ABSTRACT
In recent years, severe climate change leading to by water scarcity reduced water quality has increased the need for effective irrigation strategies for agricultural production. Among these, the reuse of reclaimed water represents a non-expensive and reliable solution. The effect of conventional or reclaimed water, applying convention or smart fertigation system, were investigated during two irrigation seasons on yield, qualitative and biochemical traits of pomegranates fruit (cv Wonderful One) at harvest, and after storage at 7 °C. The results of this study showed that using reclaimed waters with different fertigation systems did not affect the pH values, total soluble solids, and titratable acidity on pomegranates fruit showing slight decrease changes only during postharvest storage. On the other hand, the respiration rate was not affected by water quality. Furthermore, the antioxidant activity was also preserved during storage in pomegranates fruit from plants irrigated with reclaimed water by applying conventional or smart fertigation. The analysis also identified 52 compounds by UHPLC-MSn and HPLC-UV-Vis analyses. A slight decrease (about 17 %) at harvest and during storage in polyphenols content was shown in fruit grown using reclaimed water. The study demonstrates that using reclaimed water is a sustainable and effective way to limit the use of conventional water for irrigating pomegranate crops without significant reduction in yield, or in qualitative and nutritional values of the fruit at harvest and during storage.
ABSTRACT
In this study, we specifically focused on the crude methanolic leaf extract of Byrsonima coccolobifolia, investigating its antifungal potential against human pathogenic fungi and its antiviral activity against COVID-19. Through the use of high-performance liquid chromatography coupled with electrospray ionization ion trap tandem mass spectrometry, direct infusion electrospray ionization ion trap tandem mass spectrometry, and chromatographic dereplication procedures, we identified galloyl quinic acid derivatives, catechin derivatives, proanthocyanidins, and flavonoid glycosides. The broth dilution assay revealed that the methanolic leaf extract of B. coccolobifolia exhibits antifungal activity against Cryptococcus neoformans (IC50 = 4 µg/mL). Additionally, docking studies were conducted to elucidate the interactions between the identified compounds and the central residues at the binding site of biological targets associated with COVID-19. Furthermore, the extract demonstrated an in vitro half-maximum effective concentration (EC50 = 7 µg/mL) and exhibited significant selectivity (>90%) toward SARS-CoV-2.
Subject(s)
COVID-19 , Plant Extracts , Humans , Plant Extracts/pharmacology , Plant Extracts/chemistry , Antifungal Agents , Molecular Structure , SARS-CoV-2 , Spectrometry, Mass, Electrospray Ionization/methods , Methanol , Antiviral Agents/pharmacology , Chromatography, High Pressure Liquid/methodsABSTRACT
Herein, we report on the production, characterization, and antioxidant power assessment of carotenoids from the haloarchaeon Halorhabdus utahensis. It was grown at 37 °C and 180 rpm agitation in halobacteria medium supplemented with glucose, fructose, and xylose, each at concentrations of 0.2%, 1%, and 2%, and the carotenoid yield and composition were investigated. The microorganism produced the carotenoids under all the conditions tested, and their amount followed the order glucose < xylose < fructose. The highest yield was achieved in 2% fructose growth medium with 550.60 ± 7.91 µg/g dry cell and 2428.15 ± 49.33 µg/L. Separation and identification of the carotenoids were performed by RP-HPLC and HPLC/APCI-ITMSn. Bacterioruberin was the main carotenoid detected and accounted for 60.6%, 56.4%, and 58.9% in 2% glucose, 1% xylose, and 2% fructose extracts, respectively. Several geometric isomers of bacterioruberin were distinguished, and representatives of monoanhydrobacterioruberin, and bisanhydrobacterioruberin were also detected. The assignment to cis-isomers was attempted through analysis of the UV/Vis spectra, intensity of cis peaks, and spectral fine structures. The extracts exhibited superoxide scavenging activity higher than butylhydroxytoluene, ascorbic acid, and Trolox, selected as antioxidant references. The anti-hyaluronidase capacity was investigated, and the 2% fructose extract showed the highest activity reaching 90% enzyme inhibition with 1.5 µg. The overall data confirm that Hrd. utahensis can be regarded as an interesting source of antioxidants that can find applications in the food and cosmetic sectors.
ABSTRACT
The affinity of specific phenolic compounds (PCs) and capsaicinoids (CAPs) present in three Capsicum annuum varieties (Friariello, Cayenne and Dzuljunska Sipka) to the transient receptor potential vanilloid member 1 (TRPV1) was investigated by integrating an analytic approach for the simultaneous extraction and analysis through high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC/ITMS) and UV detection (HPLC-UV) of PCs and CAPs and structural bioinformatics based on the protein modelling and molecular simulations of protein-ligand docking. Overall, a total of 35 compounds were identified in the different samples and CAPs were quantified. The highest content of total polyphenols was recorded in the pungent Dzuljunska Sipka variety (8.91 ± 0.05 gGAE/Kg DW) while the lowest was found in the non-pungent variety Friariello (3.58 ± 0.02 gGAE/Kg DW). Protein modelling generated for the first time a complete model of the homotetrameric human TRPV1, and it was used for docking simulations with the compounds detected via the analytic approach, as well as with other compounds, as an inhibitor reference. The simulations indicate that different capsaicinoids can interact with the receptor, providing details on the molecular interaction, with similar predicted binding energy values. These results offer new insights into the interaction of capsaicinoids with TRPV1 and their possible actions.
Subject(s)
Capsicum , Humans , Capsicum/chemistry , Capsaicin/pharmacology , Capsaicin/analysis , Chromatography, High Pressure Liquid/methods , Plant Extracts/chemistry , Mass Spectrometry , Phenols/pharmacology , Phenols/analysis , Fruit/chemistryABSTRACT
Natural products and plant extracts exhibit many biological activities, including that related to the defense mechanisms against parasites. Many studies have investigated the biological functions of secondary metabolites and reported evidence of antiviral activities. The pandemic emergencies have further increased the interest in finding antiviral agents, and efforts are oriented to investigate possible activities of secondary plant metabolites against human viruses and their potential application in treating or preventing SARS-CoV-2 infection. In this review, we performed a comprehensive analysis of studies through in silico and in vitro investigations, also including in vivo applications and clinical trials, to evaluate the state of knowledge on the antiviral activities of secondary metabolites against human viruses and their potential application in treating or preventing SARS-CoV-2 infection, with a particular focus on natural compounds present in food plants. Although some of the food plant secondary metabolites seem to be useful in the prevention and as a possible therapeutic management against SARS-CoV-2, up to now, no molecules can be used as a potential treatment for COVID-19; however, more research is needed.
Subject(s)
Biological Products , COVID-19 , Humans , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Biological Products/pharmacology , Biological Products/therapeutic use , Plants, EdibleABSTRACT
The purpose of the current study was to determine the phenolic composition, antioxidant, and antimicrobial activities in grape cane extracts from typical cultivars of Southern Italy. Aqueous extracts at different pHs (1-13) were prepared from "Aglianico", "Fiano", and "Greco" grape canes. The results demonstrated that an alkaline pH (13.00) produced the best polyphenol-rich extracts, as the total phenolic content was more than double when compared to the respective extracts prepared at pH 1.00. "Greco" grape canes gave the highest quantity of phenolic compounds at each pH, ranging from 42.7 ± 0.4 to 104.3 ± 3.0 mg Gallic Acid Equivalents (GAE)/g Dry Extract (DE) from pH 1.00 to 13.00. The Radical Scavenging Activity (RSA) and the Ferric Reducing Antioxidant Power (FRAP) were measured. The highest antioxidant activity was showed by "Greco" extract at pH 7.00. Seventy-five compounds were identified in the extracts by HPLC-MS with six of them described for the first time in grape canes. Procyanidins were highly abundant in extracts at pH 7.00, whereas stilbenoids were the most represented compounds at pH 13.00. Very strong antiviral activity against herpes simplex viruses was recorded for the extracts at pH 7.00 and 13.00 that were active in the early stages of infection by acting directly against the viral particles. The overall results suggest that grape canes, currently underutilized, can be usefully valorised by providing active extracts to use as antioxidant and antiviral agents.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antioxidants/pharmacology , Phenols/analysis , Phytochemicals/analysis , Vitis/chemistry , Animals , Antiviral Agents/pharmacology , Bacteria/drug effects , Cell Death/drug effects , Chlorocebus aethiops , Chromatography, High Pressure Liquid , Flavonoids/analysis , Free Radical Scavengers/pharmacology , Herpesvirus 1, Human/drug effects , Hydrogen-Ion Concentration , Italy , Microbial Sensitivity Tests , Oxidation-Reduction , Tannins/analysis , Vero CellsABSTRACT
Actinidia deliciosa cv. Hayward fruit is renowned for its micro- and macronutrients, which vary in their levels during berry physiological development and postharvest processing. In this context, we have recently described metabolic pathways/molecular effectors in fruit outer endocarp characterizing the different stages of berry physiological maturation. Here, we report on the kiwifruit postharvest phase through an integrated approach consisting of pomological analysis combined with NMR/LC-UV/ESI-IT-MSn- and 2D-DIGE/nanoLC-ESI-LIT-MS/MS-based proteometabolomic measurements. Kiwifruit samples stored under conventional, cold-based postharvest conditions not involving the use of dedicated chemicals were sampled at four stages (from fruit harvest to pre-commercialization) and analyzed in comparison for pomological features, and outer endocarp metabolite and protein content. About 42 metabolites were quantified, together with corresponding proteomic changes. Proteomics showed that proteins associated with disease/defense, energy, protein destination/storage, cell structure and metabolism functions were affected at precise fruit postharvest times, providing a justification to corresponding pomological/metabolite content characteristics. Bioinformatic analysis of variably represented proteins revealed a central network of interacting species, modulating metabolite level variations during postharvest fruit storage. Kiwifruit allergens were also quantified, demonstrating in some cases their highest levels at the fruit pre-commercialization stage. By lining up kiwifruit postharvest processing to a proteometabolomic depiction, this study integrates previous observations on metabolite and protein content in postharvest berries treated with specific chemical additives, and provides a reference framework for further studies on the optimization of fruit storage before its commercialization.
ABSTRACT
Haloterrigena turkmenica was able to synthesize carotenoids when grown in halobacteria medium. These molecules have antioxidant properties and find application in food, cosmetic, and pharmaceutical fields. The carotenoids were extracted with methanol, separated by RP-HPLC, and identified by mass spectrometry and UV/Vis spectra analyses. The C50 carotenoids were the main pigments, and C30, C40, and C51 carotenoids were also detected. Seven geometric isomers were distinguished for bacterioruberin, monoanhydrobacterioruberin, and bisanhydrobacterioruberin. The assignment to a specific isomer was tentatively attempted through the analysis of the corresponding UV/Vis spectrum, the intensity of the cis peak, and its spectral fine structure. Lycopene, phytoene, and lycopersene were among the minor carotenoids further identified. The extract displayed antioxidant power higher than alpha-tocopherol, butylhydroxytoluene, and ascorbic acid used as reference compounds. Our studies identified for the first time seven geometric isomers of bacterioruberin derivatives and 30 carotenoids in a haloarchaeon.
Subject(s)
Antioxidants/chemistry , Carotenoids/chemistry , Halobacteriaceae/chemistry , Antioxidants/pharmacology , Carotenoids/pharmacology , Extreme Environments , Oxidation-Reduction , Salt ToleranceABSTRACT
Despite the encouraging results of the innovative therapeutic treatments, complete remission is uncommon in patients affected by chronic lymphocytic leukaemia, which remains an essentially incurable disease. Recently, clinical trials based on BH3-mimetic drugs showed positive outcomes in subjects with poor prognostic features. However, resistance to treatments occurs in a significant number of patients. We previously reported that the multi-kinase inhibitor quercetin, a natural flavonol, restores sensitivity to ABT-737, a BH3-mimetic compound, in both leukemic cell lines and B-cells isolated from patients. To identify the molecular target of quercetin, we employed a new cell line, HG3, obtained by immortalization of B-cells from a chronic lymphocytic leukaemia patient at the later stage of disease. We confirmed that quercetin in association with ABT-737 synergistically enhances apoptosis in HG3 (combination index < 1 for all fractions affected). We also reported that the cellular uptake of quercetin is extremely rapid, with an intracellular concentration of about 38.5 ng/106 cells, after treatment with 25 µM for 5 min. We demonstrated that the activity of protein kinase CK2, which positively triggers PI3K/Akt pathway by inactivating PTEN phosphatase, is inhibited by quercetin immediately after its addition to HG3 cells (0-2 min). PI3K activity was also inhibited by quercetin within 60 min from the treatment. The combined inhibition of CK2 and PI3K kinase activities by quercetin restored ABT-737 sensitivity and increased lethality in human leukemia cells.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Casein Kinase II/antagonists & inhibitors , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Quercetin/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Biphenyl Compounds/pharmacology , Casein Kinase II/metabolism , Cell Line, Tumor , Drug Synergism , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Nitrophenols/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Piperazines/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Quercetin/administration & dosage , Signal Transduction/drug effects , Sulfonamides/pharmacologyABSTRACT
We investigated the potential role of apple phenolic compounds in human pathologies by integrating chemical characterization of phenolic compounds in three apple varieties, computational approaches to identify potential protein targets of the compounds, bioinformatics analyses on data from public archive of gene expression data, and functional analyses to hypothesize the effects of the selected compounds in molecular pathways. Starting by the analytic characterization of phenolic compounds in three apple varieties, i.e. Annurca, Red Delicious, and Golden Delicious, we used computational approaches to verify by reverse docking the potential protein targets of the identified compounds. Direct docking validation of the potential protein-ligand interactions has generated a short list of human proteins potentially bound by the apple phenolic compounds. By considering the known chemo-preventive role of apple antioxidants' extracts against some human pathologies, we performed a functional analysis by comparison with experimental gene expression data and interaction networks, obtained from public repositories. The results suggest the hypothesis that chemo-preventive effects of apple extracts in human pathologies, in particular for colorectal cancer, may be the interference with the activity of nucleotide metabolism and methylation enzymes, similarly to some classes of anticancer drugs.
Subject(s)
Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/prevention & control , Malus/chemistry , Neoplasm Proteins/metabolism , Polyphenols/therapeutic use , Antioxidants/pharmacology , Antioxidants/therapeutic use , Chemoprevention , Chromatography, High Pressure Liquid , Computational Biology , Gene Expression Regulation, Neoplastic/drug effects , Gene Regulatory Networks/drug effects , Humans , Molecular Docking Simulation , Plant Extracts/therapeutic use , Polyphenols/pharmacologyABSTRACT
Onions (Allium cepa) are consumed worldwide and represent an important source of dietary phytochemicals with proven antioxidant properties, such as phenolic acids, flavonoids, thiosulfinates, and anthocyanins. Epidemiological and experimental data suggest that regular consumption of onions is associated with a reduced risk of degenerative disorders. Therefore, it is of interest to investigate the biological properties of different varieties of onions. Here, we characterized for the first time a variety of onion, called Ramata di Montoro (coppery onion from Montoro), grown in a niche area in southern Italy, and compared its phenolic profile and antioxidant properties to a commercial ecotype of red onion, Tropea, also present in southern Italy. An analytical method based on high-performance liquid chromatography coupled with UV detection and mass spectrometry was used to separate and characterize the phenolic fraction (anthocyanins and flavonols) extracted from both coppery and red types. The main compounds detected in the two ecotypes were quercetin and quercetin glucosides, isorhamnetin glucosides, kaempferol glucoside, and, among anthocyanins, cyanidin glucosides. Tropea ecotype onion showed a higher content of flavonols (632.82 mg/kg fresh weight) than Montoro type onion (252.91 mg/kg fresh weight). Accordingly, the antioxidant activity of the former was 2.8-fold higher compared to the latter. More pronounced were the differences existing between the four anthocyanins detected in the two ecotypes, with those in the Tropea ecotype onion present at concentrations 20-230-fold higher than in the Montoro type onion. Both extracts reduced LDL oxidation about 6-fold and protected human erythrocytes from oxidative damage induced by HClO by about 40%. In addition, as a consequence of HClO treatment, glutathione concentration in erythrocytes was reduced about 50% and pretreatment with onion extracts induced a recovery of glutathione level by about 15-22%. Qualitative differences highlighted in the chemical composition of the two phenolic extracts, especially the total content of anthocyanins, which was 30-fold higher in Montoro type onion compared to Tropea ecotype, can be associated with the protective effects measured against oxidative damage induced in human erythrocytes.
Subject(s)
Antioxidants/chemistry , Erythrocytes/metabolism , Flavonoids/chemistry , Onions/chemistry , Oxidative Stress/drug effects , Plant Extracts/chemistry , Protective Agents/chemistry , Antioxidants/pharmacology , Erythrocytes/drug effects , Flavonoids/pharmacology , Glutathione/metabolism , Humans , Italy , Onions/classification , Plant Extracts/pharmacology , Protective Agents/pharmacology , Reactive Oxygen Species/metabolismABSTRACT
Hazelnuts exhibit functional properties due to their content in fatty acids and phenolic compounds that could positively affect human health. The food industry requires precise traits for morphological, chemical, and physical kernel features so that some cultivars could be more suitable for specific industrial processing. In this study, agronomical and morphological features of 29 hazelnut cultivars were evaluated and a detailed structural characterization of kernel polyphenols was performed, confirming the presence of protocatechuic acid, flavan-3-ols such as catechin, procyanidin B2, six procyanidin oligomers, flavonols, and one dihydrochalcone in all the analyzed cultivars. In addition, an innovative methodology based on the MALDI-TOF mass spectrometric analysis of peptide/protein components extracted from kernels was developed for the authentication of the most valuable cultivars. The proposed method is rapid, simple, and reliable and holds the potential to be applied in quality control processes. These results could be useful in hazelnut cultivar evaluation and choice for growers, breeders, and food industry.
Subject(s)
Corylus/chemistry , Flavonoids/analysis , Food Quality , Nuts/chemistry , Phenols/analysis , Seeds/chemistry , Corylus/growth & development , Dietary Proteins/analysis , Dietary Proteins/chemistry , Europe , Food Inspection , Nuts/growth & development , Nuts/standards , Peptide Fragments/analysis , Peptide Fragments/chemistry , Plant Proteins/analysis , Plant Proteins/chemistry , Seeds/growth & development , Species Specificity , Turkey , United StatesABSTRACT
OBJECTIVES: Methacrylic monomers are released, from dental composite resins, either into the oral cavity or in pulpal tissues, where they can cause local or systemic adverse effects. The mechanisms of these effects are not well understood, probably because such molecules can act at different levels also inducing a depletion of intracellular glutathione (GSH). GSH can detoxify methacrylates by conjugating their α,ß-unsaturated carbon-carbon moiety to the thiol group, with the catalysis of glutathione S-transferases (GST). This reaction determines a GSH cellular depletion and belongs to the metabolism of α,ß-unsaturated esters, protecting the body against the toxic effects of electrophiles. On the basis of the above considerations, this work aim is to set up a method for the detection of the adducts formed by methacrylic monomers with GSH in cells using HPLC coupled to mass spectrometry (HPLC-MS) and micellar electrokinetic capillary chromatography (MECK) techniques. METHODS AND RESULTS: Adducts of glutathione with triethylene glycol dimethacrylate (TEGDMA) and hydroxyethyl methacrylate (HEMA) were incontrovertibly identified by HPLC-MS and MECK in human gingival fibroblasts and erythrocytes, both outside and inside cells. Molecular docking simulations of HEMA and TEGDMA in the experimental structure of glutathione S-transferase, are also reported to rationalize the effectiveness of such enzyme in the catalysis of the above described reaction. SIGNIFICANCE: The setup of a method for the identification of GSH-methacrylate adducts allows to determine when the metabolic pathway involving such compounds is employed by cells for the detoxification of monomers leached from composite resins.
Subject(s)
Chromatography, High Pressure Liquid , Chromatography, Micellar Electrokinetic Capillary , Erythrocytes/metabolism , Fibroblasts/metabolism , Gingiva/metabolism , Glutathione/metabolism , Methacrylates/metabolism , Tandem Mass Spectrometry , Biocatalysis , Cells, Cultured , Cysteine/metabolism , Cytoplasm/metabolism , Erythrocytes/enzymology , Ethacrynic Acid/metabolism , Extracellular Space/metabolism , Fibroblasts/enzymology , Gingiva/cytology , Gingiva/enzymology , Glutathione/analysis , Glutathione Transferase/metabolism , Humans , Materials Testing , Methacrylates/analysis , Molecular Conformation , Polyethylene Glycols/analysis , Polyethylene Glycols/metabolism , Polymethacrylic Acids/analysis , Polymethacrylic Acids/metabolismABSTRACT
Four novel oxylipins (1-4) were isolated from the n-butanol extract of the corms of Dracontium loretense. Their structures were assigned by 1D and 2D NMR analyses and electrospray ionization multistage ion trap mass spectrometry (ESI-ITMS(n)) data. Relative configurations were assigned on the basis of combined analysis of homonuclear and heteronuclear (2,3)J couplings, along with ROE data. Oxylipin 2 exhibited an immunostimulatory effect on human PBMC proliferation.
Subject(s)
Adjuvants, Immunologic/isolation & purification , Leukocytes, Mononuclear/drug effects , Oxylipins/isolation & purification , Plants, Medicinal/chemistry , Adjuvants, Immunologic/blood , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Humans , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Oxylipins/blood , Oxylipins/chemistry , Oxylipins/immunology , Peru , Spectrometry, Mass, Electrospray IonizationABSTRACT
Liquid chromatography-electrospray ionization multistage ion trap mass spectrometry (LC-ESI-IT-MS(n)) was used to analyze the secondary metabolites in the methanol extract of the capitulae of Eriocaulon ligulatum. The major components were mono- and diglycosides of flavonoids and naphthopyranones. Eleven compounds, including four new flavonol glycosides, were identified based on their fragmentation patterns in MS experiments and on NMR analysis of the isolated compounds. The described data may contribute to a better understanding of the taxonomic classification of the Eriocaulaceae family.
Subject(s)
Chromones/chemistry , Eriocaulaceae/chemistry , Flavonoids/chemistry , Naphthalenes/chemistry , Chromatography, High Pressure Liquid , Chromones/isolation & purification , Flavonoids/isolation & purification , Magnetic Resonance Spectroscopy , Naphthalenes/isolation & purification , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet , Tandem Mass SpectrometryABSTRACT
The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a Delta(9) desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibiotic-insensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the Delta(9) desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the "binding-type" vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.
Subject(s)
Adaptation, Physiological/genetics , Cold Temperature , Fatty Acid Desaturases/genetics , Fatty Acids/metabolism , Nicotiana/physiology , Base Sequence , DNA Primers , Electrophoresis, Polyacrylamide Gel , Nicotiana/genetics , Nicotiana/metabolism , TransgenesABSTRACT
Sixteen novel and ten known galactolipids have been isolated and characterized from the leaves of Ipomoea batatas L. (sweet potato) using an analytical method based on high-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight tandem mass spectrometry. Using this technique, the structures and regiochemistries of the fatty acyl groups and the positions of the double bonds on the acyl chains were determined. Sugar moieties were identified by analysis of one- and two-dimensional nuclear magnetic resonance spectra. The positions of the double bonds of polyunsaturated fatty acids were confirmed, and in some cases their geometries determined, by gas chromatography-mass spectrometry. This is the first report of galactolipids in the leaves of sweet potato.
Subject(s)
Chromatography, High Pressure Liquid , Galactolipids/analysis , Ipomoea batatas/chemistry , Plant Leaves/chemistry , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Fatty Acids/analysis , Galactolipids/chemistryABSTRACT
We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 degrees C and is characterized by extreme thermodynamic stability (T(m), 110 degrees C that increases to 120 degrees C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 degrees C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.
Subject(s)
Purine-Nucleoside Phosphorylase/chemistry , Purine-Nucleoside Phosphorylase/metabolism , Pyrococcus furiosus/enzymology , Amino Acid Sequence , Cloning, Molecular , Disulfides/analysis , Dithiothreitol/pharmacology , Enzyme Stability , Escherichia coli/metabolism , Hot Temperature , Kinetics , Molecular Sequence Data , Phosphates/pharmacology , Purine-Nucleoside Phosphorylase/genetics , Sequence Alignment , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity , Tandem Mass SpectrometryABSTRACT
A rapid analytical approach suitable to achieve a comprehensive characterization of the compounds present in the infusion prepared from the leaves of Byrsonima fagifolia Niedenzu (Malpighiaceae), a Brazilian plant used as an infusion to treat gastric disorders, was developed. The method was based on high-performance liquid chromatography coupled to electrospray negative ionisation multistage ion trap mass spectrometry (HPLC/ESI-ITMSn). The main ions in the ESI-ITMS spectra were attributed to a quinic acid core containing from one to five galloyl units. Quercetin derivatives containing one and two sugar moieties as well as galloyl esterification were also detected. These results indicated that HPLC/ESI-ITMSn is easily applicable to infusions of this plant and allows the rapid and direct identification of these compounds in crude plant extracts.