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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.

Plants produce up to 100,000 secondary metabolites. One of their biological functions is self-denfese, and it is referred as chemical defense, directly and/or indirectly counteracting biotic and abiotic stresses. Alkaloids constitute 12% of total secondary metabolites, and some of them exhibit detrimental effects on living organisms. Caffeine (1,3,7-trimethylxanthine) is a member of purine alkaloids, and its exogenous application to plants at relatively high concentrations (0.01-0.1%) effectively repelled herbivores and pathogenic microbes. This allowed the construction of transgenic crops that endogenously produce caffeine to tolerate stresses. Experimentally, tobacco and chrysanthemum were successfully transformed with three distinct N-methyltranferases involved in the caffeine biosynthesis pathway. They produced 0.4-5 mug caffeine/g tissue (5 x 10(-4)%), this being three magnitudes lower than values found in caffeine-producing plants and in vitro experiments. Nevertheless, they exhibited strong repellence against pest insects, and high resistance to virus and bacterial infection. They also exhibited accelerated self-defense, as estimated by constitutive expression of defense-related genes, and by elevated production of salicylic acid, a critical signaling molecule for defense response. Since caffeine content was low in transgenic lines, observed effects might not be direct, but rather indirect. We presume that, as endogenously produced caffeine could be toxic, the host plants activated its own self-defense system, which commonly occurs regarding other stresses. Eventually the host became on standby to cope with a broad range of biotic stresses. The procedure resembles mammalian vaccination, in which antigen-antibody system is critical. We propose that plants can also be vaccinated as far as proper "antigenic" chemicals are expressed in planta.

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.

Biotic and abiotic-induced wounding is one of the severest stresses that plants suffer throughout the growing period. Upon injury, plants rapidly activates a set of genes, which encode diverse proteins to cope with damages. Screening such genes that were transcriptionally activated within 30 min after mechanical wounding in tobacco plants, we identified a particular clone encoding a multfunctional protein, and designated as NtMFP (Nicotiana tabacum multifunctional protein). The deduced polypeptide is constituted of 668 amino acids with an apparent molecular mass of 72.3 kDa, and bacterially expressed protein exhibited a clear b-oxidation activity. Fusion proteins with GFP were observed in cytosol, when expressed in onion epidermal cell layers. In addition to wounding, NtMFP transcripts were induced by tobacco mosaic virus infection, and by jasmonic acid treatments. When NtMFP was suppressed by the RNAi, transgenic tobacco showed dwarfism, early senescence and reduced expresstion of jasmonic acid-responsive genes. Multifunctional protein is generally considered to catalyze multiple steps of the fatty acid b-oxidation. It is also proposed to be involved in the b-oxidation step of jasmonic acid biosynthesis. The present results suggest the possibility that NtMFP commonly functions not only in fatty acid catabolism but also in jasmonic acid biosynthesis pathway.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

A rapid and efficient genetic transformation of Panax ginseng cotyledon explants following a plasmolyzing pre-treatment was investigated. When cotyledon explants of P. ginseng were pre-treated with 1.0 M sucrose, transient expression of the β-glucuronidase (GUS) gene was strongly enhanced following co-cultivation with Agrobacteriumtumefaciens harboring the GUS gene. This enhanced expression coincided with a high frequency of stable transformation (three times higher than non-treatment). Blue-colored cells (indicative of the presence of the GUS gene) were detected over large areas of cotyledons pre-treated with sucrose. In contrast, when the plasmolyzing pre-treatment was not applied, GUS-positive cells were observed to be scattered on the cotyledons. Somatic embryos developed directly on cotyledon surfaces without intervening callus formation within 2 weeks and matured to the cotyledonary stage after about 7 weeks. Cotyledonary embryos regenerated into small transgenic plantlets on medium supplemented with gibberellic acid within 1 month. Thus, about 3 months of culture was required for small transgenic ginseng plantlets to be obtained. The presence of transformed ginseng plantlets was confirmed using the staining reaction of X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) and by the polymerase chain reaction.

The pharmacodynamics of (S)-4-[3-[4-[1-(4-methylphenyl)-3-[4-(2-methoxyphenyl)piperazine-1-yl]propoxy]benzoyl]indole-1-yl] butyric acid hydrochloride (Z-350), which has alpha(1)-adrenoceptor antagonistic and steroid 5alpha-reductase inhibitory effects, were investigated in rats. The disposition of Z-350 was a function of linear kinetics at doses from 1 to 30 mg/kg; the bioavailability was calculated to be 65.2%. The inhibition of 5alpha-reductase was dependent on the concentration of Z-350 in plasma and in the prostate. Analysis of the relationship between the concentration in the prostate and the inhibition seen after a single oral administration showed that the Hill constant was almost 1.0 and EC(50)(n(H)) was 47.4 ng/g of tissue; these parameters did not change after multiple administration. Z-350 inhibited 5alpha-reductase 1 h after oral administration at a dose of 3 mg/kg; maximum inhibition was observed after 2-4 h, and the inhibition (%) was maintained for 24 h after oral administration.

Caffeine is synthesized through sequential three-step methylation of xanthine derivatives at positions 7-N, 3-N, and 1-N. However, controversy exists as to the number and properties of the methyltransferases involved. Using primers designed on the basis of conserved amino acid regions of tea caffeine synthase and Arabidopsis hypothetical proteins, a particular DNA fragment was amplified from an mRNA population of coffee plants. Subsequently, this fragment was used as a probe, and four independent clones were isolated from a cDNA library derived from coffee young leaves. Upon expression in Escherichia coli, one of them was found to encode a protein possessing 7-methylxanthine methyltransferase activity and was designated as CaMXMT. It consists of 378 amino acids with a relative molecular mass of 42.7 kDa and shows similarity to tea caffeine synthase (35.8%) and salicylic acid methyltransferase (34.1%). The bacterially expressed protein exhibited an optimal pH for activity ranging between 7 and 9 and methylated almost exclusively 7-methylxanthine with low activity toward paraxanthine, indicating a strict substrate specificity regarding the 3-N position of the purine ring. K(m) values were estimated to be 50 and 12 microM for 7-methylxanthine and S-adenosyl-l-methionine, respectively. Transcripts of CaMXMT could be shown to accumulate in young leaves and stems containing buds, and green fluorescent protein fusion protein assays indicated localization in cytoplasmic fractions. The results suggest that, in coffee plants, caffeine is synthesized through three independent methylation steps from xanthosine, in which CaMXMT catalyzes the second step to produce theobromine.

The sulfur atom is an essential nutrient for living organisms because it plays a central role in protein folding, enzyme catalysis and maintenance of the redox status of cells. Microorganisms and plants can synthesize organic sulfur compounds, including cysteine, methionine and glutathione, from inorganic sulfur compounds such as sulfates. In contrast, animals utilize organic sulfur compounds that are mainly synthesized by plants. In the last decade, many genes whose products are involved in sulfate assimilation have been isolated from higher plants, and it has been revealed that there exist multiple isoforms for each step. The different properties of isoforms has been examined for sulfate transporters, which serve at the first step of sulfate assimilation, and for O-acetylserine(thiol)lyases, which catalyze the incorporation of sulfide into cysteine. Currently, however, it is not clear why plants developed multiple forms with similar catalytic properties. Mechanisms of regulation of the sulfate assimilation pathway have also attracted much attention, since metabolites such as cysteine and glutathione affect related enzymatic activity or corresponding gene expression. The precursor of cysteine, O-acetylserine, is a key compound in maintaining the balance between sulfate and nitrate assimilation in higher plants. In addition to nutrition, sulfate assimilation is important for conferring tolerance against environmental stresses including heavy metals. Genetic engineering of components of the sulfate assimilation pathway is a useful approach to generate useful plants for agriculture and phytoremediation.

This clinical investigation examined the effect of phosphorylated chitosan rinsing on plaque development and on the buffering capacity of plaque suspension. Three male adult subjects participated in the trial that was designed as a single blind study. Participants refrained from mechanical oral hygiene procedures during a four-day study and rinsed three times a day with 20 ml of test solutions. A wash-out period of three days was instituted between the placebo and phosphorylated chitosan rinsing period. Clinical evaluation and plaque sampling were performed at the end of each test period. We disclosed plaque accumulations on the buccal upper front teeth with a two-tone disclosing agent to distinguish between newly formed plaque and old plaque. After taking color slides, we then used a computerized image analysis. Tooth areas covered by plaque on the color slides were digitized and expressed as percentages of the tooth area. The buffering capacity of the collected plaque fluid was determined by using a beta-titrator. A mouth rinse containing 0.5% phosphorylated chitosan significantly reduced both newly formed plaque areas (red disclosed; p0.001) and old plaque areas (blue disclosed; p0.01) compared to a placebo rinsing. However there was no significant difference in the plaque buffering capacity (p0.05) between the mouth rinse containing 0.5% phosphorylated chitosan and placebo. These findings might suggest that mouth rinse containing phosphorylated chitosan would be effective in reducing plaque formation and have a slight ability to enhance plaque buffering capacity.

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.

1. The relationship between plasma levels of a new H2-receptor antagonist, Z-300, and an active sulphoxide metabolite, and inhibitory effects on gastric acid secretion after intravenous or oral administration to dog have been examined. After both routes of administration, Z-300 in plasma eliminated with two phases, and the terminal half-lives were approximately 2 h. The level of sulphoxide in plasma reached a maximum at 0.6-0.7 h after administration, then subsequently eliminated with a half-life of approximately 6 h. 2. The maximal pharmacological effect of inhibition of gastric acid secretion (90-91%) was observed at 2 h (i.v.) and 6 h (p.o.), and the action persisted until 7 h after administration. 3. Since there was no correlation between plasma levels of Z-300 and pharmacological effects, the pharmacological effects were calculated by pharmacodynamic model including the effect compartment. The inhibition of acid output could be calculated by the amounts of Z-300 and sulphoxide corrected by pharmacological efficacy in the effect compartment. 4. These findings suggest that Z-300 contributes to the rapid appearance of the pharmacological effects in dog, whereas the sulphoxide, which eliminates slowly in plasma, contributes to duration.

The cytotoxic drug tunicamycin kills cells because it is a specific inhibitor of UDP-N-acetylglucosamine:dolichol phosphateN-acetylglucosamine-1-P transferase (GPT), an enzyme that catalyzes the initial step of the biosynthesis of dolichol-linked oligosaccharides. In the presence of tunicamycin, asparagine-linked glycoproteins made in the endoplasmic reticulum are not glycosylated with N-linked glycans, and therefore may not fold correctly. Such proteins may be targeted for breakdown. Cells that are treated with tunicamycin normally experience an unfolded protein response and induce genes that encode endoplasmic reticulum chaperones such as the binding protein (BiP). We isolated a cDNA clone for Arabidopsis GPT and overexpressed it in Arabidopsis. The transgenic plants have a 10-fold higher level of GPT activity and are resistant to 1 μg/mL tunicamycin, a concentration that kills control plants. Transgenic plants grown in the presence of tunicamycin haveN-glycosylated proteins and the drug does not induce BiP mRNA levels as it does in control plants. BiP mRNA levels are highly induced in both control and GPT-expressing plants by azetidine-2-carboxylate. These observations suggest that excess GPT activity obviates the normal unfolded protein response that cells experience when exposed to tunicamycin.

Summary Peroxidases are secreted into the culture medium of BY2 suspension cells. In order to determine the role of glycosylation and glycan processing for peroxidase secretion, inhibition studies were performed. When glycosylation was inhibited by tunicamycin, secretion of peroxidases into the medium ceased totally. When glycan processing was inhibited by castanospermine, the molecular size of the peroxidases was increased concomitant with their decreased secretion into the medium. Upon inhibition treatment, pulse-labeled total secreted protein behaved similarly to the peroxidases, indicating that for secretion, glycosylation is generally essential, while glycan processing is not prerequisite but none the less necessary. These studies are supported by the observation that inhibitor treatment also induced transcript accumulation for the molecular chaperon, BiP, and for calnexin, which recognizes immature glycans. Taken together, these results strongly suggest that correct N-glycan structure is critical for protein secretion in plant cells.

Ipomoea aquatica (water spinach) is a common aquatic plant growing in lakes and wetlands in Southeast Asia. Due to its vigorous growth, they were considered to be potentially useful for remediation of polluted water with, for example, high sulfate and heavy metals. In previous studies, we successfully constructed transgenic I. aquatica plants, which simultaneously expressed two genes encoding serine acetyltransferase and cysteine synthase involved in sulfate assimilation pathways. Resulting transgenic plants were shown to rapidly grow and to accumulate sulfate at a high level. In the present study, we tested the effect of cadmium on their physiological and biochemical features. Upon hydroponical cultivation in the presence of 200 mM cadmium for 7 days, two transgenic lines (SR1 and SR2) accumulated 2- to 4-fold higher levels of cysteine and glutathione than the wild type control plants. When plantlets were exposed to 100 mM cadmium for 30 days, wild type and transgenic SR2 plantlets died, or growth was greatly retarded with reduced biomass, whereas transgenic SR1 exhibited a 1.7-fold increase in total biomass in comparison with the initial weight at day-0 of cadmium treatment. These results suggested that some transgenic plants expressing serine acetyltransferase and cysteine synthase could mitigate detrimental effects of cadmium toxicity, perhaps by efficiently producing and accumulating sulfuric compounds.

The tissue distribution of polaprezinc (an insoluble zinc complex of L-carnosine) in rats was studied by the double tracer method using [U-14C-histidine]-, 65Zn-polaprezinc. The 65Zn-radioactivity was measured with an auto-gamma counter, and the 14C containing 65Zn was converted to an absolute count according to the calibration curve for quenching with a liquid scintillation counter with the spill-over method. After the administration of 14C-, 65Zn-polaprezinc to rats, the excretion ratio and time courses in the tissues of the 14C-and 65Zn-radioactivity were different each other. We found that polaprezinc was metabolized as endogenous amino acid or zinc after dissociation in the body. The zinc concentration in plasma reached its maximum at 1 h and decreased slowly, returning to the endogenous level at 11 h after the administration of non-labeled polaprezinc. The concentrations of zinc in liver, kidney, testis, prostate, and cerebrum remained rather constant. The replacement ratios of 65Zn to zinc in the tissues at its maximum percentage were 40% in plasma, 16-20% in liver, kidney, blood, and prostate. The low replacement ratios in testis and cerebrum (2-3%) suggested that zinc uptakes in testis and brain were regulated by the blood-testis-barrier and blood-brain-barrier, respectively.

A cDNA for spermidine synthase (SPDS), which converts putrescine to spermidine using decarboxylated S-adenosylmethionine as a co-factor, has been isolated from Coffea arabica. When the SPDS cDNA is expressed in an SPDS-deficient E. coli mutant, the recombinant protein shows high SPDS activity. The C. arabica SPDS possesses the co-factor binding motifs which have been proposed for S-adenosylmethionine, and its amino acid sequence is similar to other plant SPDSs. The SPDS transcripts have been observed in roots, green stems, old and young leaves, and accumulated to a higher level in rapid growing tissues, such as green stems and younger leaves, compared to old leaves. In callus tissues, it has been expressed in all stages, even though callus growth is very slow during somatic embryogenesis.

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.

Powdery reagents obtained by complete alkaline hydrolysis of arylgermanium trichlorides were found to undergo the palladium-catalyzed cross-coupling reaction with aryl bromides and iodides in good yields. The reaction is performed in an aqueous medium taking sodium hydroxide as an activator. Some base-sensitive functionalities such as acetyl and trifluoromethyl groups survived the reaction.

In the present study we aimed to observe the intracellular responses when there was a hyperosmotic shock with a large shift in ionic strength in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates. To do this, we used the halotolerant organism Brevibacterium sp. strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations. Hyperosmotic shock was induced by transferring cells in the late exponential phase of growth in a complex medium containing 0.5 M NaCl into either old or fresh culture medium containing 2 M NaCl. Changes in the growth rate, in the pH of the medium, and in the internal cation or organic solute concentrations in the cytosol after an upshock were analyzed as a function of incubation time. The cells exhibited very different responses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became more acidic, whereas the old culture medium repressed growth and the medium became more alkaline. The intracellular free Na + concentrations remained low (80 nmol mg of protein −1 ) after an upshock in fresh culture medium, although they quickly increased twofold in the old culture medium. In contrast, K + ions immediately accumulated in the cells in fresh culture medium, whereas K + ions were taken up quite slowly in old culture medium. Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upshock, but the cells placed in old culture medium did not. Growth of the Brevibacterium strain at higher levels of salinity was supported by ectoine synthesis but was not observed after the shift to high-osmolarity conditions in the old culture. In the fresh culture, however, ectoine was vigorously synthesized in cells for more than 5 h after the upshock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein −1 at 10 h, which corresponded to a ninefold increase compared to the concentration before the shock. These findings are consistent with the results of an analysis of the extracellular medium composition before and after the upshock.

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.

The Chitinase-producing bacterium Vibrio alginolyticus H-8 isolated from mud of Hamana Lake also produced two deacetylases for (GlcNAc)2 extracellularly. Deacetylases DA1 and DA2 were purified from crude enzyme by column chromatography on Q-Sepharose FF, Phenyl Sepharose HP, Gigapite, and Superdex 200 HR. The final preparation was homogeneous in SDS-PAGE. The molecular weights were 48,000 and 46,000 for deacetylases DA1 and DA2, respectively. The pIs, optimum pHs, and optimum temperatures for deacetylases DA1 and DA2 were as follows; DA1, pI 3.3, optimum pH 8.5–9.0, optimum temperature 45°C, DA2, pI 3.5, optimum pH 8.0–8.5, optimum temperature 40°C. Both deacetylases were stable at pHs between 7.0 and 11.0 and at temperatures below 40°C. The activities of both enzymes were inhibited by Ag+ and Hg2+. 1H-NMR of the reaction product by deacetylase DA1 for (GlcNAc)2 showed that the purified deacetylase selectively hydrolyzed the 2-acetamide group at the reducing end of (GlcNAc)2.

o -Tributylstannylmethyl benzaldehyde reacts with Zn in the presence of TMSCl and 2,6 lutidine at room temperature to give α-trimethylsiloxy- o -quinodimethane, which is trapped with dienophiles to afford cycloadducts in good to high yields.

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.

DGCC(1,2-diacylglyceryl-3-(O-carboxyhydroxymethylcholine)) is a betaine lipid derived from Pavlova lutheri. The separation of this lipid from other polar lipids by HPLC was improved in order to survey the distribution of DGCC, other betaine lipids and PC (phosphatidylcholine) in marine algae. DGCC was found to be one of the common constituents of Haptophyceae; it was interesting to note that PC was not detected even as a minor component. DGTA(1,2-diacylglyceryl-O-2′-(hydroxymethyl)-(N,N,N-trimethyl)-β-alanine) was detected in five out of 16 species of Haptophyceae, whereas DGTS(1,2-diacylglyceryl-O-4′-(N,N,N-trimethyl)homoserine) was not detected. DGCC was also detected in four strains of Dinophyceae and a strain of Bacillariophyceae. Acyl moieties of DGCC in Haptophyceae were 16:0, 18:0 or 18:1 and C20 or C22 polyunsaturated fatty acids.

Novel antimicroalgal substances halymecins A (1), B (2) and C (3) were isolated from the fermentation broth of a Fusarium sp. and halymecins D (4) and E (5) from an Acremonium sp. The structures of these halymecins, Fig. 1, were determined based on extensive 2D NMR studies as well as mass spectral data. These chemical structures are conjugates of di- and trihydroxydecanoic acid. Halymecin A showed antimicroalgal activity against Skeletonema costatum.

Caffeine (1,3,7-trimethylxanthine) is a member of purine alkaloids and produced in over 80 plant species. It is one of the oldest and widely used secondary metabolites by mankind as stimulant and ingredient in drugs. Its physiological function in nature has not completely been elucidated but is thought to participate in the chemical defence against biotic attackers. To substantiate this idea, transgenic tobacco and chrysanthemum were constructed by expressing three distinct N-methyltranferases involved in the caffeine biosynthesis pathway. Resulting plants produced a low amount of caffeine (0.4–5 μg/g tissue) yet exhibited strong tolerance against herbivores and pathogens. Their self-defence system was autonomously activated without perceiving external stresses. This can be regarded as the priming of defence response, by which host plants become on standby to cope with a broad range of biotic stresses. The feature resembles mammalian immunization or vaccination, and it was proposed that plants can also be immunized by expressing a mildly toxic ‘antigenic’ chemical such as caffeine in planta. The caffeine signal was predicted to be successively transduced through phosphodiesterase, cyclic AMP, calcium flux and salicylic acid.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

23Na NMR spectroscopy was used to determine free Na+ concentrations in a halotolerant bacterium, Brevibacterium sp., and Escherichia coli. The internal Na+ concentration of both strains depended little on the growth phases and was unchanged after 5 d storage at 2 degrees C. In Brevibacterium sp. the level of intracellular sodium increased gradually at higher extracellular NaCl concentrations in both the presence and absence of yeast extract in the growth medium. E. coli cells accumulated a higher concentration of free Na+ than those of Brevibacterium sp. The change of Na+ concentration in both strains was inverse to that of growth rate. When appropriate amounts of osmoprotectants (proline, glycine betaine, or gamma-aminobutyrate) were added with the NaCl, internal free Na+ levels in Brevibacterium sp. were lowered, but those of E. coli were unchanged. While addition of KCl to medium containing NaCl increased the intracellular level of free Na+, the total sodium concentration in the cells remained unchanged, indicating that sodium that had been bound or attached was made free in the cytosol. In Brevibacterium sp. grown in the presence of 0.5 M NaCl, free and bound sodium concentrations in the cytosol were estimated to be 0.14 and 0.23 mumol (mg protein)-1, respectively. As a result, visibility by 23Na NMR was 38%.