Molecular Mechanisms of Natural Rubber Biosynthesis.

Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.

De novo induction of a DNA-histone H3K9 methylation loop on synthetic human repetitive DNA in cultured tobacco cells.

Genetic modifications in plants are crucial tools for fundamental and applied research. Transgene expression usually varies among independent lines or their progeny and is associated with the chromatin structure of the insertion site. Strategies based on understanding how to manipulate the epigenetic state of the inserted gene cassette would help to ensure transgene expression. Here, we report a strategy for chromatin manipulation by the artificial tethering of epigenetic effectors to a synthetic human centromeric repetitive DNA (alphoid DNA) platform in plant Bright-Yellow-2 (BY-2) culture cells. By tethering DNA-methyltransferase (Nicotiana tabacum DRM1), we effectively induced DNA methylation and histone methylation (H3K9me2) on the alphoid DNA platform. Tethering of the Arabidopsis SUVH9, which has been reported to lack histone methyltransferase activity, also induced a similar epigenetic state on the alphoid DNA in BY-2 cells, presumably by activating the RNA-dependent DNA methylation (RdDM) pathway. Our results emphasize that the interplay between DNA and histone methylation mechanisms is intrinsic to plant cells. We also found that once epigenetic modification states were induced by the tethering of either DRM1 or SUVH9, the modification was maintained even when the direct tethering of the effector was inhibited. Our system enables the analysis of more diverse epigenetic effectors and will help to elucidate the chromatin assembly mechanisms of plant cells.

Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

Structural-Functional Correlations between Unique N-terminal Region and C-terminal Conserved Motif in Short-chain cis-Prenyltransferase from Tomato.

Neryl diphosphate (C10) synthase (NDPS1), a homodimeric soluble cis-prenyltransferase from tomato, contains four disulfide bonds, including two inter-subunit S-S bonds in the N-terminal region. Mutagenesis studies demonstrated that the S-S bond formation affects not only the stability of the dimer but also the catalytic efficiency of NDPS1. Structural polymorphs in the crystal structures of NDPS1 complexed with its substrate and substrate analog were identified by employing massive data collections and hierarchical clustering analysis. Heterogeneity of the C-terminal region, including the conserved RXG motifs, was observed in addition to the polymorphs of the binding mode of the ligands. One of the RXG motifs covers the active site with an elongated random coil when the ligands are well-ordered. Conversely, the other RXG motif was located away from the active site with a helical structure. The heterogeneous C-terminal regions suggest alternating structural transitions of the RXG motifs that result in closed and open states of the active sites. Site-directed mutagenesis studies demonstrated that the conserved glycine residue cannot be replaced. We propose that the putative structural transitions of the order/disorder of N-terminal regions and the closed/open states of C-terminal regions may cooperate and be important for the catalytic mechanism of NDPS1.

Cell-free translation system with artificial lipid-monolayer particles as a unique tool for characterizing lipid-monolayer binding proteins.

Methods for functional analysis of proteins specifically localizing to lipid monolayers such as rubber particles and lipid droplets are limited. We have succeeded in establishing a system in which artificially prepared lipid monolayer particles are added to a cell-free translation system to confirm the properties of proteins that specifically bind to lipid monolayers in a translation-coupled manner.

Managing enzyme promiscuity in plant specialized metabolism: A lesson from flavonoid biosynthesis: Mission of a "body double" protein clarified.

Specificities of enzymes involved in plant specialized metabolism, including flavonoid biosynthesis, are generally promiscuous. This enzyme promiscuity has served as an evolutionary basis for new enzyme functions and metabolic pathways in land plants adapting to environmental challenges. This phenomenon may lead, however, to inefficiency in specialized metabolism and adversely affect metabolite-mediated plant survival. How plants manage enzyme promiscuity for efficient specialized metabolism is, thus, an open question. Recent studies of flavonoid biosynthesis addressing this issue have revealed a conserved strategy, namely, a homolog of chalcone isomerase with no catalytic activity binds to chalcone synthase, a key flavonoid pathway enzyme, to narrow (or rectify) the enzyme's highly promiscuous product specificity. Reducing promiscuity via specific protein-protein interactions among metabolic enzymes and proteins may be a solution adopted by land plants to achieve efficient operation of specialized metabolism, while the intrinsic promiscuity of enzymes has likely been retained incidentally.

Enterocloster alcoholdehydrogenati sp. nov., a Novel Bacterial Species Isolated from the Feces of a Patient with Alcoholism.

Strain C5-48T, an anaerobic intestinal bacterium that potentially accumulates acetaldehyde at levels exceeding its minimum mutagenic concentration (50 µM) in the colon and rectum, was isolated from the feces of a patient with alcoholism. The 16S rRNA gene sequence of strain C5-48T showed high similarity to the corresponding sequences of Lachnoclostridium edouardi Marseille-P3397T (95.7%) and Clostridium fessum SNUG30386T (94.7%). However, phylogenetic analysis using the sequences of the 16S rRNA, rpoB, and hsp60 genes and whole-genome analysis strongly suggested that C5-48T should be included in the genus Enterocloster. The novelty of strain C5-48T was further confirmed by comprehensive average nucleotide identity (ANI) calculations based on its whole-genome sequence, which showed appreciable ANI values with known Enterocloster species (e.g., 74.3% and 73.4% with Enterocloster bolteae WAL 16351T and Enterocloster clostridioformis ATCC 25537T, respectively). The temperature range for growth of strain C5-48T was 15-37 °C with an optimum of 37 °C. The pH range for growth was 5.5-10.5 with an optimum of 7.5. The major constituents of the cell membrane lipids of strain C5-48T were 16:0, 14:0, and 18:1 ω7c dimethyl acetal fatty acids. On the basis of the genotypic and phenotypic properties, Enterocloster alcoholdehydrogenati sp. nov. is proposed, with the type strain C5-48T (= JCM 33305T = DSM 109474T).

(+)-Sesamin, a sesame lignan, is a potent inhibitor of gut bacterial tryptophan indole-lyase that is a key enzyme in chronic kidney disease pathogenesis.

The progression of chronic kidney disease (CKD) increases the risks of cardiovascular morbidity and end-stage kidney disease. Indoxyl sulfate (IS), which is derived from dietary l-tryptophan by the action of bacterial l-tryptophan indole-lyase (TIL) in the gut, serves as a uremic toxin that exacerbates CKD-related kidney disorder. A mouse model previously showed that inhibition of TIL by 2-aza-l-tyrosine effectively reduced the plasma IS level, causing the recovery of renal damage. In this study, we found that (+)-sesamin and related lignans, which occur abundantly in sesame seeds, inhibit intestinal bacteria TILs. Kinetic studies revealed that (+)-sesamin and sesamol competitively inhibited Escherichia coli TIL (EcTIL) with Ki values of 7 µM and 14 µM, respectively. These Ki values were smaller than that of 2-aza-l-tyrosine (143 µM). Molecular docking simulation of (+)-sesamin- (or sesamol-)binding to EcTIL predicted that these inhibitors potentially bind near the active site of EcTIL, where the cofactor pyridoxal 5'-phosphate is bound, consistent with the kinetic results. (+)-Sesamin is a phytochemical with a long history of consumption and is generally regarded as safe. Hence, dietary supplementation of (+)-sesamin encapsulated in enteric capsules could be a promising mechanism-based strategy to prevent CKD progression. Moreover, the present findings would provide a new structural basis for designing more potent TIL inhibitors for the development of mechanism-based therapeutic drugs to treat CKD.

A Prospective Multicenter Phase II Study on the Feasibility and Efficacy of S-1 and Oxaliplatin Neoadjuvant Chemotherapy for Locally Advanced Rectal Cancer.

BACKGROUND: Neoadjuvant chemoradiotherapy and total mesorectal excision compose the standard of care for rectal cancer in multiple guidelines. However, neoadjuvant chemoradiotherapy has not exhibited clear survival benefits but rather has led to an increase in adverse events. Conversely, neoadjuvant chemotherapy is expected to prevent adverse events caused by radiation, yet this treatment is still controversial. OBJECTIVE: The purpose of this study was to evaluate the feasibility and efficacy of S-1 and oxaliplatin neoadjuvant chemotherapy together with total mesorectal excision for resectable locally advanced rectal cancer. DESIGN: The study was a prospective, single-arm phase II trial. SETTINGS: The study was conducted at multiple institutions. PATIENTS: Fifty-eight patients with resectable locally advanced rectal cancer were enrolled. INTERVENTION: Three cycles of S-1 and oxaliplatin were administered before surgery. S-1 was administered orally at 80 mg/m2 per day for 14 consecutive days, followed by a 7-day resting period. Oxaliplatin was given intravenously on the first day at a dose of 130 mg/m2 per day. The duration of 1 cycle was considered to be 21 days. Total mesorectal excision with bilateral lymph node dissection was carried out after neoadjuvant chemotherapy. MAIN OUTCOME MEASURES: The study was designed to detect the feasibility and efficacy of S-1 and oxaliplatin as neoadjuvant chemotherapy. RESULTS: The completion rate of 3 courses of S-1 and oxaliplatin as neoadjuvant chemotherapy was 94.8% (55/58). The reasons for discontinuation were thrombocytopenia (3.4%) and liver injury (1.7%). The most common severe (grade ≥3) adverse effect of neoadjuvant chemotherapy was thrombocytopenia (3.4%). There were no severe adverse clinical symptoms. Consequently, R0 resection was achieved in 51 (98.1%) of 52 patients. Pathologic complete response occurred in 10 patients (19.2%). LIMITATIONS: This was a single-arm, nonrandomized phase II study. CONCLUSIONS: The combination of S-1 and oxaliplatin neoadjuvant chemotherapy and total mesorectal excision is a feasible and promising treatment option for resectable locally advanced rectal cancer. See Video Abstract at http://links.lww.com/DCR/B555. UN ESTUDIO PROSPECTIVO MULTICNTRICO FASE II SOBRE LA FACTIBILIDAD Y EFICACIA DE LA QUIMIOTERAPIA NEOADYUVANTE SCON OXALIPLATINO PARA EL CNCER DE RECTO LOCALMENTE AVANZADO: ANTECEDENTES:La quimiorradioterapia neoadyuvante y la escisión mesorrectal total constituyen el estándar de atención para el cáncer de recto en varias guías. Sin embargo, la quimiorradioterapia neoadyuvante no ha mostrado beneficios claros en la sobrevida, pero si ha creado un aumento de eventos adversos. Por otro lado, se espera que la quimioterapia neoadyuvante prevenga los eventos adversos asociados a la radiación, aunque este tratamiento sigue siendo controvertido.OBJETIVO:Evaluar la factibilidad y eficacia de la quimioterapia neoadyuvante S-1 con oxaliplatino en conjunto con la escisión mesorrectal total para el cáncer de recto localmente avanzado resecable.DISEÑO:El estudio fue un ensayo prospectivo fase II de brazo único.AMBITO:Estudio realizado en múltiples instituciones.PACIENTES:Se incluyeron 58 pacientes con cáncer de recto localmente avanzado resecable.INTERVENCIÓN:Se administraron tres ciclos de S-1 con oxaliplatino antes de la cirugía. Se administró S-1 por vía oral a 80 mg / m2 / día durante 14 días consecutivos, seguido de un período de descanso de 7 días. El oxaliplatino se administró por vía intravenosa el primer día a una dosis de 130 mg / m2 / día. Se consideró la duración de un ciclo de 21 días. Posterior a la quimioterapia neoadyuvante se realizó la excisión total mesorrectal con disección ganglionar bilateral.PRINCIPALES VARIABLES EVALUDADAS:El estudio fue diseñado para conocer la factibilidad y eficacia de S-1 con oxaliplatino como quimioterapia neoadyuvante.RESULTADOS:La tasa de conclusión con tres ciclos de S-1 con oxaliplatino como quimioterapia neoadyuvante fue del 94,8% (55/58). Los motivos de interrupción fueron trombocitopenia (3,4%) y daño hepático (1,7%). El efecto adverso grave más común (grado ≥ 3) de la quimioterapia neoadyuvante fue la trombocitopenia (3,4%). No hubo síntomas clínicos adversos graves. Como resultado, la resección R0 se logró en 51 de 52 pacientes (98,1%). Una respuesta patológica completa se obtuvo en 10 pacientes (19,2%).LIMITACIONES:Fue un estudio de fase II no aleatorizado de un solo brazo.CONCLUSIONES:La combinación de S-1 con oxaliplatino como quimioterapia neoadyuvante y escisión mesorrectal total es factible y es una opción de tratamiento prometedora para el cáncer de recto localmente avanzado resecable. Consulte Video Resumen en http://links.lww.com/DCR/B555. (Traducción-Dr Juan Antonio Villanueva-Herrero).

Glycoside-specific glycosyltransferases catalyze regio-selective sequential glucosylations for a sesame lignan, sesaminol triglucoside.

Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid-related plant specialized metabolites. (+)-Sesamin and (+)-sesamolin are major hydrophobic lignans, whereas (+)-sesaminol primarily accumulates as a water-soluble sesaminol triglucoside (STG) with a sugar chain branched via ß1→2 and ß1→6-O-glucosidic linkages [i.e. (+)-sesaminol 2-O-ß-d-glucosyl-(1→2)-O-ß-d-glucoside-(1→6)-O-ß-d-glucoside]. We previously reported that the 2-O-glucosylation of (+)-sesaminol aglycon and ß1→6-O-glucosylation of (+)-sesaminol 2-O-ß-d-glucoside (SMG) are mediated by UDP-sugar-dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the ß1→2-O-glucosylation of SMG and (+)-sesaminol 2-O-ß-d-glucosyl-(1→6)-O-ß-d-glucoside [termed SDG(ß1→6)]. UGT94AG1 was phylogenetically related to glycoside-specific glycosyltransferases (GGTs) and co-ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG-deficient sesame mutant that predominantly accumulates SDG(ß1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar-sugar ß1→6-O-glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two-hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane-associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane-bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main ß-O-glucosylation sequence of STG biosynthesis is ß1→2-O-glucosylation of SMG by UGT94AG1 followed by UGT94AA2-mediated ß1→6-O-glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio-selectivity of sequential glucosylations catalyzed by GGTs.

Identification of the Genes Coding for Carthamin Synthase, Peroxidase Homologs that Catalyze the Final Enzymatic Step of Red Pigmentation in Safflower (Carthamus tinctorius L.).

Carthamin, a dimeric quinochalcone that is sparingly soluble in water, is obtained from the yellow-orange corolla of fully blooming safflower (Carthamus tinctorius L.) florets. Carthamin is a natural red colorant, which has been used worldwide for more than 4500 years and is the major component of Japanese 'beni' used for dyeing textiles, in cosmetics and as a food colorant. The biosynthetic pathway of carthamin has long remained uncertain. Previously, carthamin was proposed to be derived from precarthamin (PC), a water-soluble quinochalcone, via a single enzymatic process. In this study, we identified the genes coding for the enzyme responsible for the formation of carthamin from PC, termed 'carthamin synthase' (CarS), using enzyme purification and transcriptome analysis. The CarS proteins were purified from the cream-colored corolla of safflower and identified as peroxidase homologs (CtPOD1, CtPOD2 and CtPOD3). The purified enzyme catalyzed the oxidative decarboxylation of PC to produce carthamin using O2, instead of H2O2, as an electron acceptor. In addition, CarS catalyzed the decomposition of carthamin. However, this enzymatic decomposition of carthamin could be circumvented by adsorption of the pigment to cellulose. These CtPOD isozymes were not only expressed in the corolla of the carthamin-producing orange safflower cultivars but were also abundantly expressed in tissues and organs that did not produce carthamin and PC. One CtPOD isozyme, CtPOD2, was localized in the extracellular space. Based on the results obtained, a model for the stable red pigmentation of safflower florets during flower senescence and the traditional 'beni' manufacturing process is proposed.

[Long-Term Complete Response by Chemotherapy for Distant Lymph Node Metastases after Curative Surgery for Ascending Colon Cancer-A Case Report].

A 74-year-old woman underwent right hemicolectomy for ascending colon cancer in March 2013, after which she received adjuvant chemotherapy(UFT plus UZEL)for 6 months. In October 2014, left supraclavicular lymphadenopathy appeared, and it was diagnosed as adenocarcinoma metastasis through fine-needle aspiration cytology. CT revealed swelling of left supraclavicular lymph nodes and para-aortic lymph nodes but no metastases to other organs. Exclusion diagnosis was performed, and they were diagnosed as multiple distant lymph node metastases. Chemotherapy(mFOLFOX6 plus bevacizumab) was started in December 2014, and all swollen lymph nodes shrank and disappeared on CT in July 2015. Furthermore, there was no swelling of lymph nodes or appearance of new lesions on CT in December 2015, as the response to treatment was judged to be complete. In addition, the regimen was changed to FOLFIRI plus bevacizumab because side effects such as peripheral neuropathy were worsening. Although chemotherapy was discontinued in November 2016, there has been no recurrence and a long-term complete response has been sustained.

[A Case of Immediately-Treated Internal Hernia Occurring during Chemotherapy for Liver Metastasis after Gastric GIST Resection].

A 71-year-old man underwent total gastrectomy with Roux-en-Y reconstruction for gastric GIST in October 2017. Liver metastasis was identified in June 2019, and chemotherapy with imatinib was started in July. In December, the patient presented with acute upper abdominal pain and back pain. Abdominal contrast-enhanced CT showed that the jejunum extending from the duodenal stump was dilated. In addition, part of the jejunum had a poor wall contrast effect, with ascites also found surrounding it. We suspected a strangulated ileus and immediately performed emergency surgery. We found an internal hernia with incarceration of the afferent loop at the Petersen's defect. The time from the onset of symptoms to the surgery was relatively short, and the surgery was completed with hernial repair and closure of the hernial orifice without the development of bowel necrosis; the patient's postoperative course was good. Although the frequency of internal hernia after gastrectomy is relatively low, there is a risk that it may be severe if it occurs. Therefore, care should be taken to not cause internal hernias during surgery, and an internal hernia should be considered in the event of sudden abdominal pain after gastric surgery.

Crystal structure of chalcone synthase, a key enzyme for isoflavonoid biosynthesis in soybean.

Isoflavonoid is one of the groups of flavonoids that play pivotal roles in the survival of land plants. Chalcone synthase (CHS), the first enzyme of the isoflavonoid biosynthetic pathway, catalyzes the formation of a common isoflavonoid precursor. We have previously reported that an isozyme of soybean CHS (termed GmCHS1) is a key component of the isoflavonoid metabolon, a protein complex to enhance efficiency of isoflavonoid production. Here, we determined the crystal structure of GmCHS1 as a first step of understanding the metabolon structure, as well as to better understand the catalytic mechanism of GmCHS1.

Crystal structure of Thermobifida fusca cis-prenyltransferase reveals the dynamic nature of its RXG motif-mediated inter-subunit interactions critical for its catalytic activity.

cis-Prenyltransferases (cis-PTs) catalyze consecutive condensations of isopentenyl diphosphate to an allylic diphosphate acceptor to produce a linear polyprenyl diphosphate of designated length. Dimer formation is a prerequisite for cis-PTs to catalyze all cis-prenyl condensation reactions. The structure-function relationship of a conserved C-terminal RXG motif in cis-PTs that forms inter-subunit interactions and has a role in catalytic activity has attracted much attention. Here, we solved the crystal structure of a medium-chain cis-PT from Thermobifida fusca that produces dodecaprenyl diphosphate as a polyprenoid glycan carrier for cell wall synthesis. The structure revealed a characteristic dimeric architecture of cis-PTs in which a rigidified RXG motif of one monomer formed inter-subunit hydrogen bonds with the catalytic site of the other monomer, while the RXG motif of the latter remained flexible. Careful analyses suggested the existence of a possible long-range negative cooperativity between the two catalytic sites on the two monomeric subunits that allowed the binding of one subunit to stabilize the formation of the enzyme-substrate ternary complex and facilitated the release of Mg-PPi and subsequent intra-molecular translocation at the counter subunit so that the condensation reaction could occur in consecutive cycles. The current structure reveals the dynamic nature of the RXG motif and provides a rationale for pursuing further investigations to elucidate the inter-subunit cooperativity of cis-PTs.

Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2-hydroxyisoflavanone synthase.

Soybean (Glycine max) 5-deoxyisoflavonoids (daidzein and its conjugates) are precursors of glyceollin phytoalexins. They are also converted to equol by microbes in the human intestine, resulting in health benefits. 5-Deoxyisoflavonoids accumulate in the roots (93% mol/mol of the total root isoflavonoids) and seeds of unstressed soybean plants. Chalcone reductase (CHR) is a key enzyme mediating 5-deoxyisoflavonoid biosynthesis because it catalyzes the production of 6'-deoxychalcone through its effects on the chalcone synthase (CHS)-catalyzed reaction. The soybean genome encodes at least 11 CHR-related homologs, but it is unclear which ones are functionally important for daidzein accumulation in unstressed plants. Among the CHR homologs, the temporal and spatial expression patterns of GmCHR5 were the most correlated with the distribution patterns of 5-deoxyisoflavonoids. The CHR activity of GmCHR5 was confirmed in vitro and in planta. In the in vitro assays, the ratio of CHR products (6'-deoxychalcone) to total CHS products (R value) was dependent on GmCHR5 and CHS concentrations, with higher concentrations resulting in higher R values (i.e. approaching 90%). Subcellular localization analyses revealed that GmCHR5 was present in the cytoplasm and nucleus. Protein-protein interaction assays indicated that GmCHR5, but not GmCHR1 and GmCHR6, interacted with 2-hydroxyisoflavanone synthase (IFS) isozymes. The CHS isozymes also interacted with IFS isozymes but not with GmCHR5. The proposed micro-compartmentalization of isoflavone biosynthesis through the formation of an IFS-mediated metabolon is probably involved in positioning GmCHR5 close to CHS, resulting in an R value that is high enough for the accumulation of abundant 5-deoxyisoflavonoids in soybean roots.

Physical interactions among flavonoid enzymes in snapdragon and torenia reveal the diversity in the flavonoid metabolon organization of different plant species.

Flavonoid metabolons (weakly-bound multi-enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein-protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4-reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3'-hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage-dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co-suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long-suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.

Electrospray Deposition of {200} Oriented Regular-Assembly BaTiO3 Nanocrystal Films under an Electric Field.

Highly oriented, regularly assembled nanocrystalline films have recently emerged as attractive new functional materials. In this study, we deposited a BaTiO3 (BT) nanocube dispersion on a Si substrate by electrospraying, resulting in a dense, regularly assembled BT nanocrystalline film. X-ray diffraction analysis revealed that applying a voltage between the electrospray nozzle and the Si substrate during electrospraying caused the BT nanocubes to form a regular array in the 200 plane aligned perpendicularly to the substrate. The volume fraction of BT nanocubes in the 200 plane in the assembly was estimated by the orientation distribution function analysis to be about 50%. The formation of this regularly assembled layer was determined to be linked to the interaction between the vaporized solvent and the substrate, enabled by the enhanced wettability under the electric field. Electrospray deposition has potential applications in the manufacture of nanocrystalline assembled films for nanofunctional devices.

Purification and characterization of small and large rubber particles from Hevea brasiliensis.

Natural rubber (NR) is synthesized by the rubber transferase (RTase) on rubber particles (RPs) in latex. Due to the heterogeneity of the RPs in latex, it is difficult to precisely characterize the RTase activity. In this study, we separated the RPs of Hevea brasiliensis with different particle size distributions, via stepwise centrifugations. Analyses of protein compositions and size distributions of NR in the RPs suggest that RPs in Hevea latex can be categorized into two distinct subclasses, the larger RPs (termed 1kRP, 2kRP, and 8kRP) and the smaller RPs (termed 20kRP and 50kRP). Precise enzymatic assays using the RPs revealed that 50kRP showed the highest RTase activity, whereas the larger RPs, which had been regarded to have quite low activity, also exhibited a comparable activity to the smaller RPs. Immunological detections of cis-prenyltransferases in the RPs showed that the abundance of these enzymes correlates with the extent of RTase activity.

Identification and characterization of a novel bacterial ß-glucosidase that is highly specific for the ß-1,2-glucosidic linkage of sesaminol triglucoside.

A gene (PSTG2) coding for a novel ß-glucosidase belonging to glycoside hydrolase family 3 was identified in the vicinity of the previously identified ß-glucosidase gene [sesaminol triglucoside (STG)-hydrolyzing ß-glucosidase, PSTG1] in the genome of Paenibacillus sp. strain KB0549. Compared with PSTG1, recombinant PSTG2 more specifically acted on the ß-1,2-glucosidic linkage of the STG molecule to transiently accumulate a larger amount of 6-O-(ß-D-glucopyranosyl)-ß-D-glucopyranosylsesaminol.