Functional Properties of Rice Bran Proteins Extracted from Low-Heat-Treated Defatted Rice Bran.

Rice bran is rich in proteins with high nutritional values. However, current protein extraction methods from rice bran are greatly limited by their low yield. Therefore, in this study, we aimed to develop a feasible method to extract rice bran protein (RBP) of high purity and quality. We prepared RBP using low-heat-treated defatted rice bran (LDRB) and analyzed its functional properties. The protein solubility of LDRB increased from 25.4% to 56% upon increasing the pH level and was more than double that of heat-stabilized defatted rice bran. RBP prepared from LDRB had good functional properties, comparable to those of soy proteins. The emulsifying capacities of RBP were 424 ± 14 mL/g at pH 4 and 530 ± 21 mL/g at pH 7.0. Under acidic conditions, RBP showed a better emulsifying capacity than soy proteins (262 ± 1 mL/g at pH 4). RPB showed water-binding and oil-absorption capacities of 270 ± 35 g/100 g and 268 ± 30 g/100 g, respectively. Moreover, RBP showed better foaming capacity (610% vs. 590%) and foam stability (83% vs. 4%) than soy proteins; however, it lacked gelling properties. This study demonstrated that RBP is a potential new protein source in the food industry.

Retained binding mode of various DNA-binding molecules under molecular crowding condition.

Meso-tetrakis(N-methyl pyridinium-4-yl)porphyrin (TMPyP) intercalates between the base-pairs of DNA at a low [TMPyP]/[DNA base] ratio in aqueous solutions and molecular crowding conditions, which is induced by the addition of Poly(ethylene glycol) (PEG). Studied DNA-binding drugs, including TMPyP, 9-aminoacridine, ethidium bromide, and DAPI (4',6-diamidino-2-phenylindole) showed similar binding properties in the presence or absence of PEG molecules which is examined by circular and linear dichroism. According to the LDr (reduced linear dichroism) results of the binding drugs examined in this work, PEG molecules induced no significant change compared to their binding properties in aqueous buffering systems. These results suggest that the transition moments are not expected to be perturbed significantly by PEG molecules. In this study, the experimental conditions of PEG 8000 were maintained at 35% (v/v) of total reaction volume, which is equal to the optimal molar concentration (0.0536 M as final concentration for PEG 8000) to maintain suitable cell-like conditions. Therefore, there was no need to focus on the conformational changes of the DNA helical structure, such as forming irregular aggregate structures, induced by large quantities of molecular crowding media itself at this stage.

Enantioselective light switch effect of Δ- and Λ-[Ru(phenanthroline)2 dipyrido[3,2-a:2', 3'-c]phenazine]2+ bound to G-quadruplex DNA.

The interaction of Δ- and Λ-[Ru(phen)2DPPZ]2+ (DPPZ = dipyrido[3,2-a:2', 3'-c]phenazine, phen = phenanthroline) with a G-quadruplex formed from 5'-G2T2G2TGTG2T2G2-3'(15-mer) was investigated. The well-known enhancement of luminescence intensity (the 'light-switch' effect) was observed for the [Ru(phen)2DPPZ]2+ complexes upon formation of an adduct with the G-quadruplex. The emission intensity of the G-quadruplex-bound Λ-isomer was 3-fold larger than that of the Δ-isomer when bound to the G-quadruplex, which is opposite of the result observed in the case of double stranded DNA (dsDNA); the light switch effect is larger for the dsDNA-bound Δ-isomer. In the job plot of the G-quadruplex with Δ- and Λ-[Ru(phen)2DPPZ]2+, a major inflection point for the two isomers was observed at x ≈ .65, which suggests a binding stoichiometry of 2:1 for both enantiomers. When the G base at the 8th position was replaced with 6-methyl isoxanthopterin (6MI), a fluorescent guanine analog, the excited energy of 6-MI transferred to bound Δ- or Λ-[Ru(phen)2DPPZ]2+, which suggests that at least a part of both Ru(II) enantiomers is close to or in contact with the diagonal loop of the G-quadruplex. A luminescence quenching experiment using [Fe(CN)6]4- for the G-quadruplex-bound Ru(II) complex revealed downward bending curves for both enantiomers in the Stern-Volmer plot, which suggests the presence of Ru(II) complexes that are both accessible and inaccessible to the quencher and may be related to the 2:1 binding stoichiometry.

Nutritional quality of rice bran protein in comparison to animal and vegetable protein.

Rice bran protein (RBP) was prepared by alkali extraction and isoelectric precipitation from defatted rice bran. The protein quality of RPB was evaluated and compared to two vegetable proteins [soy protein (ISP) and rice endosperm protein (REP)] and two animal proteins [whey protein (WPI) and casein]. RPB contained 74.93% of protein and its pepsin digestibility and KOH solubility were 89.8% and 91.5%, respectively. In Sprague-Dawley rats, RBP showed protein efficiency ratio, net protein ratio, net protein utilisation, and biological value of 2.39, 3.77, 70.7, and 72.6, which were comparable to the qualities of animal proteins. The true digestibility of RBP (94.8%) was significantly higher than that of REP (90.8%), ISP (91.7%) and WPI (92.8%) and the same as that of casein. Protein digestibility corrected amino acid score (PDCAAS) of RBP was 0.90. These results suggest that rice bran protein appears to be a promising protein source with good biological values and digestibility.

Conformational analysis of genotoxic benzo[a]pyrene-7,8-dione-duplex DNA adducts using a molecular dynamics method (II).

The conformations of the benzo[a]pyrene-7,8-quinone (BPQ) modified oligonucleotide were investigated using molecular dynamic simulation. In the initial structures, the central guanine base was modified with BPQ resulting in the formation of four structurally distinguishable 10-(N2-deoxyguanosyl)-9,10-dihydro-9-hydroxy benzo[a]pyrene-7,8-dione adducts (BPQ-G3,4). Each of the oligonucleotide adduct consisted of two conformers, namely syn and anti conformations, depending on the rotation around the glycosidic bond between BPQ and the guanine base. The results revealed that the BPQ moiety was located in the major groove for all four syn conformers. The relative energies of these conformers were high, and the backbone largely deviated from the B-form. On the other hand, BPQ was located in the minor groove with relatively low energies, and backbone was retained in all of the anti conformer cases. The most conceivable BPQ-modified double stranded oligonucleotide structure was proposed from the energy calculation and the structural analysis.

DNA-binding geometry dependent energy transfer from 4',6-diamidino-2-phenylindole to cationic porphyrins.

The circular and linear dichroism (CD and LD) spectral properties of the meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP)-DNA complex at a [porphyrin]/[DNA] ratio below 0.015 showed that TMPyP intercalates between DNA base pairs. Contrarily, when cis-bis(N-methylpyridinium-4-yl)porphyrin (BMPyP) is associated with DNA, no CD spectrum was induced and a bisignate LD spectrum was observed. These spectral properties of both the TMPyP and BMPyP were essentially retained when the minor groove of the DNA was saturated with 4',6-diamidino-2-phenylindole (DAPI). The fluorescence of the DNA-bound DAPI was effectively quenched by BMPyP and TMPyP. The quenching by BMPyP can be described through a pure static mechanism while TMPyP quenching produced an upward bending curve in the Stern-Volmer plot. Quenching efficiency was by far greater than predicted by the "sphere of action model", suggesting that the DNA provides some additional processes for an effective energy transfer.

Effect of the position and number of positive charges on the intercalation and stacking of porphyrin to poly[d(G-C)2], poly[d(A-T)2], and native DNA.

The effect of the number and position of the positive charges on porphyrin with respect to the mode of binding to poly[d(G-C)2] and poly[d(A-T)2] were investigated by absorption and polarized spectroscopy, including circular and linear dichroism (CD and LD). Meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP), which possesses four positive charges on the periphery pyridinium rings, produces a negative CD and wavelength-independent reduced LD (LDr) spectra in the Soret absorption region when it associates with poly[d(G-C)2]. These spectral characteristics have been considered as diagnostic for intercalation. In contrast, both trans- and cis-bis(N-methylpyridinium-4-yl)diphenylporphyrin (trans- and cis-BMPyP), where the number of positive charges was reduced to two, multisignate CD and strong wavelength-dependence of the LDr spectra were observed, indicating that these porphyrins do not intercalate. Therefore, four positive charges are required for TMPyP intercalation. When associated with poly[d(A-T)2], trans-BMPyP exhibited a positive CD signal at a low [porphyrin]/[nucleobase] ratio with the appearance of a bisignate CD upon increase of the mixing ratio, suggestive of binding at the groove of the double helix at low mixing ratios, and stacking at increasing mixing ratios. Conversely, no monomeric binding was evident in the bis-BMPyP bisignate CD spectrum; hence, only the stacking mode was found for cis-BMPyP, even at the lowest [porphyrin]/[nucleobase] ratio, suggesting the importance of the position of the positive charges in determining monomeric groove binding or stacking. The binding geometries of trans- and cis-BMPyP were similar when associated with poly[d(A-T)2], as determined from the similar LDr spectrum. When associated with DNA, TMPyP exhibited similar spectral properties with that of the TMPyP-poly[d(G-C)2] complex, indicating intercalation of TMPyP between the DNA base pairs. Conversely, CD and LDr characteristics of both trans- and cis-BMPyP-DNA complexes resembled those that complexed with poly[d(A-T)2] at a high [porphyrin]/[DNA] ratio, suggesting that both porphyrins were stacked along the DNA stem.

Amine terminated G-6 PAMAM dendrimer and its interaction with DNA probed by Hoechst 33258.

Fluorescence characteristics of Hoechst 33258 bound to G-6 dendrimer, to the DNA-G-6 dendrimer complex, and to DNA were compared with that in an aqueous solution. The spectral properties including fluorescence emission spectrum, accessibility of anionic quencher, as well as the fluorescence decay time of the Hoechst 33258 are different for all three conditions, indicating that the environments in these conditions are different. Close analysis of the fluorescence properties led us to suggest that Hoechst 33258 located at or near the contact area of the dendrimer and DNA in the DNA-G-6 complex. In the complex, in the absence of Hoechst 33258, the shape of the circular dichroism in the DNA absorption region remained, indicating that DNA is in B form in the complex. On the other hand, the magnitude of linear dichroism (LD) decreased upon DNA-G-6 dendrimer complex formation. The decrease in LD magnitude reflects the shortening of the DNA contour length, which is expected from the fact that a large part of linear DNA is required to wrap the surface of G-6 dendrimer.

Dispersion of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin on [d(A-T)n]2 oligonucleotides.

The binding modes of the free-base meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) complexed with [d(AT)n]2 oligonucleotides (where n=3-8, corresponding to 6 to 16 AT base pairs) were studied by circular dichroism (CD). When associated with the shortest oligonucleotide, ([d(AT)3]2), a bisignate CD spectrum was produced in the Soret absorption region at the mixing ratio between 2.0 and 0.25, corresponding to one TMPyP per 0.5 to 4 oligonucleotides. Apparent bisignate CD was attributed to a stacked TMPyP along the DNA. On the other hand, when the oligonucleotide length reaches one helical turn or longer, ([d(AT)n]2, n=6,7,8), TMPyP exhibited a positive CD signal, that corresponds to the monomeric groove binding mode, at the mixing ratio below 1.0 (one TMPyP per oligonucleotide). As the mixing ratio increases, the CD signal was best accounted for by the sum of the stacked and groove-binding TMPyP. At the intermediate oligonucleotide length ([d(AT)n]2, n=4,5), the CD spectrum appeared to be the sum of the stacked and groove binding TMPyP at all mixing ratios. Therefore, it is conclusive that the full dispersion of TMPyP requires at least one helical turn of the AT sequence at a mixing ratio below 1.0. Further increase of the mixing ratio resulted in the stacking of TMPyP even at the long oligonucleotides.

Binding of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin to AT oligomers: effect of chain length and the location of the porphyrin stacking.

Circular dichroism (CD) spectra of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) that are associated with various duplex and triplex AT oligomers were investigated in this study. A strong positive CD was apparent for both the TMPyP complexed with duplex d[(A-T)(12)](2), d(A)(12).d(T)(12) and triplex d(A)(12).d[(T)(12)](2) at a low mixing ratio. As the mixing ratio increased, bisignate excitonic CD was produced for TMPyP complexed with duplexes, whereas the positive CD signal remained the same for the TMPyP-d(A)(12).d[(T)(12)](2) complex. This difference in the CD spectrum in the presence of duplex and triplex oligomers indicates that the moderate stacking of TMPyP occurs at the major groove of the duplex and the monomeric binding occurs in (or near) the minor groove. When TMPyP forms a complex with duplex d[(A-T)(6)](2) only excitonic CD was observed, even at a very low mixing ratio. Therefore, at least seven or more basepairs are required for TMPyP to exhibit a monomeric CD spectrum. After close analysis of the CD spectrum, the TMPyP-poly[d(A-T)(2)] complex could be explained by a combination of the CD spectrum of the monomeric, moderately stacked, and extensively stacked TMPyP.