Age-Related Development of γ-Aminobutyric Acid (GABA)BReceptor Functions in Various Brain Regions of Spontaneously Hypertensive Rats

The age-related development of GABA_Breceptors and their coupling to adenylate cyclase were studied in the brains of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Compared with WKY rats, the specific [³H]GABA binding to GABA_Breceptors showed a significant decrease not only in the posterior hypothalamus, midbrain, hippocampus and striatum of eleven-week-old SHR, which maintain a hypertensive state, but also in the posterior hypothalamus of four-week-old normotensive SHR. Similarly, the GABA_Breceptor agonists (baclofen and DN-2327)-induced suppression of adenylate cyclase activity showed a decrease in the posterior hypothalamus of four-week-old SHR as well as in the posterior hypothalamus and striatum of eleven-week-old SHR. These results suggest that the functions of the GABA_Breceptor in the brain of SHR may be decreased independently from the occurrence of blood pressure elevation and that such changes may even be involved in the pathogenesis of SHR.     

Evidence for a Dual Strategy in the Expression of Cauliflower Mosaic Virus Open Reading Frames I and IV

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and IV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-β-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, we detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV, and the operation of a dual strategy in the expression of two viral genes.     

A methoxyflavanone derivative from the Asian medicinal herb (<Emphasis Type="Italic">Perilla frutescens</Emphasis>) induces p53-mediated G<Subscript>2</Subscript>/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma

Perilla frutescens is an Asian dietary herb consumed as an essential seasoning in Japanese cuisine as well as used for a Chinese medicine. Here, we report that a newly found methoxyflavanone derivative from P. frutescens (Perilla-derived methoxyflavanone, PDMF; 8-hydroxy-5,7-dimethoxyflavanone) shows carcinostatic activity on human lung adenocarcinoma, A549. We found that treatment with PDMF significantly inhibited cell proliferation and decreased viability through induction of G₂/M cell cycle arrest and apoptosis. The PDMF stimulation induces phosphorylation of tumor suppressor p53 on Ser15, and increases its protein amount in conjunction with up-regulation of downstream cyclin-dependent kinase inhibitor p21^Cip1/Waf1 and proapoptotic caspases, caspase-9 and caspase-3. We also found that small interfering RNA knockdown of p53 completely abolished the PDMF-induced G₂/M cell cycle arrest, and substantially abrogated its proapoptotic potency. These results suggest that PDMF represents a useful tumor-preventive phytochemical that triggers p53-driven G₂/M cell cycle arrest and apoptosis.     

Structure-based design,synthesis, and biological evaluation of imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors

We identified novel potent inhibitors of p38 MAP kinase using structure-based design strategy. X-ray crystallography showed that when p38 MAP kinase is complexed with TAK-715 (1) in a co-crystal structure, Phe169 adopts two conformations, where one interacts with 1 and the other shows no interaction with 1. Our structure-based design strategy shows that these two conformations converge into one via enhanced protein-ligand hydrophobic interactions. According to the strategy, we focused on scaffold transformation to identify imidazo[1,2-b]pyridazine derivatives as potent inhibitors of p38 MAP kinase. Among the herein described and evaluated compounds, N-oxide 16 exhibited potent inhibition of p38 MAP kinase and LPS-induced TNF-α production in human monocytic THP-1 cells, and significant in vivo efficacy in rat collagen-induced arthritis models. In this article, we report the discovery of potent, selective and orally bioavailable imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors with pyridine N-oxide group.     

Correction to: Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate

Applied Microbiology and Biotechnology - The original publication of this paper contains mistakes for Tables 1 and 2 legends as well as the sublabels in Figs. 2, 4, 5, 6, and 7.     

Identification of 3-Hydroxy-2-indolone-3-acetylaspartic Acid as a New Indole-3-acetic Acid Metabolite in Vicia Roots

A new indole-3-acetic acid (IAA) metabolite in the root of Viciafaba L. cv. Chukyo was identified as 3-hydroxy-2-indolone-3-acetylasparticacid, with the simpler name of dioxindole-3-acetylaspartic acid,by comparison with the authentic sample. Formation of dioxindole-3-aceticacid conjugates seems to be a major route of IAA metabolismin Vicia roots. (Received October 22, 1985; Accepted January 7, 1986)     

Basal level of prostaglandin D2 in rat brain by a solid-phase enzyme immunoassay

A solid-phase enzyme immunoassay for prostaglandin D₂ (PGD₂) was developed in which PGD₂ was labeled with horseradish peroxidase. After competitive binding to the immobilized antibody between enzyme-labelled and free PGD₂, the activity of the enzyme bound to the antibody was assayed fluorometrically using 3-(p-hydroxyphenyl)- propionic acid and hydrogen peroxide as substrates. The procedure allowed determinations of 3 – 100 pg for PGD₂. The IC₅₀ value for PGD₂ in the solid-phase enzyme immunoassay was about 25 pg and the sensitivity was improved about 10 times compared to those in radioimmunoassay and in solution-phase enzyme immunoassay. The solid-phase enyzme immunoassay was applied to the measurement of PGD₂ content in rat brain and thereby an octadecylsilyl silica cartridge and a reversed-phase HPLC were sequentially used for sample preparations. Heads were immediately frozen in liquid nitrogen after decapitation to avoid a postmortem formation of PGD₂. PGD₂ contents measured by solid-phase enzyme immunoassay correlated well with the values obtained by radioimmunoassay (r = 0.966) after raising its contents by intravenous administration of PGD₂. The level of PGD₂ in rat brain was extremely low but determined to be 0.11 ± 0.03 ng/g tissue (mean ± S.E.M.) with this enzyme immunoassay. The result was equal to the value extrapolated to zero time from the postmortem change.     

A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium

Summary A human cholangiocellular carcinoma cell line, HuCC-T1, was established in vitro from the malignant cells of ascites of a 56-yr-old patient. Histologic findings of the primary liver tumor revealed a moderately differentiated adenocarcinoma. Tumor cells from the ascites have been cultured with RPMI 1640 medium containing 0.2% lactalbumin hydrolysate and the cultured cells grew as monolayers with a population doubling time of 74 h during exponential growth at Passage 25. They had an epithelial-like morphology and were positive for mucine staining. Ultrastructural studies revealed the presence of microvilli on the cell surface and poorly developed organelles in the cytoplasm. The HuCC-T1 cell was tumorigenic in nude mice. The number of chromosomes in HuCC-T1 ranged from 61 to 80. These human cholangiocellular carcinoma cells in serum-free medium secreted several tumor markers, including carbohydrate antigen 19/9, carbohydrate antigen 125, carcinoembryonic antigen, and tissue polypeptide antigen. The carbohydrate antigen 19/9 secretion level of HuCC-T1 cells cultured in PRMI 1640 medium with 1% fetal bovine serum was sixfold higher than that with 0.2% lactalbumin hydrolysate. These findings suggest that HuCC-T1 will provide useful information to clarify the mechanism of tumor marker secretion and tumor cell growth in the human cholangiocellular carcinoma.