Egg-Hatching Mechanism of Human Liver Fluke, Opisthorchis viverrini: A Role For Leucine Aminopeptidases From the Snail Host, Bithynia siamensis goniomphalos.

The human liver fluke Opisthorchis viverrini (Platyhelminthes, Trematoda, Digenea) uses snails of the genus Bithynia as first intermediate host. Peculiarly among trematodes, the eggs of O. viverrini hatch within the digestive tract of its snail host. It remains uncertain whether hatching in this species is mediated through mechanical fracture of the eggshell or by digestion with specific digestive enzymes. This study aimed to characterize enzymes with specific inhibitors and factors involved in the hatching activity of O. viverrini eggs. For measuring egg hatching in vivo, 50 O. viverrini mature eggs were fed to individual Bithynia siamensis goniomphalos snails at various temperature conditions for 24 hr. Ex vivo, mature eggs were incubated with crude snail extract and commercial leucine aminopeptidase (LAP). Egg-hatching of O. viverrini was temperature dependent, with optimal hatching occurring at 24-28 C, with a peak of hatching of 93.54% in vivo and 30.55% ex vivo occurring at these temperatures. Ex vivo hatching rates increased to 45.87% under anaerobic conditions at 28 C. Some 22.70% and 16.21% of heat-killed eggs also hatched within the snail digestive tract and snail extract, respectively, indicating that host molecules are involved in the hatching response. Most eggs hatch in the anterior regions of the digestive tract. Hatching was completely inhibited in the presence of bestatin, an inhibitor of LAP, but not in the presence of phosphatase inhibitors. Bestatin inhibition of hatching was reversible. Finally, egg hatching could be induced by addition of a porcine LAP. The results indicate that this digenean utilizes both LAP of the snail host and movement of miracidia for hatching.

Immunization and challenge shown by hamsters infected with Opisthorchis viverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke.

Here we report findings to optimize and standardize conditions to attenuate metacercariae of Opisthorchis viverrini by ionizing radiation to elicit protective immune responses to challenge infection. Metacercariae were gamma-irradiated and the ability of irradiated metacercariae to prevent patent infection of challenge metacercariae in hamsters was determined, as well as their ability to induce a host antibody response. Metacercariae irradiated in a dose-dependent manner, with 3, 5, 10, 12, 20, 25 and 50 Gray, were used to infect Syrian golden hamsters by stomach gavage to ascertain the effect of irradiation on ability of the worms to establish infection. In addition, other hamsters were infected with metacercariae irradiated with 20-50 Gray, followed by challenge with intact/wild-type (non-irradiated) metacercariae to determine the protective effect as established by the numbers of adult flukes, eggs of O. viverrini in hamster faeces and anti-O. viverrini antibody titres. Significantly fewer worms were recovered from hamsters immunized with metacercariae irradiated at 20, 25 and 50 Gray than from control hamsters infected with intact metacercariae or 0 Gray, and the worms showed damaged reproductive organs. Faecal egg numbers were decreased significantly in hamsters immunized with 25 and 50 Gray metacercariae of O. viverrini. Moreover, hamsters administered metacercariae that were protected elicited a robust, specific anti-fluke immunoglobulin G response compared to control hamsters, suggesting a role for antibody in protection elicited by radiation-attenuated metacercariae.

Mitochondrial DNA sequence variation among geographical isolates of Opisthorchis viverrini in Thailand and Lao PDR, and phylogenetic relationships with other trematodes.

The present study compared the genetic variation among 14 different geographical isolates of Opisthorchis viverrini sensu lato from Thailand and Lao PDR using sequence data for 2 mitochondrial DNA genes, the subunit 1 of NADH dehydrogenase gene (nad1) and cytochrome c oxidase gene (cox1). Four different nad1 haplotypes were detected among isolates, all of which were identical at the amino acid sequence level. Nucleotide sequence variation among 14 isolates ranged from 0 to 0.3% for nad1. Two different cox1 haplotypes were detected among isolates. These two haplotypes differed at 2 nucleotide positions, one of which resulted in a change in the amino acid sequence. Nucleotide sequence variation among isolates for cox1 ranged from 0 to 0.5%. Comparison of cox1 sequences of O. viverrini to those of other trematodes revealed nucleotide differences of 13-31%. A phylogenetic analysis of the cox1 sequence data revealed strong statistical support for a clade containing O. viverrini and 2 other species of opisthorchid trematodes; O. felineus and Clonorchis sinsensis.

Reverse phase high performance liquid chromatographic method for the analysis of letrozole in pharmaceutical dosage forms.

A rapid and sensitive reverse phase high performance liquid chromatographic method is depicted for the qualitative and quantitative assay of letrozole in pharmaceutical dosage forms. Letrozole was chromatographed on a reverse phase C18 column with a mobile phase consisting of acetonitrile and phosphate buffer (pH 7.8) in the ratio of 70:30 v/v. The mobile phase was pumped at a flow rate of 1 ml/min. Acenaphthene was used as an internal standard and the eluents were monitored at 232 nm. The retention time of the drug was 3.385 min. With this method, linearity was observed in the range of 10-100 mug/ml. The LOD and LOQ were found to be 0.51 mug/ml and 1.52 mug/ml, respectively. The method was found to be applicable for analysis of drug in tablets. The results of the analysis were validated statistically.

Carbon nanotube reinforced aluminum nanocomposite via plasma and high velocity oxy-fuel spray forming.

Free standing structures of hypereutectic aluminum-23 wt% silicon nanocomposite with multiwalled carbon nanotubes (MWCNT) reinforcement have been successfully fabricated by two different thermal spraying technique viz Plasma Spray Forming (PSF) and High Velocity Oxy-Fuel (HVOF) Spray Forming. Comparative microstructural and mechanical property evaluation of the two thermally spray formed nanocomposites has been carried out. Presence of nanosized grains in the Al-Si alloy matrix and physically intact and undamaged carbon nanotubes were observed in both the nanocomposites. Excellent interfacial bonding between Al alloy matrix and MWCNT was observed. The elastic modulus and hardness of HVOF sprayed nanocomposite is found to be higher than PSF sprayed composites.

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum.

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have named this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box. Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes.

Sjalpha elements, short interspersed element-like retroposons bearing a hammerhead ribozyme motif from the genome of the oriental blood fluke Schistosoma japonicum.

Smalpha is a short interspersed element (SINE)-like retroposon that occurs in high copy number of the genome of the human blood fluke Schistosoma mansoni. The sequence of the consensus Smalpha element includes the hallmark features of SINE-like elements including a promoter region for RNA polymerase III, an AT-rich stretch at its 3'-terminus, a short length of 500 bp or less, and short direct repeat sequences flanking the insertion site. Interestingly, the sequence of Smalpha also encodes an active ribozyme bearing a hammerhead domain. Contrary to the recent findings of Ferbeyre et al. (Mol. Cell. Biol. 18 (1998) 3880-8) that indicated that Smalpha-like elements were absent from the genome of the Oriental blood fluke Schistosoma japonicum, we report here that the genome of S. japonicum does contain a family of Smalpha-like retroposons, elements that we have named the Sjalpha family. Like Smalpha, Sjalpha elements are SINE-like in structure and sequence, are present at high copy number interspersed throughout the S. japonicum genome, and contain an ostensibly functional, hammerhead ribozyme motif. The presence of these elements in all species of Schistosoma so far examined suggests that the hammerhead domain was acquired by vertical transmission from a common schistosome ancestor.

Specific heparin preparations interfere with the simultaneous measurement of ionized magnesium and ionized calcium.

OBJECTIVE: To determine whether heparin anticoagulants used for analysis of whole blood ionized calcium would influence the measurement of ionized magnesium. METHODS: The effects of zinc heparin, lithium heparin, and electrolyte-balanced heparin on the simultaneous measurement of ionized magnesium and ionized calcium in serum were determined using ion selective electrodes. RESULTS: Time-dependent biases in ionized magnesium and calcium concentrations were apparent with zinc heparin but not with lithium or electrolyte-balanced heparin. Ionized magnesium and calcium concentrations were more significantly influenced by volume-dependent changes in zinc heparin potency than with lithium or electrolyte-balanced heparin. CONCLUSION: Zinc heparin produces a significant positive bias in the simultaneous determination of ionized magnesium and ionized calcium concentrations.

Zinc heparin introduces a preanalytical error in the measurement of ionized calcium concentration.

In this study we determined how zinc heparin affected ionized calcium concentration determinations. Zinc heparin produced a positive bias (0.020-0.067 mmol l-1) in ionized calcium measurement in serum/whole blood and a negative bias (-0.035 to -0.110 mmol l-1) with aqueous ionized calcium standards. To test if the positive bias was due to zinc ions displacing calcium from its protein-calcium complexes, we reproduced the effect by adding ZnCl2 to pooled serum. Changes in [ZnCl2] (0.1-0.5 mmol l-1) caused a dose-dependent increase, a constant bias ([ZnCl2], 0.5-1.25 mmol l-1) or a dose-dependent decrease ([ZnCl2] > 1.25 mmol l-1) in ionized calcium concentration, independent of pH. Similar results were obtained when zinc concentration of the specimen was changed by varying the volume of blood collected into syringes containing zinc heparin. We conclude that zinc heparin produces a positive bias in ionized calcium measurement in protein-based matrices.