Vertical distribution of sediment phosphorus in Lake Hachirogata related to the effect of land reclamation on phosphorus accumulation.

The focus of this work is the change in sediment properties and chemical characteristics that occur after land reclamation projects. The results indicate a higher sedimentation rate in Lake Hachirogata after reclamation, with the rate increasing with proximity to the agricultural zone. In the west-side water samples, higher levels of dissolved total nitrogen and dissolved total phosphorus (DTP) were found in both surface and bottom waters. The increase in P (39-80%) was generally greater than that for N (12-16%), regarding the nutrient supply from reclaimed farmland in the western part of the lake. In the eastern part of the lake, the pore-water Cl- profile showed a decreasing vertical gradient in the sediment core. This indicates desalination of the lake water after construction of a sluice gate in 1961. In the western sediment-core sample, a uniform Cl- profile indicates the mixing of lake water and pore water after reclamation. Considering the sedimentation of P in the last 100 years, there is a trend of increasing accumulation of P and P-activities after the reclamation project. This appears to be an impact from change in the lake environment as a result of increased agricultural nutrients, desalination, and residence. A large amount of mobile phosphorus (42-72% of TP in the western core sample) trapped in sediment increases the risk of phosphorus release and intensification of algal blooms. High sediment phosphorus and phosphorus mobility should be considered a source of pollution in the coastal environment.

Development of in-vessel components of the microfission chamber for ITER.

Microfission chambers (MFCs) will measure the total neutron source strength in ITER. The MFCs will be installed behind blanket modules in the vacuum vessel (VV). Triaxial mineral insulated (MI) cables will carry signals from the MFCs. The joint connecting triaxial MI cables in the VV must be considered because the MFCs and the MI cables will be installed separately at different times. Vacuum tight triaxial connector of the MI cable has been designed and a prototype has been constructed. Performance tests indicate that the connector can be applied to the ITER environment. A small bending-radius test of the MI cable indicates no observed damage at a curvature radius of 100 mm.

Evaluating stream water quality through land use analysis in two grassland catchments: impact of wetlands on stream nitrogen concentration.

We evaluated the impacts of natural wetlands and various land uses on stream nitrogen concentration in two grassland-dominated catchments in eastern Hokkaido, Japan. Analyzing land use types in drainage basins, measuring denitrification potential of its soil, and water sampling in all seasons of 2003 were performed. Results showed a highly significant positive correlation between the concentration of stream NO3-N and the proportion of upland area in drainage basins in both catchments. The regression slope, which we assumed to reflect the impact on water quality, was 24% lower for the Akkeshi catchment (0.012 +/- 0.001) than for the Shibetsu catchment (0.016 +/- 0.001). In the Akkeshi catchment, there was a significant negative correlation between the proportion of wetlands in the drainage basins and stream NO3-N concentration. Stream dissolved organic nitrogen (DON) and carbon (DOC) concentrations were significantly higher in the Akkeshi catchment. Upland and urban land uses were strongly linked to increases in in-stream N concentrations in both catchments, whereas wetlands and forests tended to mitigate water quality degradation. The denitrification potential of the soils was highest in wetlands, medium in riparian forests, and lowest in grasslands; and was significant in wetlands and riparian forests in the Akkeshi catchment. The solubility of soil organic carbon (SOC) and soil moisture tended to determine the denitrification potential. These results indicate that the water environment within the catchments, which influences denitrification potential and soil organic matter content, could have caused the difference in stream water quality between the two catchments.

Structure-based design of an agonistic peptide targeting Fas.

A small agonistic peptide FRAP-4 (WEWT, Fas reactive peptide-4) that binds to the human Fas molecule was discovered using our computer screening strategy named the Amino acid Complement Wave (ACW) method, which is based on the complementarities of interacting amino acids between comprehensive testing peptides and a target protein surface pocket. In silico docking studies demonstrated the specific interaction of FRAP-4 with the main Fas ligand (FasL) binding domain in the Fas molecule. An octamer of this peptide produced by carboxyl terminal linkages of polylysine branches (MAP), (FRAP-4)8-MAP, effectively induced apoptosis in human ovarian cancer cell line NOS4 cells that was associated with the activation of caspases-8, -9 and -3, and the cleavage of PARP. Alanine substitution of the N-terminal W in FRAP-4 resulted in complete loss of FasL-mimetic action of (FRAP-4)8-MAP, suggesting that the aromatic functionality at the N-terminal position W appears to play an essentially important role in Fas binding ability. These observations indicate that the FasL-mimetic peptide should serve as an excellent starting point for the design of effective compounds with FasL-mimetic activity. Furthermore, the ACW method for the structure-based design of optimized small peptides against receptor molecules such as Fas could open new avenues for the development of peptide mimetic and nonpeptidic organic forms to generate novel effective pharmaceuticals.

Granulocyte colony-stimulating factor-producing cutaneous angiosarcoma with leukaemoid reaction arising on a burn scar.

We report a 46-year-old man with a giant tumour in a burn scar on his buttock. Pathological examination revealed that the dermis was filled with anastomosing vascular channels and round- or spindle-type atypical cells, which were compatible with the diagnosis of cutaneous angiosarcoma. Based on prominent leucocytosis (up to 113 000 microL-1), we measured serum granulocyte colony-stimulating factor (G-CSF). The highly elevated serum G-CSF of 303 ng L-1 (normal, 6.1-21.5 ng L-1) and positive immunohistochemical staining of the tumour tissue for G-CSF indicated that G-CSF was produced by the cutaneous angiosarcoma. To our knowledge, this is the first reported case of G-CSF-producing cutaneous angiosarcoma.

Activation of caspase-8 is critical for sensitivity to cytotoxic anti-Fas antibody-induced apoptosis in human ovarian cancer cells.

Two ovarian cancer cell lines named NOS4 and SKOV-3 have been shown to have different sensitivities to a cytotoxic anti-Fas antibody, CH-11. Although both cell lines express Fas molecules on the cell surfaces at the same intensities, apoptosis is induced by CH-11 in NOS4 cells but not in SKOV-3 cells. In this study, the different apoptosis-sensitivities of these cells were assessed. Both cell lines express almost the same levels of FADD, RIP, c-FLIP, FAP-1, Bax, Bcl-2 and Bcl-XL. Evidence of caspase-8, caspase-9 and caspase-3 activation and of cleavage of PARP and Bid was obtained in NOS4 cells but not in SKOV-3 cells. When triggered by FasL protein, DNA fragmentation and caspase-8 activation were observed in SKOV-3 cells, though they were not as clear as in NOS4 cells. All the anti-Fas antibody-mediated signals for apoptosis induction in NOS4 cells were completely blocked by a caspase-8-specific inhibitor, Z-IETD-FMK. These results indicate that the different sensitivities to the anti-Fas antibody are solely dependent on the activation of caspase-8, which could be influenced by yet unknown qualitative or quantitative abnormalities in molecules involved in DISC formation.

Binding of the La autoantigen to the 5' untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.

Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a terminal oligopyrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5' untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5' UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5' UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.

Cepharanthine activates caspases and induces apoptosis in Jurkat and K562 human leukemia cell lines.

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy-provoked leukopenia. We here report that apoptosis was induced by low concentrations (1-5 microM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5-10 microM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr-Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas-independent pathway. CEP treatment induced activation of caspase-9 and -3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase-8 activation and Akt cleavage were observed only in Jurkat cells. The CEP-induced apoptosis was completely blocked by zVAD-fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr-Abl protein in K562 cells, and this degradation was prevented partially by zVAD-fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2-5 microM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr-Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy.

Seaweed prevents breast cancer?

To investigate the chemopreventive effects of seaweed on breast cancer, we have been studying the relationship between iodine and breast cancer. We found earlier that the seaweed, wakame, showed a suppressive effect on the proliferation of DMBA (dimethylbenz(a)anthracene)-induced rat mammary tumors, possibly via apoptosis induction. In the present study, powdered mekabu was placed in distilled water, and left to stand for 24 h at 4 degrees C. The filtered supernatant was used as mekabu solution. It showed an extremely strong suppressive effect on rat mammary carcinogenesis when used in daily drinking water, without toxicity. In vitro, mekabu solution strongly induced apoptosis in 3 kinds of human breast cancer cells. These effects were stronger than those of a chemotherapeutic agent widely used to treat human breast cancer. Furthermore, no apoptosis induction was observed in normal human mammary cells. In Japan, mekabu is widely consumed as a safe, inexpensive food. Our results suggest that mekabu has potential for chemoprevention of human breast cancer.

Genomic organization, tissue expression, and cellular localization of AF3p21, a fusion partner of MLL in therapy-related leukemia.

We previously identified the AF3p21 gene, a novel fusion partner of the MLL gene, in a patient who had developed therapy-related leukemia with t(3;11)(p21;q23). The AF3p21 gene encodes a protein consisting of 722 amino acids, which has an SH3 (Src homology 3) domain, a proline-rich domain, and a bipartite nuclear localization signal. The protein's SH3 domain has high homology with that of FYN. Analysis of the DNA from the patient's leukemic cells revealed that intron 6 of the MLL gene was fused at a point upstream of exon 1 in the AF3p21 gene, and that the der(11) chromosome formed an MLL-AF3p21 fusion transcript in leukemic cells, whereas the der(3) chromosome did not form any fusion transcript. The AF3p21 gene on chromosome band 3p21 is 19 kb long and consists of 13 exons. The size of the mRNA of the AF3p21 gene is approximately 3.5 kb. The AF3p21 gene is widely expressed in normal human tissues including the bone marrow, brain, liver, thymus, lung, and skeletal muscle. Western blot and immunocytochemical analyses showed that AF3p21 protein has an apparent molecular weight of 80 kDa and is localized exclusively in the cell nucleus. These results suggest the possibility that AF3p21 protein plays a role in signal transduction in the nucleus.

Acute megakaryoblastic leukemia in an infant with a novel t(1;9)(p32;q34).

We report a case of a 14-month-old girl with acute megakaryoblastic leukemia (AMKL). May-Giemsa staining of the bone marrow cells revealed the proliferation of two distinct types of blasts. One type of blasts had cytoplasmic blebs, and the other showed a lymphoblastic morphology without blebs. Both types of blasts were negative for peroxidase and esterase reactions. Electron microscopic platelet peroxidase (PPO) reaction also revealed the presence of two types of blasts. One had irregular-shaped nuclei and positive PPO reaction in the nuclear envelope and rough endoplasmic reticulum but not in the Golgi apparatus. These types of blasts were considered to be megakaryoblasts. The other had an immature phenotype with round nuclei and positive PPO reaction in the nuclear envelope, rough endoplasmic reticulum, and the Golgi apparatus. The origin of this type of blasts could not be defined by their morphology. Surface marker analysis indicated that most of the leukemic cells expressed platelet markers, gpIIb, gpIIb/IIIa, gpIX, and gpIbalpha. Karyotypic analysis of the bone marrow cells of this unique subset of AMKL demonstrated a novel translocation, t(1;9)(p32;q34).

Preparation of benzene, furan, and thiophene analogs of duocarmycin SA employing a newly-devised phenol-forming reaction.

Five A-ring analogs of duocarmycin SA 9a-e were synthesized in racemic form modifying our second synthetic route toward duocarmycin SA. The problem encountered at the crucial phenol forming step to secure 17a, b from 16a, b under the conventionally used Kuwajima conditions was overcome by devising a more convenient method: simple heating of 16a-c in benzene in the presence of bis(triphenylphosphine)palladium(II) chloride (10 mol%), cesium carbonate (3 eq), and triphenylphosphine (0.3 eq) gave 17a-c in high yields of 86-91%. The intermediates 17a-e were readily led to the A-ring analogs (+/-)-9a-e almost according to the reported route.

Early endosomal localization of hrs requires a sequence within the proline- and glutamine-rich region but not the FYVE finger.

Hrs is an early endosomal protein that is tyrosine-phosphorylated in cells stimulated with growth factors. Hrs is thought to play a regulatory role in endocytosis of growth factor-receptor complexes through early endosomes. Early endosomal localization of Hrs seems to be essential for Hrs to exert its function in the endocytosis. Hrs has a FYVE finger domain that binds specifically to phosphatidylinositol 3-phosphate in vitro. The FYVE finger is a likely domain that mediates membrane association of endosomal proteins. In this study, we examined whether the FYVE finger participates in early endosomal targeting of Hrs. Hrs with a zinc binding-defective FYVE finger was still localized to early endosomes. In addition, the N-terminal FYVE finger-containing fragment of Hrs showed a cytosolic distribution in mammalian cells. These results indicate that the FYVE finger is not required for the localization of Hrs to early endosomes. Furthermore, by analyzing a series of deletion mutants of Hrs, we identified a sequence of about 100 amino acids within the C-terminal proline- and glutamine-rich region as a domain essential for the targeting of Hrs to early endosomes.

Riedel's lobe of the liver evaluated by multiple imaging modalities.

An 81-year-old Japanese woman visited our hospital because of abdominal discomfort. Physical examination revealed that she had an abdominal mass. A combination of ultrasonography, computed tomography, magnetic resonance imaging (MRI), and hepatic asialoglycoprotein scintigraphy was utilized to make a diagnosis. We found that she had a downward elongated hepatic lobe or Riedel's lobe which did not appear to be common in our district. The prevalence of Riedel's lobe in the Asian population has not been studied. Furthermore, this is the first report that describes the MRI and hepatic asialoglycoprotein scintigraphy features of Riedel's lobe of the liver.

4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death.

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 microM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.