A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS

<p>Abstract</p> <p>Background</p> <p>Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI) compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices.</p> <p>Results</p> <p>A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA)) and small polar molecules (e.g., jasmonic acid (JA), salicylic acid (SA)) containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of <it>Nicotiana attenuata </it>leaf material with previously described analytical methods.</p> <p>Conclusion</p> <p>The method presented was used to detect 16 compounds in leaf extracts of <it>N. attenuata </it>plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the molecules must contain at least one free carboxyl group.</p

Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves

<p>Abstract</p> <p>Background</p> <p>Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS) which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of <it>Manduca sexta </it>OS and strong elicitors of herbivore-induced defense responses in <it>Nicotiana attenuata </it>plants.</p> <p>Results</p> <p>The metabolism of one of the major FACs in <it>M. sexta </it>OS, <it>N</it>-linolenoyl-glutamic acid (18:3-Glu), was analyzed on <it>N. attenuata </it>wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free α-linolenic acid (18:3) and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX) activity; plants silenced in the expression of <it>NaLOX2 </it>and <it>NaLOX3 </it>genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA) in <it>N. attenuata </it>leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene) in ir<it>lox2 </it>plants.</p> <p>Conclusions</p> <p>The metabolism of one of the major elicitors of herbivore-specific responses in <it>N. attenuata </it>plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential monoterpene emission.</p

Emerging Trends in Cybercrime Awareness in Nigeria

The study examined the current trend in cybercrime awareness and the relationship such trend has with cybercrime vulnerability or victimization. Selecting a sample of 1104 Internet users from Umuahia, Abia State, Nigeria, We found that: 1) awareness of information security was high in that about 2 in every 3 (68%) participants demonstrated a favorable awareness of information security and cybercrime. It was, however, revealed that such a high level of awareness could be partial and weak. 2) most Internet users demonstrated the awareness of fraud-related cybercrime categories (39%), e-theft (15%), hacking (12%), and ATM theft (10%). However, they were rarely aware of sexually related offenses, cyber-terrorism, malware attacks, spam emails, and identity theft as their proportion hovered around 8% and below. 3) Internet users significantly demonstrated more awareness of computer-assisted (M = 2.5; SD = 1.7) than that of computer-focused cybercrime categories (M = 2.2, SD = 1.3), t(1103) = 2.9, p=.000, r =.2. 4) Internet users significantly demonstrated more awareness of property cybercrime (M = 2.54; SD = 1.6) than that of violent cybercrime categories (M = 1.82, SD = 1.2), t(1103) = 5.94, p=.000, r =.3. 5) cybercrime awareness is positively correlated to cybercrime victimization experiences in that participants who demonstrated more awareness of cybercrime experienced significantly more cybercrime victimization (M = 1.66; SD = 1.7) than those who did not demonstrate awareness of cybercrime (M = .73, SD = 1.4), t(1103) = 7.55, p=.000, r =.52

SuperSAGE analysis of the Nicotiana attenuata transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses

<p>Abstract</p> <p>Background</p> <p>Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the <it>Manduca sexta </it>larvae are necessary and sufficient to elicit the herbivory-specific responses in <it>Nicotiana attenuata</it>, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.</p> <p>Results</p> <p>We used SuperSAGE combined with 454 sequencing to quantify the early transcriptional changes elicited by the FAC <it>N</it>-linolenoyl-glutamic acid (18:3-Glu) and virus induced gene silencing (VIGS) to examine the function of candidate genes in the <it>M. sexta</it>-<it>N. attenuata </it>interaction. The analysis targeted mRNAs encoding regulatory components: rare transcripts with very rapid FAC-elicited kinetics (increases within 60 and declines within 120 min). From 12,744 unique Tag sequences identified (UniTags), 430 and 117 were significantly up- and down-regulated ≥ 2.5-fold, respectively, after 18:3-Glu elicitation compared to wounding. Based on gene ontology classification, more than 25% of the annotated UniTags corresponded to putative regulatory components, including 30 transcriptional regulators and 22 protein kinases. Quantitative PCR analysis was used to analyze the FAC-dependent regulation of a subset of 27 of these UniTags and for most of them a rapid and transient induction was confirmed. Six FAC-regulated genes were functionally characterized by VIGS and two, a putative lipid phosphate phosphatase (LPP) and a protein of unknown function, were identified as important mediators of the <it>M. sexta</it>-<it>N. attenuata </it>interaction.</p> <p>Conclusions</p> <p>The analysis of the early changes in the transcriptome of <it>N. attenuata </it>after FAC elicitation using SuperSAGE/454 has identified regulatory genes involved in insect-specific mediated responses in plants. Moreover, it has provided a foundation for the identification of additional novel regulators associated with this process.</p

Ent-kaurene and ent-beyerene diterpenoids and other constituents of Thecacoris batesii

Two novel diterpenoids, thecacorins A (1) and B (2), were isolated from Thecacoris batesii and their structures were established as ent-3b,20-epoxy-16-kaurene-3a,12b-diol and ent-15-beyerene-2b,3b-diol, respectively, on the basis of extensive spectroscopic analysis, especially, 1D NMR spectra, in conjunction with 2D experiments, COSY, NOESY, HMQC and HMBC. KEY WORDS: Diterpenoids, Thecacorin A, Thecacorin B, Ent-3b,20-epoxy-16-kaurene-3a,12b-diol, Ent-15-beyerene-2b,3b-diol, Thecacoris batesii  Bull. Chem. Soc. Ethiop. 2007, 21(1), 89-94

Antimycobacterial, antibacterial and antifungal activities of Terminalia superba (Combretaceae)

The methanol extract from the stem bark of Terminalia superba (TSB), fractions (TSB1 - 7) and two compounds isolated following bio-assay guided fractionation namely 3,4'- di-O-methylellagic acid 3'-O-β-D-xylopyranoside (1) and 4'-O-galloy-3,3'-di-O-methylellagic acid 4-O-β-D-xylopyranoside (2) were evaluated for their antimycobacterial, antibacterial and antifungal activities. The broth microdilution, microplate Alamar Blue assay (MABA) and agar disc diffusion methods were used for the investigations. The results of the antimycobacterial assays showed that the crude extract, fraction TSB5-7 and compound 1 were able to prevent the growth of all the studied mycobacteria. The lowest minimal inhibitory concentration (MIC) value of 39.06 μg/ml for this extract was recorded on both M. smegmatis and M. tuberculosis MTCS2. The corresponding values were 19.53 μg/ml and 4.88 μg/ml for fractions and compounds respectively. The MIC determinations results on other organisms indicate values ranging from 19.53 to 78.12 μg/ml for TSB and compound 2 on 90.9% of the tested organisms, meanwhile compounds 1 as well as fractions TSB 6 and 7 exhibited detectable MIC values on all studied microorganisms. The overall results provide promising baseline information for the potential use of the crude extract from Terminalia superba, fractions 6-7 and the tested compounds in the treatment of tuberculosis, bacterial and fungal infections

Antihepatotoxic and Antioxidant Activities of Methanol Extract and Isolated Compounds from Ficus chlamydocarpa

Free radicals, in particular radical oxygen species (ROS), play an important role in the aetiology and pathogenesis of various diseases. Current research in many countries focuses on the use of local medicinal plants as a promising source of liver protective agents. This paper describes the hepatoprotective effects of the methanol extract and four isolated compounds from Ficus chlamydocarpa on CCl4-induced liver damage, as well as the possible antioxidant mechanisms involved in this protection. The DPPH test, along with the ß-Carotene-Linoleic Acid Model System and Ferric-Reducing Antioxidant Power assays, as well as the inhibition of microsomal lipid peroxidation were used to measure radical-scavenging and antioxidant activities. Pretreatment of rats with the methanol extract of F. chlamydocarpa before CCl4administration, significantly prevented serum increase of hepatic enzyme markers, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), in a dose-dependent manner. The hepatoprotection was also associated with a significant enhancement in hepatic reduced glutathione (GSH) and a marked decrease of liver malondialdehyde (MDA). Among the four compounds 1-4, isolated from the methanol extract, α-amyrin acetate (1) and luteolin (4) showed a significant hepatoprotective activity, as indicated by their ability to prevent liver cell death and lactate dehydrogenase (LDH) leakage during CCl4intoxication

Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions

Homeodomain-leucine zipper type I (HD-Zip I) proteins are plant-specific transcription factors associated with the regulation of growth and development in response to changes in the environment. Nicotiana attenuata NaHD20 was identified as an HD-Zip I-coding gene whose expression was induced by multiple stress-associated stimuli including drought and wounding. To study the role of NaHD20 in the integration of stress responses with changes in growth and development, its expression was silenced by virus-induced gene silencing (VIGS), and control and silenced plants were metabolically and developmentally characterized. Phytohormone profiling showed that NaHD20 plays a positive role in abscisic acid (ABA) accumulation in leaves during water stress and in the expression of some dehydration-responsive genes including ABA biosynthetic genes. Moreover, consistent with the high levels of NaHD20 expression in corollas, the emission of benzylacetone from flowers was reduced in NaHD20-silenced plants. Additionally, bolting time and the opening of the inflorescence buds was decelerated in these plants in a specific developmental stage without affecting the total number of flowers produced. Water stress potentiated these effects; however, after plants recovered from this condition, the opening of the inflorescence buds was accelerated in NaHD20-silenced plants. In summary, NaHD20 plays multiple roles in N. attenuata and among these are the coordination of responses to dehydration and its integration with changes in flower transitions