An Update of Lectins from Marine Organisms: Characterization, Extraction Methodology, and Potential Biofunctional Applications.

Lectins are a unique group of nonimmune carbohydrate-binding proteins or glycoproteins that exhibit specific and reversible carbohydrate-binding activity in a non-catalytic manner. Lectins have diverse sources and are classified according to their origins, such as plant lectins, animal lectins, and fish lectins. Marine organisms including fish, crustaceans, and mollusks produce a myriad of lectins, including rhamnose binding lectins (RBL), fucose-binding lectins (FTL), mannose-binding lectin, galectins, galactose binding lectins, and C-type lectins. The widely used method of extracting lectins from marine samples is a simple two-step process employing a polar salt solution and purification by column chromatography. Lectins exert several immunomodulatory functions, including pathogen recognition, inflammatory reactions, participating in various hemocyte functions (e.g., agglutination), phagocytic reactions, among others. Lectins can also control cell proliferation, protein folding, RNA splicing, and trafficking of molecules. Due to their reported biological and pharmaceutical activities, lectins have attracted the attention of scientists and industries (i.e., food, biomedical, and pharmaceutical industries). Therefore, this review aims to update current information on lectins from marine organisms, their characterization, extraction, and biofunctionalities.

Considerations regarding affinity determinants for aflatoxin B1 in binding cavity of fungal laccase based on in silico mutational and in vitro verification studies.

Laccase, a multicopper oxidase, is well known for its industrial potentials to remove environmental pollutants due to its low substrate specificity to oxidize phenols and thus catalytic versatility. Many efforts focused on the metabolic mechanism, yet to decipher the structural determinants responsible for the differentiation between substrates. Aflatoxin B1 (AFB1), a new substrate for laccase, is a mycotoxin with a formidable environmental threat to public health and food safety. In the present study, we combined biochemical, in silico mutational and molecular-docking data to gain an insight to the function of key residues in the active cavity close to the T1 copper site in a characterized recombinant laccase from Cerrena unicolor (rCuL). Kinetic data for computer-assisted virtual mutants established the binding affinity of hydrogen bonds and residues (Asn336, Asp207, Val391, and Thr165) in rCuL to AFB1. The augmented binding affinity to AFB1 may be related to the conformational rearrangements of the laccase and its ability to hydrogen-bond with the substrate. Furthermore, the optimal pH and temperature for rCuL and variants mediated AFB1 degradation may depend on their pH stability and thermostability. Our findings reinforce the importance of the structure-function relationship of fungal laccases in degrading AFB1, providing mechanistic guidance for future biocatalyst and bioengineering applications.

Aberrant Transferrin and Ferritin Upregulation Elicits Iron Accumulation and Oxidative Inflammaging Causing Ferroptosis and Undermines Estradiol Biosynthesis in Aging Rat Ovaries by Upregulating NF-Κb-Activated Inducible Nitric Oxide Synthase: First Demonstration of an Intricate Mechanism.

We report herein a novel mechanism, unraveled by proteomics and validated by in vitro and in vivo studies, of the aberrant aging-associated upregulation of ovarian transferrin and ferritin in rat ovaries. The ovarian mass and serum estradiol titer plummeted while the ovarian labile ferrous iron and total iron levels escalated with age in rats. Oxidative stress markers, such as nitrite/nitrate, 3-nitrotyrosine, and 4-hydroxy-2-nonenal, accumulated in the aging ovaries due to an aberrant upregulation of the ovarian transferrin, ferritin light/heavy chains, and iron regulatory protein 2(IRP2)-mediated transferrin receptor 1 (TfR1). Ferritin inhibited estradiol biosynthesis in ovarian granulosa cells in vitro via the upregulation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p65/p50-induced oxidative and inflammatory factor inducible nitric oxide synthase (iNOS). An in vivo study demonstrated how the age-associated activation of NF-κB induced the upregulation of iNOS and the tumor necrosis factor α (TNFα). The downregulation of the keap1-mediated nuclear factor erythroid 2-related factor 2 (Nrf2), that induced a decrease in glutathione peroxidase 4 (GPX4), was observed. The aberrant transferrin and ferritin upregulation triggered an iron accumulation via the upregulation of an IRP2-induced TfR1. This culminates in NF-κB-iNOS-mediated ovarian oxi-inflamm-aging and serum estradiol decrement in naturally aging rats. The iron accumulation and the effect on ferroptosis-related proteins including the GPX4, TfR1, Nrf2, Keap1, and ferritin heavy chain, as in testicular ferroptosis, indicated the triggering of ferroptosis. In young rats, an intraovarian injection of an adenovirus, which expressed iron regulatory proteins, upregulated the ovarian NF-κB/iNOS and downregulated the GPX4. These novel findings have contributed to a prompt translational research on the ovarian aging-associated iron metabolism and aging-associated ovarian diseases.

A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high-level expression in Pichia pastoris.

Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713 bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60 °C and over a broad pH range between 5.0 and 9.0, and was stable at 50 °C and over the pH range 4.0-9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of ß-N-acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

The mechanism of enriched environment repairing the learning and memory impairment in offspring of prenatal stress by regulating the expression of activity-regulated cytoskeletal-associated and insulin-like growth factor-2 in hippocampus.

BACKGROUND: Prenatal stress can cause neurobiological and behavioral defects in offspring; environmental factors play a crucial role in regulating the development of brain and behavioral; this study was designed to test and verify whether an enriched environment can repair learning and memory impairment in offspring rats induced by prenatal stress and to explore its mechanism involving the expression of insulin-like growth factor-2 (IGF-2) and activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus of the offspring. METHODS: Rats were selected to establish a chronic unpredictable mild stress (CUMS) model during pregnancy. Offspring were weaned on 21st day and housed under either standard or an enriched environment. The learning and memory ability were tested using Morris water maze and Y-maze. The expression of IGF-2 and Arc mRNA and protein were respectively measured by using RT-PCR and Western blotting. RESULTS: There was an elevation in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy. Maternal stress's offspring exposed to an enriched environment could decrease their plasma corticosterone level and improve their weight. The offspring of maternal stress during pregnancy exhibited abnormalities in Morris water maze and Y-maze, which were improved in an enriched environment. The expression of IGF-2, Arc mRNA, and protein in offspring of maternal stress during pregnancy was boosted and some relationships existed between these parameters after being exposed enriched environment. CONCLUSIONS: The learning and memory impairment in offspring of prenatal stress can be rectified by the enriched environment, the mechanism of which is related to the decreasing plasma corticosterone and increasing hippocampal IGF-2 and Arc of offspring rats following maternal chronic stress during pregnancy.

Therapeutic potential of a novel prodrug of green tea extract in induction of apoptosis via ERK/JNK and Akt signaling pathway in human endometrial cancer.

BACKGROUND: Previous studies have shown a major green tea polyphenol (-)-epigallocatechin-3-gallate ((-)-EGCG) as a powerful anti-cancer agent. However, its poor bioavailability and requirement of a high dosage to manifest activity have restricted its clinical application. Recently, our team synthesized a peracetate-protected derivative of EGCG, which can act as a prodrug of (-)-EGCG (ProEGCG) with enhanced stability and improved bioavailability in vitro and in vivo. Herein, we tested the therapeutic efficacy of this novel ProEGCG, in comparison to EGCG, toward human endometrial cancer (EC). METHODS: In this study, the effects of ProEGCG and EGCG treatments on cell growth, cell survival and modulation of intracellular signaling pathways in RL95-2 and AN3 CA EC cells were compared. The antiproliferative effect was evaluated by cell viability assay. Apoptosis was measured by annexin/propidium iodide staining. Expression of mitogen-activated protein kinases, markers of proliferation and apoptosis were measured by immunoblot analysis. In addition, the effects of ProEGCG and EGCG on tumor growth, vessel formation and gene expression profiles on xenograft models of the EC cells were investigated. RESULTS: We found that treatment with ProEGCG, but not EGCG, inhibited, in a time- and dose-dependent manner, the proliferation and increased apoptosis of EC cells. Treatment with low-dose ProEGCG significantly enhanced phosphorylation of JNK and p38 MAPK and inhibited phosphorylation of Akt and ERK which are critical mediators of apoptosis. ProEGCG, but not EGCG, elicited a significant decrease in the growth of the EC xenografts, promoted apoptotic activity of tumour cells in the EC xenografts, and decreased microvessel formation, by differentially suppressing anti-apoptotic molecules, NOD1 and NAIP. Notably, no obvious adverse effects were detected. CONCLUSIONS: Taken together, ProEGCG at a low dose exhibited anticancer activity in EC cells through its anti-proliferative, pro-apoptotic and anti-tumor actions on endometrial cancer in vitro and in vivo. In contrast, a low dose of EGCG did not bring about similar effects. Importantly, our data demonstrated the efficacy and safety of ProEGCG which manifests the potential of a novel anticancer agent for the management of endometrial cancer.

MAP30 protein from Momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis.

Increasing evidence shows that Traditional Chinese Medicine (TCM) has an obvious appeal for cancer treatment, but there is still a lack of scientific investigation of its underlying molecular mechanisms. Bitter melon or bitter gourd (Momordica charantia) is an edible fruit that is commonly consumed, and it is used to cure different diseases in various ancient folk medical practices. We report that a bioactive protein, MAP30, isolated from bitter melon seeds exhibited potent anticancer and anti-chemoresistant effects on ovarian cancer cells. Functional studies revealed that MAP30 inhibited cancer cell migration, cell invasion, and cell proliferation in various ovarian cancer cells but not normal immortalized ovarian epithelial cells. When administered with cisplatin, MAP30 produced a synergistic effect on cisplatin-induced cell cytotoxicity in ovarian cancer cells. When low doses of cisplatin and MAP30 were co-injected intraperitoneally, a remarkable reduction of tumor dissemination and tumor growth was observed in an ovarian cancer ascites mouse model. Notably, blood tests confirmed that MAP30 did not cause any adverse effects on liver and kidney functions in the treated mice. MAP30 activated AMP-activated protein kinase (AMPK) signaling via CaMKKß and induced cell cycle arrest in the S-phase. MAP30 modulated cell metabolism of ovarian cancer cells via suppression of GLUT-1/-3-mediated glucose uptake, adipogenesis, and lipid droplet formation in tumor development and progression. MAP30 also induced an increase in intracellular Ca2+ ion concentration, which triggered ROS-mediated cancer cell death via apoptosis and ferroptosis. Collectively, these findings suggest that natural MAP30 is a non-toxic supplement that may enhance chemotherapeutic outcomes and benefit ovarian cancer patients with peritoneal metastases.

Mushroom extracts and compounds with suppressive action on breast cancer: evidence from studies using cultured cancer cells, tumor-bearing animals, and clinical trials.

This article reviews mushrooms with anti-breast cancer activity. The mushrooms covered which are better known include the following: button mushroom Agaricus bisporus, Brazilian mushroom Agaricus blazei, Amauroderma rugosum, stout camphor fungus Antrodia camphorata, Jew's ear (black) fungus or black wood ear fungus Auricularia auricula-judae, reishi mushroom or Lingzhi Ganoderma lucidum, Ganoderma sinense, maitake mushroom or sheep's head mushroom Grifola frondosa, lion's mane mushroom or monkey head mushroom Hericium erinaceum, brown beech mushroom Hypsizigus marmoreus, sulfur polypore mushroom Laetiporus sulphureus, Lentinula edodes (shiitake mushroom), Phellinus linteus (Japanese "meshimakobu," Chinese "song gen," Korean "sanghwang," American "black hoof mushroom"), abalone mushroom Pleurotus abalonus, king oyster mushroom Pleurotus eryngii, oyster mushroom Pleurotus ostreatus, tuckahoe or Fu Ling Poria cocos, and split gill mushroom Schizophyllum commune. Antineoplastic effectiveness in human clinical trials and mechanism of anticancer action have been reported for Antrodia camphorata, Cordyceps sinensis, Coriolus versicolor, Ganoderma lucidum, Grifola frondosa, and Lentinula edodes.

New ribosome-inactivating proteins and other proteins with protein synthesis-inhibiting activities.

Ribosome-inactivating proteins (RIPs) consist of three varieties. Type 1 RIPs are single-chained and approximately 30-kDa in molecular weight. Type 2 RIPs are double-chained and composed of a type 1 RIP chain and a lectin chain. Type III RIPs, such as maize b-32 barley and JIP60 which are produced as single-domain proenzymes, possess an N-terminal domain corresponding to the A domain of RIPs and fused to a C-terminal domain. In addition to the aforementioned three types of RIPs originating from flowering plants, there are recently discovered proteins and peptides with ribosome-inactivating and protein synthesis inhibitory activities but which are endowed with characteristics such as molecular weights distinctive from those of the regular RIPs. These new/unusual RIPs discussed in the present review encompass metazoan RIPs from Anopheles and Culex mosquitos, antimicrobial peptides derived from RIP of the pokeweed Phytolacca dioica, maize RIP (a type III RIP derived from a precursor form), RIPs from the garden pea and the kelp. In addition, RIPs with a molecular weight smaller than those of regular type 1 RIPs are produced by plants in the Cucurbitaceae family including the bitter gourd, bottle gourd, sponge gourd, ridge gourd, wax gourd, hairy gourd, pumpkin, and Chinese cucumber. A small type II RIP from camphor tree (Cinnamomum camphora) seeds and a snake gourd type II RIP with its catalytic chain cleaved into two have been reported. RIPs produced from mushrooms including the golden needle mushroom, king tuber mushroom, straw mushroom, and puffball mushroom are also discussed in addition to a type II RIP from the mushroom Polyporus umbellatus. Bacterial (Spiroplasma) RIPs associated with the fruitfly, Shiga toxin, and Streptomyces coelicolor RIP are also dealt with. The aforementioned proteins display a diversity of molecular weights, amino acid sequences, and mechanisms of action. Some of them are endowed with exploitable antipathogenic activities.

LncRNAs with miRNAs in regulation of gastric, liver, and colorectal cancers: updates in recent years.

Long noncoding RNA (lncRNA) is a kind of RNAi molecule composed of hundreds to thousands of nucleotides. There are several major types of functional lncRNAs which participate in some important cellular pathways. LncRNA-RNA interaction controls mRNA translation and degradation or serves as a microRNA (miRNA) sponge for silencing. LncRNA-protein interaction regulates protein activity in transcriptional activation and silencing. LncRNA guide, decoy, and scaffold regulate transcription regulators of enhancer or repressor region of the coding genes for alteration of expression. LncRNA plays a role in cellular responses including the following activities: regulation of chromatin structural modification and gene expression for epigenetic and cell function control, promotion of hematopoiesis and maturation of immunity, cell programming in stem cell and somatic cell development, modulation of pathogen infection, switching glycolysis and lipid metabolism, and initiation of autoimmune diseases. LncRNA, together with miRNA, are considered the critical elements in cancer development. It has been demonstrated that tumorigenesis could be driven by homeostatic imbalance of lncRNA/miRNA/cancer regulatory factors resulting in biochemical and physiological alterations inside the cells. Cancer-driven lncRNAs with other cellular RNAs, epigenetic modulators, or protein effectors may change gene expression level and affect the viability, immortality, and motility of the cells that facilitate cancer cell cycle rearrangement, angiogenesis, proliferation, and metastasis. Molecular medicine will be the future trend for development. LncRNA/miRNA could be one of the potential candidates in this category. Continuous studies in lncRNA functional discrepancy between cancer cells and normal cells and regional and rational genetic differences of lncRNA profiles are critical for clinical research which is beneficial for clinical practice.

High-throughput amplicon sequencing demonstrates extensive diversity of xylanase genes in the sediment of soda lake Dabusu.

OBJECTIVE: To explore the diversity of glycoside hydrolase family 10 xylanase genes in the sediment of soda lake Dabusu by using high-throughput amplicon sequencing based on the Illumina HiSeq2500 platform. RESULTS: A total of 227,420 clean reads, representing approximately 49.5 M bp, were obtained. Operational taxonomic unit (OTU) classification, with a 95% sequence identity cut-off, resulted in 467 OTUs with 392 annotated as GH10 xylanase, exhibiting 35-99% protein sequence identity with their closest-related xylanases in GenBank. Above 75% of the total OTUs demonstrated less than 80% identity with known xylanases. In addition, xylanases derived from the sediment were found to be affiliated to 12 different phyla, with Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, and Basidiomycota being the dominant phyla. Moreover, barcode sequence had a major effect on abundance with only a minor effect on diversity. CONCLUSIONS: High-throughput amplicon sequencing offers insight into xylanase gene diversity at a substantially higher resolution and lesser cost than library cloning and Sanger sequencing, facilitating a more thorough understanding of xylanase distribution and ecology.

Antimicrobial Activity of Chimera Peptides Composed of Human Neutrophil Peptide 1 (HNP-1) Truncated Analogues and Bovine Lactoferrampin.

Three chimera peptides composed of bovine lactoferrampin and the analogue of truncated human neutrophil peptide 1 were synthesized by the solid-phase method. In two compounds peptide chains were connected via isopeptide bond, whereas in the third one disulfide bridge served as a linker. All three chimeras displayed significantly higher antimicrobial activity than the constituent peptides as well as their equimolar mixtures. The one with a disulfide bridge displayed selectivity toward Gram-positive bacteria and was able to penetrate bacterial cells. The chimeric peptides demonstrated low in vitro mammalian cytotoxicity, especially against benign cells. The significance of linker type was also reflected in the secondary structure and proteolytic stability of studied compounds. Presented results proved that such chimeras are good lead structures for designing antimicrobial drugs.

Hydrolysis of oligosaccharides by a fungal α-galactosidase from fruiting bodies of a wild mushroom Leucopaxillus tricolor.

A novel acidic α-galactosidase (EC 3.2.1.22) designated as Leucopaxillus tricolor α-galactosidase (LTG) has been purified to homogeneity from the fruiting bodies of L. tricolor to 855-fold with a specific activity of 956 U mg-1 by the application of chromatography and gel filtration. The molecular mass of LTG was estimated to be 60 kDa as determined by both SDS-PAGE and by gel filtration. The purified enzyme was identified by LC-MS/MS and four inner amino acid sequences were obtained. When 4-nitrophenyl α-D-glucopyranoside (pNPGal) was used as substrate, the optimal pH and optimal temperature of LTG were pH 5.0 and 50 °C, respectively. The enzyme activity was strongly inhibited by Hg2+ , Fe3 , Cu2+ , Cd2+ , and Mn2+ ions. The chemical modification agent N-bromosuccinimide (NBS) completely inhibited the enzyme activity of LTG, indicating the paramount importance of tryptophan residue(s) to its enzymatic activity. Besides, LTG displayed wide substrate diversity with activity toward a variety of substrates such as stachyose, raffinose, melibiose, locust bean gum, and guar gum. Given the good ability of degrading the non-digestible and flatulence-causing oligosaccharides, this fungus may become a useful source of α-galactosidase for multiple applications.

A hemagglutinin isolated from Northeast China black beans induced mitochondrial dysfunction and apoptosis in colorectal cancer cells.

Incidence of colorectal cancer is closely related with the lifestyle, especially the dietary habits of patients. Epidemiological researches have demonstrated a negative correlation between legume consumption and colorectal cancer incidence. Lectins/hemagglutinins are a type of carbohydrate binding proteins which are abundantly stored in legumes. Their eminent pH-stability allows them to survive digestion and remain active in the intestine where they may have direct contact with colorectal tumors. It is therefore interesting to explore the direct interaction between lectins/hemagglutinins and colorectal cancer. In the present research, we reported a detailed research on the interaction between a hemagglutinin isolated from an edible legume with two colorectal cancer cell lines. This hemagglutinin (NCBBH) was found to first bind to tumor cell membrane as early as 30min post treatment and was gradually transported inside the cytoplasm within 3h, with some of it localized in the Golgi apparatus and some in the lysosomes. After its entrance, the hemagglutinin induced aggregation of the Golgi apparatus, which in turn adversely affected the transportation of protein from endoplasmic reticulum (ER) to the Golgi apparatus, resulting in protein accumulation in ER and ER stress. The hemagglutinin-treated cells also manifested severe mitochondrial malformation and membrane depolarization, accompanied by obvious apoptosis characteristics, like chromatin condensation, phosphatidylserine exposure and caspase activation. Collectively, our results indicate that the hemaggltuinin could successfully enter the cytoplasm of colorectal cancer cells and adversely affect their growth, providing a mechanism in support of the application of edible legumes to the prevention and treatment of colorectal cancer.

CRISPR/Cas9 Technology Targeting Fas Gene Protects Mice From Concanavalin-A Induced Fulminant Hepatic Failure.

Fulminant hepatic failure is a life-threatening disease which occurs in patients without preexisting liver disease. Nowadays, there is no ideal therapeutic tool in the treatment of fulminant hepatic failure. Recent studies suggested that a novel technology termed CRISPR/Cas9 may be a promising approach for the treatment of fulminant hepatic failure. In this project, we have designed single chimeric guide RNAs specifically targeting the genomic regions of mouse Fas gene. The in vitro and in vivo effects of sgRNAs on the production of Fas protein were examined in cultured mouse cells and in a hydrodynamic injection-based mouse model, respectively. The in vivo delivery of CRISPR/Cas9 could maintain liver homeostasis and protect hepatocytes from Fas-mediated cell apoptosis in the fulminant hepatic failure model. Our study indicates the clinical potential of developing the CRISPR/Cas9 system as a novel therapeutic strategy to rescue Concanavalin-A-induced fulminant hepatic failure in the mouse model. J. Cell. Biochem. 118: 530-536, 2017. © 2016 Wiley Periodicals, Inc.

The medicinal and pharmaceutical importance of Dendrobium species.

Plants of the Dendrobium genus, one of the largest in the Orchidaceae, manifest a diversity of medicinal effects encompassing antiangiogenic, immunomodulating, antidiabetic, cataractogenesis-inhibiting, neuroprotective, hepatoprotective, anti-inflammatory, antiplatelet aggregation, antifungal, antibacterial, antiherpetic, antimalarial, aquaporin-5 stimulating, and hemagglutininating activities and also exert beneficial actions on colonic health and alleviate symptoms of hyperthyroidism. The active principles include a wide range of proteinaceous and non-proteinaceous molecules. This mini-review discusses the latest advances in what is known about the medicinal and pharmaceutical properties of members of the Dendrobium genus and explores how biotechnology can serve as a conduit to mass propagate valuable germplasm for sustainable exploration for the pharmaceutical industry.

Therapeutic potentials of short interfering RNAs.

Short interfering RNA (siRNA) is one of the members of the family of RNA interference (RNAi). Coupled with the RNA-induced silencing complex (RISC), siRNA is able to trigger the cleavage of target RNAs which serve as a defensive system against pathogens. Meanwhile, siRNA in gene silencing opens a new avenue for the treatment of various diseases. SiRNA can effectively inhibit viral infection and replication and suppress tumorigenesis and various inflammation-associated diseases and cardiovascular diseases by inactivation of viral genes and downregulation of oncogene expression. Recently, endogenous siRNAs (endo-siRNAs) were discovered in the reproductive cells of animals which may be associated with regulation of cell division. Structural modification of siRNA enhances the delivery, specificity and efficacy and bioavailability to the target cells. There are at least five categories of siRNA delivery systems including viral vectors, lipid-based nanoparticles, peptide-based nanoparticles, polymer-based nanoparticles and inorganic small molecules like metal ions, silica and carbon. Sufficient preclinical and clinical studies supported that siRNA may be a potential medicine for targeted therapy of various diseases in the near future.

Biochemical characteristics of a novel protease from the basidiomycete Amanita virgineoides.

The characterization of a novel protease from Amanita virgineoides is described. The A. virgineoides protease was purified to homogeneity using Q-Sepharose, carboxymethyl-cellulose, diethylaminoethyl-cellulose, and a gel filtration step on Superdex 75. The molecular mass of the purified protease was estimated to be 16.6 kDa. The protease was purified 32.1-fold, and its specific activity was 301.4 U/mg. The optimum pH was 4.0, and the optimum temperature was 50 °C. Kinetic constants (Km , Vmax ) were determined under the optimum reaction conditions, with Km and Vmax , being 3.74 mg/mL and 9.98 µg mL-1 Min-1 , respectively. The activity of the protease was curtailed by Cu2+ , Pb2+ , Fe3+ , Cd2+ , and Hg2+ ions but enhanced by Mg2+ , Ca2+ , and K+ ions at low concentrations. The protease activity was adversely affected by ethylene diamine tetraacetic acid, suggesting that it is a metalloprotease. Four peptide sequences were obtained from liquid chromatography-tandem mass spectrometry, including KQALSGIR, TIAMDGTEGLVR, VALTGLTVAEYFR, and AGAGSATLSMAYAGAR, which showed 86%, 64%, 60%, and 75% identity with peptides of Hypsizygus marmoreus, Dacryopinax sp. DJM-731 SS1, Trametes versicolor FP-101664 SS1, and Paxillus involutus ATCC 200175, respectively. The newly isolated protease showed good hydrolytic activity and biochemical characteristics, which expanded the knowledge of biologically active proteins and provided further insight on this poisonous fungus.

Characterization of Polysaccharides with Antioxidant and Hepatoprotective Activities from the Edible Mushroom Oudemansiella radicata.

The preliminary structure, in vitro antioxidant and in vivo hepatoprotective activities of water-soluble polysaccharides (ORWP) and alkali-soluble polysaccharides (ORAP), prepared from the mushroom Oudemansiella radicata, were investigated. Both ORWP and ORAP were heteropolysaccharides with mannose, glucose and galactose being the main monosaccharide components. Regarding the antioxidant activities, ORWP and ORAP showed effective 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, hydrogen peroxide scavenging activity and lipid peroxidation inhibitory effects, as well as moderate reducing power and Fe2+ chelating activity. For the hepatoprotective activity, administration of ORWP and ORAP prevented the increase in serum alanine aminotransferase and aspartate aminotransferase activities in a carbon tetrachloride-induced acute liver damage model, suppressed hepatic malondialdehyde formation and stimulated the activities of hepatic superoxide dismutase and glutathione peroxidase. Thus, we speculate that ORWP and ORAP may protect the liver from CCl4-induced hepatic damage via antioxidant mechanisms.

Isolation and Characterization of a Phaseolus vulgaris Trypsin Inhibitor with Antiproliferative Activity on Leukemia and Lymphoma Cells.

A 17.5-kDa trypsin inhibitor was purified from Phaseolus vulgaris cv. "gold bean" with an isolation protocol including ion exchange chromatography on DEAE-cellulose (Diethylaminoethyl-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-sepharose (Sulfopropyl-sepharose), and gel filtration by FPLC (Fast protein liquid chromatography) on Superdex 75. It dose-dependently inhibited trypsin with an IC50 value of 0.4 µM, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. It inhibited [methyl-³H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 2.3 µM and 2.5 µM, respectively. The inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 µM.