Molecular and immunological characterization of encoding gene and 14-3-3 protein 1 in Fasciola gigantica.

A cDNA encoding Fg14-3-3 protein 1 was cloned by immunoscreening of an adult-stage Fasciola gigantica cDNA library using a rabbit antiserum against tegumental antigens of the parasite. The protein has a deduced amino acid sequence of 252 residues and a calculated molecular weight of 28.7 kDa. It shows sequence identity values between 57.6 and 58.1% to the human 14-3-3 beta, zeta, theta, and eta proteins and is in a phylogenetic cluster with the 14-3-3 protein 1 of Schistosoma spp. Nucleic acid analyses indicate that the Fg14-3-3 protein 1 is encoded by a single copy gene and that this gene is expressed as a transcript of 1250 nucleotides. In adult and 4-week-old parasites the gene's transcriptional and translational products were localized in the gut epithelium, parenchyma, tegument cells, and in the reproductive organs. An antiserum against recombinant Fg14-3-3 protein 1 detected a slightly smaller 14-3-3 protein in the parasite's excretion/secretion material and showed cross-reactivity with 14-3-3 proteins in extracts of other trematodes and mouse. Antibodies against Fg14-3-3 protein were detected in the sera of rabbits as early as 2 weeks after infection with metacercariae of F. gigantica and the antibody titre increased continuously over a 10-week observation period.

Biochemical and histopathological effects of glyphosate herbicide on Nile tilapia (Oreochromis niloticus).

In Oreochromis niloticus that had been exposed for 3 months to sublethal concentrations (5 and 15 ppm) of the commercial glyphosate herbicide (C(3)H(8)NO(5)P) Roundup, the organs exhibited varying degrees of histopathological change. In the gills filament cell proliferation, lamellar cell hyperplasia, lamellar fusion, epithelial lifting, and aneurysm were observed. In the liver there were vacuolation of hepatocytes and nuclear pyknosis. Kidney lesions consisted of dilation of Bowman's space and accumulation of hyaline droplets in the tubular epithelial cells. The structural damages could be correlated to the significant increase (p

Two new HLA-B alleles, B*4422 and B*4704, identified in a study of families with autoimmunity.

HLA-B typing of approximately 1 262 individuals from a study of 372 simplex families with multiple sclerosis has led to the identification of two new alleles (HLA-B*4422 and HLA-B*4704). Sequencing confirmed that both of these new alleles represent novel combinations of previously described sequence motifs, reinforcing the notion that recombination and/or gene conversion-like events play an important role in generating HLA allelic diversity. The identification of these new alleles brings the total number of HLA-B alleles to 465.

Production and characterization of a monoclonal antibody against recombinant fatty acid binding protein of Fasciola gigantica.

In Fasciola parasites fatty acid binding proteins (FABPs) are the carrier proteins that help in the uptake of fatty acids from the hosts' fluids. Attempts have been made to utilize both native and recombinant FABP (rFABP) for immunodiagnosis and vaccine development for fasciolosis. In this study, we have produced a number of monoclonal antibodies (MoAbs) against rFABP of Fasciola gigantica. These MoAbs were initially screened against rFABP by ELISA and then tested for their specificities by immunoblotting. Five stable clones were selected and characterized further: four of them were of the isotype IgG(1) while one clone was IgG(2a). All the MoAbs reacted with rFABP which has a molecular weight (MW) of 20 kD and with at least two isoforms of native proteins at MW 14.5 kD that were present in the tegumental antigen (TA) and crude worm extracts, and the excretion-secretion materials. Immunoperoxidase staining of frozen sections of adult parasites by using these MoAbs as primary antibodies indicated that FABP were present in high concentration in the parenchymal cells and reproductive tissues, in low concentration in the tegument and caecal epithelium. All MoAbs cross-reacted with a 14.5 kD antigen present in the whole body (WB) extract of Schistosoma mansoni, while no cross-reactivities were detected with antigens from Eurytrema pancreaticum and Paramphistomum spp.

Genetic variability and linkage disequilibrium within the HLA-DP region: analysis of 15 different populations.

In order to understand the forces governing the evolution of the genetic diversity in the HLA-DP molecule, polymerase chain reaction (PCR)-based methods were used to characterize genetic variation at the DPA1 and DPB1 loci encoding this heterodimer on 2,807 chromosomes from 15 different populations including individuals of African, Asian, Amerindian, Indian and European origin. These ethnically diverse samples represent a variety of population substructures and include small, isolated populations as well as larger, presumably admixed populations. Ten DPA1 and 39 DPB1 alleles were identified and observed on 87 distinct DP haplotypes, 34 of which were found to be in significant positive linkage disequilibrium in at least one population. Some haplotypes were found in all ethnic groups while others were confined to a single ethnic group or population. Strong positive global linkage disequilibrium (Wn) between DPA1 and DPB1 was present in all 15 populations. The African populations displayed the lowest values of Wn whereas the Amerindian populations displayed near absolute disequilibrium. Analysis of the distribution of haplotypes using the normalized deviate of the Ewens-Watterson homozygosity statistic, F, suggests that DP haplotypes encoding the functional heterodimer are subject to much lower degrees of balancing selection than other loci within the HLA region. Finally, neighbor joining tree analyses demonstrate the power of haplotype diversity for inferring the relationships between the different populations.

Molecular cloning and characterization of cathepsin L encoding genes from Fasciola gigantica.

In this study cDNAs encoding cathepsin L-like proteins of Fasciola gigantica were cloned by the reverse transcription polymerase chain reaction method (RT-PCR) from total RNA of adult specimens. DNA sequence analyses revealed that six different cathepsin L cDNA fragments were isolated, which have DNA sequence identities of 87-99% towards the homologous genes from F. hepatica. Gene expression was studied at the RNA level by Northern and RNA in situ hybridizations. Northern analysis showed the cathepsin L genes to be strongly expressed in adult parasites as a group of 1050 nt sized RNAs. RNA in situ hybridization localized cathepsin L RNA to the cecal epithelial cells. Southern hybridization was used to determine the number of cathepsin L genes and indicated the presence of a family of closely related cathepsin L genes in the genome of F. gigantica.

Three new DP alleles identified in a study of 800 unrelated bone marrow donor-recipient pairs.

HLA-DP genotyping of 800 unrelated donor-recipient pairs in phase 5 of a retrospective analysis of unrelated bone marrow transplantation, sponsored by the National Marrow Donor Program (NMDP), has identified two new DPB1 alleles (DPB1*8701 and DBP1*8801) and one new DPA1 (DPA1*0108) allele. Sequencing confirmed that all three of these new alleles represent novel combinations of previously described sequence motifs, reinforcing the notion that "gene conversion-like" events play an important role in generating HLA allelic diversity. The identification of these new alleles brings the total number of DPA1 alleles to 20 and the total number of DPB1 alleles to 94.

Roles of cysteines in rat dipeptidyl peptidase IV/CD26 in processing and proteolytic activity.

The multifunctional type II transmembrane glycoprotein, dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5), is expressed by almost all mammalian cells and is identical to the adenosine deaminase binding protein CD26 on lymphocytes. The extracellular part of rat DPPIV can be divided into three domains the middle part of which harbors 10 of the 12 highly conserved cysteine residues. The cysteine-rich domain is responsible for DPPIV-binding to collagen I and to extracellular ADA. The participation of distinct cysteines in disulfide bridges is not yet known. Titration experiments have shown the presence of six free cysteines and three disulfide bridges in native rat DPPIV. To investigate the role of distinct cysteines in the structure-function relationships of rat DPPIV we constructed 12 different cysteine point mutations (C299, C326, C383, C455, C650 mutated to G; C337, C395, C445, C448, C473, C552, C763 mutated to S). Intracellular translocation to the cell surface of stable transfected Chinese hamster ovary cells was examined with antibodies against different epitopes of DPPIV. Surface expression of mutants C326G, C445S and C448S is inhibited totally; mutants C337S, C455G, C473S and C552S show weak expression only. In parallel, the half-life of these mutants is reduced to < 10% compared with wild-type enzyme. We were able to show that the specific peptidase activity of the mutant protein depends on cell-surface expression, dimerization and the existence of a 150-kDa form demonstrable by nondenaturing SDS/PAGE. We conclude that cysteine residues 326, 337, 445, 448, 455, 473 and 552 in rat DPPIV are essential for the correct folding and intracellular trafficking of this glycoprotein, and therefore for its normal biological properties.

In vivo modulation of the acidic N-glycans from rat liver dipeptidyl peptidase IV by N-propanoyl-D-mannosamine.

Derivatives of N-acyl-D-mannosamine differing in the N-acyl-side chain can be metabolically converted into neuraminic acids with corresponding N-acyl side chains. In the present study we show the in vivo modulation of sialic acids in membrane-bound dipeptidyl peptidase IV (CD 26) from rat liver after administration of N-propanoyl-D-mannosamine. Treatment of rats with this unphysiological precursor resulted in an incorporation of N-propanoylneuraminic acid into N-linked glycans of dipeptidyl peptidase IV.

Domain-specific N-glycosylation of the membrane glycoprotein dipeptidylpeptidase IV (CD26) influences its subcellular trafficking, biological stability, enzyme activity and protein folding.

Dipeptidyl peptidase IV (DPPIV, CD26) is an N-glycosylated type II plasma membrane protein. The primary structure of rat wild-type DPPIV contains eight potential N-glycosylation sites. To investigate the role of N-glycosylation in the function of DPPIV, three of its asparagine residues were separately converted to glutamine by site-directed mutagenesis. The resulting N-glycosylation mutants of rat DPPIV were studied in stable transfected Chinese hamster ovary cells. All three N-glycosylation mutants of DPPIV showed a reduced half-life, as well as differing degrees of inhibition of the processing of their N-glycans. Mutation of the first (Asn83-->Gln) or eighth (Asn686-->Gln) N-glycosylation site had only a small effect on its enzymatic activity, cell-surface expression and dimer formation, whereas the mutation of the sixth N-glycosylation site (Asn319-->Gln) abolished the enzymatic activity, eliminated cell-surface expression and prevented the dimerization of the DPPIV protein. The mutant [Gln319]DPPIV is retained in the cytoplasm and its degradation was drastically increased. Our data suggest that the N-glycosylation at Asn319 is involved in protein trafficking and correct protein folding.

Clonning and sequence analysis of the 26 kDa glutathiones-transferase gene of Schistosoma mekongi.

The number of genomic DNA or cDNA sequences of Schistosoma mekongi accessible in Genbank or EMBL is very limited up to now. Recently, two reports have appeared on the molecular phylogeny of Schistosoma species inferred from partial sequence data of rRNA genes; no further sequence data of S. mekongi is available yet. Knowledge of the molecular structure of protein coding genes of S. mekongi will provide a better understanding of gene function in the genus Schistosoma. A cDNA library of S. mekongi adult male was constructed and a cDNA encoding the 26 kDa glutathione S-transferase protein of this species was cloned. Sequence analysis of this cDNA confirmed the close phylogenetic relationship of S. mekongi to S. japonicum.