The distribution and properties of branched chain amino acid aminotransferase(EC 2.6.1.42) was investigated in adult Fasciola hepatica. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of branched chain amino acid aminotransferase was measured by the method of Ichihara and Koyama (1966) . Isozyme patterns of this enzyme was also examined by DEAE-cellulose column chromatography. The results obtained were as follows: The activity in homogenate was found to be 12.69 units/g wet tissue. The activity of this enzyme was relatively high compared with those in rat tissues. The distribution of branched chain amino acid aminotransferase in the subcellular organelles showed that 87.8 percent of the activity was in cytosolic, 10.9 percent in mitochondrial and 1.3 percent was in nuclear fraction. Cytosolic fraction of Fasciola hepatica contained Enzyme I, but not Enzyme II and III, of branched chain amino acid aminotransferase. Enzyme I was eluted by 50 mM phosphate buffer from DEAE-cellulose column and catalyzed the transamination of all three branched chain amino acids. The Enzyme I was purified about 22-folds increase in specific activity after chromatography on DEAE-cellulose. The best substrate among three amino acids (leucine, isoleucine and valine) was L-isoleucine. The optimal temperature of Enzyme I was 45 C and the optimal pH was 8.2. The Km value for leucine of Enzyme I was 4.17 mM. The Km values for alpha-ketoglutarate and pyridoxal phosphate of Enzyme I were 0.41 mM and 4.76 x 10(-3) mM, respectively.
parasitology-helminth-trematoda ; Fasciola hepatica ; biochemistry ; enzyme ; aminotransferase

Fascioliasis has not been confirmed as a human disease entity until now in Korea despite of sporadic discovery of ova of Fasciola sp. in human fecal materials being never traced to the confirmation of infection. Almost all of the cases with ova in their stool have been related with consumption of cattle liver whether eaten in raw or processed. The present authors confirmed a human fascioliasis case who was a Korean housewife of 42-year-old living in Seoul, during the exploratory laparotomy. The patient had been healthy until October 1975 when abrupt onset of urticaria, dyspepsia, epigastric discomfort developed. And the fluctuation of these symptoms was followed by epigastric colicky pain attacks from December 4, 1975. A complete worm of Fasciola sp. was removed during the bile-duct exploration with stone forceps in lower half of common bile duct, on January 20, 1976. The patient only agreed that she had eaten some raw liver of cattle on September 1975 but denied any possible sources of infection such as drinking of untreated water, handling of water flower and grass, and eating of raw watercress. The measurements of the removed worm: 35. 61 mm in body length, 14.00 mm in maximum body width(Length/width ratio, 2.54:1), distribution of testes to body length 33.9 per cent , number of branches of ovary 22, the size of intrauterine ova 157.2 x 108. 4 micrometer in average. These findings are not compatible with the classical descriptions of both Fasciola hepatica and F. gigantica, and it was concluded it is so-called Fasciola sp. which is intermediate between two species as proposed by many Japanese workers.
parasitology-helminth-trematoda ; Fasciola hepatica ; Fasciola gigantica ; Fasciola sp. ; case report ; fascioliasis ; laparotomy ; bile-duct

In attempt to investigate histone fractions and non-histones of parasites, nuclei were isolated from Fasciola hepatica by the procedure of Pogo et al. (1966). Histone fractions H1, H2a, H2b, H3 and H4 were prepared from isolated nuclei by the procedure of Johns (1964 and l967). The five histone fractions found in most tissues were also present in the Fasciola hepatica histones. These histone fractions were characterized by amino acid analysis and by polyacrylamide disc gel electrophoresis. Non-histone proteins were extracted from isolated Fasciola hepatica nuclei and separated by SDS-polyacrylamide gel electrophoresis. The results of the experiment were summarized as follows: The yield of whole histone recovered was 2.47 mg per 1 g of Fasciola hepatica. The yield of DNA was 1.02 mg per gm of tissues. Consequently the DNA to histone ratio was 1:2.44. The relative amounts of five fractions, i.e., Hl, H2a, H2b, H3 and H4 were 19.96 percent, 26.48 percent, 29.60 percent, 12.56 percent and 14.37 percent, respectively. Amino acid analysis of the individual histone fractions showed that the over-all compositions were similar but not identical to those of corresponding fraction from calf thymus. It was found that histone H2b fraction of Fasciola hepatica contained detectable amounts of epsilon-N-monomethyllysine. No evidence for the presence of methylated lysine or other side-chain derivatives was reported on this histone fraction. In SDS-polyacrylamide disc gel, it showed that 17 protein bands of nuclear acidic protein can be identified visually.
parasitology-helminth-trematoda ; Fasciola hepatica ; histone ; DNA ; biochemistry ; amino acid ; epsilon-N-monomethyllysine

The glucose uptake rate by Fasciola hepatica was a mean value of 9.62 +/- 0.54 micro-mole/hr/g, and total CO(2) production rate by the flukes averaged 24.28 +/- 4.26 micro-mole/hr/g wet wt. The relative specific activity of respiratory CO(2) showed a mean value of 79.89 +/- 1.78 per cent. The rate of CO(2) production derived from medium C(14)-glucose was a mean of 19.55 +/- 3.56 micro-mole/hr/g of we wt. Therefore, the average value of 32.72 +/- 4.8 percent of glucose utilized by the flukes from the medium C(14)-glucose was oxidized to respiratory CO(2). The tissue concentration of glycogen in F. hepatica was a mean of 38.36 +/- 2.91 mg/g or 3.84 +/- 0.29 %/g of wet wt, and the turnover rate of glycogen pool was a mean of 1.6+/-0.22 %/hr or 0.65 +/- 0.13 mg/hr/g. The average value of 37.26 +/- 3.86 per cent of glucose utilized by the fluke from the medium C(4)-glucose was incorporated to the glycogen. These data account for that approximately 70 per cent of the utilized glucose by the flukes participated in furnishing the oxidation into respiratory CO(2) and the synthetic process into glycogen.
parasitology-helminth-trematoda-Fasciola hepatica ; glucose ; biochemistry ; autoradiograhy ; glycogen ; CO(2)

The activity and distribution of aspartate aminotransferase (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2) in adult Fasciola hepatica have been studied. Fasciola hepatica was fractionated by differential centrifugation into nuclear, mitochondrial and cytosolic fractions. The activity of GOT and GPT was measured by the method of Reitman and Frankel. Isozyme patterns of those enzyme were also examined by DEAE-cellulose column chromatography. The results obtained were as follows: The activity of aspartate and alanine aminotransferase was about 0.55 unit and 0.92 unit per 1 g of Fasciola hepatica, respectively. The activity of those enzymes was relatively low compared with those in mammalian tissues. The distribution of aspartate aminotransferase in the subcellular organelles showed that 71 percent of the activity was in cytosolic, 24 percent in mitochondrial and 5 percent was in nuclear fraction. About 22 percent of the total alanine aminotransferase activity was found in the mitochondrial fraction, about 66 percent in the cytosolic fraction. Aspartate aminotransferase from cytosolic fraction was separated into two types of isozymes, whereas alanine aminotransferase from cytosolic fraction gave only one active peak on DEAE-cellulose column chromatography.
parasitology-helminth-trematoda ; Fasciola hepatica ; biochemistry ; enzyme ; aspartate aminotransferase ; alanine ; aminotransferase ; alanine aminotransferase

In order to obtain the most specific and sensitive antigen from crude antigens of Fasciola hepatica for the immunodiagnosis of bovine fascioliasis by the enzyme linked immunosorbent assay(ELISA), phosphate buffered saline extract of F. hepatica was prepared. The crude extract was fractionated into 7 antigens using Sephadex G-100 column chromatography. Seven fractionated antigens were applied to ELISA, precipitation test and intradermal test, respectively. Results obtained are as follows: The specificity (95 per cent confidence interval in negative sera of bovine fascioliasis ; Mean+2 x SD of absorbance ) of the first (MW>150,000) and the second antigens (MW 120,000) were 93.7 per cent, but those of others including crude antigen showed 100 per cen.t. The sensitivity (positive sera of bovine fascioliasis having higher values with compared to the criterion) of the first, the sixth (MW 16,000) and the seventh antigen (MW<5,000) were 91.6 per cent, 87.5 per cent and 0 per cent, respectively, but those of others showed all 100 per cent. The absorbance by ELISA using the fifth antigen (MW 26,000) was 8.43-folds higher in the positive sera than that in the negative sera. This could be used as one of the most specific antigens for the immunodiagnosis of bovine fascioliasis. In Ouchterlony test, precipitin lines were not found in the sera naturally infected with F. hepatica, but some were found in the sera of rabbits immunized with the crude antigens. The numbers of precipitin lines in the sera of rabbits were different in the different fractionated antigens. They were 6 in the crude, 2 in the second and the third antigens, 1 in the forth, the fifth and the sixth antigens and absent in the seventh antigen, respectively. The wheal size for the bovine infected with F. hepatica was 2.46+-0.15 cm in the intradermal test antigen (saline extract of F. hepatica) supplied by the Veterinary Research Institute, Rural Development Administration, Korea. The wheal size of the first, the second and the third antigens were larger than that of intradermal test antigen, whereas those of the forth, the fifth, the sixth and the seventh antigens showed smaller than that of the intradermal test antigen. The results suggest that the fifth antigen may be specific antigen for the immunodiagnosis of bovine fascioliasis.
parasitology-helminth-trematoda ; immunology ; enzyme-linke immunosorbent assay ; diagnosis ; fascioliasis ; Fasciola hepatica

In an attempt to investigate the specific antigenic substance of Fasciola hepatica, Ouchterlony tests and immunoelectrophoretic analyses were carried out. Crude Fasciola antigen was prepared and fractionated by Sephadex G-200 column to Antigen I, II and III according to protein content. Crude antigens of Paragonimus westermani, Clonorchis sinensis and Paramphistomum sp. were also prepared for control and absorption study. Antiserum was prepared by injecting 0.5 ml of crude Fasciola antigen with same amount of complete Freund's adjuvant in rabbits, 10 times at an interval of l week. The results obtained in this study were as follows: Crude Fasciola antigen reacted with antiserum with 9 precipitin bands by Ouchterlony test and with 11 bands by immunoelectrophoresis. Cross reaction was observed between Paragonimus, Clonorchis and Paramphistomum antigens and anti-Fasciola rabbit serum respectively. By Ouchterlony test, 3-4 cross reacting bands were found. Anti-Fasciola sera which were absorbed with respective Paragonimus, Clonorchis and Paramphistomum antigens, reacted with Fasciola crude antigen. Ouchterlony test gave 5-6 precipitin bands. Further reaction between Fasciola antigen and antiserum absorbed with the above 3 antigens concomitantly gave 5 precipitin bands by Ouchterlony test and 7 bands by immunoelectrophoretic analyses. Fractionated Fasciola antigens (Antigens I, II and III) reacted with anti-Fasciola rabbit serum in immunoelectrophoresis. Antigen I, II and III gave 2, 3 and 5 precipitin bands respectively. Anti-Fasciola rabbit serum which was absorbed with 3 trematodes antigens gave, by immunoelectrophoresis, 1 band with Antigen I, 2 bands with Antigen II and III of Fasciola hepatica. From the above results, it is concluded that Fasciola hepatica possessed the specific antigenic substance not cross-reacted with other trematodes.
parasitology-helminth-trematoda ; Fasciola hepatica ; Paragonimus westermani ; Clonorchis sinensis ; Paramphistomum sp. ; antigen ; immunology ; electrophoresis

Autoradiographic study was performed in order to know the distribution of exogenous radioactive substance, (3)H-6-Thymidine, by liver fluke, Fasciola hepatica. The worms were maintained in vitro for 1 hour, at 37 degrees C, in Tyrode solution containing (3)H-6-Thymidine. After the incubation, the worms were sectioned and autoradiographed by applying the techniques of microautoradiography. The black grains derived from labeled substance were highly accumulated in the vitelline duct and vitelline glands. Moderate amount of blackening showed in the subcuticular muscle fibers, testes, ovary, cirrus sac, oral sucker and pharynx. Slightly labeled particles were in the reticular tissue, ventral sucker, intestinal epithelium, prostate, uterus and ova. There can hardly be found the black grains in the cuticle.
parasitology-helminth-trematoda-Fasciola hepatica ; biochemistry ; autoradiography ; (3)H-6-Thymidine

In an attempt to investigate the antigen-antibody relations and the value of immunodiagnosis for several helminths, Ouchterlony tests and immunoelectrophoreses were carried out. Taenia saginata, Cysticercus sp. of cestodes, Clonorchis sinensis, Fasciola hepatica and Paramphistomum sp. of trematodes,and Ascaris suum of nematodes were used as antigens. On the other hand, antisera were obtained by injecting 0.5 ml each of the above antigens and the same amount of complete Freund's adjuvant into rabbits ten times at an interval of one week. The result obtained in this study are as follows: A larger number of precipitin arcs were demonstrated in homologous antigen-antibody reactions than in heterologous antigen-antibody reactions both in Ouchterlony tests and immunoelectrophoreses. Gross reactions were observed between the different species of the same class, but no cross reactions were noticed when the classes were different with one or two exceptions, such as between T. saginata, F. hepatica and A. suum. In A. suum, the difference between the male and female was more distinct in Ouchterlony test and immunoelectrophoresis than in the examination of organs such as genital organ and coeliac fluid. Immunoelectrophoresis revealed specific arcs and higher sensitive reaction than Ouchterlony test, and was considered to be a more valuable method for identifing species and immunological diagnosis.
parasitology-helminth-trematoda ; cestoda ; nematoda ; immunoelectrophoresis ; Taenia saginata ; Cysticercus ; Clonorchis sinensis ; Fasciola hepatica ; Paramphistomum sp. ; Ascaris suum ; antigen ; immunology

The adult trematode, Fasciola hepatica and Eurytrema pancreaticum, employed in this experiment were obtained from the cattle slaughtered at the local abbatoir. The worms were selected and washed several times in normal sterilized saline solution. Each ten of intact F. hepatica and about thirty to fifty of E. pancreaticum were incubated in 50 cc volume of special incubation flasks with incubation medium consisting of 50 cc of Krebs-Ringer phosohate buffer (pH 7.4). The incubation medium was added C(14)-lactate and non-radioactive carrier Na-lactate so as to contain lactate concentration of 32 mg per cent. The worms were allowed to incubate for 3 hours in the Dubnoff metabolic shaking incubator at 38 C. After incubation period, respiratory CO(2) samples from central wall of incubation flask were analysed for total CO(2) production rate and their specific activity of respiratory CO(2). The lactate uptake rate was determined by analyzing the the difference between lactate concentration in a medium before and after the incubation period, and the pyruvate appearance rate was dertermined by analyzing the pyruvate concentration in a medium after incubation. The glycogen samples isolated from worms were analyzed for the tissue concentration and their radioactivities in order to determine the turnover rate of glycogen pool. Radioactivities of these serise of experiment were counted by an endwindow Geiger-Muller counter as an infinitely thin samples. The quantative analysis of C(14)-lactate utilized by F. hepatica and E. pancreaticum were summerized and compared as following. In F. hepatica the lactate uptake rate was a mean value of 1.04+/-0.15 micromole/hr/g of wet wt. and pyruvate apperance rate was a mean value of 0.132+/-0.005 micro-mole/hr/g of wet wt. The total CO(2) production rate by the flukes averaged 13.82+/-0.75 micro-mole/hr/g of wet wt. The relative specific activities of respiratory CO(2) showed a mean value of 9.93+/-0.62 per cent. The rate of CO(2) production derived from medium C(14)-lactate was a mean of 1.38+/-0.13 micro-mole/hr/g of wet wt. Therefore the averge value of 55.27+/-5.78 per cent (R.L.D. CO(2)) and 15.35+/-1.90 per cent (R.L.D. pyr) of lactate was oxidized into respiratory CO(2) and pyruvate respectively. On the other hand, in E. pancreaticum the lactate uptake rate was a mean value of 0.61+/-0.18 micromole/hr/g of wet wt, and pyruvate appearance rate was a mean of 0.023+/-0.001 micromole/hr/g of wet wt. The total CO(2) production rate by the E. pancreaticum averaged 4.29+/-0.85 micromole/hr/g of wet wt. The relative specific activity of respiratory CO(2) (R.S.A CO2) showed a mean value of 9.20+/-0.34 per cent. Thus, a mean value of 9.20 per cent of total CO(2) production rates was originated from C14-lactate in a medium, therefore the rate of CO(2) production derived from medium C(14)-lactate was a mean value of 0.40+/-0.10 micromole/hr/g of wet wt. The average value of 23.93+/-7.11 per cent(R.L.D. CO(2)) and 3.86+/-0.45 per cent(R.L.D. pyr) of lactate was oxidized into respiratory CO(2) and pyruvate respectively. The tissue concentration of glycogen in F. hepatica was a mean of 2.63 per cent/g of wet wt, while in E. pancreaticum was a mean of 4.06 per cent/g of wet wt. The turnover rate of glycogen pool in F. hepatica yielded a value of 0.073+/-0.008 micromole/hr/g of wet wt whereas in E. pancreaticum yielded only a mean of 0.006+/-0.002 mg/hr/g of wet wt. Therefore, the half time of glycogen turnover, which is the time interval required to replace the half of glycogen pool with medium C(14)-lactate, gave value of a mean of 10.73+_0.76 days in F. hepatica. However, incorporation of C(14)-lactate into glycogen was negligible in the E. pancreaticum. Theses data impressed that the carbohydrate such as lactate may play a role of major part of their oxidative metabolism in F. hepatica, whereas minor part of lactate participates in the oxidative metabolism in E. pancreaticum.
parasitology-helminth-trematoda ; Fasciola hepatica ; Eurytrema pancreaticum ; autoradiography ; biochemistry ; pyruvate ; lactate ; glycogen ; metabolism ; Krebs-Rigner phosphate buffer