Biosynthetic Pathways of Purine and Pyridine Alkaloids in Coffee Plants.

Caffeine (1,3,7-N-trimethylxanthine) and trigonelline (IN-methylnicotinic acid) are major alkaloids in coffee plants. The key enzymes involved-in the biosyntliesis of these compounds are very closely related N-methyltransferases belonging to the motif B' family of methyltransferases. The major biosynthetic pathways of caffeine and trigonelline are summarized in this review, including new evidence obtained from recombinant enzymes. In addition, precursor supply pathways are discussed with newly obtained results. Transgenic plants produced by the modification of the expression of N-methyltransferase genes are also introduced.

De novo Biosynthesis of Trigonelline in Fenugreek (Trigonellafoenum-graecum) seedlings.

The concentration of trigonelline in dry seeds of fenugreek (Trigonellafoenum-graecum) was 29±3 [mol/g fresh weight. Trigonelline occurred in cotyledons and embryonic axes of the seedlings, but it was found mainly in the above-ground parts of the young fenugreek plants. (15)NH4⁺-feeding experiments suggest that the de novo biosynthesis of trigonelline from NH⁺4 occurs mainly in roots. Trigonelline, which is formed in roots using inorganic nitrogen, is transported to the stems and accumulates in leaves.

Effect of purine alkaloids on the proliferation of lettuce cells derived from protoplasts.

To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 µM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with > 250 µM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

Occurrence, biosynthesis and metabolism of theanine (γ-glutamyl-L-ethylamide) in plants: a comprehensive review.

Theanine (γ-glutamyl-L-ethylamide) is the most abundant non-protein amino acid in tea leaves. In addition to Camellia sinensis, theanine occurs in several plants belonging to the Ericales. Biosynthesis of theanine from glutamic acid and ethylamine by theanine synthetase is present in all organs of tea seedlings, but roots are the major site of theanine biosynthesis in adult tea trees. Theanine is transported from roots to young leaves via the xylem sap. Theanine is hydrolysed to glutamic acid and ethylamine in leaves. Ethylamine produced from theanine is predominantly used for catechin biosynthesis. Concentration of ammonia and light intensity influence the biosynthesis and degradation of theanine, respectively. Biosynthesis, translocation and degradation of theanine and related enzymes and genes are reviewed.

Occurrence and de novo biosynthesis of caffeine and theanine in seedlings of tea (Camellia sinensis).

Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3-1.1 mg N/g and 0.1-0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5-1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1-1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

Metabolism of purine alkaloids and xanthine in leaves of maté (Ilex paraguariensis).

Accumulation and metabolism of purine alkaloids in leaves of maté (Ilex paraguariensis) were investigated. In winter, leaves accumulated caffeine but not theobromine, indicating that caffeine is the end product of purine alkaloid synthesis in maté. To elucidate the purine alkaloid metabolism in maté leaves, the metabolic fate of [8-(14)C]theobromine, [8-(14)C]theophylline, [8-(14)C]caffeine and [8-(14)C] xanthine was investigated in the leaf disks of young and mature leaves. In young maté leaves, significant amounts of theobromine and theophylline were utilized for caffeine biosynthesis, but the conversion was not observed in mature leaves. A small amount of theophylline was converted to theobromine. Practically no caffeine catabolism was detected in maté leaves during a 24 h-incubation. Catabolism of theobromine and theophylline via 3-methylxanthine was observed mainly in mature leaves. Xanthine was catabolised extensively via ureides in both young and mature leaves, but limited amounts are also utilized for the synthesis of theobromine, theophylline and caffeine. Possible pathways for the metabolism of purine alkaloids in maté leaves are discussed.

Biochemical analysis of Phytolacca DOPA dioxygenase.

The biochemical analysis of Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) was carried out. The recombinant protein of PaDOD1 catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. While the reported motif conserved in DODs from betalain-producing plants was found in PaDOD1, a single amino acid residue alteration was detected in PaDOD2. A mutated PaDOD1 protein with a change of 177 Asn to Gly showed reduced specific activity compared with PaDOD1, while DOPA dioxygenase activity was not observed for a mutated PaDOD2 protein which had its conserved motif replaced with that of PaDOD. A three-dimensional (3D) structural model of PaDOD1 and PaDOD2 showed that the conserved motif in DODs was located in the N-terminal side of a loop, which was found close to the putative active site. The difference in stability of the loop may affect the enzymatic activity of PaDOD2.

Effect of caffeine on the expression pattern of water-soluble proteins in rice (Oryza sativa) seedlings.

It has been suggested that caffeine acts as an allelochemical which influences the germination and growth of plants. The effect of caffeine on the expression profiles of proteins was investigated in shoot-root axes of rice (Oryza sativa) seedlings. Two-dimensional difference gel electrophoresis combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry was employed for the separation and identification of proteins. The results indicated that amounts of 51 protein spots were reduced and 14 were increased by treatment with 1 mM caffeine. Twelve rice seedling proteins were identified. Down-regulated proteins were ß-tubulin, sucrose synthase, glyceraldehyde-3-phosphate dehydrogenase, reversibly glycosylated polypeptide/α-1,4-glucan protein synthase and cytoplasmic malate dehydrogenase. In contrast, up-regulated proteins were alanyl-aminopeptidase, acetyl-CoA carboxylase, adenine phosphoribosyltransferase, NAD-malate dehydrogenase, ornithine carbamoyltransferase, glucose-6-phosphate isomerase and nuclear RNA binding protein. Possible alternation of metabolism caused by caffeine is discussed with the protein expression data.

Short-term effect of caffeine on purine, pyrimidine and pyridine metabolism in rice (Oryza sativa) seedlings.

As part of our studies on the physiological and ecological function of caffeine, we investigated the effect of exogenously supplied caffeine on purine, pyrimidine and pyridine metabolism in rice seedlings. We examined the effect of 1 mM caffeine on the in situ metabolism of 14C-labelled adenine, guanine, inosine, uridine, uracil, nicotinamide and nicotinic acid. The segments of 4-day-old dark-grown seedlings were incubated with these labelled compounds for 6 h. For purines, the incorporation of radioactivity from [8-(14)C]adenine and [8-(14)C]guanine into nucleotides was enhanced by caffeine; in contrast, incorporation into CO2 were reduced. The radioactivity in ureides (allantoin and allantoic acid) from [8-(14)C]guanine and [8-(14)C]inosine was increased by caffeine. For pyrimidines, caffeine enhanced the incorporation of radioactivity from [2-(14)C]uridine into nucleotides, which was accompanied by a decrease in pyrimidine catabolism. Such difference was not found in the metabolism of [2-(14)C]uracil. Caffeine did not influence the pyridine metabolism of [carbonyl-14C]- nicotinamide and [2-(14)C]nicotinic acid. The possible control steps of caffeine on nucleotide metabolism in rice are discussed.

Accumulation and function of trigonelline in non-leguminous plants.

As part of our studies of the occurrence, biosynthesis, function and human use of trigonelline, we looked at trigonelline-accumulating plant species and at the distribution of trigonelline in different organs of trigonelline-accumulating non-leguminous plants. There are many trigonelline-synthesizing plant species, but apart from legume seeds only a few species accumulate high concentrations of trigonelline. We have found only three species that accumulate high levels of trigonelline: Murraya paniculata (orange jessamine), Coffea arabica (coffee) and Mirabilisjalapa (four o'clock flower). Trigonelline was found in all parts of Murraya paniculata seedlings at 4-13 micromol/g fresh weight; more than 70% was distributed in the leaves. In the coffee plant, trigonelline was found in all organs, and the concentrations in the upper stems, including tips (48 micromol/g FW) and seeds (26 micromol/g FW), were higher than in other organs. In Mirabilis jalapa plants, trigonelline was found in leaves, stems, flowers, roots and seeds; the concentration varied from 0.3 to 13 micromol/g FW and was generally higher in young tissues than in mature tissues, except for seeds. Exogenously supplied nicotinamide increases the trigonelline content. The in planta role of trigonelline and the possible use oftrigonelline-accumulating plants in herbal medicine are discussed.

Coffee: biochemistry and potential impact on health.

This review provides details on the phytochemicals in green coffee beans and the changes that occur during roasting. Key compounds in the coffee beverage, produced from the ground, roasted beans, are volatile constituents responsible for the unique aroma, the alkaloids caffeine and trigonelline, chlorogenic acids, the diterpenes cafestol and kahweol, and melanoidins, which are Maillard reaction products. The fate of these compounds in the body following consumption of coffee is discussed along with evidence of the mechanisms by which they may impact on health. Finally, epidemiological findings linking coffee consumption to potential health benefits including prevention of several chronic and degenerative diseases, such as cancer, cardiovascular disorders, diabetes, and Parkinson's disease, are evaluated.

Phytochemical profile of a Japanese black-purple rice.

Black-purple rice is becoming popular with health conscious food consumers. In the present study, the secondary metabolites in dehulled black-purple rice cv. Asamurasaki were analysed using HPLC-PDA-MS(2). The seeds contained a high concentration of seven anthocyanins (1400 µg/g fresh weight) with cyanidin-3-O-glucoside and peonidin-3-O-glucoside predominating. Five flavonol glycosides, principally quercetin-3-O-glucoside and quercetin-3-O-rutinoside, and flavones were detected at a total concentration of 189 µg/g. The seeds also contained 3.9 µg/g of carotenoids consisting of lutein, zeaxanthin, lycopene and ß-carotene. γ-Oryzanol (279 µg/g) was also present as a mixture of 24-methylenecycloartenol ferulate, campesterol ferulate, cycloartenol ferulate and ß-sitosterol ferulate. No procyanidins were detected in this variety of black-purple rice. The results demonstrate that the black-purple rice in the dehulled form in which it is consumed by humans contains a rich heterogeneous mixture of phytochemicals which may provide a basis for the potential health benefits, and highlights the possible use of the rice as functional food.

Pyridine metabolism in tea plants: salvage, conjugate formation and catabolism.

Pyridine compounds, including nicotinic acid and nicotinamide, are key metabolites of both the salvage pathway for NAD and the biosynthesis of related secondary compounds. We examined the in situ metabolic fate of [carbonyl-(14)C]nicotinamide, [2-(14)C]nicotinic acid and [carboxyl-(14)C]nicotinic acid riboside in tissue segments of tea (Camellia sinensis) plants, and determined the activity of enzymes involved in pyridine metabolism in protein extracts from young tea leaves. Exogenously supplied (14)C-labelled nicotinamide was readily converted to nicotinic acid, and some nicotinic acid was salvaged to nicotinic acid mononucleotide and then utilized for the synthesis of NAD and NADP. The nicotinic acid riboside salvage pathway discovered recently in mungbean cotyledons is also operative in tea leaves. Nicotinic acid was converted to nicotinic acid N-glucoside, but not to trigonelline (N-methylnicotinic acid), in any part of tea seedlings. Active catabolism of nicotinic acid was observed in tea leaves. The fate of [2-(14)C]nicotinic acid indicates that glutaric acid is a major catabolite of nicotinic acid; it was further metabolised, and carbon atoms were finally released as CO(2). The catabolic pathway observed in tea leaves appears to start with the nicotinic acid N-glucoside formation; this pathway differs from catabolic pathways observed in microorganisms. Profiles of pyridine metabolism in tea plants are discussed.

Distinct fluctuations in nucleotide metabolism accompany the enhanced in vitro embryogenic capacity of Brassica cells over-expressing SHOOTMERISTEMLESS.

Besides regulating meristem formation and maintenance in vivo, SHOOTMERISTEMLESS (STM) has been shown to affect embryogenesis. While the over-expression of Brassica napus (Bn)STM enhances the number of microspore-derived embryos produced in culture and their ability to regenerate viable plants, a down-regulation of this gene represses the embryogenic process (Elhiti et al., J Exp Bot, 61:4069-4085, 2010). Synthesis and degradation of pyrimidine and purine nucleotides were measured in developing microspore-derived embryos (MDEs) generated from B. napus lines ectopically expressing or down-regulating BnSTM. Pyrimidine metabolism was investigated by following the metabolic fate of exogenously supplied (14)C-uridine, uracil and orotic acid, whereas purine metabolism was estimated by using (14)C-adenine, adenosine and inosine. The improvement in embryo number and quality affected by the ectopic expression of BnSTM was linked to the increased pyrimidine and purine salvage activity during the early phases of embryogenesis and the enlargement of the adenylate pool (ATP + ADP) required for the active growth of the embryos. This was due to an increase in transcriptional and enzymatic activity of several salvage enzymes, including adenine phosphoribosyltransferase (APRT) and adenosine kinase (ADK). The highly operative salvage pathway induced by the ectopic expression of BnSTM was associated with a slow catabolism of nucleotides, suggesting the presence of an antagonist mechanism controlling the rate of salvage and degradation pathways. During the second half of embryogenesis utilization of uridine for UTP + UDPglucose (UDPG) synthesis increased in the embryos over-expressing BnSTM, and this coincided with a better post-germination performance. All these events were precluded by the down-regulation of BnSTM which repressed the formation of the embryos and their post-embryonic performance. Overall, this work provides evidence that precise metabolic changes are associated with proper embryo development in culture.

Brassinolide-improved development of Brassica napus microspore-derived embryos is associated with increased activities of purine and pyrimidine salvage pathways.

Cellular brassinolide (BL) levels regulate the development of Brassica napus microspore-derived embryos (MDEs). Synthesis and degradation of nucleotides were measured on developing MDEs treated with BL or brassinazole (BrZ), a biosynthetic inhibitor of BL. Purine metabolism was investigated by following the metabolic fate of (14)C-labelled adenine and adenosine, substrates of the salvage pathway, and inosine, an intermediate of both salvage and degradation pathways. For pyrimidine, orotic acid, uridine and uracil were employed as markers for the de novo (orotic acid), salvage (uridine and uracil), and degradation (uracil) pathways. Our results indicate that utilization of adenine, adenosine, and uridine for nucleotides and nucleic acids increased significantly in BL-treated embryos at day 15 and remained high throughout the culture period. These metabolic changes were ascribed to the activities of the respective salvage enzymes: adenine phosphoribosyltransferase (EC 2.4.2.7), adenosine kinase (EC 2.7.1.20), and uridine kinase (EC 2.7.1.48), which were induced by BL applications. The BL promotion of salvage synthesis was accompanied by a reduction in the activities of the degradation pathways, suggesting the presence of competitive anabolic and catabolic mechanisms utilizing the labelled precursors. In BrZ-treated embryos, with depleted BL levels, the salvage activity of both purine and pyrimidine nucleotides was reduced and this was associated to structural abnormalities and poor embryonic performance. In these embryos, the activities of major salvage enzymes were consistently lower to those measured in their control (untreated) counterparts.

Pyridine metabolism and trigonelline synthesis in leaves of the mangrove legume trees Derris indica (Millettia pinnata) and Caesalpinia crista.

The aim of this study was to reveal the pyridine metabolism in leaves of two mangrove legumes, Derris indica (= Millettia pinnata or Pongamia pinnata) and Caesalpinia crista. Radioactivity from [carbonyl-14C]nicotinamide supplied exogenously to young leaf disks was recovered in nicotinic acid, nicotinic acid mononucleotide, NAD, NADP, nicotinamide mononucleotide and trigonelline. These mangrove species, especially D. indica, have strong ability to convert nicotinamide to trigonelline, but not to nicotinic acid glucoside. The endogenous trigonelline content in leaves of D. indica was more than 830 microg/g dry weight. This value is 5-12 times greater than that in leaves of Glycine max. There was little short-term effect of 250 and 500 mM NaCl (equivalent to ca. 50% and 100% sea water) on nicotinamide metabolism.

Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings.

To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.

Distribution and biosynthesis of flavan-3-ols in Camellia sinensis seedlings and expression of genes encoding biosynthetic enzymes.

The distribution of phenolic compounds in young and developing leaves, stems, main and lateral roots and cotyledons of 8-week-old tea (Camellia sinensis) seedlings was investigated using HPLC-MS(2). Fourteen compounds, flavan-3-ols, chlorogenic acids, and kaempferol-O-glycosides, were identified on the basis of their retention time, absorbance spectrum, and MS fragmentation pattern. The major phenolics were (-)-epigallocatechin-3-O-gallate and (-)-epicatechin-3-O-gallate, located principally in the green parts of the seedlings. Considerable amounts of radioactivity from [ring-(14)C]phenylalanine were incorporated in (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin-3-O-gallate and (-)-epigallocatechin-3-O-gallate, by tissues of young and developing leaves and stems. Expression of genes encoding enzymes involved in flavan-3-ol biosynthesis, CHS, CHI, F3H, F3'5'H, DFR, ANS, ANR and LAR was investigated. Transcripts of all genes, except LAR, were more abundant in leaves and stems than in roots and cotyledons. No significant difference was found in the amount of transcript of LAR. These findings indicate that in tea seedlings flavan-3-ols are produced by a naringenin-chalcone-->naringenin-->dihydrokaempferol pathway. Dihydrokaempferol is a branch point in the synthesis of (-)-epigallocatechin-3-O-gallate and other flavan-3-ols which can be formed by routes beginning with either a flavonoid 3'-hydroxylase mediated conversion of the flavonol to dihydroquercetin or a flavonoid 3',5'-hydroxylase-catalysed conversion to dihydromyricetin with subsequent steps involving sequential reactions catalysed by dihydroflavanol 4-reductase, anthocyanidin synthase, anthocyanidin reductase and flavan-3-ol gallate synthase.

Transcatheter arterial infusion chemotherapy with cisplatin-lipiodol suspension in patients with hepatocellular carcinoma.

PURPOSE: The aim of this study was to investigate the antitumor efficacy of treatment, identify prognostic factors, and construct a prognostic index in patients with hepatocellular carcinoma treated by transcatheter arterial infusion chemotherapy (TAI) using cisplatin suspended in lipiodol. METHODS: We analyzed the outcomes in a total of 94 consecutive patients with previously untreated hepatocellular carcinoma who were treated by TAI using cisplatin suspended in lipiodol. RESULTS: Twenty-seven patients (29%) showed complete response and 21 patients (22%) showed partial response, with an overall response rate of 51% (95% confidence interval, 41-61%). The median survival time was 2.5 years and the proportions of survivors at 1, 2, and 5 years were 81.6, 65.2, and 18.3%, respectively. The results of multivariate analysis indicated a significant association of serum albumin > or =3.0 g/dL, maximum tumor size < or =3.0 cm, absence of ascites, and unilateral distribution of the tumors with a favorable survival. For clinical application, we also propose a prognostic index based on a combination of these prognostic factors. Based on this index, the patients were classified into three groups: those with good, intermediate, and poor prognosis. The median survival times in these three groups were 4.3, 2.7, and 1.1 years, respectively (p < 0.01). CONCLUSIONS: TAI with cisplatin suspended in lipiodol exhibited favorable tumor efficacy and survival in patients with hepatocellular carcinoma. The prognostic factors identified and the index proposed based on these factors may be useful for predicting life expectancy, determining treatment strategies, and designing future clinical trials.