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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide COVID-19 pandemic, leading to 6.8 million deaths. Numerous variants have emerged since its outbreak, resulting in its significantly enhanced ability to spread among humans. As with many other viruses, SARS‑CoV‑2 utilizes heparan sulfate (HS) glycosaminoglycan (GAG) on the surface of host cells to facilitate viral attachment and initiate cellular entry through the ACE2 receptor. Therefore, interfering with virion-HS interactions represents a promising target to develop broad-spectrum antiviral therapeutics. Sulfated glycans derived from marine organisms have been proven to be exceptional reservoirs of naturally existing HS mimetics, which exhibit remarkable therapeutic properties encompassing antiviral/microbial, antitumor, anticoagulant, and anti-inflammatory activities. In the current study, the interactions between the receptor-binding domain (RBD) of S-protein of SARS-CoV-2 (both WT and XBB.1.5 variants) and heparin were applied to assess the inhibitory activity of 10 marine-sourced glycans including three sulfated fucans, three fucosylated chondroitin sulfates and two fucoidans derived from sea cucumbers, sea urchin and seaweed Saccharina japonica, respectively. The inhibitory activity of these marine derived sulfated glycans on the interactions between RBD of S-protein and heparin was evaluated using Surface Plasmon Resonance (SPR). The RBDs of S-proteins from both Omicrion XBB.1.5 and wild-type (WT) were found to bind to heparin, which is a highly sulfated form of HS. All the tested marine-sourced sulfated glycans exhibited strong inhibition of WT and XBB.1.5 S-protein binding to heparin. We believe the study on the molecular interactions between S-proteins and host cell glycosaminoglycans provides valuable insight for the development of marine-sourced, glycan-based inhibitors as potential anti-SARS-CoV-2 agents. [ABSTRACT FROM AUTHOR]

Background: In cirrhotic patients, recurrent bleeding after the first episode of esophageal variceal bleeding (EVB) is common and lethal. The present study was aimed to compare balloon-compression endoscopic injection sclerotherapy (bc-EIS) with transjugular intrahepatic portosystemic shunt (TIPS) for the prophylaxis of variceal rebleeding. Methods: Between June 2020 and September 2022, 81 cirrhotic patients with EVB (42 in the bc-EIS group and 39 in the TIPS group) were evaluated retrospectively. The occurrence of rebleeding, hepatic encephalopathy (HE) or other complications, as well as liver functions and survival rate were compared between two groups. Results: During the 12 months of follow-up, variceal eradication was achieved in 40 (95.24%) patients of the bc-EIS group after a mean of 1.80 ± 0.94 sessions. TIPS was successfully performed in 39 (100%) patients. No significant difference in the variceal rebleeding rate was observed between bc-EIS and TIPS groups (16.67 vs. 17.95%; p = 0.111). While the bc-EIS group showed significantly decreased incidence of HE (2.38 vs. 17.95%; p < 0.001) and lower level of total bilirubin (p < 0.05) in comparison with the TIPS group. The difference in mortality between the two groups failed to reach statistical significance (0.00 vs. 7.69%; p = 0.107). Conclusion: Bc-EIS is not inferior to TIPS in the survival and control of variceal rebleeding, but associated with decreased risk of HE and liver dysfunction. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19. Suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on SARS-CoV-2 RBDs with preferential binding for Omicron RBD and inhibition of infection by the Omicron variant in vitro. [ABSTRACT FROM AUTHOR]

The strain energy density softening criterion is introduced to the peridynamic theory to reflect the failure characteristics of rock-like materials. At the same time, the critical damage condition, obeying Weibull distribution, is utilized to describe the heterogeneity of rock—making up for the deficiency that the peridynamic method cannot embody the strain-softening features and heterogeneity of rock when simulating crack propagation of rock materials. The crack extension process of rock, with a single crack under uniaxial compression, was simulated using the improved method, the results of which were then compared to the results of previous laboratory tests to verify the effectiveness of the proposed theory. Furthermore, the crack propagation patterns and failure mechanism of rock materials which contain single crack and double cracks with different prefabrication angles under uniaxial compression condition were explored in this study. The results showed that the propagation of a prefabricated single crack specimen was divided into wing crack, shear crack, and anti-wing crack, and the order of appearance was influenced by the inclination angle. The penetration modes of prefabricated double cracks were divided into wing mode (W-mode), shear mode (S-mode), and mixed mode (M-mode), which were primarily affected by the inclination angle and relative angle. [ABSTRACT FROM AUTHOR]

Complex carbohydrates (glycans) are major players in all organisms due to their structural, energy, and communication roles. This last essential role involves interacting and/or signaling through a plethora of glycan-binding proteins. The design and synthesis of glycans as potential drug candidates that selectively alter or perturb metabolic processes is challenging. Here we describe the first reported sulfur-linked polysaccharides with potentially altered conformational state(s) that are recalcitrant to digestion by heparanase, an enzyme important in human health and disease. An artificial sugar donor with a sulfhydryl functionality is synthesized and enzymatically incorporated into polysaccharide chains utilizing heparosan synthase. Used alone, this donor adds a single thio-sugar onto the termini of nascent chains. Surprisingly, in chain co-polymerization reactions with a second donor, this thiol-terminated heparosan also serves as an acceptor to form an unnatural thio-glycosidic bond ('S-link') between sugar residues in place of a natural 'O-linked' bond. S-linked heparan sulfate analogs are not cleaved by human heparanase. Furthermore, the analogs act as competitive inhibitors with > ~200-fold higher potency than expected; as a rationale, molecular dynamic simulations suggest that the S-link polymer conformations mimic aspects of the transition state. Our analogs form the basis for future cancer therapeutics and modulators of protein/sugar interactions. Heparin is a family of complex carbohydrates binding to proteins to modulate cell activities. Here the authors report the synthesis, and conformations simulations of S-linked hemi-A heparosan [GlcA-S-GlcNAc]n, a thio-glycosidic uncleavable polysaccharide, and test it as human heparanase inhibitor. [ABSTRACT FROM AUTHOR]

Synthesis of glycosaminoglycans, such as heparan sulfate (HS) and chondroitin sulfate (CS), occurs in the lumen of the Golgi, but the relationship between Golgi structural integrity and glycosaminoglycan synthesis is not clear. In this study, we disrupted the Golgi structure by knocking out GRASP55 and GRASP65 and determined its effect on the synthesis, sulfation, and secretion of HS and CS. We found that GRASP depletion increased HS synthesis while decreasing CS synthesis in cells, altered HS and CS sulfation, and reduced both HS and CS secretion. Using proteomics, RNA-seq and biochemical approaches, we identified EXTL3, a key enzyme in the HS synthesis pathway, whose level is upregulated in GRASP knockout cells; while GalNAcT1, an essential CS synthesis enzyme, is robustly reduced. In addition, we found that GRASP depletion decreased HS sulfation via the reduction of PAPSS2, a bifunctional enzyme in HS sulfation. Our study provides the first evidence that Golgi structural defect may significantly alter the synthesis and secretion of glycosaminoglycans. [ABSTRACT FROM AUTHOR]

Sponges are among the most primitive multicellular organisms and well-known as a major source of marine natural products. Cultivation of sponge cells has long been an attractive topic due to the prominent evolutionary and cytological significance of sponges and as a potential approach to supply sponge-derived compounds. Sponge cell culture is carried out through culturing organized cell aggregates called 'primmorphs.' Most research culturing sponge cells has used unfractionated cells to develop primmorphs. In the current study, a tropical marine sponge Axinella sp., which contains the bioactive alkaloids, debromohymenialdisine (DBH), and hymenialdisine (HD), was used to obtain fractionated cells and the corresponding primmorphs. These alkaloids, DBH and HD, reportedly show pharmacological activities for treating osteoarthritis and Alzheimer's disease. Three different cell fractions were obtained, including enriched spherulous cells, large mesohyl cells, and small epithelial cells. These cell fractions were cultivated separately, forming aggregates that later developed into different kinds of primmorphs. The three kinds of primmorphs obtained were compared as regards to appearance, morphogenesis, and cellular composition. Additionally, the amount of alkaloid in the primmorphs-culture system was examined over a 30-d culturing period. During the culturing of enriched spherulous cells and developed primmorphs, the total amount of alkaloid declined notably. In addition, the speculation of alkaloid secretion and some phenomena that occurred during cell culturing are discussed. [ABSTRACT FROM AUTHOR]

Astrocytes are major producers of the extracellular matrix (ECM), which is involved in the plasticity of the developing brain. In utero alcohol exposure alters neuronal plasticity. Glycosaminoglycans (GAGs) are a family of polysaccharides present in the extracellular space; chondroitin sulfate (CS)- and heparan sulfate (HS)-GAGs are covalently bound to core proteins to form proteoglycans (PGs). Hyaluronic acid (HA)-GAGs are not bound to core proteins. In this study we investigated the contribution of astrocytes to CS-, HS-, and HA-GAG production by comparing the makeup of these GAGs in cortical astrocyte cultures and the neonatal rat cortex. We also explored alterations induced by ethanol in GAG and core protein levels in astrocytes. Finally, we investigated the relative expression in astrocytes of CS-PGs of the lectican family of proteins, major components of the brain ECM, in vivo using translating ribosome affinity purification (TRAP) (in Aldh1l1-EGFP-Rpl10a mice. Cortical astrocytes produce low levels of HA and show low expression of genes involved in HA biosynthesis compared to the whole developing cortex. Astrocytes have high levels of chondroitin-0-sulfate (C0S)-GAGs (possibly because of a higher sulfatase enzyme expression) and HS-GAGs. Ethanol upregulates C4S-GAGs as well as brain-specific lecticans neurocan and brevican, which are highly enriched in astrocytes of the developing cortex in vivo. These results begin to elucidate the role of astrocytes in the biosynthesis of CS- HS- and HA-GAGs, and suggest that ethanol-induced alterations of neuronal development may be in part mediated by increased astrocyte GAG levels and neurocan and brevican expression. [ABSTRACT FROM AUTHOR]

In this paper we characterize the function of Xylosyltransferase 2 (XylT2) in different tissues to investigate the role XylT2 has in the proteoglycan (PG) biochemistry of multiple organs. The results show that in all organs examined there is a widespread and significant decrease in total XylT activity in Xylt2 knock out mice (Xylt2−/−). This decrease results in increased organ weight differences in lung, heart, and spleen. These findings, in addition to our previous findings of increased liver and kidney weight with loss of serum XylT activity, suggest systemic changes in organ function due to loss of XylT2 activity. The Xylt2−/− mice have splenomegaly due to enlargement of the red pulp area and enhanced pulmonary response to bacterial liposaccharide. Tissue glycosaminoglycan composition changes are also found. These results demonstrate a role of XylT2 activity in multiple organs and their PG content. Because the residual XylT activity in the Xylt2−/− is due to xylosyltransferase 1 (XylT1), these studies indicate that both XylT1 and XylT2 have important roles in PG biosynthesis and organ homeostasis. [ABSTRACT FROM AUTHOR]

Heparosan, the capsular polysaccharide of E. coli K5 is currently used as the starting material in the chemoenzymatic synthesis of heparan sulfate and the structurally related anticoagulant drug heparin. Base hydrolysis of N-acetyl groups and their subsequent N-sulfonation, are used to prepare N-sulfoheparosan an intermediate of biosynthesis. In the present study, when excess sulfonation reagent was used during N-sulfonation, some O-sulfation also took place in the N-sulfoheparosan product. After a nearly full digestion, a hexasaccharide fraction exhibited resistance to heparin lyase II. Excessive digestion by heparin lyase II and structural identification by NMR and mass spectroscopy indicated that the resistant hexasaccharide fraction has two structures, ΔUA-GlcNS-GlcA2S-GlcNS-GlcA-GlcNS and ΔUA-GlcNS-GlcA- GlcNS3S-GlcA-GlcNS in similar amounts. The 2-sulfated structure exhibited partial resistance to heparin lyase II; however the structure of ΔUA-GlcNS-GlcA-GlcNS3S was completely resistant to heparin lyase II. [ABSTRACT FROM AUTHOR]

Three polysaccharides (SH-1, SH-2 and SH-3) were purified from a brown macroalgea, Sargassum hemiphyllum. The autohydrolysis products from each polysaccharide were separated to three fractions (S fractions as oligomers, L fractions as low molecular weight polysaccharides and H fractions as high molecular weight polysaccharides). Mass spectroscopy of S fractions (SH-1-S, SH-2-S and SH-3-S) showed that these three polymers all contained short stretches of sulfated fucose. The structures of L fractions (SH-1-L, SH-2-L and SH-3-L) were determined by nuclear magnetic resonance (NMR). SH-1-L was composed of two units, unit A (sulfated galactofucan) and unit B (sulfated xylo-glucuronomannan). Unit A contained a backbone of (1, 6-linked β-D-Gal) n1, (1, 3-linked 4-sulfated α-L-Fuc) n2, (1, 3-linked 2, 4-di-sulfated α-L-Fuc) n3, (1, 4-linked α-L-Fuc) n4 and (1, 3-linked β-D-Gal) n5, accompanied by some branches, such as sulfated fuco-oligomers, sulfated galacto-oligomers or sulfated galacto-fuco-oligomers. And unit B consisted of alternating 1, 4-linked β-D-glucuronic acid (GlcA) and 1, 2-linked α-D-mannose (Man) with the Man residues randomly sulfated at C6 or branched with xylose (Xyl) at C3. Both SH-2-L and SH-3-L were composed of unit A and their difference was attributed to the ratio of n1: n2: n3: n4: n5. Based on monosaccharide analysis, we hypothesize that both SH-1-H and SH-2-H contained unit A and unit B while SH-3-H had a structure similar to SH-3-L. An assessment of anti-complement activities showed that the sulfated galactofucan had higher activities than sulfated galacto-fuco-xylo-glucuronomannan. These results suggest that the sulfated galactofucans might be a good candidate for anti-complement drugs. [ABSTRACT FROM AUTHOR]

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the − 973 base pair (bp) to − 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, "A" at the 22nd bp upstream of ATG, was determined with 5′-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system. [ABSTRACT FROM AUTHOR]

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states. Epithelial cells that line the gut secrete complex glycoproteins that form a mucus layer to protect the gut wall from enteric pathogens. Here, the authors provide a comprehensive characterisation of endo-acting glycoside hydrolases expressed by mucin-degrading members of the microbiome that are able to cleave the O-glycan chains of a range of different animal and human mucins. [ABSTRACT FROM AUTHOR]

Cell therapy for the injured spinal cord will rely on combined advances in human stem cell technologies and delivery strategies. Here we encapsulate homotypic spinal cord neural stem cells (scNSCs) in an alginate-based neural ribbon delivery platform. We perform a comprehensive in vitro analysis and qualitatively demonstrate graft survival and injury site retention using a rat C4 hemi-contusion model. Pre-configured neural ribbons are transport-stable modules that enable site-ready injection, and can support scNSC survival and retention in vivo. Neural ribbons offer multifunctionality in vitro including co-encapsulation of the injury site extracellular matrix modifier chondroitinase ABC (chABC), tested here in glial scar models, and ability of cervically-patterned scNSCs to differentiate within neural ribbons and project axons for integration with 3-D external matrices. This is the first extensive in vitro characterization of neural ribbon technology, and constitutes a plausible method for reproducible delivery, placement, and retention of viable neural cells in vivo. [ABSTRACT FROM AUTHOR]

Formaldehyde is a ubiquitous carcinogenic indoor pollutant. The treatment of formaldehyde has attracted increasing social attention. Over the past few decades, an increasing number of publications have reported approaches for removing indoor formaldehyde. These potential strategies include physical adsorption, chemical catalysis, and biodegradation. Although physical adsorption is widely used, it does not really remove pollution. Chemical catalysis is very efficient but adds the risk of introducing secondary pollutants. Biological removal strategies have attracted more research attention than the first two methods, because it is more efficient, clean, and economical. Plants and bacteria are the common organisms used in formaldehyde removal. However, both have limitations and shortcomings when used alone. This review discusses the mechanisms, applications, and improvements of existing biological methods for the removal of indoor gaseous formaldehyde. A combination strategy relying on plants, bacteria, and physical adsorbents exhibits best ability to remove formaldehyde efficiently, economically, and safely. When this combination system is integrated with a heating, ventilation, air conditioning, and cooling (HVAC) system, a practical combined system can be established in formaldehyde removal. Multivariate interactions of biological and non-biological factors are needed for the future development of indoor formaldehyde removal. Key Points: • Indoor gaseous formaldehyde removal is necessary especially for new residence. • Biological removal strategies have attracted increasing research attentions. • Combined system of plants, bacteria, and physical adsorbents exhibits best efficiency. • Integrated device of biological and non-biological factors will be potential practical. [ABSTRACT FROM AUTHOR]

Sclerostin (SOST) is a glycoprotein having many important functions in the regulation of bone formation as a key negative regulator of Wnt signaling in bone. Surface plasmon resonance (SPR), which allows for a direct quantitative analysis of the label-free molecular interactions in real-time, has been widely used for the biophysical characterization of glycosaminoglycan (GAG)-protein interactions. In the present study, we report kinetics, structural analysis and the effects of physiological conditions (e.g., salt concentrations, Ca2+ and Zn2+concentrations) on the interactions between GAGs and recombinant human (rh) and recombinant mouse (rm) SOST using SPR. SPR results revealed that both SOSTs bind heparin with high affinity (rhSOST-heparin, KD~36 nM and rmSOST-heparin, KD~77 nM) and the shortest oligosaccharide of heparin that effectively competes with full size heparin for SOST binding is octadecasaccharide (18mer). This heparin binding protein also interacts with other highly sulfated GAGs including, disulfated-dermatan sulfate and chondroitin sulfate E. In addition, liquid chromatography-mass spectrometry was used to characterize the structure of sulfated GAGs that bound to SOST. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) play an important role in stabilizing the gel state of eye vitreous humour. In this study, the composition of GAGs present in bovine eye vitreous was characterized through disaccharide analysis by liquid chromatography-mass spectrometry. The interaction of GAGs with collagen type II was assessed using surface plasmon resonance (SPR). The percentage of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS), of total GAG, were 96.2%, 3.5% and 0.3%, respectively. The disaccharide composition of CS consisted of 4S (49%), 0S (38%) 6S (12%), 2S6S (1.5%) and 2S4S (0.3%). The disaccharide composition of HS consisted of 0S (80%), NS2S (7%), NS (7%), 6S (4%), NS6S (2%), and TriS, 2S and 4S6S (each at 0.1%). The average molecular weights of CS and HS were 148 kDa and 204 kDa, respectively. SPR reveals that collagen type II binds to heparin (primarily composed of TriS) with a binding affinity (KD) of 755 nM and interacts with other GAGs, including CSB and CSE. Both bovine vitreous CS and HS interact with collagen type II, with vitreous HS showing a higher binding affinity. [ABSTRACT FROM AUTHOR]

The swim bladder of fish is an internal gas-filled organ that allows fish to control their buoyancy and swimming depth. Fish maws (the dried swim bladders of fish) have been used over many centuries as traditional medicines, tonics and a luxurious gourmet food in China and Southeast Asia. Little is known about the structural information of polysaccharides comprising this important functional material of fish tissue. In the present study, the total glycosaminoglycan (GAG) from fish maw was characterized. Two GAGs were identified, chondroitin sulfate (CS, having a molecular weight of 18-40 kDa) and heparan sulfate (HS), corresponding to 95% and 5% of the total GAG, respectively. Chondroitinase digestion showed that the major CS GAG was composed of ΔUA-1 → 3-GalNAc4S (59.7%), ΔUA-1 → 3-GalNAc4,6S (36.5%), ΔUA-1 → 3-GalNAc6S (2.2%) and ΔUA-1 → 3-GalNAc (1.6%) disaccharide units. 1H-NMR analysis and degradation with specific chondroitinases, both CS-type A/C and CS-type B were present in a ratio of 1.4:1. Analysis using surface plasmon resonance showed that fibroblast growth factor (FGF)-2 bound to the CS fraction (KD = 136 nM). These results suggest that this CS may be involved in FGF-signal pathway, mediating tissue repair, regeneration and wound healing. The CS, as the major GAG in fish maw, may have potential pharmacological activity in accelerating wound healing. [ABSTRACT FROM AUTHOR]

B Correction to: Surgical Endoscopy b https://doi.org/10.1007/s00464-023-10085-y The original online version of this article was revised to correct an error in Fig. The corrected figure places the box indicating 42 patients underwent bc-EIS on the left side of the flowchart, and the box indicating 39 patients underwent TIPS on the right side of the flowchart. [Extracted from the article]

In this report, we used liquid chromatography-mass spectrometry and Western blotting to analyze the content and structure of glycosaminoglycans, glycolipids and selected proteins to compare differences between patient-matched normal and cancerous lung tissues obtained from lung cancer patients. The cancer tissue samples contained over twice as much chondroitin sulfate (CS)/dermatan sulfate (DS) as did the normal tissue samples, while the amount of heparan sulfate (HS) and hyaluronan (HA) in normal and cancer tissues were not significantly different. In HS, several minor disaccharide components, including NS6S, NS2S and 2S were significantly lower in cancer tissues, while the levels of major disaccharides, TriS, NS and 0S disaccharides were not significantly different in normal and cancer tissues. In regards to CS/DS, the level of 4S disaccharide (the major component of CS-type A and DS) decreased and the level of 6S disaccharide (the major component of CS- type C) increased in cancer tissues. We also compared the content and structure of GAGs in lung tissues from smoking and non-smoking patients. Analysis of the glycolipids showed all lipids present in these lung tissues, with the exception of sphingomyelin were higher in cancer tissues than in normal tissues. Western analysis showed that syndecan 1 and 2 proteoglycans displayed much higher expression in cancer tissue/biopsy samples. This investigation begins to provide an understanding of patho-physiological roles on glycosaminoglycans and glycolipids and might be useful in identifying potential biomarkers in lung cancer. [ABSTRACT FROM AUTHOR]

There is a need for degradative enzymes in the study of glycosaminoglycans. Many of these enzymes are currently available either in their natural or recombinant forms. Unfortunately, progress in structure-activity studies of keratan sulfate (KS) have been impeded by the lack of a commercially available endo-β- N-acetylglucosaminidase, keratantase II. The current study uses a recently published sequence of a highly thermostable keratanase II identified in Bacillus circulans to clone and express a series of truncation mutants in Escherichia coli BL21. The resulting truncated forms of keratanase II exhibit activity and excellent storage and thermal stability making these useful tools for glycobiology research. [ABSTRACT FROM AUTHOR]

Heparin is a structurally complex polysaccharide used as a clinical anticoagulant. It is comprised of a heterogeneous mixture of polysaccharide chains having a variety of sequences and lengths. The production methods and regulatory controls of pharmaceutical heparins have changed over the years. This study assesses the structural and activity uniformity of the polysaccharide chains comprising two contemporary heparin products. The heparin fractions with different sizes and charges were separated with size exclusion and ion exchange chromatography. The fractions were analyzed for their molecular weight properties, di- and tetrasaccharide compositions, and anti-factor IIa and anti-factor-Xa activities. The distribution of these properties through chains of different lengths and ones with different charge density were compared. The results demonstrate that with the increase in heparin purity, activity and molecular weight required by the current pharmacopeia, the uniformity of pharmaceutical heparin products have increased. [ABSTRACT FROM AUTHOR]

Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) were prepared from the muscular stomach or gizzard of the chicken. The content of GAGs on a dry weight basis contains 0.4 wt.% a typical value observed for a muscle tissue. The major GAG components were chondroitin-6-sulfate and chondroitin-4-sulfate (~64 %) of molecular weight 21-22 kDa. Hyaluronan (~24 %) had a molecular weight 120 kDa. Smaller amounts (12 %) of heparan sulfate was also present which was made of more highly sulfated chains of molecular weight of 21-22 kDa and a less sulfated low molecular weight (< 10 kDa) heterogeneous partially degraded heparan sulfate. Chicken gizzard represents an inexpensive and readily available source of muscle tissue-derived GAGs. [ABSTRACT FROM AUTHOR]

Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins. [ABSTRACT FROM AUTHOR]

Heparosan, the capsular polysaccharide of Escherichia coli K5 having a carbohydrate backbone similar to that of heparin, has become a potential precursor for bioengineering heparin. In the heparosan biosynthesis pathway, the gene waaR encoding α-1-, 2- s the third glucosyl residues linking to the oligosaccharide chain. In the present study, a waaR deletion mutant of E. coli K5 was constructed. The mutant showed improvement of capsule polysaccharide yield. It is interesting that the heparosan molecular weight of the mutant is reduced and may become more suitable as a precursor for the production of low molecular weight heparin derived from the wild-type K5 capsular polysaccharide. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) are heterogeneous, linear, highly charged, anionic polysaccharides consisting of repeating disaccharides units. GAGs have some biological significance in cancer progression (invasion and metastasis) and cell signaling. In different cancer types, GAGs undergo specific structural changes. In the present study, in depth investigation of changes in sulfation pattern and composition of GAGs, heparan sulfate (HS)/heparin (HP), chondroitin sulfate (CS)/dermatan sulfate and hyaluronan (HA) in normal renal tissue (NRT) and renal cell carcinoma tissue (RCCT) were evaluated. The statistical evaluation showed that alteration of the HS (HS = 415.1 ± 115.3; HS = 277.5 ± 134.3), and CS (CS = 35.3 ± 12.3; CS = 166.7 ± 108.8) amounts (in ng/mg dry tissue ) were statistically significant ( p < 0.05). Sulfation pattern in NRT and RCCT was evaluated to reveal disaccharide profiles. Statistical analyses showed that RCCT samples contain significantly increased amounts (in units of ng/mg dry tissue ) of 4S (NRT = 25.7 ± 9.4; RCCT = 117.1 ± 73.9), SE (NRT = 0.7 ± 0.3; RCCT = 4.7 ± 4.5), 6S (NRT = 6.1 ± 2.7; RCCT = 39.4 ± 34.7) and significantly decreased amounts (in units of ng/mg dry tissue ) of NS6S (RCCT = 28.6 ± 6.5, RCCT = 10.2 ± 8.0), NS2S (RCCT = 44.2 ± 13.8; RCCT = 27.2 ± 15.0), NS (NRT = 68.4 ± 15.8; RCCT = 50.4 ± 21.2), 2S6S (NRT = 1.0 ± 0.4; RCCT = 0.4 ± 0.3), and 6S (NRT = 60.6 ± 17.5; RCCT = 24.9 ± 12.3). If these changes in GAGs are proven to be specific and sensitive, they may serve as potential biomarkers in RCC. Our findings are likely to help us to show the direction for further investigations to be able to bring different diagnostic and prognostic approaches in renal tumors. [ABSTRACT FROM AUTHOR]

Nattokinase (NK) is a serine protease extracted from a traditional Japanese food called natto. Due to its strong fibrinolytic and thrombolytic activity, NK is regarded as a valuable dietary supplement or nutraceutical for the oral thrombolytic therapy. In addition, NK has been investigated for some other medical applications including treatment of hypertension, Alzheimer's disease, and vitreoretinal disorders. The most widely used clinical anticoagulants are heparin and low molecular weight heparins. The interactions between heparin and proteins modulate diverse patho-physiological processes and heparin modifies the activity of serine proteases. Indeed, heparin plays important roles in almost all of NK's potential therapeutically applications. The current report relies on surface plasmon resonance spectroscopy to examine NK interacting with heparin as well as other glycosaminoglycans (GAGs). These studies showed that NK is a heparin binding protein with an affinity of ~250 nM. Examination with differently sized heparin oligosaccharides indicated that the interaction between NK and heparin is chain-length dependent and the minimum size for heparin binding is a hexasaccharide. Studies using chemically modified heparin showed the 6- O-sulfo as well as the N-sulfo groups but not the 2- O-sulfo groups within heparin, are essential for heparin's interaction with NK. Other GAGs (including HS, DS, and CSE) displayed modest binding affinity to NK. NK also interfered with other heparin-protein interactions, including heparin's interaction with antithrombin and fibroblast growth factors. [ABSTRACT FROM AUTHOR]

Bioengineered heparin is being investigated as a potential substitute for the animal-sourced anticoagulant drug. One step in the current process to prepare bioengineered heparin involves the conversion of N-sulfo heparosan, rich in → 4)GlcNS(1 → 4) GlcA(1 → sequences (where S is sulfo, GlcN is α- d-glucosamine, and GlcA is β- d-glucuronic acid), to a critical intermediate, rich in → 4)GlcNS(1 → 4) IdoA2S(1 → sequences (where S is sulfo and IdoA is α- l-iduronic acid), using 2- O-sulfotransferase (2-OST) and C5 epimerase (C-epi). Until now, these heparan sulfate biosynthetic enzymes have been expressed in Escherichia coli grown in shake flask culture as fusion proteins. The current study is focused on the high cell density fed-batch cultivation of recombinant E. coli strains expressing both enzymes. We report the high productivity expression of active 2-OST and C-epi enzymes of 6.0 and 2.2 mg/g dry cell weight, respectively. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) have numerous applications in the fields of pharmaceuticals, cosmetics, nutraceuticals, and foods. GAGs are also critically important in the developmental biology of all multicellular animals. GAGs were isolated from chicken egg components including yolk, thick egg white, thin egg white, membrane, calcified shell matrix supernatant, and shell matrix deposit. Disaccharide compositional analysis was performed using ultra high-performance liquid chromatography-mass spectrometry. The results of these analyses showed that all four families of GAGs were detected in all egg components. Keratan sulfate was found in egg whites (thick and thin) and shell matrix (calcified shell matrix supernatant and deposit) with high level. Chondroitin sulfates were much more plentiful in both shell matrix components and membrane. Hyaluronan was plentiful in both shell matrix components and membrane, but was only present in a trace of quantities in the yolk. Heparan sulfate was plentiful in the shell matrix deposit but was present in a trace of quantities in the egg content components (yolk, thick and thin egg whites). Most of the chondroitin and heparan sulfate disaccharides were present in the GAGs found in chicken eggs with the exception of chondroitin and heparan sulfate 2,6-disulfated disaccharides. Both CS and HS in the shell matrix deposit contained the most diverse chondroitin and heparan sulfate disaccharide compositions. Eggs might provide a potential new source of GAGs. [ABSTRACT FROM AUTHOR]

A capillary zone electrophoresis–laser-induced fluorescence detection (CZE-LIF) method was developed for the simultaneous analysis of disaccharides derived from heparan sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan, and keratan sulfate. Glycosaminoglycans (GAGs) were first depolymerized with the mixture of GAG lyases (heparinase I, II, III and chondroitinase ABC and chondroitinase AC II) and GAG endohydrolase (keratinase II) and the resulting disaccharides were derivatized by reductive amination with 2-aminoacridone. Nineteen fluorescently labeled disaccharides were separated using 50 mM phosphate buffer (pH 3.3) under reversed polarity at 25 kV. Using these conditions, all the disaccharides examined were baseline separated in less then 25 min. This CZE-LIF method gave good reproducibility for both migration time (≤1.03 % for intraday and ≤4.4 % for interday) and the peak area values (≤5.6 % for intra- and ≤8.69 % for interday). This CZE-LIF method was used for profiling and quantification of GAG derivative disaccharides in bovine cornea. The results show that the current CZE-LIF method offers fast, simple, sensitive, reproducible determination of disaccharides derived from total GAGs in a single run. [ABSTRACT FROM AUTHOR]

Nosiheptide is one of the EU-approved sulfur-containing peptides in feed industry to inhibit the growth of the majority of Gram-positive bacteria. The main purpose of this study is directed to breed the high nosiheptide-producers by genome shuffling and ribosome engineering in Streptomyces actuosus AW7. The starting population for shuffling was generated by combining Coγ-irradiation with LiCl mutagenesis treatments on the spores. After four rounds of protoplast fusion exposed to streptomycin as adaptive pressure, a high-yield recombinant strain D92 was obtained. In a 10-L fermenter, nosiheptide production reached 1.54 g/L which was 9.20-fold compared to that of the parental strain. Hyphae development, metabolic process, and ribosomal protein S12 sequence were investigated to characterize the differentiation among the recombinants. Several mutations in S12 were believed to be responsible to streptomycin resistance in the tested strain. The results demonstrated that the combination of genome shuffling and ribosome engineering is an efficient approach to breed high-yield industrial strains. [ABSTRACT FROM AUTHOR]

In the present study, the ability of a newly isolated strain, Methylobacterium sp. XJLW to degrade formaldehyde was investigated in shake flasks and in a bioreactor. The resting cells of Methylobacterium sp. XJLW showed high formaldehyde tolerance (60 g L) and high degradation rate (1,687.5 mg L h) in shake flasks. This biodegradation was initiated by a dismutation reaction since formic acid was formed and caused significant dropping of pH in the media. The addition of CaCO to the media was found as an effective strategy to control the pH and keep the cells in high degradation bioactivity. A three-phase fluidized bed reactor (TPFBR) was designed to test the formaldehyde-biodegrading ability of immobilized Methylobacterium sp. XJLW. Using a repeated-batch degradation mode, the immobilized cells were able to degrade 5 g L formaldehyde (with a maximal degradation rate of 464.5 mg L h under the optimum conditions) and showed stable bioactivity after 20 batches of reuse in the TPFBR. [ABSTRACT FROM AUTHOR]

Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change. [ABSTRACT FROM AUTHOR]

The structure characteristics and biosorption behaviors of heavy metals on a novel exopolysaccharide, which was secreted by the thermophilic Anoxybacillus sp. R4-33 isolated from a radon hot spring in China, were investigated. We purified the EPS-II from the culture medium of strain Anoxybacillus sp. R4-33 using a DEAE Sepharose and Sephadex G-200 column. The results of partial acid hydrolysis, gas chromatography, infrared spectrum, and nuclear magnetic resonance analysis showed that EPS-II was a heteropolysaccharide, composed of d-mannose and d-glucose as its principal monosaccharide composition in the relative proportions 1:0.45. Batch adsorption processes were conducted to characterize the kinetics, equilibrium, and mechanisms of the biosorption process of Zn(II) and Cd(II) from aqueous solution. Sorption experiments confirmed that 1.9783 mg Zn(II) and 1.4095 mg Cd(II) were adsorbed per gram EPS-II at pH 6.0, respectively. The equilibrium data was described by two isotherm models (Langmuir and Freundlich) and two kinetics models (pseudo-first order and pseudo-second order). Freundlich model provided the better correlation of Zn(II) biosorption data, while Langmuir model did to Cd(II). Pseudo-second-order kinetic equation could depict the biosorption kinetics of tested heavy metals. The EPS-II would be a potential candidate in the exopolysaccharide bioresource. [ABSTRACT FROM AUTHOR]

Placental malaria is a serious problem in sub-Saharan Africa. Young women are particular susceptible to contracting this form of malaria during their first or second pregnancy despite previously acquired immunity from past infections. Placental malaria is caused by Plasmodium falciparum parasites expressing VAR2CSA on the erythrocyte surface. This protein adheres to a low-sulfated chondroitin sulfate-A found in placental tissue causing great harm to both mother and developing fetus. In rare cases, the localization of infected erythrocytes to the placenta can even result in the vertical transmission of malaria. In an effort to better understand this infection, chondroitin sulfate was isolated from the cotyledon part of the placenta, which should be accessible for parasite adhesion, as well as two non-accessible parts of the placenta to serve as controls. The placental chondroitin sulfate structures and their VAR2CSA binding were characterized. All portions of human placenta contained sufficient amounts of the appropriate low-sulfated chondroitin sulfate-A to display high-affinity binding to a recombinant truncated VAR2CSA construct, as determined using surface plasmon resonance. The cotyledon is the only placental tissue accessible to parasites in the bloodstream, suggesting it is the primary receptor for parasite infected red blood cells. [ABSTRACT FROM AUTHOR]

A key enzyme for the biosynthesis and bioengineering of heparin, 3- O-sulfotransferase-1 (3-OST-1), was expressed and purified in Gram-positive Bacillus subtilis and Bacillus megaterium. Western blotting, protein sequence analysis, and enzyme activity measurement confirmed the expression. The enzymatic activity of 3-OST-1 expressed in Bacillus species were found to be similar to those found when expressed in Escherichia coli. The endotoxin level in 3-OST-1 from B. subtilis and B. megaterium were 10-10-fold lower than that of the E. coli-expressed 3-OST-1, which makes the Bacillus expression system of particular interest for the generation of pharmaceutical grade raw heparin from nonanimal sources. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGS) are anionic, linear, polysaccharides involved in cell signaling. The GAG content, composition and structure of human tissue have been suggested to play a role in cancer and might provide useful diagnostic or prognostic markers. The current study examines 17 stomach tissue biopsy samples taken from normal individuals and from patients with gastric cancers. An ultrasensitive liquid chromatography (LC) - mass spectrometry assay was applied to individual biopsy samples as small 250 μg providing GAG content and disaccharide composition. The results of these analyses show a significant increase in non-sulfated chondroitin/dermatan sulfate concentration in all cancer samples when compared to normal tissues. In addition in advanced gastric cancer, a significant decrease is observed in hyaluronan. [ABSTRACT FROM AUTHOR]

Genome shuffling is a powerful approach for efficiently engineering industrial microbial strains with interested phenotypes. Here we reported a high producer of nuclease P1, Penicillium citrinum G-16, that was bred by the classical physics-mutagenesis and genome shuffling process. The starting populations were generated by Co γ-irradiation mutagenesis. The derived two protoplast fractions were inactivated by heat-treatment and ultraviolet radiation respectively, then mixed together and subjected to recursive protoplast fusion. Three recombinants, E-16, F-71, and G-16, were roughly obtained from six cycles of genome shuffling. The activity of nuclease P1 by recombinant G-16 was improved up to 1,980.22 U4/ml in a 5-l fermentor, which was 4.7-fold higher than that of the starting strain. The sporulation of recombinant G-16 was distinguished from the starting strain. Random amplified polymorphic DNA assay revealed genotypic differences between the shuffled strains and the wild type strain. The close similarity among the high producers suggested that the genetic basis of high-yield strains was achieved by genome shuffling. [ABSTRACT FROM AUTHOR]

Seven commercial heparin active pharmaceutical ingredients and one commercial low molecular weight from different manufacturers were characterized with a view profiling their physicochemical properties. All heparins had similar molecular weight properties as determined by polyacrylamide gel electrophoresis ( M, 10-11 kDa; M, 13-14 kDa; polydispersity (PD), 1.3-1.4) and by size exclusion chromatography ( M, 14-16 kDa; M, 21-25 kDa; PD, 1.4-1.6). one-dimensional H- and C-nuclear magnetic resonance (NMR) evaluation of the heparin samples was performed, and peaks were fully assigned using two-dimensional NMR. The percentage of glucosamine residues with 3- O-sulfo groups and the percentage of N-sulfo groups and N-acetyl groups ranged from 5.8-7.9%, 78-82%, to 13-14%, respectively. There was substantial variability observed in the disaccharide composition, as determined by high performance liquid chromatography (HPLC)-mass spectral analysis of heparin lyase I-III digested heparins. Heparin oligosaccharide mapping was performed using HPLC following separate treatments with heparin lyase I, II, and III. These maps were useful in qualitatively and quantitatively identifying structural differences between these heparins. The binding affinities of these heparins to antithrombin III and thrombin were evaluated by using a surface plasmon resonance competitive binding assay. This study provides the physicochemical and activity characterization necessary for the appropriate design and synthesis of a generic bioengineered heparin. [ABSTRACT FROM AUTHOR]

Anticoagulant heparin has been shown to possess important biological functions that vary according to its fine structure. Variability within heparin's structure occurs owing to its biosynthesis and animal tissue-based recovery and adds another dimension to its complex polymeric structure. The structural variations in chain length and sulfation patterns mediate its interaction with many heparin-binding proteins, thereby eliciting complex biological responses. The advent of novel chemical and enzymatic approaches for polysaccharide synthesis coupled with high throughput combinatorial approaches for drug discovery have facilitated an increased effort to understand heparin's structure-activity relationships. An improved understanding would offer potential for new therapeutic development through the engineering of polysaccharides. Such a bioengineering approach requires the amalgamation of several different disciplines, including carbohydrate synthesis, applied enzymology, metabolic engineering, and process biochemistry. [ABSTRACT FROM AUTHOR]

Heparin (HP) inhibits the growth of several cell types in vitro including bovine pulmonary artery (BPA) smooth muscle cells (SMCs). In initial studies we discovered that an O-hexanoylated low-molecular-weight (LMW) HP derivative having acyl groups with 6-carbon chain length was more potent inhibitor of BPA-SMCs than the starting HP. We prepared several O-acylated LMWHP derivatives having 4-, 6-, 8-, 10-, 12-, and 18- carbon acyl chain lengths to determine the optimal acyl chain length for maximum anti-proliferative properties of BPA-SMCs. The starting LMWHP was prepared from unfractionated HP by sodium periodate treatment followed by sodium borohydride reduction. The tri- n-butylammonium salt of this LMWHP was O-acylated with butanoic, hexanoic, octanoic, decanoic, dodecanoic, and stearyl anhydrides separately to give respective O-acylated LMWHP derivatives. Gradient polyacrylamide gel electrophoresis (PAGE) was used to examine the average molecular weights of those O-acylated LMWHP derivatives. NMR analysis indicated the presence of one O-acyl group per disaccharide residue. Measurement of the inhibition of BPA-SMCS as a function of O-acyl chain length shows two optima, at a carbon chain length of 6 ( O-hexanoylated LMWHP) and at a carbon chain length 12-18 ( O-dodecanoyl and O-stearyl LMWHPs). A solution competition SPR study was performed to test the ability of different O-acylated LMWHP derivatives to inhibit fibroblast growth factor (FGF) 1 and FGF2 binding to surface-immobilized heparin. All the LMWHP derivatives bound to FGF1 and FGF2 but each exhibited slightly different binding affinity. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS), is a proteoglycan (PG) found both in the extracellular matrix and on cell surface. It may represent one of the most biologically important glycoconjugates, playing an essential role in a variety of different events at molecular level. The publication of the mouse genome, and the intensive investigations aimed at understanding the proteome it encodes, has motivated us to initiate studies in mouse glycomics focused on HS. The current study is aimed at determining the quantitative and qualitative organ distribution of HS in mice. HS from brain, eyes, heart, lung, liver, kidney, spleen, intestine and skin was purified from 6–8 week old male and female mice. The recovered yield of HS from these organs is compared with the recovered whole body yield of HS. Structural characterization of the resulting HS relied on disaccharide analysis and 1H-NMR spectroscopy. Different organs revealed a characteristic HS structure. These data begin to provide a structural understanding of the role of HS in cell-cell interactions, cell signaling and sub-cellular protein trafficking as well as a fundamental understanding of certain aspects of protein-carbohydrate interactions. [ABSTRACT FROM AUTHOR]

The adsorption and desorption of Am(III) on a calcareous soil (sierozem) and its parent material (loess) were studied by batch technique. The molarities of the Am(III) aqueous solutions were less than 5 . 10-9 mol/l. High adsorbability was found of Am(III) on the calcareous soil and its parent material. In order to decrease the adsorption and, hence, to investigate the adsorption characteristics properly, stable Eu3+ as hold back carrier and analogue was added to the aqueous solution. The relative contributions of CaCO3, organic matter (OM) to the Am(III) adsorption on calcareous soil and its parent material were investigated. The adsorption and desorption isotherms of Am(III) on untreated soil and loess and the three kinds of treated soils and three kinds of treated loesses to remove CaCO3, OM and both CaCO3 and OM were determined, respectively. It was found that all isotherms were linear, the average distribution coefficients (K-d) for the untreated soil and for the untreated loess were almost equal, while there was an obvious difference between the values of the average distribution coefficients (K-d) for the treated soil and the treated loess to remove CaCO3 or OM. The adsorption-desorption hysteresis on the untreated and treated soils and loesses actually occurred and there was an obvious difference between the hysteresis coefficients on both the corresponding treated soil and loess. It can be concluded that the adsorbability of Am(III) on calcareous soil is similar to that on its parent material, and that the contributions of CaCO3 and OM to the Am(III) adsorption by the untreated soil are different from those by the untreated parent material. [ABSTRACT FROM AUTHOR]

Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/mL produced by Sf-9. Kinetic analysis showed that V max and K m of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the V max and K m of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man2–8 GlcNAc2 and FucMan3 or 4GlcNAc2) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The proportions of these oligosaccharide structures were different in the two cell lines. Man4GlcNAc2 and FucMan4GlcNAc2 were the major rSEAP N-glycans produced in Sf-9 cells, while Man2GlcNAc2 was the major rSEAP N-glycan produced in Tn-5B1-4 cells. [ABSTRACT FROM AUTHOR]

Human secreted alkaline phosphatase (SEAP) was produced in a stablytransformed Spodoptera frugiperda Sf-9 insect cell line (Sfb4GalT) following infection with a recombinant Autographa californica multiple nuclear polyhedrovirus containing the SEAP gene under control of the polyhedrin promoter. An affinity chromatographic column prepared by linking 4-aminobenzylphosphonic acid to histidyl-expoxy-Sepharose was used to isolate SEAP from the cell supernatant following removal of cells and virus and 10-fold concentration through ultrafiltration. We found that the binding of SEAP on the affinity matrix follows the Langmuir isotherm model. In addition, either recycling SEAP sample through the column for 24 h or loading high SEAP concentrations resulted in a high-purity product. Some nonspecific binding of protein on the matrix occurred when low concentrations of SEAP sample were loaded. Finally, we found that SEAP binding occurs rapidly, i.e., within 30 min of adding the SEAP sample to the affinity matrix. [ABSTRACT FROM AUTHOR]

Complex mycelium pellets of a white rot fungus, Trametes versicolor, with activated carbon powder were prepared and investigated for decolourisation of an azo dye, Acid Violet 7. The pellets had a black core of activated carbon powder that was surrounded by a layer of white fungal mycelium. Compared to the activated carbon powder, the mycelium pellets (activated carbon free), and the mycelium pellets plus the activated carbon powder that was added into a dye solution, the complex pellets showed the highest and the most stable activity of dye decolourisation in batch cultures. The high decolourisation rate of the complex pellets was attributed not only to dye adsorption by the activated carbon in the complex pellets, but also to adsorption of extracellular enzymes and other reagents involved in dye decolourisation as well as the closeness between the dye molecules and the fungal cells. The complex pellets were further evaluated in a fluidized-bed reactor in two operation modes: a continuous flow feeding and a repeated-batch feeding. The latter gave higher and more stable decolourisation efficiency than the former. Production of laccase in flask culture and the fluidized-bed bioreactor was also compared. [ABSTRACT FROM AUTHOR]

We have explored an environmentally friendly strategy to fabricate porous carbons (JFACs) by means of KOH pre-treatment and direct pyrolysis from jacaranda flowers. The JFACs exhibit a high specific capacitance of ~ 965 F g− 1 at 1 A g− 1, outstanding capacitance retention of 98% after 10,000 cycles. The JFACs-based supercapacitor displays a high energy density of 75.28 W h kg− 1 at a power density of 1200 W kg− 1 and retains excellent cycling stability with 3.5% loss of capacitance retention after 10,000 cycles at 10 A g− 1. The facile, efficient and template-free synthesis strategy for novel JFACs from biomass sources may promote commercial application of JFACs in the fields of supercapacitors. [ABSTRACT FROM AUTHOR]