To determine the sugar binding site of the Na⁺/glucose cotransporter type1 (SGLT1), substrate specificity of a transporter which was made from rabbit SGLT1 and Xenopus SGLT1-like protein (SGLT1L) was examined. The order of the substrate specificity of SGLT1 is D-glucose:=D-galactose:=α-methyl-D-glucoside (αMDG)>>myo-inositol while that of Xenopus SGLT1L is myo-inositol>D-glucose>D-galactose:=αMDG. αMDG is a specific substrate for SGLT. A transporter was made by substituting the extracellular loop between the transmembrane domain (TM)12 and TM13 of Xenopus SGLT1L with the same portion of rabbit SGLT1. Then the was expressed in the Xenopus oocyte membrane and its transport activity was measured by the two-microelectrode voltage-clamp method. The order of the substrate specificity of the was myo-inositol:=D-glucose>D-galactose:=αMG showing that by the substitution the affinity of the with D-glucose increased but the affinities with myo-inositol, D-galactose and αMG changed little when compared to the native Xenopus SGLT1L. As a result, the substrate specificity of the transporter was similar to neither　SGLT1 nor Xenopus SGLT1L. Therefore, the extracellular loop between TM12 and TM13 is probably a part of the sugar binding site of SGLT1. [Jpn J Physiol 54 Suppl:S87 (2004)]

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We have recently reported that the extracellular loop 12, the extracellular loop between transmembrane domain (TM) 12 and TM13, is a part of the sugar translocation pathway of Na+/glucose cotransporter type 1 (SGLT1) by making a : the loop of Xenopus SGLT1-like protein (xSGLT1L) was substituted with the homologous region of rabbit SGLT1 (BBA 1758, 747-754, 2006). The showed the increased apparent affinities for D-glucose (Glc) and myo-inositol (MI) compared to wild type xSGLT1L and transported both substrates equally. Next we made the second in which the substituted region was expanded to both sides: the substituted region included about one-third of TM12 and almost of TM13. The second transported Glc better than MI, this order was the reverse of the wild type xSGLT1L. From our results we will discuss about the important amino acids in the sugar translocation pathway of SGLT1. [J Physiol Sci. 2007;57 Suppl:S133]

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In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients with characteristics such as growth retardation and short life span. Xpg null mice exhibited the same characteristics as human patients do. We generated four mutant Xpg mouse strains with different mutations in Xpg gene to identify the Xpg region that causes onset of the CS phenotype. We found that the deletion of C terminal 183 amino acids results in the CS phenotype. The primary embryonic fibroblasts isolated from Xpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization. If mice in which some tissues are replaced with Xpg-deficient cells or tissues could be generated, it seems to be very interesting to analyze the fate of such mice and/or Xpg-deficient cells in such mice. So we have generated bone marrow mice in which normal blood cells were replaced with Xpg-deficient cells and examined their life span, shortening of life span by X-irradiation and class switching of immunoglobulin heavy chain. The life span of Xpg- mice was not different from that of normal mice. The life span of Xpg- mice irradiated with X rays was shorter than that of normal mice. Though in vitro studies indicate that Xpg functions as a nuclease for class switching of immunoglobulin heavy chain, class switching was normal even in Xpg- mice.

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病害抵抗性を効率的に付与する方法を開発するために,CTVに抵抗性の‘川野なつだいだい’(N)を起原層第IIおよびIII層に組入れる方向性をも持たせた設計で,周縁キメラの効率的作出を試みた。Nと‘福原オレンジ’(F)の実生を寄せ接ぎし,胚軸の各接合部を横に切断した後,Nの胚軸を茎の方向に対して60度の角度をつけてさらに切断した。その胚軸の切断面を植物ホルモンの配合液で処理し,パラフィルムを被せた後,明るい実験室内で育てた。両品種の境界に近いN上の切断面に生じた不定芽一個を選び温室で育てた。50μMジベレリンA₃, 1μM 6-ベンジルアデニンおよび1μM α-ナフタレン酢酸の配合液を処理した区からキメラが極めて効率的に得られた。このキメラの構成品種は,筆者の開発した簡易法による分析結果から第IIおよびIII層がNから成り,第I層はFから成ると推定された。本研究により,カンキツの起原層第IIおよびIII層にかいよう病およびCTVに抵抗性の組織を簡易に導入する手法が確立されたが,本作出法を以後DHS法と呼称したい。また,このカンキツ合成周縁キメラの学名としてCitrus natsudaidai+sinensisを,品種名として‘NF-5’を提案する。

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嫌気性好熱菌Thermonanerobacter brockii由来コージビオースホスホリラーゼ (KP) は加リン酸分解反応によりコージビオースからグルコースとβ-グルコース-1-リン酸 (β-G1P) とを生成する．本酵素はまた，グルコースとβ-G1Pとからコージビオースのみならず重合度3以上のコージオリゴ糖を生成することが明らかとなっている．適当な受容体糖質の存在下で本酵素をβ-G1Pに作用させることにより新規なオリゴ糖や新規なコージオリゴ糖を調製することが可能である．本研究では，効率的なオリゴ糖の生産や新規なオリゴ糖の酵素合成を目指すため，酵素側からのアプローチとして遺伝子工学的手法を用いて野生型酵素よりもさらに有用な機能を獲得した改変酵素の創出を試みた．KP遺伝子にランダム変異を導入し，機能改変酵素のスクリーニングを行った．その結果，70，75℃における活性半減期が野生型KPの4-7倍に増大した耐熱化変異酵素D513Nと，重合度の大きい生成物を野生型よりも多く産生するDP変異酵素S676N，N687Iを得た．基質特異性改変酵素を獲得する目的で，KPと類縁酵素であるトレハロースホスホリラーゼ (TP) との間でキメラ酵素を作製した．キメラ酵素V-IIIは，全アミノ酸配列中の約84%がTPに由来するにもかかわらず，本酵素はKP活性を示したことから，置換した領域が基質特異性に大きく寄与すると考えられた．本キメラの速度論的解析の結果，コージビオースに対する親和性が野生型KPと比較して著しく増大していた．また，グルコース以外の単糖を受容体としないこともわかった．さらに，本キメラはリン酸非存在下でβ-グルコシド結合をもつオリゴ糖に作用して糖転移物を生成した．これらの結果から，キメラV-IIIの獲得した特異性変化は，触媒部位の還元末端側の構造が変化し，この部位における基質結合能力が著しく低下したためと推定された．

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The first intergeneric chimeras between radish and cabbage were successfully synthesized by in vitro grafting method. Regenerated seventy seven chimeras from the original sectorial were investigated on morphological, physiological natures comparing with other interspecific chimeras of Brassica. Chimeral structures were justified and totally classified based on the morphological characteristics, isozymatic band patterns and PCR analysis. In those chimeras, putative four-layered chimeral plants were also found for two vegetatively-propagated generations with general three-layered chimeras. Physiological interactions were typically recognized such as in flowering date, pollen fertility, and pod and seed set. However, genetic changes were not found in the progeny derived from the crosses with both parents.

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When a transparent cornea becomes opaque due to infectious diseases, trauma, or ophthalmic surgery, the impaired cornea is replaced with a donor cornea to improve visual function. In this corneal transplantation, the graft survival rate is comparatively high, partly because of lacking vascular and lymphatic vessel in cornea. However, the transplanted corneas sometimes become opaque if allograft rejection occurs. Suppression of allograft rejection is critical for favorable outcomes of corneal transplantation. The essential effects of endogenous monomeric soluble vascular endothelial growth factor receptors (VEGFRs) 1 and 2 have been reported in corneal angiogenesis and lymphangiogenesis. This study investigated the effects of dimeric soluble VEGFR2/Fc protein on corneal allograft rejection for future clinical application. Allogeneic full-thickness corneal transplantation was performed in C57BL/6 to BALB/c mice. The recipients were treated by intrastromal injection of soluble VEGFR1/Fc (sR1/Fc group), soluble VEGFR2/Fc (sR2/Fc group), or human IgG1/Fc protein (IgG/Fc group) at 0, 7, and 14 days after surgery. Both hemangiogenesis and lymphangiogenesis were significantly suppressed in the corneas of the sR2/Fc group compared with the IgG/Fc group. All grafts failed due to corneal wound rupture in the sR1/Fc group. In the sR2/Fc group, respective donor-derived MHC class II⁺/CD11c⁺ cells and CD11b-positive macrophage infiltration were reduced in the DLNs and the corneas showing a negative delayed-type hypersensitivity, compared with the IgG/Fc group. Our findings demonstrate that soluble VEGFR2/Fc protein efficiently suppresses corneal allo-rejection, while reducing hemangiogenesis and lymhangiogenesis, and immune-competent cell-trafficking and may be a powerful tool for corneal allograft survival.

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For the purpose of inducing the specific tolerance by bone marrow transplantation, the PHA non-responding cells out of the bone marrow cells were transplanted to mongrel dogs in order to avoid the acute graft-versus-host reaction after conditioning the animals with cyclophosphamide. And nine out of 13 mongrel dogs survived under the condition of successfully established chimerism. The mixed lymphocyte culture between donor and recipient showed no response after chimerism had been established, while dogs responded normally against heterologous antigens. However, the phenomenon that the degree of donor cell occupancy decreased as time went by forced us to consider the existence of immunological reaction of recipient to donor cells.

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Periclinal chimeras play important roles in vegetatively propagated plants such as chrysanthemum (Chrysanthemum morifolium). For example, periclinal chimerism causes flower color variation in chrysanthemums. In this study, a method for periclinal production in chrysanthemum was examined. A wild-type plant of chrysanthemum ‘Taihei’ and its transgenic plant carrying a yellowish-green fluorescent protein gene from the marine plankton Chiridius poppei (CpYGFP) were used as plant materials. The cut faces of the leaf explants of both materials were partially attached and then were detached for further culture. Mosaic calli consisted of transgenic and wild-type cells formed on the detached faces of the explants. We examined 996 regenerated shoots from 4,120 explants and found only a single chimeric shoot that appeared to show mericlinal chimerism. Repeated axillary bud elongation from the nodes of the mericlinal produced one L1-fluorescent and one L3-fluorescent chimeric plant. The L1 showed fluorescence in the epidermal cells and trichomes of leaf and stem. The L3 showed fluorescence in the cells of the central parts of stem and leaf, as well as in the whole root tissues. In summary, we obtained chrysanthemum periclinal chimeras through regeneration from leaf explants using the fluorescent protein transgene as a selection marker.

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Petal color change in morning glory Ipomoea tricolor cv. Heavenly Blue, from red to blue, during the flower-opening period is due to an unusual increase in vacuolar pH (pHv) from 6.6 to 7.7 in colored epidermal cells. We clarified that this pHv increase is involved in tonoplast-localized Na⁺/H⁺ exchanger (NHX). However, the mechanism of pHv increase and the physiological role of NHX1 in petal cells have remained obscure. In this study, synchrony of petal-color change from red to blue, pHv increase, K⁺ accumulation, and cell expansion growth during flower-opening period were examined with special reference to ItNHX1. We concluded that ItNHX1 exchanges K⁺, but not Na⁺, with H⁺ to accumulate an ionic osmoticum in the vacuole, which is then followed by cell expansion growth. This function may lead to full opening of petals with a characteristic blue color.

(Communicated by Ryoji NOYORI, M.J.A.)

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Cytochrome P 450 sterol 14-demethylase (P450-CYP51) is the enzyme that catalyzes 14α demethylation of lanosterol, a step in ergosterol biosynthesis, on the cytoplasmic side of the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. To investigate its localization and the localization mechanism(s), we constructed a by inserting a 30-residue segment, Leu²⁸³-Leu³¹² of P450-CYP51 containing a potential N-glycosylation site in the cytoplasmic region, into the N-terminus of the same protein and tagging the C-terminus with three repeats of a hemagglutinin epitope. This complements gene disruption on a single-copy vector and undergoes N-glycosylation, showing that it functions normally in vivo. Indirect immunofluorescence microscopy revealed that this is localized exclusively to the ER when it is expressed on either a single-copy or multicopy vector. We carried out pulse-chase experiments and found that this , when expressed on a multicopy plasmid, gradually undergoes α1→6 glycosylation, a cis-Golgi-specific modification, but not α1→3 glycosylation, a medial Golgi-specific modification. In contrast, a single-copy expression of this does not lead to the cis-Golgi specific modification. These findings suggest that, when expressed on a multicopy plasmid, a fraction of this is transported from the ER to the cis-Golgi compartment and subsequently recycled to the ER, but when expressed on a single-copy plasmid, no significant transport of this protein from the ER takes place. We thus suggest the possibility that cytochrome P 450 is retained in the ER by a saturable static mechanism.

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The antiinflammatory effect, gastrotoxicity and in vivo absorption property of drug of flurbiprofen(FP)with histamine H₂-antagonist, PPA, were compared with those of FP and FP methyl ester. FP-PPA drug and FP methyl ester were little hydrolyzed in the buffer of the pH 1.2 to 7.4 region in the absence or presence of pepsin and trypsin, in contrast to fast hydrolysis(t_1/2 : about 20 sec)in rat plasma. FP drug inhibited carrageenin induced paw swelling in the same level as FP alone. FP-PPA drug significantly reduced gastrotoxicity in comparison to equivalent dose of FP, whereas the coad-ministration of FP with PPA did not affect gastrotoxicity of FP. FP methyl ester caused slightly less damage to the gastric mucosa than FP alone. The plasma concentration of FP after administration of FP derivatives were similar to FP alone. These data suggested that drug is effective for reduction of gastric damage, compared with either FP or alkyl ester prodrug like methyl ester.

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Mutation breeding is a useful method to improve crops. More than 20 years have passed since mutation breeding with ion beams started in Japan, since which time many mutant varieties have been produced with ion beams. However, ion beams have not been sufficiently characterized in terms of mutagens for plant mutation breeding. This review introduces the characteristics of ion beams as mutagens for mutation breeding; investigated with three objectives: to obtain useful mutants with limited plant damage by irradiation treatment, the width of the mutated sector produced by irradiation, and the mutation spectrum, compared to gamma rays using rice and chrysanthemums. In addition, the optimum dose of ion beams and the criteria for an optimum irradiation dose are shown, based on which the irradiation treatment for the success of mutation breeding with ion beams is also described.

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Among SigA-dependent promoters in Bacillus subtilis, we compared the nucleotide sequences of heat shock responding and non-responding promoters. Chimeric promoter experiments revealed that the heat shock response could be ascribed to the initiation nucleotide (iNTP) of the transcription. Our in vivo reporter assay results indicated that a full response was achieved using GTP, a reduced response was observed using ATP, and no additional expression was observed using UTP or CTP. We then investigated the in vitro transcription assay in more detail. Enhanced transcription that was dependent upon the iNTP was observed when heat treatment was administered during the pre-initiation period. We next analyzed the efficiency of open complex formation using potassium permanganate footprinting, and our results revealed an increase in the ratio of open complex formation at elevated temperatures. Based on this, we suggest that the overall intensification of transcription at high temperatures was derived from the high efficiency of open complex formation together with the high affinity of RNA polymerase (RNAP) for the initiation nucleotide GTP. To determine if this mechanism observed in B. subtilis RNAP is common among bacterial species, we performed similar experiments using Escherichia coli RNAP. Our results indicated that E. coli RNAP also exhibited both temperature- and iNTP-dependent enhancement of transcription. Although the temperature ranges and the ratios of enhancement are somewhat different, the overall heat shock response mechanism mediated by the iNTP of transcription appears to be conserved among bacterial RNAP.

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Cytomorphological and phytochemical studies in a chimeric mutant obtained through colchicine treatment in mulberry were conducted. Uniform distribution of ploidal (2n+4n) was detected in different vegetative parts. Meiosis in the chimeric plant was highly irregular. Persistence of in vegetative clonal generations was observed. Distinct morphological changes along with an increase in phenol and a decrease in sugar content were observed in the mutant. Significance of ploidal in the improvement of mulberry is discussed.

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Two cytoplasmic male sterile (CMS) lines were found in the progenies of an intergeneric cybrid (Brassica oleracea L. var. capitata) derived from asymmetrical protoplast-fusion and an interspecific (Brassica rapa L. var. peruviridis) derived from in vitro grafting. Although these CMS lines were different in origin, they both had abnormal anthers and aborted pollen grains. The mitochondrial DNA in these CMS lines indicated the presence of orf138, an indicator gene for Ogura-type cytoplasm. In this study, direct sequencing of two regions near the rps16 in chloroplast DNA was performed. Although the sequence of the two regions in these two CMS lines was different from that in their parental lines, it was the same as the sequence in an Ogura-type CMS radish. Thus, these results suggest that the two CMS lines possess chloroplast DNA of the Ogura-type or a similar type as well as Ogura-type mitochondrial DNA. Furthermore, the region between rps16 and trnK was compared among species related to Brassica by PCR. The result indicated the presence of many sequence variations in this region and suggested that chloroplast DNAs could be distinguished by analyzing this region by PCR.

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There are two broad categories of : twin and dispermic. Here we report a case of a young girl who was a dispermic and had both group A₁ and B red cells in her serum simultaneously.
The patient (H. B.) was a five-year old girl with congenital nevus. She was born by normal delivery as the first child of healthy parents. She had nerve received a transfusion, and there was no evidence of a twin. Her blood contained 96% group B, type MNss red cells and 4% group A₁, type NNss red cells. The patient's saliva also contained A and B substances at normal secretor ranges. The level of D-galactosaminyltransferase activity in her serum was within the normal ranges but the level of N-acetyl-d-galactosaminyltransferase activity was at the extreme lower end of the normal range for group A₁B persons. One hundred metaphase cells from a culture of the patient's peripheral lymphocytes were all 46, XX. The phenotype of the serum group-specific component (GC) protein of the father was GC2-1S, and of the mother, GC2-1F, but the patient's GC protein was GC1S-1F-(-2). This suggests that the patient's serum contained two populations of GC protein. The sweat gland cells of her scalp tissue stained with both anti-A and anti-B reagents.
These findings demonstrate this child to be a dispermic according to Race and Sanger's classification system.

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The subject was a 28-year-old Japanese woman whose red blood cells had two populations of A, CCDee, Jk (a+b-), and AB, CcDEe, Jk (a+b+), at 95% and 5%, respectively. The karyotype for her lymphocytes was a female type of 46, XX, 100%. She was not a twin and had no history of chimeric features such as hermaphroditism, patchy skin pigmentation, or heterochromia iridis, as is often seen in dispermic chimeras. Her chimerism was further investigated using genomic DNA from leukocytes and nails as a somatic sample. Amelogenin genes specific for Y chromosome were not detected in her nails. Analyses of microsatellite and blood group genes were carried out by a PCR-SSCP method. Analysis of ABO gene revealed that A, B, and O gene coexisted in not only her peripheral blood but also her nails, and that the amount of B gene was less than that of A or O gene, corresponding to the rate of red blood cells. Rh blood group gene analyses showed the same result. Furthermore, a double contribution of alleles from her father was suggested both in her peripheral blood and nails by microsatellite analysis at the D1S102 locus. Therefore, on the basis of these findings, we considered this was a case of dispermic .

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