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A rare case of primary hyperparathyroidism associated with primary aldosteronism and breast cancer is reported. A 44-year-old woman was admitted to our hospital to undergo surgical removal of breast cancer. She had hypertension with low serum potassium, and slightly but significantly elevated serum calcium levels. Further studies demonstrated an enlarged left superior parathyroid gland and a left aldosterone-producing adrenocortical adenoma. Blood pressure was controlled with spironolactone and nifedipine, and left mastectomy was done for breast cancer. The pathological diagnosis was scirrhous breast carcinoma. Although the postoperative course was uneventful, her serum calcium gradually and progressively rose to higher levels. Left superior parathyroidectomy and left adrenalectomy were then performed simultaneously. The pathological diagnoses of the resected parathyroid gland and adrenal gland were parathyroid chief cell adenoma and adrenocortical adenoma with hyperplasia of zona glomerulosa, respectively. To clarify if the occurence of these tumors may be related to MEN1 gene mutations, we analyzed MEN1 gene in this patient, and found a loss of heterozygosity of the MEN1 locus in the parathyroid adenoma and breast cancer. Thus, we conclude that an alteration of the MEN1 gene and/or another tumor suppressor gene located at the MEN1 locus on chromosome 11q13 may be responsible for the development of parathyroid adenoma and breast cancer in our patient suggesting that the clinical spectrum of MEN1 might include breast cancer. In addition, serum calcium should be interpreted with caution in primary aldosteronism, because hypercalcemia may be masked in the presence of aldosterone excess.

Two new serratane-type triterpenoids, 1 and 2, were isolated from the stem bark of Picea jezoensis Carr. var. jezoensis (Pinaceae). Their structures were determined to be 3beta-methoxyserrat-13-en-21beta-ol (1) and 13beta, l4beta-epoxy-3beta-methoxyserratan-21beta-ol (2) on the basis of spectroscopic methods and partial syntheses. Compounds 1 and 2 and their acetates were screened as potential anti-tumor promoters by using the in vitro short-term 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced Epstein-Barr virus early antigen (EBV-EA) activation assay. IC50 value evaluation showed that compound 1 was more effective than others. In addition, compounds 1 and 2 were examined for anti-tumor promoting activities in a two-stage carcinogenesis assay of mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. Compounds 1 and 2 exhibited significant anti-tumor promoting effects on mouse skin carcinogenesis.

The potential of a dendritic cell (DC)-based vaccine against human immunodeficiency virus type 1 (HIV-1) infection in humans was explored with SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC). HIV-1-negative normal human PBMC were transplanted directly into the spleens of SCID mice (hu-PBL-SCID-spl mice) together with autologous mature DCs pulsed with either inactivated HIV-1 (strain R5 or X4) or ovalbumin (OVA), followed by a booster injection 5 days later with autologous DCs pulsed with the same respective antigens. Five days later, these mice were challenged intraperitoneally with R5 HIV-1JR-CSF. Analysis of infection at 7 days postinfection showed that the DC-HIV-1-immunized hu-PBL-SCID-spl mice, irrespective of the HIV-1 isolate used for immunization, were protected against HIV-1 infection. In contrast, none of the DC-OVA-immunized mice were protected. Sera from the DC-HIV-1- but not the DC-OVA-immunized mice inhibited the in vitro infection of activated PBMC and macrophages with R5, but not X4, HIV-1. Upon restimulation with HIV-1 in vitro, the human CD4+T cells derived from the DC-HIV-1-immunized mice produced a similar R5 HIV-1 suppressor factor. Neutralizing antibodies against human RANTES, MIP-1α, MIP-1β, alpha interferon (IFN-α), IFN-β, IFN-γ, interleukin-4 (IL-4), IL-10, IL-13, IL-16, MCP-1, MCP-3, tumor necrosis factor alpha (TNF-α), or TNF-β failed to reverse the HIV-1-suppressive activity. These results show that inactivated HIV-1-pulsed autologous DCs can stimulate splenic resident human CD4+T cells in hu-PBL-SCID-spl mice to produce a yet-to-be-defined, novel soluble factor(s) with protective properties against R5 HIV-1 infection.

A low molecular weight nonpeptide compound, KRH-1636, efficiently blocked replication of various T cell line-tropic (X4) HIV type 1 (HIV-1) in MT-4 cells and peripheral blood mononuclear cells through the inhibition of viral entry and membrane fusion via the CXC chemokine receptor (CXCR)4 coreceptor but not via CC chemokine receptor 5. It also inhibited binding of the CXC chemokine, stromal cell-derived factor 1α, to CXCR4 specifically and subsequent signal transduction. KRH-1636 prevented monoclonal antibodies from binding to CXCR4 without down-modulation of the coreceptor. The inhibitory effect against X4 viral replication by KRH-1636 was clearly reproduced in the human peripheral blood lymphocyte/severe combined immunodeficiency mouse system. Furthermore, this compound was absorbed into the blood after intraduodenal administration as judged by anti-HIV-1 activity and liquid chromatography MS in the plasma. Thus, KRH-1636 seems to be a promising agent for the treatment of HIV-1 infection.

Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. We examined F1 progeny from a crossing between the urease-negative strain (environmental isolate, serotype A, mating type alfa, haploid) and a tester strain (B 3502 from NIH of USA; urease-positive, serotype D, mating type a, haploid) for serotype, mating type, ploidy and urease activity, and performed partial sequencing of the urease gene. Phenotypes of the F1 progeny and results of SSCP analyses suggested that the serotype AD strain of the F1 progeny is a hybrid of the parental serotype A and D strains.

A new metabolite, pileotin A, was isolated from a strain of Aspergillus fumigatus originally obtained from the sea urchin Toxopneustes pileolus together with the known compound oxalicine B. The relative stereostructure of pileotin A was elucidated based on spectroscopic analyses, including 1D and 2D NMR techniques. This unique pyridino-α-pyrone sesquiterpene type metabolite has spiro-δ-lactone at C-4.

The major surface glycoprotein (Msg) of Pneumocystis jiroveci (P. jiroveci) is important in the immunopathogenesis of Pneumocystis pneumonia (PcP), but is difficult to study in humans. We generated 3 overlapping recombinant Msg fragments (MsgA, MsgB and MsgC), and analyzed their reactivity with serum samples from 95 healthy blood donors and 94 human immunodeficiency virus (HIV)-infected persons. Reactivity to the Msg fragments varied with HIV infection and prior episodes of PcP but not with geographic origin. Recognition of MsgA was lower-and recognition of MsgB was significantly lower-in HIV(+) serum compared with donor serum. Serum samples from HIV-positive patients with prior PcP recognized MsgC more frequently than did serum samples from those without PcP. None of the serum samples drawn from 9 patients before they had developed PcP recognized MsgC. These data suggest that these novel recombinant proteins are useful for the analysis of antibody responses to Msg.

Two novel skeletal triterpene aldehydes, jezananals A ( 1 ) and B ( 2 ) were isolated from the stem bark of Picea jezoensis var. jezoensis (Pinaceae). Their absolute stereo structures were determined to be 21β-hydroxy-3β-methoxy-16(15→14)abeo-13R,14S-serratan-15-al and the 3α-epimer, on the basis of spectral, single crystal X-ray analysis and chemical conversion. Compounds 1 and 2 are based on the unique 16(15→14)abeo-serratane skeleton. 3β-Methoxyserrat-14-en-21β-ol ( 3 ), 3α-methoxyserrat-14-en-21β-ol ( 4 ), and 14β,15β-epoxy-3β-methoxyserratan-21β-ol ( 5 ), which is considered to be intermediates of compounds 1 and 2 , and showed strong inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) induction, while compounds 1 and 2 were inactive.

The first synthesis of a labdane diterpenoid, (-)-13-oxo-15,16-dinorlabda-8(17),11E-dien-19-oic acid [(-)-1a], isolated from the stem bark of Thuja standishii (GORD.) CARR., from the major component trans-communic acid (3a) is described.

Trichodermanins C–E (1–3), new diterpenes with a rare fused 6-5-6-6 ring system, have been isolated from a fungus Trichoderma harzianum OUPS-111D-4 separated from a piece of a marine sponge Halichondria okadai, and these chemical structures have been established by spectroscopic analyses using IR, MASS, HRFABMS, and NMR spectra. We established their absolute stereostructures by application of the modified Mosher’s method. In addition, 1 inhibited the growth of cancer cell lines potently.

Birt–Hogg–Dube syndrome is a familial genodermatosis, of which patients frequently develop renal neoplasms, fibrofolliculomas and pneumatocele. Here, we report a mother and daughter with renal neoplasms surgically resected (69 and 46 years-of-age at surgery, respectively). The mother's tumor was diagnosed as an unclassified type renal cell carcinoma associated with microscopic tumorous nodules, whereas the daughter's tumor was a hybrid oncocytic/chromophobe tumor. The germline mutation analysis of the responsible gene, FCLN (the folliculin gene), showed a deletion of 18 bp in exon 5 (c.332_349del/p.H111_Q116del), predicting an alteration of the amino acid sequence of “HPSHPQ” replaced by a single amino acid, “L”. This is a novel germline mutation of the FCLN gene that has not been previously reported.

Birt-Hogg-Dubé syndrome (BHD) is an inherited disorder associated with a germline mutation of the folliculin (FLCN) gene. Most patients with BHD have multiple pulmonary cysts, and are at high risk of repeated pneumothorax. Although an increasing number of patients are diagnosed with BHD by genetic testing, therapeutic approaches for intractable pneumothorax have not yet been described.We treated three patients who had repeated episodes of pneumothorax. All had multiple pulmonary cysts in the lower lobes, and two had a family history of pneumothorax. Video-assisted thoracic surgery was used to perform wedge resections and partial pleural covering of the cystic lesions. The partial pleural covering technique used sheets of polyglycolic acid felt or regenerative oxidized cellulose mesh. The resected tissues underwent histopathological evaluation, and peripheral blood leukocytes were tested for FLCN mutations.The operative times were less than 2 h, and there were no complications. The resected cysts had histopathological features characteristic of BHD lung. All patients were found to have FLCN germline mutations; thus their repeated pneumothoraces were a manifestation of BHD. None of the patients developed respiratory problems after undergoing the partial pleural covering procedure, and they have all been well without pneumothorax for 30 months or more.Partial pleural covering combined with resection of protruding cysts should be a safe and effective therapeutic approach for BHD patients with intractable pneumothorax. Further investigation is needed to establish a detailed protocol for treatment of pneumothorax that results in minimal functional impairment.

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder associated with a germline mutation of folliculin (FLCN). The affected families are at a high risk for developing multiple renal cell carcinomas (RCC). Little is known about the immunostaining patterns of mutant FLCN-associated RCCs. We investigated 32 RCCs obtained from 17 BHD patients. The studied tumors included chromophobe RCCs (n = 15), hybrid oncocytic/chromophobe tumors (HOCT) (n = 14) and clear cell RCCs (n = 3). Almost all chromophobe RCCs and HOCTs revealed positive staining for S100A1, Ksp-cadherin and CD82. They stained either focally or diffusely for CK7, and were negative for CA-IX. All clear cell RCCs were positively stained for CA-IX and negative for CK7. These data confirmed that mutant FLCN-associated oncocytic and clear cell RCCs exhibited generally similar immunostaining patterns compared to their sporadic counterparts. Frequent positive staining for S100A1, Ksp-cadherin and CD82 in chromophobe RCCs and HOCTs indicated that these two types were relatively similar rather than distinctively different in their patterns of immunoreactivity. Characteristic peri-nuclear halos and polygonal cells with clear cytoplasm, which often misleads pathologists into the diagnosis of clear cell RCC, should be carefully examined using an immunohistochemical panel including CA-IX, Ksp-cadherin, CD82 and CK7.

Birt–Hogg–Dube syndrome (BHD) is an inherited disorder associated with a germline mutation of the folliculin gene (FLCN). The affected families have a high risk for developing multiple renal cell carcinomas (RCC). Diagnostic markers that distinguish between FLCN-related RCC and sporadic RCC have not been investigated, and many patients with undiagnosed BHD fail to receive proper medical care. We investigated the histopathology of 27 RCCs obtained from 18 BHD patients who were diagnosed by genetic testing. Possible somatic mutations of RCC lesions were investigated by DNA sequencing. Western blotting and immunohistochemical staining were used to compare the expression levels of FLCN and glycoprotein non-metastatic B (GPNMB) between FLCN-related RCCs and sporadic renal tumors (n = 62). The expression of GPNMB was also evaluated by quantitative RT-PCR. Histopathological analysis revealed that the most frequent histological type was chromophobe RCC (n = 12), followed by hybrid oncocytic/chromophobe tumor (n = 6). Somatic mutation analysis revealed small intragenic mutations in six cases and loss of heterozygosity in two cases. Western blot and immunostaining analyses revealed that FLCN-related RCCs showed overexpression of GPNMB and underexpression of FLCN, whereas sporadic tumors showed inverted patterns. GPNMB mRNA in FLCN-related RCCs was 23-fold more abundant than in sporadic tumors. The distinctive expression patterns of GPNMB and FLCN might identify patients with RCCs who need further work-up for BHD.

Tandyukisin (1), a novel decalin derivative with an enolic β-ketoaldehyde, has been isolated from a strain of Trichoderma harzianum OUPS-111D-4 originally derived from the marine sponge Halichondria okadai, and its structure has been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques. In addition, the absolute configuration for 1 was established by the application of CD spectrum to the tribenzoate derivative. This compounds exhibited moderate cytotoxicity against human cancer cell lines.

Background Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases, including adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Because these life-threatening and disabling diseases are not yet curable, it is important to prevent new HTLV-1 infections. Findings In this study, we have established a simple humanized mouse model of HTLV-1 infection for evaluating prophylactic and therapeutic interventions. In this model, HTLV-1-negative normal human peripheral blood mononuclear cells (PBMCs) are transplanted directly into the spleens of severely immunodeficient NOD-SCID/γcnull (NOG) mice, together with mitomycin-treated HTLV-1-producing T cells. Using this model, we tested the efficacy of monoclonal antibodies (mAbs) specific to HTLV-1 as well as human IgG isolated from HAM/TSP patients (HAM-IgG) in preventing HTLV-1-infection. One hour before and 24 h after transplantation of the human cells, each antibody sample was inoculated intraperitoneally. On day 14, human PBMCs isolated from the mouse spleens were tested for HTLV-1 infection. Whereas fresh CD4-positive and CD8-positive T cells isolated from untreated mice or mice treated with isotype control mAb, HTLV-1 non-neutralizing mAbs to envelope gp46, gag p19, and normal human IgG were all infected with HTLV-1; the mice treated with either HTLV-1 neutralizing anti-gp46 mAb or HAM-IgG did not become infected. Conclusions Our data indicate that the neutralizing function of the antibody, but not the antigen specificity, is essential for preventing the in vivo transmission of HTLV-1. The present animal model will also be useful for the in vivo evaluation of the efficacy of candidate molecules to be used as prophylactic and therapeutic intervention against HTLV-1 infection. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0074-z) contains supplementary material, which is available to authorized users.

Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs.

Three new multiflorane-type triterpene esters, i.e. 7α-hydroxymultiflor-8-ene-3α,29-diol 3-acetate-29-benzoate (1), 7α-methoxymultiflor-8-ene-3α,29-diol 3,29-dibenzoate (2), and 7β-methoxymultiflor-8-ene-3α,29-diol 3,29-dibenzoate (3), were isolated from seeds of Cucurbita maxima, along with the known compound, multiflora-7,9(11)-diene-3α,29-diol 3,29-dibenzoate (4). Compound 1 exhibited melanogenesis inhibitory activities comparable with those of arbutin. In cytotoxicity assays, compounds 1 and 3 exhibited weak cytotoxicity, with IC50 values of 34.5–93.7 μM against HL-60 and P388 cells.

OX40/OX40 ligand (OX40L) proteins play critical roles in the T cell-B cell and T cell-dendritic cell interactions. Here we describe the intercellular transfer of OX40L molecules by a non-Ag specific manner. After 2-h coculture of activated CD4+ T cell (OX40L−, OX40+) with FLAG peptide-tagged OX40L (OX40L-flag) protein-expressing COS-1 cells, the OX40L-flag protein was detected on the cell surface of the CD4+ T cells by both anti-OX40L and anti-FLAG mAbs. The intercellular OX40L transfer was specifically abrogated by pretreatment of the COS-1 cells with anti-OX40L mAb, 5A8. The OX40L transfer to OX40-negative cells was also observed, indicating an OX40-independent pathway of OX40L transfer. HUVECs, allostimulated monocytes, and human T cell leukemia virus type I-infected T cells, which all express OX40L, can potentially act as the donor cells of OX40L. The entire molecule of OX40L was transferred and stabilized on the recipient cell membrane with discrete punctate formation. The transferred OX40L on normal CD4+ T cells was functionally active as they stimulated latent HIV-1-infected cells to produce viral proteins via OX40 signaling. Therefore, these findings suggest that the intercellular molecular transfer of functional OX40L may be involved in modifying the immune responses.

Osteopontin (OPN), a secreted adhesive glycoprotein, is significantly overexpressed in a variety of malignant tumors. Moreover, increased levels of OPN have been detected in the blood of patients with metastatic carcinoma. On immunohistochemical examination, OPN was shown to be expressed in papillary thyroid carcinoma. To investigate the mechanisms of the induction of OPN expression in papillary thyroid carcinoma, we examined the expression of c-src and OPN in 30 cases of papillary thyroid carcinoma by Northern blotting, Western blottimg, in situ hybridization and immunohistochemistry. Each of 5 cases studied by Northern and Western blotting analyses revealed substantial increases of c-src and OPN mRNA and protein levels in tumor areas as compared with surrounding normal thyroid tissues. Moreover, signals of c-src and OPN mRNA were detected in both infiltrating mononuclear cells and tumor cells by in situ hybridization. Immunohistochemical localizations of c-Src and OPN proteins were the same as those of transcripts described above. These results suggested that the induction of the expression of OPN invasiveness in papillary thyroid carcinoma was one of the important downstream events of increased c-Src expression.

Nine lanostane and serratane-type triterpenes including two unknown compounds were isolated from the stem bark of Pinus luchuensis. These new compounds were characterized as 3-oxolanost-9(11)-ene-24S,25-diol (1) and 29-acetoxy-3beta-methoxyserrat-14-en-21alpha-ol (2) on the basis of spectroscopic evidence. Some of these triterpenes were tested for the inhibitory effect on DNA topoisomerase II activity. Compound 1 showed a slightly less potent inhibitory activity with an IC(50) value of 186 microM.

Seven labdane-type diterpenoids from the stem bark of Thuja standishii (Gord.) Carr. (Cupressaceae) and their analogues showed strong inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among these compounds, 15,16-bisnor-13-oxolabda-8(17), 11E-dien-19-oic acid was revealed to have the strongest inhibitory effect on the EBV-EA activation, being stronger than that of beta-carotene which has been intensively studied in cancer prevention using animal models. 15,16-bisnor-13-Oxolabda-8(17), 11E-dien-19-oic acid was also found to exhibit the excellent anti-tumor promoting activity in two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene and TPA.

A novel rearranged labdane-type diterpenoid, 19(4--3)abeo-8alpha, 13(S)-epoxylabda-4(18),14-diene (1), and two new abietane-type diterpenoids, 19-nor-abieta-4(18),8,11,13-tetraen-7-one (2) and 12-hydroxydehydroabietic acid (3) were isolated from the stem bark of Picea glehni, together with seven known diterpenoids-13-epimanoyl oxide (4), dehydroabietic acid (5), (11E)-14, 15-bisnor-8alpha-hydroxy-11-labden-13-one (6), abieta-8,11, 13-trien-7-one (7), 9alpha,13alpha-epidioxyabiet-8(14)-en-18-oic acid (8), 9,10alpha-epoxy-9,10-seco-abieta-8,11,13-trien-18-oic acid (9), and methyl 15-hydroxy-7-oxo-dehydroabietate (10). Compounds 5-8 and 10 showed potent inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate.

A new podocarpane diterpene, 7α,15-dihydroxypodocarp-8(14)-en-13-one (1) and eight new abietane diterpenes, abiet-8(14)-en-7α,13β,15,18-tetraol (2), 7α,13β,15-trihydroxyabiet-8(14)-en-18-oic acid (3), 13β,18-dihydroxyabiet-8(14)-en-7-one (4), 13β,15,18-trihydroxyabiet-8(14)-en-7-one (5), 7-oxo-13β-methoxyabiet-8(14)- en-18-oic acid (7), abiet-8(14)-en-7α,12α,13β,18-tetraol (8), 7α,12α,13β-trihydroxyabiet-8(14)-en-18-oic acid (9), and abiet-8(14)-en-7α,12α,13β,15,18-pentaol (10) were isolated from the CHCl3 extract of the cones of Larix kaempferi. Their structures were determined on the basis of chemical and spectral evidence. The inhibitory effects of compounds 1-4 and 7-9 on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), were examined for a primary screening for anti-tumor promotors. Key words: Larix kaempferi, Pinaceae, cones, abietane, EBV-EA.

Three new ergostane-type steroids, 3beta-hydroxy-4alpha, 14alpha-dimethyl-5alpha-ergosta-8,24(28)-dien-11 -one (1); 3beta, 11alpha-dihydroxy-4alpha,14alpha-dimethyl-5alpha -ergosta-8, 24(28)-dien-7-one (2); and 3beta,7alpha-dihydroxy-4alpha, 14alpha-dimethyl-5alpha-ergosta-8,24(28)-dien-11 -one (3), were isolated, together with two known triterpenoids, wrightial and lup-20(30)-ene-3beta,29-diol from the whole herb of Euphorbia chamaesyce. Compound 3 showed a potent inhibitory effect on Epstein-Barr virus early antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA).

Standishinal (1), a new irregular abietane-type diterpenoid, was isolated from the bark of Thuja standishii and the absolute stereostructure was established by 2D NMR and X-ray crystallographic analyses. It was found that treatment of 12-hydroxy-6,7-seco-abieta-8,11,13-triene-6,7-dial (2) with BF3·OEt2 at 0°C in CH2Cl2 furnished compound 1.

A CHCl 3 extract of the stem bark of Picea jezoensis var. hondoensis (Pinaceae) afforded two triterpenoids named piceanonols A and B with a novel 14(13 → 12) abeo -12α H -serratane skeleton. Extensive NMR studies, MS spectral analyses and X-ray crystallographics established their structures as 3β-methoxy-14β,21β-dihydroxy-14(13 → 12) abeo -12α H -serratan-13-one and 21α-methoxy-14β-hydroxy-14(13 → 12) abeo -12α H -serratane-3,13-dione.

Two new ergostane-type steroids were isolated together with two known compounds, obtusifoliol and 4α,14α-dimethyl-5α-ergosta-7,9(11),24(28)-trine-3β-ol, from the whole herb of Euphorbia chamaesyce . The structures of the new compounds were established as 3β-hydroxy-4α,14α-dimethyl-5α-ergosta-8,24(28)-dien-7-one and 3β-hydroxy-4α,14α-dimethyl-5α-ergosta-8,24(28)-diene-7,11-dione on the basis of chemical and spectral evidence.

Thirty-three strains of Cryptococcus neoformans were isolated from clinical specimens, including specimens from AIDS patients in Brazil, and were classified into two serotypes; we detected 31 and 2 strains of serotypes A and B, respectively. Random amplified polymorphic DNA (RAPD) fingerprint pattern analyses of these strains of serotypes A and B showed that the patterns were similar for strains of each serotype when three 10-mer primers were used as the RAPD primers. Comparative studies of the fingerprint patterns of the study isolates with those of the reference strains also showed that the RAPD patterns for strains of each serotype were related and that most of the fingerprint bands existed commonly for all strains of each serotype tested. The common RAPD bands (an approximately 700-bp band for serotype A and an approximately 450-bp band for serotype B) were extracted and the DNA sequences were determined. Using this information, we prepared two and one PCR primer pairs which were expected to be specific for C. neoformans serotypes A and B, respectively. Use of each PCR primer combination thus prepared for serotype A or B was 100% successful in identifying the respective C. neoformans serotypes, including the 33 clinical isolates tested in the present study. Among these combinations, one for serotype A was found to amplify DNA from C. neoformans serotype B as well as serotype A. Serotype B-specific PCR primer pairs amplified DNA from not only serotype B strains but also from serotype C strains. The usefulness of other serotype-specific PCR primers for clinical C. neoformans isolates is discussed.

Osteopontin (OPN) is a secreted, adhesive glycophosphoprotein that has been implicated in both normal and pathologic processes. In recent years, it has been suggested that OPN expression also plays an important role in tumorgenesis, tumor progression and tumor metastasis. To investigate OPN expression in papillary thyroid carcinoma, thirty paraffin sections of papillary thyroid carcinoma confirmed pathologically were examined by immunohistochemistry and in situ hybridization. Immunopositive reactions for OPN-protein were obtained in the nuclear and perinuclear area in all cases. OPN mRNA was expressed in the carcinoma cells around the microcalcifications. The results demonstrated that OPN mRNA is expressed not only by the infiltrated macrophages but also by the papillary thyroid cancer cells. Hence, OPN expression can be considered a pathological feature of papillary thyroid carcinoma and further study into the relation between OPN and the biological behavior of papillary thyroid carcinoma is warranted.

Two new triterpenes were isolated from the leaves of Larix kaempferi together with two known compounds, 24-methylenecycloartanone and 24-methylenecycloartanol. A new triterpene-triol, nine known compounds, n-alkyl trans-ferulates and trans-p-coumarates, rheosmin, five stigmastanes and friedelin were isolated from the stem bark. These new compounds were characterized as cycloartan-3,24-dione, 24-methylenecycloartan-3alpha-ol and lanost-9(11)-en-3alpha,24S,25-triol on the basis of chemical and spectroscopic evidence.

Several kinds of naturally occurring fernane-type triterpenoids isolated from a Euphorbia genus were tested on the inhibitory effects of DNA Topoisomerases I (Topo I) and II (Topo II) activities. A-ring cleaved 3,4-seco-8 beta H-ferna-4(23),9(11)-dien-3-oic acid and its 3-hydroxyl derivative were found to be selective inhibitors of Topo II activity without the stabilization of a DNA/Topo II cleavable complex.

Two serratane triterpenoids were isolated from the stem bark of Picea jezoenis var. hondoensis, together with two known compounds, 3β-methoxyserrat-14-en-21-one and 3β-methoxyserrat-14-en-21α-ol. The serratane triterpenoids were characterized as 14β,15β-epoxy-3α-methoxyserratan-21β-ol and 3α-methoxy-21β-hydroxyserrat-14-en-16-one, on the basis of chemical and spectroscopic evidence.

Sixty-three Paracoccidioides brasiliensis isolates obtained from three nine-banded armadillos ( Dasypus novemcinctus), one Amazonian armadillo's and 19 clinical isolates were compared by random amplified polymorphic DNA analysis with the primer OPG-19. The isolates were divided into three major clusters, I, II and III. Coincidences between human and armadillo isolates were observed in clusters I and II. Cluster III consisted only of armadillos' isolates. The results suggested that (I) humans may acquire P. brasiliensis infection by contact with armadillo's environment, (II) there may be P. brasiliensis genotypes peculiar to the animal, and (III) individual armadillos may be infected with P. brasiliensis cells with different genotypes.

We studied three different isolates of Paracoccidioides brasiliensis obtained from the mesenteric lymph node (D3LY1), the spleen (D3S1) and the liver (D3LIV1) of the same armadillo ( Dasypus novemcinctus ).Pulmonal inflammatory area was evaluated by intravenous inoculation of 106 yeast cells of each isolates in young, male, ddY mice. Moreover, the partial sequence of GP43kDa gene of P. brasiliensis was analyzed. The lung inflammatory area was greater in animals inoculated with isolate D3S1. The partial sequence of GP43kDa gene indicated that isolate D3S1 is different from isolates D3LY1 and D3LIV1. This study suggested that the same armadillo might be susceptible to multiple P. brasiliensis isolates simultaneously.

3,4-seco-8beta H-Ferna-4(23),9(11)-dien-3-oic acid (EC-2) (1) and 3,4-seco-oleana-4(23),18-dien-3-oic acid (EC-4) (2) isolated from the whole herb of Euphorbia chamaesyce L. were evaluated for their anti-tumor activities using a panel including 39 human cancer cell lines (HCC) coupled with a drug sensitivity database. Compounds 1 and 2 showed strong cytotoxicity against the HCC panel, even though alcohols 1A and 2A and methyl esters 1B and 2B were inactive.

The new diterpene acids were isolated from the leaves of Larix kaempferi, together with two known sesquiterpenes, (−)-α-cadinol and oplopanone, and 10 known diterpenes, larixol, dehydroabietinol, 7-oxo-dehydroabietinol, 7-oxodehydroabietic acid, 9α,13α-epidioxyabiet-8(14)-en-18-oic acid, 9β,13β-epidioxyabiet-8(14)-en-18-oic acid, dehydroabietic acid, 7α-hydroxydehydroabietic acid, 8α,9α,13α,14α-diepoxyabietan-18-oic acid and 15-hydroxydehydroabetic acid. The new acids and their methyl esters were characterized as methyl abieta-8,11,13,15-tetraen-18-oic acid, methyl 16-nor-15-oxodehydroabietic acid and methyl 12,15-dihydroxydehydroabietic acid, on the basis of chemical and spectroscopic evidence.

We have studied the physiological and morphological features of 17 isolates ofParacoccidioides brasiliensis in order to define their phenotypes. The isolates were cultured at room temperature on potato dextrose agar (PDA, Difco) slants for mycelial growth and in 1% dextrose brain heart infusion agar (BHIA, Difco) at 37°C for the study of yeast forms. Most mycelial and yeast forms grew well between pH 5.6–9.4. In their response to osmotic pressure the isolates were separated in three groups: intolerant, intermediate and tolerant. They also varied in carbohydrate assimilation tests, which indicated important metabolic variation. No clear differences were observed in phenol oxidase tests, KNO3, starch, casein and arbutin assimilation tests. Only 1 of the isolates, Bt-19, had gelatinase activity. No correlation was observed between the above differences and virulence. Two patterns of growth were observed in the mycelial cultures, glabrous and cottonous, the latter being correlated with increased virulence for ddY mice. Most yeast forms grew as cerebriform colonies, but Pb-HC and Bt-19 colonies had a cobblestone-like surface.

BACKGROUND Adenocarcinomas and carcinoid tumors are the most common malignant tumors of the small intestine. K-ras oncogene mutations at codon 12 are common in gastric, pancreatic, and colon carcinomas, with an incidence of 35-88%. K-ras mutations have not been extensively studied in either adenocarcinomas or carcinoid tumors of the small bowel. The purpose of this study was to determine whether ras mutations play an important role in the formation of these tumors. METHODS Archival tissues from 28 adenocarcinomas and 22 carcinoid tumors of the small bowel were studied, along with archival tissues from 32 adenocarcinomas of the large bowel, which were used as controls. DNA from the small intestine tumors was analyzed for K-ras, H-ras, and N-ras oncogene mutations at codons 12, 13, and 61, using polymerase chain reaction and sequence specific oligonucleotide hybridization techniques. Large bowel adenocarcinomas were analyzed for K-ras mutations at codons 12 and 13. RESULTS A point mutation of K-ras at codon 12 was detected in 4 of 28 (14.3%) of the small bowel adenocarcinomas, in 12 of 32 (37.5%) of the large bowel adenocarcinomas, and in 0 of 22 small intestine carcinoid tumors. No other K-ras, H-ras, or N-ras mutations were detected in any of the small bowel tumors. Each small intestine K-ras mutation was found in a duodenal adenocarcinoma (4 of 12 cases, 33%), whereas none occurred in 16 other jejunal or ileal adenocarcinomas. CONCLUSIONS K-ras mutations appear to play a significant role in the pathogenesis of duodenal adenocarcinomas, but they do not appear to be important in the development of jejunal or ileal adenocarcinomas or of carcinoid tumors of the small intestine. Cancer 1997; 79:1804-8. © 1997 American Cancer Society.

A novel pentacyclic triterpene dione was isolated from the cuticle of Picea jezoensis var. jezoensis together with the known serrat-14-ene-3,21-dione (1), and the structure of this compound was determined as 21α-methoxyserrat-13-ene-3,15-dione (2). Detailed NOESY experiments revealed that 2 has a chair form of ring A and a chairlike conformation of ring C, respectively, in CDCl3 solution. Interestingly, single-crystal X-ray analysis indicates that in the solid state 2 has a deformed boat form of ring A, in which the 3-oxo and the 25-methyl groups are arranged in flag-pole positions, and a chairlike form of ring C.

We established four new mouse strains with defective T and B cells as well as defects in innate immunological reactions using an NK cell depletion antibody and showed that all mutant mouse strains efficiently received human peripheral blood leukocyte (PBL) engraftment (hu-PBL-scid mice). Higher levels of human immunodeficiency virus type 1 (HIV-1) replication were observed in these new hu-PBL-scid mice than in conventional hu-PBL-C.B-17-scid mice. In one particular strain, hu-PBL-NOD-scid mice, high levels of HIV-1 viremia (more than 10(6) 50% infectious doses per ml) were detected after infection with HIV-1. The plasma viral load was about 100 to 1,000 times higher than that observed in other hu-PBL-scid mice infected with HIV-1. Although high-level viremia did not correlate with the total amount of HIV-1 RNA in cells from infected mice, high levels of free virions were detected only in hu-PBL-NOD-scid mice. HIV-1 viremia induced systemic HIV-1 infection involving the liver, lungs, and brain. PCR in situ hybridization confirmed that HIV-1-infected cells invaded the brain tissue of the hu-PBL-NOD-scid mice. Our results suggest that the genetic background, including innate immunity, is critical in the development of primary HIV-1 viremia and subsequent central nervous system invasion with HIV-1. The hu-PBL-NOD-scid mouse represents a useful model for the study of the pathogenesis of HIV-1 in vivo, especially brain involvement, and therapy of primary HIV-1 viremia.

本研究の目的は, 韓国におけるスポーツ参与者の増大に影響を及ぼしたスポーツ施設, スポーツ関係の法律, スポーツ組織そしてスポーツ指導者などのスポーツ環境の変化を明らかにすることである。研究の方法は, 1980年代から最近までの韓国におけるスポーツ環境に関する諸資料及び文献を総合的に検討する。スポーツ活動という社会的行為が存続し発展していくためには, パーソンズの社会理論を援用するならば, スポーツ行為が自然的・社会的環境に適応し, 目標を立ててその達成に努力し, 人々の連帯性を高めて価値を維持しなければならない。本研究では, これまでの先行研究から, スポーツ環境を構成する下位体系としてスポーツ施設, スポーツ関係の法律, スポーツ組織そしてスポーツ指導者の4つの要件を取りあげた。主な結果は以下のとおりである。1) スポーツ施設はスポーツ参与に必要な物理的な環境を提供する。韓国におけるスポーツ施設は, 1984年から最近まで大きく変化した。とくに公共施設の充実に力をいれているが, その数は1984年451ヵ所, 1988年689ヵ所, 1994年2,621ヵ所そして1996年3,307ヵ所と大幅に増えた。2) スポーツ参与の目標を策定し, その目標の達成に向かって, 諸資源を動員する機能としてスポーツ関係の法律がある。韓国のスポーツ活動を法的に支えたのは, 1962年に施行された「国民体育振興法」と1989年に施行された「体育施設の設置・利用に関する法」である。その結果, スポーツ施設とスポーツ予算の確保, スポーツ指導者の養成などに大きな目標を与えた。3) スポーツ組織は, スポーツに関連する集団やスポーツ参与者を統合する機能を果たす。韓国におけるスポーツ関係の政府組織として,「体育省」が1982年に創設されて, スポーツ振興に強力な支えとなった。また, スポーツの民間組織としてYMCAと社会体育センターもそれぞれ人々のスポーツ参与に貢献した。4) スポーツ指導者は, スポーツ文化を維持し, 発展させる機能を担っている。韓国における「生活体育指導者」は, 1986年177名, 1989年2,629名, 1992年2,951名そして1994年1,595名が養成され, 国民のスポーツ参与に貢献した。

Experimental Penicillium marneffei infection in mice was investigated histopathologically and by electron microscopy. Viable conidia (5 × 106 cells) of P. marneffei were inoculated into each mouse of group A (BALB/c mice) and group B (BALB/c-nu/nu-SIc mice) through the tail vein. All the mice were sacrificed at intervals and the livers were examined. In group A, the conidia were phagocytosed by Kupffer cells soon after inoculation, and proliferated by fission in the cytoplasm. Marked prolieration of yeast cells was observed 7 and 14 days after inoculation. With proliferation of the fungus, the number of lysosomes in Kupffer cells increased, and numerous granulomas were formed in the liver. These granulomas consisted mainly of macrophages with yeast cells, together with a few polymorphonuclear leukocytes, lymphocytes and giant cells. From 28 days on yeast cells were gradually cleared from the granulomas, and 56 days after inoculation almost all the granulomas disappeared. In group B, at an early stage of i...

It is generally recognized that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) is the predominant population during the acute and asymptomatic phases of HIV-1 infection. Here, we compared the proliferation and syncytium-inducing activities of different HIV-1 strains in primary CD4+ T cells expressing various helper T (Th)-type cytokine profiles. The macrophage-tropic HIV-1 strains HIV-1JR-CSF, HIV-1NFN-SX, and HIV-1SF162 could proliferate vigorously and generate syncytia in primary CD4+ T cells irrespective of their Th subtype, in contrast to the T-cell-line-tropic HIV-1 strains HIV-1NL4-3 and HIV-1IIIB, which favored non-type 1 Th conditions. These results indicate that macrophage-tropic HIV-1 may be more invasive and virulent, since it kills more CD4+ Th1 cells than T-cell-line-tropic HIV-1 during the early stages of HIV-1 infection, when the Th1 immune response is dominant.

One hundred and thirty-nine strains of Cryptococcus neoformans were isolated in Thailand from clinical specimens including 97 AIDS patients: 67 from Northern, 48 from Central, 17 from Northeastern and 7 from Southern regional hospitals. Six out of the 139 strains were serotype B and the remaining 133 were A. There was no correlation between serotypes and regional distribution. To our knowledge, this is the first report of serotyping studies on C. neoformans in Thailand. Studies on random amplified polymorphic DNA (RAPD) analysis showed that this method is useful for the differentiation of C. neoformans var. gattii (serotypes B and C) and C. neoformans var. neoformans (serotypes A and D). They also indicated that Thai isolates of C. neoformans var. gattii (serotype B) were a homogeneous group on the basis of their genotypes. Antifungal susceptibility tests using 5 antifungal agents including amphotericin B, fluconazole, flucytosine, itraconazole and micronazole against 50 selected strains of C. neoformans showed that they were sensitive to all of the antifungal agents tested except for one strain that was resistant to flucytosine.

Scedosporium apiospermum, a ubiquitously distributed saprophyte, is emerging as an important pathogen in immunocompromised patients. We describe a 72-year-old patient with chronic S. apiospermum infection of the lung simulating aspergilloma. His medical history was unremarkable except that he had undergone partial lung resection as a treatment for pulmonary tuberculosis several decades previously. He had no underlying immunosuppressive conditions. This patient illustrates that pulmonary scedosporiosis is not confined to immunocompromised patients and that the clinical presentation may be indistinguishable from that of aspergilloma.

Uterine tumors with sex cord-like elements are divided in two groups; uterine tumors resembling ovarian sex cord tumors (UTROSCT), and endometrial stromal tumors with sex cord-like elements (ESTSCLE). UTROSCT is currently defined as the neoplasm predominantly or exclusively composed of sex cord-like elements, and generally behaves in a benign fashion. We studied two unusual cases of UTROSCT with metastasis. One case was a 38-year-old multiparous woman presented with hypermenorrhea. The tumor grew as an intramural mass, and metastasized to a pelvic lymph node. Another case was a 57-year-old woman presented with genital bleeding. The tumor grew as a submucosal exophytic mass, and metastasized to the epiploic appendix. Microscopic examination of the 2 cases revealed that they were composed of sex cord-like cells, epithelioid cells and spindle cells. They exhibited solid pattern in predominance. Both solid and sex cord-like elements showed similar immunoreactivities for more than 3 sex cord markers, but simultaneously showed different staining patterns for some other markers. Characteristic features of endometrial stroma such as tongue-like infiltration and spiral arteries-like arterioles were not observed. RT-PCR analysis confirmed the absence of JAZF1-SUZ12 gene fusion, supporting the histopathological diagnosis of UTROSCT rather than ESTSCLE. The current cases warned the potential risk of UTROSCT whose biological behavior is still uncertain. We discuss histopathological, immunohistochemical and molecular findings of UTROSCT with metastasis.