Electrochemically driven drug metabolism by membranes containing human cytochrome P450.

Rapid analyses of drug metabolism reactions by human cytochrome P450s (CYPs) that metabolize 95% of all current drugs are very important in drug development processes and effective therapies. Since CYPs need electrons to metabolize drugs, electrons supplied from electrodes to activate CYP molecules are expected to be very useful to develop rapid assay methods for CYP reactions. Although several studies on the direct electrochemistry of isolated and purified human CYPs have been reported, the use of microsomes (membranes) containing CYP is more suitable, because they are frequently used in drug research due to their easy preparation and low cost.

Crystallization and preliminary X-ray diffraction studies of vitamin D3 hydroxylase, a novel cytochrome P450 isolated from Pseudonocardia autotrophica.

Vitamin D(3) hydroxylase (Vdh) is a novel cytochrome P450 monooxygenase isolated from the actinomycete Pseudonocardia autotrophica and consisting of 403 amino-acid residues. Vdh catalyzes the activation of vitamin D(3) via sequential hydroxylation reactions: these reactions involve the conversion of vitamin D(3) (cholecalciferol or VD3) to 25-hydroxyvitamin D(3) [25(OH)VD3] and the subsequent conversion of 25(OH)VD3 to 1alpha,25-dihydroxyvitamin D(3) [calciferol or 1alpha,25(OH)(2)VD3]. Overexpression of recombinant Vdh was carried out using a Rhodococcus erythropolis expression system and the protein was subsequently purified and crystallized.

A novel group of transcriptional repressors in Arabidopsis.

We showed previously that the ERF-associated amphiphilic repression (EAR) motif is a plant-specific repression domain that contains the conserved amino acid sequence LXLXL. In this report, we describe the identification of a novel repression domain, L/VR/KLFGVXM/V/L, which is different from known EAR motifs, in B3 DNA-binding domain transcription factors in Arabidopsis. Database analysis revealed that 29 Arabidopsis transcription factors, which included members of the RAV, ARF, Hsf and MYB families, contain the R/KLFGV conserved motif found in the novel repression domain.

Efficient synthesis of oligonucleotide conjugates on solid-support using an (aminoethoxycarbonyl)aminohexyl group for 5'-terminal modification.

Solid-support conjugation at the 5'-terminal primary amine of oligonucleotides is a convenient and powerful method for introducing various functional groups. However, conventional aliphatic amines do not necessarily provide conjugates with sufficient yields. To improve the modification efficacy, we used the amino-linker (aminoethoxycarbonyl)aminohexyl group (ssH-linker), for solid-support conjugation.

A novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U: expression, molecular features and properties of redox potential.

A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly(22)-Asn(34), in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C(15), C(16) and C(17) chain lengths and C(15) chain length, respectively, and it was considered that these fatty acids are bound to glycerol-Cys(18).

Fully active QAE isoform confers thermal hysteresis activity on a defective SP isoform of type III antifreeze protein.

Type III antifreeze protein is naturally expressed as a mixture of sulfopropyl-Sephadex (SP) and quaternary aminoethyl-Sephadex (QAE)-binding isoforms, whose sequence identity is approximately 55%. We studied the ice-binding properties of a SP isoform (nfeAFP6) and the differences from those of a QAE isoform (nfeAFP8); both of these isoforms have been identified from the Japanese fish Zoarces elongatus Kner. The two isoforms possessed ice-shaping ability, such as the creation of an ice bipyramid, but nfeAFP6 was unable to halt crystal growth and exhibited no thermal hysteresis activity.

A quinoline antibiotic from Rhodococcus erythropolis JCM 6824.

A new quinoline antibiotic, aurachin RE, was isolated and identified from a culture broth of Rhodococcus erythropolis JCM 6824. The aurachin RE structure was determined based on NMR and mass spectrometric analysis. The structure is similar to that of aurachin C antibiotics that have been identified from Stigmatella aurantiaca.

Endosymbiotic bacteria in insects: their diversity and culturability.

Many animals and plants possess symbiotic microorganisms inside their body, wherein intimate interactions occur between the partners. The Insecta, often rated as the most diverse animal group, show various types of endosymbiotic associations, ranging from obligate mutualism to facultative parasitism. Although technological advancements in culture-independent molecular techniques, such as quantitative PCR, molecular phylogeny and in situ hybridization, as well as genomic and metagenomic analyses, have allowed us to directly observe endosymbiotic associations in vivo, the molecular mechanisms underlying insect-microbe interactions are not well understood, because most of these insect endosymbionts are neither culturable nor genetically manipulatable.

Comparison of the chemical properties of a novel amino-linker with various amino modifications.

We previously reported a series of new amino-linkers, consisting of an aminoethyl carbamate structure (Komatsu, 2008). We have now examined the chemical properties of oligonucleotides modified with an ssH-linker, which is the simplest and most cost-effective derivative of the series. Although it was previously shown that monomethoxytrityl protection on a primary amine of the ssH-linker was cleaved under weakly acidic conditions (1% acetic acid), we found that the deprotection also proceeded in aqueous buffer solutions (pH 6.

Construction of an aminooxy derivative for RNA and DNA labeling.

We previously reported that the reactivity of a primary amine is increased by connecting the amine to an aromatic residue with an alkyl linker. This strategy was applied to the construction of a reagent that could form a covalent bond with an aldehyde group. We synthesized a new reagent that consisted of aminooxy and aromatic groups and biotin.

H2O2 tolerance of Vibrio rumoiensis S-1(T) is attributable to the cellular catalase activity.

The extraordinarily high level of H2O2 tolerance of Vibrio rumoiensis strain S-1(T) when compared with the tolerance levels of strain S-4, a probable catalase-deficient derivative of strain S-1(T), was demonstrated by the introduction of 0-100 mM H2O2 during the mid-exponential growth phase. The contribution of catalase to the H2O2 tolerance was also demonstrated by comparing the catalase-deficient mutant Escherichia coli strain UM2 with a UM2 strain, harboring the plasmid pBSsa1, which carried the strain S-1(T) catalase gene vktA. The decomposition rates of 23-25 mM H2O2 that was introduced in the culture fluids of strain S-1(T) and E.

Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus.

We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca(2+)-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 A resolution.

NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity.

Three distinct pattern elements of the silique are thought to contribute to its dehiscence: a separation layer, cells with a secondary wall adjacent to the separation layer, and a valve endocarp layer with secondary wall. However, the role of the secondary wall has not been proven, and the factors that regulate its formation in siliques remain to be characterized. We show here that secondary wall formation in siliques is necessary for dehiscence, and that two plant-specific transcription factors, NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1 and 3 (NST1 and NST3), regulate its formation in siliques of Arabidopsis.

Acinetobacter sp. strain Ths, a novel psychrotolerant and alkalitolerant bacterium that utilizes hydrocarbon.

A novel psychrotolerant, alkalitolerant bacterium, strain Ths, was isolated from a soil sample immersed in hot spring water containing hydrocarbons and grown on a chemically defined medium containing n-tetradecane as the sole carbon source. The isolate grew at 0 degrees C but not at temperatures higher than 45 degrees C; its optimum growth temperature was 27 degrees C. It grew in the pH range of 7-9.

Hypothermic preservation effect on mammalian cells of type III antifreeze proteins from notched-fin eelpout.

Antifreeze proteins (AFPs) can bind to the surface of ice crystals and have also been suggested to protect cells from hypothermic damage. The present study reports that type III AFPs from notched-fin eelpout, Zoarces elongatus Kner, can protect cells during hypothermic storage. This fish naturally expresses at least 13 isoforms of type III AFP (denoted NfeAFPs), the primary sequences of which were categorized into SP- and QAE-Sephadex binding groups (SP- and QAE-isoforms).

AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis.

In Arabidopsis, MYB transcription factors regulate flavonoid biosynthesis via the formation of protein complexes with a basic helix-loop-helix (bHLH) transcription factor and a WD40 repeat protein. Several R3-type single-MYB proteins (R3-MYB), such as CPC and TRY, act as negative regulators of the development of epidermal cells. However, such regulators of flavonoid biosynthesis have not yet been reported, to our knowledge.

Alkalibacterium indicireducens sp. nov., an obligate alkaliphile that reduces indigo dye.

Indigo-reducing, obligately alkaliphilic strains A11T, F11 and F12 were isolated from indigo fermentation liquor obtained from Tokushima Prefecture, Shikoku, Japan. The isolates grew at pH 9.0-12.

Effects of H2O2 under low- and high-aeration-level conditions on growth and catalase activity in Exiguobacterium oxidotolerans T-2-2T.

The effects of H2O2 under low- and high-aeration-level conditions on growth and catalase activity in Exiguobacterium oxidotolerans T-2-2T were investigated. Continuous addition of 5-200 mM H2O2 to the culture medium from the mid-exponential growth phase enhanced the growth of the strain under the low-aeration-level condition, whereas the addition of 5-50 mM H2O2 decreased intracellular specific catalase activity and extracellular total catalases activity. The detection of extracellular catalase by the cells and the decrease in intracellular specific catalase activity and extracellular total catalase activity under the high-aeration-level condition account for the stimulation of growth by the introduced H2O2 and the decrease in catalase activities induced by O(2) from H2O2 in the medium.

Bacillus polygoni sp. nov., a moderately halophilic, non-motile obligate alkaliphile isolated from indigo balls.

A moderately halophilic, obligate alkaliphile (growth range pH 8-12), designated strain YN-1(T), was isolated from indigo balls obtained from Ibaraki, Japan. The cells of the isolate stained Gram-positive, and were aerobic, non-motile, sporulating rods which grew optimally at pH 9. The strain grew in 3-14% NaCl with optimum growth in 5% NaCl.

Three Types of Antibiotics Produced from Rhodococcus erythropolis Strains.

A total of 15 Rhodococcus erythropolis strains were characterized as antibiotic producers and classified into three groups according to their antibiotic spectrum and growth compatibility (strains within a group did not inhibit each other's growth). Each of the antibiotic groups exhibited antibiotic activity against a taxonomically different breadth of bacteria: Group I exhibited antibiotic activity against a broad range of Gram-positives; Group II, mainly against the genus Rhodococcus and some other Gram-positives; and Group III, particularly against R. erythropolis.

Effect of annealing time of an ice crystal on the activity of type III antifreeze protein.

Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evaluated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8).

Novel amino linkers enabling efficient labeling and convenient purification of amino-modified oligonucleotides.

We developed new amino linker reagents for an oligonucleotide (ONT) terminus. These reagents consist of an aminoethyl carbamate main linkage and a side-chain residue, which was a naphthylmethoxymethyl, methoxymethyl, or methyl group or a hydrogen atom. The primary amine was protected with a monomethoxytrityl (MMT) group.

Bellilinea caldifistulae gen. nov., sp. nov. and Longilinea arvoryzae gen. nov., sp. nov., strictly anaerobic, filamentous bacteria of the phylum Chloroflexi isolated from methanogenic propionate-degrading consortia.

Thermophilic (strain GOMI-1(T)) and mesophilic (strain KOME-1(T)) strains were isolated from two different cultures of propionate-degrading consortia obtained from thermophilic digester sludge and rice paddy soil, respectively. The two strains were non-spore-forming, non-motile and Gram-negative. Both strains were obligately anaerobic micro-organisms, showing multicellular filamentous morphotypes more than 100 mum in length.

Crystal structure of cytochrome P450 MoxA from Nonomuraea recticatena (CYP105).

Cytochrome P450 MoxA (P450moxA) from a rare actinomycete Nonomuraea recticatena belongs to the CYP105 family and exhibits remarkably broad substrate specificity. Here, we demonstrate that P450moxA acts on several luciferin derivatives, which were originally identified as substrates of the human microsomal P450s. We also describe the crystal structure of P450moxA in substrate-free form.

Pelotomaculum propionicicum sp. nov., an anaerobic, mesophilic, obligately syntrophic, propionate-oxidizing bacterium.

An anaerobic, mesophilic, syntrophic, propionate-oxidizing bacterium, strain MGP(T), was isolated as a defined co-culture with Methanospirillum hungatei from the methanogenic sludge of a mesophilic upflow anaerobic sludge blanket (UASB) reactor. The strain grew in the presence of propionate, but only in co-culture with methanogens, suggesting that it is an obligately syntrophic bacterium. The optimum temperature for growth was 37 degrees C, and the optimum pH was between 6.