ABSTRACT
The EU's Forest Law Enforcement, Governance and Trade Action Plan (FLEGT) adopted in 2003 includes bilateral trade agreements known as Voluntary Partnership Agreements (VPAs) signed between the EU and timber-supplying countries. The EU has invested more than 1.5 billion euros in VPAs; however, only one of the seven concerned countries has managed to complete all the necessary requirements to expire FLEGT licences. Since there is no research that comprehensively integrates the scientific evidence regarding the effects of this policy, this study systematically reviews all empirical scientific studies on the effects of VPAs. We found that almost all relevant studies are case reports that use qualitative data and focus on only one country at a time, mainly Ghana, Cameroon, or Indonesia. The evidence suggests that while VPAs have contributed to the establishment of governance structures, tools, and procedures they have not been able to solve social problems (i.e., inequality and injustice) and have potentially harmed the economies of EU timber suppliers. Evidence on the effects of VPAs on illegal logging and trade and the environment remains limited. Thus, future research should focus on more countries; use a greater range of methods, including comparative experimental designs; explore possible intended effects on under-researched categories; and systematically investigate unintended effects on other categories within and outside the forestry sector.
Subject(s)
Conservation of Natural Resources , Forestry , Conservation of Natural Resources/methods , Forestry/methods , Forests , Indonesia , GhanaABSTRACT
BACKGROUND: Chitinases are plant defense-related proteins with a high biotechnological potential to be applied in agriculture. OBJECTIVES: This study aimed to purify a chitinase from the latex of Ficus benjamina. METHODS: An antifungal class I chitinase, named FbLx-Chi-1, was purified from the latex of Ficus benjamina after precipitation with 30-60% ammonium sulfate and affinity chromatography on a chitin column and antifungal potential assay against phytopathogenic fungi important to agriculture. RESULTS: FbLx-Chi-1 has 30 kDa molecular mass, as estimated by SDS-PAGE and the optimal pH and temperature for full chitinolytic activity were 5.5 and 60ºC, respectively. FbLx-Chi-1 is a high pH-, ion-tolerant and thermostable protein. Importantly, FbLx-Chi-1 hindered the growth of the phytopathogenic fungi Colletotrichum gloeosporioides, Fusarium pallidoroseum, and Fusarium oxysporum. The action mode of FbLx-Chi-1 to hamper F. pallidoroseum growth seems to be correlated with alterations in the morphology of the hyphal cell wall, increased plasma membrane permeability, and overproduction of reactive oxygen species. CONCLUSION: These findings highlight the biotechnological potential of FbLx-Chi-1 to control important phytopathogenic fungi in agriculture. In addition, FbLx-Chi-1 could be further explored to be used in industrial processes such as the large-scale environmentally friendly enzymatic hydrolysis of chitin to produce its monomer N-acetyl-ß-D-glucosamine, which is employed for bioethanol production, in cosmetics, in medicine, and for other multiple applications.
Subject(s)
Chitinases , Ficus , Antifungal Agents/pharmacology , Antifungal Agents/chemistry , Latex , Ficus/metabolism , Reactive Oxygen Species , Chitinases/pharmacology , Chitinases/chemistry , Chitinases/metabolism , Chitin/pharmacology , Chitin/chemistry , Cell Wall/metabolism , Cell Membrane/metabolismABSTRACT
Although floral nectar is a rich source of nutrients, it is rarely infected by microorganisms. Defense molecules such as proteins have been identified in this fluid, but defense peptides have been largely overlooked. Thus, the aim of this study was to perform an extensive peptidomic analysis of the ornamental tobacco floral nectar to seek peptides involved in nectar defense. Using LC-MS/MS, 793 peptides were sequenced and characterized. After extensive bioinformatics analysis, six peptides were selected for further characterization, synthesis, and evaluation of their antimicrobial properties against phytopathogenic fungi and bacteria. All six peptides had antimicrobial activity to some extent. However, the activity varied by peptide concentration and microorganism tested. An analysis of the action mechanism revealed damage in the cell membrane induced by peptides. The results show that floral nectar is rich in peptides and that, together with proteins and hydrogen peroxide, they contribute to plant defense against microorganisms during pollination.
Subject(s)
Anti-Infective Agents , Plant Nectar , Anti-Infective Agents/analysis , Anti-Infective Agents/metabolism , Antimicrobial Peptides , Chromatography, Liquid , Flowers/metabolism , Hydrogen Peroxide/metabolism , Plant Nectar/metabolism , Plant Proteins/metabolism , Pollination , Tandem Mass Spectrometry , Nicotiana/metabolismABSTRACT
Different genome editing approaches have been used to engineer resistance against plant viruses. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas; CRISPR/Cas) systems to create pinpoint genetic mutations have emerged as a powerful tool for molecular engineering of plant immunity and increasing resistance against plant viruses. This review presents (i) recent advances in engineering resistance against plant viruses by CRISPR/Cas and (ii) an overview of the potential host factors as targets for the CRISPR/Cas system-mediated broad-range resistance and immunity. Applications, challenges, and perspectives in enabling the CRISPR/Cas system for crop protection are also outlined.
ABSTRACT
BACKGROUND: In order to provide additional information on the behaviour of biochemical parameters related to stress responses to a specific long-term competition, we aimed to compare the stressful effects of a long-lasting competition on physiological variables in men and women. METHODS: This is a prospective observational analytical study. Twenty-five professional athletes, 15 men and 10 women, travelled 460âkm for 4 days in an international edition of the Ecomotion/Pro AR World. RESULTS: After the competition, we detected an increase in α-amylase and cortisol levels and a decrease in salivary immunoglobulin A (lgA) levels. The relative percentage changes in α-amylase, IgA and cortisol levels were significantly higher in women than in men, whereas women had lower relative percentage changes in glucose and lactate levels compared with men. There was a decrease in lymphocyte, eosinophil and monocyte counts, with relative percentage decreases in lymphocytes and monocytes being significantly higher in female athletes than in males. There were increases in the serum activities of total creatine kinase (CK), the creatine kinase myocardial isoform (CKMB), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) at the end of the test, with significantly higher elevations of total CK, CKMB and LDH in men and ALT in women. CONCLUSION: Long-lasting competition induced stress, muscle damage, anaemia and changes in the immune system. Women had more intense responses of cortisol and leukocytes.
Subject(s)
Biomarkers/blood , Running/physiology , Stress, Physiological/physiology , Alanine Transaminase/metabolism , Aspartate Aminotransferases/metabolism , Creatine Kinase/metabolism , Female , Humans , Male , Prospective StudiesABSTRACT
Correlations between the transcriptional responses of genes that encode superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxiredoxin (Prx) enzymes and Colletotrichum gloeosporioides development in cowpea leaves were assessed. Each of these genes is involved in the redox metabolism and hydrogen peroxide balance. Although electron microscopy revealed that conidia adhered to and germinated on the leaf cuticle, the inoculated cowpea leaves did not show any characteristic anthracnose symptoms. The adhered and germinated conidia showed irregular surfaces and did not develop further. This was apparently due to increased leaf H2O2 levels in response to inoculation with C. gloeosporioides. During the early stages post inoculation, cowpea leaves elevated the H2O2 content and modulated the defense gene expression, as well as associated pathways. During the later stages, the increased expression of the CuZnSODI and CuZnSODII genes suggested an active superoxide dismutation to further elevate H2O2 levels, which indicated that higher H2O2 content may function as a toxic agent that kills the fungus. The second increase in H2O2 production above the threshold level was correlated with the expression of the APXI, CATI, CATII, PrxIIBCD, and PrxIIE genes, which resulted in a coordinated pattern to establish an appropriate balance between H2O2 generation and scavenging. Therefore, appropriate H2O2 content in cowpea leaves inhibited C. gloeosporioides development and maintained intracellular redox homeostasis to avoid uncontrolled programmed cell death and necrosis in cowpea leaves.
Subject(s)
Colletotrichum , Disease Resistance/physiology , Hydrogen Peroxide/metabolism , Oxidation-Reduction , Vigna/microbiology , Ascorbate Peroxidases/metabolism , Catalase/metabolism , Colletotrichum/ultrastructure , Gene Expression Profiling , Gene Expression Regulation, Plant/physiology , Genes, Plant/physiology , Lipid Peroxidation , Microscopy, Electron, Scanning , Peroxiredoxins/metabolism , Plant Diseases/microbiology , Plant Leaves/metabolism , Plant Leaves/microbiology , Real-Time Polymerase Chain Reaction , Superoxide Dismutase/metabolism , Vigna/genetics , Vigna/physiologyABSTRACT
Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman-Birk protease inhibitor ( LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH2-terminal sequence has high identity with other Bowman-Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial death. This study suggests that LzaBBI is a powerful candidate for developing a new antimicrobial to overcome drug resistance toward reducing hospital-acquired infections caused by S. aureus.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Cell Membrane/drug effects , Fabaceae/chemistry , Plant Extracts/pharmacology , Protease Inhibitors/isolation & purification , Seeds/chemistry , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Chymotrypsin/antagonists & inhibitors , Microbial Sensitivity Tests , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Staphylococcus aureus/ultrastructure , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/isolation & purification , Trypsin Inhibitors/pharmacologyABSTRACT
A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.
Subject(s)
Antifungal Agents/metabolism , Chitinases/metabolism , Pichia/enzymology , Plant Proteins/metabolism , Vigna/enzymology , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Chitinases/chemistry , Chitinases/pharmacology , Hydrolysis , Penicillium/drug effects , Plant Proteins/chemistry , Plant Proteins/pharmacology , Protein BindingABSTRACT
KEY MESSAGE: The seed treatment of a CPSMV-susceptible cowpea genotype with the mutagenic agent EMS generated mutagenized resistant plantlets that respond to the virus challenge by activating biochemical and physiological defense mechanisms. Cowpea is an important crop that makes major nutritional contributions particularly to the diet of the poor population worldwide. However, its production is low, because cowpea is naturally exposed to several abiotic and biotic stresses, including viral agents. Cowpea severe mosaic virus (CPSMV) drastically affects cowpea grain production. This study was conducted to compare photosynthetic and biochemical parameters of a CPSMV-susceptible cowpea (CE-31 genotype) and its derived ethyl methanesulfonate-mutagenized resistant plantlets, both challenged with CPSMV, to shed light on the mechanisms of virus resistance. CPSMV inoculation was done in the fully expanded secondary leaves, 15 days after planting. At 7 days post-inoculation, in vivo photosynthetic parameters were measured and leaves collected for biochemical analysis. CPSMV-inoculated mutagenized-resistant cowpea plantlets (MCPI) maintained higher photosynthesis index, chlorophyll, and carotenoid contents in relation to the susceptible (CE-31) CPSMV-inoculated cowpea (CPI). Visually, the MCPI leaves did not exhibit any viral symptoms neither the presence of the virus as examined by RT-PCR. In addition, MCPI showed higher SOD, GPOX, chitinase, and phenylalanine ammonia lyase activities, H2O2, phenolic contents, and cell wall lignifications, but lower CAT and APX activities in comparison to CPI. All together, these photosynthetic and biochemical changes might have contributed for the CPSMS resistance of MCPI. Contrarily, CPI plantlets showed CPSMV accumulation, severe disease symptoms, reduction in the photosynthesis-related parameters, chlorophyll, carotenoid, phenolic compound, and H2O2 contents, in addition to increased ß-1,3-glucanase, and catalase activities that might have favored viral infection.
Subject(s)
Comovirus/physiology , Disease Resistance , Mutagenesis/genetics , Photosynthesis , Plant Diseases/virology , Vigna/physiology , Vigna/virology , Carbon Dioxide/metabolism , Carotenoids/metabolism , Chlorophyll/metabolism , Ethyl Methanesulfonate , Homeostasis , Hydrogen Peroxide/metabolism , Lignin/metabolism , Oxidation-Reduction , Phenols/metabolism , Phenylalanine Ammonia-Lyase/metabolism , Plant Leaves/enzymology , Plant Leaves/virology , Plant Proteins/metabolism , SolubilityABSTRACT
Hospital-acquired infections caused by antibiotic-resistant bacteria threaten the lives of many citizens all over the world. Discovery of new agents to hinder bacterial development would have a significant impact on the treatment of infections. Here, the purification and characterization of Rc-2S-Alb, a protein that belongs to the 2S albumin family, from Ricinus communis seed cake, are reported. Rc-2S-Alb was purified after protein extraction with Tris-HCl buffer, pH 7.5, fractionation by ammonium sulfate (50-75%), and chromatography on Phenyl-Sepharose and DEAE-Sepharose. Rc-2S-Alb, a 75 kDa peptide, displays trypsin inhibitory activity and has high in vitro antibacterial activity against Bacillus subtilis, Klebsiella pneumonia, and Pseudomonas aeruginosa, which are important human pathogenic bacteria. Atomic force microscopy studies indicated that Rc-2S-Alb disrupts the bacterial membrane with loss of the cytoplasm content and ultimately bacterial death. Therefore, Rc-2S-Alb is a powerful candidate for the development of an alternative drug that may help reduce hospital-acquired infections.
Subject(s)
2S Albumins, Plant/isolation & purification , 2S Albumins, Plant/pharmacology , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/drug effects , Seeds/chemistry , Trypsin Inhibitors/isolation & purification , Trypsin Inhibitors/pharmacology , 2S Albumins, Plant/chemistry , Anti-Bacterial Agents/chemistry , Brazil , Humans , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , Molecular Structure , Plant Proteins/chemistry , Pseudomonas aeruginosa/drug effects , Trypsin Inhibitors/chemistryABSTRACT
The physiological and biochemical responses of a drought tolerant, virus-susceptible cowpea genotype exposed to drought stress (D), infected by Cowpea severe mosaic virus (CPSMV) (V), and to these two combined stresses (DV), at 2 and 6 days post viral inoculation (DPI), were evaluated. Gas exchange parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) were reduced in D and DV at 2 and 6 DPI compared to control plants (C). Photosynthesis was reduced by stomatal and biochemical limitations. Water use efficiency increased at 2 DPI in D, DV, and V, but at 6 DPI only in D and DV compared to C. Photochemical parameters (effective quantum efficiency of photosystem II and electron transport rate) decreased in D and DV compared to C, especially at 6 DPI. The potential quantum efficiency of photosystem II did not change, indicating reversible photoinhibition of photosystem II. In DV, catalase decreased at 2 and 6 DPI, ascorbate peroxidase increased at 2 DPI, but decreased at 6 DPI. Hydrogen peroxide increased at 2 and 6 DPI. Peroxidase increased at 6 DPI and chitinase at 2 and 6 DPI. ß-1,3-glucanase decreased in DV at 6 DPI compared to V. Drought increased cowpea susceptibility to CPSMV at 2 DPI, as verified by RT-PCR. However, at 6 DPI, the cowpea plants overcome this effect. Likewise, CPSMV increased the negative effects of drought at 2 DPI, but not at 6 DPI. It was concluded that the responses to combined stresses are not additive and cannot be extrapolated from the study of individual stresses.
Subject(s)
Droughts , Mosaic Viruses/physiology , Plant Diseases/virology , Vigna/virology , Antioxidants/metabolism , Carbon Dioxide/metabolism , Chlorophyll/metabolism , Chlorophyll A , Enzymes/genetics , Enzymes/metabolism , Gene Expression Regulation, Plant , Genotype , Host-Pathogen Interactions , Hydrogen Peroxide/metabolism , Photosynthesis/genetics , Photosynthesis/physiology , Photosystem II Protein Complex/genetics , Photosystem II Protein Complex/metabolism , Plant Diseases/genetics , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/virology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Stomata/genetics , Plant Stomata/physiology , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Vigna/genetics , Vigna/metabolism , Water/metabolismABSTRACT
In this study a novel heat-stable lipid transfer protein, designated McLTP1, was purified from noni (Morinda citrifolia L.) seeds, using four purification steps which resulted in a high-purified protein yield (72 mg McLTP1 from 100g of noni seeds). McLTP1 exhibited molecular masses of 9.450 and 9.466 kDa, determined by electrospray ionisation mass spectrometry. The N-terminal sequence of McLTP1 (AVPCGQVSSALSPCMSYLTGGGDDPEARCCAGV), as analysed by NCBI-BLAST database, revealed a high degree of identity with other reported plant lipid transfer proteins. In addition, this protein proved to be resistant to pepsin, trypsin and chymotrypsin digestion. McLTP1 given intraperitoneally (1, 2, 4 and 8 mg/kg) and orally (8 mg/kg) caused an inhibition of the writhing response induced by acetic acid in mice. This protein displayed thermostability, retaining 100% of its antinociceptive activity after 30 min incubation at 80 °C. Pretreatment of mice with McLTP1 (8 mg/kg, i.p. and p.o.) also decreased neurogenic and inflammatory phases of nociception in the formalin test. Naloxone (2 mg/kg, i.p.) antagonised the antinociceptive effect of McLTP1 suggesting that the opioid mechanisms mediate the analgesic properties of this protein.
Subject(s)
Analgesics/isolation & purification , Analgesics/pharmacology , Antigens, Plant/isolation & purification , Antigens, Plant/pharmacology , Carrier Proteins/isolation & purification , Carrier Proteins/pharmacology , Morinda/chemistry , Plant Proteins/isolation & purification , Plant Proteins/pharmacology , Seeds/chemistry , Amino Acid Sequence , Analgesics/chemistry , Animals , Antigens, Plant/chemistry , Carrier Proteins/chemistry , Dose-Response Relationship, Drug , Drug Stability , Male , Mice , Plant Proteins/chemistry , Reflex/drug effectsABSTRACT
Anthracnose represents an important disease of cowpea [Vigna unguiculata L. (Walp.)] caused by the hemibiothrophic fungus Colletotrichum gloeosporioides that drastically reduces cowpea field production. In this study we investigated some biochemical aspects underlying the incompatible interaction between a resistant cowpea genotype and C. gloeosporioides using a proteomic approach. Analyses of two-dimensional gel electrophoresis patterns and protein identification indicate C. gloeosporioides infection-dependent cowpea leaf proteome changes associated with metabolism, photosynthesis, response to stress, oxidative burst and scavenging, defense signaling, and pathogenesis-related proteins. Moreover the C. gloeosporioides responsive proteins interaction network in cowpea revealed the interconnected modulation of key cellular processes involving particularly antioxidants proteins, photosynthetic apparatus forming proteins and proteins of the energetic metabolism that interact with each other suggesting that their expression changes are also important for resistance of cowpea to C. gloeosporioides.
Subject(s)
Colletotrichum/physiology , Fabaceae/metabolism , Host-Pathogen Interactions , Proteome , Electrophoresis, Gel, Two-Dimensional , Fabaceae/microbiology , Plant Leaves/metabolism , Plant Proteins/metabolism , Spectrometry, Mass, Electrospray IonizationABSTRACT
The root knot nematodes (RKN), Meloydogine spp., particularly Meloidogyne incognita and Meloidogyne javanica species, parasitize several plant species and are responsible for large annual yield losses all over the world. Only a few available chemical nematicides are still authorized for RKN control owing to environmental and health reasons. Thus, plant resistance is currently considered the method of choice for controlling RKN, and research performed on the molecular interactions between plants and nematodes to identify genes of interest is of paramount importance. The present work aimed to identify the differential accumulation of root proteins of a resistant cowpea genotype (CE-31) inoculated with M. incognita (Race 3) in comparison with mock-inoculated control, using 2D electrophoresis assay, mass spectrometry identification and gene expression analyses by RT-PCR. The results showed that at least 22 proteins were differentially represented in response to RKN challenge of cowpea roots mainly within 4-6 days after inoculation. Amongst the up-represented proteins were SOD, APX, PR-1, ß-1,3-glucanase, chitinases, cysteine protease, secondary metabolism enzymes, key enzymes involved in ethylene biosynthesis, proteins involved in MAPK pathway signaling and, surprisingly, leghemoglobin in non-rhizobium-bacterized cowpea. These findings show that an important rearrangement in the resistant cowpea root proteome occurred following challenge with M. incognita.
ABSTRACT
BACKGROUND: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). METHODS: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. RESULTS: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.564.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% ß-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. GENERAL SIGNIFICANCE: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.
Subject(s)
Antioxidants/metabolism , Cysteine/chemistry , Fabaceae/metabolism , Molecular Chaperones/metabolism , Peroxiredoxins/isolation & purification , Peroxiredoxins/metabolism , Plant Leaves/metabolism , Amino Acid Sequence , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Fabaceae/growth & development , Hydrogen Peroxide/metabolism , Hydrogen-Ion Concentration , Microscopy, Atomic Force , Molecular Sequence Data , Oxidation-Reduction , Plant Leaves/growth & development , Sequence Homology, Amino Acid , Tandem Mass SpectrometryABSTRACT
Proteins from latex of Calotropis procera (CpLP), Plumeria rubra (PrLP), Carica candamarcensis (P1G10) and Euphorbia tirucalli (EtLP) were tested for antifungal activity against phytopathogens. CpLP and P1G10 inhibited each fungi analyzed. PrLP and EtLP did not exert inhibition. CpLP and P1G10 exhibited preferential inhibitory activity towards R. solani (IC50 = 20.7 and 25.3 µg/ml, respectively). The inhibitory activity was lost after heat treatment or proteolysis, providing evidence for the involvement of proteins in the inhibitory effect. Treatment of CpLP or P1G10 with Dithiothreitol improved both, the endogenous proteolytic activity and the antifungal properties. Conversely, pre-treatment of CpLP or P1G10 with iodoacetamide drastically reduced endogenous proteolytic activities and partially abrogated antifungal activity. Similar results were observed when spores were challenged to germinate in the presence of laticifer proteins. The purified cysteine proteinase CMS2MS2 from Carica candamarcensis latex or papain (E.C. 3.4.22.2), a cysteine proteinase from latex of Carica papaya L., but not trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1), two serine proteases, replicated the results obtained with CpLP or P1G10, thus restricting the antifungal property to latex plant cysteine proteinases. CpLP, CMS2MS2 and papain induced production of reactive oxygen species in spores of F. solani, suggesting that inhibition could be linked to oxidative stress. Proteome analysis of CpLP by 2-D electrophoresis and MALDI-TOF-TOF confirmed the existence of various pathogenic-related proteins such as chitinases, peroxidases and osmotins. The results support that laticifer proteins are part of plant defense repertoire against phytopathogenic fungi.
Subject(s)
Apocynaceae/chemistry , Calotropis/chemistry , Carica/chemistry , Euphorbia/chemistry , Antifungal Agents/metabolism , Aspergillus niger/physiology , Brazil , Colletotrichum/pathogenicity , Fusarium/physiology , Latex/chemistry , Neurospora/pathogenicity , Plant Proteins/metabolism , Rhizoctonia/physiologyABSTRACT
Activity-dependent alterations of synaptic transmission important for learning and memory are often induced by Ca(2+) signals generated by depolarization. While it is widely assumed that Ca(2+) is the essential transducer of depolarization into cellular plasticity, little effort has been made to test whether Ca(2+)-independent responses to depolarization might also induce memory-like alterations. It was recently discovered that peripheral axons of nociceptive sensory neurons in Aplysia display long-lasting hyperexcitability triggered by conditioning depolarization in the absence of Ca(2+) entry (using nominally Ca(2+)-free solutions containing EGTA, "0Ca/EGTA") or the absence of detectable Ca(2+) transients (adding BAPTA-AM, "0Ca/EGTA/BAPTA-AM"). The current study reports that depolarization of central ganglia to approximately 0 mV for 2 min in these same solutions induced hyperexcitability lasting >1 h in sensory neuron processes near their synapses onto motor neurons. Furthermore, conditioning depolarization in these solutions produced a 2.5-fold increase in excitatory postsynaptic potential (EPSP) amplitude 1-3 h afterward despite a drop in motor neuron input resistance. Depolarization in 0 Ca/EGTA produced long-term potentiation (LTP) of the EPSP lasting > or = 1 days without changing postsynaptic input resistance. When re-exposed to extracellular Ca(2+) during synaptic tests, prior exposure to 0Ca/EGTA or to 0Ca/EGTA/BAPTA-AM decreased sensory neuron survival. However, differential effects on neuronal health are unlikely to explain the observed potentiation because conditioning depolarization in these solutions did not alter survival rates. These findings suggest that unrecognized Ca(2+)-independent signals can transduce depolarization into long-lasting synaptic potentiation, perhaps contributing to persistent synaptic alterations following large, sustained depolarizations that occur during learning, neural injury, or seizures.
Subject(s)
Aplysia/physiology , Calcium Signaling/physiology , Long-Term Potentiation/physiology , Synapses/physiology , Animals , Calcium Signaling/drug effects , Chelating Agents/pharmacology , Data Interpretation, Statistical , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Electrophysiology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Ganglia, Invertebrate/physiology , In Vitro Techniques , Locomotion/physiology , Long-Term Potentiation/drug effects , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/physiology , Synapses/drug effectsABSTRACT
BACKGROUND AND PURPOSE: Brain hypervascular diseases are complex and induce hemodynamic disturbances on brain parenchyma, which are difficult to accurately evaluate by using perfusion-weighted (PWI) MR imaging. Our purpose was to test and to assess the best AIF estimation method among 4 patients with brain hypervascular disease and healthy volunteers. METHODS: Thirty-three patients and 10 healthy volunteers underwent brain perfusion studies by using a 1.5T MR imaging scanner with gadolinium-chelate bolus injection. PWI was performed with the indicator dilution method. AIF estimation methods were performed with local, regional, regional scaled, and global estimated arterial input function (AIF), and PWI measurements (cerebral blood volume [CBV] and cerebral blood flow [CBF]) were performed with regions of interest drawn on the thalami and centrum semiovale in all subjects, remote from the brain hypervascular disease nidus. Abnormal PWI results were assessed by using Z Score, and evaluation of the best AIF estimation method was performed by using a no gold standard evaluation method. RESULTS: From 88% to 97% of patients had overall abnormal perfusion areas of hypo- (decreased CBV and CBF) and/or hyperperfusion (increased CBV and CBF) and/or venous congestion (increased CBV, normal or decreased CBF), depending on the AIF estimation method used for PWI computations. No gold standard evaluation of the 4 AIF estimates found the regional and the regional scaled methods to be the most accurate. CONCLUSION: Brain hypervascular disease induces remote brain perfusion abnormalities that can be better detected by using PWI with regional or regional scaled AIF estimation methods.
Subject(s)
Blood Volume , Cerebrovascular Circulation , Cerebrovascular Disorders/diagnosis , Cerebrovascular Disorders/physiopathology , Magnetic Resonance Angiography , Adolescent , Adult , Child , Female , Humans , Male , Middle Aged , Prospective StudiesABSTRACT
PURPOSE: Diffusion-weighted imaging (3 directions) and diffusion tensor imaging (9 directions) were compared for their sensitivity to detect ischemic lesion. MATERIALS AND METHODS: 41 patients (18 supposed transient ischemic attacks, 23 arterial stroke, MRISubject(s)
Cerebral Infarction/pathology
, Diffusion Magnetic Resonance Imaging/methods
, Adult
, Aged
, Anisotropy
, Female
, Humans
, Male
, Middle Aged
, Sensitivity and Specificity
ABSTRACT
The advent of new MR techniques such as perfusion and diffusion weighted imaging has revolutionized diagnostic imaging in stroke. In some institutions, MRI is used as the sole screening imaging technique for acute stroke patients. In this document, the authors will review the MR pattern of acute ischemic arterial stroke, highlight the usefulness of MRI for the identification of acute hematomas and stroke like episodes, present the potential use of MRI in the management of acute stroke patients, especially when thrombolysis is contemplated, and discuss the role of MRI for imaging transient ischemic attack.