Inhibitory Effect of Oroxylin A in a Mouse Model of Atopic Dermatitis.

Scutellaria baicalensis has long been used in Asian traditional medicine to prevent and treat suppurative dermatitis, allergic diseases, inflammation, hyperlipemia, and arteriosclerosis. Oroxylin A is a flavone present in Scutellaria baicalensis. Because the root extracts of Scutellaria baicalensis have been shown to have anti-dermatitis effects, the authors investigated the effects of oroxylin A on chemically induced atopic dermatitis (AD) in an in vivo AD model induced by 1-chloro-2,4-dinitrobenzene (CDNB) in BALB/c mice. CDNB-induced skin hypertrophy and accumulation of mast cells in the epidermis and dermis were significantly decreased by oroxylin A. Increased serum levels of immunoglobulin E, as well as pro-inflammatory chemokines and cytokines in the skin and lymph nodes, were significantly decreased by oroxylin A. Suppression of immune responses in the skin and lymph nodes by oroxylin A decreased the symptoms of AD. Thus, these results proved that oroxylin A is an effective component of Scutellaria baicalensis for treating the symptoms of AD.

Mutations in the Pectin Methyltransferase QUASIMODO2 Influence Cellulose Biosynthesis and Wall Integrity in Arabidopsis.

Pectins are abundant in the cell walls of dicotyledonous plants, but how they interact with other wall polymers and influence wall integrity and cell growth has remained mysterious. Here, we verified that QUASIMODO2 (QUA2) is a pectin methyltransferase and determined that QUA2 is required for normal pectin biosynthesis. To gain further insight into how pectin affects wall assembly and integrity maintenance, we investigated cellulose biosynthesis, cellulose organization, cortical microtubules, and wall integrity signaling in two mutant alleles of Arabidopsis (Arabidopsis thaliana) QUA2, qua2 and tsd2 In both mutants, crystalline cellulose content is reduced, cellulose synthase particles move more slowly, and cellulose organization is aberrant. NMR analysis shows higher mobility of cellulose and matrix polysaccharides in the mutants. Microtubules in mutant hypocotyls have aberrant organization and depolymerize more readily upon treatment with oryzalin or external force. The expression of genes related to wall integrity, wall biosynthesis, and microtubule stability is dysregulated in both mutants. These data provide insights into how homogalacturonan is methylesterified upon its synthesis, the mechanisms by which pectin functionally interacts with cellulose, and how these interactions are translated into intracellular regulation to maintain the structural integrity of the cell wall during plant growth and development.

Huang-Lian-Jie-Du extract ameliorates atopic dermatitis-like skin lesions induced by 2,4-dinitrobenzene in mice via suppression of MAPKs and NF-κB pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du Decoction (HLJDD), is a well-known traditional Chinese herbal formula first written in the Tang dynasty. In Chinese medicine practice, HLJDD is commonly prescribed to treat various inflammatory skin diseases, such as atopic dermatitis (AD) and psoriasis. AIM OF THE STUDY: The present study aimed at investigating the therapeutic effect of HLJDD extract (HLJDE) and to elucidate the underlying molecular mechanisms of action in the 1-chloro-2,4-dinitrobenzene (DNCB)-induced AD-like mice. MATERIALS AND METHODS: Female Balb/c mice were sensitized with DNCB for three days. After sensitization, mice were challenged with DNCB every three days and orally administrated with HLJDE (150, 300 and 600 mg/kg) daily from day 14 to day 29 for consecutive 16 days. At the end of experiment, the clinical AD scores of the mice were calculated to evaluate the therapeutic effect of HLJDE, and serum, ears and dorsal skin of the mice were collected for unravelling molecular mechanisms. RESULTS: HLJDE significantly reduced the clinical symptoms in the AD-like mice by inhibiting eosinophil and mast cell infiltration, suppressing the production of Th2-associated cytokine (IL-4) and pro-inflammatory cytokines (TNF-α). In addition, HLJDE significantly suppressed the NF-κB and MAPKs pathways. Moreover, HLJDE was able to accentuate filaggrin expression in the skin lesion when compared to the sensitized mouse without treatment. CONCLUSION: HLJDE significantly improved the AD-like symptoms on the DNCB-sensitized mice through mitigating the production of inflammatory mediators via suppressing MAPKs and NF-κB pathways. Additionally, the elevated expression of filaggrin in the skin lesion by HLJDE contributes to the recovery of dysfunctional skin barrier on the DNCB-sensitized mice.

Preprophase band formation and cortical division zone establishment: RanGAP behaves differently from microtubules during their band formation.

Correct positioning of the division plane is a prerequisite for plant morphogenesis. The preprophase band (PPB) is a key intracellular structure of division site determination. PPB forms in G2 phase as a broad band of microtubules (MTs) that narrows in prophase and specializes few-micrometer-wide cortical belt region, named the cortical division zone (CDZ), in late prophase. The PPB comprises several molecules, some of which act as MT band organization and others remain in the CDZ marking the correct insertion of the cell plate in telophase. Ran GTPase-activating protein (RanGAP) is accumulated in the CDZ and forms a RanGAP band in prophase. However, little is known about when and how RanGAPs gather in the CDZ, and especially with regard to their relationships to MT band formation. Here, we examined the spatial and temporal distribution of RanGAPs and MTs in the preprophase of onion root tip cells using confocal laser scanning microscopy and showed that the RanGAP band appeared in mid-prophase as the width of MT band was reduced to nearly 7 µm. Treatments with cytoskeletal inhibitors for 15 min caused thinning or broadening of the MT band but had little effects on RanGAP band in mid-prophase and most of late prophase cells. Detailed image analyses of the spatial distribution of RanGAP band and MT band showed that the RanGAP band positioned slightly beneath the MT band in mid-prophase. These results raise a possibility that RanGAP behaves differently from MTs during their band formation.

The microtubule cytoskeleton and pollen tube Golgi vesicle system are required for in vitro S-RNase internalization and gametic self-incompatibility in apple.

S-RNase is the female determinant of gametophytic self-incompatibility in apple and is usually considered to be the reason for rejection of pollen. In this study, we investigated the role of microtubules (MTs) in internalization of S-RNases by pollen tubes cultured in vitro. The results showed that S-RNase was imported into the pollen tube where it inhibits pollen tube growth, and that S-RNase is co-localized with the Golgi vesicle during the internalization process. Moreover, MT depolymerization is observed following accumulation of S-RNases in the pollen cytosol. On the other hand, S-RNase was prevented from entering the pollen tube when the pollen was treated with the actin filament (AF) inhibitor latrunculin A (LatA), the MT inhibitor oryzalin, or the MT stabilizer taxol at subtoxic concentrations. These hindered the construction of the MT, with pollen tubes capable of growth under these conditions. Pollen tubes showed improved growth in self-pollinated styles that were pre-treated with taxol. This suggests that cytoskeleton antagonists can prevent S-RNase-mediated inhibition of pollen tubes in vivo by blocking S-RNase internalization. These results suggest that an intact and dynamic cytoskeleton is required for the in vitro internalization of S-RNase, as shown by the effects of various cytoskeleton inhibitors. S-RNase internalization takes place via a membrane/cytoskeleton-based Golgi vesicle system, which can also affect self-incompatibility in apple.

Disruption of the microtubule network alters cellulose deposition and causes major changes in pectin distribution in the cell wall of the green alga, Penium margaritaceum.

Application of the dintroaniline compound, oryzalin, which inhibits microtubule formation, to the unicellular green alga Penium margaritaceum caused major perturbations to its cell morphology, such as swelling at the wall expansion zone in the central isthmus region. Cell wall structure was also notably altered, including a thinning of the inner cellulosic wall layer and a major disruption of the homogalacturonan (HG)-rich outer wall layer lattice. Polysaccharide microarray analysis indicated that the oryzalin treatment resulted in an increase in HG abundance in treated cells but a decrease in other cell wall components, specifically the pectin rhamnogalacturonan I (RG-I) and arabinogalactan proteins (AGPs). The ring of microtubules that characterizes the cortical area of the cell isthmus zone was significantly disrupted by oryzalin, as was the extensive peripheral network of actin microfilaments. It is proposed that the disruption of the microtubule network altered cellulose production, the main load-bearing component of the cell wall, which in turn affected the incorporation of HG in the two outer wall layers, suggesting coordinated mechanisms of wall polymer deposition.

Micronucleation by mitosis inhibitors in developing microspores of Spathiphyllum wallisii Regel.

KEY MESSAGE : We developed an efficient protocol for chromosome scattering in Spathiphyllum microspores. The effects of plant material, developmental age, genotype and antimicrotubular toxin type, exposure and concentration were evaluated. Asymmetric hybridization through microprotoplast-mediated chromosome transfer (MMCT) is a known method for overcoming sexual breeding barriers between distantly related plant species. To obtain microprotoplasts, it is necessary to induce mass micronucleation either in somatic or gametic cells. We have tested the efficiency for micronuclei induction of five mitosis inhibitors, amiprophos-methyl (APM), butamiphos (BUT), chlorpropham (CIPC), oryzalin (ORY) and propyzamide (PRO), on developing microspores of diploid Spathiphyllum wallisii Regel. Besides the used toxins, also the effect of their concentrations and incubation period as well as plant genotypes and material was tested. We observed micronuclei (MNi) in pollen mother cells, dyads and tetrads as well as other abnormalities such as ball metaphases and chromosome bridges. The flower position on the spadix and the type of starting material (dissected anthers vs. complete spadices) did not significantly influence micronucleation frequencies. The highest micronucleation index of 86 % was obtained in microspores treated with 10 µM ORY during 72 h. All six genotypes tested formed micronuclei after this particular treatment, although the efficiency varied between cultivars. Next to ORY, CIPC was also a very efficient MNi inducer. The average number of MNi found in micronucleated cells varied between 1.67-6.44 for CIPC and 0.83-5.50 for ORY. The maximal number of MNi observed was 12 for CIPC and 9 for ORY. Our results demonstrate that CIPC and ORY can be applied for mass micronucleation on developing microspores of S. wallisii as a first step of MMCT in aroid interspecific or intergeneric breeding.

Lipid nanoparticles containing oryzalin for the treatment of leishmaniasis.

Oryzalin is a dinitroaniline drug that has attracted recent interest for the treatment of leishmaniasis. Its use as an antiparasitic therapeutic agent is limited by the low water solubility associated with an in vivo rapid clearance, leading to the administration of larger and possibly toxic doses in in vivo studies, and the use of solvents that may lead to undesirable side effects. In the present work oryzalin-containing lipid nanoparticles were produced by a emulsion-solvent evaporation technique using a composition suitable for parenteral administration, i.e., tripalmitin (solid lipid) and a complex mixture of three emulsifying agents (soya lecithin, Tween® 20 and sodium deoxycholate). Physicochemical characterization included the determination of mean particle size, polydispersity index, zeta potential, encapsulation efficiency and DSC studies. Final formulations revealed values of <140 nm (PI<0.2) and zeta potential of ≈-35 mV, as well as encapsulation efficiency >75%. The effects of various processing parameters, such as lipid and surfactant and composition and concentration, as well as the stability during the harsh procedures of autoclaving (121°C/15 min) and freeze-drying were also evaluated. Formulations revealed to be stable throughout freeze-drying and moist-heath sterilization without significant variations on physicochemical properties and no significant oryzalin losses. The use of a complex surfactant mixture proved crucial for preserving formulation stability. Particularly, lecithin appears as a key component in the stabilization of tripalmitin-based oryzalin-containing lipid nanoparticles. Finally, cell viability studies demonstrated that the incorporation of oryzalin in nanoparticles decreases cytotoxicity, thus suggesting this strategy may improve tolerability and therapeutic index of dinitroanilines.

Secondary metabolite profile in induced tetraploids of wild Solanum commersonii Dun.

The main aim of this work was to study the leaf secondary metabolite profiles of artificially induced tetraploids (2n=4x=48) of Solanum commersonii, a diploid (2n=2x=24) wild potato species. The tetraploid genotypes of S. commersonii were produced by oryzalin treatment. Both HPLC-UV and LC/MS analyses revealed that there were no qualitative differences in the metabolite profiles between the diploid S. commersonii and its tetraploids. By contrast, the results showed that the phenylpropanoid content was generally significantly higher in the tetraploids than in the diploid S. commersonii. Concerning the glycoalkaloids (GAs), the results provided evidence that the content of minor GAs (solanidenediol triose, solanidadienol lycotetraose, and solanidenol lycotetraose) was higher in tetraploids than in the diploid progenitor, while the content of major GAs (dehydrodemissine and dehydrocommersonine) was significantly higher in diploid S. commersonii than in its tetraploid genotypes. The results are discussed from the practical perspective of potato biodiversity enhancement.

Synthesis and evaluation of oryzalin analogs against Toxoplasma gondii.

The synthesis and evaluation of 20 dinitroanilines and related compounds against the obligate intracellular parasite Toxoplasma gondii is reported. Using in vitro cultures of parasites in human fibroblasts, we determined that most of these compounds selectively disrupted Toxoplasma microtubules, and several displayed sub-micromolar potency against the parasite. The most potent compound was N(1),N(1)-dipropyl-2,6-dinitro-4-(trifluoromethyl)-1,3-benzenediamine (18b), which displayed an IC(50) value of 36 nM against intracellular T. gondii. Based on these data and another recent report [Ma, C.; Tran, J.; Gu, F.; Ochoa, R.; Li, C.; Sept, D.; Werbovetz, K.; Morrissette, N. Antimicrob. Agents Chemother. 2010, 54, 1453], an antimitotic structure-activity relationship for dinitroanilines versus Toxoplasma is presented.

Plant cell division is specifically affected by nitrotyrosine.

Virtually all eukaryotic alpha-tubulins harbour a C-terminal tyrosine that can be reversibly removed and religated, catalysed by a specific tubulin-tyrosine carboxypeptidase (TTC) and a specific tubulin-tyrosine ligase (TTL), respectively. The biological function of this post-translational modification has remained enigmatic. 3-nitro-L-tyrosine (nitrotyrosine, NO(2)Tyr), can be incorporated into detyrosinated alpha-tubulin instead of tyrosine, producing irreversibly nitrotyrosinated alpha-tubulin. To gain insight into the possible function of detyrosination, the effect of NO(2)Tyr has been assessed in two plant model organisms (rice and tobacco). NO(2)Tyr causes a specific, sensitive, and dose-dependent inhibition of cell division that becomes detectable from 1 h after treatment and which is not observed with non-nitrosylated tyrosine. These effects are most pronounced in cycling tobacco BY-2 cells, where the inhibition of cell division is accompanied by a stimulation of cell length, and a misorientation of cross walls. NO(2)Tyr reduces the abundance of the detyrosinated form of alpha-tubulin whereas the tyrosinated alpha-tubulin is not affected. These findings are discussed with respect to a model where NO(2)Tyr is accepted as substrate by TTL and subsequently blocks TTC activity. The irreversibly tyrosinated alpha-tubulin impairs microtubular functions that are relevant to cell division in general, and cell wall deposition in particular.

Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability.

Shoot tips of the diploid rose Thérèse Bugnet were treated in vitro to oryzalin at concentrations of 5 and 15 microM. Tetraploid shoots were obtained in highest frequencies (40%) after exposure to 5 microM oryzalin for 14 days. Thin (1 mm) nodal sections were treated with 5 microM oryzalin and the highest frequency of tetraploids (66%) was obtained after exposure for only 1 day. The shorter exposure times required to induce chromosome doubling in thin nodal sections is attributed to the more efficient delivery of oryzalin to the meristem. Tetraploids were obtained from four diploid roses and hexaploids from two triploid roses. Chromosome doubling was accompanied by increases in thickness and a darker green colouration of the leaves and, in all diploid to tetraploid and one triploid to hexaploid conversion, the breadth/length ratio of leaflets was significantly increased. Internodes were longer in tetraploids than diploids but significantly shorter in hexaploids than triploids. The number of petals per flower in the tetraploid form of Thérèse Bugnet was double that of the diploid. Significant increases in pollen viability accompanied chromosome doubling of all four diploids and one of the two triploids.

Simultaneous visualization of peroxisomes and cytoskeletal elements reveals actin and not microtubule-based peroxisome motility in plants.

Peroxisomes were visualized in living plant cells using a yellow fluorescent protein tagged with a peroxisomal targeting signal consisting of the SKL motif. Simultaneous visualization of peroxisomes and microfilaments/microtubules was accomplished in onion (Allium cepa) epidermal cells transiently expressing the yellow fluorescent protein-peroxi construct, a green fluorescent protein-mTalin construct that labels filamentous-actin filaments, and a green fluorescent protein-microtubule-binding domain construct that labels microtubules. The covisualization of peroxisomes and cytoskeletal elements revealed that, contrary to the reports from animal cells, peroxisomes in plants appear to associate with actin filaments and not microtubules. That peroxisome movement is actin based was shown by pharmacological studies. For this analysis we used onion epidermal cells and various cell types of Arabidopsis including trichomes, root hairs, and root cortex cells exhibiting different modes of growth. In transient onion epidermis assay and in transgenic Arabidopsis plants, an interference with the actin cytoskeleton resulted in progressive loss of saltatory movement followed by the aggregation and a complete cessation of peroxisome motility within 30 min of drug application. Microtubule depolymerization or stabilization had no effect.

The behaviour of the plasma membrane during plasmolysis: a study by UV microscopy.

A high resolution ultraviolet (UV) bright-field microscope was used to analyse the formation of Hechtian strands and the Hechtian reticulation that remain attached to the cell wall after plasmolysis and deplasmolysis of onion inner epidermal cells. In real time video images, UV microscopy allowed a detailed investigation of the dynamic behaviour of the plasma membrane during the processes of osmotic water loss and uptake. Furthermore, the role of cytoskeletal elements as possible linkers of the plasma membrane to the cell wall was probed by application of cytoskeletal drugs during plasmolysis. Microtubules were depolymerized in oryzalin, and latrunculin B was used to destabilize actin microfilaments. The results showed no visible changes in the formation of the Hechtian reticulation or strands. Plasmolysis forms appeared to be normal, indicating stong membrane-to-wall attachments independent of cytoskeletal elements. During re-expansion of the protoplast in deplasmolysis, the plasma membrane incorporated Hechtian strands and subprotoplasts, fused with the Hechtian reticulation and finally realigned at the cell wall.

Changes in testicular and serum hormone concentrations in the male rat following treatment with m-dinitrobenzene.

m-Dinitrobenzene (m-DNB)-induced testicular atrophy has been attributed to a direct effect upon the germinal epithelium. However, such degenerative changes in the germinal epithelium should induce shifts in the testicular hormonal milieu, which would in turn alter the hypothalamic-pituitary gonadal axis in general. This study evaluated the endocrine status of male rats (killed 3 hr, 24 hr, 1 week, and 2 weeks) following a single oral dose of m-DNB (32 mg m-DNB/kg). Serum and pituitary leuteinizing hormone, follicle-stimulating hormone (FSH), and protactin and hypothalamic gonadotropin-releasing hormone (GnRH) concentrations were determined. Testosterone and androgen-binding protein concentrations in serum, interstitial fluid, seminiferous tubule fluid, and caput epididymis were also determined. In vitro basal and hCG-stimulated testosterone release was determined in the decapsulated testis. Results of the present study indicate that pituitary hormone concentrations and hypothalamic GnRH were unaffected after a single oral dose of m-DNB. Serum FSH was elevated at 2 weeks. There was a transient decrease in serum testosterone at 24 hr, which returned to control values at 1 and 2 weeks. Interstitial fluid, seminiferous tubule fluid, and caput epididymal testosterone concentrations were increased at 1 and 2 weeks. Basal testosterone release in vitro was increased at 2 weeks, while hCG-stimulated testosterone release was increased at 1 and 2 weeks. Androgen-binding protein concentrations in serum and interstitial fluid were increased at 1 and 2 weeks. Androgen-binding protein was increased at 24 hr and 1 week in seminiferous tubule fluid, but returned to control concentrations by 2 weeks. However, the total tubular content of androgen-binding protein was dramatically decreased at 2 weeks. Androgen-binding protein in the caput epididymis was unaltered following m-DNB treatment. These data demonstrate that m-DNB exerts a direct effect on the testes and not through alterations in hypothalamic and pituitary control of gonadal function.