Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain

Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3′,4′-dihydroxylated leucocyanidin. 3′-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3′H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3′H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice.     

Synthesis and Structural Characterization of Triorganotin(IV) Derivatives with 2,2′-Thiodiglycolic Acid and 3,3′-Thiodipropionic Acid

The synthesis and structural characterization of four novel triorganotin(IV) complexes, {(R₃Sn)₂[C₂H₄S(COO)₂]} _n (R = Me: 1), {(R₃Sn)₄[C₂H₄S(COO)₂]₂} _n (R = nBu: 2), {(R₃Sn)₂[C₄H₈S(COO)₂]} _n (R = Me: 3; nBu: 4) were obtained by the reaction of 2,2′-thiodiglycolic acid, 3,3′-thiodipropionic acid and the corresponding R₃SnCl (R = Me, nBu) with potassium hydroxide in methanol. All the complexes were characterized by elemental analysis, Fourier transform infrared and nuclear magnetic resonance (¹H, ¹³C, ¹¹⁹Sn) spectroscopies, X-ray crystallography and thermogravimetric analyses. The crystal structures show that 1 has 2D network structure in which 2,2′-thiodiglycolic acid acts as a tetradentate ligand coordinating to the trimethyltin(IV) ions. Complexes 2, 3, and 4 are 3D metal-organic framework structures in which the deprotoned acids act as a tetradentate ligand afforded by four oxygen atoms.     

Syntheses and Characterization of Two New M(II)-5,5′-dimethyl-2,2′-bipyridine (M = Cd and Pb) Coordination Polymers and Study of Their Thermal and Electrochemical Properties

Two new cadmium(II) and lead(II)-thiocyanato coordination polymers with 5,5′-dimethyl-2,2′-bipyridine (5,5′-dm-2,2′-bpy) as chelating ligands were synthesized and characterized by elemental analysis, IR and ¹H NMR spectroscopy and by X-ray crystallography. Thermal and electrochemical properties were also studied as well. These complexes have formed formula [Cd(5,5′-dm-2,2′-bpy)(NCS)₂]_n (1) and [Pb(5,5′-dm-2,2′-bpy)(CH₃COO)(NCS)]_n (2). The coordination numbers of Cd^II in 1 and Pb^II in 2 are six (CdN₄S₂) and seven (PbN₃O₃S₂), respectively. In 2, “stereo-chemically active” electron lone pairs and the coordination spheres were hemidirected. Bridging properties of thiocyanato anions in 1 and 2 created one- and two-dimensional coordination polymers, respectively. The supramolecular features in these complexes were guided and controlled by weak directional intermolecular interactions.     

Do Enemies of Herbivores Influence Plant Growth and Chemistry? Evidence from a Seminatural Experiment

It is predicted that enemies of insect herbivores may influence the effects of herbivores on their host plants by affecting the choice of plant genotypes. To examine the effect of predators, we conducted two experiments, each with a different caterpillar species (Junonia coenia and Pyrrharctia isabella). Under seminatural conditions, we provided a choice between two genotypes of plantain (Plantago lanceolata) with different levels of iridoid glycosides and used Podisus maculiventris stinkbugs as predators. There were four treatments: no herbivores and no predators, low density of herbivores and no predators, high density of herbivores and no predators, and high density of herbivores plus predators. The caterpillars had little effect on plant growth but did influence the iridoid glycoside concentration. For the Junonia experiment, the concentration of iridoid glycosides was less for plots with a low density of caterpillars (with no predators) compared to the other treatments of caterpillar density. In the Pyrrharctia experiment, catalpol was induced by a high density of caterpillars (with no predators). There were no increases in total iridoid glycosides associated with either herbivore species. The presence of predators had no effect on plant growth or total iridoid glycoside pattern. The lack of effect by predators seems to reflect the relatively large variation in iridoid glycoside concentration among leaf ages, and the herbivores ability to respond to that variation, such that the difference in iridoid glycoside concentrations in the plant genotypes was less important.     

Design and Characterization of Chiral and Thermally Stable Nanostructure Poly(amide-imide)s Containing Different Trimellitylimido-Amino Acid-Based Diacids and 4,4′-Methylenebis(3-chloro-2,6-diethylaniline) Units

In this study new chiral nanostructure poly(amide-imide)s (PAI)s were synthesized via direct polycondensation of different trimellitylimido-amino acid-based diacids and 4,4′-methylenebis(3-chloro-2,6-diethylaniline), using tetra-n-butylammonium bromide and triphenyl phosphite as a green media. The formation of these nanostructure PAIs was confirmed by ¹H-NMR, Fourier transform infrared spectroscopy, specific rotation, elemental analysis, X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and thermo gravimetric analysis techniques. The FE-SEM micrographs and XRD patterns showed that, the obtained PAIs are nanostructured with different shapes and noncrystalline polymers.     

Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library

A mangrove soil metagenomic library was constructed and a β-agarase gene designated as AgaML was isolated by functional screening. The gene encoded for a 659-amino-acids polypeptide with an estimated molecular mass of 71.6 kDa. The deduced polypeptide sequences of AgaML showed the highest identity of 73% with the glycoside hydrolase family 16 β-agarase from Microbulbifer agarilyticus in the GenBank database. AgaML was cloned and highly expressed in Escherichia coli BL21(DE3). The purified recombinant protein, AgaML, showed optimal activity at 50 °C and pH 7.0. The kinetic parameters of K_m and V_max values toward agarose were 4.6 mg·mL⁻¹ and 967.5 μM·min⁻¹·mg⁻¹, respectively. AgaML hydrolyzed the β-1,4-glycosidic linkages of agar to generate neoagarotetraose (NA4) and neoagarohexaose (NA6) as the main products. These characteristics suggest that AgaML has potential application in cosmetic, pharmaceuticals and food industries.     

Synthesis and Polymerization of Bismaleimide Derived from 5(6)-amino-1(4′-aminophenyl)-1,3,3′-trimethyl indane

The acid-catalyzed dimerization of α-methyl styrene led to the formation of trimethyl phenyl indane, which on nitration followed by reduction using hydrazine hydrate gave 5(6)-amino-1(4′-aminophenyl)-1,3,3′-trimethyl indane. This aromatic diamine was used to synthesize bismaleamic acid and imidized to yield bismaleimide. The bismaleamic acid was converted to prepolymer directly by imidizing it in refluxing toluene. All the materials synthesized were characterized using FTIR, ¹H and ¹³C NMR. The direct inlet mass spectral characterizations were carried out for bismaleamic acid, bismaleimide and bismaleimide prepolymer. The fragmentation pattern was discussed in detail and the structure proposed was confirmed. The thermogravimetric studies were done for all the materials and kinetic parameters (energy of activation and frequency factor) were calculated using Dharwadkar and Kharkhanavala method. The structural changes occurring in the thermally polymerized bismaleimide and bismaleimide prepolymer were discussed.     

Viscosity behavior and chain conformation of a (1→3)-α-glucan from Ganoderma lucidum

Summary Seven fractions of a (1→3)-α-D-glucan from Ganoderma lucidum have been studied by light scattering, sedimentation equilibrium, and viscometry in dimethylsulfoxide (DMSO) containing 0.25 M lithium chloride at 25°C. The intrinsic viscosity [η] - molecular weight relation for this glucan in the mixed solvent is found to be represented approximately by [η] = 0.071 M _w ^0.60 cm³ g⁻¹ in the range of weight-average molecular weight M _w studied, i.e., from 8 × 10³ to 4.4 × 10⁵. Its analysis based on current theories for wormlike chains shows that, without excluded volume effect, the (1→3)-α-D-glucan chain is characterized by a linear mass density of 380 nm⁻¹, a Kuhn segment length of about 3 nm, and a diameter of 1.2 nm and is somewhat more extended but more flexible than amylose, a (1→4)-α-glucan, in DMSO. Received: 28 July 1998/Accepted: 18 August 1998     

Characterization of a soldering system consisting of a glass from the system CaOAl2O3B2O3SiO2 and wollastonite

In this work, the characterization of a soldering system with wollastonite as filler added as powder to the powdered base glass is shown. A soldering system consisting of the base glass and a coexisting crystalline phase is created which is then pressed to pellets and sintered to maximum density.Wollastonite increases the thermal expansion coefficient of the base glass after sintering the samples. In the present paper, the sintering behavior of the soldering system and possible interactions between glass and filler are analyzed. Also, density and porosity after sintering and the resulting crystalline phases are characterized.The mixtures with wollastonite contents up to 30% showed suitable sintering behavior and good density (low porosity). From 40% wollastonite on, the sintering of the samples was not satisfying and porosity was still high after sintering. The main crystalline phases were anorthite, wollastonite and pseudowollastonite, which occurred in all samples. This shows that all three phases are coexisting phases of the glass composition.     

Crystallisation of Wild-Type and Variant Forms of a Recombinant Plant Enzyme ��-d-Glucan Glucohydrolase from Barley (Hordeum vulgare L.) and Preliminary X-ray Analysis

Wild-type and variant crystals of a recombinant enzyme β-d-glucan glucohydrolase from barley (Hordeum vulgare L.) were obtained by macroseeding and cross-seeding with microcrystals obtained from native plant protein. Crystals grew to dimensions of up to 500 × 250 × 375 μm at 277 K in the hanging-drops by vapour-diffusion. Further, the conditions are described that yielded the wild-type crystals with dimensions of 80 × 40 × 60 μm by self-nucleation vapour-diffusion in sitting-drops at 281 K. The wild-type and recombinant crystals prepared by seeding techniques achived full size within 5–14 days, while the wild-type crystals grown by self-nucleation appeared after 30 days and reached their maximum size after another two months. Both the wild-type and recombinant variant crystals, the latter altered in the key catalytic and substrate-binding residues Glu220, Trp434 and Arg158/Glu161 belonged to the P4₃2₁2 tetragonal space group, i.e., the space group of the native microcrystals was retained in the newly grown recombinant crystals. The crystals diffracted beyond 1.57–1.95 Å and the cell dimensions were between a = b = 99.2–100.8 Å and c = 183.2–183.6 Å. With one molecule in the asymmetric unit, the calculated Matthews coefficients were between 3.4–3.5 ų·Da⁻¹ and the solvent contents varied between 63.4% and 64.5%. The macroseeding and cross-seeding techniques are advantageous, where a limited amount of variant proteins precludes screening of crystallisation conditions, or where variant proteins could not be crystallized.     

Synthesis and properties of poly(ester imide) copolymers from 3,3′,4,4′-biphenyltetracarboxylic dianhydride, 2,2′-bis(trifluoromethyl)benzidine,and 4-aminophenyl-4′-aminobenzoate

A series of poly(ester imide) (PEsI) copolymers were synthesized using 3,3′,4,4′-biphenyltetracarboxylic dianhydride (4,4′-BPDA), 2,2′-bis(trifluoromethyl)benzidine (TFMB), and 4-aminophenyl-4′-aminobenzoate (APAB) as the monomers. Wide-angle x-ray diffraction results revealed that the average interchain distances of these polymers ranged from 4.6 to 5.7 Å, increasing with the increase of TFMB contents. PEsI-0.3 and PEsI-0.4 exhibited a glass transition temperature (T_g) of 445 and 455°C, respectively, while no distinctive T_g was observed for the PEsI copolymers when the APAB content was >50 mol%. The coefficients of thermal expansion (CTE) of these PEsI copolymers ranged from 3.8 to 24.2 ppm K⁻¹, increasing with the increase of TFMB contents. The PEsI copolymers exhibited a modulus of 5.7–7.8 GPa, a tensile strength of 282–332 MPa, and an elongation-at-break of 10.2%–23.3%. Furthermore, these copolymers exhibited a dielectric constant of 2.53–2.76, and a low dissipation factor (D_f) of 0.0026–0.0032 at 10 GHz in dry state. Because of their excellent combined properties, these PEsI copolymers are promising candidates as dielectric substrate materials for the applications in next generation flexible printed circuit boards operating at high frequencies.     

Purification and Characterization of a Ginsenoside Rb1-Hydrolyzing ��-Glucosidase from Aspergillus niger KCCM 11239

Rb₁-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 was studied to develop a bioconversion process for minor ginsenosides. The specific activity of the purified enzyme was 46.5 times greater than that of the crude enzyme. The molecular weight of the native enzyme was estimated to be approximately 123 kDa. The optimal pH of the purified enzyme was pH 4.0, and the enzyme proved highly stable over a pH range of 5.0–10.0. The optimal temperature was 70 °C, and the enzyme became unstable at temperatures above 60 °C. The enzyme was inhibited by Cu²⁺, Mg²⁺, Co²⁺, and acetic acid (10 mM). In the specificity tests, the enzyme was found to be active against ginsenoside Rb₁, but showed very low levels of activity against Rb₂, Rc, Rd, Re, and Rg₁. The enzyme hydrolyzed the 20-C,β-(1→6)-glucoside of ginsenoside Rb₁ to generate ginsenoside Rd and Rg₃, and hydrolyzed 3-C,β-(1→2)-glucoside to generate F₂. The properties of the enzyme indicate that it could be a useful tool in biotransformation applications in the ginseng industry, as well as in the development of novel drug compounds.     

Characterization of trans-monounsaturated alkenyl chains in total plasmalogens (1-O-alk-1′-enyl-2-acyl glycerophospholipids) from sheep heart

In the present study, we investigated the alkenyl chains from sheep heart plasmalogens (1-O-alk-1′-enyl-2-acyl glycerophospholipids) after their conversion into trimethylene dioxyalkanyl (TMDOA) derivatives. Particular attention was given to monounsaturated alkenyl chains (C₁₈ mainly). For this purpose, a combination of silver ion TLC and GLC on highly polar, very long capillary columns was applied to TMDOA derivatives. Approximately 30 different alkenyl chains could be separated, and the main observation was that the component previously reported as a cis-9 18∶1 alkenyl chain in plasmalogens embraces in fact a wide range of trans and cis isomers, in amounts equal to 7.9 and 5.6%, respectively, of total alkenyl chains. Concerning the trans-monoenoate fraction, isomers with their ethylenic bond spanning from Δ6–Δ8 to Δ16 were tentatively identified on the basis of their distribution profile, which was similar to that of trans-18∶1 acids prepared and isolated from sheep adipose tissue. The main trans-monoenoic C₁₈ alkenyl chain in sheep heart plasmalogens would thus have its double bond in position 11, which seems logical, as alkenyl chains are derived from the corresponding alcohols, themselves issued from the corresponding FA, and in this particular case, vaccenic (trans-11 18∶1) acid. cis-Monoenoic C₁₈ alkenyl chains also appear more complex than realized earlier, showing in particular isomers with their ethylenic bond farther than the Δ9 position, in addition to the main isomer derived from oleic acid. Several trans-16∶1 alkenyl chains could be observed (totaling ca. 1%), but cis-16∶1 isomers were present in trace amounts only.     

Morphological and Thermal Properties of Poly(amide-imide)/ZnO Nanocomposites Derived from 4,4′-methylenebis(3-chloro-2,6-diethyl trimellitimidobenzene) and 3,5-diamino-N-(4-hydroxyphenyl)benzamide

A new nanostructure poly(amide-imide) (PAI) was prepared from the polymerization of 4,4′-methylenebis(3-chloro-2,6-diethyl trimellitimidobenzene) as a diacid with 3,5-diamino-N-(4-hydroxyphenyl)benzamide using triphenyl phosphite as a condensing agent and tetra-n-butylammonium bromide as a green media. The synthesized polymer was used to prepare PAI/ZnO nanocomposites (PZNC)s using nano-ZnO surface-coupled by N-trimellitylimido-L-alanine diacid as a coupling agent through ultrasonic process. The resulting PZNCs were also characterized by FT-IR, powder X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) and thermogravimetric analysis (TGA). The TEM and FE-SEM results showed a good dispersion of nanoscale inorganic particles in the polymer matrix.     

Synthesis, Characterization and Optimum Reaction Conditions of Oligo-N, N′-bis (2-hydroxy-1-naphthalidene) Thiosemicarbazone

The oxidative polycondenzation reaction conditions of N, N-bis (2-hydroxy-1-naphthalidene) thiosemicarbazone (HNTSC) using air oxygen, H₂O₂ and NaOCl were studied in an aqueous alkaline medium between 50–90°C. Oligo-N, N-bis (2-hydroxy-1-naphthalidene) thiosemicarbazone was characterized by ¹H-NMR, FT-IR, UV-Vis, size exclusion chromatography (SEC) and elemental analysis techniques. Solubility testing of oligomer was investigated using organic solvents such as DMF, THF, DMSO, methanol, ethanol, CHCl₃, CCl₄, toluene acetonitrile, ethyl acetate, concentrated H₂SO₄ and an aqueous alkaline solution. Using NaOCl, H₂O₂ and air O₂ oxidants, conversion to oligo-N, N-bis (2-hydroxy-1-naphthalidene) thiosemicarbazone (OHNTSC) of N, N-bis (2-hydroxy-1-naphthalidene) thiosemicarbazone was found to be 85, 80 and 76%, respectively, in an aqueous alkaline medium. According to the SEC analyses, the number-average molecular weight, weight-average molecular weight and polydispersity index values of OHNTSC synthesized were found to be 1050 gmol^–1 1715 gmol^–1 and 1.63, using NaOCl, and 2137, 2957 gmol^–1 and 1.38, using air O₂ and 2155 gmol^–1 4164 gmol^–1 and 1.93, using air H₂O₂, respectively. Also, TG analysis was shown to be unstable of oligo-N, N-bis (2-hydroxy-1-naphthalidene) thiosemicarbazone against thermo-oxidative decomposition. The weight loss of OHNTSC was found to be 97.29% at 900°C.     

Synthesis and Characterization of Tripodal Oxy-Schiff base (2,4,6-Tris(4-Carboxymethylenephenylimino-4′-formylphenoxy)-1,3,5-triazine) and the Thermal and Magnetic Properties of its Fe(III)/Cr(III) Complexes

Eight new trinuclear Fe(III)/Cr(III) complexes involving tetradentate (N₂O₂) and pentadentate (N₃O₂) Schiff bases of (salenH₂), (salophenH₂), (saldetaH₂) and (salpyrH₂) with 2,4,6-tris(4-carboxymethylenephenylimino-4′-formylphenoxy)-1,3,5-triazine were synthesized. The structure of ligand and complexes were identified using elemental and thermal analysis, magnetic susceptibility, and LC–MS, ICP-AES, ¹H NMR and FT-IR spectral data. All complexes are tripodal–trinuclear. The complexes are low-spin (S = 1/2) distorted octahedral salen- or salophen-capped Fe(III), high-spin (S = 5/2) distorted octahedral saldeta- or salpyr-capped Fe(III) and distorted octahedral (S = 3/2) salen-, salophen-, saldeta- or salpyr-capped Cr(III).     

A 3D Mesomeric Supramolecular Structure of a Cu(II) Coordination Polymer with 1,1′-Biphenyl-2,2′,3,3′-tetracarboxylic Acid and 5,5′-Dimethyl-2,2′-bipyridine Ligands

A novel metal–organic framework {[Cu(H₂bptc)(cbpy)(H₂O)]·(H₂O)} _n (1) has been hydrothermally synthesized through reaction of 1,1′-biphenyl-2,2′,3,3′-tetracarboxylic acid (H₄bptc) with Cu(II) salt in the presence of ancillary nitrogen ligand 5,5′-dimethyl-2,2′-bipyridine (cbpy), and its structure was determined by X-ray diffraction and characterized by elemental analysis, and IR spectrum. The title compound crystallizes in monoclinic space group P2₁/c. Both right- and left-handed helices are detected in the structure. In the ab-plane, adjacent chains are homochiral and parallel to each other, which are connected together by hydrogen bonds to form a 2D supramolecular structure. The supramolecular layers with opposite chirality are arranged alternatively along the c-axis to form a 3D mesomeric supramolecular structure through π···π interactions. The thermal stability of the complex 1 was studied by thermal gravimetric and differential thermal analysis.     

Characterization of a κ-Carrageenase from Marine Cellulophaga lytica strain N5-2 and Analysis of Its Degradation Products

A carrageenan-degrading marine Cellulophaga lytica strain N5-2 was isolated from the sediment of carrageenan production base. A κ-carrageenase (EC 3.2.1.83) with high activity was purified to electrophoretic homogeneity from the culture supernatant by a procedure of ammonium sulfate precipitation, dialyzing and gel filtration on SephadexG-200 and SephadexG-75. The purified enzyme was verified as a single protein on SDS-PAGE, and whose molecular weight was 40.8 kDa. The κ-carrageenase yielded a high activity of 1170 U/mg protein. For κ-carrageenase activity, the optimum temperature and pH were 35 °C and pH 7.0, respectively. The enzyme was stable at 40 °C for at least 2.5 h. The enzyme against κ-carrageenan gave a K_m value of 1.647 mg/mL and a V_max value of 8.7 μmol/min/mg when the reaction was carried out at 35 °C and pH 7.0. The degradation products of the κ-carrageenase were analyzed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), electrospray ionization time-of-flight mass spectroscopy (ESI-TOF-MS) and ¹³C-NMR spectroscopy, and the results indicated that the enzyme was specific of the β-1,4 linkage and hydrolyzed κ-carrageenan into κ-neocarraoctaose-sulfate and κ-neocarrahexaose-sulfate first, and then broke κ-neocarraoctaose-sulfate into κ-neocarrabiose-sulfate and κ-neocarrahexaose-sulfate.     

Hydrothermal Synthesis of Cd(II), Mn(II) and Ni(II) Coordination Polymers Derived from Benzopenone-2,4′-carboxylate and N-door Ligands

Three new coordination polymers [Cd(L)(4,4′-bipy)_0.5]_n (1), {[Mn(L)(bpp)]·H₂O}_n (2) and [Ni(L)(2,2′-bipy)(H₂O)₂]_n(3) [H₂L = Benzopenone-2,4′-dicarboxylic acid, 4,4′-bipy = 4,4′-bipy ridine, bpp = 1,3-di(4-pyridyl) propane and 2,2′-bipy = 2,2′-bipyridine] have been synthesized and structurally characterized by elemental analysis, IR and X-ray diffraction. Single-crystal X-ray analyses revealed that the H₂L ligand acts as a bridge, exhibiting three coordination modes to link metal ion: bidentate chelating, bis-monodentate, monodentate. Compound 1 has 3-connected metal–organic framework with the (6³) topology notation; Compounds 2 and 3 are one-dimensional chain structures. The luminescent properties for compound 1 was investigated.