Search

Common ragweed (Ambrosia artemisiifolia L.) is a highly allergenic annual ruderal plant and native to Northern America, but now also spreading across Europe. Air pollution and climate change will not only affect plant growth, pollen production and duration of the whole pollen season, but also the amount of allergenic encoding transcripts and proteins of the pollen. The objective of this study was to get a better understanding of transcriptional changes in ragweed pollen upon NO 2 and O 3 fumigation. This will also contribute to a systems biology approach to understand the reaction of the allergenic pollen to air pollution and climate change. Ragweed plants were grown in climate chambers under controlled conditions and fumigated with enhanced levels of NO 2 and O 3 . Illumina sequencing and de novo assembly revealed significant differentially expressed transcripts, belonging to different gene ontology (GO) terms that were grouped into biological process and molecular function. Transcript levels of the known Amb a ragweed encoding allergens were clearly up-regulated under elevated NO 2 , whereas the amount of allergen encoding transcripts was more variable under elevated O 3 conditions. Moreover transcripts encoding allergen known from other plants could be identified. The transcriptional changes in ragweed pollen upon elevated NO 2 fumigation indicates that air pollution will alter the transcriptome of the pollen. The changed levels of allergenic encoding transcripts may have an influence on the total allergenic potential of ragweed pollen., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: For efficient biofarming we attempted to enrich plant interstitial fluid (IF)/apoplastic fluid with targeted recombinant therapeutic protein. We employed a synthetic human Glucocerebrosidase (GCB), a model biopharmaceutical protein gene in this study., Results: Twenty one Nicotiana varieties, species and hybrids were initially screened for individual IF recovery and based on the findings, we selected Nicotiana tabacum NN (S-9-6), Nicotiana tabacum nn (S-9-7) and Nicotiana benthamiana (S-6-6) as model plants for raising transgenic expressing GCB via Agrobacterium mediated transformation under the control of M24 promoter; GCB specific activity in each transgenic lines were analyzed and we observed higher concentration of recombinant GCB in IF of these transgenic lines (S-9-6, S-9-7, and S-6-6) in comparison to their concentration in crude leaf extracts., Conclusion: Recovery of valuable therapeutics in plant IF as shown in the present study holds great promise for promoting plant based biofarming. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:726-736, 2017., (© 2017 American Institute of Chemical Engineers.)

Dragon's blood is a red resin mainly extracted from Dracaena plants, and has been widely used as a traditional medicine in East and Southeast Asia. The major components of dragon's blood are flavonoids. Owing to a lack of Dracaena plants genomic information, the flavonoids biosynthesis and regulation in Dracaena plants remain unknown. In this study, three cDNA libraries were constructed from the stems of D. cambodiana after injecting the inducer. Approximately 266.57 million raw sequencing reads were de novo assembled into 198,204 unigenes, of which 34,873 unique sequences were annotated in public protein databases. Many candidate genes involved in flavonoid accumulation were identified. Differential expression analysis identified 20 genes involved in flavonoid biosynthesis, 27 unigenes involved in flavonoid modification and 68 genes involved in flavonoid transport that were up-regulated in the stems of D. cambodiana after injecting the inducer, consistent with the accumulation of flavonoids. Furthermore, we have revealed the differential expression of transcripts encoding for transcription factors (MYB, bHLH and WD40) involved in flavonoid metabolism. These de novo transcriptome data sets provide insights on pathways and molecular regulation of flavonoid biosynthesis and transport, and improve our understanding of molecular mechanisms of dragon's blood formation in D. cambodiana.

Context: Rhizoma coptidis is a broadly used traditional Chinese medicine (TCM). The investigation of the influence of species and geographical origins on the phytochemicals of R. coptidis is crucial for its reasonable application and quality control., Objective: Development of an effective method to systematically study the phytochemical variations of the rhizomes of three Coptis species (Ranunculaceae) (Coptis chinensis Franch, Coptis deltoidea C.Y. Cheng et Hsiao and Coptis teeta Wall.) and a species (i.e., C. chinensis) obtained from both Daodi and non-Daodi production regions., Results: The three species had significant differences in their phytochemicals. The rhizome of C. chinensis contained more epiberberine (13.52 ± 2.65 mg/g), palmatine (18.20 ± 2.89 mg/g), coptisine (23.32 ± 4.27 mg/g) and columbamine (4.89 ± 1.16 mg/g), whereas the rhizomes of C. deltoidea and C. teeta showed the highest level of jatrorrhizine (8.52 ± 1.36 mg/g) and berberine (81.06 ± 4.83 mg/g), respectively. Moreover, the rhizome of C. chinensis from three Daodi production regions (Shizhu, Lichuan and Emeishan) contained more alkaloids than those from three non-Daodi production regions (Mianyang, Shifang and Zhenping)., Discussion and Conclusion: It is necessary to use the three R. coptidis species differentially in TCM clinical practice. Daodi C. chinensis medicinal materials have better quality than most non-Daodi ones, and so they should be preferred for TCM prescription. The combination of HPLC-based fingerprint analysis and quantification of multi-ingredients with statistical analysis provided an effective approach for species discrimination and quality evaluation of R. coptidis.

Efficient selection of potato varieties with enhanced nutritional quality requires simple, accurate and cost effective assays to obtain tuber chemical composition information. In this study, 75 Andean native potato samples from 7 Solanum species with different colors were characterized and quantified for their anthocyanin, phenolics and sugar content using traditional reference methods. IR (infrared) spectra of potato extracts were collected using a portable infrared system and partial least squares regression (PLSR) calibration models were developed. These models were validated using both full cross-validation and an independent sample set giving strong linear correlation coefficients of prediction (rPred)>0.91 and standard error of prediction (SEP) of 24mg/100g phenolics, 7mg/100g monomeric anthocyanins, 0.1g/100g reducing sugars and 0.12g/100g sucrose. Overall, portable infrared system with PLSR showed great potential to facilitate potato breeding and certain aspects of crop management, material selection for potato processing and related research by providing alternative prediction models., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Saraca asoca (Roxb.) Willd, commonly known as "Asoka" or "Ashoka," is one of the most important medicinal plants used in raw herbal trade in India. The bark extracts of the tree are used in the treatment of leucorrhea and other uterine disorders besides also having anti-inflammatory, anti-bacterial, anti-pyretic, anti-helminthic, and analgesic activity. The indiscriminate and rampant extraction of the wood to meet the ever-increasing market demand has led to a sharp decline in naturally occurring populations of the species in the country. Consequently, the species has recently been classified as "vulnerable" by the International Union for Conservation of Nature (IUCN). Increasing deforestation and increasing demand for this medicinal plant have resulted in a limited supply and suspected widespread adulteration of the species in the raw herbal trade market. Adulteration is a serious concern due to: (i) reduction in the efficacy of this traditional medicine, (ii) considerable health risk to consumers, and (iii) fraudulent product substitution that impacts the economy for the Natural Health Product (NHP) Industry and consumers. In this paper, we provide the first attempt to assess the extent of adulteration in the raw herbal trade of S. asoca using DNA barcoding validated by NMR spectroscopic techniques. Analyzing market samples drawn from 25 shops, mostly from peninsular India, we show that more than 80 % of the samples were spurious, representing plant material from at least 7 different families. This is the first comprehensive and large-scale study to demonstrate the widespread adulteration of market samples of S. asoca in India. These results pose grave implications for the use of raw herbal drugs, such as that of S. asoca, on consumer health and safety. Based on these findings, we argue for a strong and robust regulatory framework to be put in place, which would ensure the quality of raw herbal trade products and reassure consumer confidence in indigenous medicinal systems. Graphical Abstract DNA barcoding and NMR spectroscopy-based assessment of adulteration in Saraca asoca.

This is the first comprehensive study of the genetic analysis of the majority of oleoresin components of slash pine (Pinus elliottii). Pine oleoresin, the resin secreted from the pine tree, is a raw material widely used in industrial products. The objective of this study was to explore the genetic variation and correlation between the major oleoresin components of 50 open pollinated families of slash pine. The individual narrow-sense heritability of the 23 oleoresin components and genetic correlations between them were estimated using the residual maximum likelihood in the flexible mixed modeling program, ASReml-R. A high heritability of 0.424 was observed for β-pinene. Moderate levels of heritability were estimated for β-phellandrene, methyl abietate, estragole, 15-hydroxy-dehydroabietic acid, and isopimaric acid methyl ester at 0.303, 0.294, 0.27, 0.258, and 0.2, respectively. The heritabilities for pimaric acid methyl ester, abieta-8, 13-diene-18-oic acid methyl ester, sandaracopimaric acid, methyl ester, and camphene were relatively low and ranged from 0.11 to 0.17. Many negative genetic correlations were observed as unfavorable while the corresponding phenotypic correlations presented no significant relationships or positive phenotypic correlations. However, the heritabilities and genetic correlations showed that single or multiple component selections and improvement, directly or indirectly, were effective. We postulate that genetic parameters estimated in this study will work as a reference in breeding programs of oleoresin components, especially in slash pine., Competing Interests: The authors declare no conflict of interest.

Background: Ayurveda is a system of traditional medicine that originated in ancient India, and it is still in practice. Medicinal plants are the backbone of Ayurveda, which heavily relies on the plant-derived therapeutics. While Ayurveda is becoming more popular in several countries throughout the World, lack of authenticated medicinal plant raw drugs is a growing concern. Our aim was to DNA barcode the medicinal plants that are listed in the Ayurvedic Pharmacopoeia of India (API) to create a reference DNA barcode library, and to use the same to authenticate the raw drugs that are sold in markets., Methods: We have DNA barcoded 347 medicinal plants using rbcL marker, and curated rbcL DNA barcodes for 27 medicinal plants from public databases. These sequences were used to create Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL). This library was used to authenticate 100 medicinal plant raw drugs, which were in the form of powders (82) and seeds (18)., Results: Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL) was created with high quality and authentic rbcL barcodes for 374 out of the 395 medicinal plants that are included in the API. The rbcL DNA barcode differentiated 319 species (85 %) with the pairwise divergence ranging between 0.2 and 29.9 %. PCR amplification and DNA sequencing success rate of rbcL marker was 100 % even for the poorly preserved medicinal plant raw drugs that were collected from local markets. DNA barcoding revealed that only 79 % raw drugs were authentic, and the remaining 21 % samples were adulterated. Further, adulteration was found to be much higher with powders (ca. 25 %) when compared to seeds (ca. 5 %)., Conclusions: The present study demonstrated the utility of DNA barcoding in authenticating medicinal plant raw drugs, and found that approximately one fifth of the market samples were adulterated. Powdered raw drugs, which are very difficult to be identified by taxonomists as well as common people, seem to be the easy target for adulteration. Developing a quality control protocol for medicinal plant raw drugs by incorporating DNA barcoding as a component is essential to ensure safety to the consumers.

Unlabelled: Cyclotides are plant-derived, cyclic miniproteins with three interlocking disulfide bonds that have attracted great interests because of their excellent stability and potential as peptide therapeutics. In this study, we characterize the cyclotides of the medicinal plant Clitoria ternatea (butterfly pea) and investigate their biological activities. Using a combined proteomic and transcriptomic method, we identified 41 novel cyclotide sequences, which we named cliotides, making C. ternatea one of the richest cyclotide-producing plants to date. Selected members of the cationic cliotides display potent antibacterial activity specifically against Gram-negative bacteria with minimal inhibitory concentrations as low as 0.5 μm. Remarkably, they also possess prominent immunostimulating activity. At a concentration of 1 μm, cationic cliotides are capable of augmenting the secretion of various cytokines and chemokines in human monocytes at both resting and lipopolysaccharide-stimulated states. Chemokines such as macrophage inflammatory proteins 1α and 1β, interferon γ-induced protein 10, interleukin 8 and tumor necrosis factor α were among the most upregulated with up to 129-fold increase in secretion level. These findings suggest cyclotides can serve as potential candidates for novel immunomodulating therapeutics., Database: The protein sequences reported in this paper (cT13-cT21) are available in the UniProt Knowledgebase under the accession numbers C0HJS0, C0HJS1, C0HJS2, C0HJS3, C0HJS4, C0HJS5, C0HJS6, C0HJS7 and C0HJS8, respectively. The transcriptome data in this paper are available at the Sequence Read Archive database (NCBI) under accession number SRR1613316. The protein precursors reported in this paper (ctc13, ctc15, ctc17-ctc19, ctc21-ctc53) are available at GenBank under the accession numbers KT732712, KT732713, KT732714, KT732715, KT732716, KT732717, KT732718, KT732719, KT732720, KT732721, KT732722, KT732723, KT732724, KT732725, KT732726, KT732727, KT732728, KT732729, KT732730, KT732731, KT732732, KT732733, KT732734, KT732735, KT732736, KT732737, KT732738, KT732739, KT732740, KT732741, KT732742, KT732743, KT732744, KT732745, KT732746, KT732747, KT732748 and KT732749, respectively., (© 2016 Federation of European Biochemical Societies.)

RNA extraction resulting in good yields and quality is a fundamental step for the analyses of transcriptomes through high-throughput sequencing technologies, microarray, and also northern blots, RT-PCR, and RTqPCR. Even though many specific protocols designed for plants with high content of secondary metabolites have been developed, these are often expensive, time consuming, and not suitable for a wide range of tissues. Here we present a modification of the method previously described using the commercially available Concert™ Plant RNA Reagent (Invitrogen) buffer for field-grown adult pine trees with high oleoresin content.

Ragweed pollen is the main cause of allergenic diseases in Northern America, and the weed has become a spreading neophyte in Europe. Climate change and air pollution are speculated to affect the allergenic potential of pollen. The objective of this study was to investigate the effects of NO2 , a major air pollutant, under controlled conditions, on the allergenicity of ragweed pollen. Ragweed was exposed to different levels of NO2 throughout the entire growing season, and its pollen further analysed. Spectroscopic analysis showed increased outer cell wall polymers and decreased amounts of pectin. Proteome studies using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry indicated increased amounts of several Amb a 1 isoforms and of another allergen with great homology to enolase Hev b 9 from rubber tree. Analysis of protein S-nitrosylation identified nitrosylated proteins in pollen from both conditions, including Amb a 1 isoforms. However, elevated NO2 significantly enhanced the overall nitrosylation. Finally, we demonstrated increased overall pollen allergenicity by immunoblotting using ragweed antisera, showing a significantly higher allergenicity for Amb a 1. The data highlight a direct influence of elevated NO2 on the increased allergenicity of ragweed pollen and a direct correlation with an increased risk for human health., (© 2015 John Wiley & Sons Ltd.)

We have developed a novel type of biofunction-assisted aptasensor that harnesses ligand-dependent 3' processing of a premature amber suppressor tRNA and the subsequent amber suppression of a reporter gene in a wheat germ extract.

Root extract of Boerhaavia diffusa L. induced systemic resistance in tobacco against Tobacco mosaic virus. A 30 kDa protein was isolated as the active component, called BDP-30 on the basis of the molecular weight and source plant. BDP-30, a glycoprotein, was found to be temperature and protease resistant. It was basic, possessing a pI greater than 9.0. In-gel proteolytic digestion of BDP-30 generated two peptides that possessed the amino acid sequence KLYDIPPLR and KVTLPYSGNYER by LC/MS/MS. Both peptides shared absolute sequence identity with trichosanthin, a ribosome-inactivating protein from Trichosanthes kirilowii, and a 78 percent and 100 percent homology respectively with an RIP from Bryonia dioica, bryodin. Further, effort was made to look at the fate of TMV in induced resistant Nicotiana tabacum cv. Xanthi, a systemic host of the virus, at specified days after inoculation in control and treated plants. TMV coat protein (CP) was detected by immunoblot 7 days post inoculation up to 21 days in the control set, but not in treated resistant plants. TMV RNA was detected by RT-PCR using TMV-CP specific primers. Resistant tobacco did not show presence of TMV RNA up to 21 days of inoculation. This suggests that BDP-30 may be suppressing TMV replication.

In comparison to the exponential increase of genotyping methods, phenotyping strategies are lagging behind in agricultural sciences. Genetic improvement depends upon the abundance of quantitative phenotypic data and the statistical partitioning of variance into environmental, genetic, and random effects. A metabolic phenotyping strategy was adapted to increase sample throughput while saving reagents, reducing cost, and simplifying data analysis. The chemical profiles of stem extracts from maize plants grown under low nitrogen (LN) or control trial (CT) were analyzed using optimized protocols for direct-injection electrospray ionization mass spectrometry (DIESI-MS). Specific ions significantly decreased or increased because of environmental (LN versus CT) or genotypic effects. Biochemical profiling with DIESI-MS had a superior cost-benefit compared to other standard analytical technologies (e.g., ultraviolet, near-infrared reflectance spectroscopy, high-performance liquid chromatography, and gas chromatography with flame ionization detection) routinely used for plant breeding. The method can be successfully applied in maize, strawberry, coffee, and other crop species.

Context: In its powdered form, turmeric [Curcuma longa L. (Zingiberaceae)], a spice of medical importance, is often adulterated lowering its quality., Objective: The study sought to detect plant-based adulterants in traded turmeric powder using DNA barcoding., Materials and Methods: Accessions of Curcuma longa L., Curcuma zedoaria Rosc. (Zingiberaceae), and cassava starch served as reference samples. Three barcoding loci, namely ITS, rbcL, and matK, were used for PCR amplification of the reference samples and commercial samples representing 10 different companies. PCR success rate, sequencing efficiency, occurrence of SNPs, and BLAST analysis were used to assess the potential of the barcoding loci in authenticating the traded samples of turmeric., Results: The PCR and sequencing success of the loci rbcL and ITS were found to be 100%, whereas matK showed no amplification. ITS proved to be the ideal locus because it showed greater variability than rbcL in discriminating the Curcuma species. The presence of C. zedoaria could be detected in one of the samples whereas cassava starch, wheat, barley, and rye in other two samples although the label claimed nothing other than turmeric powder in the samples., Discussion and Conclusion: Unlabeled materials in turmeric powder are considered as adulterants or fillers, added to increase the bulk weight and starch content of the commodity for economic gains. These adulterants pose potential health hazards to consumers who are allergic to these plants, lowering the product's medicinal value and belying the claim that the product is gluten free. The study proved DNA barcoding as an efficient tool for testing the integrity and the authenticity of commercial products of turmeric.

The genus Lupinus is widely distributed. Its seeds are used for animal and human food, and Lupinus possesses pharmacological potential because of its high content of quinolizidine alkaloids and flavonoids; however, there is little available information about its genotoxicity. We used the comet assay and staminal nuclei of Tradescantia (clone 4430) to evaluate the in vitro genotoxicity of 4 concentrations (0.01, 0.1, 0.5, and 1.0 mM) of alkaloid extracts of Lupinus mexicanus and Lupinus montanus, flavonoids of L. mexicanus, and commercial sparteine; nitrosodiethylamine was used as a positive control and untreated nuclei were used as a negative control. All concentrations of L. mexicanus and L. montanus showed significant genotoxic activity (P ≤ 0.05). A similar behavior was observed for flavonoid extracts of L. montanus except the 1.0 mM concentration. Sparteine showed genotoxic activity only at 0.5 mM. The order of genotoxicity of the compounds studied was as follows: L. mexicanus > L. montanus > flavonoids of L. montanus > sparteine. There is evident genotoxic activity in the compounds that were studied, particularly at lower concentrations (0.01 and 0.1 mM). Given the limited information about the genotoxicity of the compounds of L. mexicanus and L. montanus, further studies are necessary.

The antioxidant and hepatoprotective effects of Heterotheca inuloides have been reported before, nevertheless its use as a possible chemopreventive agent has not been documented. The aim of this study was to evaluate the mutagenic and antimutagenic activities of H. inuloides extracts using the Ames test. Both, the methanolic and acetonic extracts, were mutagenic in the TA98 but not in TA100 or TA102 strains. On the other hand, the methanolic extract reduced the mutagenicity of norfloxacin, benzo[a]pyrene and 2-aminoanthracene. Quercetin, one of the main components in the methanolic extract, also presented a mutagenic/antimutagenic dual effect and is an inhibitor of Cytochrome P450 (CYP) 1A. The antigenotoxic properties of H. inuloides could be due to the antioxidant properties previously reported and to its CYP inhibitory effect mediated by quercetin. Further studies with in vivo systems will afford information about H. inuloides beneficial and detrimental properties.

A DNA biosensor for detection of specific oligonucleotides sequences of Plum Pox Virus (PPV) in plant extracts and buffer is proposed. The working principles of a genosensor are based on the ion-channel mechanism. The NH2-ssDNA probe was deposited onto a glassy carbon electrode surface to form an amide bond between the carboxyl group of oxidized electrode surface and amino group from ssDNA probe. The analytical signals generated as a result of hybridization were registered in Osteryoung square wave voltammetry in the presence of [Fe(CN)6]3-/4- as a redox marker. The 22-mer and 42-mer complementary ssDNA sequences derived from PPV and DNA samples from plants infected with PPV were used as targets. Similar detection limits of 2.4 pM (31.0 pg/mL) and 2.3 pM (29.5 pg/mL) in the concentration range 1-8 pM were observed in the presence of the 22-mer ssDNA and 42-mer complementary ssDNA sequences of PPV, respectively. The genosensor was capable of discriminating between samples consisting of extracts from healthy plants and leaf extracts from infected plants in the concentration range 10-50 pg/mL. The detection limit was 12.8 pg/mL. The genosensor displayed good selectivity and sensitivity. The 20-mer partially complementary DNA sequences with four complementary bases and DNA samples from healthy plants used as negative controls generated low signal.

A novel plant hairpin-like defense polypeptide named EcAMP3 was isolated from latent barnyard grass (Echinochloa crusgalli L.) seeds. The native peptide and its recombinant analogue were characterized. EcAMP3 displays antifungal and antibacterial activity in vitro. The gene family encoding EcAMPs precursor protein was also characterized; the genes and pseudogenes of this family show 97-100% homology. Every member of EcAMPs precursor family contains seven identical cysteine motifs: C1XXXC2(11-13)C3XXXC4. One of those motifs corresponds to the isolated peptide. EcAMP3 is the first member of the plant hairpin-like peptide family that inhibits the growth of phytopathogenic bacteria. Obtained results can explain the nature of the complex resistance of barnyard grass to a variety of pathogenic microorganisms., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

StCKP1 (Solanum tuberosum cytokinin riboside phosphorylase) catalyses the interconversion of the N9-riboside form of the plant hormone CK (cytokinin), a subset of purines, with its most active free base form. StCKP1 prefers CK to unsubstituted aminopurines. The protein was discovered as a CK-binding activity in extracts of tuberizing potato stolon tips, from which it was isolated by affinity chromatography. The N-terminal amino acid sequence matched the translation product of a set of ESTs, enabling a complete mRNA sequence to be obtained by RACE-PCR. The predicted polypeptide includes a cleavable signal peptide and motifs for purine nucleoside phosphorylase activity. The expressed protein was assayed for purine nucleoside phosphorylase activity against CKs and adenine/adenosine. Isopentenyladenine, trans-zeatin, dihydrozeatin and adenine were converted into ribosides in the presence of ribose 1-phosphate. In the opposite direction, isopentenyladenosine, trans-zeatin riboside, dihydrozeatin riboside and adenosine were converted into their free bases in the presence of Pi. StCKP1 had no detectable ribohydrolase activity. Evidence is presented that StCKP1 is active in tubers as a negative regulator of CKs, prolonging endodormancy by a chill-reversible mechanism.

Background: Heme oxygenase-1 (HO-1) has been shown to have antioxidant and anti-apoptotic properties. The present study transduced HO-1 protein into intestinal tissues using PEP-1, a cell-penetrating peptide, and investigated its potentiality in prevention against intestinal ischemia/reperfusion (I/R) injury., Materials and Methods: PEP-1-HO-1 fusion protein was administered intravenously to explore the time and dose characteristics through measuring serum HO-1 levels. Twenty-four male Sprague-Dawley rats were randomly divided into three groups: sham, intestinal I/R (II/R), II/R + PEP-1-HO-1 fusion protein (HO). The model was established by occluding the superior mesenteric artery for 45 min followed by 120 min reperfusion. In HO group, PEP-1-HO-1 was administered intravenously 30 min before ischemia, whereas animals in sham and II/R groups received the equal volume of physiological saline. After the experiment, the intestines were harvested for determination of histologic injury, wet/dry ratio, enzyme activity, apoptosis, and His-probe protein (one part of PEP-1-HO-1)., Results: Levels of serum HO-1 were dose- and time-dependent manner after intravenous injection of PEP-1-HO-1. I/R caused deterioration of histologic characteristics and increases in histologic injury scoring, wet/dry ratio, myeloperoxidase activity, malondialdehyde, and intestinal apoptosis. These changes were also accompanied by a decrease in superoxide dismutase activity (P < 0.05). PEP-1-HO-1 treatment significantly reversed these changes (P < 0.05). Furthermore, His-probe protein expression was only detected in PEP-1-HO-1-treated animals., Conclusion: Treatment of PEP-1-HO-1 attenuates intestinal I/R injury, which might be attributable to its antioxidant and anti-apoptotic roles of HO-1., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In April of 2013, letters addressed to the President of United States and other government officials were intercepted and found to be contaminated with ricin, heightening awareness about the need to evaluate laboratory methods for detecting ricin. This study evaluated commercial DNA purification methods for isolating Ricinus communis DNA as measured by real-time polymerase chain reaction (PCR). Four commercially available DNA purification methods (two automated, MagNA Pure compact and MagNA Pure LC, and two manual, MasterPure complete DNA and RNA purification kit and QIAamp DNA blood mini kit) were evaluated. We compared their ability to purify detectable levels of R. communis DNA from four different sample types, including crude preparations of ricin that could be used for biological crimes or acts of bioterrorism. Castor beans, spiked swabs, and spiked powders were included to simulate sample types typically tested during criminal and public health investigations. Real-time PCR analysis indicated that the QIAamp kit resulted in the greatest sensitivity for ricin preparations; the MasterPure kit performed best with spiked powders. The four methods detected equivalent levels by real-time PCR when castor beans and spiked swabs were used. All four methods yielded DNA free of PCR inhibitors as determined by the use of a PCR inhibition control assay. This study demonstrated that DNA purification methods differ in their ability to purify R. communis DNA; therefore, the purification method used for a given sample type can influence the sensitivity of real-time PCR assays for R. communis., (Published by Elsevier Ireland Ltd.)

Andrographolides, the diterpene lactones, are major bioactive phytochemicals which could be found in different parts of the medicinal herb Andrographis paniculata. A number of such compounds namely andrographolide (AG), neoandrographolide (NAG), and 14-deoxy-11,12-didehydroandrographolide (DDAG) have already attracted a great deal of attention due to their potential therapeutic effects in hard-to-treat diseases such as cancers and HIV. Recently, they have also been considered as substrates for the discovery of novel pharmaceutical compounds. Nevertheless, there is still a huge gap in knowledge on the genetic pattern of the biosynthesis of these bioactive compounds. Hence, the present study aimed to investigate the genetic mechanisms controlling the biosynthesis of these phytochemicals using a diallel analysis. The high performance liquid chromatography analysis of the three andrographolides in 210 F1 progenies confirmed that the biosynthesis of these andrographolides was considerably increased via intraspecific hybridization. The results revealed high, moderate and low heterosis for DDAG, AG and NAG, respectively. Furthermore, the preponderance of non-additive gene actions was affirmed in the enhancement of the three andrographolides contents. The consequence of this type of gene action was the occurrence of high broad-sense and low narrow-sense heritabilities for the above mentioned andrographolides. The prevalence of non-additive gene action suggests the suitability of heterosis breeding and hybrid seed production as a preferred option to produce new plant varieties with higher andrographolide contents using the wild accessions of A. paniculata. Moreover, from an evolutionary point of view, the occurrence of population bottlenecks in the Malaysian accessions of A. paniculata was unveiled by observing a low level of additive genetic variance (VA ) for all the andrographolides.

MMPs (matrix metalloproteases) are a family of zinc-dependent endopeptidases widely distributed throughout all kingdoms of life. In mammals, MMPs play key roles in many physiological and pathological processes, including remodelling of the extracellular matrix. In the genome of the annual plant Arabidopsis thaliana, five MMP-like proteins (At-MMPs) are encoded, but their function is unknown. Previous work on these enzymes was limited to gene expression analysis, and so far proteolytic activity has been shown only for At1-MMP. We expressed and purified the catalytic domains of all five At-MMPs as His-tagged proteins in Escherichia coli cells to delineate the biochemical differences and similarities among the Arabidopsis MMP family members. We demonstrate that all five recombinant At-MMPs are active proteases with distinct preferences for different protease substrates. Furthermore, we performed a family-wide characterization of their biochemical properties and highlight similarities and differences in their cleavage site specificities as well as pH- and temperature-dependent activities. Detailed analysis of their sequence specificity using PICS (proteomic identification of protease cleavage sites) revealed profiles similar to human MMPs with the exception of At5-MMP; homology models of the At-MMP catalytic domains supported these results. Our results suggest that each At-MMP may be involved in different proteolytic processes during plant growth and development.

The amount of clinically relevant, allergy-related proteins in wheat grain is still largely unknown. The application of proteomics may create a platform not only for identification and characterization, but also for quantitation of these proteins. The aim of this study was to evaluate the data-independent quantitative mass spectrometry (MS(E)) approach in combination with 76 wheat allergenic sequences downloaded from the AllergenOnline database ( www.allergenonline.org ) as a starting point. Alcohol soluble extracts of gliadin and glutenin proteins were analyzed. This approach has resulted in identification and quantification of 15 allergenic protein isoforms that belong to amylase/trypsin inhibitors, γ-gliadins, and high or low molecular weight glutenins. Additionally, several peptides carrying four previously discovered epitopes of γ-gliadin B precursor have been detected. These data were validated against the UniProt database, which contained 11764 Triticeae protein sequences. The identified allergens are discussed in relation to Baker's asthma, food allergy, wheat dependent exercise induced anaphylaxis, atopic dermatitis, and celiac disease (i.e., gluten-sensitive enteropathy). In summary, the results showed that the MS(E) approach is suitable for quantitative analysis and allergens profiling in wheat varieties and/or other food matrices.

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10⁸ α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of the present study was to elucidate whether extracts of Vigna angularis (EVA) inhibit allergic inflammatory reactions and to elucidate the possible mechanisms of action. For the assessment of allergic inflammatory response, histamine release and the expression of pro-inflammatory cytokines from human mast cells (HMC-1) were examined. To identify the underlying mechanisms of action, intracellular calcium and the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) were assayed. To confirm the effects of EVA in vivo, systemic and local allergic reaction mouse models were employed. EVA dose-dependently reduced phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-induced histamine release from mast cells. The inhibitory effects of EVA on the release of histamine from mast cells were mediated by the reduction of intracellular calcium levels. EVA decreased the PMACI-stimulated gene expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6. The inhibitory effects of EVA on pro-inflammatory cytokines were NF-κB- and MAPK-dependent. In addition, EVA inhibited compound 48/80-induced systemic anaphylaxis and immunoglobulin E (IgE)-mediated cutaneous anaphylaxis. Our findings provide evidence that EVA inhibits mast cell-derived allergic inflammation, and suggest the possible therapeutic application of EVA in allergic inflammatory disorders.

Canine atopic dermatitis (CAD) is an immune system disorder that affects 10-15% of the canine population. Short ragweed (Ambrosia artemisiifolia) pollen represents one of the major seasonal sources of allergenic pollen proteins in Europe, particularly in the Pannonian valley of the Balkan region. In Serbia, about 66% of atopic dogs showed a positive intradermal skin test with its pollen extract, which is second to house dust mites. Therefore, characterization of Ambrosia artemisiifolia pollen components, in terms of defining major and minor allergens that induce clinically manifested allergic reaction in dogs, is important for valid diagnosis and efficient therapy. This study has, for the first time, characterized and identified major Ambrosia artemisiifolia allergens in CAD, using an immunoproteomic approach. To assess the prevalence of specific IgE in electrophoretically separated ragweed pollen proteins, individual reactivity of sera from dogs with CAD was analyzed and compared to the reactivity of sera from healthy dogs in the non-reducing conditions, which were found optimal for specific canine IgE detection. A specific IgE band (38 kDa) was recognized as the most dominant allergen in CAD, occurring in 81% of positive dog's sera. 2-D immunoblotting followed by a mass spectrometry peptide fingerprint analyses with pooled canine and human atopic sera, revealed that 38 kDa major Ambrosia atremisiifolia allergens in CAD were all five isoallergens of the Amb a 1 group (antigen E), including the previously named Amb a 2 (antigen K). In contrast to canine sera, human atopic sera also recognized lower mass allergens such as the β fragment of Amb a 1 and profilins (Amb a 8 variants). The most prominent ragweed proteins in CAD, represent, as in humans, variants of all five isoallergens of the Amb a 1 group (pectate lyase): Amb a 1.0101 and its natural variant E1XUL2, Amb a 1.0202, 1.0304, 1.0402 and the natural variant of Amb a 1.0501, E1XUM0, as well as the α fragment of pollen allergen Amb a 1.0201., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 μg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: The discovery of new natural compounds with pharmacological properties is a field of interest widely growing. Recent literature shows that Brassica vegetables (Cruciferae) possess therapeutic effects particularly ascribed due to their content in glucosinolates, which upon myrosinase hydrolysis release the corresponding isothiocyanates. This study examines the potential neuroprotective and immunomodulatory effects of (RS )-glucoraphanin from Tuscan black kale (Brassica oleracea L. var. acephala sabellica) bioactivated with myrosinase (bioactive RS -GRA) (10 mg/kg/day intraperitoneally), in an experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis., Methods: EAE was induced by immunization with myelin oligodendroglial glycoprotein peptide (MOG35-55 ) in mice. After immunization, mice were observed daily for signs of EAE and weight loss. Clinical score was evaluated using a standardized scoring system., Results: By Western blot analysis of spinal cord tissues, we have demonstrated that treatment with bioactive RS -GRA significantly decreased nuclear factor (NF)-kB translocation, pro-inflammatory cytokine production such as interleukin-1β (IL-1β), and apoptosis (Bax and caspase 3 expression)., Conclusion: Our results clearly demonstrate that bioactive RS -GRA treatment may represent a useful therapeutic perspective in the treatment of this disease., (© 2013 John Wiley & Sons Ltd.)

Objective: To explore the new method of discriminating Gelsemium elegans from Lonicera japonica and its close species by using specific PCR amplification., Method: Thirteen samples of the different G. elegans materials and 58 samples of L. japonica, L. macranthoides and L. dasystyla were collected. The total DNA of the samples were extracted, and the DNA of G. elegans, L. japonica and L. macranthoides water extracts were extracted. PsbA-rnnH sequence from G. elegans was amplified by PCR and sequenced unidirectionally, ClustulW was used to align psbA-trnH sequences of the G. elegans and L. japonica and its close species from GenBank database., Result: All samples were amplified by PCR with specific primer, DNA from G. elegans would be amplified 97 bp whereas PCR products from all of Lonicera samples had not bands., Conclusion: Specific PCR amplification can be used to identify G. elegans from L. japonica and its close species successfully and is an efficient molecular marker for authentication of G. elegans and L. japonica and its close species.

Rapidly enhancing oleoresin production in conifer stems through genomic selection and genetic engineering may increase resistance to bark beetles and terpenoid yield for liquid biofuels. We integrated association genetic and genomic prediction analyses of oleoresin flow (g 24 h(-1)) using 4854 single nucleotide polymorphisms (SNPs) in expressed genes within a pedigreed population of loblolly pine (Pinus taeda) that was clonally replicated at three sites in the southeastern United States. Additive genetic variation in oleoresin flow (h(2) ≈ 0.12-0.30) was strongly correlated between years in which precipitation varied (r(a) ≈ 0.95), while the genetic correlation between sites declined from 0.8 to 0.37 with increasing differences in soil and climate among sites. A total of 231 SNPs were significantly associated with oleoresin flow, of which 81% were specific to individual sites. SNPs in sequences similar to ethylene signaling proteins, ABC transporters, and diterpenoid hydroxylases were associated with oleoresin flow across sites. Despite this complex genetic architecture, we developed a genomic prediction model to accelerate breeding for enhanced oleoresin flow that is robust to environmental variation. Results imply that breeding could increase oleoresin flow 1.5- to 2.4-fold in one generation., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Nelumbo nucifera is widely used as food, as an ornamental, in medicine, and as packing material; it is also reported to have anti-HIV effects and antioxidant capacity. We sought an improved method for extracting high-quality total RNA from different tissues of N. nucifera. Four methods for RNA extraction were assessed for their ability to recover high-quality RNA applicable for evaluation of polyphenol oxidase (PPO) gene expression profiles. The recovery and quality of the RNA obtained from five different tissues by the best CTAB-LiCl method were evaluated through UV light absorbance. Both A(260)/A(280) and A(260)/A(230) absorbance ratios were more than 2.0; the yield ranged from 59.87 to 163.75 μg/g fresh weight. The brightness of the 28S band was approximately twice that of 18S; the latter was also considered as high-quality RNA. The PPO gene fragment (606 bp) was successfully amplified by RT-PCR, demonstrating the integrity of the isolated RNA. The relative expression levels of the PPO gene based on RT-PCR in five tissues of lotus were: rhizome buds (2.66), young leaves (2.42), fresh cut rhizome (2.02), petals (1.80), and petiole (1.65), using housekeeping gene β-actin as an internal control. We concluded that the total RNA isolated by this protocol is of sufficient quality for molecular applications.

Aims: The activity of phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (Akt) is enhanced under hypertension. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of PI3K signaling, and its activity is redox-sensitive. In the rostral ventrolateral medulla (RVLM), which is responsible for the maintenance of blood pressure, oxidative stress plays a pivotal role in neurogenic hypertension. The present study evaluated the hypothesis that redox-sensitive inactivation of PTEN results in enhanced PI3K/Akt signaling in RVLM, leading to neurogenic hypertension., Results: Compared to age-matched normotensive Wistar-Kyoto (WKY) rats, PTEN inactivation in the form of oxidation and phosphorylation were greater in RVLM of spontaneously hypertensive rats (SHR). PTEN inactivation was accompanied by augmented PI3K activity and PI3K/Akt signaling, as reflected by the increase in phosphorylation of Akt and mammalian target of rapamycin. Intracisternal infusion of tempol or microinjection into the bilateral RVLM of adenovirus encoding superoxide dismutase significantly antagonized the PTEN inactivation and blunted the enhanced PI3K/Akt signaling in SHR. Gene transfer of PTEN to RVLM in SHR also abrogated the enhanced Akt activation and promoted antihypertension. Silencing PTEN expression in RVLM with small-interfering RNA, on the other hand, augmented PI3K/Akt signaling and promoted long-term pressor response in normotensive WKY rats., Innovation: The present study demonstrated for the first time that the redox-sensitive check-and-balance process between PTEN and PI3K/Akt signaling is engaged in the pathogenesis of hypertension., Conclusion: We conclude that an aberrant interplay between the redox-sensitive PTEN and PI3k/Akt signaling in RVLM underpins neural mechanism of hypertension.

One of the challenges of systems biology is to integrate multiple sources of data in order to build a cohesive view of the system of study. Here we describe the mass spectrometry based profiling of maize kernels, a model system for genomic studies and a cornerstone of the agroeconomy. Using a network analysis, we can include 97.5% of the 8,710 features detected from 210 varieties into a single framework. More conservatively, 47.1% of compounds detected can be organized into a network with 48 distinct modules. Eigenvalues were calculated for each module and then used as inputs for genome-wide association studies. Nineteen modules returned significant results, illustrating the genetic control of biochemical networks within the maize kernel. Our approach leverages the correlations between the genome and metabolome to mutually enhance their annotation and thus enable biological interpretation. This method is applicable to any organism with sufficient bioinformatic resources.

The air-dried aerial parts of Lavandula angustifolia Mill, a traditional Uygur herbal drug, is used as resuscitation-inducing therapy to treat neurodisfunctions, such as stroke. This study was designed to assess the neuroprotective effects of lavender oil against ischemia/reperfusion (IR) injury in mice. Focal cerebral ischemia was induced by the intraluminal occlusion method with a nylon string. The neurodysfuntion was evaluated by neurological deficit and the infarct area was showed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The histopathological changes were observed by hematoxylin and eosin staining. The levels of mitochondria-generated reactive oxygen species (ROS), malondialdehyde (MDA) and carbonyl, the ratio of reduced glutathione (GSH)/glutathione disulfide (GSSG), the activities of superoxide dismutase (SOD), catalase (CAT) and glutathion peroxidase (GSH-Px) in brain tissue were measured to estimate the oxidative stress state. Neurological deficit, infarct size, histopathology changes and oxidative stress markers were evaluated after 22 h of reperfusion. In comparison with the model group, treatment with lavender oil significantly decreased neurological deficit scores, infarct size, the levels of MDA, carbonyl and ROS, and attenuated neuronal damage, upregulated SOD, CAT, GSH-Px activities and GSH/GSSG ratio. These results suggested that the neuroprotective effects of lavender oil against cerebral ischemia/reperfusion injury may be attributed to its antioxidant effects.

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe-S] cluster. We determined the cluster to be a [4Fe-4S] type, which quickly oxidizes to a [2Fe-2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys(135), is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe-4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe-S cluster or Cys(135) retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe-S cluster in Planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.

A gene OsZnI encoding Cys3/His1-type zinc finger protein was isolated from the water stress-induced cDNA library of rice (Oryza sativa) cv. N-22, an early maturing, deep-rooted, drought-tolerant genotype adapted to upland conditions. The in-silico analysis revealed an insert of 800 bp with an ORF of 663 nucleotides, encoding 221 amino acids. OsZnI had three distinct features--nuclear localization signal (NLS) present in Arg152-Arg168, Zn finger domain between 185-193 amino acids and 12 amino acids conserved domain in 71-82 amino acids homologous to LEA motif, and belonged to C-type family of Zn finger protein. OsZnI showed induced expression under water deficit stress.

Angelica gigas is a medicinal plant that produces pyranocoumarins, including decursin (D) and decursinol angelate (DA), which have neuroprotective, anticancer, and antiandrogenic effects. In this study, the coumarin biosynthetic pathway was engineered to increase the production of DA. Specifically, a vector was constructed which contained the A. gigas phenylalanine ammonia-lyase (AgPAL) and cinnamate 4-hydroxylase (AgC4H) genes that were driven by the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic hairy roots that overexpressed AgPAL or AgC4H genes were obtained by using an Agrobacterium rhizogenes-mediated transformation system. Among them, only AgC4H-transgenic hairy root lines produced more DA than control transgenic hairy root lines. The enhanced gene expression corresponded to elevated C4H activities. This study showed the importance of C4H in the production of DA in A. gigas hairy root culture.

Genetic and molecular analyses mainly in Arabidopsis and in some other plants have demonstrated involvement of light signaling in cell cycle regulation. In this report, we show light-mediated activation of the promoter of AtPolλ gene, a homolog of mammalian DNA polymerase λ in Arabidopsis thaliana and an important component of DNA damage repair/recombination machinery in plants. Analyses of the light-mediated promoter activity using various deletion versions of AtPolλ promoter in transformed Arabidopsis and tobacco (Nicotiana tabaccum) plants indicate that a 130-bp promoter region between -536 and -408 of AtPolλ promoter is essential for light-induced regulation of AtPolλ expression. DNA-protein interaction studies reveal that an ATCT-motif and AE-box light-responsive elements in the light-regulated promoter region confer light responsiveness of AtPolλ promoter. DNA-binding analysis has identified a 63-kDa trans-acting protein factor which showed specific binding to ATCT-motif, while another trans-acting factor of ~52 kDa was found to bind specifically to both ATCT and AE-box sequences. The 52-kDa protein has been identified as B3-domain transcription factor by MALDI-TOF/MS analysis. Overall, our results provide novel information on the role of light signaling in regulation of expression of an important component of DNA repair machinery in plants.

In vitro-transcribed, unmodified, and non-aminoacylated amber suppressor tRNAs that are recognized by natural aminoacyl-tRNA synthetase were improved toward higher suppression efficiency in batch-mode cell-free translation in wheat germ extract. The suppression efficiency of the suppressor obtained through four sequence optimization steps (anticodon alteration of natural tRNAs (the first generation); chimerization of the efficient suppressors in the first generation; investigation and optimization of the effective parts in the second generation; combination of the optimized parts in the third generation) and by the terminal tuning was approximately 60%, which was 2.4-fold higher than that of the best suppressor in the first generation. In addition, an eRF1 aptamer further increased the efficiency up to 85%. This highly efficient suppression system also functioned well in a dialysis-based large-scale protein synthesis.

Intense efforts are currently devoted to improve plant metabolomic analyses so as to describe more accurately the whole picture of metabolic pathways. Analyses based on liquid chromatography/time-of-flight mass spectrometry (LC-TOF) are now widely distributed among plant science laboratories. However, the use of reliable, sensitive LC-TOF methods to identify and quantify micromolar or inframicromolar key metabolites is often impeded by the sensitivity of the technique to sample preparation or chromatographic conditions. Typically, the sample matrix has a substantial influence on ionization efficiency and therefore, on the detectability of such compounds. Here, we describe a new method to analyze simultaneously 23 nucleotides and cofactors from plant extracts, taking advantage of solid-phase extraction (SPE) prior to injection. The influence of common m/z fragments in several metabolites and adducts is considered. We applied this method to characterise metabolic intermediates of NAD biosynthesis in Arabidopsis thaliana, using a wild-type and an engineered transgenic plant line that produces bacterial quinolinate phosphoribosyl transferase (nadc). We show that sample pre-purification with SPE is strictly required not only for compound quantification and identification but also to allow ionization of matrix-sensitive compounds (e.g. nicotinamide) or alleviate fragmentation of others (e.g. NAD). When exogenous substrate quinolinate was infiltrated into Arabidopsis leaves to increase the natural content in downstream metabolites, a clear correlation between intermediates of NAD biosynthesis was seen, showing the accuracy of our method for quantification in biological samples. Nadc plants only showed very modest changes in NAD-related metabolites and furthermore, they were associated with slightly lower photosynthetic performance and ATP production., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

In late February 2008, law enforcement officials in Las Vegas, Nevada, discovered in a hotel room, a copy of The Anarchist Cookbook, suspected castor beans and a "white powder" thought to be a preparation of ricin. Ricin is a deadly toxin from the seed of the castor bean plant (Ricinus communis). The United States regulates the possession, use, and transfer of ricin and it is the only substance considered a warfare agent in both the Chemical and the Biological Weapons Conventions. Six samples obtained from the hotel room were analyzed by laboratories at the Centers for Disease Control and Prevention using a panel of biological and mass spectrometric assays. The biological assays (real time-PCR, time resolved fluorescence and cytotoxicity) provided presumptive evidence of active ricin in each of the samples. This initial screen was followed by an in-depth analysis using a novel, state-of-the-art mass spectrometry-based ricin functional assay and high sensitivity tandem mass spectrometry for protein identification. Mass spectrometric analysis positively identified ricin and confirmed that in each of the samples it was enzymatically active. The tandem mass spectrometry analysis used here is the most selective method available to detect ricin toxin. In each sample, ricin was unequivocally identified along with other R. communis plant proteins, including the highly homologous protein RCA120. Although database searches using tandem mass spectra acquired from the samples indicated that additional controlled substances were not present in these samples, the mass spectrometric results did provide extensive detail about the sample contents. To the best of our knowledge following a review of the available literature, this report describes the most detailed analysis of a white powder for a public health or forensic investigation involving ricin., (Published by Elsevier Ireland Ltd.)

We produced anti-ginsenoside Re (GRe)-4G10 monoclonal antibody that specifically recognizes GRe and used this to prepare an immunoaffinity column to remove GRe from American ginseng berry extract (AGBE) (thus producing GRe knock-out AGBE (GRe-KO-AGBE)). We compared the anti-diabetic and anti-obesity effects of AGBE and GRe-KO-AGBE in adult diabetic C57BL/6J ob/ob mice. Fasting blood glucose levels and body weight were measured in mice receiving daily intraperitoneal injections of 150 mg/kg of either extract for 27 consecutive days. On Day 6, the group taking AGBE had significantly lower fasting blood glucose levels compared to GRe-KO-AGBE-treated mice (70.5 mg/dl vs. 140 mg/dl). On Day 13, the glucose levels in both groups were 201 mg/dl for AGBE-treated group and 169 mg/dl for GRe-KO-AGBE, respectively. GRe (20 mg/kg) had dose-related effects on fasting blood glucose levels (reduced to 188 and 180 mg/dl on Day 5 and Day 12, respectively, compared to 229 and 235 mg/dl, respectively, in the vehicle group). This hypoglycemic effect persisted even after treatment discontinued (blood glucose levels at 3 days post-treatment: 198 and 253 mg/dl with ginsenoside and vehicle treatment, respectively). The monoclonal antibody technique can be used to prepare botanical knockout extracts for bioactivity studies.

Background: Cynanchum komarovii Al Iljinski is a desert plant that has been used as analgesic, anthelminthic and antidiarrheal, but also as a herbal medicine to treat cholecystitis in people. We have found that the protein extractions from C. komarovii seeds have strong antifungal activity. There is strong interest to develop protein medication and antifungal pesticides from C. komarovii for pharmacological or other uses., Methodology/principal Findings: An antifungal protein with sequence homology to thaumatin-like proteins (TLPs) was isolated from C. komarovii seeds and named CkTLP. The three-dimensional structure prediction of CkTLP indicated the protein has an acid cleft and a hydrophobic patch. The protein showed antifungal activity against fungal growth of Verticillium dahliae, Fusarium oxysporum, Rhizoctonia solani, Botrytis cinerea and Valsa mali. The full-length cDNA was cloned by RT-PCR and RACE-PCR according to the partial protein sequences obtained by nanoESI-MS/MS. The real-time PCR showed the transcription level of CkTLP had a significant increase under the stress of abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), NaCl and drought, which indicates that CkTLP may play an important role in response to abiotic stresses. Histochemical staining showed GUS activity in almost the whole plant, especially in cotyledons, trichomes and vascular tissues of primary root and inflorescences. The CkTLP protein was located in the extracellular space/cell wall by CkTLP::GFP fusion protein in transgenic Arabidopsis. Furthermore, over-expression of CkTLP significantly enhanced the resistance of Arabidopsis against V. dahliae., Conclusions/significance: The results suggest that the CkTLP is a good candidate protein or gene for contributing to the development of disease-resistant crops.

The currently accepted view is that the chemopreventive activity of glucosinolates is exclusively mediated by their degradation products, such as isothiocyanates. In the present study, evidence is presented for the first time that intact glucosinolates can modulate carcinogen-metabolising enzyme systems. The glucosinolates glucoraphanin and glucoerucin were isolated from cruciferous vegetables and incubated with precision-cut rat liver slices. Both glucosinolates elevated the O-dealkylations of methoxy- and ethoxyresorufin, markers for CYP1 activity; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished these effects. Moreover, both glucoerucin and glucoraphanin increased the apoprotein levels of microsomal CYP1A1, CYP1A2 and CYP1B1. At higher concentrations, both glucosinolates enhanced quinone reductase activity, whereas glucoraphanin also elevated glutathione S-transferase; in this instance, however, supplementation of the incubation medium with myrosinase exacerbated the inductive effect. Finally, both glucosinolates increased modestly cytosolic quinone reductase, GSTα and GSTμ protein levels, which became more pronounced when myrosinase was added to the incubations with the glucosinolate. It may be inferred that intact glucosinolates can modulate the activity of hepatic carcinogen-metabolising enzyme systems and this is likely to impact on the chemopreventive activity linked to cruciferous vegetable consumption., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Roasted coffee contains a complex array of volatile organic compounds (VOCs) which make an important contribution to the characteristic flavour and aroma of the final beverage. It is thought that a few of the potent coffee aroma components, such as "beta-damascenone", could be derived from carotenoid precursors. In order to further investigate the potential link between carotenoids and coffee aroma profiles, we have measured the carotenoid content in developing coffee grain. The data obtained confirms the presence of lutein in the grain, and additionally shows that the immature coffee grain also contains significant amounts of beta-carotene, alpha-carotene, violaxanthin, and neoxanthin. Complimentary quantitative gene expression analysis revealed that all the carotenoid biosynthetic genes examined are expressed in the grain, and that the transcript levels are gene and stage dependent. Furthermore, consistent with the reduction of the carotenoid levels at the last stage of grain development (mature-red), most of the transcript levels were also found to be lower at the final developmental stage. Quantitative expression analysis of the carotenoid genes was also carried out for the developing pericarp tissue of the coffee cherries. Again, all the genes examined were expressed, and in most cases, the highest transcript levels were detected around the large green-yellow stages, a period when carotenoid synthesis is probably greatest.

A total of 21 endophytic filamentous fungi were isolated from the young stems of Cinchona ledgeriana (Rubiaceae) cultivated in West Java, Indonesia. They were classified into six genera, namely nine Phomopsis spp., six Diaporthe spp., two Schizophyllum spp., two Penicillium spp., one Fomitopsis sp., and one Arthrinium sp. by using nucleotide sequence analysis of the internal transcribed spacers (ITS1 and ITS2) including 5.8S ribosomal DNA region and phylogenetic analysis.

Carotenoids and their apocarotenoid derivatives are isoprenoid molecules important for the primary and secondary metabolisms of plants and other living organisms, displaying also key health-related roles in humans and animals. Progress in the knowledge of the carotenoid pathway at the genetic, biochemical and molecular level, supported by successful genetic engineering examples for an increasing number of important plant crops have paved the way for precise molecular breeding of carotenoids. In this review, following a description of the general carotenoid pathway, select examples of plant species able to produce specialty carotenoids and apocarotenoids are illustrated. An update on plant carotenoid engineering is also provided for non-solanaceous crops and members of the Solanaceae family, by means of different strategies and making use of plant and bacterial genes.