Application of an Inter-Species Extrapolation Method for the Prediction of Drug Interactions between Propolis and Duloxetine in Humans.

Duloxetine (DLX) is a potent drug investigated for the treatment of depression and urinary incontinence. DLX is extensively metabolized in the liver by two P450 isozymes, CYP2D6 and CYP1A2. Propolis (PPL) is one of the popular functional foods known to have effects on activities of CYPs, including CYP1A2. Due to the high probability of using DLX and PPL simultaneously, the present study was designed to investigate the potent effect of PPL on pharmacokinetics (PKs) of DLX after co-administration in humans. A PK study was first conducted in 18 rats (n = 6/group), in which the plasma concentration of DLX and its major metabolite 4-hydroxy duloxetine (4-HD) with or without administration of PPL was recorded. Population PKs and potential effects of PPL were then analyzed using NONMEM software. Lastly, these results were extrapolated from rats to humans using the allometric scaling and the liver blood flow method. PPL (15,000 mg/day) exerts a statistically significant increase in DLX exposures at steady state, with a 20.2% and 24.6% increase in DLX C m a x , s s and the same 28.0% increase in DLX A U C s s when DLX (40 or 60 mg) was administered once or twice daily, respectively. In conclusion, safety issues are required to be attended to when individuals simultaneously use DLX and PPL at high doses, and the possibility of interactions between DLX and PPL might be noted.

Genetic interactions reveal the antagonistic roles of FT/TSF and TFL1 in the determination of inflorescence meristem identity in Arabidopsis.

During the transition to the reproductive phase, the shoot apical meristem switches from the developmental program that generates vegetative organs to instead produce flowers. In this study, we examined the genetic interactions of FLOWERING LOCUS T (FT)/TWIN SISTER OF FT (TSF) and TERMINAL FLOWER 1 (TFL1) in the determination of inflorescence meristem identity in Arabidopsis thaliana. The ft-10 tsf-1 mutants produced a compact inflorescence surrounded by serrated leaves (hyper-vegetative shoot) at the early bolting stage, as did plants overexpressing TFL1. Plants overexpressing FT or TSF (or both FT and TFL1) generated a terminal flower, as did tfl1-20 mutants. The terminal flower formed in tfl1-20 mutants converted to a hyper-vegetative shoot in ft-10 tsf-1 mutants. Grafting ft-10 tsf-1 or ft-10 tsf-1 tfl1-20 mutant scions to 35S::FT rootstock plants produced a normal inflorescence and a terminal flower in the scion plants, respectively, although both scions showed similar early flowering. Misexpression of FT in the vasculature and in the shoot apex in wild-type plants generated a normal inflorescence and a terminal flower, respectively. By contrast, in ft-10 tsf-1 mutants the vasculature-specific misexpression of FT converted the hyper-vegetative shoot to a normal inflorescence, and in the ft-10 tsf-1 tfl1-20 mutants converted the shoot to a terminal flower. TFL1 levels did not affect the inflorescence morphology caused by FT/TSF overexpression at the early bolting stage. Taking these results together, we proposed that FT/TSF and TFL1 play antagonistic roles in the determination of inflorescence meristem identity, and that FT/TSF are more important than TFL1 in this process.

The Rice Rolled Fine Striped (RFS) CHD3/Mi-2 Chromatin Remodeling Factor Epigenetically Regulates Genes Involved in Oxidative Stress Responses During Leaf Development.

In rice (Oryza sativa), moderate leaf rolling increases photosynthetic competence and raises grain yield; therefore, this important agronomic trait has attracted much attention from plant biologists and breeders. However, the relevant molecular mechanism remains unclear. Here, we isolated and characterized Rolled Fine Striped (RFS), a key gene affecting rice leaf rolling, chloroplast development, and reactive oxygen species (ROS) scavenging. The rfs-1 gamma-ray allele and the rfs-2 T-DNA insertion allele of RFS failed to complement each other and their mutants had similar phenotypes, producing extremely incurved leaves due to defective development of vascular cells on the adaxial side. Map-based cloning showed that the rfs-1 mutant harbors a 9-bp deletion in a gene encoding a predicted CHD3/Mi-2 chromatin remodeling factor belonging to the SNF2-ATP-dependent chromatin remodeling family. RFS was expressed in various tissues and accumulated mainly in the vascular cells throughout leaf development. Furthermore, RFS deficiency resulted in a cell death phenotype that was caused by ROS accumulation in developing leaves. We found that expression of five ROS-scavenging genes [encoding catalase C, ascorbate peroxidase 8, a putative copper/zinc superoxide dismutase (SOD), a putative SOD, and peroxiredoxin IIE2] decreased in rfs-2 mutants. Western-blot and chromatin immunoprecipitation (ChIP) assays demonstrated that rfs-2 mutants have reduced H3K4me3 levels in ROS-related genes. Loss-of-function in RFS also led to multiple developmental defects, affecting pollen development, grain filling, and root development. Our results suggest that RFS is required for many aspects of plant development and its function is closely associated with epigenetic regulation of genes that modulate ROS homeostasis.

A WUSCHEL-Independent Stem Cell Specification Pathway Is Repressed by PHB, PHV and CNA in Arabidopsis.

The homeostatic maintenance of stem cells that carry out continuous organogenesis at the shoot meristem is crucial for plant development. Key known factors act to signal between the stem cells and an underlying group of cells thought to act as the stem cell niche. In Arabidopsis thaliana the homeodomain transcription factor WUSCHEL (WUS) is essential for stem cell initiation and maintenance at shoot and flower meristems. Recent data suggest that the WUS protein may move from the niche cells directly into the stem cells to maintain stem cell identity. Here we provide evidence for a second, previously unknown, pathway for stem cell specification at shoot and flower meristems that bypasses the requirement for WUS. We demonstrate that this novel stem cell specification pathway is normally repressed by the activity of the HD-zip III transcription factors PHABULOSA (PHB), PHAVOLUTA (PHV) and CORONA (CNA). When de-repressed, this second stem cell pathway leads to an accumulation of stem cells and an enlargement of the stem cell niche. When de-repressed in a wus mutant background, this second stem cell pathway leads to functional meristems with largely normal cell layering and meristem morphology, activation of WUS cis regulatory elements, and extensive, but not indeterminate, organogenesis. Thus, WUS is largely dispensable for stem cell specification and meristem function, suggesting a set of key stem cell specification factors, competitively regulated by WUS and PHB/PHV/CNA, remain unidentified.

Leaf anatomy and its implications for phylogenetic relationships in Taxaceae s. l.

The comparative study on leaf anatomy and stomata structures of six genera of Taxaceae s. l. was conducted. Leaf anatomical structures were very comparable to each other in tissue shape and their arrangements. Taxus, Austrotaxus, and Pseudotaxus have no foliar resin canal, whereas Amentotaxus, Cephalotaxus, and Torreya have a single resin canal located below the vascular bundle. Among them, Torreya was unique with thick-walled, almost round sclerenchymatous epidermal cells. In addition, Amentotaxus and Torreya were comprised of some fiber cells around the vascular bundle. Also, Amentotaxus resembled Cephalotaxus harringtonia and its var. nana because they have discontinuous fibrous hypodermis. However, C. fortunei lacked the same kind of cells. Stomata were arranged in two stomatal bands separated by a mid-vein. The most unique stomatal structure was of Taxus with papillose accessory cells forming stomatal apparatus and of Torreya with deeply seated stomata covered with a special filament structure. Some morphological and molecular studies have already been discussed for the alternative classification of taxad genera into different minor families. The present study is also similar to these hypotheses because each genus has their own individuality in anatomical structure and stomata morphology. In conclusion, these differences in leaf and stomata morphology neither strongly support the two tribes in Taxaceae nor fairly recognize the monogeneric family, Cephalotaxaceae. Rather, it might support an alternative classification of taxad genera in different minor families or a single family Taxaceae including Cephalotaxus. In this study, we would prefer the latter one because there is no clear reason to separate Cephalotaxus from the rest genera of Taxaceae. Therefore, Taxaceae should be redefined with broad circumscriptions including Cephalotaxus.

Core pathways controlling shoot meristem maintenance.

Essential to the function of shoot meristems in plants to act as sites of continuous organ and tissue formation is the ability of cells within the meristem to remain undifferentiated and proliferate indefinitely. These are characteristics of the stem cells within meristems that are critical for their growth properties. Stem cells are found in tight association with the stem cell niche-those cells that signal to maintain stem cells. Shoot meristems are unique among stem cell systems in that the stem cell niche is a constantly changing population of recent daughter stem cells. Recent progress from Arabidopsis and other systems have uncovered a large number of genes with defined roles in meristem structure and maintenance. This review will focus on well-studied pathways that represent signaling between the stem cells and the niche, that prevent ectopic differentiation of stem cells, that regulate the chromatin status of stem cell factors, and that reveal intersection of hormone signaling and meristem maintenance.

Analysis of microflora profile in Korean traditional nuruk.

A variety of nuruk were collected from various provinces in Korea, and their microflora profiles were analyzed at the species level. A total of 42 nuruk samples were collected and when the viable cell numbers in these nuruk were enumerated, the average cell numbers of bacteria, fungi, yeast, and lactic acid bacteria from all nuruk were 7.21, 7.91, 3.49, and 4.88 log CFU/10 g, respectively. There were no significant differences in viable cell numbers of bacteria or fungi according to regions collected. Bacillus amyloliquefaciens and B. subtilis were the predominant bacterial strains in most samples. A significant portion, 13 out of 42 nuruk, contained foodborne pathogens such as B. cereus or Cronobacter sakazakii. There were various species of lactic acid bacteria such as Enterococcus faecium and Pediococcus pentosaceus in nuruk. It was unexpectedly found that only 13 among the 42 nuruk samples contained Aspergillus oryzae, the representative saccharifying fungi in makgeolli, whereas a fungi Lichtheimia corymbifera was widely distributed in nuruk. It was also found that Pichia jadinii was the predominant yeast strain in most nuruk, but the representative alcohol fermentation strain, Saccharomyces cerevisiae, was isolated from only 18 out of the 42 nuruk. These results suggested that a variety of species of fungi and yeast were distributed in nuruk and involved in the fermentation of makgeolli. In this study, a total of 64 bacterial species, 39 fugal species, and 15 yeast species were identified from nuruk. Among these strains, 37 bacterial species, 20 fungal species, and 8 yeast species were distributed less than 0.1%.

Quantitative trait loci associated with functional stay-green SNU-SG1 in rice.

During monocarpic senescence in higher plants, functional stay-green delays leaf yellowing, maintaining photosynthetic competence, whereas nonfunctional stay-green retains leaf greenness without sustaining photosynthetic activity. Thus, functional stay-green is considered a beneficial trait that can increase grain yield in cereal crops. A stay-green japonica rice 'SNU-SG1' had a good seed-setting rate and grain yield, indicating the presence of a functional stay-green genotype. SNU-SG1 was crossed with two regular cultivars to determine the inheritance mode and identify major QTLs conferring stay-green in SNU-SG1. For QTL analysis, linkage maps with 100 and 116 DNA marker loci were constructed using selective genotyping with F2 and RIL (recombinant inbred line) populations, respectively. Molecular marker-based QTL analyses with both populations revealed that the functional stay-green phenotype of SNU-SG1 is regulated by several major QTLs accounting for a large portion of the genetic variation. Three main-effect QTLs located on chromosomes 7 and 9 were detected in both populations and a number of epistatic-effect QTLs were also found. The amount of variation explained by several digenic interactions was larger than that explained by main-effect QTLs. Two main-effect QTLs on chromosome 9 can be considered the target loci that most influence the functional stay-green in SNU-SG1. The functional stay-green QTLs may help develop low-input high-yielding rice cultivars by QTL-marker-assisted breeding with SNU-SG1.

Neurotoxic pyridinium metabolites of haloperidol are substrates of human organic cation transporters.

Two neurotoxic pyridinium metabolites of haloperidol, 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxybutyl]pyridinium ion (HPP(+)) and 4-(4-(chlorophenyl)-1-4-(fluorophenyl)-4-hydroxybutyl-pyridinium (RHPP(+)), are formed in the liver and found in the brain. To understand how these neurotoxic pyridinium metabolites are distributed in the brain, HPP(+) and RHPP(+) were evaluated as substrates for human organic cation transporters (hOCTs). Both HPP(+) and RHPP(+) were accumulated in Caco-2 cells, and these accumulations were significantly inhibited by pretreatment with the hOCT inhibitors verapamil, cimetidine, phenoxybenzamine, and corticosterone. The contribution of each hOCT was evaluated based on measurements of the intracellular concentrations of haloperidol metabolites in Madin Darby canine kidney (MDCK) cells transfected with hOCT1, hOCT2, or hOCT3. HPP(+) accumulated in hOCT-overexpressing MDCK cells in a concentration-dependent manner, with estimated K(m) values of 0.99, 2.79, and 2.23 microM and V(max) values of 282.1, 256.1, and 400.2 pmol/min/microg protein for hOCT1, hOCT2, and hOCT3, respectively. RHPP(+) accumulated in hOCT1- and hOCT3-overexpressing MDCK cells, with estimated K(m) values of 5.15 and 8.21 microM and V(max) values of 1230.9 and 1348.6 pmol/min/microg protein for hOCT1 and hOCT3, respectively. On the other hand, RHPP(+) did not accumulate in the hOCT2-expressing MDCK cells. These results suggest that HPP(+) and RHPP(+) are substrates for hOCTs, with the exception of RHPP(+) for hOCT2. Thus, hOCTs seem to contribute to the disposition of these toxic metabolites in human subjects, although further in vivo studies are required to elucidate the involvement of hOCTs in the disposition of haloperidol pyridinium metabolites.

Receptor-stimulated oxidation of SHP-2 promotes T-cell adhesion through SLP-76-ADAP.

Receptor-stimulated generation of intracellular reactive oxygen species (ROS) modulates signal transduction, although the mechanism(s) is unclear. One potential basis is the reversible oxidation of the active site cysteine of protein tyrosine phosphatases (PTPs). Here, we show that activation of the antigen receptor of T cells (TCR), which induces production of ROS, induces transient inactivation of the SH2 domain-containing PTP, SHP-2, but not the homologous SHP-1. SHP-2 is recruited to the LAT-Gads-SLP-76 complex and directly regulates the phosphorylation of key signaling proteins Vav1 and ADAP. Furthermore, the association of ADAP with the adapter SLP-76 is regulated by SHP-2 in a redox-dependent manner. The data indicate that TCR-mediated ROS generation leads to SHP-2 oxidation, which promotes T-cell adhesion through effects on an SLP-76-dependent signaling pathway to integrin activation.

Chloroplast DNA phylogeny of the woody Sonchus alliance (Asteraceae: Sonchinae) in the Macaronesian Islands.

Determining accurate phylogenetic relationships among the members of the woody Sonchus alliance presents challenges because of an insufficient level of molecular variation and the convergent evolution of similar morphological traits in island settings. To obtain a better resolved phylogeny and to test the potential role of hybridization and introgression, we sequenced all members of the alliance with multiple populations for the ITS of nrDNA and over 4000 base pairs of coding and noncoding regions of cpDNA. The cpDNA phylogeny is not well resolved in the core members of the alliance (i.e., subg. Dendrosonchus and genus Taeckholmia), but like the ITS tree, it has identified basal lineages of monotypic genera. The cpDNA data set was not significantly different from that of ITS, and subsequent combined analysis provided a better resolved and supported phylogeny within the alliance. The combined ML tree identified the same basal lineages, suggested nonmonophyly of Dendrosonchus and Taeckholmia, and did not support either Boulos' or Aldridge's infrasubgeneric classification system. Assessment of the role of hybridization and introgression was limited due to poor resolution in the cpDNA phylogeny. The combined analysis supports a Gran Canaria origin for the alliance and two subsequent long distance dispersal events to Madeira and Cape Verde islands.

Phylogeny of Panax using chloroplast trnC-trnD intergenic region and the utility of trnC-trnD in interspecific studies of plants.

Sequences of the chloroplast trnC-trnD region and the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA were obtained for all species of Panax L. (the ginseng plant genus, Araliaceae) to reconstruct phylogenetic relationships. The trnC-trnD phylogeny is congruent with the ITS phylogeny for the diploid taxa of Panax. This study is the first use of the trnC-trnD sequence data for phylogenetic analysis at the interspecific level. We evaluated this DNA region for its phylogenetic utility at the lower taxonomic level for flowering plants. The trnC-trnD region includes the trnC-petN intergenic spacer, the petN gene, the petN-psbM intergenic spacer, the psbM gene, and the psbM-trnD intergenic spacer. The petN and psbM genes are small, 90 and 104-114 bp across angiosperms, respectively, and have conserved sequences. We have designed universal amplification and sequencing primers within these two genes. Using these primers, we have successfully amplified the entire trnC-trnD region for a diversity of flowering plant groups, including Aralia L. (Araliaceae), Calycanthus L. (Calycanthaceae), Corylus L. (Betulaceae), Hamamelis L. (Hamamelidaceae), Hydrocotyle L. (Apiaceae), Illigera Blume (Hernandiaceae), Nelumbo Adans. (Nelumbonaceae), Nolana L. ex L.f. (Solanaceae), Prunus L. (Rosaceae), and Staphylea L. (Staphyleaceae). In Panax, the trnC-trnD region provides a similar number of informative phylogenetic characters as the ITS regions and a slightly higher number of informative characters than the chloroplast ndhF gene. We thus demonstrate the utility of the trnC-trnD region for lower-level phylogenetic studies in flowering plants.

Reversible inactivation of the tumor suppressor PTEN by H2O2.

The tumor suppressor PTEN regulates cell migration, growth, and survival by removing the 3'-phosphate of phosphoinositides. Exposure of purified PTEN or of cells to H(2)O(2) resulted in inactivation of PTEN in a time- and H(2)O(2) concentration-dependent manner. Analysis of various cysteine mutants, including mass spectrometry of tryptic peptides, indicated that the essential Cys(124) residue in the active site of PTEN specifically forms a disulfide with Cys(71) during oxidation by H(2)O(2). The reduction of H(2)O(2)-oxidized PTEN in cells appears to be mediated predominantly by thioredoxin. Thus, thioredoxin was more efficient than glutaredoxin, glutathione, or a 14-kDa thioredoxin-like protein with regard to the reduction of oxidized PTEN in vitro. Thioredoxin co-immunoprecipitated with PTEN from cell lysates; and incubation of cells with 2,4-dinitro-1-chlorobenzene (an inhibitor of thioredoxin reductase) delayed the reduction of oxidized PTEN, whereas incubation with buthioninesulfoximine (an inhibitor of glutathione biosynthesis) did not. These results suggest that the reversible inactivation of PTEN by H(2)O(2) might be important for the accumulation of 3'-phosphorylated phosphoinositides and that the uncontrolled generation of H(2)O(2) associated with certain pathological conditions might contribute to cell proliferation by inhibiting PTEN function.

Molecular evidence for the taxonomic identity of Korean Adonis (Ranunculaceae).

Although Korean Adonis has been traditionally recognized as Adonis amurensis Regel and Radde with various infraspecific taxa described, its taxonomic identity is still in dispute. We investigated the genetic variation in 60 individuals from 12 populations in Korea to elucidate the taxonomic identity of the Korean Adonis complex. Random amplified polymorphic DNA (RAPD) analysis revealed that Korean Adonis comprises three species: A. amurensis, A. pseudoamurensis, and A. multiflora. Adonis amurensis is distributed in northern central inland regions of Korea and A. pseudoamurensis is found in southern parts of the Korean peninsula. Adonis multiflora grows only on Cheju Island, which is the southernmost part of Korea. Phylogenetic analysis of nuclear ribosomal internal transcribed spacer (ITS) sequences partially supported the presence of three Adonis taxa in Korea as detected by RAPD analysis. The Adonis population on Jangbong Island located in the West Sea, which was referred to as A. pseudoamurensison the basis of morphological examination, was separated from the other populations of A. pseudoamurensis. Otherwise, the molecular evidence is well congruent with the recent morphological study that proposes that Korean Adonis consists of these three species.