Flavonoid glycosides from the aerial parts of Curcuma comosa

Four new flavonoid glycosides, curcucomosides A–D (1–4), three known flavonoid glycosides, 5–7, and four known diarylheptanoids, 8–11, were isolated from the ethanol extract of the aerial parts of Curcuma comosa. The structures of the new compounds were established as rhamnazin 3-O-α-l-arabinopyranoside (1), rhamnocitrin 3-O-α-l-arabinopyranoside (2), rhamnazin 3-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranoside (3), and rhamnocitrin 3-O-α-l-rhamnopyranosyl-(1→2)-O-α-l-arabinopyranoside (4) by spectroscopic analysis and chemical reactions whereas those of the known compounds were identified by spectral comparison with those of the reported values.     

Transglucosylation of tertiary alcohols using cassava beta-glucosidase

We have compared the ability of beta-glucosidases from cassava, Thai rosewood, and almond to synthesize alkyl glucosides by transglucosylating alkyl alcohols of chain length C(1)-C(8). Cassava linamarase shows greater ability to transfer glucose from p-nitrophenyl-beta-glucoside to secondary alcohol acceptors than other beta-glucosidases, and is unique in being able to synthesize C(4), C(5), and C(6) tertiary alkyl beta-glucosides with high yields of 94%, 82%, and 56%, respectively. Yields of alkyl glucosides could be optimized by selecting appropriate enzyme concentrations and incubation times. Cassava linamarase required pNP-glycosides as donors and could not use mono- or di-saccharides as sugar donors in alkyl glucoside synthesis.     

The C-terminal domain of 4-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii is an autoinhibitory domain

p-Hydroxyphenylacetate (HPA) 3-hydroxylase from Acinetobacter baumannii consists of a reductase component (C(1)) and an oxygenase component (C(2)). C(1) catalyzes the reduction of FMN by NADH to provide FMNH(-) as a substrate for C(2). The rate of reduction of flavin is enhanced ～20-fold by binding HPA. The N-terminal domain of C(1) is homologous to other flavin reductases, whereas the C-terminal domain (residues 192-315) is similar to MarR, a repressor protein involved in bacterial antibiotic resistance. In this study, three forms of truncated C(1) variants and single site mutation variants of residues Arg-21, Phe-216, Arg-217, Ile-246, and Arg-247 were constructed to investigate the role of the C-terminal domain in regulating C(1). In the absence of HPA, the C(1) variant in which residues 179-315 were removed (t178C(1)) was reduced by NADH and released FMNH(-) at the same rates as wild-type enzyme carries out these functions in the presence of HPA. In contrast, variants with residues 231-315 removed behaved similarly to the wild-type enzyme. Thus, residues 179-230 are involved in repressing the production of FMNH(-) in the absence of HPA. These results are consistent with the C-terminal domain in the wild-type enzyme being an autoinhibitory domain that upon binding the effector HPA undergoes conformational changes to allow faster flavin reduction and release. Most of the single site variants investigated had catalytic properties similar to those of the wild-type enzyme except for the F216A variant, which had a rate of reduction that was not stimulated by HPA. F216A could be involved with HPA binding or in the required conformational change for stimulation of flavin reduction by HPA.     

Kinetics of a two-component p-hydroxyphenylacetate hydroxylase explain how reduced flavin is transferred from the reductase to the oxygenase

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). HPAH is composed of two proteins: a flavin mononucleotide (FMN) reductase (C1) and an oxygenase (C2). C1 catalyzes the reduction of FMN by NADH to generate reduced FMN (FMNH-) for use by C2 in the hydroxylation reaction. C1 is unique among the flavin reductases in that the substrate HPA stimulates the rates of both the reduction of FMN and release of FMNH- from the enzyme. This study quantitatively shows the kinetics of how the C1-bound FMN can be reduced and released to be used efficiently as the substrate for the C2 reaction; additional FMN is not necessary. Reactions in which O2 is rapidly mixed with solutions containing C1-FMNH- and C2 are very similar to those in which solutions containing O2 are mixed with one containing the C2-FMNH- complex. This suggests that in a mixture of the two proteins FMNH- binds more tightly to C2 and has already been completely transferred to C2 before it reacts with oxygen. Rate constants for the transfer of FMNH- from C1 to C2 were found to be 0.35 and >or=74 s-1 in the absence and presence of HPA, respectively. The reduction of cytochrome c by FMNH- was also used to measure the dissociation rate of FMNH- from C1. In the absence of HPA, FMNH- dissociates from C1 at 0.35 s-1, while with HPA present it dissociates at 80 s-1; these are the same rates as those for the transfer from C1 to C2. Therefore, the dissociation of FMNH- from C1 is rate-limiting in the intermolecular transfer of FMNH- from C1 to C2, and this process is regulated by the presence of HPA. This regulation avoids the production of H2O2 in the absence of HPA. Our findings indicate that no protein-protein interactions between C1 and C2 are necessary for efficient transfer of FMNH- between the proteins; transfer can occur by a rapid-diffusion process, with the rate-limiting step being the release of FMNH- from C1.     

Detection of Microcystis in Lake Sediment using Molecular Genetic Techniques

Summary Microcystis, which are toxic microcystin-producing cyanobacteria, normally bloom in summer and drop in numbers during the winter season in Senba Lake, Japan. Recently, this lake has been treated by ultrasonic radiation and jet circulation which were integrated with flushing with river water. This treatment was most likely sufficient for the destruction of cyanobacterial gas vacuoles. In order to confirm whether Microcystis viridis was still present, a molecular genetic monitoring technique on the basis of DNA direct extraction from the sediment was applied. Three primer sets were used for polymerase chain reaction (PCR) based on rRNA intergenic spacer analysis (RISA), the DNA dependent RNA polymerase (rpoC1) and a Microcystis sp.-specific rpoC1 fragment. The results from each primer were demonstrated on the basis of single strand conformation polymorphisms (SSCP). Using the RISA primer showed different results from the rpoC1 and Microcystis sp.-specific rpoC1 fragment; meanwhile, the rpoC1 Microcystis sp.-specific fragment was more specific than the RISA primer. Therefore, the Microcystis sp.-specific rpoC1 fragment was further analysed by denaturing gradient gel electrophoresis (DGGE). The DNA pattern representing M. viridis could not be detected in any of the sediment samples. However, the results were confirmed with another technique, terminal restriction fragment length polymorphisms (T-RFLP). Although T-RFLP patterns of 16S rDNA in sediment at 91 bp and 477 bp lengths were matched with the T-RFLP of M. viridis (HhaI and MspI endonuclease digestion, respectively), the T-RFLP pattern of 75 bp length was not matched with M. viridis (both of HhaI and MspI endonuclease digestion) which were the major T-RFLP pattern of M. viridis. Therefore, the results most likely indicated that M. viridis seems to have disappeared because of the addition of the ultrasonic radiation and jet circulation to the flushing treatment.     

Diversity of nitrogen-fixing cyanobacteria under various ecosystems of Thailand: I. Morphology,physiology and genetic diversity

The diversity among 853 isolates of nitrogen-fixing cyanobacteria obtained from soil samples collected from different ecosystems including mountainous, forest and cultivated areas in the central, northern and northeastern regions of Thailand was examined. Most isolates showed slow growth rate and had filamentous, heterocystous cells. The percentage of heterocysts in the filaments of different isolates varied from 8.3 to 9.6. Only a few strains showed high nitrogen-fixing potential, while most of the strains exhibited low capacity for nitrogen fixation. Anabaena and Nostoc were the dominant genera among these isolates. One hundred and two isolates were randomly selected from this diverse collection to determine the extent of genetic diversity on the basis of DNA fingerprinting using the PCR method. Based on the PCR products obtained by using a combination of three primers, all strains could be distinguished from one another. When a subset of 45 isolates of Nostoc and a subset of 44 isolates of Anabaena were further analysed by PCR, a wide range of diversity was observed within each of these genera.     

Cyanobacterial akinete induction and its application as biofertilizer for rice cultivation

Nostoc sp. VICCR1-1 was induced in order to form akinetes on the basis of nutrient modification. Phosphorus and iron were found to be the critical for akinete differentiation, especially when both elements were omitted. The number of akinete cells increased up to 20% when compared with culturing in BG11₀ medium (without N source). In addition, CaCl₂ played a role in heterocyst differentiation, and was able to induce heterocyst ranging between 30% and 46%. In order to prepare akinetes as inoculum, the dried form of akinetes was prepared by mixing it with montmorillonite clay. The inoculum with the amount of 2.8 × 10⁶ cells m⁻² was applied to rice (Oryza sativa) fields. After harvesting, the grain yields from chemical N fertilizer, vegetative cells, and akinete inoculum treatments were not significantly different. To monitor the persistence of Nostoc sp. VICCR1-1 after harvesting, the most probable number-denaturing gradient gel electrophoresis technique using 16S rRNA gene was employed. The results indicated that the remaining population is at 2.5 × 10⁵ and 1.62 × 10⁶ cells m⁻² in treatments supplied with vegetative cells and akinete inocula, respectively. Akinete induction might be one of the appropriate approaches for producing cyanobacterial inoculum.