The Arabidopsis NADPH oxidases RbohD and RbohF display differential expression patterns and contributions during plant immunity

Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) that perform a wide range of functions. RbohD and RbohF, two of the 10 Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes including the response to pathogens. We hypothesized that the spatio-temporal control of RbohD and RbohF gene expression might be critical in determining their multiplicity of functions. Transgenic Arabidopsis plants with RbohD and RbohF promoter fusions to β-glucuronidase and Luciferase reporter genes were generated. Analysis of these plants revealed a differential expression pattern for RbohD and RbohF throughout plant development and during immune responses. RbohD and RbohF gene expression was differentially modulated by pathogen-associated molecular patterns. Histochemical stains and in vivo expression analysis showed a correlation between the level of RbohD and RbohF promoter activity, H2O2 accumulation and the amount of cell death in response to the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 and the necrotrophic fungus Plectosphaerella cucumerina. A promoter-swap strategy revealed that the promoter region of RbohD was required to drive production of ROS by this gene in response to pathogens. Moreover, RbohD promoter was activated during Arabidopsis interaction with a non-virulent P. cucumerina isolate, and susceptibility tests with the double mutant rbohD rbohF uncovered a new function for these oxidases in basal resistance. Altogether, our results suggest that differential spatio-temporal expression of the Rboh genes contributes to fine-tune RBOH/NADPH oxidase-dependent ROS production and signaling in Arabidopsis immunity

Anti-Helicobacter pylori activity of steroidal alkaloids obtained from three Veratrum plants

Anti-Helicobacter pylori (HP) activities were examined, by disc method, on three total alkaloid fractions and fourteen steroidal alkaloids obtained from three Veratrum plants ( V. manckii, V. nigrum var. ussuriense and V. patulum) , which are used as a name of "Li-lu (藜蘆)" to treat aphasia arising from apoplexy, wind type dysentery, jaundice, headache, scabies, chronic malaria, etc. Among them, verapatulin (12) and veratramine (13) revealed anti-HP activities, and the disc-minimum inhibitory concentration (disk-MIC) value (10 μg/ml) of 12 against two standard HP strains, NCTC11637 and NCTC11916, was higher than that of a clinically used antibiotic, erythromycin (≦0.013 μg/ml) , but was comparable to those of penicillin G (3.1 μg/ml and 1.6 μg/ml, respectively). 漢薬"藜蘆"として用いられている3種のヴェラトラム属植物(V.maackii, V.nigrum var.ussuriense and V.patulum)から得た総アルカロイドフラクション3種及ぴステロイドアルカロイド14種について,抗ヘリコバクター・ピロリ活性をディスク法で測定した。調べたステロイドアルカロイドの中で,ヴェラパツリン(12)及ぴヴェラトラミン(13)が抗ヘリコバクター・ピロリ活性を示した。ヴェラパツリン(12)のヘリコバグター・ピロリ標準株2種(NCTC11637及ぴNCTC11916)に対するdisk MIC値は10μg/mlであり,臨床で用いられる抗生物質のエリスロマイシン(≦0.013μg/ml)よりは弱いが,ペニシリンG(各標準株に対して3.1μg/ml,1.6μg/ml)と同程度であった

Hypoglycemic effect of Aemotoxylon campechanum on streptozotocin (STZ)-induced diabetic rats

Aemotoxylon campechanumの血糖降下作用をSTZ誘発糖尿病ラットを用いて検討したところ,水エキスが空腹時血糖を有意かつ用量依存的に低下させた。そこで,水エキスをメタノール可溶性フラクション及び水溶性フラクションに分画したところ,メタノール可溶性フラクションがより強い作用を示し,100mg/kgの腹腔内投与で空腹時血糖値を37%低下させた。また,水エキス200mg/kg及びメタノール可溶部100mg/kgは空腹時血糖値を35.1%低下させた陽性コントロールとして用いたtolbutamide(100mg/kg)とbuformin(1mg/kg)の混合物よりも強いものであった。最後に,この活性フラクションの分離を行ない,グアイアン型セスキテルペン5種及びクマリン誘導体1種を単離・同定した。 Hypoglycemic activity of Aemotoxylon campechanum was examined in STZ-induced diabetic rats. The water extract of A. campechanum lowered fasting blood glucose level of STZ-induced diabetic rats significantly and dosedependently. The water extract further divided into MeOH and H_2O-soluble fractions. The MeOH-soluble fraction showed the strongest hypoglycemic effect, which lowered fasting blood glucose level by 37% at a dose of 100 mg/kg (i. p.). The water extract and the MeOH-soluble fraction were found to be more effective in lowering the blood glucose level of diabetic rats than the mixtures of tolbutamide (200 mg/kg) and buformin (1 mg/kg) used as positive control, which lowered blood glucose level by 35.1%. The active fraction led to isolation of five guainane-type sesquiterpenes and a coumarin derivative

Synthesis and antihepatotoxic and antiproliferative activities of di- and tri-O-caffeoylquinic acid derivatives

Methyl di- and tri-O-caffeoylquinates were synthesized by esterification of methyl quinate with di-O-acetylcaffeoyl chloride, following deprotection of the acetyl groups. Moreover, 4,5-di-O-caffeoylquinic acid was synthesized by esterification of quinide with di-O-acetylcaffeoyl chloride, followed by a hydrolysis of product quinide. These synthetic compounds were tested for their hepatoprotective activity on _D-galactosamine (_D-GalN)/tumor necrosis factor-α (TNF- α)-induced cell death in primary cultured mouse hepatocytes, which possessed significant hepatoprotective activity concentration-dependently. The activity was enhanced by the presence of caffeoyl group. On the other hand, they showed only weak antiproliferative activities against murine colon 26-L5 carcinoma, human HT-1080 fibrosarcoma, murine B16-BL6 melanoma, and human lung carcinoma A-549 cells. メチルジおよびトリカフェオイルキネート化合物1-4は,メチルキネート6をジアセチルカフェオイルクロライド7でエステル化した後,アセチル基の脱保護によって合成した。さらに,4,5-ジカフェオイルキナ酸5は,キニド8を7とエステル化して生成したキニド8aの加水分解により合成した。これらの合成した化合物1-5を用いてマウス初代培養肝細胞の_D-GalN/TNF-α誘発肝細胞死における肝保護活性の試験を行ったところ,濃度依存的な肝保護活性が認められた。その活性はカフェオイル基の存在により増強された。一方,これらの化合物1-5はマウス26-L5大腸癌腫,ヒトHT-1080繊維芽肉腫,マウスB16-BL6黒色種,及びヒトA-549肺癌腫に対し弱い細胞増殖抑制活性を示した

Protective Effects of Rosa damascena and Its Active Constituent on Aβ(25–35)-Induced Neuritic Atrophy

Dementia is a clinical syndrome characterized by multiple cognitive deficits and causes progressive neurodegeneration leading eventually to death. The incidence of dementia is increasing worldwide with the increase in ageing population. However, no effective treatment is available yet. It has been hypothesized that drugs activating neurite outgrowth might induce neuronal reconstruction and help in the recovery of brain function. Working on this hypothesis, we recently observed that the chloroform extract of the Rosa damascena significantly induced the neurite outgrowth activity and inhibited the Aβ(25–35)-induced atrophy and cell death. Further workup led the isolation of a very long polyunsaturated fatty acid having molecular formula C37H64O2 as an active constituent. The structure of this compound was established by extensive analysis of fragmentations observed in EI-MS mode. The isolated compound protected Aβ(25–35)-induced atrophy and displayed strong neurite outgrowth activity. The length of dendrite in the cells treated with this compound were comparable to those of nerve growth factor (NGF) treated cells

Antioxidative and antihepatotoxic principles of Tuocha

沱茶は中国雲南省のアッサム茶の葉を蒸して作られる緊圧茶で,抗老化,コレステロール低下,免疫増強,血圧降下等の作用があると言われている。我々は,DPPHラジカル捕捉活性を指標として成分検索を行ない,得られた成分についてDPPHラジカル捕捉活性と共にD-GalN/TNF-α誘発マウス肝細胞死阻害活性を測定した。その結果,沱茶がフェノール類,リグナン,フラボノイド,フラバン3オール類などの抗酸化成分を多く含む事を明らかにし,それら成分の構造と活性との関係について考察した。 Tuocha is one of the special varieties of fermented compressed tea leaves, praised for its important health benefits, such as anti-aging, lowering cholesterol, enhancing immune function, lowering of blood pressure, reducing heart attacks etc. In the present study, we carried out fractionation and isolation of the active constituents, guided by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. In addition, antioxidative and antihepatotoxic potency on D-galactosamine (D-GalN)/tumor necrosis factor-alpha (TNF-α)-induced cell death in primary cultured mouse hepatocytes was also examined. Our study revealed that, tuocha is rich in antioxidants such as phenolics, lignans, flavanoides and flavan-3-ols, and showed a good understanding between structure and activity relationship

Inhibitors of nitric oxide (NO) production in murine macrophage-like J774.1 cells from Brazilian propolis

ブラジル産プロポリスの水およびメタノールエキスがLPSで活性化したマウスマクロファージ様J774.1細胞の一酸化窒素(NO)産生を濃度依存的に抑制することを明らかにした。さらに,水エキスの成分検索を行ない17種のフェノール性化合物を単離したが,その中の15化合物はプロポリス水エキスからは初めて単離された化合物であった。また,methylρ-hydroxydihydrocinnamate(9)と1-(4-hydroxyphenyl)butane-1,3-dione(11)のプロポリスからの単離はこれか最初の例である。次いで各化合物のNO阻害活性を測定したところ,ラブダン型ジテルペン,フラボノイド,数種のフェノール性化合物が強いNO阻害活性を示した。特に,coniferyl aldehyde(23 ; IC_50, 18.0μM)とdimeric coniferyl acetate (33 ; IC_50, 27.1μM)は陽性コントロールのNG-monomethyl-L-arginine(L-NMMA ; IC_50, 44.5μM)よりも強い活性を示した。 Water and MeOH extracts of Brazilian propolis showed dose-dependent inhibition toward nitric oxide (NO) production in lipopolysacchalide (LPS)-activated murine macrophage-like J774.1 cells. From the water extract, 17 phenolic compounds were isolated and among them 15 are new for the water extract of propolis. Moreover, methyl ρ-hydroxydihydrocinnamate (9) and 1-(4-hydroxyphenyl)butane-1,3-dione (11) were isolated, for the first time, from propolis. Labdane-type diterpenes, flavonoids and some phenolic compounds possessed potent NO inhibitory activity. Coniferyl aldehyde (23) and dimeric coniferyl acetate (33) showed the strongest NO inhibition with IC_50 values of 18.0 and 27.1 μM, respectively, which were stronger than the positive control, N^G-monomethyl-L-arginine (L-NMMA ; IC_50, 44.5 μM)

Chitin perception in plasmodesmata characterizes submembrane immune-signaling specificity in plants

The plasma membrane (PM) is composed of heterogeneous subdomains, characterized by differences in protein and lipid composition. PM receptors can be dynamically sorted into membrane domains to underpin signaling in response to extracellular stimuli. In plants, the plasmodesmal PM is a discrete microdomain that hosts specific receptors and responses. We exploited the independence of this PM domain to investigate how membrane domains can independently integrate a signal that triggers responses across the cell. Focusing on chitin signaling, we found that responses in the plasmodesmal PM require the LysM receptor kinases LYK4 and LYK5 in addition to LYM2. Chitin induces dynamic changes in the localization, association, or mobility of these receptors, but only LYM2 and LYK4 are detected in the plasmodesmal PM. We further uncovered that chitin-induced production of reactive oxygen species and callose depends on specific signaling events that lead to plasmodesmata closure. Our results demonstrate that distinct membrane domains can integrate a common signal with specific machinery that initiates discrete signaling cascades to produce a localized response

Mechanism-based CYP2D6 inactivation by acridone alkaloids of Indonesian medicinal plant Lunasia amara

Fourteen acridone alkaloids isolated from Lunasia amara Blanco were tested for their mechanismbased inhibition on human liver microsomal dextromethorphan O-demethylation activity, a prototype marker for cytochrome P450 2D6 (CYP2D6). Among the 14 compounds, 5-hydroxygraveroline (1), 8-methoxyifflaiamine (2), lunamarine (3), and lunine (12) increased their inhibitory activity with increasing preincubation time. Then, we further examined the possibility of mechanismbased inhibition on 5-hydroxygraveroline (1) and lunamarine (3), which showed the potent inhibition. Further investigations on 1 and 3 showed that the characteristic time- and concentration-dependent inhibition, which required a catalytic step with NADPH, was not protected by nucleophiles, and was decreased by the presence of a competitive inhibitor. Thus, 1 and 3 were concluded as mechanism-based inactivators of CYP2D6

Structural and functional analysis of SGT1–HSP90 core complex required for innate immunity in plants

SGT1 (Suppressor of G2 allele of skp1), a co-chaperone of HSP90 (Heat-shock protein 90), is required for innate immunity in plants and animals. Unveiling the cross talks between SGT1 and other co-chaperones such as p23, AHA1 (Activator of HSP90 ATPase 1) or RAR1 (Required for Mla12 resistance) is an important step towards understanding the HSP90 machinery. Nuclear magnetic resonance spectroscopy and mutational analyses of HSP90 revealed the nature of its binding with the CS domain of SGT1. Although CS is structurally similar to p23, these domains were found to non-competitively bind to various regions of HSP90; yet, unexpectedly, full-length SGT1 could displace p23 from HSP90. RAR1 partly shares the same binding site with HSP90 as the CS domain, whereas AHA1 does not. This analysis allowed us to build a structural model of the HSP90–SGT1 complex and to obtain a compensatory mutant pair between both partners that is able to restore virus resistance in vivo through Rx (Resistance to potato virus X) immune sensor stabilization