Molecular phylogeography and genetic diversity of Angiostrongylus cantonensis and A. malaysiensis (Nematoda: Angiostrongylidae) based on 66-kDa protein gene.

Angiostrongylus cantonensis is the main causative agent of human angiostrongyliasis. A sibling species, A. malaysiensis has not been unequivocally incriminated to be involved in human infections. To date, there is only a single report on the application of the partial 66-kDa protein gene sequence for molecular differentiation and phylogeny of Angiostrongylus species. Nucleotide sequences of the 66-kDa protein gene of A. cantonensis and A. malaysiensis from Thailand, as well as those of the laboratory strains of A. cantonensis from Thailand and Hawaii, A. cantonensis from Japan and China, A. malaysiensis from Malaysia, and A. costaricensis from Costa Rica, were used for the reconstruction of phylogenetic tree by the maximum likelihood (ML) method and the haplotypes by the median joining (MJ) network. The ML phylogenetic tree contained two major clades with a full support bootstrap value - (1) A. cantonensis and A. malaysiensis, and (2) A. costaricensis. A. costaricensis was basal to A. cantonensis and A. malaysiensis. The genetic distance between A. cantonensis and A. malaysiensis ranged from p = .82% to p = 3.27%, that between A. cantonensis and A. costaricensis from p = 4.90% to p = 5.31%, and that between A. malaysiensis and A. costaricensis was p = 4.49% to p = 5.71%. Both A. cantonensis and A. malaysiensis possess high 66-kDa haplotype diversity. There was no clear separation of the conspecific taxa of A. cantonensis and A. malaysiensis from different geographical regions. A more intensive and extensive sampling with larger sample size may reveal greater haplotype diversity and a better resolved phylogeographical structure of A. cantonensis and A. malaysiensis.

Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hbγ-ECS1) in Latex Production in Hevea brasiliensis.

Rubber tree is a major commercial source of natural rubber. Latex coagulation is delayed by thiols, which belong to the important type of antioxidants in laticifer submembrane, and is composed of glutathione (GSH), cysteine, and methionine. The rate-limiting enzyme, γ-ECS, plays an important role in regulating the biosynthesis of glutathione under any environment conditions. To understand the relation between γ-ECS and thiols and to correlate latex flow with one-time tapping and continuous tapping, we cloned and derived the full length of one γ-ECS from rubber tree latex (Hbγ-ECS1). According to qPCR analysis, the expression levels of Hbγ-ECS1 were induced by tapping and Ethrel stimulation, and the expression was related to thiols content in the latex. Continuous tapping induced injury, and the expression of HbγECS1 increased with routine tapping and Ethrel-stimulation tapping (more intensive tapping). According to expression in long-term flowing latex, the gene was related to the duration of latex flow. HbγECS1 was expressed in E. coli Rosetta using pET-sumo as an expression vector and the recombinant enzyme was purified; then we achieved 0.827 U/mg specific activity and about 66 kDa molecular weight. The present study can help us understand the complex role of Hbγ-ECS in thiols biosynthesis, which is influenced by tapping.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum.

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5% and 47.6% less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.

Recombinant tegumental protein Shistosoma japonicum very lowdensity lipoprotein binding protein as a vaccine candidate against Schistosoma japonicum

A polyhistidine-tagged recombinant tegumental protein Schistosoma japonicum very lowdensity lipoprotein binding protein (SVLBP) from adult Schistosoma japonicum was expressed in Escherichia coli. The affinity purified rSVLBP was used to vaccinate mice. The worm numbers and egg deposition recovered from the livers and veins of the immunized mice were 33.5 percent and 47.6 percent less than that from control mice, respectively (p<0.05). There was also a marked increase in the antibody response in vaccinated mice: the titer of IgG1 and IgG2a, IgG2b in the vaccinated group was significantly higher than that in the controls (>1:6,400 in total IgG). In a comparison of the reactivity of sera from healthy individuals and patients with rSVLBP, recognition patterns against this parasite tegumental antigen varied among different groups of the individuals. Notably, the average titres of anti-rSVLBP antibody in sera from faecal egg-negative individuals was significantly higher than that in sera from the faecal egg-positives, which may be reflect SVLBP-specific protection. These results suggested that the parasite tegumental protein SVLBP was a promising candidate for further investigation as a vaccine antigen for use against Asian schistosomiasis.