Expression and serodiagnostic potential of antigen B and thioredoxin peroxidase from Taenia multiceps.

Coenurosis is a serious parasitic disease of herbivorous animals caused by the metacestode of Taenia multiceps (Coenurus cerebralis). Accordingly, a significant amount of research is currently dedicated to the development of appropriate antigens for use in rapid and accurate coenurosis diagnosis kits. In the present study, antigen B (AgB) and thioredoxin peroxidase (TPx) from T. multiceps were cloned and expressed using a prokaryotic system, molecular characterization of Tm-AgB was determined by bioinformatical analyses. The serological diagnostic potentials of rTm-AgB and rTm-TPx were evaluated by indirect ELISA and compared with those of previously reported rTm-AnxB2, rTm-HSP70, and rTm-GST. The results showed that Tm-AgB is a specific lipoprotein of cestodes with good thermal stability. The ELISA assay showed that rTm-AgB exhibited a sensitivity of 95.8% and a specificity of 87.5%, indicating its strong potential for serological diagnosis of T. multiceps.

Effect of curcuminoids and curcumin derivate products on thioredoxin-glutathione reductase from Taenia crassiceps cysticerci. Evidence suggesting a curcumin oxidation product as a suitable inhibitor.

Curcuma is a traditional ingredient of some Eastern cuisines, and the spice is heralded for its antitumoral and antiparasitic properties. In this report, we examine the effect of the curcuminoides which include curcumin, demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), as well as curcumin degradation products on thioredoxin glutathione reductase from Taenia crassiceps cysticerci Results revealed that both DMC and BDMC were inhibitors of TGR activity in the micromolar concentration range. By contrast, the inhibitory ability of curcumin was a time-dependent process. Kinetic and spectroscopical evidence suggests that an intermediary compound of curcumin oxidation, probably spiroepoxide, is responsible. Preincubation of curcumin in the presence of NADPH, but not glutathione disulfide (GSSG), resulted in the loss of its inhibitory ability, suggesting a reductive stabilizing effect. Similarly, preincubation of curcumin with sulfhydryl compounds fully protected the enzyme from inhibition. Degradation products were tested for their inhibitory potential, and 4-vinylguaiacol was the best inhibitor (IC50 = 12.9 µM), followed by feruloylmethane (IC50 = 122 µM), vanillin (IC50 = 127 µM), and ferulic aldehyde (IC50 = 180 µM). The acid derivatives ferulic acid (IC50 = 465 µM) and vanillic acid (IC50 = 657 µM) were poor inhibitors. On the other hand, results from docking analysis revealed a common binding site on the enzyme for all the compounds, albeit interacting with different amino acid residues. Dissociation constants obtained from the docking were in accord with the inhibitory efficiency of the curcumin degradation products.

Molecular cloning and characterization of leucine aminopeptidase gene from Taenia pisiformis.

Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa. TpLAP contains two characteristic motifs of the M17LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5 at 45 °C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn2+ and completely inhibited by 20 nM bestatin and 0.15 mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the M17LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T. pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention.

Two glutathione transferase isoforms isolated from juvenile cysts of Taenia crassiceps: identification, purification and characterization.

We identified and characterized the first two glutathione transferases (GSTs) isolated from juvenile cysts of Taenia crassiceps (EC 2.5.1.18). The two glutathione transferases (TcGST1 and TcGST2) were purified in a single-step protocol using glutathione (GSH)-sepharose chromatography in combination with a GSH gradient. The specific activities of TcGST1 and TcGST2 were 26 U mg-1 and 19 U mg-1, respectively, both at 25°C and pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates. The Km(CDNB) and Kcat(CDNB) values for TcGST1 and TcGST2 (0.86 µm and 62 s-1; 1.03 µm and 1.97 s-1, respectively) and Km(GSH) and Kcat(GSH) values for TcGST1 and TcGST2 (0.55 µm and 11.61 s-1; 0.3 µm and 32.3 s-1, respectively) were similar to those reported for mammalian and helminth GSTs. Mass spectrometry analysis showed that eight peptides from each of the two parasite transferases were a match for gi|29825896 glutathione transferase (Taenia solium), confirming that both enzymes are GSTs. The relative molecular masses were 54,000 ± 0.9 for the native enzymes and 27,500 ± 0.5 for the enzyme subunits. Thus, TcGST1 and TcGST2 are dimeric proteins. Optimal TcGST1 and TcGST2 activities were observed at pH 8.5 in the range of 20-55°C and pH 7.5 at 35-40°C, respectively. TcGST1 and TcGST2 were inhibited by cibacron blue (CB), bromosulphophthalein (BST), rose bengal (RB), indomethacin and haematin (Hm) with 50% inhibitory concentrations (IC50) in the µm range. TcGST1 was inhibited in a non-competitive manner by all tested inhibitors with the exception of indomethacin, which was uncompetitive. The discovery of these new GSTs facilitates the potential use of T. crassiceps as a model to investigate multifunctional GSTs.

Molecular cloing and bioinformatics analysis of lactate dehydrogenase from Taenia multiceps.

Coenurus cerebralis, the larval stage (metacestode or coenurus) of Taenia multiceps, parasitizes sheep, goats, and other ruminants and causes coenurosis. In this study, we isolated and characterized complementary DNAs that encode lactate dehydrogenase A (Tm-LDHA) and B (Tm-LDHB) from the transcriptome of T. multiceps and expressed recombinant Tm-LDHB (rTm-LDHB) in Escherichia coli. Bioinformatic analysis showed that both Tm-LDH genes (LDHA and LDHB) contain a 996-bp open reading frame and encode a protein of 331 amino acids. After determination of the immunogenicity of the recombinant Tm-LDHB, an indirect enzyme-linked immunosorbent assay (ELISA) was developed for preliminary evaluation of the serodiagnostic potential of rTm-LDHB in goats. However, the rTm-LDHB-based indirect ELISA developed here exhibited specificity of only 71.42% (10/14) and sensitivity of 1:3200 in detection of goats infected with T. multiceps in the field. This study is the first to describe LDHA and LDHB of T. multiceps; meanwhile, our results indicate that rTm-LDHB is not a specific antigen candidate for immunodiagnosis of T. multiceps infection in goats.

Characterization of glutathione S-transferase and its immunodiagnostic potential for detecting Taenia multiceps.

Taenia multiceps is a widespread zoonotic tapeworm parasite which infects cloven-hoofed animals around the world. Animal infection with Coenurus cerebralis, the coenurus larvae of T. multiceps (Tm), is often fatal, which is a major cause of economic losses in stockbreeding. This study amplified the glutathione S-transferase (GST) gene from the total RNA of C. cerebralis. The resulting protein, Tm-GST, consisted of 201 amino acids, and had a predicted molecular mass of 23.1kDa. Its amino acid sequence shares 77.61% similarity with Echinococcus granulosus GST. Recombinant Tm-GST (rTm-GST) was expressed in Escherichia coli. The protein reacted with serum from goats infected with T. multiceps. Immunofluorescence signals indicated that Tm-GST was largely localized in the parenchymatous area of adult T. multiceps; in addition, it was also apparent in the coenurus. An enzyme-linked immunosorbent assay based on rTm-GST showed specificity of 92.8% (13/14) and sensitivity of 90% (18/20) in detecting anti-GST antibodies in serum from naturally infected animals. This study suggests that Tm-GST has the potential to be used as a diagnostic antigen for Coenurosis.

Molecular characterization of enolase gene from Taenia multiceps.

Taenia multiceps is a cestode parasite with its larval stage, known as Coenurus cerebralis, mainly encysts in the central nervous system of sheep and other livestocks. Enolase is a key glycolytic enzyme and represents multifunction in most organisms. In the present study, a 1617bp full-length cDNA encoding enolase was cloned from T. multiceps and designated as TmENO. A putative encoded protein of 433 amino acid residues that exhibited high similarity to helminth parasites. The recombinant TmENO protein (rTmENO) showed the catalytic and plasminogen-binding characteristics after the TmENO was subcloned and expressed in the pET30a(+) vector. The TmENO gene was transcribed during the adult and larval stages and was also identified in both cyst fluid and as a component of the adult worms and the metacestode by western blot analysis. Taken together, our results will facilitate further structural characterization for TmENO and new potential control strategies for T. multiceps.

Identification and functional characterization of alpha-enolase from Taenia pisiformis metacestode.

Enolase belongs to glycolytic enzymes with moonlighting functions. The role of enolase in Taenia species is still poorly understood. In this study, the full length of cDNA encoding for Taenia pisiformis alpha-enolase (Tpeno) was cloned from larval parasites and soluble recombinant Tpeno protein (rTpeno) was produced. Western blot indicated that both rTpeno and the native protein in excretion-secretion antigens from the larvae were recognized by anti-rTpeno monoclonal antibodies (MAbs). The primary structure of Tpeno showed the presence of a highly conserved catalytic site for substrate binding and an enolase signature motif. rTpeno enzymatic activities of catalyzing the reversible dehydration of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) and vice versa were shown to be 30.71 ± 2.15 U/mg (2-PGA to PEP) and 11.29 ± 2.38 U/mg (PEP to 2-PGA), respectively. Far-Western blotting showed that rTpeno could bind to plasminogen, however its binding ability was inhibited by ϵ-aminocaproic acid (ϵACA) in a competitive ELISA test. Plasminogen activation assay showed that plasminogen bound to rTpeno could be converted into active plasmin using host-derived activators. Immunohistochemistry and immunofluorescence indicated that Tpeno was distributed in the bladder wall of the metacestode and the periphery of calcareous corpuscles. In addition, a vaccine trial showed that the enzyme could produce a 36.4% protection rate in vaccinated rabbits against experimental challenges from T. pisiformis eggs. These results suggest that Tpeno with multiple functions may play significant roles in the migration, growth, development and adaptation of T. pisiformis for survival in the host environment.

Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1µM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins.

Molecular identification of Taenia spp. in the Eurasian lynx (Lynx lynx) from Finland.

Cestodes of the genus Taenia are parasites of mammals, with mainly carnivores as definitive and herbivores as intermediate hosts. Various medium-sized cats, Lynx spp., are involved in the life cycles of several species of Taenia. The aim of the present study was to identify Taenia tapeworms in the Eurasian lynx (Lynx lynx) from Finland. In total, 135 tapeworms from 72 lynx were subjected to molecular identification based on sequences of 2 mtDNA regions, the cytochrome c oxidase subunit 1 and the NADH dehydrogenase subunit 1 genes. Available morphological characters of the rostellar hooks and strobila were compared. Two species of Taenia were found: T. laticollis (127 samples) and an unknown Taenia sp. (5 samples). The latter could not be identified to species based on mtDNA, and the rostellar hooks were short relative to those described among other Taenia spp. recorded in felids from the Holarctic region. In the phylogenetic analyses of mtDNA sequences, T. laticollis was placed as a sister species of T. macrocystis, and the unknown Taenia sp. was closely related to T. hydatigena and T. regis. Our analyses suggest that these distinct taeniid tapeworms represent a putative new species of Taenia. The only currently recognized definitive host is L. lynx and the intermediate host is unknown.

Quantitative screening for anticestode drugs based on changes in baseline enzyme secretion by Taenia crassiceps.

Neurocysticercosis (NCC), an infection of the brain with the larval stage of the Taenia solium tapeworm, is responsible for an estimated one-third of adult-onset epilepsy cases in regions of the world where it is endemic. Currently, anthelmintic drugs used for treatment of NCC are only partially effective, and there is, therefore, a pressing need for new therapeutic agents. Discovery of new anthelmintics with activity against T. solium has been limited by the lack of suitable sensitive assays that allow high-throughput screening. Using an in vitro culture system with Taenia crassiceps metacestodes, we demonstrate that changes in secretion of parasite-associated alkaline phosphatase (AP) and phosphoglucose isomerase (PGI) can be used to detect and quantify anthelmintic effects of praziquantel (PZQ), a drug with activity against T. solium. We applied two enzyme release assays to screen for anti-T. crassiceps activity in nonconventional antiparasitic drugs and demonstrate that nitazoxanide and artesunate induced release of both AP and PGI in differing time- and dose-related patterns. Furthermore, imatinib, a tyrosine kinase inhibitor previously reported to have parasiticidal activity against Schistosoma mansoni, also induced release of both AP and PGI in a dose-dependent manner, similar in pattern to that observed with the other anthelmintics. We also evaluated release of ATP into cyst supernatants as an indicator of drug effects but did not see any differences between treated and untreated cysts. These data provide the basis for rapid and quantitative screening assays for testing for anthelmintic activity in candidate anticestode agents.

[Cloning and sequence analysis of thioredoxin peroxidase gene from Taenia multiceps].

Protoscoleces of Taenia multiceps were collected from the naturally infected sheep and total RNA was extracted. Specific primers were designed according to TaHe2-D11 mRNA sequence and T. multiceps thioredoxin peroxidase gene (TmTPx) was amplified by RT-PCR. PCR products were ligated into pMD18-T vector and transformed to E. coli DH5alpha. The recombinant plasmids were identified by restriction digestion and sequencing. A 614 bp cDNA was amplified. The TmTPx open reading frame (591 bp) encoded a 196-amino acid protein with Mr 21,690, pI 7.61. Bioinformatics analysis indicated that TmTPx had a typical 2-Cys Prx conserved domain. Phylogenetic tree revealed that T. multiceps had the closest relationship to T. asiatica, followed by T. solium and T. crassiceps, E. granulosus and E. multilocularis.

A new MAP kinase protein involved in estradiol-stimulated reproduction of the helminth parasite Taenia crassiceps.

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17beta-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17beta-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design.

Characterization of one typical 2-Cys peroxiredoxin gene of Taenia solium and Taenia crassiceps.

The Taenia genus is capable of living for long periods within its hosts. Reports have shown that this successful establishment is related to its efficient defense mechanisms against host immune response and its high tolerance to oxidative stress. In this work, we describe the genomic sequences of one Taenia solium and Taenia crassiceps typical 2-Cys peroxiredoxins (Ts2-CysPrx, Tc2-CysPrx) genes, which are 94% identical in primary sequence with the typical 2-Cys Prxs catalytic motifs. Both genes have the same genomic architecture, showing a TATA box and Initiator (Inr) sequence in their proximal promoter, two exons split by a 67-bp type III intron and one unique transcription start site located inside the Inr. We show that T. crassiceps cysticerci are highly tolerant to H(2)O(2) presenting a lethal concentration 50 of 3.0 mM and demonstrate that the typical Tc2-CysPrx gene is not induced by H(2)O(2), showing a behavior of an antioxidant housekeeping gene. This study describes for first time the gene structure of a typical 2-Cys Prx in the Taenia genus.

Identification, expression, characterization, and immunolocalization of lactate dehydrogenase from Taenia asiatica.

From the expression sequence tags (ESTs) of Taenia asiatica, an EST containing a putative open reading frame of 993 bp was identified as lactate dehydrogenase (TaLDH) homologue. Recombinant TaLDH (rTaLDH) was expressed in Escherichia coli BL21/DE3 and purified. rTaLDH had a specific LDH activity and could be recognized in serum from swine or patient infected with T. asiatica. TaLDH was immunolocalized on the tegument of T. asiatica adult and embryonic membrane of oncosphere. Our study suggested that TaLDH is a potential drug target and candidate antigen for immunodiagnosis and vaccine for taeniasis and viscero-cysticercosis caused by T. asiatica.

[Prokaryotic expression, identification and histo-localization of the Taenia asiatica enolase gene].

OBJECTIVE: To express enolase gene of Taenia asiatica, investigate the immunoreactivity of the recombinant TaENO protein, and immuno-histo-localize the presence of the recombinant TaENO in adults of T. asiatica. METHODS: The gene encoding enolase of T. asiatica (TaENO) was cloned by high throughput sequencing from the cDNA library of adult T. asiatica. The coding region of TaENO was amplified by PCR, and cloned into a prokaryotic expression vector pET-30a (+). The recombinant plasmid was transformed into E. coli BL-21/DE3 and followed by expression of the protein induced by IPTG. The protein was purified by Ni-IDA affinity chromatography, and tested by SDS-PAGE. Its immunoreactivity was examined by Western blotting. The mice were immunized subcutaneously with purified TaENO formulated in Freund's adjuvant. Serum samples were collected and analyzed for specific antibodies by ELISA. The localization of TaENO in adult worms was demonstrated by immunofluorescent technique. RESULTS: The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml. It was recognized by antisera of SD rats immunized with TaENO, sera of taeniasis patients and sera of infected swine. The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T. asiatica adult. CONCLUSION: The TaENO gene has been expressed with immunoreactivity, and the recombinant TaENO is immunolocalized in the tegument of T. asiatica adult.

5'-p-Fluorosulfonyl benzoyl adenosine inhibits an ecto-ATP-diphosphohydrolase in the tegument surface of Taenia crassiceps cysticerci.

The tegumental membrane of Taenia crassiceps cysticerci contains an ATP-diphosphohydrolase (EC 3.6.1.5) which hydrolyzes purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates at an optimum pH of 8.5. It is Mg(2+)-dependent and insensitive to classical ATPase and phosphatase inhibitors. In solubilized tegumental membrane the Km values varied from 220 to 480 microM and the V(max) from 370 to 748 nmol of Pi release/mg/min for nucleoside triphosphates (ATP, GTP, CTP, UTP, and TTP); for nucleoside diphosphates (ADP, GDP, CDP, and UDP) the Km values were from 260 to 450 microM and the V(max) from 628 to 1134 nmol of Pi release/mg/min. An antibody specific to CD39 shows cross-reactivity with T. crassiceps ATP-diphosphohydrolase, revealing a single protein of approximately 80 kDa. Incubation of ATP-diphosphohydrolase with FSBA inhibited ATPase and ADPase activities by 85-90%. Immunoblot analyses, the competition plot, similar inhibition by free nucleotides, the lack of effect of Mg(2+) at high concentrations, and the inactivation by FSBA of ATPase and ADPase activity strongly suggest that a single enzyme catalyzes the hydrolysis of all these nucleotides. The mechanism of ATP hydrolysis shows that ATP-diphosphohydrolase releases ADP during the catalytic cycle. Incubation of intact cysticerci with FSBA caused 70-80% inhibition of ATPase and ADPase activities, indicating that the active site of the ATP-diphosphohydrolase is oriented to the external surface of the tegument of T. crassiceps. The importance of this enzyme in the parasite-host relationship is discussed.

The key steroidogenic enzyme 3beta-hydroxysteroid dehydrogenase in Taenia solium and Taenia crassiceps (WFU).

Larval and adult stages of Taenia solium and Taenia crassiceps WFU strain were analyzed by histochemical and biochemical methods to determine the existence of steroid pathways. The presence of the key enzyme 3beta-hydroxisteroid-dehydrogenase (3beta-HSD) was examined in frozen sections of cysticerci obtained from mice and segments of tapeworms obtained from the intestine of hamsters. 3beta-HSD activity was detected by nitroblue-tetrazolium products after incubation with dehydroepiandrosterone, androstendiol, or pregnenolone. Tapeworm tissues exhibited 3beta-HSD activity in the subtegumentary areas of the neck and immature proglottids following incubation with androstendiol, as well as surrounding the testes in mature proglottids. T. solium cysticerci exhibited 3beta-HSD activity in the subtegumentary tissues. The synthesis of steroid hormones involving the activity of 3beta-HSD was studied in cysticerci or tapeworms incubated in the presence of tritiated steroid precursors. The culture media were analyzed by thin layer chromatography and showed synthesis of androstendiol, testosterone, and 17beta-estradiol by cysticerci, androstendiol, and 17beta-estradiol by tapeworms. The results strongly suggest the activity of 3beta-HSD in taeniid parasites that have at least a part of the enzymatic chain required for androgen and estrogen synthesis and that the enzymes are present in the larval stage and from the early strobilar stages to the mature proglottids.

Glucose 6-phosphate dehydrogenase from larval Taenia crassiceps (cysticerci): purification and properties.

Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) was purified to homogeneity from the soluble fraction of larval Taenia crassiceps (Eucestoda: Cyclophyllidea) by a three-step protocol. Specific activity of the pure enzyme was 33.8 +/- 2.1 U mg(-1) at 25 degrees C and pH 7.8 with D: -glucose 6-phosphate and NADP+ as substrates. The activity increases to 67.6 +/- 3.9 U mg(-1) at 39 degrees C, a more physiological temperature in the intermediary host. Enzyme activity was maximal between pH 6.7 and 7.8. Km values were 14 +/- 1.7 microM and 1.3 +/- 0.4 microM for glucose 6-phosphate and NADP+, respectively. The enzyme showed absolute specificity for its sugar substrate. NAD+ was also a substrate but with a low catalytic efficiency (207 M(-1) s(-1)). No essential requirement for Mg++ or Ca++ was observed. Relative molecular mass of the native enzyme was 134,000 +/- 17,200, while a value of 61,000 +/- 1,700 was obtained for the enzyme subunit. Thus, glucose 6-phosphate dehydrogenase from T. crassiceps exists as a dimeric protein. The enzyme's isoelectric point was 4.5. The enzyme's activity dependence on temperature was complex, resulting in a biphasic Arrhenius plot. Activation energies of 9.91 +/- 0.51 and 7.94 +/- 0.45 kcal mol(-1) were obtained. Initial velocity patterns complemented with inhibition studies by product and substrate's analogues support a random bi bi sequential mechanism in rapid equilibrium. The low Ki value of 1.95 microM found for NADPH suggests a potential regulatory role for this nucleotide.

Contribution of NADH dehydrogenase subunit I and cytochrome C oxidase subunit I sequences toward identifying a case of human coenuriasis in France.

Coenuriasis is a parasitic disease induced by larval taeniid tapeworms that is rarely observed in humans. In December 2005, a case was diagnosed in Nancy, France, after surgical excision of a cyst on a 24-yr-old woman returning from the Côte d'Ivoire. Morphological and epidemiological criteria suggested that the infection was due to Taenia serialis. Molecular analysis of NADH dehydrogenase subunit I (NDI) and cytochrome c oxidase subunit I (COI) sequences was also in favor of T. serialis identification, but the absence of available genetic data on T. brauni and T. glomeratus and the small number of published sequences for T. serialis and T. multiceps must be considered with caution. The NDI partial sequences presented more variations within species of Taenia than the COI sequences, which make them more useful targets for species identification and analysis of intraspecific polymorphisms. The present study points to the usefulness of molecular biology tools to help make up for the shortcomings of the commonplace parasitological diagnosis for coenuriasis.