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Caffeine (1,3,7-trimethixanthine) is a typical purine alkaloid produced in more than 80 plant species. Its biological role is considered to strengthen plant's defense capabilities, directly as a toxicant to biotic attackers (allelopathy) and indirectly as an activator of defense system (priming). Caffeine is actively secreted into rhizosphere through primary root, and possibly affects the structure of microbe community nearby. The fungal community in coffee plant rhizosphere is enriched with particular species, including Trichoderma family, a mycoparasite that attacks and kills phytopathogens by coiling and destroying their hyphae. In the present study, the caffeine response of 8 filamentous fungi, 4 mycoparasitic Trichoderma, and 4 prey phytopathogens, was examined. Results showed that allelopathic effect of caffeine on fungal growth and development was differential, being stronger on pathogens than on Trichoderma species. Upon confronting, the prey immediately ceased the growth, whereas the predator continued to grow, indicating active mycoparasitism to have occurred. Caffeine enhanced mycoparasitism up to 1.7-fold. Caffeine thus functions in a double-track manner against fungal pathogens: first by direct suppression of growth and development, and second by assisting their natural enemy. These observations suggest that caffeine is a powerful weapon in the arms race between plants and pathogens by fostering enemy's enemy, and we propose the idea of "caffeine fostering" as the third role of caffeine.

To clarify the mechanism of the antihypertensive effect of oral Ca loading, we studied the effect of Ca supplementation on salt-induced blood pressure elevations in patients with essential hypertension and DOCA-salt hypertensive rats. When the diet was changed from low to high salt (300 mEq/day), the percent increase in mean blood pressure was smaller (p less than 0.01) in the Ca-supplemented (2,160 mg/day) patients than in the Ca-restricted (250 mg/day) ones. Oral Ca loading resulted in a smaller weight gain, a greater urinary sodium excretion, and an increase in red cell Mg. In the experimental study, high Ca (4% CaCl2) intake attenuated the blood pressure elevation in DOCA-salt-treated rats, accompanied with an increase in urinary sodium excretion, with the resultant attenuation in intra- and extracellular sodium retention. The decrease in catecholamine contents of hearts was improved, and a higher survival rate was observed in Ca-supplemented DOCA-salt rats. The results suggest that Ca supplementation may prevent a rise in BP in salt-dependent hypertension by inducing natriuresis with the resultant attenuation in sodium retention. The altered intracellular Mg level in hypertensive patients and the normalization of enhanced sympathetic nervous activity in DOCA-salt rats may, in part, be involved in its mechanism.

Background Advanced breast cancer patients have a higher risk of postoperative recurrence than early-stage breast cancer patients. Recurrence is believed to be caused by the increase in micrometases, which were not eradicated by preoperative or postoperative chemotherapy. Therefore, a new therapeutic strategy that can improve treatment efficacy is mandatory for advanced breast cancer. S-1 was shown to be effective and safe in Japanese metastatic breast cancer patients treated with previous chemotherapy, including anthracyclines. Thus, in this study, we evaluated S-1 as adjuvant chemotherapy in breast cancer patients after standard primary systemic chemotherapy. Methods The treatment consisted of 18 courses (a 2-week administration and a 1-week withdrawal; one year) administered at 80–120 mg/body/day. In cases judged to require postoperative radiotherapy, it was concurrently initiated on Day 1 of the study. If the estrogen receptor and/or human epidermal growth factor receptor 2 were positive, endocrine therapy and/or trastuzumab were permitted, concurrently. Results Of the 45 patients enrolled between September 2007 and September 2009 from 3 institutions, 43 patients were eligible. Thirty-two of the 43 (74.4%) patients received concurrent radiotherapy. Twenty-two of the 43 (51.2%) patients completed the scheduled courses of chemotherapy. The most common reasons for withdrawal of treatment were subjective symptoms, such as nausea, anorexia, or general fatigue during the first 9 courses of treatment in 9/43 (20.9%) patients, recurrence in 7/43 (16.3%) patients, and adverse events in 5/43 (11.6%) patients. The cumulative percentage of administration for 365 days was 66.4% (95% confidence interval: 50.8–79.1%). Although grade 3 neutropenia (9.3%), leukopenia (4.7%), and diarrhea (4.7%) were observed, they were manageable. No grade 4 adverse effects were observed. Conclusions The percentage of Japanese breast cancer patients completing the 18-course treatment and the cumulative percentage of administration for 365 days using S-1 after standard primary systemic chemotherapy were similar with the results of another study of adjuvant chemotherapy for the Japanese gastric cancer patients with no severe adverse effects. A phase III trial investigating the usefulness of adjuvant S-1 is now ongoing in Japan, and it is expected that S-1 will have a significant survival benefit in breast cancer patients. UMIN000013469.

Caffeine (1,3,7-trimethylxanthine) is one of the most widely used plant secondary metabolites, primarily as a stimulant and an ingredient in drugs. In nature, caffeine is believed to function in chemical defense, acting as an antiherbivory and allelopathic agent, and therefore it might be employed to protect agriculturally important crop plants. In coffee plants, caffeine is synthesized from the precursor xanthosine in four steps, three N-methylations and removal of ribose. We had previously isolated genes encoding three distinct N-methyltransferases, and we demonstrated production of recombinant enzymes that yielded caffeine in in vitro reconstitution experiments. When these caffeine biosynthetic pathway genes were simultaneously expressed in tobacco plants (Nicotiana tabacum), caffeine was successfully produced up to 5 microg/g fresh weight in leaves. The leaves were unpalatable to tobacco cutworms (Spodoptera litura). This repellent action appeared to be more widely applicable to lepidopteran caterpillars as observed with small white (Pieris rapae) fed on Chinese cabbages that had been top-treated with caffeine. Our recent results suggest a novel approach to strengthen anti-herbivore traits by producing caffeine in crop plants.

The tobacco gene NtWIN4 (Nicotiana tabacum wound-induced clone 4) is transiently up-regulated in response not only to wounding but also to pathogen attack. NtWIN4 encodes a putative basic helix-loop-helix protein with an apparent molecular mass of 28 kDa that exhibited clear nuclear transcription repression activity in Dual-Luciferase assays. However, immunoblotting indicated the existence of a 17-kDa form of NtWIN4 localized exclusively in tobacco leaf chloroplasts. Subsequent peptide dissection analyses with green fluorescent protein fusions revealed that a polypeptide of 81 amino acids starting at position 13 from the N terminus is maximally necessary for this localization. Further fine dissection analysis strongly suggested that the protein actually begins at the second Met located at position 27, yielding a signal peptide of 67 amino acids. However, the last C-terminal 15 amino acids overlap with the conserved basic region critical for DNA binding, so NtWIN4 presumably does not function as a transcription factor in planta. Transgenic tobacco plants constitutively overexpressing NtWIN4 demonstrated mortality with abnormal features, including albinism, and transient expression upon agroinfiltration resulted in distinct necrosis with a sharp decrease in chlorophyll content, consistent with the phenomenon known as chlorosis. Transgenic RNA interference tobacco plants exhibited reduced hypersensitive cell death, showing delayed tissue necrosis upon pathogen infection. These results suggest that NtWIN4 arose by divergence, becoming a chloroplast-resident factor from a nuclear transcriptional repressor by obtaining a transit peptide sequence, and that, upon translocation, it interacts with chloroplast components to induce hypersensitive cell death through chloroplast disruption, thereby contributing to plant stress responses.

NtWIF is a transcription factor activated upon phosphorylation by wound-induced protein kinase (WIPK) in tobacco plants. Transgenic tobacco plants overexpressing NtWIF exhibited constitutive accumulation of transcripts for pathogenesis-related genes, PR-1a and PR-2. Salicylic acid levels were 50-fold higher than those in wild-type plants. The levels of jasmonic acid and IAA did not significantly differ, while an increase of ABA upon wounding was delayed by 3 h in the transgenics. When challenged with tobacco mosaic virus, lesions developed faster and were smaller in the transgenic plants. The results suggest that NtWIF is likely to influence salicylic acid biosynthesis, being located downstream of WIPK.

Programmed cell death plays a critical role during the hypersensitive response in the plant defense system. One of components that triggers it is hydrogen peroxide, which is generated through multiple pathways. One example is proposed to be polyamine oxidation, but direct evidence for this has been limited. In this article, we investigated relationships among polyamine oxidase, hydrogen peroxide, and programmed cell death using a model system constituted of tobacco (Nicotiana tabacum) cultured cell and its elicitor, cryptogein. When cultured cells were treated with cryptogein, programmed cell death occurred with a distinct pattern of DNA degradation. The level of hydrogen peroxide was simultaneously increased, along with polyamine oxidase activity in apoplast. With the same treatment in the presence of α-difluoromethyl-Orn, an inhibitor of polyamine biosynthesis, production of hydrogen peroxide was suppressed and programmed cell death did not occur. A gene encoding a tobacco polyamine oxidase that resides in the apoplast was isolated and used to construct RNAi transgenic cell lines. When these lines were treated with cryptogein, polyamines were not degraded but secreted into culture medium and hydrogen peroxide was scarcely produced, with a concomitant suppression of cell death. Activities of mitogen-activated protein kinases (wound- and salicylic acid-induced protein kinases) were also suppressed, indicating that phosphorylation cascade is involved in polyamine oxidation-derived cell death. These results suggest that polyamine oxidase is a key element for the oxidative burst, which is essential for induction of programmed cell death, and that mitogen-activated protein kinase is one of the factors that mediate this pathway.

� Background and Aims Methyl CpG-binding proteins are considered to play critical roles in epigenetic control of gene expression by recognizing and interacting with 5-methylcytosine (m 5 C) in eukaryotes. However, among 13 corresponding genes in Arabidopsis thaliana, designated as featuring a methyl-binding domain (MBD), only four have so far been shown actually to bind to m 5 C. One example, AtMBD5, was selected here to screen for interacting proteins. � Methods Yeast two-hybrid assays were used for screening, and physical interaction was confirmed by pulldown and bimolecular fluorescence complementation (BiFC) assays. Cellular localization was analysed by fluorescence-tagged fusion proteins using tobacco (Nicotiana tabacum) cultured bright yellow 2 cells. � Key Results A gene finally identified was found to encode AtRAN3, a protein that belongs to the Ran GTPase family, which plays a critical role in nucleocytoplasmic transport and spindle bipolarization during cell division. AtMBD5 and AtRAN3 were clearly shown to interact in the nucleus by BiFC. On co-expression of AtMBD5-cyan fluorescence protein and yellow fluorescence protein-AtRAN3 in tobacco cells, both localized to the nucleus in the resting stage, migrating to the cytoplasm, primarily around chromatin, during mitosis, particularly at metaphase. � Conclusions These results suggest that AtMBD5 becomes localized to the vicinity of chromosomes with the aid of AtRAN3 during cell division, and may play an important role not only in maintenance of chromatin structures by binding to m 5 C, but also in progress through mitosis by detaching from m 5 C. The present findings also shed light on the physiological function of Ran GTPases, direct target proteins of which have not thus far been well defined, suggesting their key role in chromatin movements in plant cells.

Plants cope with pathogens with distinct mechanisms. One example is a gene-for-gene system, in which plants recognize the pathogen molecule by specified protein(s), this being called the R factor. However, mechanisms of interaction between proteins from the host and the pathogen are not completely understood. Here, we analyzed the mode of interaction between the N factor, a tobacco R factor, and the helicase domain (p50) of tobacco mosaic virus (TMV). To this end, domain dissected proteins were prepared and subjected to Agroinfiltration into intact leaves, followed by yeast two hybrid and pull-down assays. The results pointed to three novel features. First, the N factor was found to directly bind to the p50 of TMV, second, ATP was pre-requisite for this interaction, with formation of an ATP/N factor complex, and third, the N factor was shown to possess ATPase activity, which is enhanced by the p50. Moreover, we found that intra- and/or inter-molecular interactions take place in the N factor molecule. This interaction required ATP, and was disrupted by the p50. Based on these results, we propose a following model for the TMV recognition mechanism in tobacco plants. The N factor forms a complex with ATP, to which the helicase domain interacts, and enhances ATP hydrolysis. The resulting ADP/N factor complex then changes its conformation, thereby facilitating further interaction with the down-stream signaling factor(s). This model is consistent with the idea of 'protein machine'.

果樹剪定枝は農業系バイオマスの中では唯一の木質バイオマスであり, 果樹生産地におけるローカル資源として重要な位置を占める.しかしながら, 燃料としての価値を低減させる灰分および農地還元利用との競合については, これまで十分な検討は行われていない.和歌山県で生産されている主要な果樹を対象に, 剪定枝の発生量・発生分布を示すとともに, 灰分特性, 窒素分について検討を行い, 以下の結果を得た.灰分率は樹種に関係なく剪定枝が細くなるほど高くなる.4つの樹種に対する灰分率を平均直径の累乗関数として近似できる.灰分の多い部位は窒素も多く含まれ, 窒素含有率も平均直径の累乗関数として近似できる.堆肥利用の選択基準となるC/N比と灰分率の関係を明らかにし, エネルギー利用の際の基準 (灰分率1.5%以下, C/N比100以上) として, 直径10mm以上の剪定枝が適していることを明らかにした.うめ剪定枝はバイオペレット燃料よりも着火性がよく, ペレット代替の燃料として利用し得ることを示した.

The control efficacy of a novel fungicide, cyflufenamid, (Z)-N-[α-(cyclopropylmethoxyimino)-2,3-difluoro-6-(trifluoromethyl) benzyl]-2-phenylacetamide was studied. In field trials, a low dosage (25 ppm) of cyflufenamid (10%WG) showed excellent efficacy in almost all plants against powdery mildew caused by various pathogens in agricultural production. Cyflufenamid also had high efficacy against brown rot in stone fruits caused by Monilinia fructicola. The fungicidal properties of cyflufenamid were investigated to elucidate the high performance of the compound in the field. Pot tests against cucumber powdery mildew indicated that cyflufenamid has excellent preventive, curative, long residual, translaminar and vapor phase activities at low concentrations. © Pesticide Science Society of Japan

The activity of a novel fungicide, cyflufenamid, (Z)-N-[α-(cyclopropylmethoxyimino)-2,3-difluoro-6-(trifluoromethyl)benzyl]-2-phenylacetamide, against various fungi pathogenic to plants was investigated. In pot tests, cyflufenamid showed excellent control of powdery mildew caused by various pathogens at 0.8 to 1.6 ppm. In culture tests, several species of Ascomycetes and Deuteromycetes were sensitive to cyflufenamid. Notably, Monilinia fructicola was affected at a concentration of just 0.01 ppm. In the life cycle of pathogens causing powdery mildew in wheat, cyflufenamid did not affect infections before the formation of the appressoria, but significantly inhibited the formation of haustoria, colonies, and spores. It also affected the elongation of germ tubes after the germination of spores in M. fructicola. In ultrastructural experiments, cyflufenamid induced a reduction in the amount of highly electron-dense material in the vacuoles and immature septa in M. fructicola. © Pesticide Science Society of Japan

Azadirachta excelsa belongs to the family Meliaceae and is one of the most important silviculture trees in the tropics. In this study, we established a somatic embryogenesis system and succeeded in construction of transgenic lines expressing genes for Bar, GUS and GFP employing the Agrobacterium-mediated transformation technique. Somatic embryos were obtained after approximately two months of culture on Murashige-Skoog (MS) medium containing 5 mM 6- benzyladenine. Plantlets were then regenerated by transferring somatic embryos to modified 1/2 MS medium without phytohormones. We initially attempted genetic transformation using two different A. tumefaciens strains, and found only the strain LBA4404 to be active in infection to explants. The strain EHA101 was totally inactive, suggesting that a specific interaction between Agrobacterium sp. and host plant might be critical for gene transfer. The possibility is discussed of applying the methodology developed in this work to the practical propagation of tropical trees.

The long range goal of the present study is to practically utilize Ipomoea aquatica (water spinach) for phytoremediation of polluted water with sulfuric compounds. In higher plants, the sulfate assimilation pathway consists of 5 key enzymes, among which serine acetyltransferase (SAT) and cysteine synthase (CS) constitute one of the rate limiting steps. Subsequently we have attempted to improve the sulfur assimilation capacity of I. aquatica using genes encoding these two enzymes. Cotyledon segments of seedlings were transformed with Arabidopsis SAT and rice CS genes under the control of the cauliflower mosaic virus 35S promoter. Among 3,245 cotyledon explants, 325 regenerated shoots, and two showed a high tolerance to hygromycin, designated as SR3 and SR10. In transgenic lines, the SAT activity was over 2-fold, and the CS was 3-fold higher than those in the wild type control. The cysteine and glutathione contents were also 6- and 2-fold higher than the control, respectively. When cultured in the presence of 1 g l � 1 (7 mM) sulfate, they accumulated sulfate as much as 20 mg g � 1 fresh weight, being 5-fold higher than the control. Under standard culture conditions, transgenic lines grew faster than the control, showing a 20% increase in fresh weight within 5 weeks cultivation. These results suggested that strengthening of SAT and CS resulted in increase not only in sulfate uptake, but also in total biomass.

Hydrogen sulfide is a major environmental pollutant, highly toxic to living organisms at high concentrations. Even at low concentrations, it causes an unpleasant odor from wetlands, especially from wastewater. Plants can utilize hydrogen sulfide as a sulfur source to synthesize cysteine, which then serves as the principal substrate for synthesis of other sulfur containing compounds including glutathione and methionine. It was thus feasible to use aquatic plants, which possess high potential for sulfur assimilation, to remove hydrogen sulfide from the wetland. To this end, we have generated transgenic rice plants over-expressing cysteine synthase, a key enzyme in the sulfur assimilation pathway, and evaluated their capacity for sulfur uptake on hydrogen sulfide treatment. The obtained transgenic plants exhibited 3-fold elevated cysteine synthase activity, and incorporated more hydrogen sulfide into cysteine and glutathione than their wild type counterparts upon exposure to a high level of hydrogen sulfide. These observations suggest that over-expression of cysteine synthase in aquatic plants is a viable approach to remove hydrogen sulfide from polluted environments.

A folky mountain system (“Satoyama” in Japanese) has a peculiar feature in the view of energy system. In Japan, many of the forests exist in such steep mountains, that the large expense is needed for the collec-tion and transportation of the felled or thinned woods. On the contrary in folky mountains, we can expect to avoid the big energy loss at the process of transportation because of the short distance to the consumers. However, the folky mountain system nowadays is declining because of an aging of the laborers and subsid-ence of demand of fuelwood. Accordingly, the restoration of classical folky mountain system is quite diffi-cult without new system-tool. We proposed here several new measurements to solve the Problems: (1) rental forest, (2) household forest, (3) mountain-forestal pasture, (4) thinned wood direct use.

OBJECTIVE To characterize serial long-term histological changes in mesenchymal cells infiltrating a collagen-based matrix, as in a hollow organ with differentiated urothelial lining created intraperitoneally by grafting cultured urothelial cells, mesenchymal cells with smooth-muscle immunohistochemical characteristics infiltrated into the scaffold, despite no mesenchymal cells being seeded into the scaffold before grafting. MATERIALS AND METHODS To regenerate a urothelial lining tissue intraperitoneally, rat urothelial cells were cultured and seeded with the feeder-layer technique onto bladder acellular matrix (BAM). After 7 days of cultivation to attach urothelial cells on the BAM, the matrix was folded with the urothelial cells inside and grafted onto the mesentery of the previously partially cystectomized rat. RESULTS The grafted urothelial cells on the BAM, which formed a monolayer before grafting, stratified into three to four layers as early as 4 days after grafting. Although the regenerated urothelium became thinner with time, there was urothelial stratification and a peculiar angular appearance on the apical surface of the regenerated urothelium even after 56 days. The mesenchymal cells infiltrating the BAM showed positive immunohistochemical staining to α-smooth muscle actin or desmin at 7 days. Subsequently, the number of actin- or desmin-positive cells gradually decreased with time. On transmission electron microscopy, the infiltrating mesenchymal cells were characterized as myofibroblasts at 7 days. Smooth muscle-like cells were identified at 14 and 28 days, and fibrocytes were the main population at 56 days. CONCLUSIONS Although epithelial-mesenchymal interactions have been assumed to be one of the most critical factors in smooth-muscle development, mesenchymal cells infiltrating the scaffold in this intraperitoneal regeneration model gradually lost smooth muscle characteristics with time. These results suggest that interactions between cultured urothelial cells and infiltrating mesenchymal cells alone could not maintain the smooth muscle character of infiltrating mesenchymal cells.

Among 12,000 alkaloids which are produced in plants, caffeine (1,3,7-trimethylxanthine) is one of the best known due to it uses as an ingredient of pharmaceuticals and beverages. In coffee plants, it is synthesized from xanthosine through three successive methylation and ribose removal steps. We have isolated all genes encoding the corresponding N-methyltransferases; xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT), as well as for the 7-methylxanthosine nucleosidase. Using these genes, we have engineered caffeine production in two ways. The first is to decrease the caffeine content in coffee plants to cope with occasional health problems caused by caffeine uptake, and the other is to produce caffeine as an insect repellant in crop plants, originally not synthesizing caffeine. The first approach was performed using an RNAi for MXMT, yielding a 70% suppression of the caffeine level in leaves of transgenic coffee plants. The other approach was carried out by simultaneous introduction of three genes, XMT, MXMT and DXMT, into tobacco plants, which produced up to 5 μg caffeine per g fresh weight of leaves. This amount of caffeine was enough to repel tobacco cutworms (Spodoptera litura), suggesting the method to be practically efficient for construction of herbivore tolerant crops. The significance of the present study is discussed with reference to four topics: practical metabolic engineering; development of a genetic transformation system for tropical trees; generation of genetically modified (GM) plants with a minimal load on the environment; and providing GM foods that bring direct merits to consumers.

Adenosine phosphosulfate (APS) reductase is one of key enzymes in the sulfur assimilation pathway in higher plants, catalyzing the formation of adenosine 5′-phosphosulfate from sulfate and ATP. In order to improve sulfur uptake capacity of water spinach (Ipomea aquatica), a plant which commonly grows wild in Southern Asia and has good potential for sequestration of environmental pollutants like sulfuric compounds, an Arabidopsis gene (APR1), encoding a plastid-resident APS reductase, was introduced into cut cotyledons via Agrobacterium-mediated transformation. Among 267 regenerated shoots initially obtained from 2,119 cotyledon explants, two were found to efficiently express the introduced gene and could be grown to maturity. APS reductase activity in leaves was estimated to be over 2-fold the wild-type level. Upon cultivation in the presence of 2 mM sodium sulfate, a 2.5-fold higher sulfate uptake was observed in comparison with wild-type plants. When grown in the presence of toxic levels of sulfide or cadmium, they showed a higher tolerance with increased fresh weight as compared with controls. These results suggest that transcription from the introduced gene indeed strengthened the sulfur assimilation pathway, and that the generated plants may be practically useful for phytoremediation.

When tobacco (Nicotiana tabacum) plants are exposed to toxic level of cadmium (0.2 mM Cd), their trichomes actively excrete crystals (Choi et al., 2001). In this study, we investigated the distribution of Cd and NaCI on trichomes and leaf surfaces. Energy dispersive x-ray (EDX) analysis revealed that, under toxic Cd stress, crystals exudated from the trichomes contained high amounts of Ca, Mg, and Cd, as well as low levels of P, S, and Mn. Electron spectroscopic imaging (ESI) from trichomes and attached crystals showed that these crystals emitted denser radiation energy for Ca and Cd than did the head cells of the trichomes. However, no Cd was detected on the trichome surface itself or within the leaf epidermis. In contrast, treatment with salt (NaCI) did not stimulate crystal formation; instead, it induced the abnormal expansion of trichome cells. Although Na was not accumulated within the crystals, a considerable amount of both Na and CI was sequestered within the stalk cells of the long trichomes. Therefore, we believe that tobacco trichomes play an important role in Cd crystal exudation through crystallization, but that, under NaCI stress, the long trichomes sequester those elements within their stalks.

Transcripts that specifically accumulate in transgenic tobacco plants expressing an anti-sense construct for a tobacco type I DNA methyltransferase, NtMET1, were screened by the differential display method. Of the 31 genes identified, 16 encoded proteins with known functions; ten of these were related to biotic and abiotic stress responses, and the other six to cellular functions. In order to examine whether expression of these genes is correlated with DNA methylation status under natural stress conditions, a pathogen-responsive gene (NtAlix1) was selected as representative, and assayed for transcript induction and genomic methylation in tobacco plants infected with tobacco mosaic virus (TMV). In inoculated leaves of wild-type plants, NtAlix1 transcripts began to accumulate 12 h after the onset of the hypersensitive response (HR), and levels remained high for up to 24 h. Changes in the methylation status at the locus became obvious 24 h later, as detected by digestion of genomic DNA with a methylation-sensitive restriction enzyme. The results suggest that the level of DNA methylation may change in response to external stresses, and that this is closely related to the activation of stress-responsive genes.

The caffeine biosynthetic pathway in coffee plants has been proposed to involve three distinct N -methyltransferases, xanthosine methyltransferase (XMT), 7- N -methylxanthine methyltransferase (MXMT; theobromine synthase), and 3,7-dimethylxanthine methyltransferase (DXMT; caffeine synthase). We previously isolated all corresponding cDNAs designated as CaXMT1 , CaMXMT1 , CaMXMT2 and CaDXMT1 , respectively, and showed that caffeine was indeed synthesized in vitro by the combination of their gene products. In order to regulate caffeine biosynthesis in planta , we suppressed expression of CaMXMT1 by the double stranded RNA interference (RNAi) method. For this purpose, we first established a protocol for efficient somatic embryogenesis of Coffea arabica and C. canephora , and then Agrobacterium -mediated transformation techniques. The RNAi transgenic lines of embryogenic tissues derived from C. arabica and transgenic plantlets of C. canephora demonstrated a clear reduction in transcripts for CaMXMT1 in comparison with the control plants. Transcripts for CaXMT1 and CaDXMT1 were also reduced in the most cases. Both embryonic tissues and plantlets exhibited a concomitant reduction of theobromine and caffeine contents to a range between 30% and 50% of that of the control. These results suggest that the CaMXMT1 -RNAi sequence affected expression of not only CaMXMT1 itself, but also CaXMT1 and CaDXMT1 , and that, since the reduction in theobromine content was proportional to that for caffeine, it is involved in the major synthetic pathway in coffee plants. The results also indicate that the method can be practically applied to produce decaffeinated coffee plants.

To clarify the optimum pyrolysis condition of the semi-carbonized fuel and the effect of citric acid addition on the transportation, experiments of the “semi-carbonization” pyrolysis were conducted for cellu-lose, citric acid and their mixtures. The acid additives promoted dehydration of cellulose, and affected the weight yield of char derived from cellulose within the temperature region of the semi-carbonization pyroly-sis. The transportation analysis model of the semi-carbonized fuel was presented to evaluate the optimum weight yield for a given transportation distance. It was found that the acid additives reduced the weight yield at a given pyrolysis temperature and contributed to improving the transportation of the semi-carbon-ized fuel.

From a viewpoint of environmental preservation and resource protection, the recycling of wastes has been promoting. Expectations to new energy resource are growing by decrease of fossil fuel. Biomass is one of new energies with prevent global warming. This study is an attempt to burn pelletized woody biomass (Bio-pellet) made from sawdust and logging residue in order to thermally recycle waste products of forestry and lumbering industry. The devolatilization property of Bio-pellet were observed by the thermogravimerty and differential thermal analysis (TG/DTA) to obtained fundamental data of Bio-pellet pyrolysis. The thermogravimetric analyzer was used to measure weight loss and temperature difference. It observed that the weight of Bio-pellet decreased under three stages with endothermic reaction during pyrolysis, and with exothermic reaction during combustion. The combustion behavior of Bio-pellets was observed in the electric furnace, where the video-recording and measurements of pellet temperature and weight were carried out at sequential steps of the combustion process. The effects of furnace temperature and pellet size were exam-ined in order to elucidate the combustion characteristics of Bio-pellets, such as ignition delay, burning period, char-combustion time and the change of weight decrease and temperature rise. The results indicated that they are influenced at each step of the combustion process such as devolatilization, ignition, visible envelope flame combustion and char combustion.

Immediate early responsive genes were screened by the differential display method during the hypersensitive response upon tobacco mosaic virus infection of tobacco ( Nicotiana tabacum L.) plants carrying the N gene. Three hours after temperature shift from 30 degrees C to 20 degrees C, an increase in transcripts of a particular clone was observed. The cDNA encoded a polypeptide of 330 amino acids, whose topology indicated it to be a seven-transmembrane protein, designated as Nt7TM1. This was confirmed by direct observation of cultured tobacco cells expressing an Nt7TM1-green fluorescent protein fusion protein, which migrated exclusively to the plasma membrane and the endoplasmic reticulum. RNA blot hybridization analysis indicated that Nt7TM1 transcripts were not induced by salicylic or jasmonic acids, ethylene or hydrogen peroxide. The results suggested the presence of a unique system for pathogen response involving a novel seven-transmembrane protein.

Transgenic tobacco ( Nicotiana tabacum L.) plants constitutively expressing a rice ( Oryza sativa L.) gene encoding a small GTPase, rgp1, showed marked resistance to tobacco mosaic virus (TMV) infection compared with the wild type [H. Sano et al. (1994) Proc Natl Acad Sci USA 91:10556-10560]. In order to examine the gene expression profile, the temperature-shift method was adopted to hyper-activate the N-gene inducing the hypersensitive response (HR), and transcripts of 11 representative HR genes were analyzed. In transgenic and wild-type plants, transcripts of 10 genes were induced during the HR; however, in most cases, their expression level was higher in the former than in the latter. Mock treatment of transgenic plants also efficiently induced transcripts of 8 out of 11 genes after temperature shift, indicating that their activation is mediated by the N-gene. Salicylic acid and its glucoside-conjugates were induced in both transgenic and wild-type plants, but their quantity in the former was unusually higher than in the latter. These results suggest that expression of rgp1 positively influenced the signaling pathway of the HR, resulting in higher induction of salicylates. This possibly caused a "priming effect" that hyper-activates the HR genes through the N-gene without TMV infection. It was thus conceivable that, despite a structural similarity to the Rab-family of GTPases, which function in membrane trafficking, rgp1 might participate in the signal transduction pathway of the HR.

Arabidopsis thaliana calcineurin B-like protein (AtCBL2) is a member of a recently identified family of calcineurin B-like calcium-binding proteins in A. thaliana. The crystal structure of AtCBL2 has been determined at 2.1 A resolution. The protein forms a compact alpha-helical structure with two pairs of EF-hand motifs. The structure is similar in overall folding topology to the structures of calcineurin B and neuronal calcium sensor 1, but differs significantly in local conformation. The two calcium ions are coordinated in the first and fourth EF-hand motifs, whereas the second and third EF-hand motifs are maintained in the open form by internal hydrogen bonding without coordination of calcium ions. Both a possible site and a possible mechanism for the target binding to AtCBL2 are discussed based on the three-dimensional structure.

Caffeine is synthesized from xanthosine throughN-methylation and ribose removal steps. In the present study, three types of cDNAs encodingN-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated asCaXMT1, CaMXMT2, andCaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with aK m value of 78 μm, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a K m of 251 μm, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with aK m of 1,222 μm. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits. These results suggest that the presently identified threeN-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.

Tobacco (Nicotiana tabacum) genes regulated during the early stage of responses to wounding were screened by a modified fluorescence differential display method. Among 28 genes initially identified, a particular clone designatedNtC7 was subjected to further analysis. Its transcripts were found to accumulate rapidly and transiently within 1 h upon treatments with not only wounding but also salt and osmotic stresses. However, jasmonic and abscisic acids and ethylene did not effectively induce NtC7 transcripts. Amino acid sequence analysis suggested NtC7 to be a new type of transmembrane protein that belongs to the receptor-like protein family, and a membrane location was confirmed in onion (Allium cepa) epidermis cells transiently expressing an NtC7-green fluorescent protein fusion protein. Seeds of transgenic tobacco overexpressing NtC7normally germinated and grew in the presence of 500 mmmannitol, but not in the presence of 220 mm sodium chloride or 60 mm lithium chloride. Cuttings of mature transgenic leaf exhibited a marked tolerance upon treatment with 500 mm mannitol for 12 h, at which concentration wild-type counterparts were seriously damaged. These results suggested that NtC7 predominantly functions in maintenance of osmotic adjustment independently of ion homeostasis.

Partially normalized complementary DNA (cDNA) libraries were generated from root and leaf tissues of a diploid tobacco species, Nicotiana sylvestris. Single-pass sequences were obtained from a total of 13, 019 cDNA clones and were assembled into 6, 513 non-redundant cDNA groups. We estimate that these cDNA sequences represent approximately 3, 500 unique tobacco genes. The EST database described here will become a valuable resource for future research using Nicotiana species.(Communicated by Yasuyuki YAMADA, M. J. A., June 10, 2003)

Water spinach (Ipomoea aquatica) has high nutritional value and is considered one of the most important vegetables in Southeast Asia. Because of its quick growth and efficient absorption of various substances, it has been suggested to be useful for sequestration of environmental pollutants as well as offering a source of medical materials. We have developed and established a system for stable genetic transformation by infecting cut cotyledons with Agrobacterium harboring the GUS gene as a model case after evaluating conditions of bacterial cell density, growth phase and concentrations of acetosyringone. The resulting transgenic plants grew normally to maturity, and exhibited stable GUS activity. Thus, genetic modification of I. aquatica can be readily achieved, thereby improving its quality for whatever traits desired.

WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins. We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4. Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A. thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them. Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL). This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2. In contrast, WPK4 interacted with none of the CBLs. In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay. These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A. thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.

Using Arabidopsis thaliana, we identified the cis-element involved in the plant unfolded protein response (UPR). In transgenic plants, tunicamycin stimulated expression of a reporter gene under the control of the BiP promoter and promoter analysis identified a 24 bp sequence crucial to this induction. When fused with a minimal promoter, a hexamer of this sequence was sufficient for induction of a reporter gene in protoplasts treated with tunicamycin or dithiothreitol. Induction rate equivalent to original promoter was observed when the assay was conducted in transgenic plants. This 24 bp sequence contained two elements also responsible for the UPR in animals. Either of these elements was sufficient for the plant UPR, indicating conservation between animals and plants of cis-elements involved in the UPR.

When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence. ZmMI1 was transcribed only under cold stress. Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases. Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively. Cold stress induced severe demethylation in core regions but left linker regions relatively intact. Thus, methylation and demethylation were periodic in nucleosomes. The following biological significance is conceivable. First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues. Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H. (1990) Mol. Gen. Genet. 220, 441-447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence.

Heavy metals are potentially highly toxic for organisms. Plants possess the ability to minimize damage but the underlying molecular mechanisms have yet to be detailed. Screening Cd-responsive genes in Arabidopsis, we previously identified a gene encoding a putative metal binding protein CdI19, which, upon introduction into yeast cells, conferred marked toleration of Cd exposure. Here we describe that bacterially expressed CdI19 directly interacts with Cd at its CXXC motif, as revealed by circular dichroism analysis, and that it is exclusively localized at plasma membranes, as revealed by heterologous expression of fusion product with a green fluorescent protein in BY2 cells. Northern blot analyses indicated that CdI19 transcripts were induced not only by Cd, but also by dicationic forms of Hg, Fe and Cu. Histochemical assays using transgenic Arabidopsis expressing the CdI19 promoter::GUS showed CdI19 to be expressed in petiole, hypocotyl, peduncle and vascular bundles in root tissues. Overexpression of the CdI19 cDNA conferred Cd tolerance in transgenic Arabidopsis. These results suggest that CdI19 plays an important role in the maintenance of heavy metal homeostasis and/or in detoxification by endowing plasma membranes with the capacity to serve as an initial barrier against inflow of free heavy metal ions into cells.

The Arabidopsis AtMCP and rice OsMCP genes which encode proteins highly homologous to molybdoenzymes of the sulphite oxidase family were isolated and characterized. Both proteins seemed to possess only a molybdenum cofactor as the redox centre, unlike all the other eukaryotic molybdoenzymes. Putative MCP orthologues were identified in 17 plant species, indicating that Mo possess only a molybdenum cofactor as the redox centre, unlike all the other eukaryotic molybdoenzymes. Putative MCP orthologues were identified in 17 plant species, indicating that MCPs are widely distributed over the plant kingdom. An analysis using a green fluorescent protein fusion showed that AtMCP possesses a peroxisomal targeting signal at its C-terminus. Putative peroxisomal targeting signals were also found in all plant MCPs, suggesting the existence of a new redox pathway in this organelle.

The tbzF gene of tobacco ( Nicotiana tabacum ) encodes a basic region leucine zipper protein (bZIP) that belongs to the LIP19 subfamily. It was previously shown that tbzF transcripts accumulate on cold, abscisic acid (ABA) or ethylene treatment. They were also abundant in senescing leaves and flower buds, suggesting tbzF to possess multiple functions. In order to analyze the transcript induction profile, a 1740 bp promoter region was isolated, fused to the β-glucuronidase (GUS) gene, and introduced into tobacco plants. Resulting transgenic plants exhibited GUS activity in the stomatal guard cells of senescing leaves and in flower buds, and also in ABA- and ethylene-treated young leaves. To identify promotion responsive regions, four deletion constructs were transformed into tobacco plants. As a result no response was observed with the −720 and −220 constructs in senescing leaves, or in ABA- and ethylene-treated young leaves. In contrast, response was only reduced with the −220 construct in flower buds. These observations indicate the 500 bp between −1220 and −720 to contain cis -element(s) for senescence-associated expression, and the 500 bp between −720 and −220 to be responsible for flower-specific expression. It is concluded that, although the promoter region within −220 is essential for tbzF expression in both senescing leaves and flower buds, additional but independent elements are necessary for full activation in each organ.

Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress.

Chloroplast DNA of the green alga Chlamydomonas reinhardtii is maternally inherited. Methylation mapping directly revealed that, before mating, chloroplast DNA of maternal (mating type plus; mt + ) gametes is heavily methylated whereas that of paternal (mating type minus; mt − ) gametes is not. Indirect immunofluorescence analyses with anti-5-methylcytosine mAbs visually showed methylation to occur exclusively in chloroplast DNA of mt + gametes, and not in mt − gametes or nuclear DNA of either mt. To clarify the relationship between methylation and maternal inheritance of chloroplast DNA, we have isolated and characterized a cDNA encoding a DNA methyltransferase. The deduced protein, CrMET1, consists of 1,344 aa and contains a conserved catalytic domain at the C terminal and a nonconserved N-terminal region. The predicted N-terminal region has an arginine-rich domain, suggesting CrMET1 is transferred to chloroplasts. This finding could be directly shown by green fluorescent protein epifluorescence microscopy analyses. CrMET1 transcripts were found to be absent in both mt + and mt − vegetative cells. Upon gametogenesis, however, transcript levels clearly increased in mt + but not mt − cells. These experiments suggest that the CrMET1 protein is located in chloroplasts and that it specifically methylates cytosine residues of chloroplast DNA in mt + gametes. This conclusion was further strengthened by the observation that, during gametogenesis, CrMET1 is expressed in a mt − mutant, mat-1 , whose chloroplast DNA is heavily methylated in gametes and paternally inherited. The results provide evidence that cytosine methylation plays a critical role in maternal inheritance of chloroplast genes in C. reinhardtii .

Background: Patent ductus venosus is extremely rare with only 14 cases reported in the world literature. We present a case of patent ductus venosus. Methods and Results: A 29-year-old male was admitted with melaena stool caused by gastric haemorrhagic ulcers. Laboratory data disclosed severe anaemia; however, liver function tests were normal. Serum ammonia was also within the normal range. Serological viral markers for hepatitis B or C were all negative. The abdominal ultrasonography and computed tomography indicated a 12 mm diameter shunt located in the left lobe of the liver, which connected the portal vein with the left hepatic vein. After treatment for gastric ulcers, percutaneous transhepatic portography was performed and an enormous shunt connecting the umbilical portion of the portal vein with the left hepatic vein was revealed. Conclusions: Histological findings of the liver biopsy showed that portal venules could not be observed in the portal areas and that no fibrosis or inflammatory cell infiltration were shown. Because of the anatomical position of the shunt, the case was diagnosed as patent ductus venosus.

Jean Baptiste de Lamarck (1744-1829) maintained that characteristics that were acquired during an organism's lifetime are passed on to its offspring. This theory, known as Lamarckian inheritance, was later completely discredited. However, recent progress in epigenetics research suggests it needs to be reexamined in consideration of DNA methylation. In this article, I summarize our observations, which support Lamarckian inheritance. Initial experiments indicate that (1) artificially induced demethy-lation of rice genomic DNA results in heritable dwarfism, and (2) cold stress induces extensive demethy-lation in somatic cells of the maize root. Based on these results, I propose the hypothesis that traits that are acquired during plant growth are sometimes inherited by their progeny through persistent alteration of the DNA methylation status.

We evaluated the influence of molecular mass and degree of deacetylation of chitosan on the adsorption of Streptococcus sobrinus 6715 to saliva-treated hydroxyapatite (S-HA) by measuring the optical density of the bacterial cell suspensions released from saliva-treated hydroxyapatite. Twenty-five chitosan samples with different molecular masses (0.8-6 kDa) and degrees of deacetylation (10-95%) were prepared for the study. We found that the inhibition of adsorption of S. sobrinus 6715 to S-HA correlated positively with the molecular mass of chitosan (R = 0.876) and that the optimal degree of deacetylation was 50-60% for maximum inhibition of bacterial binding to S-HA. We also examined the effect of chitosan on zeta potentials of the oral bacteria and their surface hydrophobicities. It was observed that chitosan reduced the magnitude of the zeta potential and surface hydrophobicities of the oral bacteria. Thus, the results demonstrated that chitosan with a molecular mass of 5-6 kDa and a degree of deacetylation of 50-60% might have the potential to act as an effective anti-plaque agent because of its polycationic properties.

Transgenic chrysanthemum plants were constructed to simultaneously express three N-methyltransferases involved in caffeine biosynthetic pathways. Resulting plants produced caffeine at approximately ...

The O-acetylated and O-methylcarbonylated analogue of the methanesulfonate (I) suffer from low product yield or no product formation at all.