Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01, in human plasma or urine

We have established a highly sensitive high-performance liquid chromatographic method for the determination of an anticancer drug, UCN-01, in human plasma or urine. Using a fluorescence detector set at an excitation wavelength of 310 nm and emission monitored at 410 nm, there was a good linearity for UCN-01 in human plasma (r=0.999) or urine (r=0.999) at concentrations ranging from 0.2 to 100 ng/ml or 1 to 400 ng/ml, respectively. For intra-day assay, in plasma samples, the precision and accuracy were 1.8% to 5.6% and −10.0% to 5.2%, respectively. For inter-day assay, the precision and accuracy were 2.0% to 18.2% and 2.4% to 10.0%, respectively. In urine samples, the intra- and inter-day precision and accuracy were within 3.9% and ±2.7%, respectively. The lower limit of quantification (LLOQ) was set at 0.2 ng/ml in plasma and 1 ng/ml in urine. UCN-01 in plasma samples was stable up to two weeks at −80°C and also up to four weeks in urine samples. This method could be very useful for studying the human pharmacokinetics of UCN-01.     

Effects of blue light deficiency on acclimation of light energy partitioning in PSII and CO2 assimilation capacity to high irradiance in spinach leaves

Blue light effects on the acclimation of energy partitioningcharacteristics in PSII and CO₂ assimilation capacity in spinachto high growth irradiance were investigated. Plants were grownhydroponically in different light treatments that were a combinationof two light qualities and two irradiances, i.e. white lightand blue-deficient light at photosynthetic photon flux densities(PPFDs) of 100 and 500 µmol m^–2 s^–1. The CO₂assimilation rate, the quantum efficiency of PSII (PSII) andthermal dissipation activity / in young, fully expanded leaves were measured under 1,600 µmol m^–2 s^–1white light. The CO₂ assimilation rate and PSII were higher,while / was lower in plants grown under high irradiancethan in plants grown under low irradiance. These responses wereobserved irrespective of the presence or absence of blue lightduring growth. The extent of the increase in the CO₂ assimilationrate and PSII and the decrease in / by high growth irradiance was smaller under blue light-deficient conditions. These resultsindicate that blue light helps to boost the acclimation responsesof energy partitioning in PSII and CO₂ assimilation to highirradiance. Similarly, leaf N, Cyt f and Chl contents per unitleaf area increased by high growth irradiance, and the extentof the increment in leaf N, Cyt f and Chl was smaller underblue light-deficient conditions. Regression analysis showedthat the differences in energy partitioning in PSII and CO₂assimilation between plants grown under high white light andhigh blue-deficient light were closely related to the differencein leaf N.     

A genome-wide survey of genes for enzymes involved in pigment synthesis in an ascidian, Ciona intestinalis

The draft genome sequence and a large quantity of EST and cDNA information are now available for the ascidian Ciona intestinalis. In the present study, genes involved in pigment synthesis pathways were identified in the decoded genome of Ciona, and information about these genes was obtained from available EST and cDNA sequences. It was found that the Ciona genome contains orthologous genes for each enzyme of the melanin, pteridine, ommochrome, papiliochrome, and heme synthesis pathways. Several appear as independent duplications in the Ciona genome. Because cDNA clones for all but two of these genes have already been isolated by the cDNA project, C. intestinalis will provide an experimental system to explore molecular mechanisms underlying color patterns, through future genome-wide studies.     

2-Haloacrylate Hydratase,a New Class of Flavoenzyme That Catalyzes the Addition of Water to the Substrate for Dehalogenation

Enzymes catalyzing the conversion of organohalogen compounds are useful in the chemical industry and environmental technology. Here we report the occurrence of a new reduced flavin adenine dinucleotide (FAD) (FADH₂)-dependent enzyme that catalyzes the removal of a halogen atom from an unsaturated aliphatic organohalogen compound by the addition of a water molecule to the substrate. A soil bacterium, Pseudomonas sp. strain YL, inducibly produced a protein named Caa67_YL when the cells were grown on 2-chloroacrylate (2-CAA). The caa67_YL gene encoded a protein of 547 amino acid residues (M_r of 59,301), which shared weak but significant sequence similarity with various flavoenzymes and contained a nucleotide-binding motif. We found that 2-CAA is converted into pyruvate when the reaction was carried out with purified Caa67_YL in the presence of FAD and a reducing agent [NAD(P)H or sodium dithionite] under anaerobic conditions. The reducing agent was not stoichiometrically consumed during this reaction, suggesting that FADH₂ is conserved by regeneration in the catalytic cycle. When the reaction was carried out in the presence of H₂¹⁸O, [¹⁸O]pyruvate was produced. These results indicate that Caa67_YL catalyzes the hydration of 2-CAA to form 2-chloro-2-hydroxypropionate, which is chemically unstable and probably spontaneously dechlorinated to form pyruvate. 2-Bromoacrylate, but not other 2-CAA analogs such as acrylate and methacrylate, served as the substrate of Caa67_YL. Thus, we named this new enzyme 2-haloacrylate hydratase. The enzyme is very unusual in that it requires the reduced form of FAD for hydration, which involves no net change in the redox state of the coenzyme or substrate.Dehalogenases catalyze the removal of halogen atoms from organohalogen compounds. These enzymes have been attracting a great deal of attention partly because of their possible applications to the chemical industry and environmental technology. Several dehalogenases have been discovered and characterized (6, 11, 14, 17, 22). Some of them act on unsaturated aliphatic organohalogen compounds in which a halogen atom is bound to an sp²-hybridized carbon atom. Examples include various corrinoid/iron-sulfur cluster-containing reductive dehalogenases (1, 7), cis- and trans-3-chloroacrylic acid dehalogenases (4, 19), and LinF (maleylacetate reductase), which acts on 2-chloromaleylacetate (5).In order to gain more insight into the enzymatic dehalogenation of unsaturated aliphatic organohalogen compounds, we searched for microorganisms that dissimilate 2-chloroacrylate (2-CAA) as a sole source of carbon and energy (8). 2-CAA is a bacterial metabolite of 2-chloroallyl alcohol, an intermediate or by-product in the industrial synthesis of herbicides (26). Rats treated orally with the herbicides sulfallate, diallate, and triallate excrete urinary 2-CAA (16). Various halogenated acrylic acids are produced by a red alga (27). We obtained three 2-CAA-utilizing bacteria as a result of screening (8). For one of these bacteria, Burkholderia sp. strain WS, we previously discovered a new NADPH-dependent enzyme, 2-haloacrylate reductase (12, 13). Although this enzyme does not directly remove a halogen atom from the substrate, it is supposed to participate in the metabolism of 2-CAA by catalyzing the conversion of 2-CAA into l-2-chloropropionate, which is subsequently dehalogenated by l-2-haloacid dehalogenase.Another bacterium that we obtained, Pseudomonas sp. strain YL, also dissimilates 2-CAA. However, the metabolic fate of 2-CAA in this bacterium remains unclear. In the present study, we analyzed proteins from 2-CAA- and lactate-grown cells of Pseudomonas sp. YL by two-dimensional polyacrylamide gel electrophoresis (PAGE) and identified a 2-CAA-inducible protein. We found that the protein catalyzes the dehalogenation of 2-CAA by the addition of a water molecule to the substrate, representing a new family of dehalogenases that act on unsaturated aliphatic organohalogen compounds. Remarkably, the enzyme requires reduced flavin adenine dinucleotide (FAD) (FADH₂) for its activity, although the reaction does not involve a net change in the redox state of the coenzyme or substrate. Here we describe the occurrence and characteristics of this unusual flavoenzyme.     

Identification,characterization and interaction of <Emphasis Type="Italic">HAP</Emphasis> family genes in rice

A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5 genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB288027 to AB288048 and BR000373 to BR000375.     

Visualization of endogenous BMP signaling during Xenopus development

The TGF-beta superfamily of growth factors is known to transmit signals to the nucleus mainly through the Smads, intracellular signaling components that are highly conserved from nematodes to humans. The signaling activity of the Smads is regulated by their ligand-stimulated phosphorylation through Ser/Thr kinase receptors. Here, to examine the in vivo role of BMP, we investigated the spatio-temporal activation of BMP-regulated signals during Xenopus development, using a polyclonal antibody that specifically recognizes the phosphorylated form of BMP-regulated Smads. BMP signaling was observed uniformly in embryos as early as stage 7, but was restricted to the ventral side of the embryo at the late blastula stage, supporting the proposed role of BMP4 as a ventralizing factor in Xenopus embryos. In addition, localized staining was detected in several developing organs, consistent with the predicted function of BMP family members in organogenesis.     

Binding and dissociation of human topoisomerase I with hairpin-loop RNAs: implications for the regulation of HIV-1 replication

Cellular topoisomerase I has been reported to be present in retroviral particles and to enhance viral cDNA synthesis; however, the mechanisms involved remain unknown. In the present study, it has been demonstrated that human topoisomerase I combines with a stem-loop RNA and that the bound topoisomerase I can be dissociated from RNA substrates in the presence of ATP. In addition, in vitro cleaved synthetic RNA bound by topoisomerase I is subsequently relegated when the topoisomerase I is dissociated by ATP. A mechanism is proposed in which human topoisomerase I is carried into virions and regulates the repair of genomic RNA by its ligation activity.