The regulation of ripening in fleshy fruits directly affects the quality and shelf life of such fruit, and extensive research has aimed to understand this regulation based on its agronomic importance. The identification of the key regulatory gene in tomato has opened new horizons in our understanding of fruit ripening. encodes a MADS-box transcription factor that functions as one of the earliest acting factors in the induction of ripening, and the molecular characterization of has helped to elucidate the regulation of fruit ripening. Here I will review current advances in our understanding of the mechanisms that regulate fruit ripening in tomato, including the roles of and other MADS-box proteins, mainly based on our recent studies. First, examination of the molecular properties of the protein revealed that has activities similar to SEPALLATA type MADS-box proteins. Next, identification of the direct transcriptional targets of , via chromatin immunoprecipitation assays, demonstrated that directly regulates a broad range of ripening-associated genes. Finally, identification of MADS-box proteins that interact with revealed the functions of these proteins in ripening regulation. These studies clearly demonstrate the essential roles of MADS-box proteins in the regulation of tomato fruit ripening.

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The small G proteins including Rab5 and Rheb, which cycle between active (GTP-bound) and inactive (GDP-bound) states, play essential roles for membrane budding and trafficking in cells. Here we show novel Rab5-binding proteins ( family), which contain many functional domains shared with other members and additional Pro-rich domains. 3 displays the same biochemical properties as 2, the stimulator and stabilizer for GTP-Rab5. 3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the 3-positive vesicles when co-expressed with 3. These results indicate that 3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes. Rheb appears to be involved not only in cell growth but also in nutrient uptake. We identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression. [J Physiol Sci. 2007;57 Suppl:S43]

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トマトの追熟に及ぼす 因子の抑制作用を更に明確に調べるため, 正常種‘Rutgers’と同質遺伝子系統の 変異種の果実を用いて, 呼吸及びエチレン発生量,ABA及び幾つかの果実成分の貯蔵中の変化を調査した.
1. 貯蔵中の果実の炭酸ガスとエチレン発生量は‘Rutgers’では breaker stage から dark pink stageの間にピークがみられたが, 果実では呼吸のクライマクテリック•ライズ及びエチレン発生量の上昇はみられなかった.
2. 果肉部の遊離ABA含量は‘Rutgers’ろ で共に貯蔵9日目にピークに達し, 以後減少した. その含量は‘Rutgers’におけるよりもむしろの方が多かった. 結合ABAは遊離ABAのそれとほとんど平行的な変化を示したが, その割合は遊離型に比べて低かった.
3. 果肉部のIAA含量は‘Rutgers’においてのみ追熟中増加し, dark pink stage をピークに検出されたが, ではほとんど検出されなかった.
4. 果肉部の炭水化物含量と滴定酸度は貯蔵日数の経過につれて漸減したが, ‘Rutgers’と 両果実間に大差はみられなかった.
5. 貯蔵中の果肉部における主要な遊離アミノ酸はアスパラギン酸, スレオニン+セリン区分, グルタミン酸, フェニールアラニン及びγ-アミノ酪酸であった. これらのアミノ酸のうち, アスパラギン酸とグルタミン酸含量は‘Rutgers’と で共に貯蔵中著しく増加した. 他のアミノ酸については大きな変化はみられなかった.

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Si-Ge elements and Mo electrodes were tried to braze with 4 kinds of Ti-base brazes. Aπ-shaped module used for thermoelectric generator was constructed from one couple of p and n-type Si-Ge elements and Mo electrodes. The maximum electrical power P_max and the internal resistivity of the modules were evaluated as a function of temperature difference between the hot side and cold side of the module. The module using TZCN braze (TZCN-module) showed the highest P_max and the lowest After annealing treatment at 700°C in vacuum for 5hr, the of the TZCN-module was increased to 1.5-2 times of the initial value before annealing. After the first 5hr annealing, the was found to be stable for 53hr. Before and after annealing treatment at 700°C in vacuum for 3hr, the optical and the X-ray images of Si-Ge/Mo joint section brazed with TZCN were not observed to be changed. We thought that the increase of during the first 5hr annealing is due to the fine cracks between Si-Ge and brazing layer.

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The ripening inhibitor () mutant tomato yields non-ripening fruit, and the hybrid fruit (

RIN

/) shows an intermediate phenotype between the wild and mutant fruit, that is, red-ripe and extended shelf life. We found by a microarray analysis that the genes encoding possible allergenic proteins were expressed at a significantly lower level in the hybrid fruit than in the wild-type fruit. These allergenic proteins, which were β-fructofuranosidase and polygalacturonase 2A (PG-2A), were confirmed to accumulate at a lower level in the hybrid fruit than in the wild-type fruit. The immunoglobulin E (IgE) in serum from a tomato-allergic patient showed lower reactivity to the extract of the hybrid fruit than to that of the wild fruit. These results suggest that the gene has the potential to regulate allergen accumulation in tomato fruit.

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In an experimental field, seed vivipary occurred in one accession of tomato mutant fruit at approximately 45–50 days after pollination (DAP). In this study, the possible contributory factors to this viviparous germination were investigated. Firstly, developing seeds were freshly excised from the fruit tissue every 5 days from 25–60 DAP. Germination occurred when isolated seeds were incubated on water, but was inhibited when they remained ex situ in fruit mucilage gel. The effect of abscisic acid (ABA) and osmoticum, separate and together, on germination of developing seeds was investigated. Additionally, ABA content in the seed and mucilage gel, as well as fruit osmolality were measured. The results showed that ABA concentrations in seeds were low during early development and increased later, peaking at about 50 DAP. ABA concentrations in accession were similar to those of the control cultivar and thus are not directly associated with the occurrence of vivipary. Developing seeds of accession are more sensitive than control seeds to all inhibitory compounds. However, osmolality in fruit at later developmental stages becomes less negative that is required to permit germination of developing seeds. Hence, hypo-osmolality in fruit may be an important factor in permitting limited viviparous germination.

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In the lumber processing industry, the first priority is to secure the lumber resources to be supplied uninterruptedly. This study aims at using satoyama-(countryside coppices) not utilized so far as materials for lumber processing. We have tried to develop wooden saving boxes modeling a barrel using konara (small Japanese oaks) and kunugi (a kind of oaks) most common in countryside coppices. As a result we found that satoyama- can be utilized if processed products are small by paying due attention to drying, cutting and painting of wood. It is necessary to protection of resources for the utilization of satoyama- simultaneously, for which we are preparing materials.

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BB/Wラットの糖尿病発症機序におけるサイトカインおよびマクロファージ (Mφ) の関与を検討した. ラットインスリノーマ () 細胞増殖阻止試験でIL-1, TNF, interferon-γ (IFN-γ) の細胞障害性を検討, また, GH₃細胞を標的細胞, BB/Wラットの腹腔細胞をエフェクター細胞として⁵¹Cr放出による細胞障害試験を行った. 細胞はIL-1単独で障害されたが, TNFに対しては抵抗性を示した. 一方IL-1, TNF, IFN-γ の共存により細胞障害性に相加, 相乗作用が認められた. また急性糖尿病BB/Wラットの腹腔細胞およびMφ は細胞に対し障害作用を示したが, GH₃細胞には障害作用を示さなかった. 以上よりBB/Wラット糖尿病発症にMφ およびMφ が分泌するサイトカインの関与が示唆された.

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The present study successfully carried out microscopic imaging of the steady-state in-plane fluorescence anisotropy ( ) of rhodamine B at the free and surfactant-modified toluene/water interfaces excited under total internal reflection conditions with an inverted microscope, a laser system, a linear polarizer, and a CCD camera, which were easy to operate and inexpensive. As surfactants, sodium dodecylsulfate (SDS), Triton X-100 (TX100), and dihexadecyl hydrogen phosphate (Hdhp) were used. The microscopic images were almost homogeneous regardless of the types and concentrations of surfactants, implying homogeneous interfaces. On the other hand, the peaks of the fluorescence spectrum of interfacial rhodamine B and averaged values depended on the types and concentrations of the surfactants. This dependency was mainly attributable to the change of the rotational dynamics of interfacial rhodamine B by solvation. These results suggested that the proposed method would be a promising one to evaluate the homogeneity of liquid/liquid interfaces and the reactivity of solutes at the interface.

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RNAの分解は，臨床検体の遺伝子発現の分析を行うにあたって，重要な問題である．とくに，RNAの十分な品質は，定量PCRやマイクロアレイ解析のようなmRNA測定の精度を決定する重要な因子である．RNAサンプルのRNA収量と品質を評価し，マイクロチップ電気泳動システムによるRNA Integrity Number（）を得た．冷凍保存したRNAと培養細胞から抽出したRNAの全サンプルは，9.8以上のを示し，アガロース電気泳動においても高分子量であった．また，は，凍結融解の繰り返しによる影響を受けなかった．したがって，これらの結果は，が保存中のRNAの品質を適切に評価しうる指標であることを示唆している．

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本実験は追熟因子の欠落がエチレン生成にいかに影響を及ぼすかをみるために, 追熟性の ′Rutgers′, 追熟遅延性の nor トマトと非追熟性の トマトを用い, 色々な生育•成熟段階の果実についてACC(1-アミノシクロプロパン-1-カルボン酸)含量ならびにACC合成酵素, エチレン生成酵素(EFE)の活性の消長を調べた. 貯蔵中, ′Rutgers′ は追熟段階でエチレンを生成し, nor トマトではわずかに生成があり, トマトではほとんど生成がなかった. その原因としては ′Rutgers′ は追熟時にACCの蓄積とEFE活性の増加があり, nor トマトは生育•成熟段階の進展に伴ってACCの蓄積はあるもののEFE活性の増加はなく, トマトはACCの蓄積もなく, またEFEの活性の増加もないためであろうと考えられた.

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Recently, we have found that the accumulation of ripening inhibitor () protein increased gradually during tomato fruit ripening. Here, the recombinant protein was expressed in Escherichia coli and affinity-purified. The DNA binding activity of renatured protein was tested by electrophoretic mobility shift assay. The results indicated that an optimal expression and purification system was suitable for obtaining active with DNA binding activity.

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Monoclonal antibody 3A4 to islet cell surface antigen has been previously established in our laboratory, using hybridization of spleen lymphocytes from non-obese diabetic (NOD) mice transferred into immunologically incompetent recipient mice.
In the present study, monoclonal islet cell surface antibody 5C12 could be newly obtained in the 10 : 1 ratio of NOD mice spleen cells and mouse myeloma cells (SP2/0) without any modifications.
Protein A radioligand assay and indirect immnofluorescence on living cells showed that 5C12 antibody reacted to normal rat islet cells and cultured rat insulinoma cells (-r), but not to cultured lymphocytes (Bri-7, IM-9) and Chang-liver cells. Analysis of ¹²⁵I-labeled antibody binding revealed that unlabeled 5C12 effectively inhibited subsequent ¹²⁵I-5C12 binding to -r cells, whereas unlabeled 3A4 did not. The scatchard plot from these data showed the curvilinearity, and about 150,000 binding sites to antibody per -r cell were counted.
The treatment of -r cells with papain and neuraminidase reduced the binding of 5C12 to -r cells, whereas the effect of trypsin was not observed.
Immunoprecipitation of ¹²⁵I-labeled insulinoma cell lysates followed by SDS-PAGE and autoradiography indicated that 5C12 recognized 105K dalton cell surface protein in -r cells. Immunoblotting also showed that 5C12 antibody recognized 105Kdalton cell surface protein in -r cells.
These results demonstrated that 5C12 was an important tool for clarifying the immunoresponse against certain antigenic determinants on pancreatic B cells.
Furthermore, 5C12 has not only qualitatively and quantitatively improved diagnostic methodology, but it may also provide new reagents useful to the treatment and prevention of type 1 diabetes.

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The objective of this study is to make clear the difference of spatial conceptions of open spaces of Shinto shrines between Shasoh and its synonyms “Chinjyuno-mori ”and “Shaji-”. We approached the sides of qualitative changes and quantitative changes. By both side of research, we could make clear the meanings and their contexts considered with the social background of their words. As a result, we can mention similarities and differences between “Shasoh”, “Chinjyuno-mori” and “Shaji-”. From 1975 onward, the spaces of forest of Shinto shrines were attentioned for study site by various kinds of scientific fields. The spatial conception of “Shasoh” was intended for the space of forests only in Shinto shrines. This word was taken the Shintoism into their consideration. The spatial conception of “Chinjyuno-mori” was intended for image for gods or spiritual spaces “Geniusu Loci” in origin, and then intended for the valuable site for ecological and botanical study as space of native forest. The spatial conception of “Shaji-” was used by political stance at first, and then it was intended for the spaces involved in politics of forests and fields. This word wasn't distinguished between the space and image of Shinto shrine and Buddhism temple.

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We have previously shown a significant decrease in the ethylene production in tomato fruit from the

RIN

/ genotype. In this present study, we evaluated the amount of 1-aminocyclopropane-1-carboxylic acid (ACC) and the gene expression and enzymatic activities of ACC synthase (ACS) and ACC oxidase (ACO) to find which type of regulation influenced this low ethylene production. The results suggest that the decreased ethylene production was due to transcriptional regulation of the ACS and ACO genes by the heterozygous effect of the gene.

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Ca²⁺ overload is one of the mechanisms for H₂O₂-induced cell death in rat pancreatic β-cell line -5F cells. -5F cells express TRPM2, which is a Ca²⁺-permeable channel activated by H₂O₂, and voltage-dependent L-type Ca²⁺ channels, both of which induce Ca²⁺ entry by H₂O₂. This study examined the contribution of these channels to H₂O₂-induced Ca²⁺ entry and cell death in -5F cells. Cytosolic Ca²⁺ concentration was measured using fura-2 as a Ca²⁺ indicator. Cell death was estimated by trypan blue exclusion. Pre-treatment with poly(ADP-ribose) polymerase (PARP) inhibitors, which inhibit TRPM2 activation, strongly reduced Ca²⁺ entry by H₂O₂. The PARP inhibitors used had no effect on the Ca²⁺ elevation by voltage-dependent L-type Ca²⁺ channels. On the other hand, pre-treatment with L-type Ca²⁺ channel blockers, which did not affect TRPM2 activation, partly reduced H₂O₂-induced Ca²⁺ entry. Treatment with PARP inhibitors but not L-type Ca²⁺ channel blockers, around the early phase in H₂O₂-induced Ca²⁺ elevation, also reduced the late phase. Moreover, H₂O₂-induced -5F cell death was strongly attenuated by PARP inhibitors, in compared to L-type Ca²⁺ channel blockers. Our results suggest that TRPM2 channels rather than L-type Ca²⁺ channels primarily contribute to H₂O₂-induced Ca²⁺ entry and cell death.

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Fleshy fruits are one of important human diets and ripening is a critical step for the fruits to be tasty and higher-quality foods. During ripening, a variety of physiological changes, such as pigmentation, softening, and aroma and flavor development, occurs in a highly synchronized manner. These physiological changes at the onset of the ripening are caused by a synchronized and drastic transition of gene expression patterns in the fruit. Tomato has been used as an advantageous model plant for studies on fleshy fruits, and many advances in our understanding of ripening mechanism have been achieved by the studies of mutations suppressing the ripening phenomena entirely, such as ripening inhibitor （

）, non-ripening （nor） and Colorless non-ripening （Cnr）. These three mutation loci have been identified to encode transcription factors, indicating that these transcription factors play important roles in the drastic and synchronized changes at the onset of ripening. In this review, I will overview the function of ripening regulators including , CNR, NOR and other transcription factors identified recently, and also discuss about the interaction between these transcription factors.

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