Fern proteins show promise against crop pests.

These and other ancient plants may provide alternatives to chemical insecticides.

Isolation of Protein Complexes from Tobacco Leaves by a Two-Step Tandem Affinity Purification.

Protein purification is an essential method for understanding protein function, as many biochemical and structural techniques require a high concentration of isolated protein for analysis. Yet, many studies of protein complexes are hampered by our inability to express them recombinantly in model systems, generally due to poor expression or aggregation. When studying a protein complex that requires its host cellular environment for proper expression and folding, endogenous purification is typically required. Depending on the protein of interest, however, endogenous purification can be challenging because of low expression levels in the host and lack of knowledge working with a non-model expression system, resulting in yields that are too low for subsequent analysis. Here, we describe a protocol for the purification of protein complexes endogenous to Nicotiana benthamiana directly from leaf tissue, with yields that enable structural and biochemical characterization. The protein complex is overexpressed in Nicotiana benthamiana leaves via agroinfiltration, and the protein-packed leaves are then mechanically ground to release the complex from the cells. The protein complex is finally purified by a simple two-step tandem affinity purification using distinct affinity tags for each complex member, to ensure purification of the assembled complex. Our method yields enough protein for various biochemical or structural studies. We have previously used this protocol to purify the complex formed by an innate immune receptor native to tobacco, ROQ1, and the Xanthomonas effector XopQ, and to solve its structure by single-particle cryo-electron microscopy-we use this example to illustrate the approach. This protocol may serve as a template for the purification of proteins from N. benthamiana that require the plant's cellular environment and are expressed at low levels. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression of the protein complex in leaf tissue Basic Protocol 2: Tandem affinity purification of the ROQ1-XopQ complex.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

Leucine-Rich, Potent Anti-Bacterial Protein against Vibrio cholerae, Staphylococcus aureus from Solanum trilobatum Leaves.

A 24 kDa leucine-rich protein from ion exchange fractions of Solanum trilobatum, which has anti-bacterial activity against both the Gram-negative Vibrio cholerae and Gram-positive Staphylococcus aureus bacteria has been purified. In this study, mass spectrometry analysis identified the leucine richness and found a luminal binding protein (LBP). Circular dichroism suggests that the protein was predominantly composed of α- helical contents of its secondary structure. Scanning electron microscopy visualized the characteristics and morphological and structural changes in LBP-treated bacterium. Further in vitro studies confirmed that mannose-, trehalose- and raffinose-treated LBP completely inhibited the hemagglutination ability towards rat red blood cells. Altogether, these studies suggest that LBP could bind to sugar moieties which are abundantly distributed on bacterial surface which are essential for maintaining the structural integrity of bacteria. Considering that Solanum triolbatum is a well-known medicinal and edible plant, in order to shed light on its ancient usage in this work, an efficient anti-microbial protein was isolated, characterized and its in vitro functional study against human pathogenic bacteria was evaluated.

Expression, purification and structural characterization of resveratrol synthase from Polygonum cuspidatum.

Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.

Extraction and deproteinization process of polysaccharide from purple sweet potato.

Extraction and deproteinization process of polysaccharide from purple sweet potato (PPSP) were optimized via the response surface methodology (RSM). The results indicated that the optimal conditions of extraction in hot water of PPSP were as follows: The extraction temperature was 120℃, the extraction time was 2.5 hr, and the solid-liquid ratio was 1∶10 (g/ml). The optimal conditions of Sevage deproteinization were as under the oscillation time was 20 min, the deproteinization times was twice, and polysaccharide solution-Sevage reagent ratio was 1:1 (ml/ml). The extraction yield of PPSP was 3.32%, and the protein removal rate was 93.14% in such a condition.

Physicochemical and functional properties of Alcalase-extracted protein hydrolysate from oil palm leaves.

BACKGROUND: The oil palm tree produces 90% of wastes and the limited usage of these wastes causes a major disposal problem in the mills. Nevertheless, these by-products have a large amount of nutritional components. Thus, the present study aimed to determine the physicochemical and functional properties of protein hydrolysates (PH) from oil palm leaves (OPL) extracted using different concentrations of Alcalase (0-10%) at 2 h of hydrolysis time. RESULTS: Fourier transform infrared spectral analyses showed that the enzymatic hydrolysis altered functional groups of OPL where a secondary amine was present in the PH. Changes were also observed in the thermal stability where the enthalpy heat obtained for PH (933.93-1142.57 J g-1 ) was much lower than OPL (7854.11 J g-1 ). The results showed that the PH extracted by 8% Alcalase exhibited absolute zeta potential, as well as a high emulsifying activity index (70.64 m2  g-1 of protein) and emulsion stability index (60.58 min). Furthermore, this PH showed higher solubility (96.32%) and emulsifying properties compared to other PHs. It is also comparable with commercial plant proteins, indicating that 8% Alcalase is an optimum concentration for hydrolysis. CONCLUSION: In summary, the physicochemical and functional properties of PH extracted from OPL showed good functional properties, suggesting that it can be used as an alternative plant protein in food industries. © 2021 Society of Chemical Industry.

Comparative structural and techno-functional elucidation of full-fat and defatted flaxseed extracts: implication of atmospheric pressure plasma jet.

BACKGROUND: The relatively inferior techno-functionality of flaxseed protein/polysaccharide complexes, especially regarding emulsifying and antioxidant activities, has partially limited their implication in the health food system. The present study aimed to investigate the effects of an atmospheric pressure plasma jet (APPJ) on the physicochemical, structural and selected techno-functional properties of flaxseed extracts. RESULTS: The results obtained showed that the full-fat and defatted flaxseed extract solutions (5 mg mL-1 ) displayed a sustainable decline in pH (-54.06%, -48.80%, P < 0.05) and zeta potential values (-29.42%, -44.28%, P < 0.05), but a gradual increase in particle sizes, as visualised by an optical microscope, during 0-120 s of APPJ treatment. Moreover, the APPJ led to initial decrease but subsequent increase in protein carbonyls and secondary lipid oxidation products, and concurrently changed the spatial conformation and microstructure of flaxseed extracts, as indicated by endogenous fluorescence properties and scanning electron microscopy (SEM). Additionally, the protein subunit remodeling and gum polysaccharides depolymerization were different for full-fat and defatted flaxseed extracts after 30 s of APPJ exposure. Importantly, the emulsifying and antioxidant activities of defatted flaxseed extract were particularly improved, as assessed by cyro-SEM and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity following 15-30 s of APPJ treatment, as a result of the changing interactions between protein and gum polysaccharides, as well as the release of specific phenolic compounds. CONCLUSION: APPJ could serve as a promising strategy for tailoring the specific techno-functionality of flaxseed extracts based on mild structural modification. © 2021 Society of Chemical Industry.

Pulse protein ingredient modification.

Increasing population and depletion of resources have paved the way to find sustainable and nutritious alternative protein sources. Pulses have been identified as a nutritious and inexpensive alternative source of protein that can meet this market demand. Pulses can be converted into protein concentrates and isolates through dry and wet separation techniques. Wet extraction results in relatively pure protein isolates but less sustainable due to higher energy requirements and high waste generation. Dry separation focuses on ingredient functionality rather than molecular level purity. These extracted pulse protein ingredients can be incorporated into different food systems to increase the nutritional value and to achieve the desired functionality. But many plant-based alternative proteins including pulses, face several formulation challenges especially in nutritional, sensory, and functional aspects. Native pulse protein ingredients can contain antinutrients, beany flavor, and undesirable functionality. Modification by biological (enzymatic, fermentation), chemical (acylation, deamidation, glycosylation, phosphorylation), and physical (cold plasma, extrusion, heat, high pressure, ultrasound) methods or a combination of these can improve pulse protein ingredients at the macro and micro level for their desired use. These modification processes will thermodynamically change the structural and conformational characteristics of proteins and expect to improve the quality. © 2021 Society of Chemical Industry.

Isolation and functional characterization of a glucose-6-phosphate/phosphate translocator (IbG6PPT1) from sweet potato (Ipomoea batatas (L.) Lam.).

Sweet potato (Ipomoea batatas (L.) Lam.) is a good source of carbohydrates, an excellent raw material for starch-based industries, and a strong candidate for biofuel production due to its high starch content. However, the molecular basis of starch biosynthesis and accumulation in sweet potato is still insufficiently understood. Glucose-6-phosphate/phosphate translocators (GPTs) mediate the import of glucose-6-phosphate (Glc6P) into plastids for starch synthesis. Here, we report the isolation of a GPT-encoding gene, IbG6PPT1, from sweet potato and the identification of two additional IbG6PPT1 gene copies in the sweet potato genome. IbG6PPT1 encodes a chloroplast membrane-localized GPT belonging to the GPT1 group and highly expressed in storage root of sweet potato. Heterologous expression of IbG6PPT1 resulted in increased starch content in the leaves, root tips, and seeds and soluble sugar in seeds of Arabidopsis thaliana, but a reduction in soluble sugar in the leaves. These findings suggested that IbG6PPT1 might play a critical role in the distribution of carbon sources in source and sink and the accumulation of carbohydrates in storage tissues and would be a good candidate gene for controlling critical starch properties in sweet potato.

Eruca sativa seed napin structural insights and thorough functional characterization.

A potent napin protein has been thoroughly characterized from seeds of rocket salad (Eruca sativa). Eruca sativa napin (EsNap) was purified by ammonium sulfate precipitation (70%) and size-exclusion chromatography. Single intact 16 kDa EsNap band was reduced to 11 and 5 kDa bands respectively on SDS-PAGE. Nano LC-MS/MS yielded two fragments comprising of 26 residues which showed 100% sequence identity with napin-3 of Brassica napus. CD spectroscopy indicated a dominant α-helical structure of EsNap. Monodispersity of EsNap was verified by dynamic light scattering, which also confirmed the monomeric status with a corresponding hydrodynamic radius of 2.4 ± 0.2 nm. An elongated ab initio shape of EsNap was calculated based on SAXS data, with an Rg of 1.96 ± 0.1 nm. The ab initio model calculated by DAMMIF with P1 symmetry and a volume of approx. 31,100 nm3, which corresponded to a molecular weight of approximately 15.5 kDa. The comparison of the SAXS and ab initio modeling showed a minimized χ2-value of 1.87, confirming a similar molecular structure. A homology model was predicted using the coordinate information of Brassica napus rproBnIb (PDB ID: 1SM7). EsNap exhibited strong antifungal activity by significantly inhibiting the growth of Fusarium graminearum. EsNap also showed cytotoxicity against the hepatic cell line Huh7 and the obtained IC50 value was 20.49 µM. Further, strong entomotoxic activity was experienced against different life stages of stored grain insect pest T. castaneum. The result of this study shows insights that can be used in developing potential antifungal, anti-cancerous and insect resistance agents in the future using EsNap from E. sativa.

A Proteomic Approach to Identify Zein Proteins upon Eco-Friendly Ultrasound-Based Extraction.

Zein is a type of prolamin storage protein that has a variety of biomedical and industrial applications. Due to the considerable genetic variability and polyploidity of the starting material, as well as the extraction methods used, the characterization of the protein composition of zein requires a combination of different analytical processes. Therefore, we combined modern analytical methods such as mass spectrometry (MS), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), atomic force microscopy (AFM), or Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) for a better characterization of the extracted zein. In this study, we present an enhanced eco-friendly extraction method, including grinding and sieving corn seeds, for prolamins proteins using an ultrasonic extraction methodology. The use of an ultrasonic homogenizer, 65% ethanol extraction buffer, and 710 µm maize granulation yielded the highest protein extraction from all experimental conditions we employed. An SDS PAGE analysis of the extracted zein protein mainly revealed two intense bands of approximatively 20 and 23 kDa, suggesting that the extracted zein was mostly α-zein monomer. Additionally, MS analysis revealed as a main component the α-zein PMS2 (Uniprot accession no. P24450) type protein in the maize flour extract. Moreover, AFM studies show that extracting zein with a 65% ethanol and a 710 µm granulation yields a homogeneous content that could allow these proteins to be employed in future medical applications. This research leads to a better understanding of zeins content critical for developing new applications of zein in food and pharmaceutical industries, such as biocompatible medical vehicles based on polyplexes complex nanoparticles of zein with antimicrobial or drug delivery properties.

Lupinus albus Protein Components Inhibit MMP-2 and MMP-9 Gelatinolytic Activity In Vitro and In Vivo.

Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are regarded as important clinical targets due to their nodal-point role in inflammatory and oncological diseases. Here, we aimed at isolating and characterizing am MMP-2 and-9 inhibitor (MMPI) from Lupinus albus and at assessing its efficacy in vitro and in vivo. The protein was isolated using chromatographic and 2-D electrophoretic procedures and sequenced by using MALDI-TOF TOF and MS/MS analysis. In vitro MMP-2 and 9 inhibitions were determined on colon adenocarcinoma (HT29) cells, as well as by measuring the expression levels of genes related to these enzymes. Inhibitory activities were also confirmed in vivo using a model of experimental TNBS-induced colitis in mice, with oral administrations of 15 mg·kg-1. After chromatographic and electrophoretic isolation, the L. albus MMP-9 inhibitor was found to comprise a large fragment from δ-conglutin and, to a lower extent, small fragments of ß-conglutin. In vitro studies showed that the MMPI successfully inhibited MMP-9 activity in a dose-dependent manner in colon cancer cells, with an IC50 of 10 µg·mL-1 without impairing gene expression nor cell growth. In vivo studies showed that the MMPI maintained its bioactivities when administered orally and significantly reduced colitis symptoms, along with a very significant inhibition of MMP-2 and -9 activities. Overall, results reveal a novel type of MMPI in lupine that is edible, proteinaceous in nature and soluble in water, and effective in vivo, suggesting a high potential application as a nutraceutical or a functional food in pathologies related to abnormally high MMP-9 activity in the digestive system.

Solid-state fermented brewer's spent grain enzymatic extract increases in vitro and in vivo feed digestibility in European seabass.

Brewer's spent grain (BSG) is the largest by-product originated from the brewery industry with a high potential for producing carbohydrases by solid-state fermentation. This work aimed to test the efficacy of a carbohydrases-rich extract produced from solid-state fermentation of BSG, to enhance the digestibility of a plant-based diet for European seabass (Dicentrarchus labrax). First, BSG was fermented with A. ibericus to obtain an aqueous lyophilized extract (SSF-BSG extract) and incorporated in a plant-based diet at increasing levels (0-control; 0.1%, 0.2%, and 0.4%). Another diet incorporating a commercial carbohydrases-complex (0.04%; Natugrain; BASF) was formulated. Then, all diets were tested in in vitro and in vivo digestibility assays. In vitro assays, simulating stomach and intestine digestion in European seabass, assessed dietary phosphorus, phytate phosphorus, carbohydrates, and protein hydrolysis, as well as interactive effects between fish enzymes and dietary SSF-BSG extract. After, an in vivo assay was carried out with European seabass juveniles fed selected diets (0-control; 0.1%, and 0.4%). In vitro digestibility assays showed that pentoses release increased 45% with 0.4% SSF-BSG extract and 25% with Natugrain supplemented diets, while amino acids release was not affected. A negative interaction between endogenous fish enzymes and SSF-BSG extract was observed in both diets. The in vivo digestibility assay corroborated in vitro data. Accordingly, the dietary supplementation with 0.4% SSF-BSG increased the digestibility of dry matter, starch, cellulose, glucans, and energy and did not affect protein digestibility. The present work showed the high potential of BSG to produce an added-value functional supplement with high carbohydrases activity and its potential contribution to the circular economy by improving the nutritional value of low-cost and sustainable ingredients that can be included in aquafeeds.

Silencing a Simple Extracellular Leucine-Rich Repeat Gene OsI-BAK1 Enhances the Resistance of Rice to Brown Planthopper Nilaparvata lugens.

Many plant proteins with extracellular leucine-rich repeat (eLRR) domains play an important role in plant immunity. However, the role of one class of eLRR plant proteins-the simple eLRR proteins-in plant defenses against herbivores remains largely unknown. Here, we found that a simple eLRR protein OsI-BAK1 in rice localizes to the plasma membrane. Its expression was induced by mechanical wounding, the infestation of gravid females of brown planthopper (BPH) Nilaparvata lugens or white-backed planthopper Sogatella furcifera and treatment with methyl jasmonate or abscisic acid. Silencing OsI-BAK1 (ir-ibak1) in rice enhanced the BPH-induced transcript levels of three defense-related WRKY genes (OsWRKY24, OsWRKY53 and OsWRKY70) but decreased the induced levels of ethylene. Bioassays revealed that the hatching rate was significantly lower in BPH eggs laid on ir-ibak1 plants than wild-type (WT) plants; moreover, gravid BPH females preferred to oviposit on WT plants over ir-ibak1 plants. The exogenous application of ethephon on ir-ibak1 plants eliminated the BPH oviposition preference between WT and ir-ibak1 plants but had no effect on the hatching rate of BPH eggs. These findings suggest that OsI-BAK1 acts as a negative modulator of defense responses in rice to BPH and that BPH might exploit this modulator for its own benefit.

Identification and Capture of Phenolic Compounds from a Rapeseed Meal Protein Isolate Production Process By-Product by Macroporous Resin and Valorization Their Antioxidant Properties.

In this study, phenolic compounds from an aqueous protein by-product from rapeseed meal (RSM) were identified by HPLC-DAD and HPLC-ESI-MS, including sinapine, sinapic acid, sinapoyl glucose, and 1,2-di-sinapoyl gentibiose. The main phenolic compound in this by-product was sinapine. We also performed acid hydrolysis to convert sinapine, and sinapic acid derivatives present in the permeate, to sinapic acid. The adsorption of phenolic compounds was investigated using five macroporous resins, including XAD4, XAD7, XAD16, XAD1180, and HP20. Among them, XAD16 showed the highest total phenolic contents adsorption capacities. The adsorption behavior of phenolic compounds was described by pseudo-second-order and Langmuir models. Moreover, thermodynamics tests demonstrated that the adsorption process of phenolic compounds was exothermic and spontaneous. The highest desorption ratio was obtained with 30% (v/v) and 70% (v/v) ethanol for sinapine and sinapic acid, respectively, with a desorption ratio of 63.19 ± 0.03% and 94.68 ± 0.013%. DPPH and ABTS tests revealed that the antioxidant activity of the hydrolyzed fraction was higher than the non-hydrolyzed fraction and higher than the one of vitamin C. Antioxidant tests demonstrated that these phenolic compounds could be used as natural antioxidants, which can be applied in the food industry.

Cationic Clitoria ternatea Seed Peptide as a Potential Novel Bioactive Molecule.

BACKGROUND: While several biologics have been reported from different parts of Clitoria ternatea, a herbaceous climber of the family Fabaceae, specific production of cationic peptides other than cyclotides (<3.7 kDa) has barely been investigated, or their bioactive potential been looked into. OBJECTIVE: The study aims to uncover potential bioactivities and characteristics of novel cationic peptides from C. ternatea seeds. METHODS: C. ternatea seed cationic peptide purified by simple and cost-effective procedures was analyzed by electrophoresis and mass spectrometry. Antimicrobial efficacy was evaluated against bacterial and fungal pathogens. Antioxidant potential was quantified by in vitro antioxidant assays. Physicochemical characterization and Tandem mass spectrometry were performed. RESULTS: An 8.5 kDa cationic peptide purified from C. ternatea seeds was active against Candida albicans, Staphylococcus aureus, Aeromonas hydrophila and Escherichia coli at a minimum inhibitory concentration in the range of 8-32 µg/ml. This activity was totally uncompromised at pH 5-8 or after 1 h of heat treatment at 70-80ºC, but was sensitive to protease treatment. Concentration-dependent free-radical scavenging activity and ferric-reducing capacity demonstrated the antioxidant potential of the peptide. Tandem MS analysis of trypsin-digested peptide based on shotgun proteomics detected matching peptide sequences with one or two cysteine residues but had low sequence coverage (≤17%) to known sequences in the C. ternatea protein database. Taken together, the distinct characteristics of this novel 8.5 kDa peptide clearly distinguish it from known cyclotides of C. ternatea. CONCLUSIONS: Insights have been obtained into the functional characteristics of what appears to be a novel cationic peptide from C. ternatea seeds, exhibiting significant antimicrobial and antioxidant activities.

Expression, purification and crystallization of TGW6, which limits grain weight in rice.

Rice is the staple food for over half the world's population. Genes associated with rice yield include THOUSAND GRAIN WEIGHT 6 (TGW6), which negatively regulates the number of endosperm cells as well as grain weight. The 1-bp deletion allele of tgw6 cloned from the Indian landrace rice cultivar Kasalath, which has lost function, enhances both grain size and yield. TGW6 has been utilized as a target for breeding and genome editing to increase the yield of rice. In the present study, we describe an improved heterologous expression system of TGW6 in Escherichia coli to enable purification of the recombinant protein. The best expression was achieved using codon optimized TGW6 with a 30 amino acid truncation at the N-terminus (Δ30TGW6) in the Rosetta-gami 2(DE3) host strain. Furthermore, we found that calcium ions were critical for the purification of stable Δ30TGW6. Crystals of Δ30TGW6 were obtained using the sitting-drop vapor-diffusion method at 283 K, which diffracted X-rays to at least 2.6 Å resolution. Herein, we established an efficient procedure for the production and purification of TGW6 in sufficient quantities for structural and functional studies. Detailed information concerning the molecular mechanism of TGW6 will enable the design of more efficient ways to control the activity of the enzyme.

Bidentatide, a Novel Plant Peptide Derived from Achyranthes bidentata Blume: Isolation, Characterization, and Neuroprotection through Inhibition of NR2B-Containing NMDA Receptors.

Increasing attention is being focused on the use of polypeptide-based N-methyl-d-aspartate (NMDA) receptor antagonists for the treatment of nervous system disorders. In our study on Achyranthes bidentata Blume, we identified an NMDA receptor subtype 2B (NR2B) antagonist that exerts distinct neuroprotective actions. This antagonist is a 33 amino acid peptide, named bidentatide, which contains three disulfide bridges that form a cysteine knot motif. We determined the neuroactive potential of bidentatide by evaluating its in vitro effects against NMDA-mediated excitotoxicity. The results showed that pretreating primary cultured hippocampal neurons with bidentatide prevented NMDA-induced cell death and apoptosis via multiple mechanisms that involved intracellular Ca2+ inhibition, NMDA current inhibition, and apoptosis-related protein expression regulation. These mechanisms were all dependent on bidentatide-induced inhibitory regulation of NR2B-containing NMDA receptors; thus, bidentatide may contribute to the development of neuroprotective agents that would likely possess the high selectivity and safety profiles inherent in peptide drugs.