Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors

OBJECTIVES@#To determine exon/intron organization of the Toxocara canis (T. canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants.@*METHODS@#Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1200 bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C for the forward reaction (phosphagen synthesis).@*RESULTS@#Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K value of the mutant (Serine to Glycine) increased to 0.19 mM. The K value (0.19 mM) of the double mutant (Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type (0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica (A. indica), an aminoglycoside antibiotic (aminosidine), a citrus flavonoid glycoside (rutin) and a commercially available catechin mixture against TCAK. Green and black tea (1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK.@*CONCLUSIONS@#Arginine kinase in T. canis has a seven-exon/six-intron gene structure. However, further studies are needed to identify a specific compound within the extract causing the inhibitory effect and also to determine the molecular mechanisms behind inhibition of arginine kinase in T. canis.

Arginine kinase in Toxocara canis: Exon–intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors

Objectives To determine exon/intron organization of the Toxocara canis (T. canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Methods Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1 200 bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C for the forward reaction (phosphagen synthesis). Results Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K

[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.

Aureobasidium-Derived Soluble Branched (1,3-1,6) beta-Glucan (Sophy beta-glucan) Enhances Natural Killer Activity in Leishmania amazonensis-Infected Mice

The beta-glucans derived from yeast cell walls have been reported for having many immunomodulatory activities in vivo and in vitro. In this study, Aureobasidium-derived soluble branched (1,3-1,6) beta-glucan (Sophy beta-glucan) was checked for natural killer (NK) activity and for the production of IFN-gamma and IL-4 in Leishmania amazonensis infection. The main experiment was performed with a group of female C57BL/6 and BALB/c mice, orally supplemented with 5% of Sophy beta-glucan and infected with promastogotes of L. amazonensis (1 x 10(7)) into the footpad. Increase in the footpad thickness with time was observed in BALB/c mice in spite of the oral Sophy beta-glucan supplement, but it was less in C57BL/6 mice. The difference in overall mean footpad thickness between 'infection only' versus 'infection + glucan' groups was statistically significant (P < 0.001). High NK activity in C57BL/6 than BALB/c mice was observed in 'glucan only' group compared to the control group and also in 'infection + glucan' group compared to 'infection only' group. The difference in the NK activity among these groups was significant (P < 0.05). The IFN-gamma level increased at weeks 7 and 8 post-infection in C57BL/6 mice and was significantly high in 'infection + glucan' group compared to the 'infection only' group (P < 0.05). IL-4 levels did not increase up to detectable levels throughout the study. The results led a conclusion that Sophy beta-glucan enhances NK activity and cellular immunity in L. amazonensis-infected mice.