Ganoderma lucidum polysaccharide peptide alleviates hyperuricemia by regulating adenosine deaminase and urate transporters.

Hyperuricemia (HUA) affects human health and is involved in the pathogenesis of common chronic diseases. Previous studies showed that Ganoderma lucidum extract lowered HUA in animals. However, the active ingredient and pharmacological mechanism of Ganoderma lucidum extract in the improvement of HUA are unknown. The purpose of this study was to determine the anti-HUA efficacy and related mechanism of Ganoderma lucidum polysaccharide peptide (GLPP) using a potassium oxonate (PO)-induced mouse model and an adenosine-induced cell model. The experimental results showed that blood uric acid (UA) was decreased up to 40.6% by GLPP in HUA mice in a dose-dependent manner. Additionally, GLPP significantly reduced UA production by inhibiting the hepatic and blood adenosine deaminase (ADA) activity and increased UA excretion by decreasing the expression of glucose transporter 9 (GLUT9) and increasing the expression of organic anion transporter 1 (OAT1) in kidney. The adenosine-induced cell model showed that the inhibitory effect of GLPP on ADA activity may be the main reason for the alleviation of HUA by GLPP. Furthermore, PO-induced renal histopathological damage was also alleviated by GLPP in a dose-dependent manner. The experimental results in this study indicated that GLPP exerted anti-HUA effects via regulating the UA production and excretion, suggesting that GLPP could be developed into a therapeutic agent for HUA.

Low-field and variable-field NMR relaxation studies of H2O and D2O molecular dynamics in articular cartilage.

Osteoarthritis (OA) as the main degenerative disease of articular cartilage in joints is accompanied by structural and compositional changes in the tissue. Degeneration is a consequence of a reduction of the amount of macromolecules, the so-called proteoglycans, and of a corresponding increase in water content, both leading to structural weakening of cartilage. NMR investigations of cartilage generally address only the relaxation properties of water. In this study, two-dimensional (T1-T2) measurements of bovine articular cartilage samples were carried out for different stages of hydration, complemented by molecular exchange with D2O and treatment by trypsin which simulates degeneration by OA. Two signal components were identified in all measurements, characterized by very different T2 which suggests liquid-like and solid-like dynamics. These measurements allow the quantification of separate hydrogen components and their assignment to defined physical pools which had been discussed repeatedly in the literature, i.e. bulk-like water and a combination of protein hydrogens and strongly bound water. The first determination of 2H relaxation dispersion in comparison to 1H dispersion suggests intramolecular interactions as the dominating source for the pronounced magnetic field dependence of the longitudinal relaxation time T1.

Isolation, characterization and HepG-2 inhibition of a novel proteoglycan from Flammulina velutipes.

Flammulina velutipes has anti-inflammatory, immunomodulatory, antioxidant and many bioactive properties with high contents of carbohydrate, proteins and fibers. In this study, a novel proteoglycan with polysaccharide complexes and protein chain, named PGD1-1, was isolated from F. velutipes. The structural characteristics of PGD1-1 were then determined, and its anti-proliferation and pro-apoptotic activities against HepG-2 cells were demonstrated in vitro. Results proved that the average molecular weight of PGD1-1 was 32.71 kDa, and the carbohydrate and protein contents were 93.35 and 2.33%, respectively. The protein moiety was bonded to a polysaccharide chain via O-glycosidic linkage. The monosaccharides consisted of d-glucose, D-galactose and D-xylose in a molar ratio of 21.90:2.84:1.00. PGD1-1 significantly inhibited the proliferation of HepG-2 cells by affecting cell lipid peroxidation and nitric oxide production. In addition, PGD1-1 promoted the apoptosis of HepG-2 cells, especially the early apoptosis. These findings proved that PGD1-1 was a novel potent ingredient against the proliferation of HepG-2, which will provide a theoretical basis for the development and utilization of the functional ingredients of the F. velutipes.

Modulatory properties of extracellular matrix glycosaminoglycans and proteoglycans on neural stem cells behavior: Highlights on regenerative potential and bioactivity.

Despite the poor regenerative capacity of the adult central nervous system (CNS) in mammals, two distinct regions, subventricular zone (SVZ) and the subgranular zone (SGZ), continue to generate new functional neurons throughout life which integrate into the pre-existing neuronal circuitry. This process is not fixed but highly modulated, revealing many intrinsic and extrinsic mechanisms by which this performance can be optimized for a given environment. The capacity for self-renewal, proliferation, migration, and multi-lineage potency of neural stem cells (NSCs) underlines the necessity of controlling stem cell fate. In this context, the native and local microenvironment plays a critical role, and the application of this highly organized architecture in the CNS has been considered as a fundamental concept in the generation of new effective therapeutic strategies in tissue engineering approaches. The brain extracellular matrix (ECM) is composed of biomacromolecules, including glycosaminoglycans, proteoglycans, and glycoproteins that provide various biological actions through biophysical and biochemical signaling pathways. Herein, we review predominantly the structure and function of the mentioned ECM composition and their regulatory impact on multiple and diversity of biological functions, including neural regeneration, survival, migration, differentiation, and final destiny of NSCs.

Purification and antioxidant and anti-Inflammatory activity of extracellular polysaccharopeptide from sanghuang mushroom, Sanghuangporus lonicericola.

BACKGROUND: Sanghuang mushrooms are medicinal fungi widely used in eastern Asia. In this study, the antioxidant and anti-inflammatory activity of a novel extracellular polysaccharopeptide, sanghuang extracellular polysaccharopeptide (SePSP) was investigated. The SePSP was purified from the submerged fermentation broth of a sanghuang mycelium, Sanghuangporus lonicericola strain CBS17, which was isolated from a wild sanghuang fruiting body. RESULTS: The SePSP was extracted using an ethanol precipitation procedure, followed by diethylaminoethanol (DEAE) anion-exchange and size-exclusion chromatography. The mass ratio of the polysaccharide and peptide components in the purified SePSP was approximately 4.87:1. By determining its free radical scavenging abilities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), the hydroxyl free radical, and the superoxide anion free radical, as well as its total reducing power, SePSP was shown to have strong concentration-dependent antioxidant activity in vitro. Further, SePSP effectively alleviated dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in mice. Administration of 200 mg kg-1 SePSP by gavage for 7 days prevented body weight loss; significantly reduced the mRNA levels of proinflammatory cytokines, including TNF-α and IL-1ß; increased mRNA level of the anti-inflammatory cytokine IL-10 in the colon, and decreased the malondialdehyde concentration from 6.42 to 4.82 µmol L-1 in the blood in UC mice. CONCLUSION: The SePSP had strong concentration-dependent antioxidant activity in vitro and effectively alleviated DSS-induced UC in mice. The in vivo therapeutic efficacy in DSS-induced UC may be mediated by modulating the expression of inflammatory cytokines and inhibiting oxidative stress. The findings provide a scientific rationale for the use of bioactive nutraceuticals from sanghuang mushrooms to develop functional foods for the prevention and treatment of UC. © 2020 Society of Chemical Industry.

Proteoglycan from Bacillus sp. BS11 Inhibits the Inflammatory Response by Suppressing the MAPK and NF-κB Pathways in Lipopolysaccharide-Induced RAW264.7 Macrophages.

Inflammation is involved in the pathogenesis of many debilitating diseases. Proteoglycan isolated from marine Bacillus sp. BS11 (EPS11) was shown to have anticancer activity, but its anti-inflammatory potential remains elusive. In the present study, the anti-inflammatory effects and mechanism of EPS11 were evaluated using a lipopolysaccharide (LPS)-induced RAW264.7 macrophage model. Biochemical characterization showed that the total sugar content and protein content of EPS11 were 49.5% and 30.2% respectively. EPS11 was composed of mannose, glucosamine, galactosamine, glucose, galactose, rhamnose, and glucuronic acid. Its molecular weight was determined to be 3.06 × 105 Da. The protein determination of EPS11 was also performed. EPS11 displayed a strong anti-inflammatory effect on LPS-stimulated RAW264.7 macrophages in vitro, which significantly suppressed inflammatory cytokines and mediators (such as NO, TNF-α, IL-6 and IL-1ß, and COX-2). Western blot analysis indicated that EPS11 could downregulate the expression of many key proteins in mitogen-activated protein kinases (MAPKs) and transcription factor nuclear factor-κB (NF-κB) signaling pathways. In particular, EPS11 almost completely inhibited the expression of NF-κB P65, which indicated that EPS11 acted primarily on the NF-κB pathways. These findings offer new insights into the molecular mechanism underlying the anti-inflammatory effect of EPS11.

A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins.

Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).

Embryonic proteoglycans regulate monoamines in the rat frontal cortex and hippocampus in Alzheimer's disease-like pathology.

Using the rat Alzheimer's disease (AD)-like model we have analyzed the hippocampal short-term potentiation, levels of monoamines, and morphological changes in the hippocampal and cortical neurons after the administration of proteoglycans of embryonic origin (PEG). Results showed that the levels of monoamines and especially norepinephrine in the target AD brain structures were found elevated, except serotonin, which was unaffected in the hippocampus, but decreased in the frontal cortex. These changes were accompanied by the substantial structural damage of cortical and hippocampal neurons. PEG was able to reverse most of these changes. In addition, PEG administration had regime-dependent effects on a short-term potentiation pattern of hippocampal neurons. The elevated levels of key elements of brain monoaminergic system in the model of AD support the hypothesis of the important role of monoamines in the excessive synaptic excitation resulting in cognitive dysfunction in AD brain. The neuroprotective effect of PEG, as manifested by the recovery of the monoaminergic system, suggests this bioactive substance as a perspective therapeutic agent for the treatment of AD.

A proteoglycan extract from Ganoderma Lucidum protects pancreatic beta-cells against STZ-induced apoptosis.

The pancreatic ß-cell death or dysfunction induced by oxidative stress plays an important effect on the development and progression of diabetes mellitus. Based on our previous findings, a natural proteoglycan extracted from Ganoderma Lucidum, named FYGL, could treat T2DM in vivo. In this study, we investigated the effects of FYGL on STZ-induced apoptosis of INS-1 cells and its underlying mechanisms. The results showed that FYGL significantly improved the cell viability and alleviated the apoptosis in STZ-treated INS-1 cells. Moreover, FYGL markedly decreased the intracellular ROS accumulation and NO release, and deactivated NF-κB, JNK, and p38 MAPK signaling pathways in STZ-induced INS-1 cells. Furthermore, FYGL improved the insulin secretion through inhibiting the activation of JNK and improving the expression of Pdx-1 in INS-1 cells damaged by STZ. These results indicated that FYGL could protect pancreatic ß-cells against apoptosis and dysfunction, and be used as a promising pharmacological medicine for diabetes management. Abbreviations: T2DM: type 2 diabetes mellitus; FYGL: Fudan-Yueyang G. lucidum; ROS: reactive oxygen species; NO: reactive oxygen species; NF-κB: nuclear factor kappa beta; JNK: c-jun N-terminal kinase; MAPK: mitogen-activated protein kinase; Pdx-1: Pancreatic duodenal homeobox 1.

Production of a codonopsis polysaccharide iron complex and evaluation of its properties.

A water extraction and alcohol precipitation method was applied to extract polysaccharides from Codonopsis pilosula (CPP), response surface methodology was used to optimize the extraction conditions and synthesis of C. pilosula polysaccharide iron (CPPI), and the properties of CPPI were evaluated. The optimum extraction conditions for CPP were as follows: liquid-solid ratio of 29.39 mL/g, time of 1.25 h and temperature of 62.84 °C. The optimum synthesis conditions for CPPI were pH 8.9, temperature 70.30 °C and the ratio of citric acid to CPP1 of 2.95. An HPSEC-MALLS-RID system, UV spectroscopy, FT-IR spectroscopy and NMR were used for characterization of the polysaccharide. CPPI exhibited antioxidant activity in vitro and a relatively strong inhibitory effect on A2780 cells growth. After CPPI treatment, the reactive oxygen species increased, the mitochondrial membrane potential decreased, and DNA damage was observed in A2780 cells. Therefore, CPPI should be explored as a potential antioxidant and an antitumor drug in a clinical setting.

Production, partial purification and characterization of a proteoglycan bioemulsifier from an oleaginous yeast.

In this study, Meyerozyma caribbica, an indigenously isolated oleaginous yeast, produced in media containing glucose a bioemulsifier that was partially characterized as a proteoglycan based on preliminary analysis. Optimization of carbon:nitrogen (C:N) ratio revealed 30:1 as the suitable ratio for enhanced production. Apart from higher emulsification activity (E24: 70-80%), this molecule showed strong emulsion stability over a wide range of pH (2.0-9.0), salinity (0.05%-10%, w/v) and temperature (- 80 °C to + 50 °C). The current study emphasizes on the determination of critical media parameters for improved and stable bioemulsifier production coupled with partial characterization and identification of the molecule. Thus, a proteoglycan-based bioemulsifier with such a stable emulsifying property can serve as a versatile and potential component in food, cosmetics and pharmaceutical formulations.

Bioactive structural basis of proteoglycans from Sarcandra glabra based on spectrum-effect relationship.

ETHNOPHARMACOLOGICAL RELEVANCE: Proteoglycans are one of the active ingredients of great importance in Sarcandra glabra. The biological activities of proteoglycans extracted from Sarcandra glabra including suppressing tumor growth and antioxidant activity were studied. However, raw materials from different regions may cause differences in the activity of natural extracts, especially for bioactive biomacromolecules. Conventional identification of S.glabra cannot accurately reflect the distinguishing relationship between internal components and the pharmacological activity. The identification of biologically active structures was obtained by constructing multiple fingerprint and spectrum-effect relationship. AIM OF THE STUDY: To evaluate the bioactive structural basis of proteoglycans from S.glabra based on spectrum-effect relationship and chemometric methods. MATERIALS AND METHODS: Multiple fingerprinting including HPSEC, PMP-HPLC, and FT-IR of proteoglycans was established from 18 batches of samples based on the structural characteristics. Both antitumor activity and antioxidant activity were determined. Mathematical analysis was used to analyze the spectrum-effect relationship. RESULTS: PCA results showed monosaccharides including Xly, Rha, and GlcA, carboxyl group in acidic sugars, peptide bond in proteins, and methylene groups could be used as markers for distinguishing the samples from different sources. The results of the spectrum-effect relationship analysis indicated that the bioactive markers of inhibitory activity on MG63 and U2OS cells by PLS-DA were related to GlcA, Xyl, Fuc, ß-glycosidic bonds, peptide linkage, and methylene groups. Markers composing monosaccharide for antioxidant activity were Xyl, GlcA, and GlcN. Meanwhile, the group markers were pyranose ring, carboxyl group, peptide linkage, and methylene structure. CONCLUSIONS: The material basis that affects the pharmacological efficacy could be found according to the spectrum-effect relationship analysis. This study could lay a foundation for further exploring the relationship between structural characteristics and pharmacodynamics of macromolecular glycoconjugates in Traditional Chinese Medicine.

Mechanism of atopic cataract caused by eosinophil granule major basic protein.

Atopic cataracts develop under the ages of 40 years, after which visual acuity rapidly declines. However, the mechanism underlying the development of atopic cataracts is not yet clear. We focused on the eosinophil granule major basic protein (MBP), which was detected in the aqueous humor of atopic cataracts previously, and which was cytotoxic. Specifically, we investigated its origin in this fluid and its effects on lens epithelial cells (LECs). MBP immunostaining was positive in atopic cataract-derived LECs, but negative in age-related cataract-derived LECs. MBP mRNA was not detected in either type of cataract, but protein was detected in the aqueous humor. Furthermore, the flare values associated with atopic cataracts were higher than those with age-related cataracts. When MBP was purified from eosinophils or recombinant MBP was added to LEC culture medium, cell viability decreased in a concentration-dependent manner, but an MBP antibody neutralized the cytotoxic effect of this protein towards these cells. These results were consistent with the flow of MBP into the aqueous humor from the blood due to a compromised blood-aqueous barrier. Thus, MBP could further penetrate the lens capsule and adhere to LECs, resulting in decreased cell viability and the development of atopic cataracts.

Proteoglycan isolated from Corbicula fluminea exerts hepato-protective effects against alcohol-induced liver injury in mice.

Corbicula fluminea (Asian clam), a freshwater bivalve mollusk, has been consumed in China for centuries as a health food and traditional Chinese medicine for treating liver diseases and alcoholism. This study aimed to evaluate the hepato-protective effects and potential mechanisms of a proteoglycan (PSP) from C. fluminea on alcohol-induced liver injury in mice. Results showed that PSP pretreatment significantly antagonized the increases in serum alanine aminotransferase, aspartate aminotransferase, triacylglycerides, and hepatic malondialdehyde levels; elevated the antioxidant enzyme activities and hepatic glutathione levels; and suppressed the levels of hepatic inflammatory cytokines in alcohol-induced liver injury in mice (P < 0.05). Histopathological observation further revealed the potential hepato-protective effect of PSP against alcohol damage. Particularly, PSP pretreatment resulted in significantly decreased expression of cytochrome P450 2e1 (CYP2E1) while significantly upregulating the expression of hemeoxygenase-1 (HO-1) (P < 0.05). These results suggested that PSP could protect the liver from hepatocyte injury induced by alcohol possibly by alleviating hepatic lipid metabolism, elevating antioxidant-enzyme activity, suppressing the immune inflammatory response, and reversing the expression levels of CYP2E1 and HO-1. Therefore, PSP may be developed as a food supplement that can be used to prevent liver diseases.

Polysaccharide peptide (PsP) Ganoderma lucidum: a potential inducer for vascular repair in type 2 diabetes mellitus model.

INTRODUCTION: The increasing blood glucose level due to insulin resistance which occurs in diabetes mellitus (DM) may cause vascular damage. This study aims to prove the effect of the polysaccharide peptide (PsP) Ganoderma lucidum on improving vascular damage through an increase of circulating endothelial cells and circulating endothelial cells (CEC) ratio, decreased H2O2, triglyceride (TG), total cholesterol (TC) and insulin resistance in type 2 DM. METHODS: Our study is a true experimental study with randomized posttest control group design that used 35 Wistar rats divided into five groups: normal, control (+) and three groups of different variant PsP doses 50, 150 and 300 mg/kg BW (n=7). RESULTS: By using one-way ANOVA and post-hoc Duncan test, the results show a significant increase of endothelial progenitor cell (EPC) concentration (p=0.000) and ratio EPC:CEC (0.000) by dose-dependent fashion and also reduced CEC concentration (p=0.001), H2O2 (p=0.03), TG (p=0.001), TC (p=0.01) and insulin resistance (p=0.003). CONCLUSION: In this study, PsP induced endothelial repairing process and reduced the risk factor with 300 mg/kg BW as optimum dose. However, further research on EPC and CEC detection markers is important. Further research on PsP and clinical trial for commercial uses is also needed.

Endocan regulates acute lung inflammation through control of leukocyte diapedesis.

Acute respiratory distress syndrome is a severe form of respiratory failure, occurring in up to 20% of patients admitted to the intensive care unit with sepsis. Dysregulated leukocyte diapedesis is a major contributor to acute respiratory distress syndrome. Endocan is a circulating proteoglycan that binds to the leukocyte integrin leukocyte functional antigen-1 and blocks its interaction with its endothelial ligand, ICAM-1. The objective of this study was to evaluate the role of endocan in the control of acute lung inflammation. In vitro, endocan inhibited human leukocyte transendothelial migration as well as ICAM-1-dependent migration but had a very mild effect on ICAM-1-dependent adhesion. Endocan also acted as an inhibitor of transendothelial migration of mouse leukocytes. The effect of systemic administration of recombinant human endocan was assessed in a model of acute lung inflammation in BALB/c mice. Treatment with endocan 1 h after intratracheal LPS challenge reduced the alveolar inflammatory response, diminished histological features of acute lung injury, and improved respiratory function. These results highlight the anti-inflammatory role of human endocan and its protective effect against acute lung injury.NEW & NOTEWORTHY We show here that endocan inhibits ICAM-1-dependent human leukocyte transendothelial migration and ICAM-1-dependent adhesion. We also found that in BALB/c mice with tracheal LPS-induced acute lung injury treatment with recombinant human endocan reduces lung inflammation, notably through reduction of neutrophilic recruitment, and restores normal lung function. These results confirm the hypothesis that human endocan may have a protective effect against acute lung inflammation.

In Depth Quantification of Extracellular Matrix Proteins from Human Pancreas.

Extracellular matrix (ECM) is an important component of the pancreatic microenvironment which regulates ß cell proliferation, differentiation, and insulin secretion. Protocols have recently been developed for the decellularization of the human pancreas to generate functional scaffolds and hydrogels. In this work, we characterized human pancreatic ECM composition before and after decellularization using isobaric dimethylated leucine (DiLeu) labeling for relative quantification of ECM proteins. A novel correction factor was employed in the study to eliminate the bias introduced during sample preparation. In comparison to the commonly employed sample preparation methods (urea and FASP) for proteomic analysis, a recently developed surfactant and chaotropic agent assisted sequential extraction/on pellet digestion (SCAD) protocol has provided an improved strategy for ECM protein extraction of human pancreatic ECM matrix. The quantitative proteomic results revealed the preservation of matrisome proteins while most of the cellular proteins were removed. This method was compared with a well-established label-free quantification (LFQ) approach which rendered similar expressions of different categories of proteins (collagens, ECM glycoproteins, proteoglycans, etc.). The distinct expression of ECM proteins was quantified comparing adult and fetal pancreas ECM, shedding light on the correlation between matrix composition and postnatal ß cell maturation. Despite the distinct profiles of different subcategories in the native pancreas, the distribution of matrisome proteins exhibited similar trends after the decellularization process. Our method generated a large data set of matrisome proteins from a single tissue type. These results provide valuable insight into the possibilities of constructing a bioengineered pancreas. It may also facilitate better understanding of the potential roles that matrisome proteins play in postnatal ß cell maturation.

A hyaluronan-based polysaccharide peptide generated by a genetically modified Streptococcus zooepidemicus.

Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs.

A polysaccharide-peptide with mercury clearance activity from dried fruiting bodies of maitake mushroom Grifola frondosa.

Mercury is considered to be "a global pollutant" and raises concern worldwide. Once mercury enters the body, it will be distributed all over the body but will accumulate in the brain, kidney and liver. To date, no substance originating from edible fungi capable of adsorbing mercury has been reported. We found that the mushroom Grifola frondosa exhibited mercury adsorption capacity. A polysaccharide-peptide (GFPP), displaying the unique N-terminal amino acid sequence of APPGMHQKQQ and 7 partial sequences with high reliability obtained by LC-MS/MS, was isolated by hot-water extraction of its fruiting bodies followed by ion exchange chromatography and gel filtration chromatography. Two rat models were employed to determine the dose and the duration of HgCl2 treatment (given by acute administration or continuous treatment) to test if G. frondosa could promote mercury elimination. For rats subjected to acute treatment with HgCl2, both GFPP and G. frondosa fruiting bodies (GFFF) could accelerate the decline of blood mercury level, which fell precipitously by 50% on the second day. GFPP and GFFF also promoted elimination of the burden of mercury in the liver and kidneys. For rats receiving continuous HgCl2 treatment, G. frondosa prevented the progressive increase of blood mercury level, and kept the blood mercury level within a relatively stable range.

Proteoglycan Isolation and Analysis.

Proteoglycans can be difficult molecules to isolate and analyze due to large mass, charge, and tendency to aggregate or form macromolecular complexes. This unit describes detailed methods for purification of matrix, cell surface, and cytoskeleton-linked proteoglycans. Methods for analysis of glycoaminoglycan size and type and of core protein species are described. © 2018 by John Wiley & Sons, Inc.