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Cytochrome b(561) is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles in the central and peripheral nervous systems of higher animals. We succeeded in cloning a full-length cDNA encoding planarian cytochrome b(561). The deduced amino acid sequence shows a very similar six transmembrane topology to those of cytochromes b(561) of higher vertebrates and contains both putative ascorbate- and monodehydro ascorbate-binding sites. Among the six totally-conserved His residues of cytochrome b(561) in higher vertebrates, one is substituted with an Asn residue, indicating that His88 and His161 of bovine cytochrome b(561) play roles as heme b ligands at the extravesicular side. Northern- and Western-blot analyses confirmed the expression of the mRNA and protein with the expected sizes in planarians. The distributions of the mRNA and apoprotein were analyzed by in situ hybridization and immunohistochemical staining, respectively, showing two morphologically distinct structures, a pair of ventral nerve cords and the cephalic ganglion cluster in the head region. The present results suggest that the usage of ascorbate to supply electron equivalents to neuroendocrine-specific copper-containing monooxygenases is likely to have originated in organisms with a very simple nervous system.

Neuropeptides in the brain, in the nervous system, and in various endocrine cells are synthesized in the rough endoplasmic reticulum as large precursor proteins. After transit to vesicles and during axonal transportation along axons, several processing enzymes residing in the vesicles process the peptides to convert them to mature forms. C-terminal a-amidation of the peptides occurs in the late stage [1] and is probably a rate-limiting step in many instances [2]. Over half of peptide hormones or neuropeptides are amidated in vertebrates; in insects, greater than 90% of such peptides show the presence of a C-terminal amide moiety [3]. This C-terminal amide is very important in their functions, as its absence often disrupts the activity or receptor-binding properties of the peptide ligands [4]. Indeed, most neurotransmitters thus far identified are amidated peptides in cnidarians Planarians are one of the simplest animal groups with a central nervous system. Their primitive central nervous system produces large quantities of a variety of neuropeptides, of which many are amidated at their C terminus. In vertebrates, peptide amidation is catalyzed by two enzymes [peptidylglycine a-hydroxylating monooxygenase (PHM) and peptidyl-ahydroxylglycine a-amidating lyase] acting sequentially. In mammals, both enzymatic activities are contained within a single protein that is encoded by a single gene. By utilizing PCR with degenerate oligonucleotides derived from conserved regions of PHM, we succeeded in cloning a full-length cDNA encoding planarian PHM. The deduced amino acid sequence showed full conservation of five His residues and one Met residue, which bind two Cu atoms that are essential for the activity of PHM. Northern blot analysis confirmed the expression of a PHM mRNA of the expected size. Distribution of the mRNA was analyzed by in situ hybridization, showing specific expression in neurons with two morphologically distinct structures, a pair of the ventral nerve cords and the brain. The distribution of PHM was very similar to that of cytochrome b 561 . This indicates that the ascorbate-related electron transfer system operates in the planarian central nervous system to support the PHM activity and that it predates the emergence of Plathelminthes in the evolutionary history.Abbreviations AsA, ascorbic acid; CNS, central nervous system; DBH, dopamine b-hydroxylase; DBHL, dopamine b-hydroxylase-like protein; PAL, peptidyla-hydroxylglycine a-amidating lyase; PAM, peptidylglycine a-amidating monooxygenase; PHM, peptidylglycine a-hydroxylating monooxygenase; MDA, monodehydroascorbic acid; TBH, tyramine b-hydroxylase; VNC, ventral nerve cords.

In recent times, oncolytic viruses expressing an extraneous gene have attracted great interest; in fact, they have been engaged in multiple applications, such as medicine for cancer. Our group made an oncolytic adenovirus, namely, OBP-301, for use in treating solid cancers and press clinical trial to get approval for a pharmaceutical product. In this study, we applied a flow cytometry-based method to determine the titer of adenoviruses expressing an extraneous gene as well as assess their quality. We considered using the green fluorescent protein (GFP) 50 titer as a measure of viral quality. The GFP 50 titer (GFP 50 /mL) is the viral load required to render the HeLa S3 cell line 50% GFP-positive by analysing flow cytometry data. We measured the GFP 50 titers for three types of recombinant adenoviruses (OBP-401, OBP-1101, and OBP-1106). We compared GFP 50 /mL and tissue culture infectious dose (TCID 50 /mL), a conventional titration index, and found that these titers showed a linear correlation, with a correlation coefficient of >0.9. Moreover, GFP 50 /mL showed high repetitive accuracy. We expect this flow cytometry-based method to be useful in case of clinically relevant viruses expressing an extraneous gene, in particular, to control viral quality.

We have developed a new method to analyze structure of substrate, which is bound to enzyme, by detecting its resonance Raman (RR) spectra. We have applied the new methods to indoleamine 2,3-dioxygenase (IDO) which catalyses direct incorporation of two oxygen atoms into tryptophan (Trp) and whose reaction mechanism is not fully understood due to the lack of structural information. We have successfully obtained UVRR spectra of the bound-substrate in IDO and found some spectral change in Trp bands by binding to IDO; 10 cm -1 down-shift of W3 mode and 10 cm -1 up-shift of W18 mode. These results clearly show that the bound Trp to IDO as substrate has a specific conformation as compared to Trp in solution. We developed a real-time analysis system for biological membranes in the whole-cell recording configuration: two micro-fabricated flow channels connected with a micro aperture (2 μm) where a large-sized liposome is captured. To validate the system, we assessed the proton pump activity of complex I (CI) of the respiratory chain as follows. First, a large-sized protoplast from E. coli (10 μm) was trapped and perforated at the aperture with an electrical pulse. Then, one of the channels was infused with NADH, a substrate for CI. As NADH diffused into the E. coli via the aperture, pH fluorescent indicator started to become bright at another channel. It suggests proton was transferred across the membrane by CI, as observed in bulk biochemical assays. 2P099 ブタ心臓由来ミトコンドリア調製法の確立と活性測定Establishment of a new process for preparation of porcine heart mitochondria, and their activity measurementsWe have been developing a novel real time monitoring device for membrane protein activities. As a membrane sample, we chose the mitochondrial inner membrane (IM) prepared from porcine hearts. Porcine heart, which is not required any assessment of BSE, is extremely fresher than bovine heart, because the hearts can be obtained immediately after the animals are slaughtered.We confirmed the proton pumping activity of the IM proteins in bulk experiment by using a fluorescent pH indicator. The respiratory complexes were selectively activated depending on various substrates, which is consistent with bovine IM assay. By fusing the IM, we also successfully prepared a giant vesicle (>10 μm), which will allow us to image membrane protein activities using our device. Ras is one of small G-protein known as a molecular switch mediating cellular signalling. We performed basic study to control the function of Ras reversibly using photochromic molecules, 4-phenylazophenyl maleimide (PAM) upon visible (VIS) light and ultra-violet (UV) light irradiation. We have prepared the Ras mutants which have a single cysteine at functional sites and modified with PAM stoichiometrically. The GTPase activities of PAM-Ras were reversibly altered upon VIS and UV light irradiations. In order to monitor the effect on GTPase kinetic pathway by the photoisomerization of PAM, We synthesized a novel fluorescent GTP analogue, NBD-GTP. The Kinetic studies suggested that initial ...