In several solanaceow plants, hyoscyamine is first hydroxylated at the 6j%position, and then epoxidized to scopolamine. We expressed hyoscyamine 6,Bhydroxylase (W6H) in Esclrerc&a coil as a fusion protein with maltose-biding protein. The crude cell extract from the bacterium that expressed the soluble fusion protein showed a strong hydroxyiase activity and a weak epoxidase activity. When 100 PM of hyoscyamine was fed to the recombinant bacterium, the alkaloid was first converted to dg-hydroxyhyoscyamine, and then to scopolamine, which was almost the only alkaloid found in the culture after one week. Therefore, H6H catalyzes two consecutive reactions that oxidize hyoscyamine to scopolamine.Hyoscyamine 6/?-hydroxylase; Bacterial expression; Epoxidation; Scopolamine Hyoscyamine and its epoxide sctopolamine are typical tropane alkaloids found in several soianaceous plants. Previous feeding experiments with alkaloid precursors [1,2] have suggested that the 6,7-epoxide bridge of scopolamine is formed by hyoscyamine by way of B/?-hydroxyhyoscyamine {Fig. 1). We discovered a 2-oxoglutarate-dependent dioxygenase that hydroxyiates hyoscyamine at the @-position in alkaloid-producing root cultures, and named it hyoscyamine S&hydroxylase (H6H; EC 1.14,i 1.11) [3,4]. Later, we also found that a similar 2-oxo~~tarate~e~ndent dioxygenase converts 6~-hydroxyhyo~y~ne tu scopolamine by dehydrogenation of the 7/&hydrogen f5]. This epoxidase activity was relatively weak and represented only l-108 of the hydroxylase activity in partially purified enzyme preparations. Our subsequent observations indicated that H6H may be a bifunctionai dioxygenase endowed with strong hydroxyiase activity and comparatively weak epoxidase activity; these two activities were observed in the same fractions during partial purification [5], and transgenic tobacco and Atropa MZadonna that expressed H6H under the cauliflower mosaic virus promoter efficiently converted hyoseyamine that was supplied either exogenousiy f6] or endogenously [?] to scupolamine.Although these observations are very suggestive, we need direct evidence that H6H has both hydroxyiase Correspondence addwx T. Hashimoto, Department of Agriculttural Chemistry, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan. Fax: (81) (75) 753 6398. and epoxidase activities, and that the relatively weak epoxidase activity, together with the strong hydroxyiase activity, is sufficient to oxidize hyoscyamine completely to scopolamine, without having the conversion blocked at 6/&hydroxyhyoscyamine. In this study, we first found that the monoclonai antibody mAb5 that was raised against homogeneous hydroxyiase [8] inhibited both enzyme activities to the same extent, Next, we constructed a prokaryotic expression vector in which the H6H cDNA from Hy~scy~~lss niger [9] was fused to the maltose-binding protein (MBP) gene, and expressed the fusion protein in an enzymatically active form in Escheri hia coli. Measurement of enzyme activities in the bacterial extracts and feeding of hyos&y~ine to ...

Intraoperative radioactivity-guided sentinel node mapping was accurate for patients with colorectal cancer with T1 or T2 tumors. The results suggest that sentinel node mapping and intraoperative biopsy may be a sensitive and specific diagnostic method for detecting metastasis in regional lymph nodes in patients with colorectal cancer.

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Hepatitis C virus (HCV), a major causative agent of post transfusion non-A, non-B hepatitis (NANBH), can only infect humans and chimpanzees. We produced nine transgenic mouse lines carrying a full-length HCV cDNA with the human serum amyloid P component (hSAP) promoter that can direct liver-specific expression. In one of these lines HCV mRNA and HCV core protein were detected in the liver of the transgenic mouse, although the levels of expression were very low. In addition, HCV-related antibody was detected in the serum.Key words: Hepatitis C virus -Transgenic mouse -Human serum amyloid P component (hSAP) promoter Hepatitis C virus (HCV) is the most important etiological agent of post-transfusion non-A, non-B hepatitis (NANBH) 1) and is a major cause of chronic liver disease and hepatocellular carcinoma. HCV is distantly related genetically to both the pestiviruses and flaviviruses. The HCV genome is about 9.4 kb in length and consists of a highly conserved 5′ untranslated region followed by a single open reading frame that encodes a polyprotein of 3010 to 3033 amino acids.2-4) The host signal peptidase mediates the cleavage of a basic, putative nucleocapsid protein (C) from the N terminal of the polyprotein precusor, followed by two glycoproteins (E1, E2), both of which represent potential components of the viral envelope. 5-7)Various putative nonstructural proteins (NS2-5) are processed from the downstream region of the polyprotein, mediated in part by a viral protease encoded within the NS3 domain. [7][8][9][10] There is a need to develop vaccines and other antiviral therapies against HCV. A major reason for the lack of rapid progress is the absence of a convenient or inexpensive animal model for propagating HCV. As host animals are limited to humans and chimpanzees, there have been several attempts to develop in vitro HCV replication systems and in vivo infection or expression models. The former include in vitro replication systems using human T cells, [11][12][13][14][15][16][17][18][19] human fibroblasts, 20) human peripheral blood mononuclear cells, 21) non-neoplastic human hepatocytes, 12,22) human hepatoblastoma cells, 23) chimpanzee primary hepatocytes 24) and other cells. [25][26][27] Such HCVinfected culture systems will be useful for various biological studies, including investigations into the mechanisms of HCV replication of the viral genome and cellular tropism of HCV 12,15,16,19,[25][26][27] and into the structure of viral particles.17, 18, 24) Advances in in vivo infection models for reproducible propagation of HCV and hepatitis C viremia have been achieved either by transplantation of HCVinfected liver fragments from patients into BNX (beige/ nude/X-linked immunodeficient) mice 28) or intravenous injection of HCV DNA using gene delivery systems. 29,30) However, there is not enough information about the mechanism of viral persistence, the cellular tropism of HCV, or its pathogenicity in vivo. Another approach to elucidate these questions, including in vivo behavior, would be to establish a tran...