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S Goldenberg

Publications and source records attributed to S Goldenberg.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and characterization of the gene encoding Trypanosoma cruzi DNA topoisomerase II.

The gene encoding Trypanosoma cruzi type II topoisomerase (TcTOP2) was isolated from a genomic library with a heterologous probe corresponding to part of the Trypanosoma brucei type II topoisomerase (TBrTOP2) gene. Nucleotide sequencing of TcTOP2 showed that the gene consists of an open reading frame of 3696 nucleotides (1232 amino acids), predicting a polypeptide product of 138,413 Da. Comparison of the amino acid sequence with that of type II topoisomerases from T. brucei (TBrTOP2) and Crithidia fasciculata (CfaTOP2), shows a high degree of conservation with estimated identities of 78% and 69%, respectively. TcTOP2 is a single copy gene in the genome of T. cruzi Dm28c and is expressed as a 4.5-kb mRNA. PCR mapping showed two distinct mini-exon addition sites at positions 225 and 203 upstream from the initiator AUG.

Amino Acid Sequence↗
PubMed1992

Trypanosoma cruzi flagellar repetitive antigen expression by recombinant baculovirus: towards an improved diagnostic reagent for Chagas' disease.

We constructed a recombinant baculovirus that expressed part of a Trypanosoma cruzi flagellar repetitive antigen (FRA). Both cell- associated and secreted forms of recombinant FRA were detected in cultures of virus-infected Spodoptera frugiperda (Sf9) cells. These forms show a complex pattern after polyacrylamide gel electrophoresis and Western blot analysis using either an anti-FRA rabbit serum or human Chagasic sera. Competitive Western-blot experiments revealed that all bands react with the same antibodies as a bacterially-derived FRA. Polymerase chain reaction and Southern blots of the recombinant viral DNA also showed a complex pattern, suggesting the presence of more than one repeat unit in the viral genome. When tested against a panel of human sera from an endemic area for Chagas' disease, FRA recombinant-Sf9 culture supernatant showed the same reactivity as purified FRA produced in bacteria.

Animals↗
PubMed1992

Expression of the eukaryotic Trypanosoma cruzi CRA gene in Yersinia enterocolitica and induction of an immune response against CRA in mice.

The 70-kb plasmid pYV of Yersinia enterocolitica directs the secretion of a set of proteins, called Yops, that are produced during infection of humans and animals. Trypanosoma cruzi, the agent of American trypanosomiasis, synthesizes a cytoplasmic protein termed CRA that is considered to be T. cruzi specific. To produce CRA in Y. enterocolitica, we constructed a hybrid yopE-CRA gene that we integrated into plasmid pYV by homologous recombination. Recombinant Y. enterocolitica produced a chimeric Yop-CRA protein that was secreted in large amounts in the surrounding medium. This protein reacted with sera directed against either CRA or YopE. To test the ability of the recombinant strain to induce an immune response against CRA, we inoculated C57BL/6J mice by gastric intubation with live recombinant bacteria. A clear antibody response directed against CRA was detected in the mouse serum. The CRA-presenting Y. enterocolitica strain also carried a bioluminescence detection marker, which allowed us to monitor colonization of the intestinal lumen of infected mice. No significant differences were observed between the infectivity of the CRA antigen-producing and -nonproducing Y. enterocolitica strains, despite the fact that one of them no longer produced YopE.

Animals↗
PubMed1992

Use of recombinant antigens for the accurate immunodiagnosis of Chagas' disease.

We tested two Trypanosoma cruzi recombinant antigens in a diagnostic test for Chagas' disease. These antigens were a cytoplasmic repetitive antigen (CRA) and a flagellar repetitive antigen (FRA). The results indicate that the recombinant antigens give better results when used in combination than when used separately, and that the removal of the beta-galactosidase portion of the recombinant fusion proteins increases the specificity of the diagnostic test for Chagas' disease. In addition, a direct enzyme-linked immunosorbent assay (ELISA), which involves the use of peroxidase-labeled antigens to detect the immune-complexes, was developed and compared with a conventional ELISA. The results indicate that the recombinant (CRA+FRA) ELISA is better than the conventional ELISA in the diagnosis of Chagas' disease, providing 100% specificity and sensitivity in all sera tested to date. The recombinant ELISA was compared with conventional serologic tests (hemagglutination and immunofluorescence) for Chagas' disease diagnosis, and the results show that the recombinant ELISA does not give rise to false-positive results that are observed with the two other tests. The use of the recombinant ELISA should be useful in the prevention of transmission of Chagas' disease by blood transfusions.

Animals↗
PubMed1992

Glucose absorption by the interposed colon segment after intestinal resection.

One of the proposed surgical treatments of Short Bowel Syndrome is the interposition of a distal colon segment between two portions of the remnant small intestine. This method proved to reverse the nutritional disorders caused by this morbid entity. Surgical technique consisted in an 80% small bowel resection and the interposition of a 3 cm segment of distal colon between the remaining jejunum and ileum. After 70 days, the animals were reoperated and the interposed and the distal colon were isolated and tied. By using the method of rapid and successive absorptions of a glucose solution through the intestinal lumen, the relations between the absorption curves of the interposed and the normal colon could be drawn. Results show that the interposed colon segment absorbs more glucose (mean = 1.43 +/- 1.16 mg/dl) than the distal colon (mean = 0.37 +/- 0.29 mg/dl) and that its absorption pattern is similar to the small bowel rather than the colon. These results allow the use of this method for further studies in which the interposed colon adaptation is studied with other nutrients and/or under specific conditions.

Animals↗
PubMed1991

The multidrug resistance phenotype: 31P nuclear magnetic resonance characterization and 2-deoxyglucose toxicity.

In order to identify changes in 31P nuclear magnetic resonance (NMR) spectra associated with multiple drug resistance (MDR), a number of wild type and drug-resistant cancer cell lines were studied. The resistant cells included cells selected with various drugs, mainly Adriamycin, as well as cells transfected with the human multidrug resistance gene (MDR1 gene), which encodes P-glycoprotein. In most cases, 31P NMR spectra were significantly different from those of parental, drug-sensitive lines. The spectra of resistant cells generally indicated increased levels of ATP and phosphocreatine in the cytoplasm. These changes are compatible with the increased glucose utilization rate previously described for resistant cells. Major changes were also observed in the levels of glycerophosphocholine and glycerophosphoethanolamine. Changes in cellular metabolism reflected by 31P NMR spectra depend on the drug used to select the cells for MDR. The direction of these changes was not consistent for all cell lines studied and could not be directly attributed to expression of P-glycoprotein, suggesting that the changes may be related to alterations in metabolism and membrane function associated with other mechanisms of MDR. The results demonstrate the suitability of 31P NMR for studies of biochemical changes associated with MDR. The toxicity of 2-deoxyglucose, a glucose antimetabolite, was investigated in addition to the NMR studies and was found to be consistently higher in multidrug-resistant cells than in the parental drug-sensitive lines. For MCF-7 breast cancer cells, where several sublines with different levels of resistance were available, the toxicity was highest for the most resistant lines.

Adenosine Triphosphate↗
PubMed1991

Further characterization of Trypanosoma cruzi GP57/51 as the major antigen expressed by differentiating epimastigotes.

Study of Trypanosoma cruzi metacyclogenesis under chemically defined conditions showed that the expression of a group of acidic polypeptides with a molecular weight of 45-50 kDa is markedly increased on adhesion of epimastigotes to the culture vessels. Immunochemical analysis revealed that these developmentally regulated polypeptides are structurally related to, and possibly homologous with, a major T. cruzi antigen, namely, GP57/51, a glycoprotein that has recently been characterized as a cysteine proteinase. The differentiation of epimastigotes into metacyclic trypomastigotes is accompanied by a 2- to 5-fold reduction in the concentration of GP57/51 antigen as determined by radioimmunoassays using monoclonal antibodies. Two-dimensional polyacrylamide gel electrophoretic analysis of metabolically labelled parasites indicated that this antigen is synthesized as a precursor with a molecular weight of 60 kDa, which is then processed to a level of 45-50 kDa via the formation of at least one intermediate processing product. The observation that expression of GP57/51-related products increases during epimastigote differentiation suggests an important role for this proteinase during the life cycle of T. cruzi.

Animals↗
PubMed1991

Bacteriology of the anal wound after open hemorrhoidectomy. Qualitative and quantitative analysis.

The purpose of this study is to analyze the size of the bacterial colonies in anal wounds after open hemorrhoidectomy. Twenty patients were studied during predetermined postoperative time periods. Material was collected from the surface and from within the tissue of each patient's three open wounds, intraoperatively, on the 6th, 13th and 20th postoperative days for bacteriologic examination in aerobic, microaerophilic, and anaerobic media. The bacterium most commonly identified was Escherichia coli, followed by Staphylococcus aureus and Staphylococcus epidermidis. Pseudomonas aeruginosa, Enterococcus faecalis, Klebsiella pneumoniae, Proteus vulgaris, and Proteus mirabilis were also identified. Critical indexes of colonization were present since the intraoperative stage (greater than 10(5) bacteria/g of tissue and greater than 10(6) bacteria/ml); obligate anaerobic bacteria were not identified; neither the species nor the number of bacteria, even when critical indexes were present, prevented proper healing. The same bacteria were not necessarily present on the surface and in the tissue; the bacterial load observed among the three wounds (left lateral, right posterior, and right anterior), was the same.

Adult↗
PubMed1991

Characterization and expression of proteases during Trypanosoma cruzi metacyclogenesis.

Investigation of protease activities during the transformation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigoes (metacyclo-genesis) revealed three major components with apparent molecular weights of 65, 52, and 40 kDa. The 65-kDa protease is a metacyclic trypomastigote stage-specific protease with an isoelectric point of 5.2 whose activity is inhibited by 1,10-phenanthroline, suggesting that it might be a metalloprotease. The 52-kDa component is also a metalloprotease which is constitutively expressed in epimastigotes and metacyclic trypomastigoes. On the other hand, the 40-kDa component is apparently made up of several isoforms of a cysteine protease which is expressed in much higher levels in epimastigotes than in metacyclic trypomastigote forms. The fact that the 65- and 40-kDa proteases are developmentally regulated suggests that proteases might be important for T. cruzi differentiation. Accordingly, T. cruzi metacyclogenesis is blocked by metallo- and cysteine-protease inhibitors.

Animals↗
PubMed1991

Changes in cell-surface carbohydrates of Trypanosoma cruzi during metacyclogenesis under chemically defined conditions.

Highly purified lectins with specificities for receptor molecules containing sialic acid, N-acetylglucosamine (D-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose (D-Gal), mannose-like residues (D-Man) or L-fucose (L-Fuc), were used to determine changes in cell-surface carbohydrates of the protozoal parasite Trypanosoma cruzi during metacyclogenesis under chemically defined conditions. Of the D-GalNAc-binding lectins, BS-I selectively agglutinated metacyclic trypomastigotes, MPL was selective for replicating epimastigotes, whereas SBA strongly agglutinated all developmental stages of T. cruzi. WGA (sialic acid and/or D-GlcNAc specific) was also reactive with differentiating epimastigotes and metacyclic trypomastigotes but displayed a higher reactivity with replicating epimastigote forms. A progressive decrease in agglutinating activity was observed for jacaline (specific for D-Gal) during the metacyclogenesis process; conversely, a progressive increase in affinity was observed for RCA-I (D-Gal-specific), although the reactivity of other D-Gal-specific lectins (PNA and AxP) was strong at all developmental stages. All developmental stages of T. cruzi were agglutinated by Con A and Lens culinaris lectins (specific for D-Man-like residues); however, they were unreactive with the L-fucose-binding lectins from Lotus tetragonolobos and Ulex europaeus. These agglutination assays were further confirmed by binding studies using 125I-labelled lectins. Neuraminidase activity was detected in supernatants of cell-free differentiation medium using the PNA hemagglutination test with human A erythrocytes. The most pronounced differences in lectin agglutination activity were observed between replicating and differentiating epimastigotes, suggesting that changes in the composition of accessible cell-surface carbohydrates precede the morphological transformation of epimastigotes into metacyclic trypomastigotes.

Agglutination Tests↗
PubMed1991

Small bowel and colon glucose absorption study in rats by an adaptation of Sols and Ponz method.

An adaptation of the Sols and Ponz method for the study of glucose intestinal absorption was developed by considering the special conditions of our line research. The glucose absorption was studied in proximal jejunum, distal ileum and distal colon in Wistar rat. The main adaptations in the method for successive absorptions with intestinal perfusions in vivo were the length of the intestinal segment and the change of the pumping system. The results are very similar to those obtained with the original method.

Anastomosis, Surgical↗
PubMed1990

Expression and polymorphism of a Trypanosoma cruzi gene encoding a cytoplasmic repetitive antigen.

The study of the expression of a Trypanosoma cruzi gene encoding a cytoplasmic repetitive antigen (CRA) during the metacyclogenesis process shows that this gene is not expressed in metacyclic trypomastigote forms of the parasite. However, a slight increase in CRA expression was observed following the nutritional stress of epimastigotes which precedes T. cruzi metacyclogenesis in vitro. The comparison of the expression of CRA in different T. cruzi strains shows that this gene is highly polymorphic: some strains display one and others display two polypeptides reacting with a CRA antiserum. The comparison of T. cruzi G-49 strain and Dm 28c clone shows that they display rather different Northern and Southern blot profiles when probed with a clone corresponding to the repetitive region of the CRA gene. A similar polymorphism was also observed for the gene encoding a flagellar repetitive antigen, suggesting that gene polymorphism might be a common feature of many T. cruzi genes.

Animals↗
PubMed1990

Characterization of a Trypanosoma cruzi specific nuclear repeated sequence.

We report the isolation of three different clones from a Trypanosoma cruzi genomic library bearing a common repeated sequence. This sequence is not tandemly repeated, and is dispersed on many chromosomes. All of the T. cruzi strains tested share this element. On the other hand, it is absent from the genome of other Kinetoplastida. The size of this element is about 10-12 kb, and its copy number is 220 in the T. cruzi Dm 28c genome. A transcript homologous to this sequence is detected in epimastigote forms of the parasite.

Animals↗
PubMed1990

Tumour necrosis factor (TNF) as a mediator of macrophage helminthotoxic activity.

Lymphokine-activated macrophages are cytotoxic for larvae of the helminth parasite Schistosoma mansoni. That soluble secreted factors may mediate this cytotoxicity was suggested by the observation that culture supernatant fluids from stimulated macrophages also exhibited larvacidal activity. These fluids contain the monokine tumour necrosis factor (TNF). Several observations indicated that TNF is directly toxic to schistosome larvae. Cytotoxic sera taken from BCG- or S. mansoni-immunized mice after endotoxin challenge killed schistosomula in vitro, and upon gel filtration the larvacidal factor(s) in the sera co-eluted with the tumoricidal activity defined as TNF. Recombinant-derived TNF exhibited direct toxicity to schistosomula at high concentrations, or at lower concentrations in the presence of IFN gamma. The larvacidal activity of macrophage supernatant fluids was abrogated by addition of either anti-TNF antisera or Zn+2, which has been shown to inhibit TNF-induced damage of tumour cells. Anti-TNF and Zn+2 likewise suppressed schistosomulum killing by lymphokine-activated peritoneal macrophages or the IC-21 macrophage line, indicating that TNF also plays a role in the effector mechanism of larval killing by whole cells.

Animals↗
PubMed1990

Trypanosoma cruzi proliferation and differentiation are blocked by topoisomerase II inhibitors.

Bacterial topoisomerase II inhibitors (ofloxacin and its commercial derivative Tarivid, nalidixic acid, and novobiocin) were tested as blockers of Trypanosoma cruzi differentiation and proliferation. The transformation of either epimastigotes into metacyclic trypomastigotes or amastigotes into trypomastigotes was inhibited by the drugs in a dose-dependent manner. The inhibition of epimastigote differentiation was also dependent on the time of drug addition to the medium. Proliferation of T. cruzi was also blocked in a dose-dependent manner by the drugs, with the exception of novobiocin, which did not inhibit epimastigote replication and resulted in cell lysis when it was used at high concentrations. On the other hand, the transformation of amastigotes into epimastigotes in axenic culture was not inhibited; this process did not require either kinetoplast (mitochondrial) DNA replication or changes in the DNA network organization. Electron microscopy of cells treated with Tarivid (ofloxacin) showed damage to the kinetoplast, suggesting that this organelle might be the target of the drug. These results indicate that a bacterial-like topoisomerase II plays an important role in T. cruzi proliferation and differentiation.

Animals↗
PubMed1989

Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia.

We studied transepithelial transport of 3H-labeled hydrophobic cationic drugs in epithelia formed by wild-type and by drug-resistant Madin-Darby canine kidney (MDCk) cells that had been infected with a retrovirus carrying the multidrug-resistance (MDR1) cDNA which encodes the P-glycoprotein. P-glycoprotein is an ATP consuming plasma membrane multidrug transporter responsible for the efflux of cytotoxic chemotherapeutic drugs from resistant cancer cells. Wild-type MDCK cells have small amounts of P-glycoprotein detected by immunoprecipitation. Net transepithelial transport across wild-type MDCK epithelia was demonstrated. Basal to apical flux of 100 nM vinblastine was about six times higher than apical to basal flux. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. Daunomycin, vincristine, and actinomycin D were also actively transported and at 20 microM these agents inhibited transport of vinblastine, suggesting that wild-type MDCK cells have a common transporter for all these drugs. Vinblastine transport was also inhibited by 20 microM verapamil, which inhibits the multidrug transporter and reverses multidrug-resistance in non-polarized cells. Net transepithelial transport of all these cytotoxic drugs and of verapamil was much higher in epithelia formed by MDCK cells infected with a human MDR1 virus (MDR-MDCK) which is expressed on the apical surface of MDR-MDCK monolayers. Because the transport of these cytotoxic drugs and verapamil is increased in MDR-MDCK epithelia compared to wild-type MDCK epithelia, transport in both these cell populations can be attributed to P-glycoprotein. These results are consistent with a role for P-glycoprotein in multidrug secretory transport across the epithelium of the proximal tubule since P-glycoprotein is normally expressed on the apical membrane of proximal tubule cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗