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B H Kalinna

Publications and source records attributed to B H Kalinna.

14 recordsLinked to original sources
PubMed1998

Differential antigen-stimulated proliferation of human mononuclear cells by recombinant Schistosoma japonicum antigens in a Chinese population.

Peripheral blood mononuclear cells (PBMC) from 117 individuals living on two islands in an area (Dongting Lake) endemic for schistosomiasis japonica in China, and 15 control individuals from a non-endemic area of China, were assessed for antigen-stimulated proliferation against five recombinant Schistosoma japonicum antigens of recognized interest in the development of immunity to schistosomiasis. Two recombinant antigens, paramyosin and 28-kD glutathione-S-transferase, stimulated cellular proliferation (stimulation index > or = 3.0) in 38.5% and 42.5% of subjects, respectively, a level similar to that induced by a soluble whole parasite extract (51.3%). In contrast, three other recombinant antigens tested--a fatty acid binding protein, 22-kD tegumental membrane-associated antigen, and glyceraldehyde-3-phosphate dehydrogenase--stimulated PBMC proliferation in only 3-8% of subjects. Moreover, we also identified a positive association between the degree of exposure, and cellular proliferation following stimulation with recombinant paramyosin or whole parasite extract. Highly significant differences in antigen-stimulated proliferation were also observed between the two islands, Niangashan and Qingshan. The whole parasite extract stimulated proliferation in 90% of subjects from Niangashan island compared with only 42.1% of subjects from Qingshan island (chi2 = 16.88, P = 0.00004), while glutathione-S-transferase stimulated proliferation in 77.3% of subjects from Niangashan island compared with only 34.7% of subjects from Qingshan island (chi2 = 13.09, P = 0.003). A similar, but not significant, trend was observed for paramyosin and the fatty-acid binding protein. The identification of differential cellular proliferative responses to specific schistosome antigens within an infected human population may have important practical implications for vaccine development against schistosomiasis japonica.

Adult↗
PubMed1997

Engineering and expression of a full length cDNA encoding Schistosoma japonicum paramyosin. Purification of the recombinant protein and its recognition by infected patient sera.

A cDNA encoding the complete open reading frame of the Schistosoma japonicum paramyosin has been constructed and cloned. The 2600 bp cDNA was engineered by PCR using a N-terminally truncated paramyosin clone (pmy25) as template and a 57-mer primer that introduced the eight missing amino acids and matched the 5' sequence of pmy25 in conjunction with a pmy specific reverse primer. After cloning and expression, the recombinant protein was purified by affinity chromatography under non-denaturing conditions and was shown to have a molecular mass of 99 kDa which is equivalent to the expected size of the full length recombinant fusion protein, comprising the 97 kDa paramyosin plus an additional 2 kDa for the N-terminal fusion peptide incorporating the six histidine residues required for purification. In Western blot assays it reacted specifically with anti-paramyosin antibodies in sera from vaccinated animals and patients with Asian schistosomiasis. The engineering of the full-length cDNA encoding Schistosoma japonicum paramyosin, its bacterial expression and purification will facilitate future studies aimed at determining its efficacy as an anti-schistosomiasis vaccine.

Animals↗
PubMed1997

A vaccine against the Asian schistosome, Schistosoma japonicum: an update on paramyosin as a target of protective immunity.

Paramyosin from parasitic worms of the genus Schistosoma has shown promise as a vaccine target and it is one of the candidates selected by WHO for the development of a vaccine against schistosomiasis. Here we discuss the literature of the past decade and report on different recombinant paramyosin constructs we are using in our laboratory to develop a vaccine against the Asian schistosoma, Schistosoma japonicum.

Amino Acid Sequence↗
PubMed1997

Cloning and expression of a cDNA encoding a nonintegrin laminin-binding protein from Echinococcus granulosus with localization of the laminin-binding domain.

We have isolated a cDNA from the hydatid tapeworm, Echinococcus granulosus, encoding a protein that binds laminin. This is the first report of a helminth parasite laminin-binding protein and the first description of a cDNA encoding a laminin-binding protein from a parasite. The cDNA clone (egmo3) was isolated from an E. granulosus protoscolex cDNA expression library, and identified on the basis of sequence homology to the nonintegrin mammalian metastasis-associated 67-kDa laminin receptor (67-LR). The amino acid sequence predicted from the cDNA sequence is 268 residues long with a calculated molecular mass of 29.9 kDa. Southern blot analysis suggested that many copies of the gene may occur in the E. granulosus genome. A Northern blot revealed that the gene is expressed as a single transcript of approximately 1 kb consistent with the size of the cDNA insert. Antibodies raised to the purified protein interacted with a 30 kDa protein in whole E. granulosus protoscoleces. A Western blot of the purified and refolded recombinant protein specifically bound 125I-labelled laminin, as did a synthetic peptide derived from the inferred amino acid sequence of egmo3 which is similar in homology to peptide G, the active ligand-binding site of 67-LR. We also isolated the 3' end of the cDNA encoding the homologous protein from the closely related species, E. multilocularis. The polypeptide encoded by egmo3 also shares substantial identity with the acidic class of ribosomal proteins which are involved in protein synthesis. As such, the egmo3 protein may be multifunctional in E. granulosus, acting as a laminin-binding molecule but also playing a role in cell division and growth.

Amino Acid Sequence↗
PubMed1997

Proteolytic degradation of host hemoglobin by schistosomes.

Schistosomes acquire amino acids for growth, development, and reproduction by catabolizing hemoglobin obtained from ingested host erythrocytes. While the biochemical pathway(s) involved has not been determined definitively, a number of proteases including schistosome legumain and cathepsin L-, D-, B- and C-like enzymes have been ascribed roles in the degradation of hemoglobin to diffusible peptides. Transcripts encoding these schistosome proteases, which appear to be expressed in the gastrodermis and cecum of the schistosome, have been reported. Because these enzymes are candidate targets at which to direct novel anti-schistosomal therapies, the comparative biochemistry of these and their counterpart mammalian proteases is now the focus of research in a number of laboratories. This paper reviews reports dating from 40 years ago to the present on how schistosomes digest host-derived hemoglobin, and interprets apparent anomalies in some earlier compared to later reports, the latter having benefited from the availability of PCR and gene cloning technologies. More specifically, the review concentrates on five proteolytic enzymes, and their associated genes, which have been ascribed key roles in the pathway of hemoglobin degradation.

Animals↗
PubMed1997

DNA-based vaccination using Schistosoma japonicum (Asian blood-fluke) genes.

We have examined the efficacy of nucleic acid vaccination in inducing immunity to the multicellular parasite, Schistosoma japonicum, a trematode worm responsible for causing schistosomiasis in humans and other mammalian species. A panel of Schistosoma japonicum cDNAs were cloned into eukaryotic expression vectors, injected into animals, and tested for immunogenicity. The cDNAs tested encoded 26- and 28-kDa glutathione-S-transferases, calreticulin, glyceraldehyde-3-phosphate dehydrogenase, a 22.6 kDa membrane-associated antigen, a 14 kDa fatty-acid binding protein, fragments of paramyosin, full-length paramyosin, and a novel gene comprising the 26 kDa glutathione-S-transferase fused to a fragment of paramyosin cDNA. The paramyosin gene constructs, including the fusion, were all able to induce anti-paramyosin antibodies; with the fragments of paramyosin these were of the IgG1, IgG2a and IgG2b isotypes. In contrast, none of the other schistosome cDNAs tested were able to induce detectable antibody responses. The anti-paramyosin antibodies did not protect mice challenged with cercariae of S. japonicum.

Animals↗
PubMed1997

DNA vaccines for parasitic infections.

There has been a tremendous explosion in the area of DNA vaccine research over the last 4 years, particularly in relation to antiviral vaccines. This report discusses the development and application of this new technology with regard to parasitic infections. Progress has been made towards the development of a vaccine against malaria, cryptosporidiosis, leishmaniasis, toxoplasmosis and schistosomiasis. In the future, nucleic acid vaccines will be a useful tool to help control these and other parasitic infections.

Animals↗
PubMed1996

Cloning and characterization of a ribosomal P protein from Taenia solium, the aetiological agent of human cysticercosis.

A cDNA encoding the complete open reading frame of the Taenia solium acidic ribosomal phosphoprotein P2 has been cloned. The 417 bp cDNA (arpP2) was isolated from a T. solium cDNA expression library by PCR using P protein specific pimers. The ORF encodes a protein of 121 amino acids exhibiting 40-55% identity to mammalian, fungal, insect, and parasite P2 proteins. The inferred molecular mass of the protein is 12,592 Daltons, similar to that reported for other ribosomal P2 proteins. After subcloning and expression, the recombinant protein was purified by affinity chromatography under non-denaturing conditions and was shown to have a molecular mass of 15 kDa which is equivalent to the expected size of the full length recombinant fusion protein, comprising the 12.5 kDa arpP2 plus an additional 2 kDa for the N-terminal fusion peptide incorporating the six histidine residues required for purification.

Amino Acid Sequence↗
PubMed1995

Cloning and characterization of the Schistosoma japonicum aspartic proteinase involved in hemoglobin degradation.

A cDNA encoding a Schistosoma japonicum aspartic proteinase was cloned, sequenced, and found to encode a zymogen of 380 amino acid residues, and its gene was shown to be present as a single copy in the S. japonicum genome. Identity comparisons showed that the enzyme (Sjpasp) was most closely related to the cathepsin Ds. The deduced amino acid sequence has four potential glycosylation sites, two of which are in identical positions to the two glycosylation sites of human kidney lysosomal cathepsin D. Furthermore, all four disulfide bonds found in mammalian cathepsin D sequences are present in Sjpasp, although the beta-hairpin (loop 3), which is cleaved during maturation of vertebrate cathepsin Ds to yield light and heavy chain subunits, is absent from Sjpasp. While most residues involved in substrate specificity and catalysis of aspartic proteinases are preserved in Sjpasp, several residues in these regions exhibit changes that may result in a novel substrate specificity. Aspartic proteinase activity is present in extracts of adult S. japonicum and Schistosoma mansoni and in culture media in which schistosomes were maintained and was capable of digesting hemoglobin. The schistosome aspartic proteinase may play a pivotal role in the catabolism of hemoglobin obtained from host erythrocytes.

Amino Acid Sequence↗
PubMed1995

Schistosoma japonicum: heterogeneity in paramyosin genes.

Paramyosin is an integral muscle protein found in many invertebrates including schistosomes, and is considered an important candidate vaccine antigen in schistosomiasis. The characterisation of natural molecular variation in vaccine antigens including paramyosin is important because strain-specific vaccination may be necessary against schistosomiasis japonica. We have isolated partial cDNAs encoding paramyosin from an adult, Chinese strain Schistosoma japonicum gene library. Two of these cDNAs (B6 and Y6) encode the same region of paramyosin but their nucleotide sequences differ at eight positions and their deduced amino acid sequences differ by an arginine/cysteine substitution, demonstrating intrastrain variation in paramyosin. Southern blot analysis of genomic DNA from the Chinese and Philippine strains of S. japonicum demonstrated strain-related RFLPs at the paramyosin locus, and suggested that more than one copy of the paramyosin gene was present in the S. japonicum genome. PCR-based RFLP analysis which exploited restriction site differences between B6 and Y6 showed that paramyosin genotype B6 was much more common in schistosome populations and verified the existence of introns in the paramyosin gene(s) of S. japonicum.

Amino Acid Sequence↗
PubMed1995

Gene cloning and complete nucleotide sequence of philippine Schistosoma japonicum paramyosin.

The development of an effective vaccine is recognised as a necessary adjunct to the control of schistosomiasis japonica, a disease affecting several million people in China and the Philippines. Currently, recombinant Schistosoma japonicum molecules are considered most suitable for large scale vaccine production and a number of genes encoding vaccine candidate polypeptides have been cloned and expressed (see Waine et al., 1993a). One of the molecules providing most promise as a vaccine target is paramyosin (Butterworth, 1992), a major structural protein of thick filaments in the muscle of most invertebrates; paramyosin genes have now been cloned from a range of parasitic helminths, including schistosomes (Limberger and McReynolds, 1990; Laclette et al., 1991; Dahmen et al., 1993; Landa et al., 1993; Mühlschlegel et al., 1993, Nara et al., 1994). The cloning and nucleotide sequence of S. Japonicum paramyosin is described.

Animals↗
PubMed1994

Gene cloning, overproduction and purification of a functionally active cytoplasmic fatty acid-binding protein (Sj-FABPC) from the human blood fluke Schistosoma japonicum.

We report the gene cloning, molecular characterisation and purification of a 14.7-kDa functionally active recombinant (re) cytoplasmic fatty acid-binding protein (Sj-FABPC) from the Chinese strain of the human bloodfluke Schistosoma japonicum (Sj). As schistosomes are unable to synthesise long chain fatty acids and sterols de novo and must, therefore, take up these lipids from the host, Sj-FABPC is an attractive vaccine and/or drug target. Clone 39 (C39), which contains the entire Sj-FABPC gene, was isolated from a Sj lambda ZAPII cDNA expression library immunoscreened with hyperimmune rabbit serum (HRS) raised against soluble adult Sj proteins. The complete ORF (open reading frame) of Sj-FABPC encodes a protein of 132 amino acids (aa) of 14.7 kDa. The aa sequence of Sj-FABPC exhibits 91% identity to a FABP of S. mansoni (Sm14) and 45% identity to a FABP of Fasciola hepatica (Fh15), putative vaccine candidates for schistosomiasis. Sj-FABPC was subcloned into the QIAexpress vector, pQE-10, and subsequently expressed in Escherichia coli. The re-Sj-FABPC, purified under non-denaturing conditions, was recognized by sera from patients with acute and chronic schistosomiasis japonica. The purified re-Sj-FABPC was also shown to bind to palmitic acid with high affinity. The functional expression of Sj-FABPC will facilitate studies on re-Sj-FABPC to assess its potential as a drug and/or vaccine candidate.

Amino Acid Sequence↗
PubMed1994

Purification of a recombinant Schistosoma japonicum antigen homologous to the 22-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate against schistosomiasis.

We describe the cDNA cloning, overproduction and purification of a 22.6-kDa antigen from the human blood fluke Schistosoma japonicum. A 777-bp cDNA (C32) was isolated from a S. japonicum lambda ZAPII cDNA expression library immuno-screened with hyperimmune rabbit serum (HRS) raised against soluble adult S. japonicum proteins. The open reading frame of C32 encodes a protein of 191 amino acids (aa) which exhibits 71% identity to a 22.6-kDa membrane-associated antigen of S. mansoni, a putative vaccine candidate for schistosomiasis. We have identified a sequence motif known as an EF-hand calcium-binding domain in both the S. japonicum and S. mansoni aa sequences, suggesting that the 22.6-kDa antigens are able to bind Ca2+. Further, we have, for the first time, obtained the 22.6-kDa antigen in purified, non-denatured, recombinant form, and in sufficient quantity to assess the protective value of the molecule in vaccination/challenge experiments. This was achieved by synthesizing the schistosome antigen with a short polyhistidine tag fused to the N-terminus which was then used for subsequent affinity purification. The recombinant protein was purified under non-denaturing conditions using nickel-chelate affinity chromatography.

Amino Acid Sequence↗