Novel Antioxidant Peptides from Crassostrea Hongkongensis Improve Photo-Oxidation in UV-Induced HaCaT Cells

Enzymatic hydrolysates from Oysters (OAH) display multiple biological activities. Previously, a 3~5 KDa oyster ultrafiltration component (OUP) showed a high property of preventing skin oxidation. Subsequently, we identified specific peptides with such activity. OUP was fractionated stepwise by Sephadex-G25 and RP-HPLC, and active fractions were screened using UV-irradiated HaCaT cells. The most active fractions (OP5-3) were analyzed by LC-MS/MS and a total of 17 peptides were identified. Results from mass spectrometry showed that OP5-3 consisted of peptides with a molecular weight range of 841.51–1786.92 Da. Six of these peptides were synthesized for validating the activity of resisting skin oxidation in the same cell model. All six peptides showed varying degrees of antioxidant activity, while pretreatment of HaCaT cells with AIVAEVNEAAK alleviated UV cytotoxicity, inhibited metalloproteinase 1 (MMP-1) expression, and showed the highest activity to resist UV-induced skin photo-oxidation among these peptides. In addition, results from molecular docking analysis of MMP-1 with AIVAEVNEAAK showed that AIVAEVNEAAK suppresses its enzymatic activity by directly interacting with MMP-1 and thus exhibit anti-photoaging activity.     

茶尺蠖普通气味结合蛋白EoblGOBP2与茶树挥发物的结合功能研究

茶尺蠖是茶园中一种重要的鳞翅目害虫,主要以幼虫取食危害茶树叶片,给茶叶生产带来了严重威胁。茶尺蠖在寻找配偶、产卵地以及觅食活动中其嗅觉系统发挥了非常重要的作用。本研究以茶尺蠖Ectropis obliqua Prout嗅觉系统中的一个普通气味结合蛋白——EoblGOBP2为研究对象,在克隆得到的EoblGOBP2基因基础上,通过对EoblGOBP2进行原核表达、分离纯化后,利用1-NPN的荧光竞争结合法研究了体外重组EoblGOBP2与茶树挥发物的结合生理功能。Scatchard方程显示EoblGOBP2与1-NPN的解离常数K_D为2.310 μmol·L^-1。在荧光竞争结合实验中,7种待测配基均能使1-NPN的相对荧光强度降低到50%以下,表明与EoblGOBP2均有较强的结合能力,而其中邻苯二甲酸二丁酯结合能力最强,K_D值达到4.353 μmol·L^-1。本研究7种配基均为茶树叶片挥发物,表明EoblGOBP2在茶尺蠖嗅觉系统对茶树挥发物的信息识别结合功能中发挥了重要作用。     

Monoclonal Antibody Production and Immunolocalization of a Salinity Stress-Related Protein in Rice (Oryza sativa)

Among various physiological responses to salt stress, the synthesis of a lectin-related protein of 14.5 kDa was observed in rice plants (Oryza sativa L.) under the treatment of 170 mmol/L NaCl. In order to better understand the role of the SALT protein in the physiological processes involving salinity, it was immunolocalized in mesophilic cells of leaf sheath and blade of a rice variety IAC-4440 following monoclonal antibodies produced by hybridome culture technique. This variety turned out to be an excellent model for that purpose, since it accumulates SALT protein even in absence of salt treatment and it has been classified as moderately sensitive to salinity and a superior grain producer. This feature was relevant for this work since it allowed the use of plants without the deleterious effects caused by salinity. Immunocytochemistry assays revealed that the SALT protein is located in the stroma of chloroplasts under non-stressing condition. Since the chloroplast is the main target affected by salinity and considering that the SALT protein does not present any apparent signal peptide for organelle localization, its lectin-like activity seems to play an important role in the establishment of stable complexes, either to other proteins or to oligosaccharides that are translocated to the chloroplast.     

Identification and Characterization of a Novel Lectin from the Clam Glycymeris yessoensis and Its Functional Characterization under Microbial Stimulation and Environmental Stress

Lectin from the bivalve Glycymeris yessoensis (GYL) was purified by affinity chromatography on porcine stomach mucin–Sepharose. GYL is a dimeric protein with a molecular mass of 36 kDa, as established by SDS-PAGE and MALDI-TOF analysis, consisting of 18 kDa subunits linked by a disulfide bridge. According to circular dichroism data, GYL is a β/α-protein with the predominance of β-structure. GYL preferentially agglutinates enzyme-treated rabbit erythrocytes and recognizes glycoproteins containing O-glycosidically linked glycans, such as porcine stomach mucin (PSM), fetuin, thyroglobulin, and ovalbumin. The amino acid sequences of five segments of GYL were acquired via mass spectrometry. The sequences have no homology with other known lectins. GYL is Ca²⁺-dependent and stable over a range above a pH of 8 and temperatures up to 20 °C for 30 min. GYL is a pattern recognition receptor, as it binds common pathogen-associated molecular patterns, such as peptidoglycan, LPS, β-1,3-glucan and mannan. GYL possesses a broad microbial-binding spectrum, including Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Vibrio proteolyticus), but not the fungus Candida albicans. Expression levels of GYL in the hemolymph were significantly upregulated after bacterial challenge by V. proteolyticus plus environmental stress (diesel fuel). Results indicate that GYL is probably a new member of the C-type lectin family, and may be involved in the immune response of G. yessoensis to bacterial attack.     

橡胶树叶柄愈伤组织乳管细胞的组织化学和分子生物学研究

橡胶树乳管分化研究主要以树皮为研究材料。本研究通过组织化学染色法、尼罗红荧光染色法、免疫组织化学法和分子生物学方法证实橡胶树叶柄来源的愈伤组织中存在乳管细胞。乳管细胞在叶柄愈伤组织中随机分布,分布模式类似橡胶树初生乳管;叶柄愈伤组织乳管细胞在发育早期时橡胶粒子较少,在发育后期时充满橡胶粒子。RT-PCR扩增出叶柄愈伤组织乳管细胞的橡胶延伸因子(REF)、小橡胶粒子蛋白(SRPP)、橡胶凝集素(hevein)和橡胶转移酶(CPT)等胶乳特异基因的转录本,经比对它们序列与所报道的橡胶树树干胶乳中表达的基因序列一致,表明橡胶树叶柄愈伤组织乳管细胞拥有树皮乳管细胞相似的功能,为叶柄愈伤组织作为一种新型的研究乳管分化的模式提供了科学依据。     

Preliminary Characterization of Extracellular Allelochemicals of the Toxic Marine Dinoflagellate Alexandrium tamarense Using a Rhodomonas salina Bioassay

Members of the marine dinoflagellate genus Alexandrium are known to exude allelochemicals, unrelated to well-known neurotoxins (PSP-toxins, spirolides), with negative effects on other phytoplankton and marine grazers. Physico/chemical characterization of extracellular lytic compounds of A. tamarense, quantified by Rhodomonas salina bioassay, showed that the lytic activity, and hence presumably the compounds were stable over wide ranges of temperatures and pH and were refractory to bacterial degradation. Two distinct lytic fractions were collected by reversed-phase solid-phase extraction. The more hydrophilic fraction accounted for about 2% of the whole lytic activity of the A. tamarense culture supernatant, while the less hydrophilic one accounted for about 98% of activity. Although temporal stability of the compounds is high, substantial losses were evident during purification. Lytic activity was best removed from aqueous phase with chloroform-methanol (3:1). A “pseudo-loss” of lytic activity in undisturbed and low-concentrated samples and high activity of an emulsion between aqueous and n-hexane phase after liquid-liquid partition are strong evidence for the presence of amphipathic compounds. Lytic activity in the early fraction of gel permeation chromatography and lack of activity after 5 kD ultrafiltration indicate that the lytic agents form large aggregates or macromolecular complexes.     

Structural and Molecular Characterization of Squalene Synthase Belonging to the Marine Thraustochytrid Species Aurantiochytrium limacinum Using Bioinformatics Approach

The marine microorganisms thraustochytrids have been explored for their potential in the production of various bioactive compounds, such as DHA, carotenoids, and squalene. Squalene is a secondary metabolite of the triterpenoid class and is known for its importance in various industrial applications. The bioinformatic analysis for squalene synthase (SQS) gene (the first key enzyme in the tri-terpenoid synthesis pathway), that is prevailing among thraustochytrids, is poorly investigated. In-silico studies combining sequence alignments and bioinformatic tools helped in the preliminary characterization of squalene synthases found in Aurantiochytrium limacinum. The sequence contained highly conserved regions for SQS found among different species indicated the enzyme had all the regions for its functionality. The signal peptide sequence and transmembrane regions were absent, indicating an important aspect of the subcellular localization. Secondary and 3-D models generated using appropriate templates demonstrated the similarities with SQS of the other species. The 3-D model also provided important insights into possible active, binding, phosphorylation, and glycosylation sites.     

Fucoxanthin Pretreatment Ameliorates Visible Light-Induced Phagocytosis Disruption of RPE Cells under a Lipid-Rich Environment via the Nrf2 Pathway

Fucoxanthin, a special xanthophyll derived from marine algae, has increasingly attracted attention due to its diverse biological functions. However, reports on its ocular benefits are still limited. In this work, the ameliorative effect of fucoxanthin on visible light and lipid peroxidation-induced phagocytosis disruption in retinal pigment epithelium (RPE) cells was investigated in vitro. Marked oxidative stress, inflammation, and phagocytosis disruption were evident in differentiated RPE cells following their exposure to visible light under a docosahexaenoic acid (DHA)-rich environment. Following pretreatment with fucoxanthin, however, the activated nuclear factor erythroid-derived-2-like 2 (Nrf2) signaling pathway was observed and, furthermore, when the fucoxanthin -pretreated RPE cells were irradiated with visible light, intracellular reactive oxygen species (ROS), malondialdehyde (MDA) levels and inflammation were obviously suppressed, while phagocytosis was significantly improved. However, following the addition of Nrf2 inhibitor ML385, the fucoxanthin exhibited no ameliorative effects on the oxidative stress, inflammation, and phagocytosis disruption in the RPE cells, thus indicating that the ameliorative effect of fucoxanthin on the phagocytosis of RPE cells is closely related to the Nrf2 signaling pathway. In conclusion, these results suggest that fucoxanthin supplementation might be beneficial to the prevention of visible light-induced retinal injury.     

Biogenic Synthesis of Copper Nanoparticles Using Bacterial Strains Isolated from an Antarctic Consortium Associated to a Psychrophilic Marine Ciliate: Characterization and Potential Application as Antimicrobial Agents

In the last decade, metal nanoparticles (NPs) have gained significant interest in the field of biotechnology due to their unique physiochemical properties and potential uses in a wide range of applications. Metal NP synthesis using microorganisms has emerged as an eco-friendly, clean, and viable strategy alternative to chemical and physical approaches. Herein, an original and efficient route for the microbial synthesis of copper NPs using bacterial strains newly isolated from an Antarctic consortium is described. UV-visible spectra of the NPs showed a maximum absorbance in the range of 380–385 nm. Transmission electron microscopy analysis showed that these NPs are all monodispersed, spherical in nature, and well segregated without any agglomeration and with an average size of 30 nm. X-ray powder diffraction showed a polycrystalline nature and face centered cubic lattice and revealed characteristic diffraction peaks indicating the formation of CuONPs. Fourier-transform infrared spectra confirmed the presence of capping proteins on the NP surface that act as stabilizers. All CuONPs manifested antimicrobial activity against various types of Gram-negative; Gram-positive bacteria; and fungi pathogen microorganisms including Escherichia coli, Staphylococcus aureus, and Candida albicans. The cost-effective and eco-friendly biosynthesis of these CuONPs make them particularly attractive in several application from nanotechnology to biomedical science.     

Characterization of a Novel Conus bandanus Conopeptide Belonging to the M-Superfamily Containing Bromotryptophan

A novel conotoxin (conopeptide) was biochemically characterized from the crude venom of the molluscivorous marine snail, Conus bandanus (Hwass in Bruguière, 1792), collected in the south-central coast of Vietnam. The peptide was identified by screening bromotryptophan from chromatographic fractions of the crude venom. Tandem mass spectrometry techniques were used to detect and localize different post-translational modifications (PTMs) present in the BnIIID conopeptide. The sequence was confirmed by Edman’s degradation and mass spectrometry revealing that the purified BnIIID conopeptide had 15 amino acid residues, with six cysteines at positions 1, 2, 7, 11, 13, and 14, and three PTMs: bromotryptophan, γ-carboxy glutamate, and amidated aspartic acid, at positions “4”, “5”, and “15”, respectively. The BnIIID peptide was synthesized for comparison with the native peptide. Homology comparison with conopeptides having the III-cysteine framework (–CCx₁x₂x₃x₄Cx₁x₂x₃Cx₁CC–) revealed that BnIIID belongs to the M-1 family of conotoxins. This is the first report of a member of the M-superfamily containing bromotryptophan as PTM.     

Antimetastatic Effect of Halichondramide,a Trisoxazole Macrolide from the Marine Sponge Chondrosia corticata,on Human Prostate Cancer Cells via Modulation of Epithelial-to-Mesenchymal Transition

Halichondramide (HCA), a trisoxazole-containing macrolide isolated from the marine sponge Chondrosia corticata has been shown to exhibit cytotoxicity and antifungal activities. In our previous study, HCA was also found to exhibit antiproliferative activity against a variety of cancer cells. However, the precise mechanism of action of HCA in the antitumor activity remains to be elucidated. In the present study, we identified the antimetastatic activity of HCA in the highly metastatic PC3 human prostate cancer cells. HCA showed potent growth inhibitory activity of the PC3 cells with an IC₅₀ value of 0.81 µM. Further analysis revealed that HCA suppressed the expression of a potential metastatic biomarker, phosphatase of regenerating liver-3 (PRL-3), in PC3 cells. The suppression of PRL-3 by HCA sequentially down-regulates the expression of phosphoinositide 3-kinase (PI3K) subunits p85 and p110. The antimetastatic effect of HCA was also correlated with the down-regulation of matrix metalloproteases (MMPs) and the modulation of cadherin switches N-cadherin and E-cadherin. In addition, HCA also effectively suppressed the migration and invasion of PC3 cells. These findings suggest that halichondramide might serve as a potential inhibitor of tumor cell metastasis with the modulation of PRL-3.     

Production of a Novel Fucoidanase for the Green Synthesis of Gold Nanoparticles by Streptomyces sp. and Its Cytotoxic Effect on HeLa Cells

Marine actinobacteria-produced fucoidanases have received considerable attention as one of the major research topics in recent years, particularly for the medical exploitation of fucoidans and their degradation products. The present study describes the optimization and production of a novel fucoidanase for the green synthesis of gold nanoparticles and its biological applications. The production of fucoidanase was optimized using Streptomyces sp. The medium components were selected in accordance with the Plackett-Burman design and were further optimized via response surface methodology. The fucoidanase was statistically optimized with the most significant factors, namely wheat bran 3.3441 g/L, kelp powder 0.7041 g/L, and NaCl 0.8807 g/L, respectively. The biosynthesized gold nanoparticles were determined by UV-vis spectroscopy and were further characterized by X-ray diffraction analysis, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis, and high-resolution transmission electron microscopy. Furthermore, the biosynthesized gold nanoparticles exhibited a dose-dependent cytotoxicity against HeLa cells and the inhibitory concentration (IC₅₀) was found to be 350 µg/mL at 24 h and 250 µg/mL at 48 h. Therefore, the production of novel fucoidanase for the green synthesis of gold nanoparticles has comparatively rapid, less expensive and wide application to anticancer therapy in modern medicine.     

Anti-Allergic Effect of 3,4-Dihydroxybenzaldehyde Isolated from Polysiphonia morrowii in IgE/BSA-Stimulated Mast Cells and a Passive Cutaneous Anaphylaxis Mouse Model

In this study, we investigated the anti-allergic effects of 3,4-dihydroxybenzaldehyde (DHB) isolated from the marine red alga, Polysiphonia morrowii, in mouse bone-marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-sensitized mice. DHB inhibited IgE/bovine serum albumin (BSA)-induced BMCMCs degranulation by reducing the release of β-hexosaminidase without inducing cytotoxicity. Further, DHB dose-dependently decreased the IgE binding and high-affinity IgE receptor (FcεRI) expression and FcεRI-IgE binding on the surface of BMCMCs. Moreover, DHB suppressed the secretion and/or the expression of the allergic cytokines, interleukin (IL)-4, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF)-α, and the chemokine, thymus activation-regulated chemokine (TARC), by regulating the phosphorylation of IκBα and the translocation of cytoplasmic NF-κB into the nucleus. Furthermore, DHB attenuated the passive cutaneous anaphylactic (PCA) reaction reducing the exuded Evans blue amount in the mouse ear stimulated by IgE/BSA. These results suggest that DHB is a potential therapeutic candidate for the prevention and treatment of type I allergic disorders.     

Metabolomic Characterization of a cf. Neolyngbya Cyanobacterium from the South China Sea Reveals Wenchangamide A,a Lipopeptide with In Vitro Apoptotic Potential in Colon Cancer Cells

Metabolomics can be used to study complex mixtures of natural products, or secondary metabolites, for many different purposes. One productive application of metabolomics that has emerged in recent years is the guiding direction for isolating molecules with structural novelty through analysis of untargeted LC-MS/MS data. The metabolomics-driven investigation and bioassay-guided fractionation of a biomass assemblage from the South China Sea dominated by a marine filamentous cyanobacteria, cf. Neolyngbya sp., has led to the discovery of a natural product in this study, wenchangamide A (1). Wenchangamide A was found to concentration-dependently cause fast-onset apoptosis in HCT116 human colon cancer cells in vitro (24 h IC₅₀ = 38 μM). Untargeted metabolomics, by way of MS/MS molecular networking, was used further to generate a structural proposal for a new natural product analogue of 1, here coined wenchangamide B, which was present in the organic extract and bioactive sub-fractions of the biomass examined. The wenchangamides are of interest for anticancer drug discovery, and the characterization of these molecules will facilitate the future discovery of related natural products and development of synthetic analogues.     

Using the Gluten Peak Tester as a tool to measure physical properties of gluten

The functional properties of wheat are largely dictated by composition and interactions of the gluten proteins. All flours contain gliadin and glutenin, but produce baked products of varying quality, which provides evidence that gluten proteins from different wheats possess different properties. A common method to study differences in gluten properties, which is utilized in this study, is fractionation/reconstitution experiments to understand how various gliadin to glutenin ratios and how fractions from different wheat sources affect gluten aggregation properties. Gliadin and glutenin from a vital wheat gluten were fractionated with 70% ethanol and reconstituted at various gliadin to glutenin ratios. Gliadin and glutenin from a Canadian eastern soft, eastern hard and western hard wheat (14% moisture) were fractionated and substituted between flours at the native gliadin to glutenin ratio. Gluten combinations were evaluated with a Gluten Peak Tester at constant temperature and mixing. Varying gliadin to glutenin ratio showed that 50:50 is optimal for fast gluten aggregation while amount of glutenin dictates strength. Substitution experiments showed that replacing good quality gluten fractions with those from a lower quality wheat decreases gluten quality, and vice versa. Data also showed that cultivar specific differences in gliadin and glutenin are more important in dictating gluten strength (torque), while gliadin to glutenin ratio dictates aggregation time (PMT) independent of the source of fractions. The study demonstrated the ability of the improved method to evaluate gluten aggregation by controlling for all variables except the one being tested. The data also revealed information about gluten aggregation properties never before seen.     

Antiradical Capacity and Induction of Apoptosis on HeLa Cells by a <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Extract

Jamapa bean is a black Phaseolus vulgaris variety rich in condensed tannins, anthocyanins and flavonols with interesting biological activities. The objective of this work was to evaluate the antiradical capacity (ARC) of a Jamapa bean methanolic extract (BME) and some of the proanthocyanidin-rich fractions derived from it, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The effect of the BME on some proteins involved in apoptosis on HeLa cells was also evaluated. A strong correlation between proanthocyanidin concentration in BME and antiradical capacity was found, suggesting that these compounds contribute significantly to antiradical activity. BME was a better radical scavenger than butylated hydroxytoluene (45.6 and 33.9% ARC at 400 μM, respectively). Two proanthocyanidin-rich fractions obtained after a preliminary separation of the BME using Toyopearl (TP4 and TP6) exhibited a higher antiradical activity than the parent extract. The treatment of HeLa cells with 35 μg BME/ml/24 h increased the expression of Bax and Caspase-3, pro-apoptotic proteins (6.13 and 1.2 times for Caspase-3 and Bax, respectively). The mechanism of action of some proteins involved in apoptosis was also evaluated, and the results suggest that black Jamapa bean could be an important source of polyphenolic compounds with potential biological use as antioxidant and anticancer agents.     

Structural Characterization of New Peptide Variants Produced by Cyanobacteria from the Brazilian Atlantic Coastal Forest Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Tandem Mass Spectrometry

Cyanobacteria from underexplored and extreme habitats are attracting increasing attention in the search for new bioactive substances. However, cyanobacterial communities from tropical and subtropical regions are still largely unknown, especially with respect to metabolite production. Among the structurally diverse secondary metabolites produced by these organisms, peptides are by far the most frequently described structures. In this work, liquid chromatography/electrospray ionization coupled to high resolution quadrupole time-of-flight tandem mass spectrometry with positive ion detection was applied to study the peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest. A total of 38 peptides belonging to three different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected in the extracts. Of the 38 peptides, 37 were detected here for the first time. New structural features were proposed based on mass accuracy data and isotopic patterns derived from full scan and MS/MS spectra. Interestingly, of the 40 surveyed strains only nine were confirmed to be peptide producers; all of these strains belonged to the order Nostocales (three Nostoc sp., two Desmonostoc sp. and four Brasilonema sp.).     

A Simple and Low-Cost Method to Classify Amylose Content of Rice Using a Standard Color Chart

Abstract

An alternative method to classify amylose content in rice was developed. In this method, based on amylose-iodine colorimetry, a standard color chart was used instead of a spectrophotometer to successfully classify a wide range of cultivars with known and unknown amylose content. This is a rapid low-cost method that may be useful for prediction of amylose content in rice for breeding purposes.     

SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.