Identification of a putative eggshell precursor protein of the female Schistosoma japonicum

In adult worms of Schistosoma japonicum, a prominent radiolabelled female-specific protein (34 kDa) was demonstrated on fluorography of SDS gels with the pulse incorporation of 14C-tyrosine in vitro, though it was difficult to detect major female-specific proteins by direct staining methods. This female-specific protein was demonstrated to localize exclusively in the vitelline cells by indirect immunofluorescence using the rabbit anti-34 kDa female protein antiserum. It was shown that 14C-tyrosine was selectively incorporated into the vitelline cells by the pulse labelled autoradiographs. Two days after the exposure of worms to radio-tyrosine, the shells of eggs in the uterus were demonstrated to have become radioactive, indicating that 14C-tyrosine-labelled protein was used as a material for the eggshell. In the fluorograph of proteins extracted from newly laid eggs in vitro, the prominent band was not found at the 34 kDa region, but a lot of radioactivity appeared at higher than 100 kDa. The results suggested that a 34 kDa female protein was a precursor of the eggshell and became a much larger protein molecule as a result of cross-linking during eggshell hardening.     

Schistosoma mansoni and Schistosoma japonicum: Utilization of amino acids

, , and 1972. Schistosoma mansoni and Schistosoma japonicum: utilization of amino acids. International Journal for Parasitology 2: 425–430. The production of ¹⁴CO₂ from 12 labeled amino acids by S. mansoni and S. japonicum was studied. No ¹⁴CO₂ was detected from incubations with glycine, isoleucine, leucine, lysine or phenylalanine. Differences were found between sexes and/or species for the other amino acids studied. Species related differences included a greater rate of metabolism of glutamic and aspartic acid by S. mansoni than by S. japonicum. Proline and histidine were utilized by S. mansoni males and females, respectively. S. japonicum male worms did not utilize proline, while histidine was not utilized by the female of this species. Major sex related differences included greater ¹⁴CO₂ production from glutamic acid, aspartic acid and arginine by S. mansoni males than by females, and the utilization of histidine by male S. japonicum but not by females. Incubation in tyrosine resulted in the release of only small amounts of ¹⁴CO₂ by female worms of both species but no ¹⁴CO₂ production by male worms.     

Membrane transport in Schistosoma mansoni: transport of amino acids by adult males.

The mechanisms by which adult male Schistosoma mansoni transport amino acids have been investigated using radioactive amino acids during 2-min incubation times. The transport constants (K_t) for mediated uptake of glycine, proline, methionine, arginine, glutamate, and tryptophan were calculated to be 0.60-1.05, 1.67-1.98, 2.0, 0.10-0.35, 0.30-0.50, and 0.5-1.0 mM, respectively. Maximal velocities (V_max) were 5.5–7.5, 25, 6.4, 1.5-2.0, 2.5, and 3.0–6.0 μmoles absorbed/g worm protein/2 min, respectively. Cysteine is taken up solely by diffusion. Proline uptake is unique in that no significant diffusion component was found. The other amino acids studied were absorbed by diffusion as well as by specific transport systems. In the 2-min incubation periods employed glycine, proline, glutamate, and methionine were not significantly metabolized indicating that the uptake studies using these substrates reflect transport. Metabolism of the other amino acids used in these studies was not examined. The specificity of the transport systems was studied by testing the inhibitory effects of various amino acids on the uptake of each of the amino acids studied. The results suggest the presence of at least five transport systems. There is a highly specific transport locus for proline, and one for acidic amino acids. There are probably at least two transport systems, each of broad and overlapping specificity, for most of the neutral amino acids. Basic amino acids also appear to be taken up by complex transport systems, at least one of which overlaps with the neutral sites. The results are discussed with respect to the nutrition of the parasite and the host-parasite relationship.     

Uptake and excretion of amino acids and utilization of glucose by Schistosoma japonicum eggs

The utilization of amino acids and glucose in the external nutrients and the excretion of nitrogenous compounds by Schistosoma japonicum eggs were investigated with the eggs cultured in a chemically defined medium (MEMSE-J). Of the 15 amino acids in MEMSE-J, arginine and glutamine markedly decreased in concentration during cultivation of S. japonicum eggs. The nitrogenous excretory products of developing eggs were demonstrated to be at least four amino acids (alanine, proline, glutamic acid and ornithine), urea and ammonia. Glucose was consumed at an estimated rate of 32 ng/living egg/day during the period of egg growth and differentiation. When 14C-labelled glucose was included in the culture medium, the radioactivity was incorporated into three amino acids (alanine, proline and glutamic acid), which were excreted by S. Japonicum eggs. The results were discussed with reference to the possible role in stimulating fibrosis in the granuloma of schistosomiasis.     

Incorporation of amino acids into protein by cell-free extracts of penicillium chrysogenum

Summary The preparation of ribosomal particles from P. chrysogenum has been described. After correction to 20°C and extrapolation to infinite dilution, they had sedimentation coefficients of approximately 80S, 60 S, and 40 S. The washed ribosomes plus purified supernatant fraction, KCl and sucrose incorporated C¹⁴-l-amino acids into protein. There was no stimulation of incorporation by ATP, GTP, CTP, UTP, or Mg ions. Incorporation was inhibited by streptomycin and chloramphenicol but not by ribonuclease.The amino acid incorporating system from P. chrysogenum did not resemble bacterial and yeast systems in many respects and reasons for this are discussed.     

Schistosoma japonicum: immunological characterization and detection of circulating polysaccharide antigens from adult worms

The antigenic constituents of a trichloroacetic acid (TCA)-soluble fraction of adult Schistosoma japonicum were studied with immunoelectrophoresis, and compared with those of Schistosoma mansoni. Eight TCA-soluble antigens of S. japonicum were demonstrated, five of which showed immunological identity with S. mansoni antigens. Of the eight antigens, five antigens with anodic motility were found as circulating antigens in S. japonicum-infected hamster and rabbit sera; the major circulating antigen was the circulating anodic antigen (CAA). Two other antigens, with cathodic motility, including the circulating cathodic antigen (CCA), were demonstrable as circulating antigens in S. mansoni infections, but not in S. japonicum infections. Most of the circulating antigens were shown to be gut-associated. Only one antigen, line 2, which was not demonstrable as circulating antigen and which was present in the parenchyma of the worms, was found to be specific for S. japonicum. Using an ELISA for the detection of CAA in the sera of S. japonicum-infected rabbits, a lower detection level of 100 ng CAA/ml serum was achieved. Moreover, at 7-8 weeks after infection, a direct relationship between worm burden and CAA level was demonstrated.     

Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.

As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.     

Incorporation of amino acids into protein of foetal rabbit diaphragm: effect of insulin.

The incorporation of six L-amino acids into the protein fraction of the diaphragm of foetal and neonatal rabbits has been measured. Insulin stimulates the incorporation of lysine from the 29th day and of glycine from the 31st day of gestation onwards. The incorporation of histidine, serine and threonine is only enhanced in the diaphragm of 16-20 hour-old rabbits; no effect of insulin on the incorporation of leucine has been shown for the period under study.     

Schistosoma mansoni: incorporation and metabolism of protein amino acids in vitro

1. The amino acid composition of total proteins in six stages of the life cycle of Schistosoma mansoni was determined by routine autoanalysis of acid hydrolysates. Aspartate, glutamate and glycine were consistently the most abundant protein amino acids in all stages. 2. Incorporation of each of the protein amino acids into adult and egg proteins was determined using 72 hr cultures in complex media. Incorporation rates varied widely and there was no correlation between abundance in protein and the rate of incorporation. 3. Only five amino acids were interconverted to other amino acids which were themselves incorporated into worm and egg proteins (ala, arg, asp, gly, ser); of these only two (glu from ala and pro from arg) appeared to be of quantitative significance. Exogenous glucose yielded only three protein amino acids (ala, asp, glu). 4. The data are considered in the light of differences in egg and adult protein synthesis and with particular regard to potential chemotherapy at this level.     

Incorporation of ammonium in amino acids by Trypanosoma cruzi

Ammonium ions were incorporated into L-glutamate and alpha-ketoglutarate in epimastigote forms of Trypanosoma cruzi through the following enzymatic systems: NADPH and NADH-dependent glutamate dehydrogenase, NADPH-dependent glutamate synthase, L-glutamine synthetase and NADH-dependent glutamate synthase in order of decreasing specific activity (mumoles of product formed/min/mg protein). The pH optima and Km's for the glutamate dehydrogenase system were determined. Disc electrophoresis showed the presence of cathodic bands of GDH activity, which were highly dependent on NADP+.     

Schistosoma japonicum: a method for transformation by electroporation

Despite of our knowledge of genetic make up of schistosomes, a number of genes have not been characterized largely due to lack of effective transformation protocols. Here we present electroporation as a strategy for effective introduction of plasmids DNA into schistosomula and adults. Using plasmids of pEGFP-C1 as an expression vector, we first verified that the CMV promoter could direct EGFP to express in primary culture cells from Schistosoma japonicum. Subsequently, the plasmids were introduced into schistosomula and adults by electroporation and EGFP expression was demonstrated using molecular and microscopical methods. Our findings indicate that electroporation is an effective method for transformation of S. japonicum.     

Schistosoma haematobium: analysis of eggshell protein genes and their expression

Eggshell protein genes of Schistosoma mansoni that encode a 14 kDa protein have been shown to be highly conserved and expressed in a sex-, tissue-, and temporal-specific manner. To initiate studies on the eggshell protein genes of S. haematobium, a cDNA probe, pSMf 61-46, representing a S. mansoni eggshell protein mRNA was used to screen a S. haematobium genomic library. Of the seven independent recombinant clones isolated, two (lambda SH 2-1 and lambda SH 6-1) were analyzed and compared to those of S. mansoni. lambda SH 2-1 and lambda SH 6-1 each contain a different genomic copy of the gene encoding a 19.8 and 17.6 kDa protein, respectively. This is due to an additional 78 bp present in the coding region of lambda SH 2-1 relative to lambda SH 6-1. The rest of the coding sequences are identical, and the 5' and 3' untranslated regions are nearly identical. The deduced amino acid sequences of S. haematobium eggshell proteins are very rich in glycine (47 and 50%) when compared to 43.5% glycine in the protein encoded by S. mansoni. Long stretches of glycines, as many as 15 in a row, occur in the S. haematobium sequence. DNA comparison of the eggshell protein genes of the two schistosome species yielded an overall homology of 83.1%. The homology is much higher in the 5' and 3' untranslated regions than in the protein-coding regions. Genomic clones of both species contained second open reading frames, which appeared to be kept open as a consequence of the amino acid composition of the other. There are no introns in S. haematobium or S. mansoni eggshell protein genes, and the genomic Southern data indicated a similar arrangement of these genes in the genome of both species. Primer extension experiments and dideoxynucleotide sequencing of the RNA determined the mRNA cap site sequence as ATCAT and ATCAC in lambda SH 2-1 and lambda SH 6-1, respectively. Northern blot analysis determined the size of the mRNA to be about 1.0 kp. Expression of the RNA from these genes appears to be regulated in a manner similar to the corresponding genes in S. mansoni. mRNA is found only in mature females and first appears at 70 days after infection of hamsters. DNA sequence comparisons of the 5' flanking regions of S. haematobium and S. mansoni eggshell protein genes to each other and to those of silkmoth and Drosophila revealed several short sequence elements that are shared.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protein synthesis in tomato-fruit locule tissue: Incorporation of amino acids into protein by aseptic cell-free systems

1. Osmotically disrupted protoplasts and isolated plastids from tomato-fruit locule tissue were found capable of incorporating (14)C-labelled amino acids under aseptic conditions into an exhaustively washed trichloroacetic acid-insoluble protein fraction. 2. The disrupted protoplast system incorporated 20-45mumumoles of amino acid/mg. of protein in 10min. The isolated plastid system incorporated 10-20mumumoles of amino acid/mg. of protein; 40-150mumug. of carbon/mg. of protein was incorporated in 10min. from (14)C-labelled amino acid mixture. 3. Incorporation is stimulated by added ATP in the dark, but no added ATP is required when the system is illuminated. The cell-free plastid system is to some extent self-sufficient and does not normally require an added supernatant fraction or unlabelled amino acids. 4. Amino acid incorporation by plastids is inhibited by chloramphenicol, puromycin, actinomycin D, ribonuclease and deoxyribonuclease. It is suggested that the mechanism of protein synthesis in the cell-free plastids, and in the tissue generally, is basically the same as established for bacteria. Ribosomes and highspeed supernatant from this tissue were to some extent interchangeable with Escherichia coli ribosomes and supernatant in cell-free incubations. 5. Incorporation of amino acids by isolated plastids was stimulated by indol-3-ylacetic acid and kinetin, and, whereas incorporation normally proceeds for only 10-20min., the time-course was extended in the presence of these growth substances. It is suggested that hormones may be involved in the regulation of protein synthesis in plants.     

Comparative genomics of human-like Schistosoma japonicum genes indicates a putative mechanism for host-parasite relationship

Schistosoma japonicum causes schistosomiasis in humans and livestock in the Asia-Pacific region. We assembled more than 43,700 S. japonicum expressed sequence tags and conducted comparative genomic analyses between S. japonicum and its human host. Some schistosome genes showed exceptionally high similarity in nucleotide sequence to their human homologues, of which five exhibited anomalous phylogeny and human codon usage bias. The most plausible explanation for their presence is horizontal gene transfer from host to parasite. Functional evidence suggests that S. japonicum might exploit host endocrine and immune signals for cell development and maturation via these host-like genes.