The combined recombinant cathepsin L1H and cathepsin B3 vaccine against Fasciola gigantica infection

Protections against Fasciola gigantica infection in mice immunized with the individual and combined cathepsin L1H and cathepsin B3 vaccines were assessed. The vaccines comprised recombinant (r) pro-proteins of cathepsin L1H and B3 (rproFgCatL1H and rproFgCatB3) and combined proteins which were expressed in Pichia pastoris. The experimental trials were performed in ICR mice (n = 10 per group) by subcutaneous injection with 50 μg of the recombinant proteins combined with Alum or Freund's adjuvants. At two weeks after the third immunization, mice were infected with 15 F. gigantica metacercariae per mouse by oral route. The percents of protection of rproFgCatL1H, rproFgCatB3 and combined vaccines against F. gigantica were approximately 58.8 to 75.0% when compared with adjuvant-infected control. These protective effects were similar among groups receiving vaccines with Alum or Freund's adjuvants. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th1 and Th2 immune responses, it was found that both Th1 and Th2 humoral immune responses were significantly increased in vaccinated groups compared with the control groups, with higher levels of IgG1 (Th2) than IgG2a (Th1). Mice in vaccinated groups showed reduction in liver pathological lesions when compared with control groups. This study indicates that the combined rproFgCatB3 and rproFgCatL1H vaccine had a high protective potential than a single a vaccine, with Alum and Freund's adjuvants showing similar level of protection. These results can serve as guidelines for the testing of this F. gigantica vaccine in larger economic animals.     

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between na?ve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.     

Characterization and expression of cathepsin B2 in Fasciola gigantica

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2 week-old juveniles, but not in 3, 4 week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2 week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites’ antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.     

Molecular cloning and characterization of a cDNA encoding caltractin from Dunaliella salina.

We cloned a cDNA encoding caltractin, a 20 kDa calcium-binding protein, from Dunaliella salina (DSCALT). The Ca(2+)-bound mobility shift detected in Chlamydomonas caltractin was hardly detectable in DSCALT. Also, some differences were found in the electrophoretic mobility between the native DSCALT and the bacterial-expressed DSCALT. This difference may have resulted from the posttranslational modification. Immunoblot analysis revealed that this protein might be localized mainly in the basal body complex, the major microtubule organizing center (MTOC) in D.salina and the functional homologue of the centrosome of the animal cell.     

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.     

Molecular characterization revealed Fasciola specimens in Ecuador are all Fasciola hepatica,none at all of Fasciola gigantica or parthenogenic Fasciola species

All 225 Fasciola flukes obtained from domestic animals (73 cattle, 7 sheep and 1 pig) of 18 distinct geographic areas in Ecuador-South America, were identified as Fasciola hepatica, based on molecular analyses of nuclear pepck and pold genes, and mitochondrial nad1gene as well as the morphological observation of sperm within the seminal vesicles. Fasciola gigantica and parthenogenic Fasciola forms endemic to Asian countries were not found in this study, although zebu cattle and water buffalos have introduced into South America from Asia; this could be due to the absence of suitable intermediate host snails. The results of pepck analysis using multiplex PCR developed previously showed that 32 of the flukes could not be confirmed as F. hepatica, suggesting that the method is unreliable for the accurate discrimination of F. hepatica, and that pepck gene of the species consists of multiple loci, not a single locus. The results of genetic diversity, phylogenetic, and network analyses based on mitochondrial nad1 sequences suggest that F. hepatica populations in South America, including Ecuador, formed from the ancestral F. hepatica individuals introduced into the continent along with anthropogenic movement of livestock infected with the species.     

Molecular cloning and characterization of cathepsin F gene from olive flounder Paralichthys Olivaceus

We isolated a homologue of cathepsin F from cDNA library of olive flounder liver. A 2,077 kb full-length cDNA encoding a predicted polypeptide of 474 amino acids was sequenced. The flounder cathepsin F exhibits a domain structure typical for papain-like cysteine proteases, a 17 amino acid N-terminal hydrophobic signal sequence followed by an extraordinarily long propeptide of 244 amino acids and the domain of the mature protease comprising 213 amino acids. The mature region contains all features characteristic of a papain-like cysteine protease, including the highly conserved cysteine, histidine and asparagine residues of the ‘catalytic triad’. The cathepsin F protein showed 49–99% amino acid sequence identity with other known cathepsin F sequences. An in vivo expression study showed that cathepsin F mRNA was expressed predominantly in brain, liver, eye and heart, and moderately in other tissues. The accumulation of cathepsin F mRNA in early stage of development increased with development. This expression pattern suggests that flounder cathepsin F has been implicated in the growth and reproduction regulation.     

Molecular cloning of streptomyces genes encoding vanillate demethylase

The vanillate demethylase genes from Streptomyces sp. NL15-2K were cloned and sequenced. The vanA and vanB gene homologs, which encode the terminal oxygenase subunit (VanA) and the ferredoxin-type reductase subunit (VanB) of the enzyme respectively, were found in the sequenced 7.5-kb DNA region. Expression of the vanAB genes in Streptomyces lividans 1326 resulted in in vivo demethylation of veratric acid to vanillic acid.     

Fasciola gigantica: purification and characterization of adenosine deaminase

Nucleotidase cascades (apyrase, 5′ nucleotidase, and adenosine deaminase (ADA) were investigated in the parasitic trematode Fasciola gigantica. ADA had the highest activity in the nucleotidase cascades. Adenosine deaminase was purified from F. gigantica through acetone precipitation and chromatography on CM-cellulose. Two forms of enzyme (ADAI, ADAII) were separated. ADAII was purified to homogeneity after chromatography on Sephacryl S-200. The molecular mass was 29 KDa for the native and denatured enzyme using gel filtration and SDS-PAGE, respectively. The enzyme (ADAII) had a pH optimum at 7.5 and a K_m 1.0 mM adenosine, a temperature optimum at 40 °C and heat stability up to 40 °C. The order of effectiveness of metals as inhibitors was found to be Hg²⁺ > Mn²⁺ > Cu²⁺ > Ca²⁺ > Zn²⁺ > Ni²⁺ > Ba²⁺.     

Molecular cloning and characterization of homeo-box-containing genes from Atlantic salmon

As the most primitive group among vertebrates, fish might serve as a model system when studying the genetic regulation of embryogenesis in higher animals. To identify genes important for early development, we have constructed a genomic library from Atlantic salmon (Salmo salar) and screened it with homeobox-containing probes from Drosophila melanogaster. Five different salmon homeoboxes were isolated. Two of these were located in the same clone, separated by only 7.5 kb. This demonstrates the presence of clustered homeobox genes in fish. The two clustered homeoboxes were sequenced and shown to be closely related to the ANT-C/BX-C class of Drosophila, being about 80% homologous to the Ultrabithorax gene (Ubx) homeobox. One of the clustered genes appears to be the salmon equivalent of the mouse Hox-2.1 gene, indicating that some of the vertebrate homeobox-containing genes are conserved in evolution. A more diverged homeobox that shares only 60% homology with Ubx, was also sequenced. In analogy to Drosophila, therefore, the salmon genome contains more than one class of homeoboxes. In addition, Northern-blot experiments demonstrated that two of the homeobox genes are expressed in salmon embryos, suggesting their importance for proper development.     

Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica.

Nucleotide sequences of two regions, cytochrome c-oxidase subunit 1 (CO1) and NADH dehydrogenase subunit 1 (ND1) of the mitochondrial DNA and two regions, internal-transcribed spacer 2 (ITS2) and the D2 region in the 28S rDNA (28S) of the nuclear DNA were obtained from five Korean worms of the genus Fasciola in order to elucidate their taxonomic status. The CO1 and ND1 regions are all monomorphic in the Korean worms and similar to those of F. gigantica. On the other hand, the ITS2 and D2 regions were found to be polymorphic; that is, out of five worms, two possessed a F. gigantica-type sequence, one, a F. hepatica-type sequence and two possessed sequences of both types indicating an existence of different alleles at the loci. It should be noted that these variations of the ITS2 and D2 regions co-occur at the same individual worms. This was confirmed by sequencing five to six cloned PCR products for each worm. The present study strongly suggests interspecific cross-hybridization between the two species coexisting in Korea.     

Molecular cloning and characterization of hetR genes from filamentous cyanobacteria

HetR, a serine type protease, plays an important role in heterocyst differentiation in filamentous cyanobacteria. We isolated and sequenced the hetR genes from different heterocystous and filamentous nonheterocystous cyanobacteria. The hetR gene in the heterocyst forming Anabaena variabilis ATCC 29413 FD was interrupted by interposon mutagenesis (mutant strain WSIII8). This mutant does not form heterocysts and shows no diazotrophic growth under aerobic conditions. However, under anaerobic N(2)-fixing conditions, the WSIII8 cells are able to grow, and high nitrogenase (Nif2) activity is detectable. Nif2 expression was demonstrated in each vegetative cell of the filament by immunolocalization 4 h after nitrogen step-down.