Singlet oxygen is essential for neutrophil extracellular trap formation

Neutrophil extracellular traps (NETs) that bind invading microbes are pivotal for innate host defense. There is a growing body of evidence for the significance of NETs in the pathogenesis of infectious and inflammatory diseases, but the mechanism of NET formation remains unclear. Previous observation in neutrophils of chronic granulomatous disease (CGD) patients, which defect NADPH oxidase (Nox) and fail to produce reactive oxygen species (ROS), revealed that ROS contributed to the formation of NETs. However, the active species were not identified. In this study, we discovered that singlet oxygen, one of the ROS, mediated Nox-dependent NET formation upon stimulation with phorbol myristate acetate. We also revealed that singlet oxygen itself could induce NET formation by a distinct system generating singlet oxygen with porfimer sodium (Photofrin) in CGD neutrophils, as well as healthy neutrophils. This was independent of Nox activation. These results show that singlet oxygen is essential for NET formation, and provide novel insights into the pathogenesis of infectious and inflammatory diseases.     

Murine glycosyltransferases responsible for the expression of globo-series glycolipids: cDNA structures, mRNA expression, and distribution of their products

Biological functions of globo-series glycosphingolipids are not well understood. In this study, murine cDNAs of two glycosyltransferases responsible for the synthesis of globo-series glycolipids and mRNA expression of those genes were analyzed. Distribution of their products was also analyzed. Murine cDNAs for Gb3/CD77 synthase and Gb4 synthase predicted that both of them are type II membrane proteins with 348 and 331 amino acids, respectively. In northern blotting, Gb3/CD77 synthase gene was mainly expressed in kidney and lung but also detected in many other tissues. Gb4 synthase was expressed in brain, heart, kidney, liver, skin, and testis. In the immunohistological analysis, Gb3/CD77 was mainly expressed in the proximal tubules as revealed with coincidental expression with angiotensin-converting enzyme (ACE). In spleen, it was detected in pre-B cells in the peripheral region of the white pulp, as suggested with coincidental expression with CD10. It was also expressed on the endothelia of the alveolar capillaries in lung and on the sebaceous ducts aside of the hair follicles. Gb4 was also detected mainly on the proximal tubules in kidney and on the endothelia of the alveolar capillaries in lung as Gb3/CD77. But it was also detected on the epithelium of the bronchus, seminiferous tubules and tails of spermatozoa in testis, blood vessels of choroids plexus and endothelial cells in brain, and central and hepatoportal veins in liver. The expression patterns of two genes and their products almost corresponded with some exception. The results would provide essential information for the functional studies of globo-series glycolipids.     

Molecular Taxonomic Study and Revision of the Three Japanese Species of Asplenium sect. Thamnopteris

Asplenium sect. Thamnopteris or A. nidus L. complex is defined by the synapomorphic character peculiar to Aspleniaceae, an anastomosing vein near the margin of the simple lamina. Thus, it is easily recognizable and its monophyly is quite clear. In spite of its naturalness as the whole group, species delimitation is very confusing. Three species of sect. Thamnopteris, A. antiquum Makino, A. australasicum (J. Smith) Hooker and A. nidus L. have been recognized in Japan, but the naturalness of each species is still not clear because their morphology is too simple to find good qualitative taxonomical characters. In the present work, we examined the intraspecific variation of allozymes and rbcL sequences in the Japanese plants of sect. Thamnopteris and compared them with those from other paleotropical localities in order to recognize natural units in such morphologically simple plants. We found a large amount of genetic variation in this section and inferred that A. antiquum is a species of ancient origin, though morphologically it is not so different from other species of the sect. Thamnopteris. It was also discovered that the so called “A. australasicum” in Japan has a very different rbcL sequence from A. australasicum sensu Holttum, which is distributed in Australia and South Pacific Islands. Based on these molecular data, we described the Japanese “A. australasicum” as a new species, Asplenium setoi N. Murak. et Seriz. Received 9 September 1998/ Accepted in revised form 22 December 1998     

Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes

Development and preclinical testing of new cancer therapies is limited by the scarcity of in vivo models that authentically reproduce tumor growth and metastatic progression. We report new models for breast tumor growth and metastasis in the form of transplantable tumors derived directly from individuals undergoing treatment for breast cancer. These tumor grafts illustrate the diversity of human breast cancer and maintain essential features of the original tumors, including metastasis to specific sites. Co-engraftment of primary human mesenchymal stem cells maintains phenotypic stability of the grafts and increases tumor growth by promoting angiogenesis. We also report that tumor engraftment is a prognostic indicator of disease outcome for women with newly diagnosed breast cancer; orthotopic breast tumor grafting is a step toward individualized models for tumor growth, metastasis and prognosis. This bank of tumor grafts also serves as a publicly available resource for new models in which to study the biology of breast cancer.     

Enhancement by streptozotocin of O2- radical generation by the xanthine oxidase system of pancreatic beta-cells

Spin-trapping techniques and electron spin resonance (ESR) spectroscopy were used to study the relationship between the effect of streptozotocin (STZ) on pancreatic beta-cells and free radical formation by these cells. Results showed that STZ enhanced generation of the DMPO-OH radical adduct, which is a degradation product of the superoxide anion (O2-) in the presence of cellular components, in a hypoxanthine-xanthine oxidase (XOD) system with a homogenate of beta-cells. This enhancing effect was also observed in a system without cellular components; STZ increased the signal height due to the O2- radical in a concentration-dependent manner and caused a maximum of 150% enhancement at a concentration of 1.5 mM. Thus, STZ seemed to enhance the generation of the O2- radical in the XOD system, probably by some mechanism of its interaction with XOD. Pancreatic beta-cells exhibited a high XOD activity and a very low superoxide dismutase activity. Therefore, the present result supports the possibility that the cytotoxic effect of STZ is closely related to free radical generation in pancreatic beta-cells.     

Discovery of a potent,metabolically stabilized resorcylic lactone as an anti-inflammatory lead

With bioactivity-guided phenotype screenings, a potent anti-inflammatory compound f152A1 has been isolated, characterized and identified as the known natural product LL-Z1640-2. Metabolic instability precluded its use for the study on animal disease models. Via total synthesis, a potent, metabolically stabilized analog ER-803064 has been created; addition of the (S)-Me group at C4 onto f152A1 has resulted in a dramatic improvement on its metabolic stability, while preserving the anti-inflammatory activities.     

Further chemical modification of trehalase inhibitor trehazolin: Structure and inhibitory-activity relationship of the inhibitor

Eight analogues of trehazolin were synthesized and tested for trehalase inhibitors. Deoxygenation of the cyclopentanepolyol moiety all decreased the inhibitory activity. Epimerization at the branching point of the cyclopentane ring did not so affect the potency. The cyclic isourea part was shown to be replaced with guanidine structure with a considerable decrease of activity. The 6′-fluoro-6′-deoxy derivative was still a strong inhibitor. It seems that trehazolin strictly mimics the substrate ,-trehalose and any structural change and/or removal of the hydroxyl functions appreciably influence its potency. The present results led to finding 5-aminocyclopentane-1,2,3,4-tetraols to be new lead compounds for glycohydrolase inhibitors.     

New indanone compounds from Onychium japonicum

A new indanone glucoside pteroside M has been isolated from fronds of Onychium japonicumPteridaceae. The structure of its aglycone pterosin M has been established by ¹³C NMR, PMR spectra and degradation with nitric acid.