Annual Report 2006

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Fig. 3 Inhibition of MRSA proliferation by fibroin films containing the 9-mer peptide, ALYLAIRRR-NH2 MRSA seeded on the culture plates was covered with the fibroin films containing 0 (a) and 300 mg/cm 2 (b)ofthe peptide. The plates were incubated at 37for 24h. The transparent moist films were gradually dried in an a incubator and formed creases. Note that many MRSA colonies are detected under the film containing no peptides (a), whereas no MRSA colonies are seen under the films containing the peptides (b). females. However, not only defensin A, but also three other defensin isoforms showed gene expression in the midgut, suggesting the possibility that these antibacterial peptides are secreted into the midgut lumen. To further understand tick immune mechanisms, the involvement of antibacterial peptides in midgut defense was investigated. Three antibacterial peptides with molecular masses near defensin isoforms B, C and D were detected in the midgut contents of blood-fed females. Enzymelinked immunosorbent assay analysis revealed that the antibacterial peptides in the midgut contents cross-reacted with defensin A antibodies and increased as a response to blood feeding. Simultaneously, the antibacterial activity of the midgut contents was enhanced by blood feeding. Secretion of antibacterial peptides into the midgut lumen and an increase in the peptide concentration following blood feeding was also confirmed. These findings further support the hypothesis that antibacterial peptides play an important role in the midgut defense of ticks. An antibacterial peptide was isolated from a lepidopteran insect, The molecular mass of this peptide was determined to be 4489.55 by matrix assisted laser desorption/ ionization-time of flight mass (MALDI-TOF-MS) spectrometry. The peptide consists of 42 amino acids and the sequence has 69-98% identity to those of moricin-related peptides, antibacterial peptides from lepidopteran insects. Thus, the peptide was designated (Sl) moricin. Sl moricin showed a broad antibacterial spectrum against Gram-positive and negative bacteria. Sl moricin gene was inducible by bacterial injection and expressed tissue specifically in the fat body and hemocytes. Furthermore, the solution structure of Sl moricin was determined by two-dimensional (2D) 1 H-nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometrysimulated annealing calculation. The tertiary structure revealed a long a-helix containing eight turns along nearly the full length of the peptide like that of moricin, confirming that Sl moricin is a new moricin-like antibacterial peptide. These results suggest that moricin is present not only in but also in other lepidopteran insects forming a gene family. Development of mammalian selection markers We have developed a series of new mammalian cell surface marker fusion genes using a gene from as an antigen. The fusion genes are intended to use as selection markers to separate transformed mammalian cells rapidly without any toxic effect on cell growth. Two different length of the gene was used: a longer fragment contains the native bacterial signal sequence which the shorter fragment lacks. To express the streptavidin antigen on mammalian cell surface, the streptavidin gene was sandwiched by a mammalian signal sequence and a transmembrane sequence at N-terminus and C-
terminus, respectively. A signal sequence and a trans-membrane sequence from either the mouse gene or the mouse gene were used. Some constructs contains the gene sequence next to the gene sequence. Totally eight kinds of fusion genes were constructed in this study. To examine the expression of the fusion genes on mammalian cell surface, a series of plasmids encoding the fusion protein were transfected into the HeLa cells. Expression of the fusion protein on cell surface was observed for any of the constructs. Then, an antibody-mediated immunomagnetic separation methodology was applied to separate transformed cells. Transfected cells were incubated with a polyclonal antibody against streptavidin, and the antibody bound cells were pulled out using a paramagnetic beads coupled with the corresponding secondary antibody. Highly pure population of transformed cells was separated (Fig. 4). Then, effects of fusion proteins on cell growth were assayed. Cell proliferation rate of transformed HeLa cells were compared with that of the untransformed HeLa cells. No significant difference of cell growth was observed in any of the fusion genes. The property of eight fusion genes developed in this study was found to be similar. These results suggest that the fusion genes and the immunomagnetic separation protocol are useful for various transformation applications. Fig. 4 Expression of fusion protein on HeLa cells transfected with a plasmid encoding the FSAH2K-EGFP fusion protein (A) Localization of streptavidin antigen on the surface of a transformed cell. Serial images captured at 5-µm intervals on the z-axis. (B) Transfected HeLa cells. Red staining indicates transformed cells. (C-E) Immunomagnetically separated cells, same field. (C) Light-interference view. (D) Cells immunostained with anti-streptavidin antibody. (E) EGFP-positive cells. Scale bar: 10 mm (A) and 100 mm (B-E).
Page 1 and 2: 2006
Page 3 and 4: Teruo Ishige, President The Nationa
Page 5 and 6: List of Publication Original paper
Page 7 and 8: Topics of Research in This Year Pos
Page 9 and 10: studies. To construct a fully satur
Page 11 and 12: Fig. 2 Geographical distribution of
Page 13 and 14: (ABR0015, ABR0257, ABR0417, ABR0495
Page 15 and 16: Mulberry latex defense mulberry tre
Page 17 and 18: Fig. 2 Growth inhibitory effects of
Page 19 and 20: Fig. 3 Change of the levels of luci
Page 21 and 22: Next, we examined the effect of tra
Page 23 and 24: G enome Research Department Introdu
Page 25 and 26: The Rice Annotation Project Databas
Page 27 and 28: industrial and agricultural concern
Page 29 and 30: One of the main problems involving
Page 31 and 32: Fig. 3 Mapping of QTL for rachis in
Page 33 and 34: two genes, XOO2263 and XOO4134 cont
Page 35 and 36: G enebank Genebank Project The NIAS
Page 37 and 38: using dormant winter buds. Also, we
Page 39 and 40: Insect and Animal Sciences Division
Page 41 and 42: Expression patterns of two novel ge
Page 43 and 44: Fig. 5 PGC and EBC fused cells PGCs
Page 45 and 46: cells. The epidermal cells maintain
Page 47: Animal and cell culture models for
Page 51 and 52: Fig. 2 Trehalose content of body pa
Page 53 and 54: Fig. 6 Birth weight of 11 adult SNT
Page 55 and 56: The role of glucose as a metabolic
Page 57 and 58: analysis using microsatellite marke
Page 59 and 60: Fig. 2 Recorded Electromyograms (le
Page 61 and 62: Table 1 Menu of Commercial silkworm
Page 63 and 64: Fig. 3 Expression of GFP gene in th
Page 65 and 66: also surveyed in the genetic stocks
Page 67 and 68: esearch were located in or near gen
Page 69 and 70: elatives results in severe reductio
Page 71 and 72: and circadian clocks may control th
Page 73 and 74: B iochemistry Department Structural
Page 75 and 76: Proteomic analysis Based on the pro
Page 77 and 78: photoautotrophic medium (Fig. 1B).
Page 79 and 80: Fig. 4 Blast resistance of OsWRKY45
Page 81 and 82: phosphatase activity by SIPK depend
Page 83 and 84: the endosperm in co-operation with
Page 85 and 86: isolated genes in transgenic Anal
Page 87 and 88: Fig. 1 Gene structures and mutation
Page 89 and 90: cytochimera among the mulberries (
Page 91 and 92: 15 G. Hariraj, Kinoshita Haruo, T.
Page 93 and 94: 49 Kameda Tsunenori, Teramoto Hidet
Page 95 and 96: 85 Matsuyama Shuichi, Ohkura Satosh
Page 97 and 98: 120 Ozawa Kenjiro, Kawahigashi Hiro
Page 99 and 100:
155 Taguchi-Shiobara Fumio, Yamamot
Page 101 and 102:
192 Ueno Osamu, Agarie Sakae, (2005
Page 103 and 104:
8 Murakami Ritsuko, Koyama Akio, Sh
Page 105 and 106:
20 Todoroki Junichi, Kaneko Hiroyuk
Page 107 and 108:
Executive Members and Research Staf
Page 109 and 110:
Developmental Biology Department Di
Page 111 and 112:
Research Leader Hayasaka Shoji Rese
Page 113 and 114:
Mutation Genetics Laboratory Head N
Page 115 and 116:
thousands of yen TOTAL BUDGET OPERA
Page 117:
Annual Report 2006 (Apr. 2005Mar. 2
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Annual Report 2006

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