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.

Incorporation of radiolabelled amino acids by adult Schistosoma japonicum: further characterization of a putative eggshell precursor protein.

The synthesis patterns of female-specific proteins of Schistosoma japonicum were further investigated with particular reference to the 34 kDa putative eggshell precursor protein. Adult male and female worms of S. japonicum were metabolically labelled with 14C-tyrosine, 14C-glycine and 35S-methionine in vitro. The rates of amino acid incorporation for female worms were significantly higher than for males in all radiolabelling experiments. Labelled proteins were resolved by two-dimensional gel electrophoresis and visualized by fluorography. By using 14C-tyrosine and 14C-glycine, the 34 kDa female protein band resolved into three major spots with pI 6.0, 5.8 and 5.6. On the other hand, labelling studies using 35S-methionine failed to reveal synthesis of any corresponding spots at Mr 34 kDa. These results, together with the observations that eggshell hydrolysates are very rich in glycine but poor in methionine, suggested that the 34 kDa putative eggshell precursor protein of S. japonicum consists of at least three isoelectric forms. In addition, we have demonstrated several other female-specific polypeptides synthesized by this worm.

A novel cDNA clone of Schistosoma japonicum encoding the 34,000 Dalton eggshell precursor protein.

A cDNA clone encoding the 34 kDa eggshell protein of Schistosoma japonicum was isolated from an adult female cDNA library with a rabbit antiserum raised against the 34 kDa female worm fraction. A 230 bp-insert of this clone (Sj23A) was introduced in frame into the expression plasmid vector, pMAL-c2, and the recombinant fusion protein of the Sj23A transiation product was induced in Escherichia coli. The antiserum raised against the recombinant protein reacted only with the native 34 kDa protein of mature female worms, which localized in the vitelline cells of the vitelline glands. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was found that the gene corresponding to the Sj23A was expressed exclusively in mature female worms. The clone Sj23A showed a high degree of homology to the genes for the eggshell precursor proteins of Fasciola hepatica. At the deduced polypeptide level, the Sj23A also had similarities with the F. hepatica-protein sequence, the amino acid composition [high glycine (16%), lysine (12%) and tyrosine (11%)] and the presence of tyrosine residues flanked by glycine. The clone Sj23A also shared an extensive sequence homology with 3 S. mansoni expression sequence tags (ESTs). The present results suggest that the protein encoded by the female-specific Sj23A gene of S. japonicum is widely conserved in trematodes and plays a significant role as a precursor involved in eggshell formation.

Chromosomal differentiation of the Schistosoma japonicum complex.

The C-banding pattern, location of telomere sequence and chiasma frequency of four species of the Schistosoma japonicum complex were compared with those of two African species, Schistosoma mansoni and Schistosoma haematobium. In the six species, C-banding patterns of seven autosomes and the two sex chromosomes (Z and W) showed relatively species-specific and geographical (Asian and African) differences. Particularly, a plausible pathway of alteration of chromosome 2 revealed a direction from the A-chromosome to the M- chromosome in terms of rearrangements of pericentric inversion and elimination of constitutive heterochromatin (AM inversion). This chromosome change suggested hypothetically that the S. japonicum complex is the original type, and the African species represents the derived type. Moreover, the mosaic construct of the Asian and African types in Schistosoma sinensium chromosomes prompted us to propose that the species might have been formed by hybrid speciation of the genomes of Asian and African species. Localisation of telomeric repeats enabled Asian and African schistosomes to be distinguished clearly by simple terminal location and by terminal and interstitial locations, respectively. Change of chiasma frequency in the S. japonicum complex might be caused by the reduction of interstitial chiasmate (Xi) in the larger chromosomes, 1 and Z (or W), and the change seems to have progressed to Japan from South East Asia. These data enabled us to predict a tentative evolutionary pathway of schistosomes at the cytogenetic level.

Molluscicidal saponins from Anagallis arvensis against schistosome intermediate hosts.

The molluscicidal activity of saponins isolated from the plant Anagallis arvensis (Primulaceae) was studied against schistosome intermediate hosts, Biomphalaria glabrata and Oncomelania quadrasi. @Strong molluscicidal activity was found in two compounds called desglucoanagalloside B and anagalloside B. Their structures were identified on the basis of chemical and spectroscopic analyses and their activities are comparable to that of the synthetic molluscicide, niclosamide.

Nutritional requirements of Schistosoma japonicum eggs.

Newly laid eggs of Schistosoma japonicum were cultured in a serum-free, chemically defined medium, RPMI 1640, which contained 20 amino acids, glutathione, 11 vitamins, and glucose in a balanced salt solution. The requirements for these components in the nutrition of the eggs was investigated by the deletion of single component from the medium. The following 14 amino acids were shown to be essential for the full development of the egg in the medium: L-arginine, L-cystine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. Choline chloride was the essential vitamin. The omission of nicotinamide from the medium affected maturation adversely. Glucose was also required by the eggs. Minimal concentration of glucose for maturation of the eggs was 0.02 mM, but concentrations ranging from 0.16 to 20.00 mM gave better results while the concentration of the other elements of the medium were kept constant.

C282Y and H63D mutations in the HFE gene have no effect on iron overload disorders in Japan.

OBJECTIVE: The gene responsible for hereditary hemochromatosis close to the human leukocyte antigen A locus was previously identified and designated as HFE. This study was performed to evaluate the clinical significance of two mutations, C282Y and H63D of HFE, in Japanese patients with hepatic iron overload. PATIENTS AND METHODS: We examined C282Y and H63D in 11 patients with primary hemochromatosis, 94 patients with chronic hepatitis C, 54 patients with miscellaneous liver diseases, and 151 healthy volunteers. The HFE gene region of DNA samples extracted from peripheral leukocytes was amplified by polymerase chain reaction. Restriction enzyme analysis was performed using SnaBI for C282Y and BclI for H63D. Direct sequence analysis was then performed when products suggested the presence of a mutation. RESULTS: All the subjects studied were free from C282Y. None of the patients with hemochromatosis had H63D. One patient with chronic hepatitis C was homozygous, and 4 patients were heterozygous for H63D. Two patients with alcoholic liver disease were heterozygous for H63D. The prevalence of chromosomes with H63D was 6/188 (3.2%) in patients with chronic hepatitis C, 2/108 (1.9%) in patients with miscellaneous liver diseases, and 8/302 (2.6%) in healthy volunteers. These differences were not significant. CONCLUSION: Our results suggested that neither C282Y nor H63D in HFE affect Japanese patients with hemochromatosis or chronic hepatitis C.

Thyroid imaging in a typical case of acute suppurative thyroiditis with abscess formation due to infection from a persistent thyroglossal duct.

The clinical evaluation of thyroid imaging with 99mTc, 201Tl, and 67Ga in the uncommon, but potentially serious, disorder of acute suppurative thyroiditis (AST) with abscess formation due to infection from a persistent thyroglossal duct is reported. The 99mTc image showed functioning areas of the diseased thyroid gland and the 201Tl image demonstrated abscess formation in the thyroid gland of this patient. In addition, marked 67Ga accumulation was demonstrated in a wide area covering not only the area of the thyroid gland involved, but also associated circumferential inflammatory lesions in a patient with AST. The net thyroid uptake of 67Ga at 72 hours was calculated to be 13.8% of the injected dose.

Plummer's disease with spontaneous progression to hypothyroidism.

A case of Plummer's disease that spontaneously progressed to hypothyroidism is presented. A 49-year-old female visited our hospital because of a 3 kg decrease in body weight during the previous month and a painless nodule in the right anterior area of her neck. A diagnosis of Plummer's disease was made based on the results of thyroid function tests, thyroid scintigrams, and an ultrasonogram, but the patient's disease followed an usual clinical course. About two months later, she gradually developed manifestations of permanent hypothyroidism, and anti-thyroid autoantibodies became positive. In spite of continuous administration of levothyroxine sodium, uptake of 99mTcO4- to the nodule was unchanged or rather increased according to the consecutive thyroid scintigraphies. These results suggested that this case represented an autonomously functioning nodule with underlying silent thyroiditis and Hashimoto's disease.

Clinical application of SPECT in adrenal imaging with iodine-131 6 beta-iodomethyl-19-norcholesterol.

Forty-one patients with or without adrenocortical disorders were studied to evaluate the clinical usefulness of SPECT in adrenal imaging with I-131 Adosterol. In the SPECT images from this study, all glands with either normally functioning or hyperfunctioning adrenal cortices could be detected, while those glands with hypofunctioning adrenal cortices could not be detected. Particularly in transaxial and sagittal slices, the adrenal gland was identified posteriorly and was clearly distinguished from the gallbladder. In preliminary results using SPECT by a standard method, uptake in 68 detectable glands ranged from 1.7% to 4.9% in four glands with Cushing's syndrome, from 1.1% to 1.3% in seven glands with primary aldosteronism, and were distributed below 1.0% in the remaining glands with normally functioning adrenal cortices. These data show that it is possible to evaluate the adrenocortical functioning status simply by analyzing the SPECT images of the adrenal.

Glucose metabolism of adult Schistosoma japonicum as revealed by nuclear magnetic resonance spectroscopy with D-[13C6]glucose.

Excretory end-products of adult Schistosoma japonicum, fed D-[13C6]glucose in vitro under aerobic and anaerobic conditions, were studied using 1H- and 13C-nuclear magnetic resonance (NMR) spectroscopy. The glucose in the medium is degraded to produce lactate and alanine aerobically and succinate and acetate as well as lactate and alanine anaerobically. Succinate and acetate have not been previously recorded as excretory products resulting from the metabolism of glucose for schistosomes. The presence of [13C3] and [2,3-13C2]lactate, and [1,2,2'-13C3] and [2,2'-13C2]succinate as end-products suggests that a partial reversed tricarboxylic acid (TCA) cycle is active in adult Schistosoma japonicum under anaerobic conditions. The physiological role of this pathway in adult schistosomes remains obscure.

[Evaluation of BEKI TPS radioassay kit as a one-step immunoradiometric assay utilizes monoclonal and polyclonal antibodies specific for serum tissue polypeptide antigen].

We evaluated the usefulness of the BEKI TPS radioassay kit as a one-step immunoradiometric assay (IRMA) utilizes monoclonal and polyclonal antibodies specific for serum tissue polypeptide antigen (TPA). This IRMA was found to be highly sensitive to serum TPA; minimum detectable concentration of TPA was 20 U/L. There were no problems in the intraassay and interassay reproducibility and recovery test. However, dilution test of the patient's serum with higher TPA concentrations showed no linear relationship between TPA concentrations and diluted serum samples. The antigen measured by this IRMA was immunologically similar to TPA, and the TPS concentration was closely correlated (r = 0.835, p < 0.01) with the TPA concentration in 101 patient's serum. Four out of 77 healthy subjects (5.2%) and 24 out of 74 patients with benign diseases (32.4%) showed a serum concentration over cut-off value of 71 U/L. The serum TPS concentration was elevated in 80 of 232 patients with malignant diseases (34.5%) including 21 of 26 with hepatocellular carcinoma (80.8%) and 9 of 15 with cholangiocarcinoma (60.0%). In addition, the serum TPA level during the clinical course of patients with malignant diseases was a very useful indicator for the effect of treatment. Thus, our findings suggested that BEKI TPS IRMA kit is a useful assay system for serum TPA as a tumor marker that can be performed by a simple assay operation within about 2 hours.