[Study on molecular phylogeny of Schistosoma bovis based on mitochondrial DNA sequence and gene order].

OBJECTIVE: To determine the nucleotide sequence of the partial mitochondrial (mt) genome and the order of the mitochondrial protein-coding genes for Schistosoma bovis for analysis of possible phylogenetic position of this species in the genus Schistosoma. METHODS: The genomic DNA of adult worms were extracted by the GNT-K method. The target regions were amplified by PCR using a degenerated primer and specific primer. The PCR products were purified before ligating into the pGEM1 T-vector system. Recombinant plasmids were amplified in Escherichia coli, extracted and purified using routine methods. The nucleotide sequences were determined with an ABI PRISM 3100-Avant DNA sequencer using a BigDye Terminators v3.1 Cycle Sequencing Kit (Applied Bio-systems, CA, U.S.A.) with two T-vector specific primers (T7 and SP6). Positive colonies were sequenced with two internal specific primers to obtain the full sequence of each fragment on both strands by means of primer walking. Sequences of related schistosomes were retrieved from GenBank and aligned with our data. Gene trees were constructed using neighbor joining methods. RESULTS: The nucleotide sequence was determined and the gene order of this region in S. bovis was found as follows: NADHdehydrogenase4 (nad4)-trnQ (Gln)-trnK(Lys)-NADH dehydrogenase 3(nad3)-trnD (Asp)-NADH dehydrogenase 1(nad1). The gene order covering such region of S. bovis was similar to that of the African Schistosoma species, but strikingly different from the Asian species. Phylogenetic trees inferred from the alignment including partial nad4, nad3, partial nad1 and partial nad4+nad3+nad1 sequence for other 8 Schistosoma spp., respectively, revealed that S. bovis is placed proximally to S. haematobium in the African sub-group, which is identical with those placed by gene order in the African clade. CONCLUSION: The mtDNA analysis based on mitochondrial DNA sequence and the gene order strongly support the hypothesis that S. bovis belongs to the African schistosome clade rather than the Asian Schistosoma species.

[Evaluation of immunogenicity and protection efficacy of the recombinant hypoxanthine-guanine-xanthine of plasmodium falciparum in mice].

OBJECTIVE: To investigate immunogenicity and protection efficacy of the recombinant hypoxanthine-guanine-xanthine (HGXPRT) of Plasmodium falciparum expressed in Pichia pastoris. METHODS: 35 BALB/c mice were divided randomly into five groups: HGXPRT+ISA720 experiment group, HGXPRT+Freund experiment group, ISA720 adjuvant control group, Freund adjuvant control group, and blank control group. BALB/c mice were subcutaneously immunized three times with the HGXPRT protein formulated by either Freund or ISA720 adjuvants at a three weeks interval. Mice were bled via tail vein at 2 weeks after each immunization. Specific antibodies were detected by ELISA as well as IFAT using cultured parasites. The immunized mice were challenged with 10(5) P. yoelii 10 days after the third immunization and parasitemia was monitored daily by examining Giemsa-stained thin film. RESULTS: Strong immune response was induced by the HGXPRT antigen formulated with the adjuvant. Antibody titers of more than 1:10(5) were detected after the third immunization while no specific antibody was detected in the mice immunized with adjuvants only. The antibodies against HGXPRT recognized the cultured parasite by IFAT. Four days after mice were challenged with P. yoelii, high parasitemia appeared in the two control groups, which were 24 h earlier than experiment groups. The mean parasitemia of HGXPRT+ISA720 experiment group (29.3%) was significantly lower than that of control groups (70.0%) (P<0.05). The mean parasitemia of HGXPRT+Freund experiment group (51.0%) was significantly lower than that of adjuvant control (60.7%) and blank control(70.0%) (P<0.05). CONCLUSION: HGXPRT of P. falciparum expressed in Pichia pastoris was highly immunogenic in mice. Antibody against the recombinant protein recognizes the cultured parasites, and immunization of mice with the recombinant protein provides partial protection against the challenge of P. yoelii.

Sequence variability in four mitochondrial genes among pinworm Aspicularis tetraptera isolates from laboratory mice in four provinces, China.

This research aimed at exploring sequence variability in four mitochondrial (mt) genes, namely, cytochrome c oxidase subunit 1 (cox1), cytochrome b (cytb) and NADH dehydrogenase subunits 1 and 5 (nad1 and nad5), among pinworm Aspicularis tetraptera isolates from laboratory mice in four different provinces, China. A part of the cox1 (pcox1), cytb (pcytb), nad1 and nad5 genes (pnad1 and pnad5) were amplified separately from individual pinworms by polymerase chain reaction (PCR) and sequenced to determine sequence variations and examine their phylogenetic relationships. Herein, the intra-specific sequence variations within A. tetraptera were 0-0.5% for pcox1, 0-1.4% for pcytb, 0-1.8% for pnad1 and 0-1.7% for pnad5, respectively. In contrast, the inter-specific sequence differences among members of the Oxyuridae were significantly higher, being 13.7-17.0% for pcox1, 24.5-34.7% for pcytb, 26.6-29.6% for pnad1 and 24.4-25.5% for pnad5, respectively. Three methods, namely, Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP), were used for phylogenetic analyses based on the combined sequences of the four mt gene sequences, and the results indicated that all A. tetraptera samples form monophyletic groups, but samples from the same geographical origin did not always cluster together. This study demonstrated the existence of low-level intra-specific variation in four mtDNA sequences among A. tetraptera isolates from laboratory mice in different geographic regions in China, indicating no obvious geographical distinction among A. tetraptera isolates in China. These findings have important implications for studying systematics, molecular epidemiology and population genetics of A. tetraptera.

[Studies on the relationship between the level of cytokine and liver function in patients with clonorchiasis sinensis].

OBJECTIVE: To explore the relationship between cytokine level and liver function among patients infected with Clonorchis sinensis. METHODS: 47 patients were divided into three groups according to the degree of Child-Pugh liver function grade: 20 in group A (3-4 scores), 15 in group B (5-6 scores) and 12 in group C (7-9 scores). Interleukin 2 (IL-2), soluble IL-2 receptor (sIL-2R), interleukin 8 (IL-8) and tumor necrosis factor (TNF-alpha) were examined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Automatic biochemical analyzer was employed for the determination of serum level of total bilirubin (TBL), albumin (ALB) and alanine aminotransferase (ALT). Data were analyzed with SAS statistic software. RESULTS: Serum levels of sIL-2R, IL-8 and TNF-alpha from patients were significantly higher than those obtained from healthy people (P<0.05, P<0.05, P<0.01), whereas the IL-2 level was significantly lower than the former (P<0.01). With the affected degree of the liver, serum levels of sIL-2R, IL-8 and TNF-alpha increased, in contrast to the decrease of IL-2 level. The differences were significant between groups A and C (P<0.05). The level of sIL-2R and TNF-alpha directly correlated with that of TBL (r=0.331 P<0.05, r=0.518 P<0.01) and ALT (r=0.475 P<0.01, r=0.285 P<0.05) respectively, but inversely correlated with the level of ALB (r=-0.319 P<0.05, r=-0.665 P<0.01). CONCLUSION: The infection of Clonorchis sinensis results in the reduction of cellular immune function of the patients. Certain relationship exists between serum cytokine level and liver function. Two cytokines, sIL-2R and TNF-alpha, are involved in the process of pathology.

Molecular cloning and characterization of taurocyamine kinase from Clonorchis sinensis: a candidate chemotherapeutic target.

BACKGROUND: Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK) constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK) of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK) gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. CONCLUSION: CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart, creatine.

Genetic variation among Clonorchis sinensis isolates from different hosts and geographical locations revealed by sequence analysis of mitochondrial and ribosomal DNA regions.

The present study examined genetic variability among Clonorchis sinensis isolates from four different geographical localities (Guangzhou, Nanning, Jiamusi and Daqing) and host species (cats, dogs, human and rabbits) in Mainland China by sequence analyses of two mitochondrial DNA (mtDNA) genes, namely NADH dehydrogenase subunits 2, 5 (nad2 and nad5) and ribosomal internal transcribed spacer 1 (ITS1). A portion of the ITS1, nad2 (pnad2) and nad5 (pnad5) was amplified by polymerase chain reaction separately from adult C. sinensis individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of ITS1, pnad2 and pnad5 was 643, 666 and 771 bp, respectively. The intraspecific sequence variations within C. sinensis were 0-1.7% for ITS1, 0-1.4% for pnad2 and 0-0.9% for pnad5. The interspecific sequence variations within other zoonotic trematodes, which were published previously, were 4.5-84.9% for ITS1, 21.9-43.6% for pnad2 and 19.2-48.9% for pnad5. The A+T contents of the sequences were 45.26-45.88% (ITS1), 62.91-63.51% (pnad2) and 58.24-58.63% (pnad5). Phylogenetic analyses using ribosomal and mitochondrial sequence data set, with three different computational algorithms (Bayesian inference, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support. These findings demonstrated the existence of low-level intraspecific variations in ribosomal DNA (rDNA) and mtDNA sequences among C. sinensis isolates from four different regions and hosts in China and elucidated that mtDNA sequences and rDNA sequences provided reliable genetic markers for phylogenetic studies of zoonotic trematodes.

miR-761 regulates the mitochondrial network by targeting mitochondrial fission factor.

Mitochondria are dynamic organelles that constantly undergo fission and fusion. The balance between fission and fusion determines the fate of the cell. In this study, we show that mitochondrial fission factor (MFF) is upregulated upon hydrogen peroxide treatment or ischemia/reperfusion (I/R) injury. Knockdown of MFF attenuated hydrogen peroxide- and I/R injury-induced cardiomyocyte apoptosis and myocardial infarction. We found that MFF is a direct target of miR-761, and miR-761 inhibits mitochondrial fission and cardiomyocyte apoptosis by repressing MFF. This study reveals a novel model of mitochondrial fission regulation, which is composed of miR-761 and MFF. Modulation of their levels may provide a new approach for tackling apoptosis and myocardial infarction.