Nematicidal activity of 'major royal jelly protein'-containing glycoproteins from Acacia honey.

Parasitic nematodes infect more than two billion people worldwide particularly in developing countries. We previously reported nematicidal activity of natural honey using model nematode Caenorhabditis elegans. In this study, characterization of nematicidal effects of natural honey and its glycoproteins has been carried out. Chromatographically separated honey glycoproteins showed potent anti-C. elegans activity (LD50 = 100 ng proteins/µL). Honey glycoproteins with molecular masses of ∼260 kD and ∼160 kD comprised of 'major royal jelly protein-1'-containing complexes. In these complexes, MRJP1 was present in different glycosylation forms. Quantitative PCR based gene expression assays described molecular functions of C. elegans affected by honey and honey glycoproteins. Expression of 14 gene transcripts associated with key cellular and molecular functions including energy metabolism, cytoskeleton, cell division, transcription and translation was analyzed. Acacia honey exerted a concentration-dependent alteration of gene transcripts involved in the citric acid cycle (mdh-1 and idhg-1) and cytoskeleton (act-1, act-2, and arp6). Likewise, MRJP1-containing glycoproteins caused down-regulation of arp-6 and idhg-1; and up-regulation of act-1 and mdh-1 gene transcripts. Consistent down-regulation of isocitrate dehydrogenase encoding idhg-1 gene which is among the rate-controlling enzymes of the citric acid cycle was considered as main biochemical factor involved in the nematicidal activity of honey and MRJP-containing glycoproteins. Acacia honey suppressed the expression of gene transcripts encoding actin-2, while honey glycoproteins did not. Hence, honey partly exerted anti-C. elegans activity by decreasing the transcription of actin-2 gene transcripts, demonstrated by a defect in the movement and egg laying. Moreover, arp-6 gene transcripts encoding actin-related protein 6 was significantly and constantly down-regulated by honey and honey proteins.

Isolation and characterization of Cu/Zn-superoxide dismutase in Fasciola gigantica.

A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed.

Heat shock protein 60 of filarial parasite Brugia malayi: cDNA cloning, expression, purification and in silico modeling and analysis of its ATP binding site.

We report here cloning and expression of full length mitochondrial HSP60 gene of Brugia malayi adult worm (mtHSP60bm), purification of the gene product by affinity chromatography, its in silico 3D structure and the sequence homology of the protein with Escherichia coli GroEL/ES and human HSP60. The ATP binding pocket of human HSP60 and mtHSP60bm were analyzed and compared using in silico models. The distribution of HSP60 in different life-stages of the parasite was determined using antibodies raised against recombinant mtHSP60bm (rmtHSP60bm). mtHSP60bm was present in all life-stages of the parasite except third stage infective larvae, in which it could be induced by heat-shock, and showed high degree of homology with E. coli GroEL/ES. The ATP binding pocket of HSP60 in humans, E. coli and B. malayi were also found structurally conserved. This similarity between human and mtHSP60bm might be useful in understanding the host-parasite interactions. This is the first ever report on distribution, cloning, sequence homology and ATP binding site of mtHSP60bm.

Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus.

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2-70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus.

Molecular analysis of zinc transporters in Schistosoma japonicum.

Members of the zinc-regulated transporter/iron-regulated transporter-like protein (ZIP) family of proteins transport metal ions across cell membranes. Genes encoding ZIPs are present in the genomes of schistosomes. Here, we describe molecular characterisation of six ZIPs (SjZIPA-F) from Schistosoma japonicum. Quantitative PCR analyses of these ZIPs through the lifecycle showed that each is expressed predominantly during the intramammalian stage and are particularly enriched in adult females. Using laser microdissected tissue as template, SjZIPA-D were transcriptionally enriched in female reproductive tissues, SjZIPE was not expressed in specific tissues and SjZIPF was expressed similarly in each tissue. Whole mount in situ hybridization revealed that SjZIPA and SjZIPB were localised to the oesophageal gland of adults and the vitellaria. We have shown that multiple ZIPs are expressed by schistosomes during the intramammalian parasitic phases and propose that the encoded products perform diverse cellular functions related to metal transport in different cells of S. japonicum.