1-Naphthaleneacetic Acid Improved the In Vitro Cell Culturing by Inhibiting Apoptosis.

In vitro cell culturing witnessed its applications in scientific research and industrial activities. Attempts to shorten the doubling time of cultured cells have never ceased. In plants, auxin is applied to promote plant growth, the synthetic derivative 1-Naphthaleneacetic acid (NAA) is a good example. Despite the auxin's naturally occurring receptors are not present in mammalian cells, studies suggested they may affect cell culturing. Yet the effects and mechanisms are still unclear. Here, an up to 2-fold increase in the yield of in vitro cultured human cells is observed. Different types of human cell lines and primary cells are tested and found that NAA is effective in all the cells tested. The PI staining followed by FACS suggested that NAA do not affect the cell cycling. Apoptosis-specific dye staining analysis implicated that NAA rescued cell death. Further bulk RNA sequencing is done and it is identified that the lipid metabolism-engaging and anti-apoptosis gene, ANGPTL4, is enhanced in expression upon NAA treatment. Studies on ANGPTL4 knockout cells indicated that ANGPTL4 is required for NAA-mediated response. Thus, the data identified a beneficial role of NAA in human cell culturing and highlighted its potency in in vitro cell culturing.

Role of naphthaleneacetic acid in the degradation of bisphenol A and wastewater treatment by microalgae: Enhancement and signaling.

Coupling microalgae cultivation with wastewater treatment is a promising environmentally sustainable development strategy. However, toxics such as Bisphenol A (BPA) in wastewater damage microalgae cells and reduces bioresources production. Phytohormone regulation has the potential to solve this issue. However, phytohormone research is still in its infancy. In this work, 0.2 µM naphthyl acetic acid (NAA) significantly enhanced Chlorella vulgaris BPA detoxification by 127.3% and Chlorella biomass production by 46.4%. NAA helps Chlorella convert bisphenol A into small non-toxic intermediates by enhancing the expression of associated enzymes. Simultaneously, NAA promoted carbon fixation and photosynthetic metabolism. Activation of the mitogen-activated protein kinase (MAPK) pathway strengthened the downstream antioxidant system while improving photosynthesis and intracellular starch and lipid synthesis. Carbohydrates, pigment, and lipid production was significantly enhanced by 20.0%, 46.9%, and 21.8%, respectively. A new insight is provided into how phytohormones may increase microalgae in wastewater's bioresource transformation and toxicity resistance.

Combined Metabolite and Transcriptome Profiling Reveals the Norisoprenoid Responses in Grape Berries to Abscisic Acid and Synthetic Auxin.

The abscisic acid (ABA) increase and auxin decline are both indicators of ripening initiation in grape berry, and norisoprenoid accumulation also starts at around the onset of ripening. However, the relationship between ABA, auxin, and norisoprenoids remains largely unknown, especially at the transcriptome level. To investigate the transcriptional and posttranscriptional regulation of the ABA and synthetic auxin 1-naphthaleneacetic acid (NAA) on norisoprenoid production, we performed time-series GC-MS and RNA-seq analyses on Vitis vinifera L. cv. Cabernet Sauvignon grape berries from pre-veraison to ripening. Higher levels of free norisoprenoids were found in ABA-treated mature berries in two consecutive seasons, and both free and total norisoprenoids were significantly increased by NAA in one season. The expression pattern of known norisoprenoid-associated genes in all samples and the up-regulation of specific alternative splicing isoforms of VviDXS and VviCRTISO in NAA-treated berries were predicted to contribute to the norisoprenoid accumulation in ABA and NAA-treated berries. Combined weighted gene co-expression network analysis (WGCNA) and DNA affinity purification sequencing (DAP-seq) analysis suggested that VviGATA26, and the previously identified switch genes of myb RADIALIS (VIT_207s0005g02730) and MAD-box (VIT_213s0158g00100) could be potential regulators of norisoprenoid accumulation. The positive effects of ABA on free norisoprenoids and NAA on total norisoprenoid accumulation were revealed in the commercially ripening berries. Since the endogenous ABA and auxin are sensitive to environmental factors, this finding provides new insights to develop viticultural practices for managing norisoprenoids in vineyards in response to changing climates.

Role of human flavin-containing monooxygenase (FMO) 5 in the metabolism of nabumetone: Baeyer-Villiger oxidation in the activation of the intermediate metabolite, 3-hydroxy nabumetone, to the active metabolite, 6-methoxy-2-naphthylacetic acid in vitro.

Nabumetone (NAB) is a non-steroidal anti-inflammatory drug used clinically, and its biotransformation includes the major active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA). One of the key intermediates between NAB and 6-MNA may be 3-hydroxy nabumetone (3-OH-NAB). The aim of the present study was to investigate the role of flavin-containing monooxygenase (FMO) isoform 5 in the formation of 6-MNA from 3-OH-NAB. To elucidate the biotransformation of 3-OH-NAB to 6-MNA, an authentic standard of 3-OH-NAB was synthesised and used as a substrate in an incubation with human liver samples or recombinant enzymes. The formation of 3-OH-NAB was observed after the incubation of NAB with various cytochrome P450 (CYP) isoforms. However, 6-MNA itself was rarely detected from NAB and 3-OH-NAB. Further experiments revealed a 6-MNA peak derived from 3-OH-NAB in human hepatocytes. 6-MNA was also detected in the extract obtained from 3-OH-NAB by a combined incubation of recombinant human FMO5 and human liver S9. We herein demonstrated that the reaction involves carbon-carbon cleavage catalyzed by the Baeyer-Villiger oxidation (BVO) of a carbonyl compound, the BVO substrate, such as a ketol, by FMO5. Further in vitro inhibition experiments showed that multiple non-CYP enzymes are involved in the formation of 6-MNA from 3-OH-NAB.

Colonization of endophyte Acremonium sp. D212 in Panax notoginseng and rice mediated by auxin and jasmonic acid.

Endophytic fungi can be beneficial to plant growth. However, the molecular mechanisms underlying colonization of Acremonium spp. remain unclear. In this study, a novel endophytic Acremonium strain was isolated from the buds of Panax notoginseng and named Acremonium sp. D212. The Acremonium sp. D212 could colonize the roots of P. notoginseng, enhance the resistance of P. notoginseng to root rot disease, and promote root growth and saponin biosynthesis in P. notoginseng. Acremonium sp. D212 could secrete indole-3-acetic acid (IAA) and jasmonic acid (JA), and inoculation with the fungus increased the endogenous levels of IAA and JA in P. notoginseng. Colonization of the Acremonium sp. D212 in the roots of the rice line Nipponbare was dependent on the concentration of methyl jasmonate (MeJA) (2-15 µmol/L) and 1-naphthalenacetic acid (NAA) (10-20 µmol/L). Moreover, the roots of the JA signaling-defective coi1-18 mutant were colonized by Acremonium sp. D212 to a lesser degree than those of the wild-type Nipponbare and miR393b-overexpressing lines, and the colonization was rescued by MeJA but not by NAA. It suggests that the cross-talk between JA signaling and the auxin biosynthetic pathway plays a crucial role in the colonization of Acremonium sp. D212 in host plants.

ZmEHD1 Is Required for Kernel Development and Vegetative Growth through Regulating Auxin Homeostasis.

The roles of C-terminal Eps15 homology domain (EHD) proteins in clathrin-mediated endocytosis in plants are poorly understood. Here, we isolated a maize (Zea mays) mutant, designated ehd1, which showed defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that ehd1 encodes an EHD protein. Internalization of the endocytic tracer FM4-64 was substantially reduced in the ehd1 mutant and ZmEHD1 knockout mutants. We further demonstrated that ZmEHD1 and the ZmAP2 σ subunit physically interact at the plasma membrane. Auxin distribution and ZmPIN1a-YFP localization were altered in the ehd1 mutant. Kernel indole-3-acetic acid levels were substantially lower in the ehd1 mutant than in wild-type maize. Exogenous application of 1-naphthaleneacetic acid, but not GA3 or 2-naphthaleneacetic acid, rescued the seed germination and seedling emergency phenotypic defects of ehd1 mutants. Taken together, these results indicate that ZmEHD1 regulates auxin homeostasis by mediating clathrin-mediated endocytosis through its interaction with the ZmAP2 σ subunit, which is crucial for kernel development and vegetative growth of maize.

A metabolic pathway for the prodrug nabumetone to the pharmacologically active metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA) by non-cytochrome P450 enzymes.

The pathway for the transformation of the prodrug nabumetone, 4-(6-methoxynaphthalen-2-yl)butan-2-one, to the active metabolite 6-methoxy-2-naphthylacetic acid (6-MNA), a potent cyclooxygenase-2 inhibitor, has not yet been clarified in humans.To confirm the activation pathway, authentic standards of the nabumetone intermediates, 2-(6-methoxynaphthalen-2-yl)ethyl acetate (6-MNEA), 2-(6-methoxynaphthalen-2-yl)ethan-1-ol (6-MNE-ol) and 2-(6-methoxynaphthalen-2-yl)acetaldehyde (6-MN-CHO) were synthesized. High performance liquid-chromatography and gas chromatography-mass spectrometry on nabumetone oxidation revealed the generation of three metabolites.The formation of 6-MNA after a 60-min incubation of nabumetone was detected and 6-MNE-ol, an alcohol-related intermediate, was also generated by in cryopreserved hepatocytes. However, 6-MNA was below detection limit, but 4-(6-methoxynaphthalen-2-yl)butan-2-ol (MNBO) and 4-(6-hydroxynaphthalen-2-yl)butan-2-one (M3) peak were found in both the microsomes and S9 extracts with any cofactors.Nabumetone has recently been proposed as a typical substrate of flavin-containing monooxygenase isoform 5 (FMO5) and was shown to be efficiently oxidized in vitro to 6-MNEA. 6-MNA was detected in the extract obtained from a combined incubation of recombinant FMO5 and S9 fractions.The specificity of FMO5 towards catalyzing this Baeyer-Villiger oxidation (BVO) was demonstrated by the inhibition of the BVO substrate, 4-methoxyphenylacetone. Further in vitro inhibition studies demonstrated that multiple non-cytochrome P450 enzymes are involved in the formation of 6-MNA.

NAA at a high concentration promotes efficient plant regeneration via direct somatic embryogenesis and SE-mediated transformation system in Ranunculus sceleratus.

The novel methods for efficient plant regeneration via direct somatic embryogenesis (SE) and SE-mediated transformation system under high concentration of NAA in Ranunculus sceleratus were established. On MS media containing a high concentration of NAA (10.0 mg/L) in the dark, all inoculated explants (root, stem and leaf) formed somatic embryos at high frequencies, respectively, 66.03, 126.47 and 213.63 embryoids per explant, and 100% of the embryoids developed into plantlets on 1/2 MS rooting media. Morphological and histological analyses revealed that SE in R. sceleratus followed a classical pattern. All inoculated explants can be used as receptors for genetic transformation in R. sceleratus, through direct SE-mediated method after Agrobacterium infection. RcLEC1-B, as a marker gene, changed the number and morphology of flower organs and the development of cuticle in R. sceleratus, which indicated that the efficient transgenic system of R. sceleratus was established. To our knowledge, this is the first observation that both direct SE and transgenic transformation system, via induction of a single plant growth regulator, have been successfully constructed in R. sceleratus.

Gut microbiota metabolizes nabumetone in vitro: Consequences for its bioavailability in vivo in the rodents with altered gut microbiome.

1. The underlying microbial metabolic activity toward xenobiotics is among the least explored factors contributing to the inter-individual variability in drug response. 2. Here, we analyzed the effect of microbiota on a non-steroidal anti-inflammatory drug nabumetone. 3. First, we cultivated the drug with the selected gut commensal and probiotic bacteria under both aerobic and anaerobic conditions and analyzed its metabolites by high-performance liquid chromatography (HPLC) with UV detection. To analyze the effect of microbiota on nabumetone pharmacokinetics in vivo, we administered a single oral dose of nabumetone to rodents with intentionally altered gut microbiome - either rats treated for three days with the antibiotic imipenem or to germ-free mice. Plasma levels of its main active metabolite 6 methoxy-2-naphthylacetic acid (6-MNA) were analyzed at pre-specified time intervals using HPLC with UV/fluorescence detection. 4. We found that nabumetone is metabolized by bacteria to its non-active metabolites and that this effect is stronger under anaerobic conditions. Although in vivo, none of the pharmacokinetic parameters of 6-MNA was significantly altered, there was a clear trend towards an increase of the AUC, Cmax and t1/2 in rats with reduced microbiota and germ-free mice.

Novel Strategy to Decipher the Regulatory Mechanism of 1-Naphthaleneacetic Acid in Strawberry Maturation.

1-Naphthaleneacetic acid (NAA) has long been used to regulate strawberry growth. However, its regulatory mechanisms are unclear. Here, a nuclear magnetic resonance (NMR)-based metabolomics approach was utilized to capture differential metabolites, then matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and transcriptomics as assisted methods to validate the significant findings of metabolomics. The metabolomics results suggested that NAA regulated strawberry growth via multiple metabolic pathways, and different NAA application times also influenced these regulatory effects. We also found an interesting phenomenon that citric acid had completely opposite changes when NAA was sprayed at two different ripening stages of the strawberries. Furthermore, MALDI-TOF MS validated the changes of citric acid and transcriptomics identified the related genes. The study demonstrated that the novel strategy of "metabolomics capture-MALDI-TOF MS and transcriptomics assisted validation" could offer a fresh insight for understanding the mechanism of the plant growth regulator in strawberry maturation.

A supramolecular hydrogel for spatial-temporal release of auxin to promote plant root growth.

Short peptide-based hydrogels have attracted extensive research interests in drug delivery because of their responsive properties. So far, most drug molecules have been conjugated with short peptides via an amide bond, restricting the release of the native drug molecules. In this study, we demonstrated the effectiveness of an auxin-based hydrogelator linked by a hydrolysable ester bond. Hydrogel I, formed by the gelator (NAA-G'FFY) linked with an ester bond, was able to release 1-naphthaleneacetic acid (NAA), whereas hydrogel II, formed by the gelator without an ester bond (NAA-GFFY), was not. By mixing NAA-G'FFY with Fmoc-GFFY to form a two-component hydrogel, the spatial and temporal release of NAA was achieved, promoting on-site auxin responses including primary root elongation and lateral root formation in the model plant Arabidopsis thaliana. The strategy of using a hydrolysable ester bond to connect drug molecules and self-assembling peptides could lead to the development of supramolecular hydrogels with more controllable drug release profiles.

The enhanced biomass and lipid accumulation in Coccomyxa subellipsoidea with an integrated treatment strategy initiated by brewery effluent and phytohormones.

Brewery effluent (BE) as an appreciable and sustainable resource presented new possibilities in low-cost algal biomass production, whereas the relatively low essential macronutrients hindered extensive applications as growth medium for microalgae cultivation. The objective of this study was to investigate the feasibility of an integrated treatment strategy initiated by BE coupling phytohormones in augmenting biomass and lipid accumulation in Coccomyxa subellipsoidea. Results revealed that BE coupling synthetic 1-naphthaleneacetic acid (NAA) accomplished the favorable lipid productivity of 481.76 mg/L/days, representing 6.80- to 9.71-fold more than that of single BE as well as standard Basal media. BE coupling NAA feeding also heightened the proportions of C16-C18 fatty acids (over 96%) and mono-unsaturated C18:1 (approximate 45%) which were prone to high-quality biofuels-making. Such profound lipids accumulation might be attributable to that BE coupling NAA treatment drove most of metabolic flux (i.e. acetyl-CoA) derived from TCA cycle and glycolysis flowing into lipid accumulation pathway. Concurrently, the complete removal of total nitrogen and total phosphorus by C. subellipsoidea with assistance of NAA were easily complied with the permissible dischargeable limits for BE. These present results strongly demonstrated that BE coupling NAA was a potential feeding strategy in boosting algal lipid productivity and further provided great possibilities in linking affordable algal biomass production with high-efficient biological contaminants removal.

Expression Profiling of Strawberry Allergen Fra a during Fruit Ripening Controlled by Exogenous Auxin.

Strawberry fruit contain the allergenic Fra a proteins, members of the pathogenesis-related 10 protein family that causes oral allergic syndrome symptoms. Fra a proteins are involved in the flavonoid biosynthesis pathway, which might be important for color development in fruits. Auxin is an important plant hormone in strawberry fruit that controls fruit fleshiness and ripening. In this study, we treated strawberry fruits with exogenous auxin or auxin inhibitors at pre- and post-harvest stages, and analyzed Fra a transcriptional and translational expression levels during fruit development by real-time PCR and immunoblotting. Pre-harvest treatment with 1-naphthaleneacetic acid (NAA) alone did not affect Fra a expression, but applied in conjunction with achene removal NAA promoted fruit pigmentation and Fra a protein accumulation. The response was developmental stage-specific: Fra a 1 was highly expressed in immature fruit, whereas Fra a 2 was expressed in young to ripe fruit. In post-harvest treatments, auxin did not contribute to Fra a induction. Auxin inhibitors delayed fruit ripening; as a result, they seemed to influence Fra a 1 expression. Thus, Fra a expression was not directly regulated by auxin, but might be associated with the ripening process and/or external factors in a paralog-specific manner.

Adaptation of root growth to increased ambient temperature requires auxin and ethylene coordination in Arabidopsis.

KEY MESSAGE: A fresh look at the roles of auxin, ethylene, and polar auxin transport during the plant root growth response to warmer ambient temperature (AT). The ambient temperature (AT) affects plant growth and development. Plants can sense changes in the AT, but how this change is transduced into a plant root growth response is still relatively unclear. Here, we found that the Arabidopsis ckrc1-1 mutant is sensitive to higher AT. At 27 °C, the ckrc1-1 root length is significantly shortened and the root gravity defect is enhanced, changes that can be restored with addition of 1-naphthaleneacetic acid, but not indole-3-acetic acid (IAA). AUX1, PIN1, and PIN2 are involved in the ckrc1-1 root gravity response under increased AT. Furthermore, CKRC1-dependent auxin biosynthesis was critical for maintaining PIN1, PIN2, and AUX1 expression at elevated temperatures. Ethylene was also involved in this regulation through the ETR1 pathway. Higher AT can promote CKRC1-dependent auxin biosynthesis by enhancing ETR1-mediated ethylene signaling. Our research suggested that the interaction between auxin and ethylene and that the interaction-mediated polar auxin transport play important roles during the plant root growth response to higher AT.

The effects of exogenous hormones on rooting process and the activities of key enzymes of Malus hupehensis stem cuttings.

Malus hupehensis is an excellent Malus rootstock species, known for its strong adverse-resistance and apomixes. In the present study, stem cuttings of M. hupehensis were treated with three types of exogenous hormones, including indole acetic acid (IAA), naphthalene acetic acid (NAA), or green growth regulator (GGR). The effects and mechanisms of exogenous hormone treatment and antioxidant enzyme activity on adventitious root formation were investigated. The results showed that the apparent morphology of the adventitious root had four stages, including root pre-emergence stage (S0), early stage of root formation (S1), massive root formation stage (S2), and later stage of root formation (S3). The suitable concentrations of the three exogenous hormones, IAA, NAA and GGR, were 100 mg·L-1, 300 mg·L-1, and 300 mg·L-1, respectively. They shortened the rooting time by 25-47.4% and increased the rooting percentages of cuttings by 0.9-1.3 times, compared with that in the control. The dispersion in S0 stage was 3.6 times of that in the S1 stage after exogenous hormone application. The earlier the third critical point (P3) appeared, the shorter the rooting time and the greater the rooting percentage of the cuttings. During rhizogenesis, the activities of three antioxidant enzymes (POD, SOD, and PPO) showed an A-shaped trend. However, peak values of enzyme activity appeared at different points, which were 9 d before the P3, P3, and the fourth critical point (P4), respectively. Exogenous hormone treatment reduced the time to reach the peak value by 18 days, although the peak values of the enzymatic activities did not significantly changed. Our results suggested that exogenous hormone treatment mainly acted during the root pre-emergence stage, accelerated the synthesis of antioxidant enzymes, reduced the rooting time, and consequently promoted root formation. The three kinds of antioxidant enzymes acted on different stages of rooting.

SlTIR1 is involved in crosstalk of phytohormones, regulates auxin-induced root growth and stimulates stenospermocarpic fruit formation in tomato.

TIR1 and its homologs act as auxin receptors and play important roles in plant growth and development in Arabidopsis thaliana. An auxin receptor homolog Solanum lycopersicum TIR1 (SlTIR1) has been isolated from tomato cultivar Micro-Tom, and SlTIR1 over-expression results in parthenocarpic fruit formation. In this study, the promoter driving the ß-glucuronidase (GUS) expression vector was constructed and stably transformed into Micro-Tom seedlings. The SlTIR1 promoter driving GUS expression accumulated predominantly in the leaf and vasculature in transgenic seedlings. Promoter analysis identified an auxin-response element (AuxRE) and two gibberellic acid (GA)-response elements in the SlTIR1 promoter. Quantitative PCR showed that SlTIR1 transcript level was down-regulated by naphthaleneacetic acid, ethephon and abscisic acid and up-regulated by GA. Furthermore, because of the lack of ability to form reproductive seeds in SlTIR1 over-expressing Micro-Tom, this limits further exploration of potential roles of SlTIR1 in auxin signaling. Here, an antisense vector and an over-expression vector of the SlTIR1 gene were stably transformed into Micro-Tom and Ailsa Craig tomato, respectively. Phenotypes and physiological analyses indicated that SlTIR1 regulated primary root growth and auxin-associated lateral root formation in Micro-Tom. Meanwhile, SlTIR1 also stimulated abnormal seed development, so-called stenospermocarpy, in Ailsa Craig. Transcript accumulations of auxin-signaling genes determined by quantitative PCR were consistent with the idea that SlTIR1 regulated plant growth and development, partially mediated by controlling the mRNA levels of auxin-signaling genes. Our work demonstrates that SlTIR1 regulated auxin-induced root growth and stimulated stenospermocarpic fruit formation. SlTIR1 may be a key mediator of the crosstalk among auxin and other hormones to co-regulate plant growth and development.

Auxin-cytokinin interaction and variations in their metabolic products in the regulation of organogenesis in two Eucomis species.

In the current study, we evaluated the effect of α-naphthaleneacetic acid (NAA) individually or in combination with different cytokinins (CKs) including benzyladenine (BA), meta-topolin (mT) and isopentenyladenine (iP) on organogenesis, auxin and CK content in Eucomis autumnalis subspecies autumnalis (EA) and Eucomis zambesiaca (EZ). These species were used as model plants due to their ornamental and medicinal properties. Three leaf explants were inoculated in screw-cap jars containing 30mL Murashige and Skoog (MS) media supplemented with 5µM NAA alone or in combination with 5µM CK (BA, mT or iP). After 10 weeks (EA) or 15 weeks (EZ), parameters including shoot and root growth as well as plant fresh weight were recorded. For analysis of auxin and CK content, whole plantlets were harvested, pooled and freeze-dried for the different treatments. In both species, shoot and root proliferation as well as plant biomass were generally higher when NAA was combined with the individual CK than in NAA or CK treatment. The highest concentration of indole-3-acetic acid (IAA, 619pmolg-1 DW) and 2-oxindole-3-acetic acid (OxIAA, 2381pmolg-1 DW) were observed in EA-treated with NAA alone while mT treatment (without NAA) had the most abundant indole-3-acetyl-l-aspartic acid (IAAsp, 904 and 582pmolg-1 DW for EA and EZ, respectively) in both species. A significant concentration of total endogenous CK accumulated in both Eucomis regenerants from mT and mT+NAA when compared to the other treatments. The majority of the detected CKs were of the aromatic CK-type, mainly free bases. The potential physiological roles of these quantified phytohormones in relation to the observed morphological responses are discussed.

Interactions of Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN reveal a novel intracellular auxin transport mechanism.

Little is known about the transport mechanism of intracellular auxin. Here, we report two vacuole-localized proteins, Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN (OsCLIP), that regulate intracellular auxin transport and homoeostasis. Overexpression of OsCOLE1 markedly increased the internode length and auxin content of the stem base, whereas these parameters were decreased in RNA interference (RNAi) plants. OsCOLE1 was localized on the tonoplast and preferentially expressed in mature tissues. We further identified its interacting protein OsCLIP, which was co-localized on the tonoplast. Protein-protein binding assays demonstrated that the N-terminus of OsCOLE1 directly interacted with OsCLIP in yeast cells and the rice protoplast. Furthermore, (3) H-indole-3-acetic acid ((3) H-IAA) transport assays revealed that OsCLIP transported IAA into yeast cells, which was promoted by OsCOLE1. The results indicate that OsCOLE1 affects rice development by regulating intracellular auxin transport through interaction with OsCLIP, which provides a new insight into the regulatory mechanism of intracellular transport of auxin and the roles of vacuoles in plant development.

In Vitro Propagation of Sambong (Blumea balsamifera Linn.).

Terminal shoot tips of sambong (Blumea balsamifera Linn.) are cultured to initiate and regenerate shoots on Murashige and Skoog (MS) medium containing 1.0 mg/L benzyl adenine (BA). After 1 month, shoots, usually 4.5 cm long are separated and subcultured for multiplication. Regenerated shoots, about 6 cm long are rooted on MS medium supplemented with 1.0 mg/L naphthalene acetic acid (NAA). Exposure of shoots to high humidity for the first 2 weeks and equal proportion (1:1:1) of sterile sand, compost, and coir dust as potting mix favors the development of whole sambong plants. Young shoots from in vitro-derived sambong plants could also be used for propagation.

A novel injection strategy of flurbiprofen axetil by inhibiting protein binding with 6-methoxy-2-naphthylacetic acid.

Flurbiprofen axetil (FPA) is an injection product and a prodrug of a non-steroidal anti-inflammatory drug (NSAID). After injection, it is rapidly hydrolyzed to the active form, flurbiprofen (FP). Since frequent injections of FPA can lead to abnormal physiology, an administration strategy is necessary to ensure there is enhancement of the analgesic efficiency of FP after a single dose and to reduce the total number of doses. FP strongly binds to site II of albumin, and thus the free (unbound) FP concentration is low. This study focused on 6-methoxy-2-naphthylacetic acid (6-MNA), the active metabolite of nabumetone (a prodrug of NSAID). We performed ultrafiltration experiments and pharmacokinetics analysis in rats to investigate whether the inhibitory effect of 6-MNA on FP binding to albumin increased the free FP concentration in vitro and in vivo. Results indicated that 6-MNA inhibited the binding of FP to albumin competitively. When 6-MNA was injected in rats, there was a significant increase in the free FP concentration and the area under concentration-time curve (AUC) calculated from the free FP concentration, while there was a significant decrease in the total (bound + free) FP concentration and the AUC calculated from the total FP concentration. These findings indicate that 6-MNA inhibits the protein binding of FP in vivo. This suggests that the frequency of FPA injections can be reduced when administered with nabumetone, as there is increase in the free FP concentration associated with pharmacological effect.