Heparan sulfates (HSs) 1 are highly sulfated polysaccharides, present on the surface of mammalian cells and in the extracellular matrix in large quantities. HSs play critical roles in a variety of important biological processes, including assisting viral infection, regulating blood coagulation and embryonic development, suppressing tumor growth, and controlling the eating behavior of mice by interacting with specific regulatory proteins (1-5). HS polysaccharides carry negative charges under physiological pH, and the disaccharide repeating units consist of 134-linked sulfated glucosamine and uronic acid. The unique sequences determine to which specific proteins HSs bind, thereby regulating biological processes.The biosynthesis of HS occurs in the Golgi apparatus. It is initially synthesized as a copolymer of glucuronic acid and N-acetylated glucosamine by D-glucuronyl and N-acetyl-D-glucosaminyltransferase, followed by various modifications (6). These modifications include N-deacetylation and N-sulfation of glucosamine, C 5 epimerization of glucuronic acid to form iduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid, as well as 6-O-sulfation and 3-O-sulfation of glucosamine. Several enzymes that are responsible for the biosynthesis of HS have been cloned and characterized (see review by Esko and Lindahl (7). These enzymes have become essential tools for investigating the relationship between the structures and functions of HS.What is still unknown is the detailed mechanism for regulating the biosynthesis of HS with a defined saccharide sequence. A recent report (8) suggests that the expression levels of various HS biosynthetic enzyme isoforms contribute to the synthesis of specific saccharide sequences in specific tissues. HS N-deacetylase/N-sulfotransferase, 3-O-sulfotransferase, and 6-O-sulfotransferase are present in multiple isoforms. Each isoform is believed to recognize a saccharide sequence around the modification site in order to generate a specific sulfated saccharide sequence (8 -10). For instance, HS D-glucosaminyl 3-O-sulfotransferase (3-OST) isoforms generate 3-Osulfated glucosamine residues that are linked to different sulfated uronic acid residues. 3-OST-1 transfers sulfate to the 3-OH position of an N-sulfated glucosamine residue that is linked to a glucuronic acid residue at the nonreducing end

Fractional derivatives and integrals are convolutions with a power law. Multiplying by an exponential factor leads to tempered fractional derivatives and integrals. Tempered fractional diffusion equations, where the usual second derivative in space is replaced by a tempered fractional derivative, govern the limits of random walk models with an exponentially tempered power law jump distribution. The limiting tempered stable probability densities exhibit semi-heavy tails, which are commonly observed in finance. Tempered power law waiting times lead to tempered fractional time derivatives, which have proven useful in geophysics. The tempered fractional derivative or integral of a Brownian motion, called a tempered fractional Brownian motion, can exhibit semi-long range dependence. The increments of this process, called tempered fractional Gaussian noise, provide a useful new stochastic model for wind speed data. A tempered difference forms the basis for numerical methods to solve tempered fractional diffusion equations, and it also provides a useful new correlation model in time series.

Metablastin (also called p19, stathmin, prosolin, p18, Lap18, and oncoprotein 18) is a highly conserved, cytosolic 149-amino acid polypeptide that is expressed in immature vertebrate cells and undergoes extracellular factor-and cell cycle-regulated serine phosphorylation. The protein was shown recently to destabilize microtubules in vitro (Belmont, L., and Mitchison, T. J. (1996) Cell 84, 623-631). Here we demonstrate that microinjection of recombinant metablastin induces a loss of microtubules in COS-7 cells. This effect is enhanced by serineto-alanine mutations at several phosphorylation sites and virtually abolished by aspartate substitution at a single site, Ser-63. We also show that stoichiometric amounts of metablastin prevent assembly and promote disassembly of microtubules in vitro. Interestingly, the phosphorylation site mutations of metablastin that have dramatic differential effects in intact cells do not alter the ability of metablastin to block tubulin assembly in vitro. The data suggest that phosphorylation of metablastin controls its microtubule-destabilizing activity in vivo but that this regulation may require additional cellular factors. This control mechanism is poised to play a critical role in the dynamic reorganization of the cellular microtubule network that occurs during morphogenesis and mitosis.Serine phosphorylation of metablastin is stimulated in mammalian cells by a diverse group of extracellular factors, which include cAMP-linked agonists (1-3), factors known to activate protein kinase C (3, 4), growth factors that initiate signaling through tyrosine kinase receptors (5, 6), heat shock (7), and, in some cells, agents that induce calcium flux (8). Furthermore, the phosphorylation state of metablastin fluctuates during the cell cycle, achieving its highest level in M phase (9 -11).The known phosphorylation sites of metablastin are . Although the specific protein kinases that directly phosphorylate metablastin in vivo have not been identified, the available evidence suggests that, in cells, Ser-63 and, to a lesser extent, Ser-16 are phosphorylated by cAMP-dependent protein kinase (14), Ser-25 and Ser-38 by mitogen-activated protein kinase(s) and cyclin-dependent kinase(s) (14 -16), and Ser-16 by Ca 2ϩ /calmodulin kinase-GR (8).To test the microtubule-destabilizing activity of metablastin in intact cells and to explore the potential role of phosphorylation in regulating this activity, we have introduced recombinant metablastin and phosphorylation site mutants into COS-7 cells by microinjection and assessed changes in the cellular microtubule array by immunocytochemistry. EXPERIMENTAL PROCEDURESPreparation of Recombinant Proteins-Metablastin and mutated forms of the protein were expressed as GST 1 fusion proteins in Escherichia coli. Metablastin cDNA (17) encoding amino acids 6 -145 was amplified using the polymerase chain reaction (forward primer, 5Ј-AGGGATCCAGGTGAAAGAGCTGGAGAAG-3Ј; reverse primer, 5ЈCA-AAGACCCCGCGGACGAGAATTCCA3Ј; 30 temperature cycles: 94°C, 1 min; 60°C, 45 s; 72°C, 30...

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3-phosphoadenosine 5-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates. Heparan sulfate (HS)3 is a ubiquitous component of the cell surface and extracellular matrix. It regulates a wide range of physiologic and pathophysiologic functions, including embryonic development and blood coagulation and can facilitate viral infection (1, 2). HS exerts its biological effects by interacting with the specific proteins involved in a given process (3). HS is a highly charged polysaccharide consisting of 1 3 4-linked glucosamine and glucuronic/iduronic acid units that contain both N-and O-sulfo groups. Unique saccharide sequences within HS determine the specificity of the binding of HS to its target proteins (4). Heparin, a specialized form of HS, is a commonly used anticoagulant drug. Thus, new methods for the synthesis of heparin and HS attract considerable interest for those developing anticoagulant drugs having improved pharmacological effects.Chemical synthesis has been the major route to obtain structurally defined heparin and HS oligosaccharides (5). The most important example involves the structure antithrombin-binding pentasaccharide. A synthetic pentasaccharide, based on this structure, has been marketed in the United States under the trade name Arixtra. Arixtra is a specific factor Xa inhibitor that is used clinically to prevent venous thromboembolic incidents during surgery. Unfortunately, the total synthesis of heparin and HS oligosaccharides, larger than pentasaccharides, is extremely difficult. HS analogues with 14 saccharide units inhibit the activity of thrombin, but these synthetic analogues are simplified hybrid molecules of HS oligosaccharides and highly sulfated glucose units (6, 7) and are not the naturally occurring structures. Although our laboratory and others continue to pursue the synthesis of heparin and HS oligosaccharides (8), it has become clear that chemical synthesis alone is currently incapable of generating most larger oligosaccharide structures. The application of HS biosynthetic enzymes for generating large heparin and HS oligosaccharides with desired biological activities o...

Recently, many studies have shown the potential use of circulating exosomes as novel biomarkers for monitoring and predicting a number of complex diseases, including cancer. However, reliable and cost-effective detection of exosomes in routine clinical settings, still remain a difficult task, mainly due to the lack of adequately easy and fast assay platforms. Therefore, we demonstrate here the development of a visible and simple method for the detection of exosomes by integrating single-walled carbon nanotubes that being excellent water solubility (s-SWCNTs) and aptamer. Aptamers, specific to exosomes transmembrane protein CD63, are absorbed onto the surface of s-SWCNTs and improve the minic peroxidase activity of s-SWCNTs, which can efficiently catalyze HO-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and lead to a change from colorless to blue in solution. However, after adding exosomes, the aptamers are bound with CD63, leaving from the surface of s-SWCNTs through conformational changes, which results the color of solution from deep to moderate, and this can be observed by the naked eye and monitored by UV-vis spectrometry. Under optimal conditions, the linear range of exosomes is estimated to be 1.84×10 to 2.21×10 particles/μL with a detection of limit (LOD) of 5.2×10 particles/μL. Consequently, a visible and simple approach detecting exosomes is successfully constructed. Moreover, this proposed colorimetric aptasensor can be universally applicable for the detection of other targets by simple change the aptamer.

Membrane fusion induced by herpes simplex virus (HSV) is required for both entry and cell-to-cell spread. It is mediated by the viral glycoprotein gB, gD, gH-gL and gD receptors. Although 3-O-sulfated heparan sulfate (3-OS HS) is a receptor for HSV-1 entry, the requirement for heparan sulfate in the fusion process has been ruled out. Here, it is demonstrated that cells expressing 3-OS HS, generated by D-glucosaminyl 3-O-sulfotransferase isoforms-3 and/or -5 (3-OST-3 and 3-OST-5), fused with cells expressing the four glycoproteins. The cell fusion observed exhibited similar requirements but was independent of protein receptors, HVEM or nectin-1. Additionally, removal of 3-OS HS from the cell surface by heparinase-I treatment and, in separate experiments, the presence of soluble 3-OST-3-and 3-OST-5-modified HS, significantly inhibited fusion. Taken together, these results indicate that 3-OS HS can play a crucial role in virus entry and cell fusion.

Heparan sulfate (HS) represents a major class of glycans that perform central physiological functions. Emerging HS and glycosaminoglycan microarray techniques are used to interrogate the structure and function relationship to develop novel therapeutic agents. Availability of HS with specific sulfation patterns has been a limiting factor and impedes the accuracy of HS glycomics studies. Although organic synthesis provides oligosaccharides, these may not fully represent the biological functions of polysaccharides. Here, we present a study for developing an enzyme-based approach to synthesize a polysaccharide library with different sulfation patterns. Using different combinations of biosynthetic enzymes, we synthesized eight unique polysaccharides. We discovered that polysaccharides without the iduronic acid residue displayed strong binding affinity to antithrombin and high anti-Xa and anti-IIa activities. The enzyme-based synthetic approach could become a general method for discovering new HS structures with unique biological functions.

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