Intercomparison study on 152Eu gamma ray and 36Cl AMS measurements for development of the new Hiroshima–Nagasaki Atomic Bomb Dosimetry System 2002 (DS02)

In the process of developing a new dosimetry system for atomic bomb survivors in Hiroshima and Nagasaki (DS02), an intercomparison study between (152)Eu and (36)Cl measurements was proposed, to reconcile the discrepancy previously observed in the Hiroshima data between measurements and calculations of thermal neutron activation products. Nine granite samples, exposed to the atomic-bomb radiation in Hiroshima within 1,200 m of the hypocenter, as well as mixed standard solutions containing known amounts of europium and chlorine that were neutron-activated by a (252)Cf source, were used for the intercomparison. Gamma-ray spectrometry for (152)Eu was carried out with ultra low-background Ge detectors at the Ogoya Underground Laboratory, Kanazawa University, while three laboratories participated in the (36)Cl measurement using accelerator mass spectrometry (AMS): The Technical University of Munich, Germany, the Lawrence Livermore National Laboratory, USA and the University of Tsukuba, Japan. Measured values for the mixed standard solutions showed good agreement among the participant laboratories. They also agreed well with activation calculations, using the neutron fluences monitored during the (252)Cf irradiation, and the corresponding activation cross-sections taken from the JENDL-3.3 library. The measured-to-calculated ratios obtained were 1.02 for (152)Eu and 0.91-1.02 for (36)Cl, respectively. Similarly, the results of the granite intercomparison indicated good agreement with the DS02 calculation for these samples. An average measured-to-calculated ratio of 0.98 was obtained for all granite intercomparison measurements. The so-called neutron discrepancy that was previously observed and that which included increasing measured-to-calculated ratios for thermal neutron activation products for increasing distances beyond 1,000 m from the hypocenter was not seen in the results of the intercomparison study. The previously claimed discrepancy could be explained by insufficient understanding of the measured data.     

Synthesis and structure-activity relationships of novel IKK-beta inhibitors. Part 3: Orally active anti-inflammatory agents

A series of 2-amino-3-cyano-4-alkyl-6-(2-hydroxyphenyl)pyridine derivatives was synthesized and evaluated as I kappaB kinase beta (IKK-beta) inhibitors. Modification of a novel IKK-beta inhibitor 1 (IKK-beta IC(50)=1500 nM, Cell IC(50)=8000 nM) at the 4-phenyl ring and 6-phenol group on the pyridine core ring resulted in a marked increased in biological activities. An optimized compound, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile, exhibited excellent in vitro profiles (IKK-beta IC(50)=8.5 nM, Cell IC(50)=60 nM) and a strong oral efficacy in in vivo anti-inflammatory assays (significant effects at 1mg/kg, po in arachidonic acid-induced ear edema model in mice).     

Effect of anticoagulants in colorimetric assay for basic carboxypeptidases

Carboxypeptidases (CP) in plasma and sera serve as regulators of anaphylatoxins such as C3a and C5a. The activity of CP can be measured by determining hippuric acid after cleavage of the small synthetic substrate hippuryl-L-arginine. Although a colorimetric assay is convenient for determining hippuric acid generated by CP, we noticed that some anticoagulants, such as citrate, interfere with the color development of the reagents used. EDTA and heparin provide an appropriate value. EGTA used as anticoagulant also provides an appropriate value. Therefore, concentration of citrate in samples should be controlled to be constant for background subtraction.     

Insulin-like growth factor-1 protects peroxynitrite-induced cell death by preventing cytochrome c-induced caspase-3 activation

We investigated the effect of IGF-1 on cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Exposure of the cells to 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, caused cytochrome c release from the mitochondria, caspase-3-like activation, and cell death. Pre-incubation of the cells with the caspase-3 inhibitor partially prevented SIN-1-induced cell death. Simultaneous addition of IGF-1 reduced SIN-1-induced caspase-3-like activation and cell death, whereas IGF-1 failed to reduce the release of cytochrome c. IGF-1 increased Akt phosphorylation, and Akt phosphorylation was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. In addition, wortmannin prevented IGF-1-evoked inhibition of cell death and caspase-3-like activation. In a cell-free system, addition of cytochrome c to cytosolic fraction resulted in caspase-3-like activation. The activation was reduced when the cytosolic fraction prepared from IGF-1-treated cells was used. These results suggest that IGF-1 protects peroxynitrite-induced cell death downstream of cytochrome c release through the inhibition of caspase-3-like activation.     

An immune-responsive Serpin regulates the melanization cascade in Drosophila

In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO), which, in turn, catalyzes the synthesis of melanin. Here we report the biochemical and genetic characterization of a Drosophila serine protease inhibitor protein, Serpin-27A, which regulates the melanization cascade through the specific inhibition of the terminal protease prophenoloxidase-activating enzyme. Our data demonstrate that Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization.     

The 99 and 170 loop-modified factor VIIa mutants show enhanced catalytic activity without tissue factor

To elucidate the functions of the surface loops of VIIa, we prepared two mutants, VII-30 and VII-39. The VII-30 mutant had all of the residues in the 99 loop replaced with those of trypsin. In the VII-39 mutant, both the 99 and 170 loops were replaced with those of trypsin. The k(cat)/K(m) value for hydrolysis of the chromogenic peptidyl substrate S-2288 by VIIa-30 (103 mm(-)1s(-)1) was 3-fold higher than that of wild-type VIIa (30.3 mm(-)1 s(-)1) in the presence of soluble tissue factor (sTF). This enhancement was due to a decrease in the K(m) value but not to an increase in the k(cat) value. On the other hand, the k(cat)/K(m) value for S-2288 hydrolysis by VIIa-39 (17.9 mm(-)1 s(-)1) was 18-fold higher than that of wild-type (1.0 mm(-)1 s(-)1) in the absence of sTF, and the value was almost the same as that of wild-type measured in the presence of sTF. This enhancement was due to not only a decrease in the K(m) value but also to an increase in the k(cat) value. These results were in good agreement with their susceptibilities to a subsite 1-directed serine protease inhibitor. In our previous paper (Soejima, K., Mizuguchi, J., Yuguchi, M., Nakagaki, T., Higashi, S., and Iwanaga, S. (2001) J. Biol. Chem. 276, 17229-17235), the replacement of the 170 loop of VIIa with that of trypsin induced a 10-fold enhancement of the k(cat) value for S-2288 hydrolysis as compared with that of wild-type VIIa in the absence of sTF. These results suggested that the 99 and the 170 loop structures of VIIa independently affect the K(m) and k(cat) values, respectively. Furthermore, we studied the effect of mutations on proteolytic activity toward S-alkylated lysozyme as a macromolecular substrate and the activation of natural macromolecular substrate factor X.     

Browning and decomposed products of model orange juice

A model solution of orange juice containing sugars, ascorbic acid, and citric acid was prepared and its browning during storage was examined. The solution gradually turned brown. Ascorbic acid (AsA) most contributed to the browning. Citric acid and such amino acids as Arg and Pro promoted the browning. DTPA, a strong chelator, inhibited the browning. 3-Hydroxy-2-pyrone (3OH2P), 5-hydroxymethylfurfural (HMF), furfural, 5-hydroxymaltol, and 2-furoic acid were identified as decomposed products in the stored solution. When 3OH2P was stored, the solution turned slightly brown. Furfural solution added with amino acids turned yellow. 3OH2P showed a positive relation with the browning of retail orange juice during storage.     

Ultraviolet light exposure suppresses contact hypersensitivity by abrogating endothelial intercellular adhesion molecule-1 up-regulation at the elicitation site

Hapten sensitization through UV-exposed skin induces systemic immune suppression, which is experimentally demonstrated by inhibition of contact hypersensitivity (CHS). Although this UV-induced effect has been shown to be mediated by inhibition of the afferent phase of the CHS, the UV effects on the efferent (elicitation) phase remain unknown. In this study, UV effects on endothelial ICAM-1 expression at elicitation sites were first examined. Mice were sensitized by hapten application onto UV-exposed back skin, and ears were challenged 5 days later. ICAM-1 up-regulation at nonirradiated elicitation sites following hapten challenge was eliminated by UV exposure on sensitization sites distant from elicitation sites. To assess whether loss of the ICAM-1 up-regulation at elicitation sites contributed to UV-induced immunosuppression, we examined CHS responses in UV-exposed ICAM-1-deficient (ICAM-1(-/-)) mice that genetically lacked the ICAM-1 up-regulation. ICAM-1(-/-) mice exhibited reduced CHS responses without UV exposure, but UV exposure did not further reduce CHS responses in ICAM-1(-/-) mice. Furthermore, ICAM-1 deficiency did not affect the afferent limb, because ICAM-1(-/-) mice had normal generation of hapten-specific suppressor and effector T cells. This UV-induced immunosuppression was associated with a lack of TNF-alpha production after Ag challenge at elicitation sites. Local TNF-alpha injection before elicitation abrogated the UV-induced CHS inhibition with increased endothelial ICAM-1 expression. TNF-alpha production at elicitation sites was down-regulated by IL-10, a possible mediator produced by hapten-specific suppressor T cells that are generated by UV exposure. These results indicate that UV exposure inhibits CHS by abrogating up-regulation of endothelial ICAM-1 expression after Ag challenge at elicitation sites.     

Role of the indigenous microbiota in maintaining the virus-specific CD8 memory T cells in the lung of mice infected with murine cytomegalovirus

The potent role of indigenous microbiota in maintaining murine CMV (MCMV)-specific memory T cells, which were measured by multimer staining, was investigated using germfree (GF) mice. When the BALB/c mice bred under specific pathogen-free (SPF) conditions were i.p. infected with 0.2 LD(50) of MCMV, high frequencies of CD69(+)/CD44(+) MCMV-specific CD8 T cells were noted in the lungs even at 6-12 mo after infection (11.1 +/- 3.2 and 9.8 +/- 0.9%, respectively). In contrast, even though the viral load and expression levels of mRNA of such cytokines as IL-2, IL-7, IL-15, and IFN-gamma in the lungs of MCMV-infected GF mice were comparable to those of infected SPF mice, the frequencies of MCMV-specific CD8 T cells in the lungs of infected GF mice were kept lower than 1% at 6-12 mo after infection. In addition, the reconstitution of microbiota of MCMV-infected GF mice by orally administering a fecal suspension prepared from SPF mice restored the frequencies of both CD8(+)/multimer(+) and CD8(+)/multimer(-) T cells to levels similar to those found in SPF mice. These results suggested the indigenous microbiota to play a crucial role in the expansion and maintenance of viral-specific CD8 memory T cells, probably by cross-reactivity between the antigenic epitope of the MCMV-specific memory T cells and the variety of peptides derived from the members of the microbiota. Such cross-reactivity may thus be a major feature of those cells.