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PubMed | Complex Carbohydrate Research Center and Oklahoma Medical Research Foundation
Type: | Journal: Glycobiology | Year: 2016

Determining the correct enzymatic activity of putative glycosyltransferases (GTs) can be challenging as these enzymes can utilize multiple donor and acceptor substrates. Upon initial determination of the donor-sugar nucleotide(s), a GT utilizes various acceptor molecules that can then be tested. Here, we describe a quick method to screen sugar-nucleotide donor specificities of GTs utilizing a sensitive, nonradioactive, commercially available bioluminescent uridine diphosphate detection kit. This in vitro method allowed us to validate the sugar-nucleotide donor-substrate specificities of recombinantly expressed human, bovine, bacterial and protozoan GTs. Our approach, which is less time consuming than many traditional assays that utilize radiolabeled sugars and chromatographic separations, should facilitate discovery of novel GTs that participate in diverse biological processes.

Cui J.,University of Georgia | Chen Y.,Jilin University | Chou W.-C.,University of Georgia | Sun L.,Jilin University | And 18 more authors.
Nucleic Acids Research | Year: 2011

This report describes an integrated study on identification of potential markers for gastric cancer in patients' cancer tissues and sera based on: (i) genome-scale transcriptomic analyses of 80 paired gastric cancer/reference tissues and (ii) computational prediction of blood-secretory proteins supported by experimental validation. Our findings show that: (i) 715 and 150 genes exhibit significantly differential expressions in all cancers and early-stage cancers versus reference tissues, respectively; and a substantial percentage of the alteration is found to be influenced by age and/or by gender; (ii) 21 co-expressed gene clusters have been identified, some of which are specific to certain subtypes or stages of the cancer; (iii) the top-ranked gene signatures give better than 94 classification accuracy between cancer and the reference tissues, some of which are gender-specific; and (iv) 136 of the differentially expressed genes were predicted to have their proteins secreted into blood, 81 of which were detected experimentally in the sera of 13 validation samples and 29 found to have differential abundances in the sera of cancer patients versus controls. Overall, the novel information obtained in this study has led to identification of promising diagnostic markers for gastric cancer and can benefit further analyses of the key (early) abnormalities during its development. The Author(s) 2010. Published by Oxford University Press.2010This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( 5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2010.

Guo J.,University of Sheffield | Guo J.,Complex Carbohydrate Research Center | Mayers P.C.,University of Sheffield | Breault G.A.,Astrazeneca | Hunter C.A.,University of Sheffield
Nature Chemistry | Year: 2010

The advent of template-directed synthesis has provided access to a range of new interlocked molecular architectures. Although many syntheses of molecular catenanes and rotaxanes have been reported, molecular knots are a class of molecules with topologically non-planar graphs that are rather rare. Here we report a synthetic strategy for the preparation of a molecular trefoil knot from a flexible bipyridine oligomer and a zinc(II) octahedral coordination template. The oligomer folds into a stable open-knot conformation in the presence of the template, and trapping of this arrangement through esterification or ring-closing metathesis produces the closed-knot complex. Subsequent removal of the template from the metathesis product results in a molecular trefoil knot. © 2010 Macmillan Publishers Limited.

Wolfert M.A.,Complex Carbohydrate Research Center | Boons G.-J.,Complex Carbohydrate Research Center
Nature Chemical Biology | Year: 2013

Major histocompatibility complex (MHC) class I and II are glycoproteins that can present antigenic peptides at the cell surface for recognition and activation of circulating T lymphocytes. Here, the importance of the modification of protein antigens by glycans on cellular uptake, proteolytic processing, presentation by MHC and subsequent T-cell priming is reviewed. Antigen glycosylation is important for a number of diseases and vaccine design. All of the key proteins involved in antigen recognition and the orchestration of downstream effector functions are glycosylated. The influence of protein glycosylation on immune function and disease is covered. © 2013 Nature America, Inc. All rights reserved.

Kirkland J.J.,Advanced Materials Technology, Inc. | Schuster S.A.,Advanced Materials Technology, Inc. | Johnson W.L.,Advanced Materials Technology, Inc. | Boyes B.E.,Advanced Materials Technology, Inc. | Boyes B.E.,Complex Carbohydrate Research Center
Journal of Pharmaceutical Analysis | Year: 2013

The advent of superficially porous particles (SPPs) for packed HPLC columns has changed the way that many practitioners have approached the problem of developing needed separations. The very high efficiency of such columns, combined with convenient operating conditions, modest back pressures and the ability to use conventional HPLC instruments has resulted in intense basic studies of SPP technology, and widespread applications in many sciences. This report contains an overview of the SPP technology first developed in 2006 by Advanced Materials Technology, Inc., for sub-3-μm particles, then expanded into a family of SPP products with different particle sizes, pore sizes and other physical parameters. This approach was designed so that each particle of the family could be optimized for separating a particular group of compounds, usually based on solute size. © 2013 Xi'an Jiaotong University.

Nguyen T.-M.,U.S. Department of Agriculture | Chang S.,U.S. Department of Agriculture | Condon B.,U.S. Department of Agriculture | Thomas T.P.,Complex Carbohydrate Research Center | Azadi P.,Complex Carbohydrate Research Center
Journal of Analytical and Applied Pyrolysis | Year: 2014

There has been a great scientific interest in exploring the great potential of the piperazine-phosphonates in flame retardant (FR) application on cotton fabric by investigating the thermal decomposition of cotton fabric treated with them. This research tries to understand the mode of action of the two piperazine-phosphonates derivatives on cotton fabric. The investigation proceeds by preparing Tetraethyl piperazine-1,4-diyldiphosphonate (TEPP) and Diethyl 4-methyl piperazin-1-ylphosphoramidate (DEPP), engrafting them on cotton fabric and studying their mechanism of thermal degradation on fabric. In studying the mechanism, we learned the chemical functional groups of the chemicals on the surface of the treated fabrics and of the evolved gases produced thermogravimetrically and pyrolytically by using different analytical techniques such as attenuated total reflection infrared (ATR-IR), thermogravimetric analysis-Fourier transform infrared (TGA-FTIR) spectroscopy and pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). The experiment's results showed some distinctive details in the thermal degradation of the fabric when applied with these additives.

PubMed | California State University, Fresno and Complex Carbohydrate Research Center
Type: | Journal: Glycobiology | Year: 2016

In cancer cells, the glycoprotein Mucin 1 (MUC1) undergoes abnormal, truncated glycosylation. The truncated glycosylation exposes cryptic peptide epitopes that can be recognized by antibodies. Since these immunogenic regions are cancer specific, they represent ideal targets for therapeutic antibodies. We investigated the role of tumor-specific glycosylation on antigen recognition by the therapeutic antibody AR20.5. We explored the affinity of AR20.5 to a synthetic cancer-specific MUC1 glycopeptide and peptide. The antibody bound to the glycopeptide with an order of magnitude stronger affinity than the naked peptide. Given these results, we postulated that AR20.5 must specifically bind the carbohydrate as well as the peptide. Using X-ray crystallography, we examined this hypothesis by determining the structure of AR20.5 in complex with both peptide and glycopeptide. Surprisingly, the structure revealed that the carbohydrate did not form any specific polar contacts with the antibody. The high affinity of AR20.5 for the glycopeptide and the lack of specific binding contacts support a hypothesis that glycosylation of MUC1 stabilizes an extended bioactive conformation of the peptide recognized by the antibody. Since high affinity binding of AR20.5 to the MUC1 glycopeptide may not driven by specific antibody-antigen contacts, but rather evidence suggests that glycosylation alters the conformational equilibrium of the antigen, which allows the antibody to select the correct conformation. This study suggests a novel mechanism of antibody-antigen interaction and also suggests that glycosylation of MUC1 is important for the generation of high affinity therapeutic antibodies.

PubMed | Complex Carbohydrate Research Center and Iowa State University
Type: | Journal: Journal of visualized experiments : JoVE | Year: 2016

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcRIIIa and the rat 2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

PubMed | Complex Carbohydrate Research Center, Harvard University, University of Georgia and University of South Carolina
Type: | Journal: The Journal of biological chemistry | Year: 2017

To study the regulation of colorectal adenocarcinoma progression by O-GlcNAc, we have focused on the O-GlcNAc-mediated epigenetic regulation of human colon cancer stem cells (CCSC). Xenograft tumors from colon tumor cells with OGT knockdown grew significantly slower than those formed from control cells, indicating a reduced proliferation of tumor cells due to inhibition of OGT expression. Significant reduction of CCSC population was observed in the tumor cells after OGT knockdown, while tumor cells treated with O-GlcNAcase inhibitor showed an increased CCSC population, indicating that O-GlcNAc levels regulated the CCSC compartment. When grown in suspension, tumor cells with OGT knockdown showed a reduced ability to form tumorspheres, indicating a reduced self-renewal of CCSC due to reduced levels of O-GlcNAc. ChIP-seq experiments using an anti-O-GlcNAc antibody revealed significant chromatin enrichment of O-GlcNAc modified proteins at the promoter of the transcription factor MYBL1, which was also characterized by the presence of H3K27me3. RNA-seq analysis showed an increased expression of MYBL1 in tumor cells with OGT knockdown. Forced overexpression of MYBL1 led to a reduced population of CCSC and tumor growth in vivo, similar to the effects of OGT silencing. Moreover, two CpG islands near the TSS site of MYBL1 were identified, and O-GlcNAc levels regulated their methylation status. These results strongly argue that O-GlcNAc epigenetically regulates MYBL1, functioning similarly to H3K27me3. The aberrant CCSC compartment observed after modulating O-GlcNAc levels, therefore, is likely to result, at least in part, from the epigenetic regulation of MYBL1 expression by O-GlcNAc, thereby significantly affecting tumor progression.

News Article | February 15, 2017

The study, published this month in Nature Communications, finds that the transition from a non-parenting state to a parenting state reflects differences in neuropeptides generally associated with mating, feeding, aggression and increased social tolerance. Neuropeptides are small proteins that allow neurons in the brain to communicate with each other; they also influence behavior. The team's research-tested on an insect, the burying beetle Nicrophorus vespilloides-provides a predictive framework for studying the genetics of parenting and social interactions. The burying beetle is intimately involved in raising its children, including regurgitating food to its begging offspring. "We tested the idea that we could predict the genetic pathways involved in parenting based on old predictions from ethologists in the 1960s and 1970s," said the study's lead author Allen Moore, Distinguished Research Professor and head of the department of genetics. "When [burying beetle] parents feed their babies, they are feeding others rather than themselves and so genes that influence food-seeking behavior are likely to be involved." Behavioral scientists predicted that genetic changes occur over time to develop parenting in a species. Based on this hypothesis, Moore's team sequenced and assembled the genome of the burying beetle and measured the abundance of neuropeptides. They theorized that behaviors related to parenting stemmed from alterations in existing genes rather than the evolution of new ones. By looking at parenting and non-parenting beetles, their tests indicated that neuropeptides changed in abundance during parenting. "When new traits evolve, evolution tends to modify existing genetic pathways rather than create new genes," Moore said. The research, Moore said, suggests that many of the genes influencing parenting will be the same across many species. The commonality among organisms will help researchers identify genetic pathways important to parenting. "It is exciting science when you take a step toward predicting the genetic changes involved in a behavior as complicated as parental care," he said. "And it was pleasing to collaborate with colleagues in genetics and Complex Carbohydrate Research Center, which allowed us to apply techniques that wouldn't otherwise be available to test our ideas." Explore further: Beetles provide clues about the genetic foundations of parenthood More information: Christopher B. Cunningham et al. Ethological principles predict the neuropeptides co-opted to influence parenting, Nature Communications (2017). DOI: 10.1038/ncomms14225

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