Physicochemical and biological properties of 2-O-α-d-galactosyl-cyclomaltohexaose (α-cyclodexterin) and -cyclomaltoheptaose (β-cyclodextrin)

The physicochemical and biological properties of the new branched cyclomaltooligosaccharides (cyclodextrins; CDs), 2-O-α-d-galactosyl-cyclomaltohexaose (2-O-α-d-galactosyl-α-cyclodextrin, 2-Gal-αCD) and 2-O-α-d-galactosyl-cyclomaltoheptaose (2-O-α-d-galactosyl-β-cyclodextrin, 2-Gal-βCD), were investigated. The formation of inclusion complexes of 2-Gal-CDs with various kinds of guest compounds (clofibrate, cholesterol, cholecalciferol, digitoxin, digitoxigenin, and prostaglandin A₁) was examined by a solubility method, and the results were compared with those of non-branched CDs and other 6-O-glycosyl-CDs such as 6-O-α-d-galactosyl-CDs, 6-O-α-d-glucosyl-CDs, and 6-O-α-maltosyl-CDs. The inclusion abilities of 2-Gal-αCD for clofibrate and prostaglandin A₁, and 2-Gal-βCD for clofibrate, cholecalciferol, cholesterol, and digitoxigenin were markedly weaker than those of non-branched CD and other 6-O-glycosyl-CDs in each series, probably because of a steric hindrance caused by the α-(1→2)-galactoside linkage. The hemolytic activities of 2-Gal-CDs on human erythrocytes were the lowest among each CD series, and the compounds showed negligible cytotoxicity towards Caco-2 cells up to at least 100 mM.     

Extraction of cellulose-synthesizing activity of Gluconacetobacter xylinus by alkylmaltoside

This study reinvestigated the synthesis of cellulose in vitro with a well-known cellulose-producing bacterium, Gluconacetobacter xylinus. Alkylmaltoside detergents, which are more frequently used in recent structural biological researches, are uniquely used in this study to solubilize cellulose-synthesizing activity from the cell membrane of G. xylinus. Activity comparable to that previously reported is obtained, while the synthesized cellulose is crystallized into a non-native polymorph of cellulose (cellulose II) as well as the previous studies. In spite of this failure to recover the native activity to synthesize cellulose I microfibril in vitro, the product is a polymer with a degree of polymerization greater than 45 as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was thus concluded that the established protocol can solubilize cellulose-synthesizing activity of G. xylinus with polymerizing activity.     

Autecology, distributional expansion and negative effects of Amorpha fruticosa L. on a river ecosystem: a case study in the Sendaigawa River, Tottori Prefecture

Amorpha fruticosa L., introduced from the eastern part of North and Central America as a revegetation material for artificial slopes, has escaped into riverbeds in Japan, and its negative effects are of concern. In this study, an attempt has been made to clarify the actual extent of escape and establishment of A. fruticosa, through a case study of the Sendaigawa River, Tottori Prefecture, coupled with a survey of national distribution using the National Censuses on River Environments. The autecology of A. fruticosa, including phenology, germination, allelopathy and coppicing ability, was also studied by observation and experiments. The following conclusions were drawn: A. fruticosa preferred sunny sites; the total established area in the study site was 7,500 m²; A. fruticosa showed strong coppicing ability; there was lower species diversity in and around areas populated by A. fruticosa; and A. fruticosa has been expanding its area both in the watershed of Sendaigawa River region and nationwide. The authors emphasize the importance of quick control of A. fruticosa.     

Homologous Pairing Activities of Two Rice RAD51 Proteins,RAD51A1 and RAD51A2

In higher eukaryotes, RAD51 functions as an essential protein in homologous recombination and recombinational repair of DNA double strand breaks. During these processes, RAD51 catalyzes homologous pairing between single-stranded DNA and double-stranded DNA. Japonica cultivars of rice (Oryza sativa) encode two RAD51 proteins, RAD51A1 and RAD51A2, whereas only one RAD51 exists in yeast and mammals. However, the functional differences between RAD51A1 and RAD51A2 have not been elucidated, because their biochemical properties have not been characterized. In the present study, we purified RAD51A1 and RAD51A2, and found that RAD51A2 robustly promotes homologous pairing in vitro. RAD51A1 also possesses homologous-pairing activity, but it is only about 10% of the RAD51A2 activity. Both RAD51A1 and RAD51A2 bind to ssDNA and dsDNA, and their DNA binding strictly requires ATP, which modulates the polymer formation activities of RAD51A1 and RAD51A2. These findings suggest that although both RAD51A1 and RAD51A2 have the potential to catalyze homologous pairing, RAD51A2 may be the major recombinase in rice.     

Cellular Localization of t-Cinnamic Acid 4-Hydroxylase Produced in Sweet Potato in Response to Cut Injury

The acid-catalyzed rearrangement of 3-alkyl-3-phenylglycidic esters with ethyl, propyl or isopropyl substituents at 3-position (Ib, Ic and Id) gave substituted 2-hydroxy-3-butenoic esters (IIb, IIe and IId) and substituted pyruvic esters (IIIb, IIIc and IIId). The mechanism of their formation is thought to be the ring cleavage of the oxirane followed by competition of proton elimination at 4-position with hydride shift at 2-position. Prolonged treatment of IId gave a dienoic ester (IV) resulting from dehydration, whereas the same treatment of IIb and IIc gave the unchanged starting materials.     

Thiol modification by bioactivated polyphenols and its potential role in skin inflammation

In the present study, we evaluated the modifying behavior of simple phenolic compounds on the sulfhydryl groups of glutathione and proteins. The catechol-type polyphenols, including protocatechuic acid, but neither the monophenols nor O-methylated catechol, can modify the sulfhydryl groups in a phenol oxidase-dependent manner. The possible involvement of polyphenol bioactivation in the enhancement of skin inflammation was also suggested.     

Chemical Structure of Mating Pheromone Peptides, αsk1 and αsk3 Pheromones,Produced by Mating Type α Cells of Saccharomyces kluyveri

Three peptides, α^sk1, α^sk2 and α^sk3 pheromones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, α^sk2 pheromone, having already been determined. The unknown N-terminus of α^sk1 pheromone was elucidated to be 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from α^sk1 pheromone, and the primary structure of α^sk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of α^sk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of α^sk2 pheromone.     

Chemical Modification of Sulfhydryl Groups in Porcine Pancreatic α-Amylase,and Purification and Properties of the Modified Amylase

The behavior of SH groups of porcine pancreatic α-amylase, called PPA II, was studied by chemical modification with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). Only two SH groups in PPA II reacted, in a pseudo-first-order reaction, and the modification was accompanied with the inactivation of the amylase. The reactivity of SH groups with DTNB was influenced by the ionic strength of the medium. The SH groups were protected against modification by the addition of some substrate analogs; maltopentaitol, maltotetraitol, maltotriitol and cyclomaltohexaose were effective analogs, whereas maltitol, d-glucitol and methyl α-d-glucoside did not protect these groups. The modified enzymes (M₁ and M₂), in which one and two SH groups reacted with DTNB, respectively, were purified in an electrophoretically homogeneous state by chromatography on Bio-Gel P-2 and TSK-Gel DEAE-Toyopearl 650S. The optimum pH of the modified enzyme (M₂) was 6.9~7.0, which was the same as that of the native PPA II. The isoelectric points of M₁ and M₂ were estimated to be 5.8 and 5.2, respectively, by the method of Catsimpoolas. The CD spectrum of PPA II was altered partially by the modification of SH groups with DTNB. Moreover, a precipitin line with a spur was observed in a double immunodiffusion test of PPA II and M₂ to rabbit antiserum of PPA II. It is concluded that the free SH group(s) in PPA II, located near the substrate binding site, don’t participate directly in its catalytic activity, but that the SH group(s) are involved in the antigenicity of PPA II.