TNP-470 Suppresses the Tumorigenicity of HT1080 Fibrosarcoma Tumor Through the Inhibition of VEGF Secretion From the Tumor Cells

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer. TNP-470, a systemic analogue of fumagillin, is an angiogenesis inhibitor capable of suppressing the tumorigenicity in several animal models even though the mechanisms of action have not been completely clarified. In the current study, we investigated the effects of TNP-470 on human fibrosarcoma cells in vivo and in vitro. The administration of TNP-470 could suppress the tumorigenicity of HT1080 fibrosarcoma tumor. The conditioned medium from HT1080 fibrosarcoma cells treated with TNP-470 inhibited the proliferation and migration of human endothelial cell line, HUVEC and ECV304. The concentration of VEGF in the conditioned medium from HT1080 cells treated with TNP-470 was lower than that of the cells without TNP-470 treatment, indicating that TNP-470 downregulates the secretion of VEGF from HT1080 cells. These findings strongly suggest that the direct action of TNP-470 on sarcoma cells inhibits angiogenesis through the downregulation of VEGF secretion and this angiogenesis suppression resulted in the inhibition of tumorigenicity of HT1080 fibrosarcoma tumo

Substitution of Glu122 by Glutamine Revealed the Function of the Second Water Molecule as a Proton Donor in the Binuclear Metal Enzyme Creatininase.

Creatininase is a binuclear zinc enzyme and catalyzes the reversible conversion of creatinine to creatine. It exhibits an open-closed conformational change upon substrate binding, and the differences in the conformations of Tyr121, Trp154, and the loop region containing Trp174 were evident in the enzyme-creatine complex when compared to those in the ligand-free enzyme. We have determined the crystal structure of the enzyme complexed with a 1-methylguanidine. All subunits in the complex existed as the closed form, and the binding mode of creatinine was estimated. Site-directed mutagenesis revealed that the hydrophobic residues that show conformational change upon substrate binding are important for the enzyme activity. We propose a catalytic mechanism of creatininase in which two water molecules have significant roles. The first molecule is a hydroxide ion (Wat1) that is bound as a bridge between the two metal ions and attacks the carbonyl carbon of the substrate. The second molecule is a water molecule (Wat2) that is bound to the carboxyl group of Glu122 and functions as a proton donor in catalysis. The activity of the E122Q mutant was very low and it was only partially restored by the addition of ZnCl(2) or MnCl(2). In the E122Q mutant, k(cat) is drastically decreased, indicating that Glu122 is important for catalysis. X-ray crystallographic study and the atomic absorption spectrometry analysis of the E122Q mutant-substrate complex revealed that the drastic decrease of the activity of the E122Q was caused by not only the loss of one Zn ion at the Metal1 site but also a critical function of Glu122, which most likely exists for a proton transfer step through Wat2

ガンバンナイ ノ オウリョク ヘンカ ノ タンサシン ト シテノ ゲンバ ニ オケル ダイチヒ テイコウ モニタリング

京都大学0048新制・課程博士博士(理学)甲第9959号理博第2620号新制||理||1335(附属図書館)UT51-2003-H380京都大学大学院理学研究科地球惑星科学専攻(主査)助教授 柳谷 俊, 教授 大志万 直人, 教授 町田 忍学位規則第4条第1項該当Doctor of ScienceKyoto UniversityDA

STUDIES ON "KASEN" OF HORSES IN HOKKAIDO : II. RESULTS OBTAINED IN 1954

Etiological and therapeutical studies on "Kasen" of horses in Hokkaido were made by the authors from the early spring to the late autumn in 1954 in the same manner as in 1953. The results thus obtained may be summarized as follows : 1. Clinical and hematological observations were almost similar to those of the preceding year. 2. Living microfilariae were detected from pieces of cutaneous tissues in 2 patients (11% out of 18 cases) and these microfilariae were identified with those of Onchocerca cervicalis by the morphological examinations. The detection of microfilariae from the biting insects was unsuccessful. 3. Histopathologically, 2 cases (11%) showed eczematous lesions, whilst infiltrations of eosinophilic cells were observed in all cases by employing serial sections, however no microfilariae were found at all. 4. In the therapeutic treatments, the result of the subcutaneous injections of "Neostinal" was somewhat effective, however the manner of its operation was not clarified. Use of arsenic compound ("Solminol") and piperazine derivative ("Supatonin" and "Hetrazan" syrup) did not yield beneficial results. Use of "Allergin" (anti-histamine preparation) alone and of "Allergin" and "Solminol" at the same time did obtain beneficial resunlts. So far as the above described data are concerned, it is thought that the patients of "Kasen" were placed in allergic conditions. Also, it is interesting fact that this disease seems to be similar to "Queensland Itch" or "Allergic dermatitis" reported by RIEK

Evidence that multiple P2X purinoceptors are functionally expressed in rat supraoptic neurones

The expression, distribution and function of P2X purinoceptors in the supraoptic nucleus (SON) were investigated by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and Ca2+-imaging and whole-cell patch-clamp techniques, respectively.RT-PCR analysis of all seven known P2X receptor mRNAs in circular punches of the SON revealed that mRNAs for P2X2, P2X3, P2X4, P2X6 and P2X7 receptors were expressed in the SON, and mRNAs for P2X3, P2X4 and P2X7 were predominant.In situ hybridization histochemistry for P2X3 and P2X4 receptor mRNAs showed that both mRNAs were expressed throughout the SON and in the paraventricular nucleus (PVN).ATP caused an increase in [Ca2+]i in a dose-dependent manner with an ED50 of 1.7 × 10−5m. The effects of ATP were mimicked by ATPγS and 2-methylthio ATP (2MeSATP), but not by AMP, adenosine, UTP or UDP. αβ-Methylene ATP (αβMeATP) and ADP caused a small increase in [Ca2+]i in a subset of SON neurones.The P2X7 agonist 2′- & 3′-O-(4-benzoylbenzoyl)-ATP (BzATP) at 10−4m increased [Ca2+]i, but the potency of BzATP was lower than that of ATP. In contrast, BzATP caused a more prominent [Ca2+]i increase than ATP in non-neuronal cells in the SON.The effects of ATP were abolished by extracellular Ca2+ removal or by the P2 antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), and inhibited by extracellular Na+ replacement or another P2 antagonist, suramin, but were unaffected by the P2X7 antagonist oxidized ATP, and the inhibitor of Ca2+-ATPase in intracellular Ca2+ stores cyclopiazonic acid.Two patterns of desensitization were observed in the [Ca2+]i response to repeated applications of ATP: some neurones showed little or moderate desensitization, while others showed strong desensitization.Whole-cell patch-clamp analysis showed that ATP induced cationic currents with marked inward rectification. The ATP-induced currents exhibited two patterns of desensitization similar to those observed in the [Ca2+]i response.The results suggest that multiple P2X receptors, including P2X3, are functionally expressed in SON neurones, and that activation of these receptors induces cationic currents and Ca2+ entry. Such ionic and Ca2+-signalling mechanisms triggered by ATP may play an important role in the regulation of SON neurosecretory cells