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Background Plants are exposed to various forms of environmental stress. Penetration by pathogens is one of the most serious environmental insults. Wounding caused by tissue damage or herbivory also affects the growth and reproduction of plants. Moreover, wounding disrupts physical barriers present at the plant surface and increases the risk of pathogen invasion. Plants cope with environmental stress by inducing a variety of responses. These stress responses must be tightly controlled, because their unnecessary induction is detrimental to plant growth. In tobacco, WIPK and SIPK, two wound-responsive mitogen-activated protein kinases, have been shown to play important roles in regulating wound responses. However, their contribution to downstream wound responses such as gene expression is not well understood. Results To identify genes regulated by WIPK and SIPK, the transcriptome of wounded WIPK/SIPK-suppressed plants was analyzed. Among the genes down-regulated in WIPK/SIPK-suppressed plants, the largest group consisted of those involved in the production of antimicrobial phytoalexins. Almost all genes involved in the biosynthesis of capsidiol, a major phytoalexin in tobacco, were transcriptionally induced by wounding in WIPK/SIPK-dependent and -independent manners. 5-epi-aristolochene synthase (EAS) is the committing enzyme for capsidiol synthesis, and the promoter of EAS4, a member of the EAS family, was analyzed. Reporter gene analysis revealed that at least two regions each 40-50 bp length were involved in activation of the EAS4 promoter by wounding, as well as by artificial activation of WIPK and SIPK. Unlike transcripts of the capsidiol synthesis genes, accumulation of EAS protein and capsidiol itself were not induced by wounding; however, wounding significantly enhanced their subsequent induction by a pathogen-derived elicitor. Conclusions Our results suggest a so-called priming phenomenon since the induction of EAS by wounding is only visible at the transcript level. By inducing transcripts, not the proteins, of EAS and possibly other capsidiol synthesis genes at wound sites, plants can produce large quantities of capsidiol quickly if pathogens invade the wound site, whereas plants can minimize energy loss and avoid the cytotoxic effects of capsidiol where pathogens do not gain entry during wound healing., Article, BMC Plant Biology 19(1) : 576, (2019)

The oomycete Phytophthora infestans is the causal agent of potato late blight, one of the most devastating pathogens for potato. To investigate the plant mechanisms for resistance against P. infestans, the Solanaceae model plant Nicotiana benthamiana was employed in this study. Previously, we reported that NbNup75, a nuclear pore complex protein, is required for the resistance against P. infestans, suggesting that nuclear-pore-mediated transport is involved in the induction of defense responses. In this study, we investigated the role of N. benthamiana exportin 1 NbXpo1 in disease resistance. Mammalian and yeast exportin 1 proteins are known as a regulator for nuclear-pore-mediated export of proteins and RNAs. NbXpo1-silenced N. benthamiana showed minor growth defects and significantly decreased resistance to P. infestans. Gene silencing of NbXpo1 compromised defense responses induced by the elicitor INF1 (a secretory protein of P. infestans), such as production of the phytoalexin capsidiol and induction of cell death. In NbXpo1-silenced plants, the genes for capsidiol biosynthesis, NbEAS, and defense-related MAP kinase, NbWIPK, were significantly downregulated, while genes encoding the plant defensin, NbPDF, and anti-microbial protein thionin, NbTHI, were upregulated. These results indicate that N. benthamiana Xpo1 is involved in activating a specific group of defense-related genes.

Treatment of epilepsy remains difficult because patients suffer from pharmacoresistant forms of the disease and drug side-effects. Thus, there is an urgent need to identify not only new antiepileptic drug candidates but also novel epileptic animal models. Here, we characterize seizures induced with kainic acid (KA) in the common marmoset (Callithrix jacchus). Adult marmosets received 0.1, 1, or 10 mg/kg of KA subcutaneously. All animals exhibited early convulsive behavior (seizure scores of I and II on the Racine scale). Seizure scores were low at lower KA doses, but the highest dose of KA tested triggered generalized seizures (scores IV and V on the Racine scale). We next performed preliminary evaluation of the efficacy of the antiepileptic drug diazepam. This drug at 1 mg/kg (delivered subcutaneously) prevented 10 mg/kg KA-induced stage V seizures. KA administration to marmosets reliably triggers generalized seizures; therefore, the marmoset is a useful animal model in which to analyze the seizures of a nonhuman primate brain and to develop new treatments for epilepsy.

Plant cells enhance the tolerances to abiotic and biotic stresses via recognition of the stress, activation and nuclear import of signaling factors, up-regulation of defense genes, nuclear export of mRNA and translation of defense proteins. Nuclear pore-mediated transports should play critical roles in these processes, however, the regulatory mechanisms of nuclear-cytoplasmic transport during stress responses are largely unknown. In this study, a regulator of nuclear export of RNA and proteins, NbRanBP1-1 (Ran-binding protein1-1), was identified as an essential gene for the resistance of Nicotiana benthamiana to potato blight pathogen Phytophthora infestans. NbRanBP1-1-silenced plants showed delayed accumulation of capsidiol, a sesquiterpenoid phytoalexin, in response to elicitor treatment, and reduced resistance to P. infestans. Abnormal accumulation of mRNA was observed in NbRanBP1-1-silenced plants, indicating that NbRanBP1-1 is involved in the nuclear export of mRNA. In NbRanBP1-1-silenced plants, elicitor-induced expression of defense genes, NbEAS and NbWIPK, was not affected in the early stage of defense induction, but the accumulation of NbWIPK protein was reduced. Nuclear export of the small G-protein NbRan1a was activated during the induction of plant defense, whereas this process was compromised in NbRanBP1-1-silenced plants. Silencing of genes encoding the nuclear pore proteins, Nup75 and Nup160, also caused abnormal nuclear accumulation of mRNA, defects in the nuclear export of NbRan1a, and reduced production of capsidiol, resulting in decreased resistance to P. infestans. These results suggest that nuclear export of NbRan is a key event for defense induction in N. benthamiana, and both RanBP1-1 and nucleoporins play important roles in the process.

Nicotiana benthamiana ABCG1 and ABCG2 are ABC transporters which are probably involved in the export of capsidiol, the major phytoalexin of Nicotiana species. While capsidiol export by these transporters plays an essential role in post-invasion defense against Phytophthora infestans, they also export unidentified antimicrobial compound(s) involved in pre-invasion defense. In this study, promoter activity of NbABCG2 (Pabcg2a) was analyzed using a GFP marker. Expression of GFP under the control of Pabcg2a was significantly increased by co-expression with the INF1 elicitor from P. infestans. Disruption of the ethylene-responsive GCC box in Pabcg2a compromised INF1-induced activation of Pabcg2a. Consistently, penetration by P. infestans was increased by gene-silencing of NbEIN2, the key ethylene-signaling component, suggesting the involvement of ethylene for pre-invasion defense of N. benthamiana.

A glycosidically bound fraction was prepared by adsorbing a 80% methanol extract from fresh rhizomes of ginger onto a column of Amberlite XAD-2 resin and successively eluting with ethyl acetate or methanol. Enzymatic hydrolysis of this fraction with an acetone powder prepared from fresh ginger and commercial glycosidase liberated such alcohols as geraniol, 2-heptanol, α-terpineol, nerol, linalool, and citronellol, suggesting that fresh ginger included glycosides and glycosidase. The ethyl acetate eluate was chromatographed by an ODS flash column and then HPLC to isolate the β-glucopyranosides of 5-hydroxyborneol, 1,8-epoxy-p-menthan-3-ol, (2E, 6E)- and (2E, 6Z)-3,7-dimethyl-8-hydroxyoctadien-1-ol, 2-heptanol, geraniol, nerol, (R)-linalool, and citronellol. All the glucosides, except for 5-hydroxybornyl-O-β-D-glucopyranoside, were identified for the first time in the rhizome of ginger, and many of their aglycons were major constituents of the essential oil. The results indicate that these glucosides are aroma precursors of fresh ginger.

The phase behavior of the aqueous cholesteric mesophase composed of potassium N-dodecanoyl-l-alaninate (L-KDDA) was studied in the presence of various cations such as a cobalt(III) complex and cesium ions using multinuclear magnetic resonance and laser and X-ray scatterings. The cobalt(III) complexes used are [Co(NH3)6]Cl3, [Co(en)3]Cl3 (en = ethylenediamine), Δ-[Co(chxn)3]Cl3 (chxn = (R,R)-1,2-cyclohexanediamine), [Co(tn)3]Cl3 (tn = trimethylenediamine), and [Co(phen)3]Cl3 (phen = 1,10-phenanthroline). Transition temperatures from anisotropic to isotropic phases determined by 2H2O NMR spectra were appreciably dependent on the kinds and concentrations of the added cations. The NH3-, en-, tn-, and phen-complex ions were more favorable for stabilizing the aqueous mesophase of L-KDDA compared to the chxn-complex ion. The change in the order parameters for the surfactant aggregates was monitored by anisotropic splittings of the 1H NMR spectra envelope and was compared to structural parameters of the mesophase...

Interactions of tripositive cobalt(III) complex ions, such as Δ(lel3)-[Co(chxn)3]3+ (chxn = (R,R)-1,2-cyclohexanediamine), [Co(oct-en)(2,3,2-tet)]3+ (oct-en = N-octylethylenediamine, 2,3,2-tet = 3,7-diazanonane-1,9-diamine), [Co(sep)3]3+ (sep = sepulchrate = 1,3,6,8,10,13,16,19-octaazabicyclo[6.6.6]eicosane), and [Co(tn)3]3+ (tn = trimethylenediamine), in various solvents were studied using 59Co chemical shifts together with the electronic absorption spectra. The results are discussed in comparison with those of [Co(en)3]3+, whose 59Co chemical shift in various solvents had been previously reported to be fairly proportional to the donor numbers. There was a trend that the 59Co chemical shift changes greatly if the acidic NH-hydrogen atoms of the ligands are favorable to form hydrogen bonding with the solvents. The magnitudes of the chemical-shift changes for [Co(tn)3]3+ were significantly smaller than those for the en and chxn complexes. This difference can be attributed to the more conformationally labil...

Interactions between micelles and tripositive ions of metal complexes such as [M(en)3]3+ (M = Co or Cr, en = ethylenediamine), [M(chxn)3]3+ (chxn = (R,R)-1,2-cyclohexanediamine), and [M(phen)3]3+ (phen = 1,10-phenanthroline), were studied using 59Co NMR spectroscopies and paramagnetic Cr(III)-induced 13C NMR relaxations of the surfactants. The magnitudes of the changes in 59Co chemical shifts and relaxation rates for [Co(en)3]3+ (= en complex) and [Co(chxn)3]3+ (= chxn complex) were dependent on the type of the head group: The carboxylate and alaninate surfactants caused larger changes in the 59Co NMR parameters than the sulfonate and sulfate ones did. The 59Co longitudinal and transverse relaxation rates were measured for the typical systems; these results gave effective 59Co electric field gradients (efgs) and effective correlation times for the three complex ions in the micellar solutions. The effective 59Co efgs obtained for the en and chxn complexes were larger in the carboxylate micelles than in th...

59Co NMR relaxation rates for the [Co(chxn)3]3+ (chxn = trans-(1R,2R)-1,2-diaminocyclohexane) and [Co(en)3]3+ (en = ethylenediamine) ions were measured in the presence of sulfate or oxalate ions. 1H relaxation rates for the chxn ligands in the same systems were also measured. The ratios of the 59Co relaxation rates to the 1H ones indicate that the 59Co electric field gradient of [Co(chxn)3]3+ is appreciably decreased by the formation of the 1:1 ion pair with the sulfate ion. The difference of the concentration dependence of the 59Co NMR relaxations between the sulfate and the oxalate ions can be attributed to the faster change in the configuration of the 1:1 ion pair of the oxalate ion.

Formation of diastereomers of Δ- and Λ-[Co(en)3]3+ was investigated in aqueous chiral-electrolyte solutions using 59Co NMR spectroscopy. The chiral electrolytes used were alkali salts for L- and D-tartrates, bis(μ-(−)589-tartrato)diantimonate(III), N-dodecanoyl-L-(and D-)alaninate, N-dodecanoyl-L-threoninate, and N-hexadecanoyl-L-prolinate. The 59Co NMR parameters for the [Co(en)3]3+ group were different between the enantiomers in most cases. The NMR parameters used for chiral discrimination were chemical shifts (in simple electrolyte and micellar solutions), relaxation rates (in simple electrolyte and micellar solutions), peak areas (in micellar solutions), and quadrupole splittings (in liquid crystalline solutions). The larger values of the relaxation rates suggest stronger interactions. On the other hand, the magnitude of the chemical shift change was not simply related to the extent of the interactions. For a chemical shift, the contribution of a geometrical factor seems to be dominant in systems havi...

Bovine mammary epithelial cells (BMEC) were isolated as acinous fragments from a mammary gland of a lactating cow. They grew well on plastic substratum, showed the characteristic cobblestone morphology of epithelial cells, and secreted alpha s1-, beta-, and kappa-caseins even when grown on plastic substratum. A plasmid containing the bacterial chloramphenicol acetyl transferase (CAT) gene was transfected to the isolated BMEC by calcium phosphate precipitation and electroporation methods. The transfection efficiency of BMEC by the calcium phosphate method was greatly improved by post-transfection osmotic shock with glycerol or polyethylene glycol. An about 700 bp DNA fragment containing 5'-flanking sequence of bovine alpha s1-casein gene showed promoter activity in the transfected BMEC. The primary culture of BMEC might be useful for studies on regulation of bovine milk-protein gene expression.

59Co and 1H NMR relaxation studies for the Δ-[Co(chxn)3]3+(chxn ≡ trans-(1R,2R)-1,2-diaminocyclohexane) ion revealed that the decrease in the 59Co NMR relaxation rates with an addition of multivalent anions such as sulfate and phosphate can be attributed to an appreciable decrease in the electric field gradient at 59Co nucleus by the ion pairing.

An estimation of the peak areas of the 59Co NMR spectra for cobalt(III) complex ions and of the 25Mg spectra for magnesium aqua ions gave population ratios of their hidden ions, which are defined as too strongly interacting with nematic lyomesophases (composed of cationic or anionic surfactants) to contribute to the NMR spectra. The cobalt(III) complex ions used were [Co(NH3)6]3+, [Co(en)3]3+ (en = ethylenediamine), [Co(phen)3]3+ (phen = 1,10-phenanthroline), and [Co(CN)6]3−. It was revealed that appreciable amounts of multivalent ions are present as hidden ions on the lyomesophase, whose sign of charge is opposite to that of the ion in question. For such hydrophilic ions as [Co(NH3)6]3+, [Co(en)3]3+, [Co(CN)6]3−, and magnesium ions, the ions binding to the mesophases are classified into two states based on the NMR spectra: One is an outer-sphere complex (ion pair); the other is a hidden ion. In these systems the hidden ion/total ion population ratios depending on the composition of the surfactant are rou...

Chiral discrimination of [Co(en)3]3+ was investigated by using 59Co NMR spectroscopy in liquid crystals comprising cesium N-dodecanoyl-L-(or D)-alaninate. The 59Co quadrupole splitting was distinguishable by diastereomeric interactions while a change in the chemical shift was the same.