The Jasmonic Acid Pathway Positively Regulates the Polyphenol Oxidase-Based Defense against Tea Geometrid Caterpillars in the Tea Plant (Camellia sinensis).

Polyphenol oxidases (PPOs) as inducible defense proteins, contribute to tea (Camellia sinensis) resistance against tea geometrid larvae (Ectropis grisescens), and this resistance has been associated with the jasmonic acid (JA) signaling by testing geometrid performance in our previous work. However, the regulation of PPO-based defense by JA and other hormone signaling underlying these defense responses is poorly understood. Here, we investigated the role of phytohormones in regulating the PPO response to tea geometrids. We profiled levels of defense hormones, PPO activity and CsPPO genes in leaves infested with tea geometrids. Then, hormone levels were manipulated by exogenous application of methyl jasmonate (MeJA), gibberellin acid (GA3), abscisic acid (ABA), JA biosynthesis inhibitors (sodium diethyldithiocarbamate trihydrate, DIECA and salicylhydroxamic acid, SHAM) and GA inhibitor (uniconazole, UNI). Upon geometrid attack, JA levels significantly increased, whereas GA levels notably decreased and ABA level was slightly decreased. And the PPO activity significantly increased in line with the transcript levels of CsPPO2 and CsPPO4 but not CsPPO1. There were an obvious antagonistic cross-talk between JA and GA signals and an association among JA signals, PPO response and herbivore resistance in tea plants. Pretreatment with MeJA increased PPO activity by activating the transcripts of CsPPO2 and CsPPO4, whereas application of JA inhibitor DIECA suppressed PPO activity. GA3 strongly enhanced PPO activity, but ABA did not alter PPO activity. These findings strongly suggest that JA is a central player in PPO-mediated tea resistance against tea geometrids in a manner that prioritizes defense over growth.

Arabidopsis JASMONATE-INDUCED OXYGENASES down-regulate plant immunity by hydroxylation and inactivation of the hormone jasmonic acid.

The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JA-responsive gene expression by the bioactive form JA-isoleucine have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. Because they are induced by JA we named them JASMONATE-INDUCED OXYGENASES (JOXs). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in overaccumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses.

Vitamin E Is Superior to Vitamin C in Delaying Seedling Senescence and Improving Resistance in Arabidopsis Deficient in Macro-Elements.

Nitrogen (N), phosphorus (P), and potassium (K) are three essential macro-elements for plant growth and development. Used to improve yield in agricultural production, the excessive use of chemical fertilizers often leads to increased production costs and ecological environmental pollution. Vitamins C and E are antioxidants that play an important role in alleviating abiotic stress. However, there are few studies on alleviating oxidative stress caused by macro-element deficiency. Here, we used Arabidopsis vitamin E synthesis-deficient mutant vte4 and vitamin C synthesis-deficient mutant vtc1 on which exogenous vitamin E and vitamin C, respectively, were applied at the bolting stage. In the deficiency of macro-elements, the Arabidopsis chlorophyll content decreased, malondialdehyde (MDA) content and relative electric conductivity increased, and reactive oxygen species (ROS) accumulated. The mutants vtc1 and vte4 are more severely stressed than the wild-type plants. Adding exogenous vitamin E was found to better alleviate stress than adding vitamin C. Vitamin C barely affected and vitamin E significantly inhibited the synthesis of ethylene (ETH) and jasmonic acid (JA) genes, thereby reducing the accumulation of ETH and JA that alleviated the senescence caused by macro-element deficiency at the later stage of bolting in Arabidopsis. A deficiency of macro-elements also reduced the yield and germination rate of the seeds, which were more apparent in vtc1 and vte4, and adding exogenous vitamin C and vitamin E, respectively, could restore them. This study reported, for the first time, that vitamin E is better than vitamin C in delaying seedling senescence caused by macro-element deficiency in Arabidopsis.

NINJA connects the co-repressor TOPLESS to jasmonate signalling.

Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. On perception of JA-Ile, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the Arabidopsis JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress-related and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.

HIV-1 Vpu utilizes both cullin-RING ligase (CRL) dependent and independent mechanisms to downmodulate host proteins.

BACKGROUND: Hijacking of the cullin-RING E3 ubiquitin ligase (CRL) machinery is a common mechanism employed by diverse groups of viruses for the efficient counteraction and degradation of host proteins. In particular, HIV-1 Vpu usurps the SCF(ß-TrCP) E3 ubiquitin ligase complex to mark CD4 for degradation by the 26S proteasome. Vpu also interacts with and downmodulates a number of other host proteins, including the restriction factor BST-2. However, whether Vpu primarily relies on a cullin-dependent or -independent mechanism to antagonize its cellular targets has not been fully elucidated. RESULTS: We utilized a sulphamate AMP analog, MLN4924, to effectively block the activation of CRLs within infected primary CD4(+) T cells. MLN4924 treatment, in a dose dependent manner, efficiently relieved surface downmodulation and degradation of CD4 by NL4-3 Vpu. MLN4924 inhibition was highly specific, as this inhibitor had no effect on Nef's ability to downregulate CD4, which is accomplished by a CRL-independent mechanism. In contrast, NL4-3 Vpu's capacity to downregulate BST-2, NTB-A and CCR7 was not inhibited by the drug. Vpu's from both a transmitted founder (T/F) and chronic carrier (CC) virus preserved the ability to downregulate BST-2 in the presence of MLN4924. Finally, depletion of cellular pools of cullin 1 attenuated Vpu's ability to decrease CD4 but not BST-2 surface levels. CONCLUSIONS: We conclude that Vpu employs both CRL-dependent and CRL-independent modes of action against host proteins. Notably, we also establish that Vpu-mediated reduction of BST-2 from the cell surface is independent of ß-TrCP and the CRL- machinery and this function is conserved by Vpu's from primary isolates. Therefore, potential therapies aimed at antagonizing the activities of Vpu may need to address these distinct mechanisms of action in order to achieve a maximal effect.

Necrotrophic pathogens use the salicylic acid signaling pathway to promote disease development in tomato.

Plants use different immune pathways to combat pathogens. The activation of the jasmonic acid (JA)-signaling pathway is required for resistance against necrotrophic pathogens; however, to combat biotrophic pathogens, the plants activate mainly the salicylic acid (SA)-signaling pathway. SA can antagonize JA signaling and vice versa. NPR1 (noninducible pathogenesis-related 1) is considered a master regulator of SA signaling. NPR1 interacts with TGA transcription factors, ultimately leading to the activation of SA-dependent responses. SA has been shown to promote disease development caused by the necrotrophic pathogen Botrytis cinerea through NPR1, by suppressing the expression of two JA-dependent defense genes, proteinase inhibitors I and II. We show here that the transcription factor TGA1.a contributes to disease development caused by B. cinerea in tomato by suppressing the expression of proteinase inhibitors I and II. Finally, we present evidence that the SA-signaling pathway contributes to disease development caused by another necrotrophic pathogen, Alternaria solani, in tomato. Disease development promoted by SA through NPR1 requires the TGA1.a transcription factor. These data highlight how necrotrophs manipulate the SAsignaling pathway to promote their disease in tomato.

Jasmonic acid is involved in the signaling pathway for fungal endophyte-induced volatile oil accumulation of Atractylodes lancea plantlets.

BACKGROUND: Jasmonic acid (JA) is a well-characterized signaling molecule in plant defense responses. However, its relationships with other signal molecules in secondary metabolite production induced by endophytic fungus are largely unknown. Atractylodes lancea (Asteraceae) is a traditional Chinese medicinal plant that produces antimicrobial volatiles oils. We incubated plantlets of A. lancea with the fungus Gilmaniella sp. AL12. to research how JA interacted with other signal molecules in volatile oil production. RESULTS: Fungal inoculation increased JA generation and volatile oil accumulation. To investigate whether JA is required for volatile oil production, plantlets were treated with JA inhibitors ibuprofen (IBU) and nordihydroguaiaretic acid. The inhibitors suppressed both JA and volatile oil production, but fungal inoculation could still induce volatile oils. Plantlets were further treated with the nitric oxide (NO)-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the H2O2 inhibitors diphenylene iodonium (DPI) and catalase (CAT), and the salicylic acid (SA) biosynthesis inhibitors paclobutrazol and 2-aminoindan-2-phosphonic acid. With fungal inoculation, IBU did not inhibit NO production, and JA generation was significantly suppressed by cPTIO, showing that JA may act as a downstream signal of the NO pathway. Exogenous H2O2 could reverse the inhibitory effects of cPTIO on JA generation, indicating that NO mediates JA induction by the fungus through H2O2-dependent pathways. With fungal inoculation, the H2O2 scavenger DPI/CAT could inhibit JA generation, but IBU could not inhibit H2O2 production, implying that H2O2 directly mediated JA generation. Finally, JA generation was enhanced when SA production was suppressed, and vice versa. CONCLUSIONS: Jasmonic acid acts as a downstream signaling molecule in NO- and H2O2-mediated volatile oil accumulation induced by endophytic fungus and has a complementary interaction with the SA signaling pathway.

Whiteflies interfere with indirect plant defense against spider mites in Lima bean.

Plants under herbivore attack are able to initiate indirect defense by synthesizing and releasing complex blends of volatiles that attract natural enemies of the herbivore. However, little is known about how plants respond to infestation by multiple herbivores, particularly if these belong to different feeding guilds. Here, we report the interference by a phloem-feeding insect, the whitefly Bemisia tabaci, with indirect plant defenses induced by spider mites (Tetranychus urticae) in Lima bean (Phaseolus lunatus) plants. Additional whitefly infestation of spider-mite infested plants resulted in a reduced attraction of predatory mites (Phytoseiulus persimilis) compared to attraction to plants infested by spider mites only. This interference is shown to result from the reduction in (E)-beta-ocimene emission from plants infested by both spider mites and whiteflies. When using exogenous salicylic acid (SA) application to mimic B. tabaci infestation, we observed similar results in behavioral and chemical analyses. Phytohormone and gene-expression analyses revealed that B. tabaci infestation, as well as SA application, inhibited spider mite-induced jasmonic acid (JA) production and reduced the expression of two JA-regulated genes, one of which encodes for the P. lunatus enzyme beta-ocimene synthase that catalyzes the synthesis of (E)-beta-ocimene. Remarkably, B. tabaci infestation concurrently inhibited SA production induced by spider mites. We therefore conclude that in dual-infested Lima bean plants the suppression of the JA signaling pathway by whitefly feeding is not due to enhanced SA levels.

Chestnut species and jasmonic acid treatment influence development and community interactions of galls produced by the Asian chestnut gall wasp, Dryocosmus kuriphilus.

Jasmonic acid (JA) is a plant-signaling hormone involved in defenses against insects and pathogens as well as the regulation of nutrient partitioning. Gall wasps (Hymenoptera: Cynipidae) induce the formation of galls on their host plants, which house immature wasps and provide them with nutrition and protection. The goal of this study was to investigate the effects of JA application on gall development and defenses. Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) galls on American chestnut, Castanea dentata (Marsh.) Borkhausen (Fagales: Fagaceae), and Chinese chestnut, C. mollissima Blume, were treated with JA or a JA- inhibitor, diethyldithiocarbamic acid (DIECA), to determine the effects of these treatments on gall characteristics and defenses. Chinese chestnut galls treated with JA had greater volume and dry weight, thicker sclerenchyma layers, and fewer external fungal lesions compared with controls. Galls from both chestnut species treated with JA contained a lower proportion of empty chambers, and elevated tannin levels compared with controls. The effects of DIECA on galls were generally opposite from those of JA. American chestnut galls treated with DIECA had lower dry weight and fewer feeding punctures caused by the lesser chestnut weevil compared with controls. Galls from both chestnut species that were treated with DIECA were smaller and had more external fungal lesions compared with controls. Compared to American chestnut galls, Chinese chestnut galls had increased parasitism rates and fewer gall wasps. This study is the first to investigate the effects of JA on an insect gall, and indicates that JA treatments benefit gall wasps by increasing gall size and defenses.

Structure-guided analysis of Arabidopsis JASMONATE-INDUCED OXYGENASE (JOX) 2 reveals key residues for recognition of jasmonic acid substrate by plant JOXs.

The jasmonic acid (JA) signaling pathway is used by plants to control wound responses. The persistent accumulation of JA inhibits plant growth, and the hydroxylation of JA to 12-hydroxy-JA by JASMONATE-INDUCED OXYGENASEs (JOXs, also named jasmonic acid oxidases) is therefore vital for plant growth, while structural details of JA recognition by JOXs are unknown. Here, we present the 2.65 Å resolution X-ray crystal structure of Arabidopsis JOX2 in complex with its substrate JA and its co-substrates 2-oxoglutarate and Fe(II). JOX2 contains a distorted double-stranded ß helix (DSBH) core flanked by α helices and loops. JA is bound in the narrow substrate pocket by hydrogen bonds with the arginine triad R225, R350, and R354 and by hydrophobic interactions mainly with the phenylalanine triad F157, F317, and F346. The most critical residues for JA binding are F157 and R225, both from the DSBH core, which interact with the cyclopentane ring of JA. The spatial distribution of critical residues for JA binding and the shape of the substrate-binding pocket together define the substrate selectivity of the JOXs. Sequence alignment shows that these critical residues are conserved among JOXs from higher plants. Collectively, our study provides insights into the mechanism by which higher plants hydroxylate the hormone JA.

Forskolin inhibits and reverses the effects of brefeldin A on Golgi morphology by a cAMP-independent mechanism.

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the ER, leaving no definable Golgi apparatus, and blocks transport of proteins into post-Golgi compartments in the cell. In this study we follow the disassembly of the Golgi apparatus in BFA-treated, living cells labeled with NBD-ceramide and demonstrate that forskolin can both inhibit and reverse this process. Long, tubular processes labeled with NBD-ceramide were observed emerging from Golgi elements and extending out to the cell periphery in cells treated with BFA for 5 min. With longer incubations in BFA, the NBD label was dispersed in a fine reticular pattern characteristic of the ER. Treatment with forskolin inhibited these effects of BFA as well as BFA's earliest morphologic effect on the Golgi apparatus: the redistribution to the cytosol of a 110-kD Golgi peripheral membrane protein. In addition, forskolin could reverse BFA's block in protein secretion. Forskolin inhibition of BFA's effects was dose dependent and reversible. High concentrations of BFA could overcome forskolin's inhibitory effect, suggesting forskolin and BFA interact in a competitive fashion. Remarkably, in cells already exposed to BFA, forskolin could reverse BFA's effects causing the 110-kD Golgi peripheral membrane protein to reassociate with Golgi membrane and juxtanuclear Golgi complexes to reassemble. Neither membrane permeant cAMP analogues nor cAMP phosphodiesterase inhibitors could replicate or enhance forskolin's inhibition of BFA. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, was equally as effective as forskolin in antagonizing BFA. A derivative of forskolin, 7-HPP-forskolin, that is less potent than forskolin at binding to adenylyl cyclase, was also equally effective as forskolin in antagonizing BFA. In contrast a similar derivative, 6-HPP-forskolin, that is equipotent with forskolin at binding to adenylyl cyclase, did not inhibit BFA's effects. These results suggest that forskolin acts as a competitive antagonist to BFA, using a cAMP-independent mechanism to prevent and reverse the morphologic effects induced by BFA.

PDMP blocks brefeldin A-induced retrograde membrane transport from golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism.

In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not "rescue" the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1, 3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Effects of corticosteroid and neuraminidase inhibitors on survival in patients with respiratory distress induced by influenza virus.

BACKGROUND/PURPOSE: Neuraminidase inhibitors (NAIs) including oseltamivir and peramivir are used for influenza treatment. A systemic corticosteroid is usually administrated for acute respiratory distress syndrome. The aim of this study was to investigate the effect of a systemic corticosteroid and its interaction with NAIs in patients with influenza infection and respiratory distress. METHODS: A retrospective survey of hospitalized patients infected with influenza from January 2012 to May 2014 was conducted in a medical center in Taiwan. RESULTS: Eighty-six patients were hospitalized during the study period. Forty-eight patients had respiratory distress and 39 of them (81.3%, 39/48) were supported by a mechanical ventilator. All patients with respiratory distress received oseltamivir; 60.4% (29/48) and 31.3% (15/48) of them received a corticosteroid and salvage intravenous peramivir, respectively. All-cause mortality was 29.1% (14/48), 20% (3/15), and 31% (9/29) in patients with respiratory distress, patients who received salvage peramivir, and patients who received a systemic corticosteroid, respectively. Salvage peramivir seemed to improve prognosis in patients with H1pdm09 or type B virus infection and respiratory distress (p = 0.05). Early initiating corticosteroid had a worse prognosis than initiation after 72 hours of NAI treatment (p = 0.024). In particular, a systemic corticosteroid seemed to lead to a shorter survival time in patients with chronic lung disease (p = 0.05). CONCLUSION: Salvage peramivir provided a better prognosis than monotherapy with oseltamivir in patients who were infected with H1pdm09 or type B virus and who developed respiratory distress. A systemic corticosteroid should be administered after initiating NAI therapy, especially in patients with chronic lung disease.

NF-kappaB: a stress-regulated switch for cell survival.

Nuclear factor-kappaB (NF-kappaB), a stress-regulated transcription factor belonging to the Rel family, has a pivotal role in the control of the inflammatory and the innate immune responses. Its activation rapidly induces the transcription of a variety of genes encoding cell adhesion molecules, inflammatory and chemotactic cytokines, cytokine receptors, and enzymes that produce inflammatory mediators. More recently, NF-kappaB activation has been connected with multiple aspects of oncogenesis, including the control of cell proliferation, migration, cell cycle progression, and apoptosis. Interestingly, NF-kappaB is constitutively activated in several types of cancer cells, including hematological and epithelial malignancies. In addition, activation of NF-kappaB in cancer cells by chemotherapy or radiation therapy has been associated with the acquisition of resistance to apoptosis, which has emerged as a significant impediment to effective cancer treatment. Selective cyclopentenone inhibitors of the IkappaB kinase, the key enzyme controlling NF-kappaB activation, were recently shown to be potent inducers of apoptosis in chemoresistant lymphoid malignancies. Increasing evidence, summarized in this review, indicates that the development of selective NF-kappaB inhibitors may represent a promising therapeutic tool to sensitize tumor cells to apoptosis and increase the efficacy of conventional anticancer drugs in a wide spectrum of malignancies.

Jasmonic acid is a crucial signal transducer in heat shock induced sesquiterpene formation in Aquilaria sinensis.

Agarwood, a highly valuable resinous and fragrant heartwood of Aquilaria plants, is widely used in traditional medicines, incense and perfume. Only when Aquilaria trees are wounded by external stimuli do they form agarwood sesquiterpene defensive compounds. Therefore, understanding the signaling pathway of wound-induced agarwood formation is important. Jasmonic acid (JA) is a well-characterized molecule that mediates a plant's defense response and secondary metabolism. However, little is known about the function of endogenous JA in agarwood sesquiterpene biosynthesis. Here, we report that heat shock can up-regulate the expression of genes in JA signaling pathway, induce JA production and the accumulation of agarwood sesquiterpene in A. sinensis cell suspension cultures. A specific inhibitor of JA, nordihydroguaiaretic acid (NDGA), could block the JA signaling pathway and reduce the accumulation of sesquiterpene compounds. Additionally, compared to SA and H2O2, exogenously supplied methyl jasmonate has the strongest stimulation effect on the production of sesquiterpene compounds. These results clearly demonstrate the central induction role of JA in heat-shock-induced sesquiterpene production in A. sinensis.

Targeting Hormone-Related Pathways to Improve Grain Yield in Rice: A Chemical Approach.

Sink/source relationships, regulating the mobilization of stored carbohydrates from the vegetative tissues to the grains, are of key importance for grain filling and grain yield. We used different inhibitors of plant hormone action to assess their effects on grain yield and on the expression of hormone-associated genes. Among the tested chemicals, 2-indol-3-yl-4-oxo-4-phenylbutanoic acid (PEO-IAA; antagonist of auxin receptor), nordihydroguaiaretic acid (NDGA; abscisic acid (ABA) biosynthesis inhibitor), and 2-aminoisobutyric acid (AIB; ethylene biosynthesis inhibitor) improved grain yield in a concentration dependent manner. These effects were also dependent on the plant developmental stage. NDGA and AIB treatments induced an increase in photosynthesis in flag leaves concomitant to the increments of starch content in flag leaves and grains. NDGA inhibited the expression of ABA-responsive gene, but did not significantly decrease ABA content. Instead, NDGA significantly decreased jasmonic acid and jasmonic acid-isoleucine. Our results support the notion that the specific inhibition of jasmonic acid and ethylene biosynthesis resulted in grain yield increase in rice.

Guanosine nucleotides modulate the inhibitory effect of brefeldin A on protein secretion.

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the endoplasmic reticulum (ER), leaving no definable Golgi-apparatus, and blocks transport of proteins from the ER to distal secretory compartments of the cell. Using pulse-chase experiments the present study shows that BFA (1 microgram/ml) inhibits basal and CCK-stimulated protein secretion in isolated pancreatic acinar cells by 65 +/- 6% and 84 +/- 5%, respectively. In isolated permeabilized cells higher concentrations of BFA (30 micrograms/ml) were necessary to obtain inhibition of protein secretion. In parallel experiments protein secretion was stimulated by GTP (1 mM). BFA had no inhibitory effect on protein secretion in the presence of GTP, indicating that BFA might act on a GTP-binding protein. Investigating the effect of BFA on small molecular weight GTP-binding proteins we observed that [alpha-32P]GTP binding to a 21 kDa protein in a subcellular fraction enriched in ER was increased in the presence of BFA. We conclude that this 21 kDa and possibly also other GTP-binding proteins may be the molecular target of Brefeldin A in pancreatic acinar cells.

Wounding and chemicals induce expression of the Arabidopsis thaliana gene Thi2.1, encoding a fungal defense thionin, via the octadecanoid pathway.

In seedlings of Arabidopsis thaliana the thionin gene Thi2.1 is inducible by methyl jasmonate, wounding, silver nitrate, coronatine, and sorbitol. We have used a biochemical and genetic approach to test the signal transduction of these different inducers. Both exogenously applied jasmonates and jasmonates produced endogenously upon stress induction, lead to GUS expression in a Thi2.1 promoter-uidA transgenic line. No GUS expression was observed in a coil mutant background which lacks jasmonate perception whereas methyl jasmonate and coronatine but not the other inducers were able to overcome the block in jasmonic acid production in a fad3-2 fad7-2 fad8 mutant background. Our results show conclusively that all these inducers regulate Thi2-1 gene expression via the octadecanoid pathway.

Protection of mitochondrial respiration activity by bilobalide.

Mitochondria alteration is an early event in ischemia-induced damage, and its prevention improves tissue survival upon reperfusion. Adenine translocase and complex I activities are rapidly affected by ischemia. Ginkgo biloba extract demonstrates anti-ischemic properties attributable to the terpenoid fraction, mainly due to the presence of bilobalide. The mechanism of the protection afforded by bilobalide is not yet known. In this work, the effects of bilobalide on mitochondrial respiration were investigated. Mitochondria isolated from rats treated with bilobalide (2 to 8 mg/kg) showed a dose-dependent increase in the respiratory control ratio, due to a lower oxygen consumption during state 4. Bilobalide also decreased the sensitivity of oxygen consumption to inhibition of complex I by Amytal or to inhibition of complex III by antimycin A or myxothiazol. There was no protection of complexes IV and V. It also increased the activity of complex I but not of adenine translocase. Similar effects were also obtained in vitro when control mitochondria were preincubated for 1 hr with 0.8 microg/mL bilobalide. Treatment of the rats with 8 mg/kg bilobalide also prevented the ischemia-induced decrease in state 3 of the mitochondrial respiration and thus the decrease in RCR. The protective effect of bilobalide on cellular ATP content observed under ischemic conditions can be correlated with the above observations. By protecting complex I and III activities, bilobalide allows mitochondria to maintain their respiratory activity under ischemic conditions as long as some oxygen is present, thus delaying the onset of ischemia-induced damage. This mechanism provides a possible explanation for the anti-ischemic properties of bilobalide and of Ginkgo biloba extract in therapeutic interventions.

Studies on the mechanism of synergistic action with synergisidin and imidazole antimycotics.

The investigation of mechanism of synergistic action with SYN and ECZ was performed using C. albicans SC5314 so that SYN was confirmed to show strong synergistic effects against Candida sp. in particular with addition of extremely small quantities under the MICs of imidazole antimycotics such as ECZ, MCZ and CTZ. The synergistic effect of antifungal activity against C. albicans SC5314 with a combination of SYN and ECZ (SYN + ECZ) showed fungistatic action. Effect of SYN + ECZ on osmotic resistance was not recognized and protoplast was not observed under a microscope. Accordingly, SYN + ECZ was considered not to take part in cell wall synthesis directly. For effect of SYN + ECZ on release of intracellular components, slow release of 260 nm-absorbing substances was occurred, so that SYN + ECZ was seemed not to affect cytoplasmic membrane damage directly. Also, it was suggested clearly that SYN + ECZ affected lipid metabolism and glycolysis including TCA cycle from the investigation on antagonism by growth recovery of C. albicans SC5314 by 106 kinds of substances such as fatty acids, isoprenoids, phospholipids, vitamins, amino acids, nucleic acid-related substances and TCA cycle-related substances. From the above results, it was suggested that the mechanism of synergistic action with SYN and ECZ against C. albicans SC5314 was due to affect the different reactions in lipid metabolism and the similar reactions in glycolysis including TCA cycle, respectively, in consideration of respective mechanism of actions of SYN alone and ECZ alone. A part of this work was presented at the Annual Meeting of the Agricultural Chemical Society of Japan, 1981 (Kyoto).