Abraham Landa

Alternative splicing of the Schistosoma mansoni gene encoding a homologue of epidermal growth factor receptor

The complete coding DNA for a Schistosoma mansoni homologue of the epidermal growth factor receptor (SER) was characterized from cDNA clones obtained by homology to the tyrosine kinase domain of erbB. The DNA sequence predicts a 200-kDa translation product that contains a secretory leader, a cysteine-rich extracellular domain, a hydrophobic transmembrane sequence, and an intracellular tyrosine kinase domain. The SER transcript is present in cercariae and adult schistosomes. In addition to SER transcripts, schistosomes produce at least 3 variant transcripts encoding truncated SER products that include the secretory leader and a small portion of the extracellular domain followed by short sequences of unrelated, C-terminal amino acids. Based on these sequences, 2 of the variant mRNAs (class 2 and 5) appear to encode soluble, secreted proteins while one (class 4) encodes an SER variant protein with a hydrophobic C-terminus that may serve as a membrane anchor. Class 2 SER variant transcripts are present at levels comparable to SER transcripts in adult worms but are not detected in cercariae. Class 4 and 5 SER variant transcripts are also found within adult worms but at lower levels. Genomic cloning and characterization demonstrate that the variant SER transcripts arise through alternative splicing of the SER gene.

Paramyosin is the Schistosoma mansoni (Trematoda) homologue of antigen B from Taenia solium (Cestoda).

Antigen B, a major antigen of the cestode parasite Taenia solium, has been purified and a portion of amino acid sequence obtained. Paramyosin of the trematode parasite Schistosoma mansoni, an immunogenic protein that has shown promise as a vaccine candidate, has several biochemical and immunological properties in common with antigen B. A full-length cDNA clone of S. mansoni paramyosin has been obtained and the predicted translation product contains a sequence that is highly homologous to the sequence obtained for antigen B. The predicted amino acid composition and isolectric point of paramyosin are nearly identical to those established for antigen B. Recombinant S. mansoni paramyosin, expressed in Escherichia coli as a fusion protein with beta-galactosidase, was recognized by antisera against T. solium antigen B. We conclude from these results that S. mansoni paramyosin and T. solium antigen B are homologous proteins. Since S. mansoni paramyosin is thought to be a muscle protein and T. solium antigen B a secreted glycoprotein with anti-complement activity, this conclusion raises some interesting questions regarding the role of this class of proteins in the host-parasite relationship.

4-Hydroxycoumarin disorganizes the actin cytoskeleton in B16–F10 melanoma cells but not in B82 fibroblasts, decreasing their adhesion to extracellular matrix proteins and motility

This study determined the in vitro effects of 4-hydroxycoumarin (4-HC) employing the melanoma cell line B16-F10 and the non-malignant fibroblastic cell line B82. 4-HC disorganized the actin cytoskeleton in B16-F10 cells, but not in B82 fibroblasts. Cytoskeletal disorganization correlated with reductions in cell adhesion to four extracellular matrix proteins and inhibition of random motility. 4-HC did not modify cell viability or actin expression, but decreased tyrosine phosphorylation of several proteins in melanoma cells. Because adhesion of tumor cells to extracellular matrix is required during the metastatic process, 4-HC might be useful as an adjuvant therapy for melanoma.

Cloning and sequencing of two actin genes from Taenia solium (Cestoda)

Genomic and cDNA actin clones were isolated from Taenia solium gene libraries. The actin genes are interrupted by intervening sequences. Protein coding regions of both genes predict the same amino acid sequence. cDNA sequence data indicate that at least one gene is expressed at the larval stage. Results from Northern and Western blots showed that T. solium expresses an actin transcript of about 1,400 bases and a protein of 45,000 Da.

Characterization of glutathione S-transferase of Taenia solium.

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.

Cloning, production and characterisation of a recombinant Cu/Zn superoxide dismutase from Taenia solium

A full-length complementary DNA clone encoding a cytosolic Cu/Zn superoxide dismutase with a M(r) of 15,588 Da was isolated from a Taenia solium larvae complementary DNA library. Comparison analysis of its deduced amino acid sequence revealed a 71% identity with Schistosoma mansoni, 57.2-59.8% with mammalian and less than 54% with other helminth cytosolic Cu/Zn superoxide dismutase. The characteristic motifs and the amino acid residues involved in coordinating copper and zinc enzymatic function are conserved. The T. solium Cu/Zn superoxide dismutase was expressed in the pRSET vector. Enzymatic and filtration chromatographic analysis showed a recombinant enzyme with an activity of 2,941 U/mg protein and a native M(r) of 37 kDa. Inhibition assays using KCN, H(2)O(2), NaN(3) and SDS indicated that Cu/Zn is the metallic cofactor in the enzyme. Thiabendazole (500 microM) and albendazole (300 microM) completely inhibited the activity of T. solium Cu/Zn superoxide dismutase. Thiabendazole had no effect on bovine Cu/Zn superoxide dismutase; in contrast, albendazole had a moderate effect on it at same concentrations. Antibodies against T. solium Cu/Zn superoxide dismutase did not affect the enzymatic function; nevertheless, it cross reacts with several Taenia species, but not with trematodes, nematodes, pig, human and bovine Cu/Zn superoxide dismutase enzymes. Western blot analysis indicated the enzyme was expressed in all stages. These results indicate that T. solium possesses a Cu/Zn superoxide dismutase enzyme that can protect him from oxidant-damage caused by the superoxide anion.

Molecular and functional characterization and tissue localization of 2 glucose transporter homologues (TGTP1 and TGTP2) from the tapeworm Taenia solium

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers D-glucose and D-mannose but not by L-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

Potential role of the cannabinoid receptor cb1 in rapid eye movement sleep rebound

Sleep is an unavoidable activity of the brain. The delay of the time to sleep (sleep deprivation), induces an increase of slow-wave sleep and rapid-eye-movement (REM) sleep (rebound) once the subject is allowed to sleep. This drive to sleep has been hypothesized to be dependent on the accumulation of sleep-inducing molecules and on the high expression of these molecule receptors. In this study we selectively deprived rats of REM sleep for 24 h by using the flowerpot technique. One group deprived of REM sleep was treated with SR141716A, a cannabinoid receptor 1 (CB1) receptor antagonist and then allowed to sleep for the next 4 h. Two other groups were killed, one immediately after the REM sleep deprivation period and the other after 2 h of REM sleep rebound (REM sleep deprivation plus 2 h of rebound). In both groups we determined the expression of the CB1 receptor and its mRNA. Results indicated that SR141716A prevents REM sleep rebound and REM sleep deprivation does not modify the expression of the CB1 protein or mRNA. However, REM sleep deprivation plus 2 h of sleep rebound increased the CB1 receptor protein and, slightly but significantly, decreased mRNA expression. These results suggest that endocannabinoids may be participating in the expression of REM sleep rebound.

Characterization of a recombinant mu-class glutathione S-transferase from Taenia solium

Abstract. A Taenia solium larval glutathione S-transferase fraction (SGSTF), composed of two proteins with Mr 25,500 (SGSTM1) and 26,500 (SGSTM2), was purified by GSH-sepharose. Its N-terminal sequence analysis revealed that both proteins are related to mammalian mu-class GST enzymes. A cDNA clone coding for SGSTM1 was isolated and the amino acid sequence analysis showed close identity with two Echinococcus GSTs and also high identity with several mu-class GSTs that have been reported. In addition, SGSTM1 presents a similar structure to mu-class GSTs, including the mu loop. The recombinant SGSTM1 is a dimeric protein with enzymatic properties clearly related to mammalian mu-class GSTs. Western blot studies indicated that SGSTM1 is not antigenically related to SGSTM2 or mammalian GSTs from rabbit, pig and rat livers. Immunization with SGSTF and SGSTM2 was highly effective in reducing cysticerci load in murine cysticercosis. In contrast, no protection was obtained using native SGSTM1 and recombinant SGSTM1 as immunogens.

Cloning, expression and characterisation of a recombinant triosephosphate isomerase from Taenia solium ☆

We isolated and characterised the cDNA that encodes the glycolytic enzyme, triosephosphate isomerase from Taenia solium. A 450 bp DNA fragment was obtained by the polymerase chain reaction using a cDNA from larval stage as template and degenerate oligonucleotides designed from conserved polypeptide sequences from TPIs of several organisms. The fragment was used to screen a T. solium larval stage cDNA library. The isolated cDNA, encoding a protein of 250 amino acids shares 44.8-59.6% positional identity with other known TPIs, in which the catalytic enzyme residues were conserved. The complete coding sequence of the T. solium TPI cDNA was cloned into the expression vector pRSET and expressed as a fusion protein with an N-terminal tail of six histidine residues. The catalytic activity of the purified protein was similar to other TPI enzymes. Northern and Southern blot analysis suggest that in T. solium, single gene exists for triosephosphate isomerase and that the gene is expressed in all stages of the parasite.