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Based on 2-methyl-4-nitro-2H-pyrazole-3-carboxylic acid[2-(cyclohexanecarbonylamino)benzothiazol-6-yl]amide (1), which shows selective cytotoxicity against tumorigenic cell lines, 2,6-dichloro-N-[2-(cyclopropanecarbonylamino)benzothiazol-6-yl]benzamide (13b) was designed and synthesized as a biologically stable derivative containing no nitro group. The highly potent derivative 13b exhibited excellent in vivo inhibitory effect on tumor growth.

For the construction of new combinatorial libraries, a lead compound was created by replacing the core structure of a hit compound discovered by screening for cytotoxic agents against a tumorigenic cell line. The newly designed compound maintained biological activity and allowed alternative library construction for antitumor drugs.

A molecular and morphometric investigation is reported on the species complex of mammalian lungflukes of which Paragonimus skrjabini Chen, 1959 and P. miyazakii Kamo, Nishida, Hatsushika & Tomimura, 1961 are the best-known examples. This species complex (here called the P. skrjabini complex) is shown to be monophyletic using DNA sequences from the nuclear ITS2 region and the mitochondrial cox1 gene. The latter marker permits the discrimination of populations, some previously named as distinct species, from various geographical locations in China and Japan. Morphometric analysis of a number of variables accords remarkably closely with the molecular results. Main findings are that (1) nominal P.skrjabini from Fujian Province in eastern China is phylogenetically very close to P. miyazakii from Japan. It is proposed that both taxa should be referred to the same subspecies as P. krjabini miyazakii. (2) Populations from Guangdong Province, Guangxi Zhuang Autonomous Region, Sichuan and Hubei should be referred to P. skrjabini skrjabini. Specimens of P. skrjabini from the Yangtze basin in Sichuan and Hubei are remarkably similar to one another according to genetic and morphometric data, but are not so different from Guangdong populations that they should be referred to a different subspecies at this stage. Specimens of P. skrjabini from Yunnan have not been assigned to a subspecies at this stage. (3) Partly following earlier studies, we regard the following as synonyms of P. skrjabini: P. miyazakii (reduced to subspecific status); P. szechuanensis Chung & Tsao, 1962 (probably belongs to P. s. skrjabini); and P. hueitungensis Chung, Hsu, Ho, Kao, Shao, Chiu, Pi, Liu, Ouyang, Shen, Yi & Yao, 1975 (probably belongs to P.s.skrjabini ); P. veocularis (Chen & Li, 1979) (specimens from Fujian regarded as belonging to this species should be referred to P.skrjabini miyazakii: the subspecific status of specimens from the type-locality in northern Sichuan is unclear). A number of questions remain unresolved. The name P. hokuoensis Ho & Chung, 1964 was proposed for two individual metacercariae of distinctive appearance from southern Yunnan. DNA sequences from very similar metacercariae from the same locality place this nominal species within, or sister to, the P. skrjabini complex. As yet, nothing is known regarding adult morphology or biology of this taxon and we retain it here as a distinct species. P. heterorchis (Zhou, Pang & Hsiang, 1982) might be a synonym of P. skrjabini: the form of the metacercaria provides evidence against this view and further work is required. P. macrorchis Chen, 1962 has probably been confused with P. skrjabiniin China. Within China, the former probably occurs only on Hainan Island, although P. fukienensis Tang & Tang, 1962 from Fujian Province could be a synonym.

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb-e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb-e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb-e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.

As a result of a hit-to-lead program using a technique of solution-phase parallel synthesis, a highly potent (2,4-dimethoxyphenyl)-[6-(3-fluorophenyl)-4-hydroxy-3-methylbenzofuran-2-yl]methanone (15b) was synthesized as an optimized derivative of 4-hydroxy-3-methyl-6-phenylbenzofuran-2-carboxylic acid ethyl ester (1), which was discovered as a screening hit from small-molecule libraries and exhibited selective cytotoxicity against a tumorigenic cell line.

(3-Amino-6-thiophen-2-yl-thieno[2,3-b]pyridin-2-yl)phenylmethanone (3) was discovered as a new type of cytotoxic agent selective against a tumorigenic cell line. The molecular structure of a previously reported compound, (4-hydroxy-3-methyl-6-phenylbenzofuran-2-yl)phenylmethanone (2), had remarkably similar bioisosteric substructures to that of compound 3. Although the relationship between the molecular structure and biological activity of each derivative synthesized from these two hit compounds (2 and 3) were studied, unexpectedly no correlation was observed. However, after further synthetic study from 3, one of the most potent derivative (10k) having a different SAR profile from 2, was discovered.

Bivitellobilharzia nairi (Mudaliar and Ramanujachar, 1945) Dutt and Srivastava, 1955 was first recorded in India. A number of adult worm specimens of this schistosome species were recovered from a domestic elephant, which died in 1999 in Sri Lanka. This is the first report of this schistosome from Sri Lanka. In the present study, in order to clarify the phylogenetic relationship with other species of schistosomes, sequences from the second internal transcribed spacer (ITS2) of the ribosomal gene repeat, part of the 28S ribosomal RNA gene (28S), and part of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene from B. nairi were analyzed. Two intraspecific variations were seen within 13 individuals in the ITS2 region. In the CO1 region of the mitochondrial DNA, there were four haplotypes in the nucleotide sequences and two haplotypes in the amino acid sequences. Phylogenetic analysis using the nuclear DNA showed that B. nairi was basal to all of species of the genus Schistosoma. The 28S tree also showed that the mammalian lineage was monophyletic. However, phylogenetic analysis using the mitochondrial DNA showed that B. nairi was nested within the genus Schistosoma. The taxonomical position for this species as well as the contradiction between the results from the nuclear and mitochondrial genes were discussed.

Based on the structure of 4-hydroxy-3-methyl-6-phenylbenzofuran-2-carboxylic acid ethyl ester (1), which exhibits selective cytotoxicity against a tumorigenic cell line, (2,4-dimethoxyphenyl)-(4-hydroxy-3-methyl-6-phenylbenzofuran-2-yl)-methanone (18m) was designed and synthesized as a biologically stable derivative containing no ester group. Although the potency of 18m was almost the same as our initial hit compound 1, 18m is expected to last longer in the human body as an anticancer agent.

In his hypothesis on the coevolution of Asian schistosomes and snails, Davis implies that the ancestors of the Schistosoma japonicum and S. indicum species group were African and arrived in Asia via the Indian plate. This paper briefly reviews molecular phylogenetic relationships among species of the genus Schistosoma to test Davis' theory about the origin and evolution of S. japonicum. All analyses using DNA base sequences, mitochondrial genome gene order and C-banding patterns suggest that Schistosoma originated in Asia and not Africa.

EI-2128-1, a novel interleukin-1beta converting enzyme (ICE) inhibitor, was isolated from the culture broths of Penicillium sp. E-2128. EI-2128-1 selectively inhibited human recombinant ICE activity with IC50 value of 0.59 microM, without inhibiting elastase and cathepsin B. EI-2128-1 also inhibited mature interleukin-1beta secretion from THP-1 cells induced by LPS with IC50 value of 0.28 microM.

Three dog heartworms (Dirofilaria immitis) were detected in the lumen of the right cardiac ventriculus and of the pulmonary artery of a captive female snow leopard (Uncia uncia) that died of pancreatic carcinoma at a zoo in Japan. Neither clinical respiratory nor circulatory symptoms caused by the heartworm infection were observed. The filarial worms were identified as D. immitis from the morphologic characteristics of the esophagus, the presence of faint longitudinal ridges on the cuticular surface, the situation of vulva posterior to the esophagus, and the measurements of the body. The heartworms from the snow leopard were identical to that of D. immitis from dogs in the sequence of the cytochrome oxidase I region in the mitochondrial DNA. This host record is the first of D. immitis in U. uncia.

The proline-rich motif in proteins is known to function as a ligand sequence that binds to protein modules such as SH3, WW, and several other protein interaction domains. These proline-rich ligand-mediated protein-protein interactions (abbreviated PLPI) are important in many signaling pathways that are involved in various diseases. Our previous studies showed that UCS15A, produced by Streptomyces species, inhibited PLPI. Here we report on synthetic analogs of UCS15A that show more potent activity than UCS15A in inhibiting PLPI. A synthetic analog, compound 2c, blocked in vitro PLPI of Sam68-Fyn-SH3 as well as in vivo PLPI of Grb2-Sam68 and Grb2-Sos1. Activation of MEK was also inhibited by compound 2c. Unlike UCS15A, compound 2c was an order of magnitude less cytotoxic and did not cause morphological changes in treated cells.

We present an emergent surgical case of massive pulmonary embolism (MPE) that happened during orthopedic surgery. A 38-year-old man, who had bone fractures with his lumbar vertebra and ankle, underwent the internal fixation of the tibial bone with tourniquet under general anesthesia in our hospital. During this surgery, the pulse oxymeter showed a drop of arterial oxygen saturation suddenly. Immediately we installed a transesophageal echo (TEE) probe in the patient, and detected enlargement of the right ventricle and right atrial thrombus. Half an hour later, the thrombus disappeared from the right atrium and the patient showed hemodynamic shock. We performed emergent embolectomy immediately under moderate hypothermic complete cardiopulmonary bypass. The postoperative course was uneventful, and one month later, his orthopedic surgery underwent completely. We conclude that TEE was a useful devise for the diagnosis of intraoperative MPE.

Novel halohydrin and oxime derivatives of radicicol (1) were prepared and evaluated for their v-src tyrosine kinase inhibitory, antiproliferative, and antitumor activities. Some of the resulting derivatives showed significantly improved antitumor activities than those of 1 in vitro as tested in a cell proliferation assay and in vivo using sc-inoculated human breast carcinoma and epidermoid tumor models. Design and synthesis of radicicol-based novel affinity probes are also described.

RP-1776, a novel cyclic peptide, was isolated from the culture broth of Streptomyces sp. KY11784. RP-1776 selectively inhibited the binding of PDGF BB to the extracellular domain of the PDGF beta-receptor with an IC50 value of 11 +/- 6 microM. Detailed binding experiments suggested that RP-1776 directly interacts with PDGF BB. RP-1776 inhibited the phosphorylation of the PDGF beta-receptor induced by PDGF BB. These results suggested that RP-1776 antagonizes the signaling of PDGF BB probably through the inhibition of PDGF BB binding to the PDGF beta-receptor.

The task of using partial ND1 sequences to infer a phylogeny for species of the genus Paragonimus (Trematoda: Digenea) was complicated by the discovery of at least two ND1 lineages within individual worms. The divergence of the ND1 lineages is shown by phylogenetic analysis not only to predate the divergence of the three Paragonimus species or species groups investigated but also the divergence of some trematode families. Some sequences are clearly pseudogenes as they contain single base deletions and/or premature termination codons. The presence of both pseudogenes and/or mitochondrial heteroplasmy are invoked to explain the presence of multiple and divergent ND1 lineages in these trematodes, which have two distinct cytochemical types of mitochondria. The implications for phylogenetic studies generally and of parasitic helminths specifically, using ND1 sequence data, are discussed. The ability of these organisms to adapt their metabolic processes to the variable availability of oxygen as an electron acceptor are proposed to explain some of the molecular diversity observed in parasitic helminths and possibly also in other anaerobically adapted eukaryotes.

Paragonimus westermani is a medically important foodborne trematode occurring throughout southeast Asia. We have used molecular techniques to test the hypothesis that the parthenogenetic triploid form of P. westermani has arisen only once. Sources of data for comparison were: (a) restriction fragment length polymorphisms (RFLPs) of ribosomal internal transcribed spacers (ITS); and (b) 'fingerprint' patterns observed when genomic digests were probed with simple sequence repeats (ATT)10 and (ATGT)7. In all cases there were distinct differences among triploid isolates from southwest Japan, northeast China and Korea. These findings are considered in the context of previous cytogenetic, allozyme, mitochondrial-RFLP and partial cytochrome c-oxidase subunit I (COI) sequence studies and indicate that triploid lineages may have arisen independently on more than one occasion. We favour this view. An alternative explanation is that the triploids did have a single origin, but that different clonal lineages have undergone subsequent mutations.

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.

Radicicol, a macrocyclic antifungal antibiotic, has been shown to bind to the heat shock protein 90 (Hsp90) chaperone, interfering with its function. Hsp90 family chaperones have been shown to associate with several signaling molecules and play an essential role in signal transduction, which is important for tumor cell growth. Because radicicol lacks antitumor activity in vivo in experimental animal models, we examined the antitumor activity of a novel radicicol oxime derivative, radicicol 6-oxime (KF25706), on human tumor cell growth both in vitro and in vivo. KF25706 showed potent antiproliferative activities against various human tumor cell lines in vitro and inhibited v-src- and K-ras-activated signaling as well as radicicol. In addition, Hsp90 family chaperone-associated proteins, such as p185erbB2, Raf-1, cyclin-dependent kinase 4, and mutant p53, were depleted by KF25706 at a dose comparable to that required for antiproliferative activity. KF25706 was also shown to compete with geldanamycin for binding to Hsp90. KF29163, which is an inactive derivative of radicicol, was less potent both in p185erbB2 depletion and Hsp90 binding. More importantly, KF25706 showed significant growth-inhibitory activity against human breast carcinoma MX-1 cells transplanted into nude mice at a dose of 100 mg/kg twice daily for five consecutive i.v. injections. KF25706 was also shown to possess antitumor activity against human breast carcinoma MCF-7, colon carcinoma DLD-1, and vulval carcinoma A431 cell lines in vivo in an animal model. Finally, we confirmed the depletion of Hsp90-associated signaling molecules (Raf-1 and cyclin-dependent kinase 4) with ex vivo Western blotting analysis using MX-1 xenografts. In agreement with in vivo antitumor activity, KF25706 depleted Hsp90-associated molecules in vivo, whereas KF29163 and radicicol did not show this activity in vivo. Taken together, these results suggest that antitumor activity of KF25706 may be mediated, at least in part, by binding to Hsp90 family proteins and destabilization of Hsp90-associated signaling molecules.

We investigated the utility of the ribosomal first internal transcribed spacer (ITS1) for phylogenetic studies on trematodes of the genus Paragonimus. Numerous clones containing ITS1 PCR products were sequenced for P. miyazakii, P. macrorchis, and members of the P. ohirai and P. westermani species complexes. Some additional data were obtained by direct sequencing of PCR products. The ITS1 is composed of three distinct regions: the short 5' end, followed by a tract of approximately 120 nucleotides which occurs a variable number of times in tandem, and the 3' region, which lacks repeats and is referred to as the "post-repeat" fragment. Sequences from all three regions can be aligned among the species studied. Our initial hypothesis, that the post-repeat region would be valuable for phylogenetic studies within the P. westermani complex, was proved wrong. Intraindividual sequence variation in P. westermani was sometimes greater than between individuals of the species complex. In the P. ohirai species complex, however, sequence variation within individuals was minimal. Possible reasons for these observations are discussed. We also wished to determine whether the length variants sequenced were the dominant variants present in Paragonimus species. This was done by probing Southern blots of genomic digests with an ITS1 fragment which lacks repeat sequences. There is generally greater abundance of large variants, with much lower abundance of small variants, such as those sequenced. Differences in ITS1 lengths are attributed largely to differing numbers of repeats, though some exceptions (which are discussed) were found., (Copyright 1999 Academic Press.)

The review concentrates on literature that has appeared since the 1960s. Since then, numerous species of Paragonimus have been described, mainly from Asia but also from Africa and the Americas. Some of these cause disease in humans. Recent information on life cycles and routes of transmission is summarized. All described species and their hosts are listed, with synonyms where known. For well-known species such as Paragonimus westermani, subspecific taxa and strains are reviewed and genetic studies discussed. Paragonimiasis in humans and experimental animals is discussed with emphasis on clinical manifestations and pathology, diagnosis, immune interactions with the host, treatment and public health issues.

A series of hexadeoxyribonucleotides (6-mers), d(TGGGAG), substituted with a variety of aromatic groups at the 5'-end were synthesized and tested for anti-human immunodeficiency virus type 1 (HIV-1) activity. While unmodified d(TGGGAG) (31) had no anti-HIV-1 activity, compound 23 with a 3,4-di(benzyloxy)benzyl (DBB) group at the 5'-end potently inhibited the HIV-1IIIB-induced cytopathicity of MT-4 cells in vitro (IC50 = 0.37 microM) without cytotoxicity up to 40 microM. A thermal denaturation study on the 5'-end-substituted 6-mers by means of the circular dichroism (CD) spectra demonstrated that the aromatic substituent attached at the 5'-end of the 6-mer strongly enhanced the formation of a parallel helical structure consisting of four strands (quadruplex). On the contrary, compound 36, in which one of the guanosines of 23 was replaced by a thymidine, did not form a quadruplex, thus exhibiting no anti-HIV-1 activity. Moreover, both compound 15, with a tert-butyldiphenylsilyl group solely at its 3'-end, and compound 21, with a relatively small substituent, a benzyl group, at the 5'-end, formed quadruplexes but had no anti-HIV-1 activity. These findings led us to the conclusion that both the quadruplex structure and the aromatic substituent with adequate size at the 5'-end are crucial for the interaction of the 5'-end-substituted 6-mers with the V3 loop as well as the CD4 binding site on viral gp120, resulting in anti-HIV-1 activity.

Radicicol, a macrocyclic anti-fungal antibiotic, has the ability to suppress transformation by diverse oncogenes such as Src, Ras and Mos. Despite this useful property, the mechanism by which radicicol exerts its anti-transformation effects is currently unknown. To understand the transformation-suppressing effects of radicicol, a biotinylated derivative of radicicol was chemically synthesized and used as a probe in a Western-blot format to visualize cellular proteins that interact with radicicol. In transformed and untransformed mouse fibroblasts, the most prominent cellular protein that bound to radicicol had a molecular weight of approximately 90 kDa. Further analysis revealed that this protein was the mouse homologue of the 90 kDa heat shock protein (HSP90). This was confirmed by demonstrating the ability of radicicol to specifically bind purified human HSP90. Specificity of binding was demonstrated by the inhibition of binding of biotinylated radicicol by the native drug. Taken together with other studies the present observations suggest that the anti-transformation effects of radicicol may be mediated, at least in part, by the association of radicicol with HSP90 and the consequent dissociation of the Raf/HSP90 complex leading to the attenuation of the Ras/MAP kinase signal transduction pathway.

GE3, a novel cyclic hexadepsipeptide antibiotic, was isolated from the culture broth of Streptomyces sp. GE3. GE3 was weakly active against some Gram-positive and Gram-negative bacteria and showed potent cytotoxicity against human tumor cell lines. GE3 also exhibited antitumor activity against human pancreatic carcinoma, PSN-1, in vivo. GE3B, a linear peptide form of GE3, which was isolated from the same culture broth with GE3, showed no antibiotic and cytotoxic activities, suggesting the necessity of the cyclic structure of GE3 for its biological activities.

It has been shown in previous reports that a guanine-rich phosphodiester oligonucleotide bearing a dimethoxytrityl (DmTr) residue on its 5'-terminal. DmTr-TGGGAGGTGGGTCTG (SA-1042), is an inhibitor of HIV-1 infection in vitro. SA-1042 interfered with the attachment of gp120 to the CD4 receptor and the subsequent entry stage of viral infection. We investigated the structure-activity relationship of the DmTr-conjugated oligomer by using 15-mer oligonucleotides with various nucleotide sequences. Results show that location of guanine nucleosides at the 5'-terminal and modification of the 5'-terminal with DmTr are essential for anti-HIV-1 activity. First, substitution of the guanine nucleoside close to the 5'-terminal of SA-1042 with another nucleotide prevented antiviral activity. Second, the existence of at least three consecutive guanine nucleosides adjacent to the 5'-terminal was required for the activity. Finally, modification of the 5'-terminal was essential for the activity. Based on these findings, the hexanucleotide, DmTr-TGGGAG, was identified as a potent inhibitor of HIV-1 infection. The hexamer was found to be capable of inhibiting the binding of gp120 to its receptor CD4 molecule, and it was also capable of inhibiting accessibility of anti-V3 monoclonal antibody to its ligand V3 peptide.

The structures of three novel interleukin-1 beta converting enzyme inhibitors, EI-1511-3, -5 and EI-1625-2 were determined by spectroscopic methods. Their absolute configurations were also determined by analyses of the CD spectra of the inhibitors and their oxidation products with chromium trioxide.

Large changes in the responsiveness of target organs to oxytocin are thought to originate from alteration of the number of oxytocin receptors (OTR). To elucidate the molecular mechanisms regulating the synthesis of the OTR, we developed a competitive reverse transcription-PCR protocol to measure OTR mRNA. We synthesized cRNA comprising a small stuffer introduced into the target mRNA. Using this cRNA as an internal standard, we made a quantitative estimation of OTR mRNA. Application of this method to the rat uterus revealed that the mean levels of OTR mRNA remained unchanged until 1030-1100 h on day 21 of pregnancy, increased significantly after 2200-2230 h on the same day and declined rapidly after parturition. A similar rapid increase in uterine OTR mRNA content was observed in rats given prostaglandin on day 18, inducing premature delivery on day 19 of pregnancy. All parturient rats had higher OTR mRNA levels regardless of whether parturition was spontaneous or prostaglandin induced. However, in a few rats, OTR mRNA remained as low as that observed during mid pregnancy even on day 22 of gestation, the expected day of parturition in about 70% of the rats in our colony. A similar increase in uterine OTR mRNA content to that observed at parturition was induced by oestrogen treatment for 3 days in ovariectomized virgin rats, but concomitant injection of progesterone did not influence the effect of oestrogen. The present results revealed that the large increase of uterine OTR at the peripartum period is accompanied by an increase in OTR mRNA content that may be brought about, at least in part, by increased oestrogen secretion following luteolysis.

A comparative study of the cellular response to Schistosoma japonicum eggs was conducted in order to explore its significance, using hosts with differing susceptibilities to the parasite. In experimentally induced, synchronized hepatic granuloma formation, animal species formed each characteristic feature of the granulomas, and the magnitude of tissue reaction was significantly larger in highly susceptible hosts, such as mice and hamsters, while less susceptible hosts, such as rats and quails, formed smaller granulomas. Confluent neutrophils were seen within the tissue lesions for mice and hamsters, while rats and quails showed obviously scanty neutrophils. Guinea pigs failed to develop any granulomas. When splenic cells and bone marrow cells were used for in vitro granuloma formation, bone marrow cells showed markedly higher reactions than splenic cells from naive or sensitized animals and the reactivities of bone marrow cells from susceptible hosts, mice and hamsters, were clearly higher than those from rats, indicating similar results to those of in vivo granuloma formation. This study indicates that the in vivo and in vitro cellular response to S. japonicum eggs varies greatly according to the host's susceptibility, independent of whether the host is a naive or sensitized animal. Our results seem to support the concept that the parasites exploit the host immune system in order to complete their life-span.

Previous studies have shown that a guanine-rich oligonucleotide SA-1042, DmTr-TGGGAGGTGGGTCTG, neutralizes HIV-1 infectivity, blocks syncytium formation and inhibits the binding of recombinant gp120 to immobilized soluble CD4 in vitro (Furukawa et al., 1994). We have now investigated the precise mode of action of SA-1042. We show here that SA-1042 specifically antagonizes the binding of anti-V3 loop antibodies or anti-CD4 binding-site antibodies to recombinant gp120, and also blocks the binding of an anti-V3 loop antibody to the V3 peptide (gp120IIIB: aa302-324). In contrast, SA-1042 does not inhibit gp120 binding of monoclonal antibodies directed to other regions of gp120, such as the conserved N-terminal regions (gp120IIIB: aa35-108 or gp120IIIB: aa72-130) or the C-terminal region (gp120IIIB: aa481-496). Furthermore, SA-1042 does not interfere with the binding of monoclonal antibodies directed to other molecules, gp41, CD4, CD11a, CD18, CD26, CD44 or CD54. These data suggest that SA-1042 exerts its antiviral effects by targeting the V3 loop as well as the CD4 binding site on gp120.

EI-1507-1 and -2, novel interleukin-1 beta converting enzyme (ICE) inhibitors, were isolated from the culture broths of Streptomyces sp. E-1507. EI-1507-1 and EI-1507-2 selectively inhibited the recombinant human ICE activity with IC50 values of 0.23 and 0.42 microM, respectively. EI-1507-1 and EI-1507-2 also inhibited mature interleukin-1 beta secretion from THP-1 cells with IC50 values of 1.1 and 1.4 microM, respectively.

Wortmannin inhibited phosphatidylinositol 3-kinase (P13-kinase) and Fc epsilon RI-mediated histamine secretion in RBL-2H3 cells to a similar degree, with IC50 values of 3 and 2 nM, respectively. Although P13-kinase is an acknowledged regulator of intracellular trafficking and secretion, wortmannin has proved to be a difficult drug to use in assessing the role of P13-kinase because it inhibits another important enzyme, myosin light-chain kinase (MLCK; IC50 = 200 nM). In the present study we synthesized a unique derivative of wortmannin, O-acetyl-delta 16-wortmannin-17-ol (KT7692), that has an inhibitory potency against PI3-kinase one-hundredth that of wortmannin, but retains a similar potency to wortmannin against MLCK. Histamine secretion was influenced 100-fold more by wortmannin than by KT7692.2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a structurally different PI3-kinase inhibitor from wortmannin, inhibited PI3-kinase with an IC50 of 2 microM but had little effect on MLCK activity in this concentration range. LY294002 also inhibited histamine secretion in RBL-2H3 cells with an IC50 of 5 microM. These results provide further evidence that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion after stimulation of Fc epsilon RI. Furthermore KT7692 in combination with wortmannin and LY294002 would be a powerful tool for clarifying the involvement of PI3-kinase as distinct from that of MLCK in signal transduction systems of various cellular responses.

The technique of isoenzyme electrophoresis was applied to Japanese wild populations of Taenia taeniaeformis (isolated from Norway rats) and three laboratory reared isolates (KRN isolated from a Malaysian Norway rat, BMM from a Belgian house mouse and ACR from a Japanese gray red-backed vole). The average heterozygosities of Japanese wild populations were fairly small and total genetic variability was 0.0499. The genetic make-up of T. taeniaeformis in Norway rats was rather uniform in the whole of Japan. In KRN isolate, each of all 10 loci examined possessed the allele which was predominant in Japanese wild populations. Similarly, each of 9 loci in BMM isolate possessed the same alleles, but one of 2 alleles at HK locus was different from that in the others. T. taeniaeformis parasitizing house mice and rats were considered to be genetically closely related to each other. In ACR isolate, 7 out of 10 loci possessed different alleles from those in the other populations. It was considered that ACR isolate was genetically distant and its phylogenetic origin in Japan should be different from worms parasitizing Norway rats.

Two groups of Paragonimus westermani (Tematoda: Platyhelminthes) exist in nature: diploids and triploids. Generally, these two groups live allopatrically, but in Kuandian, Liaoning Province, in the Republic of China, they live sympatrically. In our Chinese experiment on Paragonimus we used metacercariae of P. westermani, which we collected in Kuandian, Xigutai, and performed a cytological analysis. The results were as follows: (1) the P. westermani in Xigutai lived sympatrically as diploids and triploids; (2) all of the small metacercariae were diploids; (3) the large metacercariae were in large proportion triploids; (4) we found one tetraploid specimen in both the medium and the large metacercariae--this was the first time tetraploid lung flukes were discovered; (5) the somatic chromosomes of the tetraploids were different in numbers (4n = 44), but we could not find any difference in the karyotype of haploid sets and that of the diploids and the triploids; (6) unlike the triploids, during their meiosis the tetraploids produced a chromosome pairing, and we found a tendency of the large chromosomes to become quadrivalent; and (7) also unlike the triploids, a great number of spermatids were found in the tetraploid testes. Because of these findings, we can consider tetraploids to be autotetraploids, and these are probably produced by the fertilization of diploids and triploids. We also think that the gametes of tetraploids have a fertilization capability.

Oligodeoxyribonucleotides (ODN) linked at their 5'-end with dimethoxytrityl (DmTr) residue were examined for antiviral activities against human immunodeficiency virus type 1 (HIV-1). We found that guanine-rich oligonucleotides exhibit anti-HIV activity upon 5'-end modification with DmTr. One oligonucleotide, DmTr-TGGGAGGTGGGTCTG (SA-1042), showed potent anti-HIV activity in vitro. A greater than 95% reduction of infectivity was observed if the cells were treated with 10 micrograms/ml of SA-1042 at the time of viral infection, PCR analysis confirmed that there was a significant reduction of provirus in the cells exposed to virus in the presence of SA-1042. By contrast, no inhibition was observed if the cells were treated with the oligomer 1 h after virus adsorption. SA-1042 prevented syncytium formation between chronically infected cells and CD4 positive uninfected cells. Furthermore, the oligomer interfered the interaction of purified gp120 to the CD4 receptor. By contrast, SA-1042 had no inhibitory effect on chronically HIV-infected cells. These results strongly suggest that the DMTr-ODNs with appropriate base sequences antagonize HIV-1 infection during the stage of virus-cell interaction.

Leishmania parasites isolated from two patients with cutaneous leishmaniasis from geographically different localities in Paraguay have been characterized by enzyme electrophoresis (zymodeme) and digestion profiles of kinetoplast DNA with restriction enzymes (schizodeme). Both Paraguayan isolates showed identical zymodeme profiles to each other using 14 enzymes (glutamic pyruvate transaminase, glutamic oxaloacetic transaminase, enolase, fumarate hydratase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, malic enzyme, mannose phosphate isomerase, nucleoside phosphorylase, peptidase-D, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and pyruvate kinase). Although two Paraguayan isolates showed different zymodeme profiles from those of six Leishmania reference strains of Old and New World Leishmania species, they showed identical zymodeme profiles to those of an L. major-like parasite from Ecuador. These observations were confirmed by schizodeme analysis using three restriction endonucleases (Msp I, Hae III, and Taq I). These results indicate that Leishmania parasites isolated in Paraguay are identified as an L. major-like parasite, and it is necessary to consider the existence of L. major-like parasites when classifying Leishmania isolates from the New World.

Twelve enzymes (encoded by 14 loci) in liver flukes of Fasciola species originating from Japan (parthenogenetic triploids), Korea (parthenogenetic diploids), the United States of America (USA) and Australia (all sexual diploids) were analysed using starch gel electrophoresis. Variation in electrophoretic patterns between samples was detected at fic enzyme loci (Ak, Got, Gpi, 6-Pgd and Pgm-2). Japanese worms (31, of which six were established as uniparental laboratory strains), which reproduce by parthenogenesis, exhibited three different isozyme patterns. This indicates that triploidy has arisen more than once in Japanese flukes. Japanese Fasciola sp. can be separated into three types on morphological grounds. For the six laboratory strains of Japanese worms, the parental morphological type was known. Each of the three isozyme patterns observed was restricted to one morphological type. Most alleles detected in the Japanese triploids were also found in diploid worms from the other countries: the only alleles not represented elsewhere were four at the Got locus and two at the Pgm locus. Flukes from a laboratory strain derived from a single Korean diploid worm resembled the Japanese worms in genotype more closely than did America (seven uniparental laboratory strains) or Australian (30 worms) specimens. Worms from the last two countries were closely related.

Restriction endonuclease analysis of mitochondrial DNA was carried out in order to differentiate diploid and triploid types of Paragonimus westermani originating from Japan. It was found that out of 16 restriction enzymes. PstI, HaeIII and RsaI revealed consistently different cleavage patterns between the two types.

Five new chlorinated phenyl ethers, russuphelins B (2), C (3), D (4), E (5) and F (6) have been isolated from the mushroom Russula subnigricans Hongo, and their structures were elucidated by spectroscopic and chemical means. Russuphelins B (2), C (3) and D (4) exhibited cytotoxic activity in vitro against P388 leukemia cells.

A new cytotoxic substance, designated russuphelin A (1), has been isolated from the mushroom Russula subnigricans Hongo (Basidiomycetes). The structure was elucidated as 2,6-bis(2,6-dichloro-4-hydroxyphenyloxy)-1,4-dimethoxy-benzene on the basis of spectroscopic data and confirmed by total synthesis.

C-banding patterns have been observed in 14 specimens of Paragonimus westermani from Jaro, Leyte, the Philippines. One of them showed mutations of 2 chromosomes in spreads. Comparative analysis of C-banding patterns between the individual and standard type clarified that the mutant resulted from a reciprocal translocation between the long arms of chromosomes 4 and 6.

A total of 18 enzymes (encoded by 20 loci) from Paragonimus heterotremus collected at a local area in Saraburi, Thailand, were electrophoretically compared with those from the three Japanese species P. ohirai, P. miyazaki and P. westermani using horizontal starch-gel electrophoresis. Relative genetic distances between these species were quantified. The Thai P. heterotremus was found to be most closely related to P. miyazaki and rather distantly related to P. westermani. On the other hand, among the three Japanese species, the closest relationship was observed between P. miyazaki and P. ohirai.

Lymphokine-activated killer (LAK) cells were induced from C57BL/6 mouse spleen cells and the effects of culture time on the expression of cell surface phenotypes and cytotoxic activity of LAK cells were determined. The expression of CD2 remarkably decreased after culture of LAK cells for 30 days, while LFA-1, a principal adhesion molecule in LAK cells, and CD3 were not changed by the culture. LAK cells cultured for 90 days completely lost CD2. In accordance with the decrease of CD2, the cytotoxic activity of LAK cells declined but a certain leven was retained even after the complete loss of CD2. The established LAK cell clones were also strongly positive for the expression of LFA-1 but negative for CD2. When the LAK cell clones were transfected with the CD2 cDNA, they started to express CD2 on their cell surface and to show greater binding ability and stronger cytotoxicity to target tumor cells. These results indicated that CD2 plays a role as an adhesion molecule responsible for target cell killing in murine LAK cells.

To assess the properties of lymphokine-activated killer (LAK) cells, we established mouse LAK cell clones from LAK cell lines induced from C57BL/6 mouse spleen cells. Although these clones expressed similar phenotypes to the parent LAK cells, Lyt-2 was expressed in a restricted portion of the clones. All clones were found to express T3 CD2 and T cell receptor (TcR) alpha beta on their cell surface. Rearrangement patterns of TcR beta were the same among the clones derived from the same parent cell line but differed in those from different cell lines as determined by using C beta 1 and J beta 2 probes. The molecules responsible for LAK-target cell binding were examined by using a monoclonal antibody (mAb) against lymphocyte function associated antigen 1 (LFA-1). This mAb (termed KBA) showed inhibitory effects on both LAK-target cell binding and cytolytic activity of LAK cell clones, indicating a principal role of LFA-1 in LAK cell clones. The magnitude of perforin mRNA expression in LAK cell clones was unrelated to their cytolytic activities.