Site-Directed Mutagenesis Study Revealed Three Important Residues in Hc-DAF-22, a Key Enzyme Regulating Diapause of Haemonchus contortus.

Haemonchus contortus (H. contortus) is one of the most important parasites of small ruminants, especially goats and sheep. The complex life cycle of this nematode is a main obstacle for the control and prevention of haemonchosis. So far, a special form of arrested development called diapause different from the dauer stage in Caenorhabditis elegans (C. elegans) has been found in many parasitic nematodes. In our previous study, we have characterized a novel gene Hc-daf-22 from H. contortus sharing high homology with Ce-daf-22 and functional analysis showed this gene has similar biological function with Ce-daf-22. In this study, Hc-daf-22 mutants were constructed using site-directed mutagenesis, and carried out rescue experiments, RNA interference (RNAi) experiments and in vitro enzyme activity analysis with the mutants to further explore the precise function site of Hc-DAF-22. The results showed that Hc-daf-22 mutants could be expressed in the rescued ok693 worms and the expression positions were mainly in the intestine which was identical with that of Hc-daf-22 rescued worms. Through lipid staining we found that Hc-daf-22 could rescue daf-22 mutant (ok693) from the fatty acid metabolism deficiency while Hc-daf-22 mutants failed. Brood size and body length analyses in rescue experiment along with body length and life span analyses in RNAi experiment elucidated that Hc-daf-22 resembled Ce-daf-22 in effecting the development and capacity of C. elegans and mutants impaired the function of Hc-daf-22. Together with the protease activity assay, this research revealed three important active resides 84C/299H/349H in Hc-DAF-22 by site-directed mutagenesis.

Characterization and function analysis of a novel gene, Hc-maoc-1, in the parasitic nematode Haemonochus contortus.

BACKGROUND: Enoyl-CoA hydratase (MAOC) is required for the biosynthesis of the fatty acid-derive side chains of the ascaroside via peroxisome ß-oxidation in the free-living nematode Caenorhabditis elegans. The derivative of dideoxy-sugar, ascarylose is used as dauer pheromones or daumones to induce development of the stress-resistant dauer larvae stage. METHODS: Hc-maoc-1 gene was obtained by searching the Wellcome Trusts Sanger Institute's H. contortus genomic database. qRT-PCR was performed to analyse the transcriptional levels of Hc-maoc-1 with different developmental stages as templates. IFA was carried out to determine the expression pattern in L3 larvae and micro-injection was used to verify the promoter activity of 5'-flanking region of Hc-maoc-1. Overexpression and RNAi experiments were applied in N2 strain to ascertain the gene function of Hc-maoc-1. RESULTS: The full-length cDNA of Hc-maoc-1 was 900 bp in length, which contained eight exons separated by seven introns and possessed the Hotdog domain and the MaoC-like domain, together with several other residues and a hydratase 2 motif. It was transcribed throughout the lifecycle and peaked in the fourth-stage larvae (L4) of H. contortus; however, its transcription level decreased in diapausing L4. The protein expression and location of Hc-MAOC-1 were mainly in the intestine of L3 larvae. Overexpression of Ce-maoc-1 and Hc-maoc-1 in C. elegans showed extended lifespan and increased body size. The protein Ce-MAOC-1 and Hc-MAOC-1 were localized in the intestine with a punctate pattern. In C. elegans, knockdown of Ce-maoc-1 conferred shortened lifespan and body lengths, decreased brood size and increased lipid storage. CONCLUSION: Caenorhabditis elegans was used as a model organism to ascertain the function of Hc-maoc-1 in H. contortus. Our results showed the similar characteristics and functions with Ce-maoc-1 and provided evidences of the potential functions of Hc-maoc-1 in biosynthesis of daumones in H. contortus.

Structural and functional characterization of a novel gene, Hc-daf-22, from the strongylid nematode Haemonchus contortus.

BACKGROUND: The strongylid nematode Haemonchus contortus is a parasite of major concern for modern livestock husbandry because hostile environmental conditions may induce diapause in the early fourth-stage larvae. METHODS: A new gene Hc-daf-22 was identified which is the homologue of Ce-daf-22 and human SCPx. Genome walking and RACE were performed to obtain the whole cDNA and genomic sequence of this gene. Using qRT-PCR with all developmental stages as templates to explore the transcription level and micro-injection was applied to confirm the promoter activity of the 5'-flanking region. Overexpression, rescue and RNA interference experiments were performed in N2, daf-22 mutant (ok 693) strains of C. elegans to study the gene function of Hc-daf-22. RESULTS: The full length gene of Hc-daf-22 (6,939 bp) contained 16 exons separated by 15 introns, and encoded a cDNA of 1,602 bp (533 amino acids, estimated at about 59.3 kDa) with a peak in L3 and L4 in transcriptional level. The Hc-DAF-22 protein was consisted of a 3-oxoacyl-CoA thiolase domain and a SCP2 domain and evolutionarily conserved. The 1,548 bp fragment upstream of the 5'-flanking region was confirmed to have promoter activity compared with 5'-flanking region of Ce-daf-22. The rescue experiment by micro-injection of daf-22 (ok693) mutant strain showed significant increase in body size and brood size in the rescued worms with significantly reduced or completely absent fat granules confirmed by Oil red O staining, indicating that Hc-daf-22 could partially rescue the function of Ce-daf-22. Furthermore, RNAi with Hc-daf-22 could partially silence the endogenous Ce-daf-22 in N2 worms and mimic the phenotype of daf-22 (ok693) mutants. CONCLUSION: The gene Hc-daf-22 was isolated and its function identified using C. elegans as a model organism. Our results indicate that Hc-daf-22 shared similar characteristics and function with Ce-daf-22 and may play an important role in peroxisomal ß-oxidation and the development in H. contortus.

Rapid Identification of unstable acyl glucoside flavonoids of Oxytropis racemosa Turcz by high-performance liquid chromatography-diode array detection-electrospray ionisation/multi-stage mass spectrometry.

INTRODUCTION: Oxytropis racemosa Turcz is an important minority medicine that is used mainly to improve children's indigestion, especially in inner Mongolia and Tibet. Previous studies indicated that the characteristic constituents of this plant are acylated flavonoids. OBJECTIVE: Rapidly identify the characteristic chemical constituents of O. racemosa by high-performance liquid chromatography-diode array detection-electrospray ionisation/multi-stage mass spectrometry (HPLC-DAD-ESI/MS(n) ) and suggest a useful method to control the quality of this medicinal plant. METHODS: In the HPLC fingerprint, 32 flavonoids were tentatively identified by a detailed analysis of their mass spectra, UV spectra and retention times. Furthermore, 13 flavonoids were confirmed by comparison with previously isolated compounds obtained from O. racemosa. RESULTS: In total, 32 flavonoids, including 13 flavonoids with 3-hydroxy-3-methylglutaric acid (HMG) moieties and four flavonoids with 3-malonyl moieties, were identified in the extract of O. racemosa. Among the compounds identified, 10 were characterised as new compounds for their particular acylated sugar moieties. CONCLUSIONS: The method described is effective for obtaining a comprehensive phytochemical profile of plants containing unstable acylated flavonoids. The method is also useful for constructing the chromatographic fingerprint of the minority medicine -O. racemosa Turcz for quality control.

3-Hydroxymethylglutaryl flavonol glycosides from a Mongolian and Tibetan medicine, Oxytropis racemosa.

Three new flavonol 3-O-glycosides, rhamnetin 3-O-[(S)-3-hydroxy-3-methyl-glutaroyl(1→6)]-ß-D-glucopyranoside (1), rhamnocitrin 3-O-[(S)-3-hydroxy-3-methylglutaroyl(1→6)]-ß-D-glucopyranoside (2), and isorhamnetin 3-O-[(S)-3-hydroxy-3-methylglutaroyl(1→6)]-α-L-rhamnopyranosyl(1→2)-ß-D-glucopyranoside (3), along with 13 known compounds, were isolated from Oxytropis racemosa TURCZ. Their structures were deduced by means of spectroscopic methods and chemical evidence. 2 and 6 showed cytotoxic activities against HCT-8 (IC50 6.38 µM) and A549 (IC50 5.20 µM), respectively.

Flavonol glycosides and monoterpenoids from Potentilla anserina.

Potentilin A (1), a rare diflavonol ester of mu-truxinic acid and a new normonoterpenoid, 2,6-dimethyl-2,3-dihydro-4-oxo-4H-pyran-2-acetic acid (2), was isolated from Potentilla anserina, together with 19 known flavonol glycosides (3-21) and 2 known monterpenoids (22,23). Their structures were elucidated by means of UV, IR, HR-ESI-MS, 1D and 2D NMR spectroscopic data.