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Previous research has shown that the effective use of inertial motion (i.e., less or no torque input at the knee joint) plays an important role in achieving a smooth gait of transfemoral prostheses in the swing phase. In our previous research, a method for generating a timed knee trajectory close to able-bodied individuals, which leads to sufficient clearance between the foot and the floor and the knee extension, was proposed using the inertial motion. Limb motions are known to correlate with each other during walking. This phenomenon is called kinematic synergy. In the present study, we measure gaits in level walking of able-bodied individuals with a wide range of walking velocities. We show that this kinematic synergy also exists between the motions of the intact limbs and those of the knee as determined by the inertial motion technique. We then propose a new method for generating the motion of the knee joint using its inertial motion close to the able-bodied individuals in mid-swing based on its kinematic synergy, such that the method can adapt to the changes in the motion velocity. The numerical simulation results show that the proposed method achieves prosthetic walking similar to that of able-bodied individuals with a wide range of constant walking velocities and termination of walking from steady-state walking. Further investigations have found that a kinematic synergy also exists at the start of walking. Overall, our method successfully achieves knee motion generation from the initiation of walking through steady-state walking with different velocities until termination of walking.

2-[(Trimethylsilyl)methyl]benzyl methanesulfonates react with potassium fluoride in the presence of a catalytic amount of 18-crown-6 at room temperature to give o-quinodimethanes, which are trapped with electron-deficient olefins to afford [4+2] cycloadducts in good yields.

Background Controversy surrounds the reliability of sentinel lymph node biopsy after primary systemic chemotherapy. In this study, we assessed axillary ultrasound for selecting patients most likely to optimally benefit from biopsy. Methods The study included 87 patients who received primary systemic chemotherapy and underwent a sentinel lymph node biopsy followed by axillary lymph node dissection. Lymph nodes >10 mm in diameter, irregularly swollen, round, and homogeneously hypoechoic without an echo-rich center were considered axillary ultrasound positive. Results In axillary ultrasound–negative patients before and after primary systemic chemotherapy, identification, sensitivity, and false-negative rates were 81%, 100%, and 0%, respectively. However, in patients whose lymph nodes converted from positive to negative after primary systemic chemotherapy, these values were 83%, 70.8%, and 29.2%, respectively. Conclusions Axillary ultrasound–negative patients before and after primary systemic chemotherapy were suitable for sentinel lymph node biopsy. Axillary ultrasound should be used during primary systemic chemotherapy and before surgery.

Catalytic asymmetric Diels–Alder reaction of α-amino-o-quinodimethane with α,β-unsaturated aldehydes was achieved with high diastereo- and enantioselectivities in the presence of l-proline, which acts as a promoter to generate the quinodimethane from the corresponding precursor as well as a chiral catalyst for the enantioselective Diels–Alder reaction.

A novel approach was introduced for breast surgery using the BiClamp, a new bipolar thermal energy device, to avoid complications and to shorten the time required for the dissection of the axillary lymph nodes.Thirty-six patients with early breast cancer were assessed. The surgical parameters were compared between the procedures performed using the BiClamp technique (n = 14) and conventional surgery with suture ligation (n = 22). The parameters included the operation time, blood loss, and discharge on the first postoperative day. In addition, each of those parameters was compared between the patients with a high body mass index (BMI) (22) and a low BMI (or =22). The sealed vessels were examined histologically and heat-associated morphological vessel wall alterations were evaluated.The operation time was significantly shorter in the BiClamp group than in the control group (P = 0.017, 90 +/- 18 vs 115 +/- 33 min). In addition, the blood loss in the BiClamp group tended to be smaller than in the control group, but the difference was not significant (P = 0.54, 61 +/- 47 vs 74 +/- 67 g). No other parameters showed any significant differences between the two groups.The BiClamp thermofusion technique was safe and useful in breast surgery involving axillary dissection.

Caffeine is synthesized from the precursor xanthosine through three methylation and one nucleoside removal steps. Methylation is catalyzed by N-methyltransferases, designated as CaXMT1, CaMXMT1 and CaDXMT1, which, respectively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into 3,7-dimethylxanthine, and 3,7-dimethylxanthine into 1,3,7-trimethylxanthine (caffeine). In the present study, we examined their cytological and biochemical properties using fusion proteins with fluorescent proteins. All three enzymes were found to localize in cytosol as visualized by green fluorescence protein fusions. The possibility of dimer formation among these enzyme proteins was examined in vivo by transient expression of bimolecular fluorescence complementation of yellow fluorescent protein (YFP) using onion epidermal cell layers. Results showed that each enzyme protein formed a homo-dimer in cytosol as seen by a clear reconstituted YFP fluorescence. In addition, each enzyme also formed a hetero-dimer with each of the other two enzymes in cytosol. The biological significance of dimerization among structurally resembling methyltransferases involved in caffeine biosynthesis is discussed.

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.

ATPase associated with various cellular activities (AAA) proteins are commonly distributed among eukaryotes, and are involved in a multitude of cellular functions. NtAAA1 is one such example, being involved in pathogen response in tobacco plants. When its activity was suppressed in RNAi transgenic tobacco plants, an elevated resistance to the pathogenic bacterium Pseudomonas syringae was observed in comparison with the wild type. As AAA proteins function through interaction with specific partners, NtAAA1-interacting proteins were screened by the yeast two-hybrid assay, and one particular gene encoding a small GTPase, an ADP ribosylation factor, was identified and designated as NtARF. Its specific binding to NtAAA1 was confirmed by in vitro pull-down assay, and their interaction was predominant between active forms of NtARF and NtAAA1, each bound to GTP and ATP, respectively. Their physical interaction in vivo around the plasma membrane was shown by fluorescence resonance energy transfer assays, suggesting their role in membrane trafficking. Transgenic tobacco plants constitutively expressing NtARF under the control of a cauliflower mosaic virus 35S promoter exhibited spontaneous and wound-induced lesion formation, and enhanced resistance to pathogen attack. Expression of NtAAA1 in leaves of NtARF transgenic plants attenuated lesion and suppressed pathogen resistance. In wild-type tobacco plants, transcripts of NtAAA1 and NtARF could be induced by ethylene and salicylic acid, respectively. These results suggest that NtAAA1 balances plant resistance through suppression of NtARF, and that the molecular basis for the known antagonistic actions of ethylene and salicylic acid in defense response could be partly attributable to these two proteins.

To examine the relationship between gene expression and DNA methylation, transcriptionally activated genes were screened in hypomethylated transgenic tobacco plants expressing an anti-DNA methyltransferase sequence. Among 16 genes initially identified, one clone was found to encode a glycerophosphodiesterase-like protein (NtGPDL), earlier reported to be responsive to aluminium stress. When detached leaves from wild type tobacco plants were treated with aluminium, NtGPDL transcripts were induced within 6 h, and corresponding genomic loci were demethylated at CCGG sites within 1 h. Direct bisulfite methylation mapping revealed that CG sites in coding regions were selectively demethylated, and that promoter regions were totally unmethylated regardless of the stress. Salt and low temperature treatments also induced similar demethylation patterns. Such effects could be attributable to oxidative stress, since reactive oxygen species generated by paraquat efficiently induced the same pattern of demethylation at coding regions. Pathogen infection induced neither transcripts nor genomic demethylation. These results suggested a close correlation between methylation and expression of NtGPDL upon abiotic stresses with a cause-effect relationship. Since DNA methylation is linked to histone modification, it is conceivable that demethylation at coding regions might induce alteration of chromatin structure, thereby enhancing transcription. We propose that environmental responses of plants are partly mediated through active alteration of the DNA methylation status.

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.

� 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'.

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.

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.

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.

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.

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.

Since Lamarck proposed the idea of inheritance of acquired traits 200 years ago, much has been said for and against it, but the theory was finally declined after the 1930s. Despite of the negative opinions of the majority of geneticists, botanists and plant breeders have long recognized that altered properties during the growth were occasionally transmitted to the offspring. This was also the case with artificially altered properties such as dwarfism, flowering timing and plant stature, which were induced by a non-mutagenic chemical, 5-azacytidine and its derivatives. As these drugs are powerful inhibitors of DNA methylation in vivo, a close correlation between methylation and phenotypic expression was suggested. Subsequent studies showed that rice plants acquired disease resistance upon demethylation of the corresponding resistant gene, and that both resistant trait and hypomethylated status were inherited by the progeny up to nine generations. Whether or not the methylation pattern changes under natural condition was then questioned, and recent studies have indicated that it indeed naturally changes in response to environmental stresses. Whether or not the altered methylation pattern during the vegetative growth is heritable was also questioned, and studies on toadflax and rice affirmed the question, showing stable maintenance of hypermethylation in the former and hypomethylation in the latter for 250 and 10 years, respectively. The observation strongly suggested that acquired traits can be heritable as far as the acquired methylation pattern is stably transmitted. This concept is consistent with the Lamarck's theory of the inheritance of acquired traits, which therefore should be carefully reevaluated to reestablish his impaired reputation.

This paper proposes a new synthesis method for methanol as a future alternative fuel, by the combination of carbon supplied from wood and hydrogen supplied from the electrolysis of water using a solar power generation system in the desert. In the developed countries, more than half of the potential forest area has already been converted into other land uses, while existing forests are well organized and available for wood production. In the developing countries, potential forest sites are expected to be available for wood production, even though they are presently grasslands or secondary forests, while natural tropical forests will not be allowed to be converted into artificial forests. The area available for plantations within 500 km distant from deserts was estimated to be 65 Mha in the world, except for the mountain areas. Biomass production from these sites will be converted annually into 980 Mt methanol with hydrogen from the deserts. This amount is equal to 34% of the world's fuel consumption by vehicles.

A cDNA fragment encoding part of a DNA methyltransferase was isolated from maize. The putative amino acid sequence identically matched that deduced from a genomic sequence in the database (accession no. AF063403), and the corresponding gene was designated as ZmMET1. Bacterially expressed ZmMET1 actively methylated DNA in vitro. Transcripts of ZmMET1 could be shown to exclusively accumulate in actively proliferating cells of the meristems of mesocotyls and root apices, suggesting ZmMET1 expression to be associated with DNA replication. This was confirmed by simultaneous decrease of transcripts of ZmMET1 and histone H3, a marker for DNA replication, in seedlings exposed to wounding, desiccation and salinity, all of which suppress cell division. Cold stress also depressed both transcripts in root tissues. In contrast, however, accumulation of ZmMET1 transcripts in shoot mesocotyls was not affected by cold stress, whereas those for H3 sharply decreased. Such a differential accumulation of ZmMET1 transcripts was consistent with ZmMET1 protein levels as revealed by western blotting. Expression of ZmMET1 is thus coexistent, but not completely dependent on DNA replication. Southern hybridization analysis with a methylation-sensitive restriction enzyme revealed that cold treatment induced demethylation of DNA in the Ac/Ds transposon region, but not in other genes, and that such demethylation primarily occurred in roots. These results suggested that the methylation level was decreased selectively by cold treatment, and that ZmMET1 may, at least partly, prevent such demethylation.

A cDNA encoding a DNA methyltransferase, with a predicted polypeptide of 1556 amino acid residues containing all motifs conserved in this enzyme family, was isolated from tobacco plants, and the corresponding gene was designated as NtMET1. RNA blot analysis indicated NtMET1 transcripts to accumulate in dividing tissues of tobacco plants, and they could be detected during the S phase in synchronized dividing BY2 cells. In situ hybridization revealed the transcripts to be localized exclusively in actively proliferating tissues around axillary apical meristem. In order to ascertain physiological roles, transgenic tobacco plants that had the antisense construct were made and examined for phenotypes. Methylation levels of genomic DNA from transgenic plants significantly decreased in comparison with wild-type levels, and distinct phenotypic changes including small leaves, short internodes and abnormal flower morphology were noted. Microscopic observation revealed that leaf structure differed between transgenic and wild-type plants. These results suggest that NtMET1 functions during DNA replication, and that DNA methylation plays an important role in plant morphogenesis.

Callus of the mangrove plant, Sonneratia alba J. Smith, established from pistils of flower buds were cultured on solid Murashige and Skoog medium supplemented with 0 to 500 mM NaCl. Maximum growth was observed with 50 mM NaCl, and net growth of callus occurred for concentrations up to 200 mM NaCl. At 500 mM NaCl, growth of callus was completely inhibited, although a part of the tissue was still alive after 30 d. Cellular levels of Na+ and Cl− were greatly increased by the treatment with NaCl. Uptake of K+ was also enhanced and was accompanied by increasing levels of Na+ and Cl− so that the Na+/K+ ratio was almost constant (4.1–4.2) in callus grown with 50–200 mM NaCl. Levels of Mg2+ and Ca2+ were not changed significantly with 50–200 mM NaCl, whereas levels of free NH 4 + , NO 3 − and SO 4 2− ions, which are convertible to organic compounds, were lowest in callus grown with 50 mM NaCl. The rate of conversion of 15NH 4 + into macromolecules during 30 d culture with 0–100 mM NaCl did not vary greatly, but 200 mM NaCl reduced the biosynthesis of macromolecules from this ion. The highest rate of conversion of 15NO 3 − into macromolecules was observed at 50 mM NaCl. Identification of compatible solutes with NMR-spectroscopy indicated that mannitol is the compatible solute for intact plants of Sonneratia alba, but no accumulation of mannitol was found in calluses, not even in those grown at high concentrations of NaCl.

Wounding induces expression of genes encoding defense-related proteins involved in wound healing. An intensive survey has been carried out to clarify the initial signal transduction pathways that mediate this stress to expression of genes. In this context, signal molecules that intermediate in the wound signal to cellular response have been actively searched for. Jasmonic acid (JA) has been considered to be a key signal molecule in this pathway. Systemin, ABA, ethylene, and electrical current have been suggested to function by transmitting the wound signal to JA. A mitogen-activated protein kinase has been shown to respond rapidly to wounding, and proposed to function as one of the key enzymes involved in JA biosynthesis. Transgenic plants overexpressing a gene encoding a Rab-type, small GTP-binding protein contained 6-fold higher levels of cytokinins than wild-type plants, and responded to wounding by rapidly producing JA and, uncommonly, accumulating salicylic acid (SA), a pathogenic signal. These phenomena observed in the transgenic plants were reproduced when wild-type plants were wounded in the presence of the synthetic cytokinin, benzylaminopurine, suggesting that cytokinins are indispensable in the control of endogenous levels of JA and SA.

Plants respond differentially to wounding and pathogens using distinct signaling pathways, so that wound signals are transmitted to jasmonic acid (JA) which induces basic pathogenesis-related (PR) proteins, whereas pathogenic signals cause, in addition to JA, accumulation of salicylic acid (SA) which stimulates production of acidic PR proteins. Transgenic tobacco plants expressing a gene for a small GTP binding protein respond abnormally to mechanical wounding to produce SA and consequently acidic PR proteins, suggesting that wound signals cross witli pathogen signaling pathways [Sano et al. (1994) Proc. Natl. Acad. Sci. USA 91: 10556]. This unusual signal crossing is associated with a highly sensitive wound-response of transgenic plants which, upon wounding, produce JA at least eighteen hours earlier than wild-type plants. When wildtype plants are wounded in the presence of the synthetic cytokinin, benzylaminopurine, production of JA begins six hours earlier than in untreated samples, and also SA begins to accumulate. The cytokinin antagonist, 2-chloro-4-cyclohexylamino-6-ethylamino-s-triazine, erases these effects. Because transgenic plants constitutively produce four- to six-fold higher amounts of endogenous cytokinins than wild-type plants, it is concluded that cytokinins are indispensable for control of endogenous levels of SA and JA.

We proposed a CO 2 -storage using by the articicial ice-caving on Antarctic ice sheet, which enables the inside to keep colder than outside average temperature of the Antarctic climate. It makes possible to store the dry-ice CO 2 in the vast area on the Antarctic ice sheet.

O-acetylserine (thiol) lyase (EC4.2.99.8) is the key enzyme in the cysteine biosynthetic pathway of plants and prokaryotes. The gene, cys1, encoding this enzyme was isolated from a wheat (Triticum aestivum L.) cDNA library, and its deduced amino acid sequence found to show 53% sequence identity with the O-acetyl-serine (thiol) lyase of Escherichia coli and Salmonella typhimurium. The deduced peptide consists of 325 amino acids (34.1 kDa), contains a conserved motif for the binding of pyridoxal phosphate, a co-factor required for enzymatic activity, and an N-terminal region of 37 amino acid residues resembling chloroplast transit peptides. The identity of cys1 was further demonstrated through complementation of an E. coli cysteine auxotroph, which lacks O-acetylserine (thiol) lyase, by expression of the wheat gene. Northern blot analysis showed that cys1 is highly expressed in green vegetative and reproductive tissues and in the roots of wheat, as well as in the leaves of several plant species. Southern blot analysis demonstrated that the gene exists as a single copy in the wheat genome. Tobacco plants transformed with cys1 in the sense orientation (sense plants) or antisense orientation (antisense plants), under the control of the CaMV 35S promoter, showed high levels of transcripts. The O-acetylserine (thiol) lyase activity in transgenic plants was determined, and found to be three- to fivefold higher in sense plants than in control plants, but unaffected in antisense transformants. Fumigation experiments with toxic levels of hydrogen sulphide (H2S) gas showed that while sense transformants were highly resistant, control and antisense plants were severely damaged by the treatment.

The efficacy of a test dentifrice containing nano-sized (several tens to hundreds of nm) calcium carbonate (hereafter NC) on enamel lesion remineralization was studied in an in vitro system that employed collagen-coated wells for cell culture, as a model of oral surfaces for NC retention. The well surfaces were treated with the test dentifrice and briefly rinsed with distilled water. Thin sections of enamel with artificial subsurface demineralization were remineralized in the plate wells containing remineralizing solution. The dentifrice treatment was repeated twice a day (in the morning and evening) for 20 days. After remineralization, microradiographic analysis was performed to evaluate the rate of lesion remineralization on the sections. The test dentifrice showed a statistically significant mineral gain (48.8% decrease in DeltaZ % x microm from the baseline value), indicating lesion remineralization, whereas the placebo dentifrice without NC did not. An elevated Ca concentration in the remineralizing solution was also observed after a single treatment with the test dentifrice. We conclude that the test dentifrice has potential to remineralize incipient enamel lesions due to the unique properties of NC, which is retained on oral surfaces, thereafter releasing Ca ions into oral fluids (saliva, plaque).

A single exposure of germinated rice seeds (Oryza sativa) to either of the DNA demethylating agents 5-azacytidine (azaC) or 5-azadeoxycytidine (azadC) induced dwarf plants. At maturity, seeds treated with azaC exhibited normal morphological characteristics in comparison with untreated controls except that their height (total stem length) was reduced by about 15%. The M1 progeny, obtained by self-fertilization of an azaC-induced dwarf plant, segregated into dwarf (35%) and apparently tall types (65%). The M2 progenies, obtained by self-fertilization of dwarf M1 plants, were also dwarf, while those from tall M1 plants were only tall. Genomic DNA isolated from mature leaves of azaC-treated seeds showed about a 16% reduction in the 5-methylcytosine (m5C) content in comparison with DNA from untreated samples. A similar reduction in the m5C content was also observed in the M1 and M2 progenies. Thus, both undermethylation and dwarfism induced by azaC treatment were heritable. The results suggest that azaC induced demethylation of genomic DNA, which caused an altered pattern of gene expression and consequently a reduction in plant stem length.

ATPase associated with various cellular activities (AAA) proteins are commonly distributed among eukaryotes, and are involved in a multitude of cellular functions. NtAAA1 is one such example, being involved in pathogen response in tobacco plants. When its activity was suppressed in RNAi transgenic tobacco plants, an elevated resistance to the pathogenic bacterium Pseudomonas syringae was observed in comparison with the wild type. As AAA proteins function through interaction with specific partners, NtAAA1-interacting proteins were screened by the yeast two-hybrid assay, and one particular gene encoding a small GTPase, an ADP ribosylation factor, was identified and designated as NtARF. Its specific binding to NtAAA1 was confirmed by in vitro pull-down assay, and their interaction was predominant between active forms of NtARF and NtAAA1, each bound to GTP and ATP, respectively. Their physical interaction in vivo around the plasma membrane was shown by fluorescence resonance energy transfer assays, suggesting their role in membrane trafficking. Transgenic tobacco plants constitutively expressing NtARF under the control of a cauliflower mosaic virus 35S promoter exhibited spontaneous and wound-induced lesion formation, and enhanced resistance to pathogen attack. Expression of NtAAA1 in leaves of NtARF transgenic plants attenuated lesion and suppressed pathogen resistance. In wild-type tobacco plants, transcripts of NtAAA1 and NtARF could be induced by ethylene and salicylic acid, respectively. These results suggest that NtAAA1 balances plant resistance through suppression of NtARF, and that the molecular basis for the known antagonistic actions of ethylene and salicylic acid in defense response could be partly attributable to these two proteins.

Mitogen-activated protein kinases (MAPKs) constitute one of the most critical signaling components in plants. A typical example is wound-induced protein kinase (WIPK), which functions during pathogen responses in tobacco plants (Nicotiana tabacum). Searching for direct down-stream components, we previously isolated a novel transcription factor, which was activated upon phosphorylation by WIPK and designated as N. tabacum WIPK-interacting factor (NtWIF). Overexpression of NtWIF in tobacco plants enhanced the hypersensitive response (HR) upon tobacco mosaic virus infection and cryptogein treatment, while its silencing by RNAi suppressed such HR. NtWIF contains a specific motif similar to the B3 DNA binding domain, which recognizes the core TGTCTC motif called the auxin-responsive element (ARE). Using synthetic ARE sequences, NtWIF was also shown to recognize the ARE motifs and to transactivate the Luciferase (Luc)-reporter gene driven by such AREs in tobacco BY2 cultured cells. Subsequent microarray screening of NtWIF overexpressing tobacco identified 49 stress-responsive genes, and in silico analyses of available promoter regions of these genes revealed beta-1,3-glucanase, ACS2, P-450, and WIPK itself to contain the ARE core motif consisted of either TGTCTC or TGTCCT. Gel shift assay showed NtWIF to efficiently bind to both sequences. Assays with 1.5-kb PR-Q and 1.2 kb WIPK promoter regions, each fused to the Luc-reporter gene, indicated NtWIF to exhibit a clear transactivation activity, which was increased up to 3-fold upon phosphorylation by WIPK. These results revealed that NtWIF directly regulates multiple stress-responsive genes containing the ARE motif in their promoters, thereby partly filling up the last step of the MAPK cascade.

Objectives Expenditures on long-term care insurance (LTCI) in Japan have been increasing with the aging of the population, which has led to an increase in premiums. To optimize resource allocation, we aim to clarify the priorities of the functions of long-term care facilities from the viewpoint of future beneficiaries. Design The present study was conducted using a cross-sectional study design. Setting/Participants We conducted a mail-in survey targeting 2400 adults aged 50–65 in 8 cities in Japan, and 371 persons responded. Measurements Conjoint analysis was applied to measure participants' preferences for long-term care facility services. Participants read 1 of 2 vignettes of an 80-year-old person with either dementia or a fracture, and were asked to envision it as a possible future scenario for themselves. Participants then completed 8 or 9 tasks to select suitable long-term care facilities for the person described. The questionnaire also contained common questions on participants' personal profiles: age, gender, family situation, education, approximate yearly family income, experience as a family caregiver, dwelling status, present health status, and possibility of requiring long-term care services in the future. Results The results focused mainly on (1) possibilities of individual choice for daily schedules/meals; (2) availability of regular care staff; (3) room; (4) main daily interactions; (5) necessity of relocation associated with medical deterioration; 6) Waiting time; 7) distance from present residence; and (8) monthly fees. Necessity of relocation associated with medical deterioration was consistently given the greatest importance. Participants with experience as a family caregiver showed significantly greater preference for individualized care and communication. Conclusions The option of avoiding relocation was highly valued by participants compared with private rooms and individualized care. The present situation of high demand for intensive care homes for the elderly, provoked by anxiety about future residence, will not change unless a robust system is built to support residents even when their health has deteriorated. Individualized care has been promoted by long-term care insurance policies, but further advances will require efforts to obtain the understanding of the insured.

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.

In the green alga, Chlamydomonas, chloroplast DNA is maternally transmitted to the offspring. We previously hypothesized that the underlying molecular mechanism involves specific methylation of maternal gamete DNA before mating, protecting against degradation. To obtain direct evidence for this, we focused on a DNA methyltransferase, DMT1, which was previously shown to be localized in chloroplasts. The full-length DMT1 protein with a molecular mass of 150 kD was expressed in insect cells, and its catalytic activity was determined. In vitro assays using synthetic DNA indicated methylation of all cytosine residues, with no clear selectivity in terms of the neighboring nucleotides. Subsequently, transgenic paternal cells constitutively expressing DMT1 were constructed and direct methylation mapping assays of their DNA showed a clear nonselective methylation of chloroplast DNA. When transgenic paternal cells were crossed with wild-type maternal cells, the frequency of biparental and paternal offspring of chloroplasts increased up to 23% while between wild-type strains it was ∼3%. The results indicate that DMT1 is a novel type of DNA methyltransferase with a nonselective cytosine methylation activity, and that chloroplast DNA methylation by DMT1 is one of factors influencing maternal inheritance of chloroplast genes.

Exposure of normal, tall rice (Oryza sativa) seedlings to 5-azacytidine, a powerful inhibitor of DNA methylation in vivo, induced both demethylation of genomic DNA and dwarf plants. Genes that had been affected by treatment were identified by differential screening of a cDNA library, and a ras-related gene, rgp1, was subsequently isolated. The cDNA of rgp1 was found to encode a deduced protein sequence of 226 amino acids with a relative molecular mass of 24850, which was most closely related to the ras-related ypt3 protein of fission yeast, Schizosaccharomyces pombe. The rgp1 protein, expressed in transformed Escherichia coli, clearly showed GTP-binding activity. During seedling rgp1 expression was first observed 14 days after germination, reaching a maximum level between 28 and 42 days, and gradually decreased thereafter until 63 days when it attained the same level of expression as in 14-day-old seedlings. Expression of rgp1 was found to be markedly reduced throughout the growth period of both 5-azacytidine-induced dwarf plants and their progenies, relative to levels in untreated tall control plants. These results suggest that expression of rgp1 may be influenced, either directly or indirectly, by DNA methylation, and that the rgp1 protein may play an important role in plant growth and development.