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Enzyme inhibitory peptides with a loop structure stabilized using an antiparallel β-sheet scaffold (β-loop peptide) were obtained from a designed peptide phage library. Human dihydrofolate reductase (hDHFR) was used as the target enzyme. The obtained β-loop peptides were competitive inhibitors of hDHFR with micromolar inhibition constants and dissociation constants.

Background Lectins are carbohydrate binding proteins and related to various biological events and diseases including virus infection and cancer metastasis. In particular, galactose-binding lectins have attracted attention as targets for drug delivery and cancer markers. We, previously, demonstrated that sugar-modified peptides (glycopeptides) were useful ligands for the detection and characterization of lectins compared to the sugar unit alone. Gold nanoparticles (GNPs) conjugated with mannose-modified glycopeptides were useful in detection of concanavalin A, a mannose binding lectin. Objectives The main objective of this study was to expand our glycopeptide-GNP conjugates for detection and imaging of galactose-binding lectins. Methods Four galactose-modified peptides (glycopeptides) were synthesized by Fmoc-based solid peptide synthesis method. Synthesized glycopeptides were conjugated with PEG-coated GNPs using thiol-maleimide chemistry. The interaction between glycopeptide-GNPs (GP/GNPs) (0.5 nM) and RCA120, a galactose binding lectin, (0.5-1000 nM) was evaluated by mesuring absorption spectra of GNPs. The inhibition experiment in the interaction between GP/GNPs (0.5 nM) and RCA120 (100 nM) was performed in the presence of 60 mM α- methyl mannose or 60 mM lactose. HepG2 and MCF7 cells were placed on 22×22 mm cover slip in 6 well cell culture plates (2×105 cells / well) and cultured overnight at 37°C under 5% CO2 condition. 1 mL of GP/GNPs (0.2 nM) were added in each well and incubated for 18 h at 37°C under 5% CO2 condition. After incubation, cells were washed twice with PBS and fixed with 4% paraformaldehyde solution. The cover slips were coated with 90% glycerol and sealed to slide glass. Dark-field images based on elastic light scattering were taken using a Nikon microscope (TieU) with an immersion dark field condenser. Results In the titration experiment of RCA120, GP/GNPs showed a decrease of absorbance according to the addition of RCA120, suggesting that the aggregation of GP/GNPs is induced through the binding to RCA120. The EC50 values of AA(Gal)/GNP, WF(Gal)/GNP, TS(Gal)/GNP and ED(Gal)/ GNP were estimated as 66.2 nM, 43.2 nM, 38.6 nM and 104.4 nM, respectively. TS(Gal)/GNP showed the lowest EC50 value among GP/GNPs. RCA120 has several binding sites for the galactose, and there are hydrophilic amino acids (Thr24, Glu26, Gln35, Asn42 and Asp44) around one of galactose binding sites. This result indicates that the hydrogen bonds between these amino acids and Thr/Ser residues of TS(Gal) contribute to the efficient aggregation of TS(Gal)/GNP. Next, inhibition experiments in the aggregation of WF(Gal)/GNP with RCA120 revealed that lactose inhibits the WF(Gal)/GNP binding with RCA120, but α-methyl mannose does not, and that WF(Gal)/GNP selectively interacts with RCA120 and forms the aggregate. Finally, a galactose binding protein on the surface of HepG2 cells was successfully visualized by using GP/GNPs as optical probes. Conclusion Our results demonstrated that GP/GNPs could detect RCA120 by the selective binding and the aggregation formation. Furthermore, a galactose binding protein on the surface of HepG2 cells is successfully visualized using WF(Gal)/GNP as an optical probe. Thus, GNPs conjugated with glycopeptides will be useful probes for the selective detection and imaging of lectins.

A monosaccharide-modified β-loop peptide library displayed on phage has been constructed and used for the screening of glycopeptide ligands against a carbohydrate-binding protein. The β-loop peptide library was designed and modified with a mannose derivative on phage. The glycopeptide ligands to concanavalin A (ConA), a mannose-binding protein, were obtained from the mannose-modified peptide phage library. The amino acids neighboring the mannose unit of glycopeptides not only reinforced the binding affinity but also gave diverse binding characteristics.

Novel calcium ion (Ca2+)-responsive hydrogels composed of designed β-sheet peptides were constructed. As the novel designed peptide, E1Y9, has a Glu residue to interact with Ca2+, the peptide in the sol-state self-assembled into hydrogels in the presence of Ca2+. The hydrogelation did not occur in the absence of Ca2+; therefore, Ca2+-dependent hydrogelation was achieved by the molecular design. The hydrogelation from the viscous sol-state solution can be induced by a slower self-assembly process of the β-sheet peptide involving a rapid process of Ca2+binding. When the sol-state peptide solution was injected with Ca2+, gel drops and strings with desired shapes could be constructed. Different cell lines can be cultured on the hydrogel, demonstrating its low toxicity, which is comparable to commercially available microtiter plate surfaces for cell culture. Furthermore, the hydrogels showed a high cell-adhesive ability that was similar in magnitude to fibronectin, which is a native cell-adhesive protein. The Ca2+-responsive peptide nanofiber-based hydrogelation system will facilitate novel studies exploiting self-assembling peptide nanomaterials that will lead to cell-based technology, such as three-dimensional cell culturing. Newly designed β-sheet peptides bearing the calcium ion binding Glu residue were self-assembling into self-supporting hydrogels. The hydrogelation from the viscous sol-state solution can be induced by a slower self-assembly process followed by a rapid injection to Ca2+ solution. Peptide gel strings can be easily constructed. Cell culture experiments demonstrated low toxicity and high-adhesive abilities of the peptide hydrogels.

Structure-activity relationship studies were conducted on HIV integrase (IN) inhibitory peptides which were found by the screening of an overlapping peptide library derived from HIV-1 gene products. Since these peptides located in the second helix of Vpr are considered to have an alpha-helical conformation, Glu-Lys pairs were introduced into the i and i+4 positions to increase the helicity of the lead compound possessing an octa-arginyl group. Ala-scan was also performed on the lead compound for the identification of the amino acid residues responsible for the inhibitory activity. The results indicated the importance of an alpha-helical structure for the expression of inhibitory activity, and presented a binding model of integrase and the lead compound.

Anti-HIV peptides with inhibitory activity against HIV-1 integrase (IN) have been found in overlapping peptide libraries derived from HIV-1 gene products. In a strand transfer assay using IN, inhibitory active peptides with certain sequential motifs related to Vpr- and Env-derived peptides were found. The addition of an octa-arginyl group to the inhibitory peptides caused a remarkable inhibition of the strand transfer and 3'-end-processing reactions catalyzed by IN and significant inhibition against HIV replication.

In the postgenome era, G-protein-coupled receptor families have been recognized as significant drug targets in medicinal chemistry. A specific chemokine receptor, CXCR4, has multiple critical functions in normal physiologies including embryonic development of the cardiovascular, hemopoietic and central nervous systems, and underlies problematic pathologies such as HIV infection, cancer metastasis, leukemia progression and rheumatoid arthritis.A tetradecamer peptide, T140, derived from the horseshoe crab, and its biologically stable derivative, 4F-benzoyl-TN14003, were found to be powerful CXCR4 antagonists that block HIV entry to cells. These peptides have also shown remarkable inhibitory activity against cancer metastasis and progression in a variety of cancers. Slow release administration of 4F-benzoyl-TN14003, for example, was found to significantly reduce pulmonary metastasis of breast cancer cells in severe combined immunodeficient mice. This peptide also shows inhibitory effects against melanoma metastasis and Epstein-Barr virus-associated lymphoproliferation in mice, suppresses the delayed-type hypersensitivity response induced by sheep red blood cells and reduced collagen-induced arthritis in both mouse models of arthritis.T140 analogues have the potential to become promising agents for chemotherapy of AIDS, cancer and rheumatoid arthritis. This review summarizes the development of low molecular weight CXCR4 antagonists based on pharmacophore identification in T140 analogues and also provides an opinion on the future of the development of CXCR4 antagonists.

The chemokine receptor, CXCR4, is a GPCR that transduces signals of its endogenous ligand, CXCL12 (stromal cell-derived factor-1, SDF-1). The CXCL12-CXCR4 system plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, etc. This system has recently been proven to be involved in several problematic diseases, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA) and pulmonary fibrosis. Thus, CXCR4 is thought to be an important therapeutic target to overcome the above diseases. Fourteen-mer peptides, T140 and its analogs, were previously found to be specific CXCR4 antagonists that were characterized as HIV-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents and anti-RA agents. Based on our knowledge of pharmacophores of T140, CXCR4 antagonists, such as FC131, were previously found by the efficient utilization of cyclic pentapeptide libraries. This review article focuses on our recent research on the development of low molecular weight CXCR4 antagonists including FC131 analogs, in which structural tuning of the cyclic peptide ring and chemical modifications were performed for an increase in potency and a reduction of the peptide character.

The chemokine receptor CXCR4 possesses multiple critical functions in normal and pathologic physiology. CXCR4 is a G-protein-coupled receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-1). The interaction between CXCL12 and CXCR4 plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, and so on. This interaction is also known to be involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA), and pulmonary fibrosis. It is conjectured that this interaction may be a critical therapeutic target in all of these diseases, and several CXCR4 antagonists have been proposed as potential drugs. Fourteen-mer peptides, T140 and its analogues, were previously developed in our laboratory as specific CXCR4 antagonists that were identified as HIV-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents, and anti-RA agents. Cyclic pentapeptides, such as FC131 [cyclo(D-Tyr-Arg-Arg-L-3-(2-naphthyl)alanine-Gly)], were also previously found as CXCR4 antagonist leads based on pharmacophores of T140. This review article describes the elucidation of multiple functions of CXCR4 antagonists and the development of a number of low-molecular weight CXCR4 antagonists involving FC131 analogues and other compounds with different scaffolds including linear-type structures.

Protein phosphorylation is ubiquitously involved in living cells, and it is one of the key events controlling protein-protein surface interactions, which are essential in signal transduction cascades. We now report that the small molecular receptors bearing binuclear Zn(II)-Dpa can strongly bind to a bis-phosphorylated peptide in a cross-linking manner under neutral aqueous conditions when the distance between the two Zn(II) centers can appropriately fit in that of the two phosphate groups of the phosphorylated peptide. The binding property was quantitatively determined by ITC (isothermal titration calorimetry), induced CD (circular dichroism), and NMR. On the basis of these findings, we demonstrated that these types of small molecules were able to effectively disrupt the phosphoprotein-protein interaction in a phosphorylated CTD peptide and the Pin1 WW domain, a phosphoprotein binding domain, at a micromolar level. The strategy based on a small molecular disruptor that directly interacts with phosphoprotein is unique and should be promising in developing a designer inhibitor for phosphoprotein-protein interaction.

A new and simple method to tether a functional molecule at the proximity of the active site of an enzyme has been successfully developed without any activity loss. The one-pot sequential reaction was conducted on a surface of human carbonic anhydrase II (hCAII) based on the affinity labeling and the subsequent hydrazone/oxime exchange reaction. The reaction proceeds in a greater than 90% yield in the overall steps under mild conditions. The enzymatic activity assay demonstrated that the release of the affinity ligand from the active site of hCAII concurrently occurred with the replacement by the aminooxy derivatives, so that it restored the enzymatic activity from the completely suppressed state of the labeled hCAII. Such restoring of the activity upon the sequential modification is quite unique compared to conventional affinity labeling methods. The peptide mapping experiment revealed that the labeling reaction was selectively directed to His-3 or His-4, located on a protein surface proximal to the active site. When the fluorescent probe was tethered using the present sequential chemistry, the engineered hCAII can act as a fluorescent biosensor toward the hCAII inhibitors. This clearly indicates the two advantages of this method, that is (i) the modification is directed to the proximity of the active site and (ii) the sequential reaction re-opens the active site cavity of the target enzyme.

Artificial functional peptides are valuable tools in variousfields of chemical biology. Small peptides, such as anoligohistidine tag (His tag), can be genetically incorporatedinto target proteins and used for purification of recombinantproteins, immobilization of proteins on microplates, andbioimaging of proteins on the surface of living cells with theircomplementary partner molecules, such as Ni

The human immunodeficiency virus type 1 (HIV-1) uses CD4 and the co-receptor CCR5 or CXCR4 in the process of cell entry. The negatively charged extracellular domains of CXCR4 (CXCR4-ED) interact with positive charges on the V3 loop of gp120, facilitating binding via electrostatic interactions. The presence of highly conserved positively charged residues in the V3 loop suggests that CXCR4-ED-derived inhibitors might be broadly effective inhibitors. Synthetic peptide derivatives were evaluated for anti-HIV-1 activity. The 39-mer extracellular N-terminal region (NT) was divided into three fragments with 10-mer overlapping sites (N1-N3), and these linear peptides were synthesized. Peptide N1 contains Met 1-Asp 20 and shows significant anti-HIV-1 activity. Extracellular loops 1 and 2 (ECL1 and 2) were mimicked by cyclic peptides C1 and C2, which were synthesized by chemoselective cyclization. Cyclic peptides C1 and C2 show higher anti-HIV-1 activity than their linear peptide counterparts, L1 and L2. The cytotoxicities of C1 and C2 are lower than those of L1 and L2. These results indicate that Met 1-Asp 20 segments of the NT and cyclic peptides of ECL1 and ECL2 are potent anti-HIV-1 drug candidates.

The present study aimed at investigating whether neuromotor development, from birth to 14 months of age, shows seasonal, cyclic patterns in association with months of birth. Study participants were 742 infants enrolled in the Hamamatsu Birth Cohort (HBC) Study and followed-up from birth to the 14th month of age. Gross motor skills were assessed at the ages of 6, 10, and 14 months, using Mullen Scales of Early Learning. The score at each assessment was regressed onto a trigonometric function of months of birth, with an adjustment for potential confounders. Gross motor scores at the 6th and 10th months showed significant 1-year-cycle variations, peaking among March- and April-born infants, and among February-born infants, respectively. Changes in gross motor scores between the 10th and 14th months also showed a cyclic variation, peaking among July- and August-born infants. Due to this complementary effect, gross motor scores at the 14th month did not show seasonality. Neuromotor development showed cyclic seasonality during the first year of life. The effects brought about by month of birth disappeared around 1 year of age, and warmer months seemed to accelerate the neuromotor development.

Antibiotic resistance is increasingly seen as a serious problem that threatens public health and erodes our capacity to effectively combat disease. So-called non-iron metalloporhyrins have shown promising antibacterial properties against a number of pathogenic bacteria including Staphylococcus aureus. However, little is known about the molecular mechanism(s) of action of these compounds and in particular how they reach the interior of the bacterial cells. A popular hypothesis indicates that non-iron metalloporphyrins infiltrate into bacterial cells like a "Trojan horse" using heme transport systems. Iron-regulated surface determinant (Isd) is the best characterized heme transport system of S. aureus. Herein we studied the molecular mechanism by which the extracellular heme-receptor IsdH-NEAT3 of Isd recognizes antimicrobial metalloporphyrins. We found that potent antibacterial porphyrins Ga(III)-protoporphyrin IX (PPIX) and Mn(III)-PPIX closely mimicked the properties of the natural ligand heme, namely (i) stable binding to IsdH-NEAT3 with comparable affinities for the receptor, (ii) nearly undistinghuishable three-dimensional structure when complexed with IsdH-NEAT3, and (iii) similar transfer properties to a second receptor IsdA. On the contrary, weaker antibacterial porphyrins Mg(II)-PPIX, Zn(II)-PPIX, and Cu(II)-PPIX were not captured effectively by IsdH-NEAT3 under our experimental conditions and displayed lower affinities. Moreover, reduction of Fe(III)-PPIX to Fe(II)-PPIX with dithionite abrogated stable binding to receptor. These data revealed a clear connection between oxidation state of metal and effective attachment to IsdH-NEAT3. Also, the strong correlation between binding affinity and reported antimicrobial potency suggested that the Isd system may be used by these antibacterial compounds to gain access to the interior of the cells. We hope these results will increase our understanding of Isd system of S. aureus and highlight its biomedical potential to deliver new and more efficient antibacterial treatments.

Nitrogen-fixing (acetylene-reducing) activity of intact saplings ofAlnus maximowiczii was measured under natural conditions in the subalpine zone of Mt Fuji. The nitrogen-fixing activity was detected from the middle of June when expansion of leaves had just begun to the end of October when the shedding of leaves was almost completed. Diurnal changes in the activity were almost parallel with those of ground temperature. The measured nitrogen-fixing activity was related to ground temperature and total leaf area. Using this relation, annual nitrogen fixation was estimated from the data of ground temperature and leaf area measured in the field. The amount of annual nitrogen fixation was almost the same as that of nitrogen used for annual growth. It was concluded that nitrogen fixation by nodules made a considerable contribution to the nitrogen economy in the saplings ofA. maximowiczii.

In human cases and experimental animals of schistosomiasis japonica, schistosomal eggs are often detected in the lungs. On a derivation of the intrapulmonary eggs many reviews are reported. Though the major opinions are that they flow into the lungs from the portal vein and the hepatic vessels in which the flukes mainly inhabit, the route by which they reach the lungs is not known with certainty. In order to study the problem, rabbits were infected with cercariae of Schistosoma japonicum, and observed for 2 or 32 weeks thereafter, and histological specimens were cut into serious sections and stained, and then stereographical scrutiny was carried out by the sketching reconstruction method. Some worms lying within the pulmonary artery occasionally had mature eggs in their uterus, moreover, there were thrombi with eggs or egg nodules in its peripheral branch. These findings suggest with positive fact that the eggs in the lung are produced not only owing to an inflow from the portal vein, but also owing to a direct egg-laying in the lungs.

The effect of high pressure, 10 Kg/cm2, was examined with respect to Schistosoma japonicum cercariae. High pressure affected about 20% of cercariae in the pressurizing period, having them lose their tails and sedimenting them to the bottom of the test tubes. These cercariae might cause lower infectivity at 2 hours after pressurization and high death rate at 24 hours. At 24 hours after pressurization the infection rate were practically same for the pressurized and the control cercariae.

Rabbits which were experimentally infected with Clonorchis sinensis demonstrated gross anatomic changes in liver tissue characterized by proliferation of connective tissue beginning at the margin of the liver and by nodule formation along impaired intrahepatic bile ducts containing the worms. Hepatomegaly, liver cirrhosis, swelling of the gall bladder, dilatation of the extrahepatic bile duct and ascites were also distinctive findings. Liver function tests, SGOT, SGPT, ALP and total cholesterol, showed abnormally higher values in the infected rabbits than in the control group from the 13th to the 20th day after infection. After the 35th day, as gross anatomic changes became obvious, the liver function tests had almost completely returned to the normal range. Changes in the serum protein fractions, however, maintained the pattern of decreased albumin and increased globulin during the experiment. These data suggest confirmed injury to liver parenchyma and disturbance of liver function.

Using counter immunoelectrophoresis, an antigen in circulation of rabbits infected with Schistosoma japonicum was investigated. The sensitivity of counter immunoelectrophoresis was found to be 16 to 32 fold higher than that of ouchterlony test. The rapidity and simplicity seemed to be much superior to other serological tests. The antigen was detected in 22 of 24 infected rabbits with various periods of infection. The worm burden in negative cases were very low. In all infected rabbits, the antigen was constantly found since the appearance. The antigen titer in serum of each infected rabbits was significantly correlated with the worm burdens per body weight (r=0.70, p

Challenge infections of Schistosoma japonicum were carried out on rabbits. The animals were infected with 100 to 150 cercariae by subcutaneous injection every 4 to 5 weeks. In the serum there could be seen immunoglobulin to some degree, but a conclusive evidence that immunity has some relation not only with the advance of liver lesions but with the resistance to recurrent infection could not be confirmed. The experimental studies in rabbits led us to make a conclusion that repeated infections of Schistosoma japonicum might play an important role in keeping up or inducing the condition of liver cirrhosis due to the present parasite.

The basin of the River Chikugo around Kurume is still one of the prominent areas in Japan infected with schistosomiasis japonica. Therefore, autopsies done in this district often reveal schistosome ova in the tissue specimens of each organ. Furthermore, as a residue of the present disease, very high pathological changes of the liver, advancing to a stage of liver cirrhosis with schistosomal ova, can be detected.During the seven years from 1955-1961 the authors found the ova of schistosoma japonicum in 34 cases of all the liver specimens prepared in their department. In this first report, macroscopical observations on the morphology of liver are described. Full particulars will be published later by Watanabe.

Pathological studies on human cases of schistosomiasis mansoni in Puerto Rico were conducted. Materials used were the autopsied cases of schistosomiasis mansoni encountered at the Department of Pathology, Puerto Rico Medical Center, from January 1967 to September 1971. There were 83 casesof schistosomiasis mansoni which contained schistosoma eggs in their organs. On the basis of thesecases, morbidity of the present disease was investigated, and histological examinations of the liver lesions were critically performed. Typical 3 cases of liver cirrhosis due to Schistosoma mansoni were subjoined and different points between schistosomiasis mansoni in Puerto Rico and schistosomiasis japonica in Kurume, Japan, were also discussed.

Chemokine receptors have attracted a good deal of public attention as important therapeutic targets for many diseases and disorders. In this issue of Chemistry & Biology, Kumar and colleagues propose a new concept of synthetic modular modifications to generate unnatural chemokines, which exhibit high receptor selectivity [1].

Using fluorescent antibody techniques, immunoglobulins (Ig's), mainly IgG class, were detected inside Schistosoma japonicum eggs lodged in mouse liver. Ig's were observed as a focal pattern between the miracidium and the eggshell during early infection (5-7 wk), particularly in lightly infected mice (20 cercariae). With advancement of time of infection (8-18 wk), a diffuse pattern of staining over the miracidial body developed and became predominant. The diffuse pattern of staining could be observed in the eggs taken from heavily infected mice (50 cercariae), during early stage. Eggs showing the focal pattern in a restricted area appeared to be morphologically intact, whereas eggs showing the diffuse pattern exhibited some types of eggshell destruction. We conclude that the focal pattern reflects disintegration of eggs in the initial stage and the diffuse pattern in the advanced stage. This spatial relationship between Ig's and eggs is discussed in relation to destruction of eggs. When Schistosoma eggs are incubated with sera from infected animals or patients, a precipitation reaction occurs outside the eggshells (Oliver- Gonzalez, 1954). In eggs lodged in tissue, an analogous phenomenon to the circumoval pre- cipitation occurs in the presence of immune se- rum. This reaction, known as the "Hoeppli phe- nomenon" (Hoeppli, 1932), has been shown to

The effect of a hydrochloric acid solution on the circumoval precipitin (COP) reactivity of Schistosoma japonicum eggs was studied. When eggs, recovered by trypsin digestion from tissues of an 8 week-infected rabbit, were treated with a pepsin HCl solution, they had significantly higher reactivity than the saline-treated eggs (control). A similar effect was obtained with HCl solution (0.008 N), alone. In contrast, a sodium hydroxide solution (0.05 N) decreased the reactivity. Treatment with different concentrations of HCl (0.01 to 6N) for 2 days revealed that the reactivity was highest at 0.1 N, and was diminished by the higher concentrations (3 and 6N). Preservation in an HCl solution (0.01N) yielded a peak reactivity at 2 days. While the exact role of the HCl solution was not studied, this study contributes to the preparation of egg specimens and to our understanding of the low reactivity of eggs.

A circulating anodic antigen (CAA) was detected in Schistosoma japonicum-infected mice using counter- immunoelectrophoresis. The incidence of CAA increased with the total number of female and male worms and reached 100% in mice with 12 worms. The worm burdens, expressed as male worm equivalents based on the CAA content, also showed a close correlation with CAA frequency. On the other hand, the incidence of antibodies had an inverse relationship with CAA frequency. No reactions were seen in mice with more than 13 worms or 30 male worms equivalents. It was notable that mice infected with only male and/or immature worms had a significantly higher degree of reaction (54.5%) than bisexually infected mice (18.2%). For comparison, S. mansoniinfected mice were investigated using S. japonicum worm -derived antigen or antiserum. However, no CAA was detected in the sera from 18 mice, even though one group of mice had a large number of worms (X ± SE, 49.7 ± 11.7). In contrast, antibodies were detected in sera of all mice sacrificed at 14 and 20 weeks after the infection.

Production of pulmonary schistosomiasis in rabbits was attempted by frequent intravenous injections of massive numbers of Schistosoma japonicum ova. The relationship between the extent of pulmonary involvement and the dose of ova injected was investigated histologically. The extent of parenchymatous and arterial involvement can be correlated to a gradual accumulation of egg emboli. However, they were essentially focal in distribution. It was concluded that S. japonicum ova that were frequently injected into the pulmonary microvasculature of rabbits did not evoke the diffuse and severe lesions leading to cor pulmonare.

Prevalence of anti-S. japonicum antibodies in persons living in the Chikugo River basin, Kyushu, previously an endemic area of schistosomiasis japonica, was investigated by circumoval precipitin (COP) and indirect haemagglutination (IHA) tests. Of 327 specimens of sera collected, 51 (15.6 %) and 112 (34.3 %) were positive by COP and IRA tests, respectively. Reactivities of the COP test were predominantly weak and 82.1 % of IHA-positive sera were of low titers-1:40 to 1:80. Between the results of COP and IHA tests no relation was observed. When indirect fluorescent antibody (IFA) test was applied to randomly sampled 67 sera, using S. japonicum adult worms fixed with Rossman's fixative higher concordance (64.2 %) was observed between IFA and IHA tests in comparison to that (32.8 %) between IFA and COP tests. However, the relationship between results of IFA and IHA tests was not statistically significant. The differences among the results obtained by those serological tests are believed to result from differences in antigen components as well as by the low levels of antibody titers. The results show that anti-S. japonicum antibodies are still prevalent in this district, but since most sera tested are only slightly positive, active infection with S. japonicum is not apparently a problem at present.

Ultrasound diagnosis of chronic schistosomiasis japonica was assessed by comparison with the results of serological tests using enzyme-linked immunosorbent assay (ELISA) with egg and adult worm antigen, and the circumoval precipitation test. The subjects were persons resident in the Chikugo River Basin in Kyushu, southwest Japan, where schistosomiasis used to be highly prevalent. Of 93 individuals with a past history of infection, 37 (39.8%) presented the characteristic echo pattern of the disease (network, sieve, mottled or mixed pattern). In the serological tests, the ultrasound (US)-positive subjects showed a significantly higher IgG antibody-positive rate in ELISA (81.1% for egg-ELISA), than the US-negative subjects (37.5% for egg-ELISA). Individuals bearing dead Schistosoma japonicum eggs proven by tissue biopsy showed a similar antibody-positive rate (76.5% for egg-ELISA) to that of the US-positive subjects. Sera of a group which had no schistosomiasis characteristic echo pattern, but had liver fibrosis, hepatomegaly or liver cirrhosis as shown by US, were also highly positive (71.4% for egg-ELISA). The present serological studies thus confirmed the usefulness of ultrasound diagnosis for chronic schistosomiasis japonica. Furthermore, we were able to determine certain indications of liver abnormality currently undefined in the normally utilized US classifications.

Rabbits were experimentally infected with Scbistosoma japonicum to observe morphological alterations of the liver to the 60th week of infection in chronological sequence. In the present report, the developing process of liver cirrhosis due to Schistosoma japonicum was elucidated together with the repairing process occurring after its cirrhotic state. It is concluded that the experimental liver cirrhosis due to Schistosoma japonicum is repairable. Consequently, in the persistence and progress of a cirrhotic state in the liver, repeated infections appear to play an important role.

An epidemiological study on Clonorchis sinensis infection along the Nam-river (total length; 186km) flowing in Gyeongsang-nam-do, southern part of Korea, was carried out. Formalin-ether concentration technique and Stoll's egg counting method were employed to figure out the prevalence of C. sinensis infection. For the detection of cercariae from Parafossarulus sp., the snail host of C. sinensis, each snail was placed in aerated tap water, and examined for expelled cercariae. For observing the metacercariae the fresh water fish favorably eaten in raw conditions were prepared by means of pressing the muscles between tow slide glasses and/or by digesting them with artificial gastric juice. The fresh water fish were fed to the rabbits to get the worms and to identify the morphology of adult C. sinensis. The results are summarized as follows: 1. Overall prevalence of C. sinensis infection was 38.7% form 5,291 examinees; 44.1% (1,408 out of 3,196) in male and 30.4% (637 out of 2,095) in female. 2. The prevalence rates were 42.0% at the upper stream, 41.2% around the vicinity of Jinyang-lake, 34.2% at Jinju city, 34.2% at middle stream and 40.3% at down stream regions, respectively. 3. By age, the highest positive rate (53.4~54.3%) was observed in 30 to 59 years of age. In this age group, the rate in males was 59.7~62.2%, and in females 42.2~44.4%. In the age group of less than 19years it was 7.5~20.9%. 4. By social strata, the positive rate was 16.5% in the primary school children, 22.6% in school students, 46.2% in teachers and local officers and 49.6% in the general inhabitants. 5. The quantitative examinations with the stool collected from clonorchiasis cases revealed that the light infection (less than 4,000/EPG) was 53.6%, moderate infection (4,001~10,000/EPG) 30.3% and heavy infection (more than 10,001/EPG) 16.1, respectively. More than half of total cases examined were light infection, and 73.2% of female examinees were lightly infected with this fluke. 6. The average value of EPG was 4,963 (male, 6,057; female, 2,557 and the highest value was obtained from the age group of 30 to 59 years (5,240~6,454). 7. The prevalence of Metagonimus yokogawai infection in humans was 5.5%, and 89.8% of metagonimiasis cases were double-infected with C. sinensis. The highest prevalence rate was observed in JInju city(11.2%). 8. Total of 5,005 Parafossarulus sp., the snail intermediate host of C. sinensis, were examined for the detection of cercariae. The cercarial expulsion rate was 0.34%, and the snails collected in Jin-yang-lake side and in the down stream expelled mainly the cercariae of C. sinensis. 9. About 788 cercariae/day (range: 127~1,503) were expelled daily from a snail naturally infected with C. sinensis. The snails which released more than 1,000 cercariae/day were 30.8% out of total collected. A snail uniquely released 5,840 cercariae/day in this study. 10. The other trematode cercariae besides C. sinensis were also detected, and the rates out of total snails were the cercariae of Loxogenes liberum 6.71%, Cyathocotyle orientalis 0.26%, Notocotylus attenuatus 2.52% and Mucobucaris 0.54%. 11. Ten out of 18 species of fresh water fish caught along the river harbored the vetacercariae of C. sinensis. The highest rate of metacercarial infection in fish wa detected in Pseudorabora parva(85.9%). The fish mainly eaten by the inhabitants along the Nam-river, and the metacercarial infection rates were: Zacco platypus 8.0%, Hemibarbus sp. 18.2~26.7%, Gnathopogon sp. 37.5%, Ischikauia steenackeri 42.9% and Pseudogobio esocinus 16.7%. 12. Out of 36 P. parva, the number of metacercariae were about 109 (range; 18~446) per fish and 27 per gram of flesh. The fish caught in spring harbored the highest number of metacercariae. As indecated above, the prevalence of C. sinensis infection in the inhabitants resding around the Nam-river was relatively high. The farther toward the upper stream areas, the higher was the positive rate of C. sinensis infection, and most of clonorchiasis cases were lightly infected. The snail hosts of C. sinensis distributed all around the Nam-river. Several species of freshwater fish were infected with the metacercariae of C. sinensis and the infection rates were relatively high. To prevent C. sinensis infection in the endemic areas, the effective health education system is suggested as a control measure, althought mass treatment is also expected to be useful, using chemotherapeutic agents such as "praziquantel", a recently developed anthelminthic for C. sinensis infection.