-replicative trypomastigotes. However, slot blot analysis of RNAs extracted from epimastigotes and metacyclic trypomastigotes showed that the mRNA encoding the enzyme is present in both developmental stages of the parasite. Confocal laser microscopy using the antiserum raised against recombinant TctopoII showed that the enzyme is located exclusively in the nucleus of the parasite. Similar results were obtained by immunofluorescence analysis of Crithidia fasciculata. However, monoclonal antisera against the corresponding enzyme extracted from C. fasciculata recognizes a kinetoplast protein in both T. cruzi and Crithidia.

Animals↗