The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity.

Science.gov (United States)

Christensen, L L; Jensen, U B; Bross, P; Orntoft, T F

2000-09-01

The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

Does alpha 1-acid glycoprotein act as a non-functional receptor for alpha 1-adrenergic antagonists?

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Qin, M; Oie, S

1994-11-01

The ability of a variety of alpha 1-acid glycoproteins (AAG) to affect the intrinsic activity of the alpha 1-adrenergic antagonist prazosin was studied in rabbit aortic strip preparations. From these studies, the activity of AAG appears to be linked to their ability to bind the antagonist. However, a capability to bind prazosin was not the only requirement for this effect. The removal of sialic acid and partial removal of the galactose and mannose residues by periodate oxidation of human AAG all but eliminated the ability of AAG to affect the intrinsic pharmacologic activity of prazosin, although the binding of prazosin was not significantly affected. The presence of bovine AAG, a protein that has a low ability to bind prazosin, reduced the effect of human AAG on prazosin activity. Based upon these results, we propose that AAG is able to bind in the vicinity of the alpha 1-adrenoceptors, therefore extending the binding region for antagonists in such a way as to decrease the ability of the antagonist to interact with the receptor. The carbohydrate side-chains are important for the binding of AAG in the region of the adrenoceptor.

INFLUENCE OF ALPHA-1-ACID GLYCOPROTEIN UPON PRODUCTION OF CYTOKINES BY PERIPHERAL BLOOD MONONUCLEARS

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М. V. Osikov

2007-01-01

Full Text Available Abstract. Alpha-1-acid glycoprotein (orosomucoid is a multifunctional acute phase reactant belonging to the family of lipocalines from plasma alpha-2 globulin fraction. In present study, we investigated dosedependent effects of orosomucoid upon secretion of IL-1â, IL-2, IL-3, IL-4 by mononuclear cells from venous blood of healthy volunteers. Mononuclear cells were separated by means of gradient centrifugation, followed by incubation for 24 hours with 250, 500, or 1000 mcg of orosomucoid per ml RPMI-1640 medium (resp., low, medium and high dose. The levels of cytokine production were assayed by ELISA technique. Orosomucoid-induced secretion of IL-1â and IL-4 was increased, whereas IL-3 secretion was inhibited. IL-2 production was suppressed at low doses of orosomucoid, and stimulated at medium and high doses. The effect of alpha-1-acid glycoprotein upon production of IL-2, IL-3 and IL-4 was dose-dependent. Hence, these data indicate that orosomucoid is capable of modifying IL-1â, IL-2, IL-3, and IL-4 secretion by blood mononuclear cells.

N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

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Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

2015-01-01

N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

susceptible PAI-1 variant was not necessarily the one used when raising the antibody. This and other observations indicated that the carbohydrate moieties or the glycosylation sites are unlikely to be part of the epitopes for these antibodies. The antibody susceptibility characteristic for non......Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...

aguA, the gene encoding an extracellular alpha-glucuronidase from Aspergillus tubingensis, is specifically induced on xylose and not on glucuronic acid.

Science.gov (United States)

de Vries, R P; Poulsen, C H; Madrid, S; Visser, J

1998-01-01

An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.

Glycosylation analysis and protein structure determination of murine fetal antigen 1 (mFA1)--the circulating gene product of the delta-like protein (dlk), preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs

DEFF Research Database (Denmark)

Krogh, T N; Bachmann, E; Teisner, B

1997-01-01

By means of sequence analysis, murine fetal antigen 1 (mFA1) isolated from Mus musculus amniotic fluid was shown to be the circulating protein of the delta-like protein, stromal-cell-derived protein 1 (SCP-1) and preadipocyte factor 1 (Pref-1) gene products. The protein contains 36 cysteine...... residues arranged in six epidermal-growth-factor-like domains. The purification of several C-terminal peptides of varying lengths showed mFA1 to be C-terminal heterogeneous. O-linked glycosylations of the NeuNAc alpha2-3Gal beta1-3(NeuNAc alpha2-6)GalNAc type were present on all C-terminal peptides...... at residues Thr235, Thr244 and Thr248, although glycosylation on Thr244 was only partial. Three N-linked glycosylations were localized in mFA1 (Asn77, Asn142 and Asn151), two of which (Asn142 and Asn151) were in the unusual Asn-Xaa-Cys motif. Fucosylated biantennary complex-type and small amounts (less than 5...

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

Science.gov (United States)

Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian; Wagtberg Sen, Jette; Rasmussen, Søren Kofoed; Kontoravdi, Cleo; Weilguny, Dietmar; Andersen, Mikael Rørdam

2015-03-01

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans

Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein.

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Lingappa, V R; Lingappa, J R; Prasad, R; Ebner, K E; Blobel, G

1978-05-01

mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized alpha-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat alpha-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of alpha-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communis lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature alpha-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by alpha-methyl mannoside, but not to R. communis lectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.

Identification of a novel bile acid in swans, tree ducks, and geese: 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid.

Science.gov (United States)

Kakiyama, Genta; Iida, Takashi; Goto, Takaaki; Mano, Nariyasu; Goto, Junichi; Nambara, Toshio; Hagey, Lee R; Schteingart, Claudio D; Hofmann, Alan F

2006-07-01

By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by (1)H and (13)C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes. 3alpha,7alpha,15alpha-Trihydroxy-5beta-cholan-24-oic acid is a new primary bile acid that is present in the biliary bile acids of swans, tree ducks, and geese and may be termed 15alpha-hydroxy-chenodeoxycholic acid.

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

DEFF Research Database (Denmark)

Behrendt, N; Rønne, E; Ploug, M

1990-01-01

-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid......, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported...

Mechanisms of activation of muscle branched-chain alpha-keto acid dehydrogenase during exercise in man

DEFF Research Database (Denmark)

Van Hall, Gerrit; MacLean, D A; Saltin, B

1996-01-01

1. Exercise leads to activation (dephosphorylation) of the branched-chain alpha-keto acid dehydrogenase (BCKADH). Here we investigate the effect of low pre-exercise muscle glycogen content and of branched-chain amino acid (BCAA) ingestion on the activity of BCKADH at rest and after 90 min of one......-leg knee-extensor exercise at 65% maximal one-leg power output in five subjects. 2. Pre-exercise BCAA ingestion (308 mg BCAAs (kg body wt)-1) caused an increased muscle BCAA uptake, a higher intramuscular BCAA concentration and activation of BCKADH both at rest (9 +/- 1 versus 25 +/- 5% for the control...... and BCAA test, respectively) and after exercise (27 +/- 4 versus 54 +/- 7%). 3. At rest the percentage active BCKADH was not different, 6 +/- 2% versus 5 +/- 1%, in the normal and low glycogen content leg (392 +/- 21 and 147 +/- 34 mumol glycosyl units (g dry muscle)-1, respectively). The post...

Diagnostic serum glycosylation profile in patients with intellectual disability as a result of MAN1B1 deficiency

DEFF Research Database (Denmark)

Van Scherpenzeel, Monique; Timal, Sharita; Rymen, Daisy

2014-01-01

Congenital disorders of glycosylation comprise a group of genetic defects with a high frequency of intellectual disability, caused by deficient glycosylation of proteins and lipids. The molecular basis of the majority of the congenital disorders of glycosylation type I subtypes, localized...... in the cytosol and endoplasmic reticulum, has been solved. However, elucidation of causative genes for defective Golgi glycosylation (congenital disorders of glycosylation type II) remains challenging because of a lack of sufficiently specific diagnostic serum methods. In a single patient with intellectual...... disability, whole-exome sequencing revealed MAN1B1 as congenital disorder of glycosylation type II candidate gene. A novel mass spectrometry method was applied for high-resolution glycoprofiling of intact plasma transferrin. A highly characteristic glycosylation signature was observed with hybrid type N...

High abundance of Serine/Threonine-rich regions predicted to be hyper-O-glycosylated in the secretory proteins coded by eight fungal genomes

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González Mario

2012-09-01

Full Text Available Abstract Background O-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk. Results By comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length. Conclusions About one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends.

Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

DEFF Research Database (Denmark)

Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

1996-01-01

A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...

Age Differences of Salivary Alpha-Amylase Levels of Basal and Acute Responses to Citric Acid Stimulation Between Chinese Children and Adults.

Science.gov (United States)

Yang, Ze-Min; Chen, Long-Hui; Zhang, Min; Lin, Jing; Zhang, Jie; Chen, Wei-Wen; Yang, Xiao-Rong

2015-01-01

It remains unclear how salivary alpha-amylase (sAA) levels respond to mechanical stimuli in different age groups. In addition, the role played by the sAA gene (AMY1) copy number and protein expression (glycosylated and non-glycosylated) in sAA activity has also been rarely reported. In this study, we analyzed saliva samples collected before and after citric acid stimulation from 47 child and 47 adult Chinese subjects. We observed that adults had higher sAA activity and sAA glycosylated levels (glycosylated sAA amount/total sAA amount) in basal and stimulated saliva when compared with children, while no differences were found in total or glycosylated sAA amount between them. Interestingly, adults showed attenuated sAA activity levels increase over those of children after stimulation. Correlation analysis showed that total sAA amount, glycosylated sAA amount, and AMY1 copy number × total sAA amount were all positively correlated with sAA activity before and after stimulation in both groups. Interestingly, correlation r between sAA levels (glycosylated sAA amount and total sAA amount) and sAA activity decreased after stimulation in children, while adults showed an increase in correlation r. In addition, the correlation r between AMY1 copy number × total sAA amount and sAA activity was higher than that between AMY1 copy number, total sAA amount, and sAA activity, respectively. Taken together, our results suggest that total sAA amount, glycosylated sAA amount, and the positive interaction between AMY1 copy number and total sAA amount are crucial in influencing sAA activity before and after stimulation in children and adults.

Age differences of salivary alpha-amylase levels of basal and acute responses to citric acid stimulation between Chinese children and adults

Directory of Open Access Journals (Sweden)

Zemin eYang

2015-11-01

Full Text Available It remains unclear how salivary alpha-amylase (sAA levels respond to mechanical stimuli in different age groups. In addition, the role played by the sAA gene (AMY1 copy number and protein expression (glycosylated and non-glycosylated in sAA activity has also been rarely reported. In this study, we analyzed saliva samples collected before and after citric acid stimulation from 47 child and 47 adult Chinese subjects. We observed that adults had higher sAA activity and sAA glycosylated levels (glycosylated sAA amount/total sAA amount in basal and stimulated saliva when compared with children, while no differences were found in total or glycosylated sAA amount between them. Interestingly, adults showed attenuated sAA activity levels increase over those of children after stimulation. Correlation analysis showed that total sAA amount, glycosylated sAA amount, and AMY1 copy number×total sAA amount were all positively correlated with sAA activity before and after stimulation in both groups. Interestingly, correlation r between sAA levels (glycosylated sAA amount and total sAA amount and sAA activity decreased after stimulation in children, while adults showed an increase in correlation r. In addition, the correlation r between AMY1 copy number×total sAA amount and sAA activity was higher than that between AMY1 copy number, total sAA amount and sAA activity, respectively. Taken together, our results suggest that total sAA amount, glycosylated sAA amount, and the positive interaction between AMY1 copy number and total sAA amount are crucial in influencing sAA activity before and after stimulation in children and adults.

Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary

DEFF Research Database (Denmark)

Fenger, M; Johnsen, A H

1988-01-01

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well...... (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders...... amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests...

Oleic acid and peanut oil high in oleic acid reverse the inhibitory effect of insulin production of the inflammatory cytokine TNF-alpha both in vitro and in vivo systems.

Science.gov (United States)

Vassiliou, Evros K; Gonzalez, Andres; Garcia, Carlos; Tadros, James H; Chakraborty, Goutam; Toney, Jeffrey H

2009-06-26

Chronic inflammation is a key player in pathogenesis. The inflammatory cytokine, tumor necrosis factor-alpha is a well known inflammatory protein, and has been a therapeutic target for the treatment of diseases such as Rheumatoid Arthritis and Crohn's Disease. Obesity is a well known risk factor for developing non-insulin dependent diabetes melitus. Adipose tissue has been shown to produce tumor necrosis factor-alpha, which has the ability to reduce insulin secretion and induce insulin resistance. Based on these observations, we sought to investigate the impact of unsaturated fatty acids such as oleic acid in the presence of TNF-alpha in terms of insulin production, the molecular mechanisms involved and the in vivo effect of a diet high in oleic acid on a mouse model of type II diabetes, KKAy. The rat pancreatic beta cell line INS-1 was used as a cell biological model since it exhibits glucose dependent insulin secretion. Insulin production assessment was carried out using enzyme linked immunosorbent assay and cAMP quantification with competitive ELISA. Viability of TNF-alpha and oleic acid treated cells was evaluated using flow cytometry. PPAR-gamma translocation was assessed using a PPRE based ELISA system. In vivo studies were carried out on adult male KKAy mice and glucose levels were measured with a glucometer. Oleic acid and peanut oil high in oleic acid were able to enhance insulin production in INS-1. TNF-alpha inhibited insulin production but pre-treatment with oleic acid reversed this inhibitory effect. The viability status of INS-1 cells treated with TNF-alpha and oleic acid was not affected. Translocation of the peroxisome proliferator- activated receptor transcription factor to the nucleus was elevated in oleic acid treated cells. Finally, type II diabetic mice that were administered a high oleic acid diet derived from peanut oil, had decreased glucose levels compared to animals administered a high fat diet with no oleic acid. Oleic acid was found to

Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger.

Science.gov (United States)

Chill, Liat; Trinh, Loc; Azadi, Parastoo; Ishihara, Mayumi; Sonon, Roberto; Karnaukhova, Elena; Ophir, Yakir; Golding, Basil; Shiloach, Joseph

2009-02-15

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla......The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous...... with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

Pentavalent Bismuth-Mediated Glycosylation Methods to Activate Sialic and Uronic Acids and the Incorporation of Sialic Acids Into Insulin

Science.gov (United States)

Kabotso, Daniel Elorm Kwame

The negative charge at physiological pH of carboxylic acid-containing monosaccharides modulate the properties of many natural biomolecules such as oligosaccharides and glycoconjugates. Unfortunately, these altered electronic properties also make the incorporation of such acidic sugars more challenging as compared to the more commonly studied neutral sugars. Herein are reported the first demonstration of glycosylation reactions mediated by triphenylbis(1,1,1-trifluoromethanesulfonato)-bismuth with thioglycosides containing carboxylic acid substituents protected as esters. Unlike with many neutral sugar substrates, the addition of 1-propanethiol to the reactions proved critical to obtaining good yields of the desired glycosylation products using sialic acid, galacturonic acid, and glucuronic acid. The protocol was demonstrated to be amenable to automation using a liquid-handling platform. The consequences of artificially incorporating carboxylic-acid-containing sugars into proteins were tested by the design of a linker containing 1 to 4 sialic acids--a sugar found in many human proteins and brain tissues--that was attached via reductive amination of trityl thiopropylaldehyde at the phenyl alanine terminal end of the protein insulin produced through solid-phase peptide synthesis. Removal of the trityl group with neat trifluoroacetic acid furnished the thiol-free modified insulin that was ligated via a disulfide bond to the peptide scaffold bearing acetyl protected sialic acids. A 14-15% ammonium hydroxide solution was found to be effective in deprotecting the acetyl groups without degradation of the disulfide bond. In addition to maintaining the potency and bioactivity of insulin, the sialic acid-containing linker rendered insulin more resistant to aggregation due to heat and mechanical agitation compared to the unmodified protein.

Control of mucin-type O-glycosylation

DEFF Research Database (Denmark)

Bennett, Eric P; Mandel, Ulla; Clausen, Henrik

2012-01-01

residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue...... in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The Gal...

Analysis of urinary PSA glycosylation is not indicative of high-risk prostate cancer.

Science.gov (United States)

Barrabés, Sílvia; Llop, Esther; Ferrer-Batallé, Montserrat; Ramírez, Manel; Aleixandre, Rosa N; Perry, Antoinette S; de Llorens, Rafael; Peracaula, Rosa

2017-07-01

The levels of core fucosylation and α2,3-linked sialic acid in serum Prostate Specific Antigen (PSA), using the lectins Pholiota squarrosa lectin (PhoSL) and Sambucus nigra agglutinin (SNA), can discriminate between Benign Prostatic Hyperplasia (BPH) and indolent prostate cancer (PCa) from aggressive PCa. In the present work we evaluated whether these glycosylation determinants could also be altered in urinary PSA obtained after digital rectal examination (DRE) and could also be useful for diagnosis determinations. For this purpose, α2,6-sialic acid and α1,6-fucose levels of urinary PSA from 53 patients, 18 biopsy-negative and 35 PCa patients of different aggressiveness degree, were analyzed by sandwich ELLA (Enzyme Linked Lectin Assay) using PhoSL and SNA. Changes in the levels of specific glycosylation determinants, that in serum PSA samples were indicative of PCa aggressiveness, were not found in PSA from DRE urine samples. Although urine is a simpler matrix for analyzing PSA glycosylation compared to serum, an immunopurification step was necessary to specifically detect the glycans on the PSA molecule. Those specific glycosylation determinants on urinary PSA were however not useful to improve PCa diagnosis. This could be probably due to the low proportion of PSA from the tumor in urine samples, which precludes the identification of aberrantly glycosylated PSA. Copyright © 2017 Elsevier B.V. All rights reserved.

A basic research of gadolinium hydrogen [alpha], [alpha]', [alpha]'', [alpha]'''-tetramethly- 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetate with high complex stability as a contrast agent for MRI

Energy Technology Data Exchange (ETDEWEB)

Seri, Shigemi; Hashiguchi, Yuji; Kubomura, Kan; Abe, Yukiko; Iguchi, Toshio; Iwai, Kumiko [Nihon Medi-Physics Co., Ltd., Sodegaura, Chiba (Japan); Watanabe, Tokuko

1993-05-01

Gadolinium hydrogen [alpha], [alpha]', [alpha]'', [alpha]'''-tetramethyl- 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetate (abbreviated Gd-DOTMA) was developed as a new contrast agent for magnetic resonance imaging. Our study focused on the evaluation of the pharmaceutical properties as in vivo agent. The new modified process by which Gd-DOTMA was synthesized resulted in high yields of this agent. A high stability constant of 10[sup 26] fro Gd-DOTMA was determined at physiological pH. It is more stable than Gd complex with tetraazacyclododecanetetraacetic acid (which is regarded as the most stable Gd complex). The strong T[sub 1] relaxivities of 4.0 and 3.7 (mM [center dot] s)[sup -1] at 0.5 tesla and 1.5 tesla were measured in the aqueous solution. The osmolarity of 0.5 M solution, dissolved with equal amounts of meglumine as a solubilizer is 1020 mOsmol/kg. This contrasting agent was studied in vivo by using rats as the experimental group. The agent showed strong enhancement of transplanted tumors within the rat population studied. This compound is rapidly excreted by the kidneys, and has a half-life of 26 min in blood. The median lethal dose (LD[sub 50] value) of the stable Gd-DOTMA has a favorable tolerance of over 12.3 mmol/kg. (author).

Prediction of mucin-type O-glycosylation sites in mammalian proteins using the composition of k-spaced amino acid pairs

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Sheng Zhi-Ya

2008-02-01

Full Text Available Abstract Background As one of the most common protein post-translational modifications, glycosylation is involved in a variety of important biological processes. Computational identification of glycosylation sites in protein sequences becomes increasingly important in the post-genomic era. A new encoding scheme was employed to improve the prediction of mucin-type O-glycosylation sites in mammalian proteins. Results A new protein bioinformatics tool, CKSAAP_OGlySite, was developed to predict mucin-type O-glycosylation serine/threonine (S/T sites in mammalian proteins. Using the composition of k-spaced amino acid pairs (CKSAAP based encoding scheme, the proposed method was trained and tested in a new and stringent O-glycosylation dataset with the assistance of Support Vector Machine (SVM. When the ratio of O-glycosylation to non-glycosylation sites in training datasets was set as 1:1, 10-fold cross-validation tests showed that the proposed method yielded a high accuracy of 83.1% and 81.4% in predicting O-glycosylated S and T sites, respectively. Based on the same datasets, CKSAAP_OGlySite resulted in a higher accuracy than the conventional binary encoding based method (about +5.0%. When trained and tested in 1:5 datasets, the CKSAAP encoding showed a more significant improvement than the binary encoding. We also merged the training datasets of S and T sites and integrated the prediction of S and T sites into one single predictor (i.e. S+T predictor. Either in 1:1 or 1:5 datasets, the performance of this S+T predictor was always slightly better than those predictors where S and T sites were independently predicted, suggesting that the molecular recognition of O-glycosylated S/T sites seems to be similar and the increase of the S+T predictor's accuracy may be a result of expanded training datasets. Moreover, CKSAAP_OGlySite was also shown to have better performance when benchmarked against two existing predictors. Conclusion Because of CKSAAP

Identification of a new defective SERPINA1 allele (PI*Zla palma) encoding an alpha-1-antitrypsin with altered glycosylation pattern.

Science.gov (United States)

Hernández-Pérez, José M; Ramos-Díaz, Ruth; Pérez, José A

2017-10-01

Alpha-1-antitrypsin (AAT) deficiency is a genetic condition that arises from mutations in the SERPINA1 gene and predisposes to develop pulmonary emphysema and, less frequently, liver disease. Occasionally, new defective SERPINA1 alleles are detected as an outcome of targeted-screening programs or case-findings. This study began with a female patient showing bronchial hyperreactivity. Serum level and phenotype for AAT was analysed by immunonephelometry and isoelectric focusing electrophoresis. The SERPINA1 gene of the proband was genotyped by PCR amplification and DNA sequencing. Analysis of AAT deficiency was extended to the proband's family. An abnormal AAT variant that migrated to a more cathodal position than PiZ AAT was detected in the proband's serum. Genetic analysis demonstrated that proband is heterozygous for a new defective SERPINA1 allele (PI*Z la palma ) characterized by the c.321C > A (p.Asn83Lys) mutation in the M1Val213 background. This mutation abolishes the N-glycosylation site in position 83 of the mature AAT. Eight relatives of the proband are carriers of the PI*Z la palma allele and four of them have shown symptoms of bronchial asthma or bronchial hyperreactivity. The mean α1AT level in the serum of PI*MZ la palma individuals was 87.1 mg/dl. The reduction in circulating AAT levels associated to the PI*Z la palma allele was similar to that of PI*Z allele, representing a risk of impairment in lung function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Links between CD147 Function, Glycosylation, and Caveolin-1

OpenAIRE

Tang, Wei; Chang, Sharon B.; Hemler, Martin E.

2004-01-01

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-cataly...

Hypothalamic PGC-1 alpha Protects Against High-Fat Diet Exposure by Regulating ER alpha

NARCIS (Netherlands)

Morselli, Eugenia; Fuente-Martin, Esther; Finan, Brian; Kim, Min; Frank, Aaron; Garcia-Caceres, Cristina; Navas, Carlos Rodriguez; Gordillo, Ruth; Neinast, Michael; Kalainayakan, Sarada P.; Li, Dan L.; Gao, Yuanqing; Yi, Chun-Xia; Hahner, Lisa; Palmer, Biff F.; Tschöp, Matthias H.; Clegg, Deborah J.

2014-01-01

High-fat diets (HFDs) lead to obesity and inflammation in the central nervous system (CNS). Estrogens and estrogen receptor alpha (ER alpha) protect premenopausal females from the metabolic complications of inflammation and obesity-related disease. Here, we demonstrate that hypothalamic PGC-1 alpha

alpha-Ketoglutarate application in hemodialysis patients improves amino acid metabolism.

Science.gov (United States)

Riedel, E; Nündel, M; Hampl, H

1996-01-01

In hemodialysis patients, free amino acids and alpha-ketoacids in plasma were determined by fluorescence HPLC to assess the effect of alpha-ketoglutarate administration in combination with the phosphate binder calcium carbonate on the amino acid metabolism. During 1 year of therapy in parallel to inorganic phosphate, urea in plasma decreased significantly, histidine, arginine and proline as well as branched chain alpha-ketoacids, in particular alpha-ketoisocaproate, a regulator of protein metabolism, increased. Thus, administration of alpha-ketoglutarate with calcium carbonate effectively improves amino acid metabolism in hemodialysis patients as it decreases hyperphosphatemia.

Microwave Effect for Glycosylation Promoted by Solid Super Acid in Supercritical Carbon Dioxide

Directory of Open Access Journals (Sweden)

Takahiko Maeda

2009-12-01

Full Text Available The effects of microwave irradiation (2.45 GHz, 200 W on glycosylation promoted by a solid super acid in supercritical carbon dioxide was investigated with particular attention paid to the structure of the acceptor substrate. Because of the symmetrical structure and high diffusive property of supercritical carbon dioxide, microwave irradiation did not alter the temperature of the reaction solution, but enhanced reaction yield when aliphatic acceptors are employed. Interestingly, the use of a phenolic acceptor under the same reaction conditions did not show these promoting effects due to microwave irradiation. In the case of aliphatic diol acceptors, the yield seemed to be dependent on the symmetrical properties of the acceptors. The results suggest that microwave irradiation do not affect the reactivity of the donor nor promoter independently. We conclude that the effect of acceptor structure on glycosylation yield is due to electric delocalization of hydroxyl group and dielectrically symmetric structure of whole molecule.

Indirect approach to C-3 branched 1,2-cis-glycofuranosides: synthesis of aceric acid glycoside analogues.

Science.gov (United States)

de Oliveira, Marcelo T; Hughes, David L; Nepogodiev, Sergey A; Field, Robert A

2008-02-04

Aceric acid (3-C-carboxy-5-deoxy-alpha-l-xylofuranose) residues are present in pectic polysaccharide rhamnogalacturonan II (RG II) in the form of synthetically challenging 1,2-cis-glycofuranosides. To access synthetic fragments of RG II incorporating aceric acid, a four-step procedure based on C-2 epimerisation of initially prepared 1,2-trans-glycofuranoside was developed. Readily available derivatives of branched-chain l-lyxofuranose bearing a 3-C-vinyl group as a masked 3-C-carboxyl group were investigated as potential precursors of aceric acid units. In the first step of the procedure, installation of a participating group at C-2 of the furanose ring ensured stereocontrol of the O-glycosylation, which was carried out with the thioglycoside of 2-O-acetyl-3,5-di-O-benzyl-3-C-vinyl-L-lyxofuranose. After the glycosylation step, the 2-O-acetyl group was removed, the free 2-OH group was oxidised and the resulting ketone was finally reduced to form the C-3-vinyl-L-xylofuranoside. The use of L-Selectride in the key reduction reaction was essential to achieve the required stereoselectivity to generate 1,2-cis-furanoside.

Improved synthesis with high yield and increased molecular weight of poly(alpha,beta-malic acid) by direct polycondensation.

Science.gov (United States)

Kajiyama, Tetsuto; Kobayashi, Hisatoshi; Taguchi, Tetsushi; Kataoka, Kazunori; Tanaka, Junzo

2004-01-01

The development of synthetic biodegradable polymers, such as poly(alpha-hydroxy acid), is particularly important for constructing medical devices, including scaffolds and sutures, and has attracted growing interest in the biomedical field. Here, we report a novel approach to preparing high molecular weight poly(malic acid) (HMW--PMA) as a biodegradable and bioabsorbable water-soluble polymer. We investigated in detail the reaction conditions for the simple direct polycondensation of l-malic acid, including the reaction times, temperatures, and catalysts. The molecular weight of synthesized alpha,beta-PMA is dependent on both the reaction temperature and time. The optimum reaction condition to obtain alpha,beta-PMA by direct polycondensation using tin(II) chloride as a catalyst was thus determined to be 110 degrees C for 45 h with a molecular weight of 5300. The method for alpha,beta-PMA synthesis established here will facilitate production of alpha,beta-PMA of various molecular weights, which may have a potential utility as biomaterials.

Glycosylation in HIV-1 envelope glycoprotein and its biological implications

KAUST Repository

Ho, Yung Shwen

2013-08-01

Glycosylation of HIV-1 envelope proteins (Env gp120/gp41) plays a vital role in viral evasion from the host immune response, which occurs through the masking of key neutralization epitopes and the presentation of the Env glycosylation as \\'self\\' to the host immune system. Env glycosylation is generally conserved, yet its continual evolution plays an important role in modulating viral infectivity and Env immunogenicity. Thus, it is believed that Env glycosylation, which is a vital part of the HIV-1 architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may assist in effectively designing Env-based immunogens and in clearly understanding HIV vaccines. This review provides an in-depth perspective of various aspects of Env glycosylation in the context of HIV-1 pathogenesis. © 2013 Future Medicine Ltd.

Influence of dietary long-chain n-3 fatty acids from menhaden fish oil on plasma concentrations of alpha-tocopherol in geriatric beagles.

Science.gov (United States)

Hall, Jean A; Tooley, Katie A; Gradin, Joseph L; Jewell, Dennis E; Wander, Rosemary C

2002-01-01

To determine effects of dietary n-3 fatty acids from Menhaden fish oil on plasma alpha-tocopherol concentrations in Beagles. 32 female Beagles. For 82 days, dogs were fed diets that contained 1 of 2 ratios of n-6:n-3 fatty acids (40:1 [low n-3] and 1.4:1 [high n-3]) and 1 of 3 concentrations of all-rac-alpha-tocopheryl acetate (low, 17 mg/kg of diet; medium, 101 mg/kg; and high, 447 mg/kg) in a 2 X 3 factorial study. Diets high in n-3 fatty acids significantly increased total content of n-3 fatty acids in plasma (17.0 g/100 g of fatty acids), compared with low n-3 diets (2.02 g/100 g of fatty acids). Mean +/- SEM plasma concentration of cholesterol was significantly lower in dogs consuming high n-3 diets (4.59 +/- 0.48 mmol/L), compared with dogs consuming low n-3 diets (5.71 +/- 0.48 mmol/L). A significant interaction existed between the ratio for n-6 and n-3 fatty acids and amount of alpha-tocopheryl acetate in the diet (plasma alpha-tocopherol concentration expressed on a molar basis), because the plasma concentration of alpha-toco-pherol was higher in dogs consuming low n-3 diets, compared with those consuming high n-3 diets, at the 2 higher amounts of dietary alpha-tocopheryl acetate. Plasma alpha-tocopherol concentration expressed relative to total lipid content did not reveal effects of dietary n-3 fatty acids on concentration of alpha-tocopherol. Plasma alpha-tocopherol concentration is not dependent on dietary ratio of n-6 and n-3 fatty acids when alpha-tocopherol concentration is expressed relative to the total lipid content of plasma.

Performance of Alpha Fetoprotein in Combination with Alpha-1-acid Glycoprotein for Diagnosis of Hepatocellular Carcinoma Among Liver Cirrhosis Patients

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Rino A Gani

2016-05-01

Full Text Available Aim: to evaluate the use of alpha-1-acid glycoprotein (AAG for diagnosing hepatocellular carcinoma (HCC, and to combine with alpha fetoprotein (AFP as part of routine examination in liver cirrhosis patients. Methods: this is a diagnostic study using cross-sectional design. A hundred and six patients were included in this study. Baseline data such as age, gender, AFP, AAG, peripheral blood count, AST and ALT were consecutively collected from liver cirrhosis patients with or without HCC. Serum AAG were measured quantitatively using immunoturboditimetric assay and AFP with enzyme immune assay (EIA. Statistical analysis were done using SPSS 13.0. Data comparisons between group were done using Mann-Whitney test. Diagnostic performance for each marker alone was compared to the surrogate use of both markers (combined parallel approach in HCC cases. Results: receiver operating characteristic (ROC analysis showed that area under the curve for AFP AAG combination was 88.1% and higher than AFP only (86.2% or AAG only (76.5% with sensitivity of 83%, 73% and 44%, respectively, at specificity of >80%. Conclusion: our study showed that combination of AFP and AAG is superior than either marker alone in diagnosing HCC in liver cirrhosis patients. Combination of AFP and AAG may be used to prompt early diagnosis screening of HCC. Key words: alpha fetoprotein, alpha-1-acid glycoprotein, biomarker, liver cancer

Two lectin-like receptors for alpha 1-acid glycoprotein in mouse testis

DEFF Research Database (Denmark)

Andersen, U O; Kirkeby, S; Bøg-Hansen, T C

1997-01-01

Three glycoforms of alpha 1-acid glycoprotein (AGP) were biotinylated to examine their binding in mouse testis by light microscopy. The transition from one stage to another in the spermatogenic cycle is marked with an appearance of a receptor for the Concanavalin A (Con A) non-reactive glycoform...

Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent

Science.gov (United States)

Wadzinski, Tyler J.; Steinauer, Angela; Hie, Liana; Pelletier, Guillaume; Schepartz, Alanna; Miller, Scott J.

2018-06-01

Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

Oral benfotiamine plus alpha-lipoic acid normalises complication-causing pathways in type 1 diabetes.

Science.gov (United States)

Du, X; Edelstein, D; Brownlee, M

2008-10-01

We determined whether fixed doses of benfotiamine in combination with slow-release alpha-lipoic acid normalise markers of reactive oxygen species-induced pathways of complications in humans. Male participants with and without type 1 diabetes were studied in the General Clinical Research Centre of the Albert Einstein College of Medicine. Glycaemic status was assessed by measuring baseline values of three different indicators of hyperglycaemia. Intracellular AGE formation, hexosamine pathway activity and prostacyclin synthase activity were measured initially, and after 2 and 4 weeks of treatment. In the nine participants with type 1 diabetes, treatment had no effect on any of the three indicators used to assess hyperglycaemia. However, treatment with benfotiamine plus alpha-lipoic acid completely normalised increased AGE formation, reduced increased monocyte hexosamine-modified proteins by 40% and normalised the 70% decrease in prostacyclin synthase activity from 1,709 +/- 586 pg/ml 6-keto-prostaglandin F(1alpha) to 4,696 +/- 533 pg/ml. These results show that the previously demonstrated beneficial effects of these agents on complication-causing pathways in rodent models of diabetic complications also occur in humans with type 1 diabetes.

Characterization of kallikrein-related peptidase 4 glycosylations.

Science.gov (United States)

Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C; Simmer, James P

2011-12-01

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. © 2011 Eur J Oral Sci.

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan.

Science.gov (United States)

Bozzi, Manuela; Bianchi, Marzia; Sciandra, Francesca; Paci, Maurizio; Giardina, Bruno; Brancaccio, Andrea; Cicero, Daniel O

2003-11-25

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.

Linkage specific fucosylation of alpha-1-antitrypsin in liver cirrhosis and cancer patients: implications for a biomarker of hepatocellular carcinoma.

Directory of Open Access Journals (Sweden)

Mary Ann Comunale

2010-08-01

Full Text Available We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT. To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC.Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (alpha-1,3 fucosylation. Increases in core (alpha-1,6 fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL, specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific.This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

Science.gov (United States)

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

2016-05-15

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. Copyright © 2015 Elsevier B.V. All rights reserved.

Synthesis and conformational analysis of hybrid α/β-dipeptides incorporating S-glycosyl-β(2,2)-amino acids.

Science.gov (United States)

García-González, Iván; Mata, Lara; Corzana, Francisco; Jiménez-Osés, Gonzalo; Avenoza, Alberto; Busto, Jesús H; Peregrina, Jesús M

2015-01-12

We synthesized and carried out the conformational analysis of several hybrid dipeptides consisting of an α-amino acid attached to a quaternary glyco-β-amino acid. In particular, we combined a S-glycosylated β(2,2)-amino acid and two different types of α-amino acid, namely, aliphatic (alanine) and aromatic (phenylalanine and tryptophan) in the sequence of hybrid α/β-dipeptides. The key step in the synthesis involved the ring-opening reaction of a chiral cyclic sulfamidate, inserted in the peptidic sequence, with a sulfur-containing nucleophile by using 1-thio-β-D-glucopyranose derivatives. This reaction of glycosylation occurred with inversion of configuration at the quaternary center. The conformational behavior in aqueous solution of the peptide backbone and the glycosidic linkage for all synthesized hybrid glycopeptides was analyzed by using a protocol that combined NMR experiments and molecular dynamics with time-averaged restraints (MD-tar). Interestingly, the presence of the sulfur heteroatom at the quaternary center of the β-amino acid induced θ torsional angles close to 180° (anti). Notably, this value changed to 60° (gauche) when the peptidic sequence displayed aromatic α-amino acids due to the presence of CH-π interactions between the phenyl or indole ring and the methyl groups of the β-amino acid unit. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous improvement in production of microalgal biodiesel and high-value alpha-linolenic acid by a single regulator acetylcholine.

Science.gov (United States)

Parsaeimehr, Ali; Sun, Zhilan; Dou, Xiao; Chen, Yi-Feng

2015-01-01

Photoautotrophic microalgae are a promising avenue for sustained biodiesel production, but are compromised by low yields of biomass and lipids at present. We are developing a chemical approach to improve microalgal accumulation of feedstock lipids as well as high-value alpha-linolenic acid which in turn might provide a driving force for biodiesel production. We demonstrate the effectiveness of the small bioactive molecule "acetylcholine" on accumulation of biomass, total lipids, and alpha-linolenic acid in Chlorella sorokiniana. The effectiveness exists in different species of Chlorella. Moreover, the precursor and analogs of acetylcholine display increased effectiveness at higher applied doses, with maximal increases by 126, 80, and 60% over controls for biomass, total lipids, and alpha-linolenic acid, respectively. Production of calculated biodiesel was also improved by the precursor and analogs of acetylcholine. The biodiesel quality affected by changes in microalgal fatty acid composition was addressed. The chemical approach described here could improve the lipid yield and biodiesel production of photoautotrophic microalgae if combined with current genetic approaches.

Zonal variation in the distribution of an alpha 1-acid glycoprotein glycoform receptor in human adrenal cortex

DEFF Research Database (Denmark)

Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

1999-01-01

receptor was located in the cytoplasm of glomerulosa and outer fasciculata cells. The intensity of the reaction product decreased in the fasciculata, and no staining was seen in inner fasciculata and reticularis. Inhibition with the simple sugars, mannose and GlcNAc confirmed a lectin-like reaction...... specific receptor. The binding of alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C to the glycoform specific receptor is inhibited by the steroid hormones cortisone, aldosterone, estradiol and progesterone but not by testosterone. The pronounced changes in the distribution...

Development of on-chip fully automated immunoassay system "μTASWako i30" to measure the changes in glycosylation profiles of alpha-fetoprotein in patients with hepatocellular carcinoma.

Science.gov (United States)

Kurosawa, Tatsuo; Watanabe, Mitsuo

2016-12-01

Glycosylation profiles significantly change during oncogenesis. Aberrant glycosylation can be used as a cancer biomarker in clinical settings. Different glycoforms can be separately detected using lectin affinity electrophoresis and lectin array-based methods. However, most methodologies and procedures need experienced technique to perform the assays and expertise to interpret the results. To apply glycomarkers for clinical practice, a robust assay system with an easy-to-use workflow is required. Wako's μTASWako i30, a fully automated immunoanalyzer, was developed for in vitro diagnostics based on microfluidic technology. It utilizes the principles of liquid-phase binding assay, where immunoreactions are performed in a liquid phase, and electrokinetic analyte transport assay. Capillary electrophoresis on microfluidic chip has enabled the detection of different glycoform types of alpha-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma. AFP with altered glycosylation can be separated based on the reactivity to Lens culinaris agglutinin on electrophoresis. The glycoform AFP-L3 was reportedly more specific in hepatocellular carcinoma. This assay system can provide a high sensitivity and rapid results in 9 min. The test results for ratio of AFP-L3 to total AFP using μTASWako i30 are correlated with those of conventional methodology. The μTASWako assay system and the technology can be utilized for glycosylation analysis in the postgenomic era. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Alpha Hydroxy Acids

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... or tenderness (8), chemical burns (6), and increased sunburn (3). The frequency of such reports for skin ... bear a statement that conveys the following information: Sunburn Alert: This product contains an alpha hydroxy acid ( ...

Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha.

Science.gov (United States)

Dietrich, Christoph G; Martin, Ina V; Porn, Anne C; Voigt, Sebastian; Gartung, Carsten; Trautwein, Christian; Geier, Andreas

2007-09-01

Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.

Influence of ascorbic acid on in vivo amidation of alpha-melanocyte stimulating hormone in guinea pig pituitary

DEFF Research Database (Denmark)

Fenger, M; Hilsted, L

1988-01-01

The effect of ascorbic acid depletion on the amidation of alphamelanocyte stimulating hormone (alpha MSH) was studied in vivo in guinea pig pituitary. After four weeks, the concentration of ascorbic acid was 1.20 +/- 0.11 mumol/g tissue (mean +/- SD) in the pituitary and 0.34 +/- 0.07 mumol......-39) immunoreactivity was observed in the depleted guinea pigs. Gel chromatography and reversed-phase high-performance luquid chromatography showed that the alpha MSH and ACTH (1-14) immunoreactivity was of low molecular weight and partly mono- or diacetylated. Depletion of ascorbic acid had no influence on the degree...... of acetylation of alpha MSH and ACTH (1-14). It is concluded that depletion of ascorbic acid reduces the in vivo amidation of ACTH (1-14) in the guinea pig pituitary....

C595 antibody: A potential vector for targeted alpha therapy

International Nuclear Information System (INIS)

Perkins, A.C.; Allen, B.J.

2005-01-01

Full text: Mucins are high molecular-weight heavily glycosylated glycoproteins with many oligosaccharide side-chains, linked to a protein backbone called apomucin. A total of 19 different mucin genes (MUC1-MUC4, MUC5B, MUC5AC, MUC6-MUC18) have been identified to date. Mucins are present on the surface of most epithelial cells and play a role in their protection and lubrication. In cancer cells the mucin molecule becomes altered, thus representing an important target for diagnosis and therapy. Urinary epithelial mucin1 (MUC1) is found to be frequently up-regulated and abnormally glycosylated in a number of common malignancies, including breast, bladder, colon, ovarian and gastric cancer. The monoclonal antibody C595 is an IgG3 murine MAb raised against the protein core of human MUC1. Epitope mapping has shown that C595 recognizes a tetrapeptide motif (RPAP) within the protein core of MUC1 mucin that contains a large domain of multiples of a highly conserved 20-amino-acid-repeat sequence (PDTRPAPGSTAPPAHGVTSA). This antibody has previously been radiolabelled with 99m Tc and 111 In and used for imaging a range of tumour types including ovary, breast and bladder. The antibody has also been radiolabelled with 67 Cu and 188 Re for the therapy of superficial bladder cancer. More recently we have investigated the pre-clinical use of the C595 antibody for targeted alpha therapy using 213 Bi which emits alpha particles with high linear energy transfer (LET), short range (80 m) radiation and has a short physical half-life of 45.6 minutes. Alpha particles are some 7300 times heavier than beta particles and in theory, following binding of an alpha immunocongugates to the target, a large fraction of the alpha particle energy is delivered to cancer cells, with minimal concomitant radiation of normal tissues. 213 Bi was produced from the 225 Ac/ 213 Bi generator. For antibody conjugation the chelator, cyclic diethylenetriaminepentacetic acid anhydride (DTPA) was used. Initial

Quantitation of alpha-linolenic acid elongation to eicosapentaenoic and docosahexaenoic acid as affected by the ratio of n6/n3 fatty acids

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Somoza Veronika

2009-02-01

Full Text Available Abstract Background Conversion of linoleic acid (LA and alpha-linolenic acid (ALA to their higher chain homologues in humans depends on the ratio of ingested n6 and n3 fatty acids. Design and methods In order to determine the most effective ratio with regard to the conversion of ALA to eicosapentaenoic acid (EPA and docosahexaenoic acid (DHA, human hepatoma cells were incubated with varying ratios of [13C] labeled linoleic acid ([13C]LA- and alpha-linolenic acid ([13C]ALA-methylesters. Regulative cellular signal transduction pathways involved were studied by determinations of transcript levels of the genes encoding delta-5 desaturase (D5D and delta-6 desaturase (D6D, peroxisome proliferator-activated receptor alpha (PPARα and sterol regulatory element binding protein 1c (SREBP-1c. Mitogen-activated protein kinase kinase 1 (MEK1 and mitogen-activated protein kinase kinase kinase 1 (MEKK1 were also examined. Results Maximum conversion was observed in cells incubated with the mixture of [13C]LA/[13C]ALA at a ratio of 1:1, where 0.7% and 17% of the recovered [13C]ALA was converted to DHA and EPA, respectively. Furthermore, differential regulation of enzymes involved in the conversion at the transcript level, dependent on the ratio of administered n6 to n3 fatty acids in human hepatocytes was demonstrated. Conclusion Formation of EPA and DHA was highest at an administered LA/ALA ratio of 1:1, although gene expression of PPARα, SREBP-1c and D5D involved in ALA elongation were higher in the presence of ALA solely. Also, our findings suggest that a diet-induced enhancement of the cell membrane content of highly unsaturated fatty acids is only possible up to a certain level.

Glucosamine derived DISAL donors for stereoselective glycosylations under neutral conditions

DEFF Research Database (Denmark)

Grathe, S.; Thygesen, M.B.; Larsen, K.

2005-01-01

DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report the synthe......DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report...... the synthesis of new glucosamine DISAL donors, carrying N-TCP, -Troc, or -TFAc protecting groups, and their use in beta-(1,2-trans) selective glycosylations, primarily in NMP in the absence of any added Lewis acids, or in CH3NO2 with LiClO4. Finally, precise microwave heating proved effective in promoting...

Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

Science.gov (United States)

Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

2006-01-01

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

Effect of the Administration of Alpha-Lipoic Acid on Contrast Sensitivity in Patients with Type 1 and Type 2 Diabetes

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Anna Gębka

2014-01-01

Full Text Available The aim of this study was to estimate the effects of oral supplementation of alpha-lipoic acid (ALA on contrast sensitivity (CS in patients with type 1 diabetes mellitus (T1DM and type 2 diabetes mellitus (T2DM. The study included 12 patients with T1DM aged 43±12 years, 48 patients with T2DM aged 59±10 years, and 20 control subjects aged 33±8 years. Patients from each studied group, including the control group, were randomly assigned to receive 300 mg of ALA orally once daily for 3 months. CS was evaluated with the Functional Acuity Contrast Test (FACT, Stereo Optical. In the group of patients with T1DM receiving ALA for 3 months CS remained stable and improved in those with T2DM. Reduction of CS in both T1DM and T2DM patients without alpha-lipoic acid supplementation was observed. In the control group on alpha-lipoic acid supplementation, CS improvement was noticed at one spatial frequency. Changes in the CS were observed, despite stable visual acuity and eye fundus image in all studied subjects. Our study demonstrated that oral administration of alpha-lipoic acid had influence on CS in both T1DM and T2DM patients.

Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Tartaglio, Virginia [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Rennie, Emilie A. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Cahoon, Rebecca [Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Wang, George [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Baidoo, Edward [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Mortimer, Jennifer C. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Cahoon, Edgar B. [Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Scheller, Henrik V. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology

2016-09-19

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. In conclusion, this study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important role s for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.

Enantioselective synthesis of alpha,beta-disubstituted-beta-amino acids.

Science.gov (United States)

Sibi, Mukund P; Prabagaran, Narayanasamy; Ghorpade, Sandeep G; Jasperse, Craig P

2003-10-01

Highly diastereoselective and enantioselective addition of N-benzylhydroxylamine to imides 17 and 20-30 produces alpha,beta-trans-disubstituted N-benzylisoxazolidinones 19 and 31-41. These reactions proceed in 60-96% ee with 93-99% de's using 5 mol % of Mg(NTf2)2 and ligand 18. The product isoxazolidinones can be hydrogenolyzed directly to provide alpha,beta-disubstituted-beta-amino acids.

Serum alpha-tocopherol and ascorbic acid concentrations in Type 1 and Type 2 diabetic patients with and without angiopathy.

Science.gov (United States)

Skrha, Jan; Prázný, Martin; Hilgertová, Jirina; Weiserová, Hana

2003-03-01

Alpha-tocopherol and ascorbic acid form a part of scavenger system influencing the level of oxidative stress in diabetes mellitus. The aim of this study was to evaluate serum concentrations of alpha-tocopherol and ascorbic acid in Type 1 and Type 2 diabetes mellitus and to compare them with the presence of vascular complications as well as with oxidative stress and endothelial dysfunction. A total of 38 Type 1 and 62 Type 2 diabetic patients were subdivided into those with and without angiopathy. Serum alpha-tocopherol and ascorbic acid concentrations were estimated in all patients and in 38 healthy persons. Their results were compared with diabetes control, with oxidative stress measured by plasma malondialdehyde and with endothelial dysfunction estimated by serum N-acetyl-beta-glucosaminidase activity. In addition, the differences in biochemical variables were compared between patients with and without angiopathy. Serum alpha-tocopherol related to the sum of cholesterol and triglyceride concentrations (AT/CHT ratio) was significantly lower in diabetic patients with macroangiopathy than in those without vascular changes (pascorbic acid levels were significantly lower only in Type 2 diabetic patients with macroangiopathy as compared with healthy controls as well as with patients without vascular disease (pcholesterol or triglyceride concentrations in both Type 1 and Type 2 diabetic patients. The presence of oxidative stress together with endothelial dysfunction measured by N-acetyl-beta-glucosaminidase activity was accompanied by lower AT/CHT ratio (pascorbic acid concentration in serum. Their low concentrations may participate at the increased level of oxidative stress in these individuals.

Regulation of hepatic branched-chain alpha-keto acid dehydrogenase complex in rats fed a high-fat diet

Science.gov (United States)

Objective: Branched-chain alpha-keto acid dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of branched chain alpha-ketoacid (BCKA) catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal...

Activation of peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes

Energy Technology Data Exchange (ETDEWEB)

Kimura, Rino [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Takahashi, Nobuyuki, E-mail: nobu@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murota, Kaeko [Department of Life Science, School of Science and Engineering, Kinki University, Osaka 770-8503 (Japan); Yamada, Yuko [Laboratory of Physiological Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Niiya, Saori; Kanzaki, Noriyuki; Murakami, Yoko [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Moriyama, Tatsuya [Department of Applied Cell Biology, Graduate School of Agriculture, Kinki University, Nara 631-8505 (Japan); Goto, Tsuyoshi; Kawada, Teruo [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

2011-06-24

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of fatty acid oxidation-related genes in human intestinal epithelial Caco-2 cells. {yields} PPAR{alpha} activation also increased oxygen consumption rate and CO{sub 2} production and decreased secretion of triglyceride and ApoB from Caco-2 cells. {yields} Orally administration of bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and CO{sub 2} production in small intestinal epithelial cells. {yields} Treatment with bezafibrate decreased postprandial serum concentration of triglyceride after oral injection of olive oil in mice. {yields} It suggested that intestinal lipid metabolism regulated by PPAR{alpha} activation suppresses postprandial lipidemia. -- Abstract: Activation of peroxisome proliferator-activated receptor (PPAR)-{alpha} which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPAR{alpha} activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPAR{alpha} activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPAR{alpha} agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and

Intestinal epithelial cell surface glycosylation in mice. I. Effect of high-protein diet.

Science.gov (United States)

Gupta, R; Jaswal, V M; Meenu Mahmood, A

1992-01-01

The effects of variation in dietary protein content have been investigated on brush border glycosylation and enzyme activities in mice small intestine. The comparison of different parameters was made between the mice fed 30% (high protein, HP) and 18% protein (pair-fed, PF, and ad libitum-fed) for 21 days. The activities of brush border sucrase, lactase, p-nitrophenyl (PNP)-beta-D-glucosidase and PNP-beta-D-galactosidase were reduced in the HP diet-fed mice compared to PF and ad libitum-fed controls. Alkaline phosphatase and leucine amino-peptidase activities were significantly enhanced while gamma-glutamyl transpeptidase activity was unaltered under these conditions. Total hexoses and sialic acid content in the brush borders were reduced significantly in the test group compared to the controls while hexosamine and fucose contents remained essentially similar in different groups. The results on the binding of wheat germ agglutinin and Ulex europaeus agglutininI to microvillus membranes corroborated the chemical analysis data on sialic acid and fucose contents of the membranes. Peanut agglutinin binding was enhanced in mice from the HP group. Incorporation of (14C)-mannose into membranes was significantly less in HP diet-fed mice. These results indicate that the feeding of HP diet to mice brings about marked alterations in small intestinal epithelial cell surface glycosylation and enzyme functions.

Thioesterase activity and acyl-CoA/fatty acid cross-talk of hepatocyte nuclear factor-4{alpha}.

Science.gov (United States)

Hertz, Rachel; Kalderon, Bella; Byk, Tamara; Berman, Ina; Za'tara, Ghadeer; Mayer, Raphael; Bar-Tana, Jacob

2005-07-01

Hepatocyte nuclear factor-4alpha (HNF-4alpha) activity is modulated by natural and xenobiotic fatty acid and fatty acyl-CoA ligands as a function of their chain length, unsaturation, and substitutions. The acyl-CoA site of HNF-4alpha is reported here to consist of the E-F domain, to bind long-chain acyl-CoAs but not the respective free acids, and to catalyze the hydrolysis of bound fatty acyl-CoAs. The free acid pocket, previously reported in the x-ray structure of HNF-4alpha E-domain, entraps fatty acids but excludes acyl-CoAs. The acyl-CoA and free acid sites are distinctive and noncongruent. Free fatty acid products of HNF-4alpha thioesterase may exchange with free acids entrapped in the fatty acid pocket of HNF-4alpha. Cross-talk between the acyl-CoA and free fatty acid binding sites is abrogated by high affinity, nonhydrolyzable acyl-CoA ligands of HNF-4alpha that inhibit its thioesterase activity. Hence, HNF-4alpha transcriptional activity is controlled by its two interrelated acyl ligands and two binding sites interphased in tandem by the thioesterase activity. The acyl-CoA/free-acid and receptor/enzyme duality of HNF-4alpha extends the paradigm of nuclear receptors.

Effects of alpha-lipoic acids on sperm membrane integrity during liquid storage of boar semen

Directory of Open Access Journals (Sweden)

Laura Parlapan

2015-05-01

Full Text Available Preliminary studies have shown that sperm membrane from swine shows high sensitivity to cryopreservation process, causing a dramatic reduction in sperm quality. This has been attributed to the production of reactive oxygen species, that cause lipid peroxidation in sperm membranes. The aim of the present study was to minimize the oxidative attack by adding different concentration of alpha-lipoic acid into the sperm liquid storage at 17ºC for 7 days. Freshly ejaculated boar semen was diluted with Beltsville Thawing Solution (BTS and supplemented with 5 levels of alpha-lipoic  acid (0.015, 0.02, 0.05, 0.1, 0.15 mmol/ml. The membrane integrity was evaluated at days 0, 1, 3, 5 and 7 of liquid preservation, using flow cytometer FACSCanto II (BD Biociencias systems. The experiment indicate that supplementation of alpha-lipoic  acid to the semen liquid storage extender improve sperm membrane

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Science.gov (United States)

Avni, A; Avital, S; Gromet-Elhanan, Z

1991-04-25

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.

Genotoxicity evaluation of alpha-linolenic acid-diacylglycerol oil

Directory of Open Access Journals (Sweden)

Hiroshi Honda

Full Text Available The alpha-linolenic acid (ALA-diacylglycerol (DAG oil is an edible oil enriched with DAG (>80% and ALA (>50%. Although DAG oil, which mainly consists of oleic and linoleic acids has no genotoxic concerns, the fatty acid composition could affect the chemical property of DAG. Therefore, the purpose of this study was to evaluate the genotoxicity of ALA-DAG oil using standard genotoxicity tests in accordance with the OECD guidelines. ALA-DAG oil showed negative results in the bacterial reverse mutation test (Ames test and in vitro micronucleus test in cultured Chinese hamster lung cells with and without metabolic activation, and in the in vivo bone marrow micronucleus test in mice. Our results did not show any genotoxicity, suggesting that the fatty acid composition had no deleterious effects. We conclude that ALA-DAG oil had no genotoxicity concerns under the testing conditions. Keywords: Alpha-linolenic acid-rich diacylglycerol, Diacylglycerol, Alpha-linolenic acid, Fatty acid composition, Genotoxicity

Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

2014-11-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H. J.; Hensbergen, Paul J.; Reiding, Karli R.; Hazes, Johanna M. W.; Dolhain, Radboud J. E. M.; Wuhrer, Manfred

2014-01-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. PMID:25004930

Endoplasmic reticulum stress and N-glycosylation modulate expression of WFS1 protein

International Nuclear Information System (INIS)

Yamaguchi, Suguru; Ishihara, Hisamitsu; Tamura, Akira; Yamada, Takahiro; Takahashi, Rui; Takei, Daisuke; Katagiri, Hideki; Oka, Yoshitomo

2004-01-01

Mutations of the WFS1 gene are responsible for two hereditary diseases, Wolfram syndrome and low frequency sensorineural hearing loss. The WFS1 protein is a glycoprotein located in the endoplasmic reticulum (ER) membrane but its function is poorly understood. Herein we show WFS1 mRNA and protein levels in pancreatic islets to be increased with ER-stress inducers, thapsigargin and dithiothreitol. Another ER-stress inducer, the N-glycosylation inhibitor tunicamycin, also raised WFS1 mRNA but not protein levels. Site-directed mutagenesis showed both Asn-663 and Asn-748 to be N-glycosylated in mouse WFS1 protein. The glycosylation-defective WFS1 protein, in which Asn-663 and Asn-748 had been substituted with aspartate, exhibited an increased protein turnover rate. Consistent with this, the WFS1 protein was more rapidly degraded in the presence of tunicamycin. These data indicate that ER-stress and N-glycosylation play important roles in WFS1 expression and stability, and also suggest regulatory roles for this protein in ER-stress induced cell death

Asimetric synthesis of alpha-phenyl amino acids; Sintesis asimetrica de alfa-fenilaminoacidos

Energy Technology Data Exchange (ETDEWEB)

Caltiviela, C.; Diaz de Villegas, M.D.; Galvez, J.A.; Lapena, Y. [Departamento de Quimica Organica, Universidad de Zaragoza, Zaragoza (Spain)

1994-12-31

A new synthetic strategy to obtain alpha-phenyl amino acids in enantiomerically pure form is described. The new route is based on the highly diastereoselective alkylation of (1S,2R,4R)-10-dicyclohexylsulfamoylisobornyl 2-phenylcianoacetate and subsequent curtis type rearrangement of the alkylated compound with total retention of configuration. 17 refs

Tetranuclear copper(II) complexes bridged by alpha-D-glucose-1-phosphate and incorporation of sugar acids through the Cu4 core structural changes.

Science.gov (United States)

Kato, Merii; Sah, Ajay Kumar; Tanase, Tomoaki; Mikuriya, Masahiro

2006-08-21

Tetranuclear copper(II) complexes containing alpha-D-glucose-1-phosphate (alpha-D-Glc-1P), [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(bpy)4(H2O)2]X3 [X = NO3 (1a), Cl (1b), Br (1c)], and [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(phen)4(H2O)2](NO3)3 (2) were prepared by reacting the copper(II) salt with Na2[alpha-D-Glc-1P] in the presence of diimine ancillary ligands, and the structure of 2 was characterized by X-ray crystallography to comprise four {Cu(phen)}2+ fragments connected by the two sugar phosphate dianions in 1,3-O,O' and 1,1-O mu4-bridging fashion as well as a mu-hydroxo anion. The crystal structure of 2 involves two chemically independent complex cations in which the C2 enantiomeric structure for the trapezoidal tetracopper(II) framework is switched according to the orientation of the alpha-D-glucopyranosyl moieties. Temperature-dependent magnetic susceptibility data of 1a indicated that antiferromagnetic spin coupling is operative between the two metal ions joined by the hydroxo bridge (J = -52 cm(-1)) while antiferromagnetic interaction through the Cu-O-Cu sugar phosphate bridges is weak (J = -13 cm(-1)). Complex 1a readily reacted with carboxylic acids to afford the tetranuclear copper(II) complexes, [Cu4{mu-(alpha-D-Glc-1P)}2(mu-CA)2(bpy)4](NO3)2 [CA = CH3COO (3), o-C6H4(COO)(COOH) (4)]. Reactions with m-phenylenediacetic acid [m-C6H4(CH2COOH)2] also gave the discrete tetracopper(II) cationic complex [Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)(CH2COOH))2(bpy)4](NO3)2 (5a) as well as the cluster polymer formulated as {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)2)(bpy)4](NO3)2}n (5b). The tetracopper structure of 1a is converted into a symmetrical rectangular core in complexes 3, 4, and 5b, where the hydroxo bridge is dissociated and, instead, two carboxylate anions bridge another pair of Cu(II) ions in a 1,1-O monodentate fashion. The similar reactions were applied to incorporate sugar acids onto the tetranuclear copper(II) centers. Reactions of 1a with delta

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation

DEFF Research Database (Denmark)

Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian

2015-01-01

optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylationrelated product quality. In this work, different fed-batch processes with two chemically defined proprietary media......Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process...... and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality....

Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

Energy Technology Data Exchange (ETDEWEB)

Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

2011-04-22

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

Science.gov (United States)

Saito, M; Takenouchi, Y; Kunisaki, N; Kimura, S

2001-05-01

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1)*

Science.gov (United States)

Zhuo, You; Yang, Jeong-Yeh; Moremen, Kelley W.; Prestegard, James H.

2016-01-01

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions. PMID:27471271

High frequency of HIF-1 alpha overexpression in BRCA1 related breast cancer

NARCIS (Netherlands)

van der Groep, Petra; Bouter, Alwin; Menko, Fred H.; van der Wall, Elsken; van Diest, Paul J.

2008-01-01

Hypoxia is a hallmark of cancer. Hypoxia inducible factor-1 alpha (HIF-1 alpha) is the key regulator of the hypoxia response. HIF-1 alpha is overexpressed during sporadic breast carcinogenesis and correlated with poor prognosis. Little is known on the role of HIF-1 alpha in hereditary breast

Cytokine vaccination: neutralising IL-1alpha autoantibodies induced by immunisation with homologous IL-1alpha

DEFF Research Database (Denmark)

Svenson, M; Hansen, M B; Thomsen, Allan Randrup

2000-01-01

with IL-1alpha coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1alpha. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1beta. The induced anti-IL-1alpha aAb inhibited binding...... in mice by vaccination with recombinant murine IL-1alpha conjugated to PPD. Studies of the effects of IL-1alpha aAb in such animals may help clarify the importance of naturally occurring IL-1alpha aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients...

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

Energy Technology Data Exchange (ETDEWEB)

Velho, Albertina M. [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); Jarvis, Simon M., E-mail: S.M.Jarvis@westminster.ac.uk [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); University of Westminster, School of Biosciences, London W1W 6UW (United Kingdom)

2009-08-01

The Na{sup +}-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 ({approx} 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated ({approx} 68-90 kDa) with only 31-65% of WT L-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered ({approx} 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at {approx} 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 {sup o}C or 37 {sup o}C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.

Nutritional Therapies in Congenital Disorders of Glycosylation (CDG

Directory of Open Access Journals (Sweden)

Peter Witters

2017-11-01

Full Text Available Congenital disorders of glycosylation (CDG are a group of more than 130 inborn errors of metabolism affecting N-linked, O-linked protein and lipid-linked glycosylation. The phenotype in CDG patients includes frequent liver involvement, especially the disorders belonging to the N-linked protein glycosylation group. There are only a few treatable CDG. Mannose-Phosphate Isomerase (MPI-CDG was the first treatable CDG by high dose mannose supplements. Recently, with the successful use of d-galactose in Phosphoglucomutase 1 (PGM1-CDG, other CDG types have been trialed on galactose and with an increasing number of potential nutritional therapies. Current mini review focuses on therapies in glycosylation disorders affecting liver function and dietary intervention in general in N-linked glycosylation disorders. We also emphasize now the importance of early screening for CDG in patients with mild hepatopathy but also in cholestasis.

[Dietary supplementation of obese children with 1000 mg alpha-linolenic acid per day: a placebo-controlled double blind study].

Science.gov (United States)

Lohner, Szimonetta; Marosvölgyi, Tamás; Burus, István; Schmidt, János; Molnár, Dénes; Decsi, Tamás

2007-08-12

Enhanced dietary intake of omega-3 fatty acids may benefit persons with increased cardiovascular risk, among them obese subjects. Incorporation of omega-3 fatty acids into the plasma lipids is a prerequisite to achieve the favorable effects; however, only very few data are available on the dose of omega-3 fatty acid supplementation in children. The aim of our study was to examine the effects of the consumption of a diet supplemented with 1000 mg alpha-linolenic acid daily on plasma lipids in obese children. In this two times six-week-long, placebo-controlled, crossover study, 9 obese children (age: 13.1 [2.5] years, body mass index: 31.2 [6.2] kg/m 2 ), median [IQR]) incorporated into their diet one egg and one meatball (50 g) per day from hens fed diets containing flaxseed oil, i.e. supplementary dietary intake of 1000 mg alpha-linolenic acid per day was provided. The fatty acid composition of plasma lipids was determined by high-resolution gas-liquid chromatography. Tendencies of increase were observed in the alpha-linolenic acid content of plasma lipids in the phospholipid, triacyl-glycerine and sterol-ester fractions after the supplementation with alpha-linolenic acid. In the non-esterified fatty acid fraction, the values of alpha-linolenic acid were significantly higher after the supplementation (0.11 [0.08] versus 0.14 [0.20], % weight/weight, p < 0.05), indicating the beginning of the accumulation of alpha-linolenic acid in plasma lipids. In obese children a six-week-long supplementation of the diet with 1000 mg alpha-linolenic acid per day increased significantly the contribution of omega-3 fatty acids only to the non-esterified fatty acids of plasma lipids, but had no significant effect on the esterified fractions. Increase of the dose of supplementation may be needed to influence omega-3 fatty acid status in obese children.

Crystal Structures of Glycosyltransferase UGT78G1 Reveal the Molecular Basis for Glycosylation and Deglycosylation of (Iso)flavonoids

Energy Technology Data Exchange (ETDEWEB)

Modolo, Luzia V.; Li, Lenong; Pan, Haiyun; Blount, Jack W.; Dixon, Richard A.; Wang, Xiaoqiang; (SRNF)

2010-09-21

The glycosyltransferase UGT78G1 from Medicago truncatula catalyzes the glycosylation of various (iso)flavonoids such as the flavonols kaempferol and myricetin, the isoflavone formononetin, and the anthocyanidins pelargonidin and cyanidin. It also catalyzes a reverse reaction to remove the sugar moiety from glycosides. The structures of UGT78G1 bound with uridine diphosphate or with both uridine diphosphate and myricetin were determined at 2.1 {angstrom} resolution, revealing detailed interactions between the enzyme and substrates/products and suggesting a distinct binding mode for the acceptor/product. Comparative structural analysis and mutagenesis identify glutamate 192 as a key amino acid for the reverse reaction. This information provides a basis for enzyme engineering to manipulate substrate specificity and to design effective biocatalysts with glycosylation and/or deglycosylation activity.

Efficient synthesis of glycosylated phenazine natural products and analogs with DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors

DEFF Research Database (Denmark)

Laursen, Jane B.; Petersen, Lars; Jensen, K.J.

2003-01-01

Inspired by the occurrence and function of phenazines in natural products, new glycosylated analogs were designed and synthesized. DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors were used in an efficient and easily-handled glycosylation protocol compatible with combinatorial chemistry....... Benzoylated D-glucose, D-galactose and L-quinovose DISAL glycosyl donors were synthesized in high yields and used under mild conditions to glycosylate methyl saphenate and 2-hydroxyphenazine. The glycosides were screened for biological activity and one compound showed inhibitory activity towards topoisomerase...

Alpha 1 B- but not alpha 1 A-adrenoceptors mediate inositol phosphate generation

NARCIS (Netherlands)

Michel, M. C.; Hanft, G.; Gross, G.

1990-01-01

We used novel highly subtype-selective antagonists to study whether alpha 1A- and/or alpha 1B-adrenoceptors mediate the stimulation of inositol phosphate generation by noradrenaline in rat cerebral cortex. Phentolamine (10 microM) and prazosin (100 nM) completely abolished the stimulated inositol

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla...

Pharmacologically relevant receptor binding characteristics and 5alpha-reductase inhibitory activity of free Fatty acids contained in saw palmetto extract.

Science.gov (United States)

Abe, Masayuki; Ito, Yoshihiko; Oyunzul, Luvsandorj; Oki-Fujino, Tomomi; Yamada, Shizuo

2009-04-01

Saw palmetto extract (SPE), used widely for the treatment of benign prostatic hyperplasia (BPH) has been shown to bind alpha(1)-adrenergic, muscarinic and 1,4-dihydropyridine (1,4-DHP) calcium channel antagonist receptors. Major constituents of SPE are lauric acid, oleic acid, myristic acid, palmitic acid and linoleic acid. The aim of this study was to investigate binding affinities of these fatty acids for pharmacologically relevant (alpha(1)-adrenergic, muscarinic and 1,4-DHP) receptors. The fatty acids inhibited specific [(3)H]prazosin binding in rat brain in a concentration-dependent manner with IC(50) values of 23.8 to 136 microg/ml, and specific (+)-[(3)H]PN 200-110 binding with IC(50) values of 24.5 to 79.5 microg/ml. Also, lauric acid, oleic acid, myristic acid and linoleic acid inhibited specific [(3)H]N-methylscopolamine ([(3)H]NMS) binding in rat brain with IC(50) values of 56.4 to 169 microg/ml. Palmitic acid had no effect on specific [(3)H]NMS binding. The affinity of oleic acid, myristic acid and linoleic acid for each receptor was greater than the affinity of SPE. Scatchard analysis revealed that oleic acid and lauric acid caused a significant decrease in the maximal number of binding sites (B(max)) for [(3)H]prazosin, [(3)H]NMS and (+)-[(3)H]PN 200-110. The results suggest that lauric acid and oleic acid bind noncompetitively to alpha(1)-adrenergic, muscarinic and 1,4-DHP calcium channel antagonist receptors. We developed a novel and convenient method of determining 5alpha-reductase activity using LC/MS. With this method, SPE was shown to inhibit 5alpha-reductase activity in rat liver with an IC(50) of 101 microg/ml. Similarly, all the fatty acids except palmitic acid inhibited 5alpha-reductase activity, with IC(50) values of 42.1 to 67.6 microg/ml. In conclusion, lauric acid, oleic acid, myristic acid, and linoleic acid, major constituents of SPE, exerted binding activities of alpha(1)-adrenergic, muscarinic and 1,4-DHP receptors and inhibited 5

Nonenzymatic glycosylation of human hemoglobin at multiple sites

International Nuclear Information System (INIS)

Shapiro, R.; McManus, M.; Garrick, L.; McDonald, M.J.; Bunn, H.F.

1979-01-01

The most abundant minor hemoglobin component of human hemolysate is Hb A1c, which has glucose bound to the N-terminus of the beta chain by a ketoamine linkage. Hb A1c is formed slowly and continuously throughout the 120 day lifespan of the red cell. It can be synthesized in vitro by incubating purified hemoglobin with 14C-glucose. Other minor components, Hb A1a1 and Hb A1a2 are adducts of sugar phosphates at the N-terminus of the beta chain. Hb A1b contains an unidentified nonphosphorylated sugar at the beta N-terminus. In addition, a significant portion of the major hemoglobin component (Hb Ao) is also glycosylated by a glucose ketoamine linkage at other sites on the molecule, including the N-terminus of the alpha chain and the epsilon-amino group of several lysine residues on both the alpha and the beta chains. The results indicate that the interaction of glucose and hemoglobin is rather nonspecific and suggests that other proteins are modified in a similar fashion

Comprehensive evaluation of the flavonol anti-oxidants, alpha-glycosyl isoquercitrin and isoquercitrin, for genotoxic potential.

Science.gov (United States)

Hobbs, Cheryl A; Koyanagi, Mihoko; Swartz, Carol; Davis, Jeffrey; Kasamoto, Sawako; Maronpot, Robert; Recio, Leslie; Hayashi, Shim-Mo

2018-03-01

Quercetin and its glycosides possess potential benefits to human health. Several flavonols are available to consumers as dietary supplements, promoted as anti-oxidants; however, incorporation of natural quercetin glycosides into food and beverage products has been limited by poor miscibility in water. Enzymatic conjugation of multiple glucose moieties to isoquercitrin to produce alpha-glycosyl isoquercitrin (AGIQ) enhances solubility and bioavailability. AGIQ is used in Japan as a food additive and has been granted generally recognized as safe (GRAS) status. However, although substantial genotoxicity data exist for quercetin, there is very little available data for AGIQ and isoquercitrin. To support expanded global marketing of food products containing AGIQ, comprehensive testing of genotoxic potential of AGIQ and isoquercitrin was conducted according to current regulatory test guidelines. Both chemicals tested positive in bacterial reverse mutation assays, and exposure to isoquercitrin resulted in chromosomal aberrations in CHO-WBL cells. All other in vitro mammalian micronucleus and chromosomal aberration assays, micronucleus and comet assays in male and female B6C3F1 mice and Sprague Dawley rats, and Muta™ Mouse mutation assays evaluating multiple potential target tissues, were negative for both chemicals. These results supplement existing toxicity data to further support the safe use of AGIQ in food and beverage products. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Multiple forms of the human tyrosine phosphatase RPTP alpha. Isozymes and differences in glycosylation

DEFF Research Database (Denmark)

Daum, G; Regenass, S; Sap, J

1994-01-01

Among all the receptor-linked protein-tyrosine-phosphatase RPTP alpha clones described from mammalian tissues, one differed in that it encoded a 9-amino-acid insert 3 residues upstream from the transmembrane segment (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R. Ravera, M., Ricca, G...

Anticonvulsant activity of a mGlu(4alpha) receptor selective agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid.

Science.gov (United States)

Chapman, A G; Talebi, A; Yip, P K; Meldrum, B S

2001-07-20

The metabotropic Group III agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1), selective for the mGlu(4alpha) receptor, suppresses sound-induced seizures in DBA/2 mice following its intracerebroventricular (i.c.v.) administration (ED(50) 5.6 [2.9-10.7], nmol i.c.v., 15 min, clonic phase) and in genetically epilepsy-prone (GEP) rats following focal administration into the inferior colliculus (ED(50) 0.08 [0.01-0.50], nmol, 60 min, clonic phase). ACPT-1 also protects against clonic seizures induced in DBA/2 mice by the Group I agonist, (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG) (ED(50) 0.60 [0.29-1.2], nmol i.c.v.) and by the Group III antagonist, (RS)-alpha-methylserine-O-phosphate (MSOP) (ED(50) 49.3 [37.9-64.1], nmol i.c.v.). Another Group III agonist, (RS)-4-phosphonophenyl-glycine (PPG), preferentially activating the mGlu(8) receptor, previously shown to protect against sound-induced seizures in DBA/2 mice and GEP rats, also protects against seizures induced in DBA/2 by 3,5-DHPG (ED(50) 3.7 [2.4-5.7], nmol i.c.v.) and by the Group III antagonist, MSOP (ED(50) 40.2 [21.0-77.0], nmol i.c.v.). At very high doses (500 nmol i.c.v. and above), Group III antagonists have pro-convulsant and convulsant activity. The anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(4) receptor agonist ACPT-1, is partially reversed by the co-administration of the Group III antagonists, MSOP, (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) or (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), in the 20-50 nmol dose range. At doses of 50-200 nmol, MPPG and MAP4 cause further reversal of the ACPT-1 anticonvulsant protection, while the MSOP effect on ACPT-1 protection is abolished at higher doses. In contrast, the anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(8) receptor agonist PPG, is not

Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors.

Science.gov (United States)

Jayaraman, Akila; Koh, Xiaoying; Li, Jing; Raman, Rahul; Viswanathan, Karthik; Shriver, Zachary; Sasisekharan, Ram

2012-06-15

The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus. In the present study, it was demonstrated that one such conserved glycosylation site at Asn(91) in H1N1 HA critically governs the glycan receptor-binding specificity and hence would potentially impinge on the host adaptation of the virus.

Impact of O-glycosylation on the molecular and cellular adhesion properties of the Escherichia coli autotransporter protein Ag43.

Science.gov (United States)

Reidl, Sebastian; Lehmann, Annika; Schiller, Roswitha; Salam Khan, A; Dobrindt, Ulrich

2009-08-01

Antigen 43 (Ag43) represents an entire family of closely related autotransporter proteins in Escherichia coli and has been described to confer aggregation and fluffing of cells, to promote biofilm formation, uptake and survival in macrophages as well as long-term persistence of uropathogenic E. coli in the murine urinary tract. Furthermore, it has been reported that glycosylation of the Ag43 passenger domain (alpha(43)) stabilizes its conformation and increases adhesion to Hep-2 cells. We characterized the role of Ag43 as an adhesin and the impact of O-glycosylation on the function of Ag43. To analyze whether structural variations in the alpha(43) domain correlate with different functional properties, we cloned 5 different agn43 alleles from different E. coli subtypes and tested them for autoaggregation, biofilm formation, adhesion to different eukaryotic cell lines as well as to purified components of the extracellular matrix. These experiments were performed with nonglycosylated and O-glycosylated Ag43 variants. We show for the first time that Ag43 mediates bacterial adhesion in a cell line-specific manner and that structural variations of the alpha(43) domain correlate with increased adhesive properties to proteins of the extracellular matrix such as collagen and laminin. Whereas O-glycosylation of many alpha(43) domains led to impaired autoaggregation and a significantly reduced adhesion to eukaryotic cell lines, their interaction with collagen was significantly increased. These data demonstrate that O-glycosylation is not a prerequisite for Ag43 function and that the different traits mediated by Ag43, i.e., biofilm formation, autoaggregation, adhesion to eukaryotic cells and extracellular matrix proteins, rely on distinct mechanisms.

Corrosion inhibition of mild steel in 1 M HCl solution by henna extract: A comparative study of the inhibition by henna and its constituents (Lawsone, Gallic acid, {alpha}-D-Glucose and Tannic acid)

Energy Technology Data Exchange (ETDEWEB)

Ostovari, A. [Technical Inspection Engineering Department, Petroleum University of Technology, Abadan (Iran, Islamic Republic of)], E-mail: A.Ostovari@gmail.com; Hoseinieh, S.M.; Peikari, M. [Technical Inspection Engineering Department, Petroleum University of Technology, Abadan (Iran, Islamic Republic of); Shadizadeh, S.R. [Petroleum Engineering Department, Petroleum University of Technology, Abadan (Iran, Islamic Republic of); Hashemi, S.J. [Technical Inspection Engineering Department, Petroleum University of Technology, Abadan (Iran, Islamic Republic of)

2009-09-15

The inhibitive action of henna extract (Lawsonia inermis) and its main constituents (lawsone, gallic acid, {alpha}-D-Glucose and tannic acid) on corrosion of mild steel in 1 M HCl solution was investigated through electrochemical techniques and surface analysis (SEM/EDS). Polarization measurements indicate that all the examined compounds act as a mixed inhibitor and inhibition efficiency increases with inhibitor concentration. Maximum inhibition efficiency (92.06%) is obtained at 1.2 g/l henna extract. Inhibition efficiency increases in the order: lawsone > henna extract > gallic acid > {alpha}-D-Glucose > tannic acid. Also, inhibition mechanism and thermodynamic parameters are discussed.

Glycosylation of DMP1 Is Essential for Chondrogenesis of Condylar Cartilage.

Science.gov (United States)

Weng, Y; Liu, Y; Du, H; Li, L; Jing, B; Zhang, Q; Wang, X; Wang, Z; Sun, Y

2017-12-01

The mandibular condylar cartilage (MCC) shoulders force for the subchondral bone during mastication. The cartilage matrix contains various large molecules, such as type I, II, and X collagens and proteoglycans (PGs), which jointly play essential roles in maintaining cartilage characteristics. PGs play key roles in maintaining the elasticity of cartilage and providing a cushion against mastication forces. In addition to the well-known PGs, DMP1-PG, which is the PG form of dentin matrix protein 1 (DMP1), is a newly identified PG. DMP1 is proteolytically processed in vivo, and the N-terminus is glycosylated into its PG form-that is, DMP1-PG, which is highly expressed not only in tooth and bone but also in the matrix of the MCC. However, the specific functions of DMP1-PG in the MCC remain unclear. In human temporomandibular joint osteoarthritis and hyperocclusion model rat specimens, PGs are significantly downregulated, and DMP1-PG is the most prominently affected PG. To further investigate the role of DMP1-PG in condylar chondrogenesis, a glycosylation site mutant (S 89 -G 89 ) mouse model was established with knock-in methods. In the MCC of the S89G-DMP1 mice, the glycosylation level of DMP1 was significantly downregulated, and a series of abnormal developmental and pathologic changes could be observed. The morphologic changes included thinner cartilage layers, deformations of the MCC, and disordered arrangements of the chondrocytes, and an earlier onset of temporomandibular joint osteoarthritis-like changes was observed. In addition, markers of chondrogenesis were downregulated, and the matrix of the MCC displayed OA phenotypes in the S89G-DMP1 mice. Further investigations showed that the transforming growth factor β signaling molecules were affected in the MCC after the loss of DMP1-PG. In addition, the loss of DMP1-PG significantly accelerated the progression of cartilage injuries in the hyperocclusion models. Given these findings, we investigated the significant

Charge and Polarity Preferences for N-Glycosylation: A Genome-Wide In Silico Study and Its Implications Regarding Constitutive Proliferation and Adhesion of Carcinoma Cells.

Science.gov (United States)

Manwar Hussain, Muhammad Ramzan; Iqbal, Zeeshan; Qazi, Wajahat M; Hoessli, Daniel C

2018-01-01

The structural and functional diversity of the human proteome is mediated by N - and O- linked glycosylations that define the individual properties of extracellular and membrane-associated proteins. In this study, we utilized different computational tools to perform in silico based genome-wide mapping of 1,117 human proteins and unravel the contribution of both penultimate and vicinal amino acids for the asparagine-based, site-specific N -glycosylation. Our results correlate the non-canonical involvement of charge and polarity environment of classified amino acids (designated as L, O, A, P, and N groups) in the N -glycosylation process, as validated by NetNGlyc predictions, and 130 literature-reported human proteins. From our results, particular charge and polarity combinations of non-polar aliphatic, acidic, basic, and aromatic polar side chain environment of both penultimate and vicinal amino acids were found to promote the N -glycosylation process. However, the alteration in side-chain charge and polarity environment of genetic variants, particularly in the vicinity of Asn-containing epitope, may induce constitutive glycosylation (e.g., aberrant glycosylation at preferred and non-preferred sites) of membrane proteins causing constitutive proliferation and triggering epithelial-to-mesenchymal transition. The current genome-wide mapping of 1,117 proteins (2,909 asparagine residues) was used to explore charge- and polarity-based mechanistic constraints in N -glycosylation, and discuss alterations of the neoplastic phenotype that can be ascribed to N -glycosylation at preferred and non-preferred sites.

Prion propagation in cells expressing PrP glycosylation mutants.

Science.gov (United States)

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Hydrogen/deuterium exchange of cross-linkable alpha-amino acid derivatives in deuterated triflic acid

OpenAIRE

Wang, Lei; Murai, Yuta; Yoshida, Takuma; Okamoto, Masashi; Masuda, Katsuyoshi; Sakihama, Yasuko; Hashidoko, Yasuyuki; Hatanaka, Yasumaru; Hashimoto, Makoto

2014-01-01

In this paper we report here a hydrogen/deuterium exchange (H/D exchange) of cross-linkable alpha-amino acid derivatives with deuterated trifluoromethanesulfonic acid (TfOD). H/D exchange with TfOD was easily applied to o-catechol containing phenylalanine (DOPA) within an hour. A partial H/D exchange was observed for trifluoromethyldiazirinyl (TFMD) phenylalanine derivatives. N-Acetyl-protected natural aromatic alpha-amino acids (Tyr and Trp) were more effective in H/D exchange than unprotect...

Pre-column derivatisation method for the measurement of glycosylated hydroxylysines of collagenous proteins

NARCIS (Netherlands)

Bank, R.A.; Beekman, B.; Tenni, R.; Tekoppele, J.M.

1997-01-01

Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Energy Technology Data Exchange (ETDEWEB)

Kilcoyne, Michelle; Sharma, Shashank [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McDevitt, Niamh; O' Leary, Claire [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland); Joshi, Lokesh [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McMahon, Siobhan S., E-mail: siobhan.mcmahon@nuigalway.ie [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland)

2012-04-13

Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment

Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-06-01

Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

Ultrasound-Assisted Synthesis of 1-N-glycosyl-1H-1,2,3-Triazole Derivatives and their Anti-inflammatory Activity

Directory of Open Access Journals (Sweden)

Gilson Bezerra Silva

2012-06-01

Full Text Available We have been synthesized various glycosyl triazoles from the reaction between glycosyl azide (1 and terminal alkynes (2a-g. The glycopiranosyl 1,2,3-triazoles (3-9 have been obtained in moderate-to-excellent yields (63-99% through the copper (I-catalyst 1,3-dipolar cycloaddition reaction at room temperature using ultrasound irradiation. In addition, preliminary anti-inflammatory tests have been performed in the compounds conjugates with benzoheterocycles (3-7 moieties that shown moderate activity

Identification of high-mannose and multiantennary complex-type N-linked glycans containing alpha-galactose epitopes from Nurse shark IgM heavy chain.

Science.gov (United States)

Harvey, David J; Crispin, Max; Moffatt, Beryl E; Smith, Sylvia L; Sim, Robert B; Rudd, Pauline M; Dwek, Raymond A

2009-11-01

MALDI-TOF mass spectrometry, negative ion nano-electrospray MS/MS and exoglycosidase digestion were used to identify 36 N-linked glycans from 19S IgM heavy chain derived from the nurse shark (Ginglymostoma cirratum). The major glycan was the high-mannose compound, Man(6)GlcNAc(2) accompanied by small amounts of Man(5)GlcNAc(2), Man(7)GlcNAc(2) and Man(8)GlcNAc(2). Bi- and tri-antennary (isomer with a branched 3-antenna) complex-type glycans were also abundant, most contained a bisecting GlcNAc residue (beta1-->4-linked to the central mannose) and with varying numbers of alpha-galactose residues capping the antennae. Small amounts of monosialylated glycans were also found. This appears to be the first comprehensive study of glycosylation in this species of animal. The glycosylation pattern has implications for the mechanism of activation of the complement system by nurse shark IgM.

Dissociation of branched-chain alpha-keto acid dehydrogenase kinase (BDK) from branched-chain alpha-keto acid dehydrogenase complex (BCKDC) by BDK inhibitors.

Science.gov (United States)

Murakami, Taro; Matsuo, Masayuki; Shimizu, Ayako; Shimomura, Yoshiharu

2005-02-01

Branched-chain alpha-keto acid dehydrogenase kinase (BDK) phosphorylates and inactivates the branched-chain alpha-keto acid dehydrogenase complex (BCKDC), which is the rate-limiting enzyme in the branched-chain amino acid catabolism. BDK has been believed to be bound to the BCKDC. However, recent our studies demonstrated that protein-protein interaction between BDK and BCKDC is one of the factors to regulate BDK activity. Furthermore, only the bound form of BDK appears to have its activity. In the present study, we examined effects of BDK inhibitors on the amount of BDK bound to the BCKDC using rat liver extracts. The bound form of BDK in the extracts of liver from low protein diet-fed rats was measured by an immunoprecipitation pull down assay with or without BDK inhibitors. Among the BDK inhibitors. alpha-ketoisocaproate, alpha-chloroisocaproate, and a-ketoisovalerate released the BDK from the complex. Furthermore, the releasing effect of these inhibitors on the BDK appeared to depend on their inhibition constants. On the other hand, clofibric acid and thiamine pyrophosphate had no effect on the protein-protein interaction between two enzymes. These results suggest that the dissociation of the BDK from the BCKDC is one of the mechanisms responsible for the action of some inhibitors to BDK.

Glycosylation Engineering

DEFF Research Database (Denmark)

Clausen, Henrik; Wandall, Hans H.; Steentoft, Catharina

2017-01-01

Knowledge of the cellular pathways of glycosylation across phylogeny provides opportunities for designing glycans via genetic engineering in a wide variety of cell types including bacteria, fungi, plant cells, and mammalian cells. The commercial demand for glycosylation engineering is broad......, including production of biological therapeutics with defined glycosylation (Chapter 57). This chapter describes how knowledge of glycan structures and their metabolism (Parts I–III of this book) has led to the current state of glycosylation engineering in different cell types. Perspectives for rapid...

Endocytosis of GPI-linked membrane folate receptor-alpha.

Science.gov (United States)

Rijnboutt, S; Jansen, G; Posthuma, G; Hynes, J B; Schornagel, J H; Strous, G J

1996-01-01

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region.

Science.gov (United States)

van de Bovenkamp, Fleur S; Derksen, Ninotska I L; Ooijevaar-de Heer, Pleuni; van Schie, Karin A; Kruithof, Simone; Berkowska, Magdalena A; van der Schoot, C Ellen; IJspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E M; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S Marieke; Vidarsson, Gestur; Rispens, Theo

2018-02-20

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N -linked glycans, a process conditional on the introduction of consensus amino acid motifs ( N -glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

Stannylene‐Mediated Regioselective 6‐O‐Glycosylation of Unprotected Phenyl 1‐Thioglycopyranosides

DEFF Research Database (Denmark)

Maggi, Agnese; Madsen, Robert

2013-01-01

acetal, and then subjected to selective glycosylation at the 6‐position with the Koenigs–Knorr protocol. Peracylated glycosyl bromides of D‐glucose, D‐galactose, D‐mannose and D‐glucosamine were employed as the donors to give the corresponding (1→6)‐linked disaccharides in moderate to good yields......‐thio‐β‐D‐glucopyranoside gave rise to the corresponding (1→6)‐linked trisaccharides in moderate yields....

Modeling the mechanism of glycosylation reactions between ethanol, 1,2-ethanediol and methoxymethanol.

Science.gov (United States)

Azofra, Luis Miguel; Alkorta, Ibon; Toro-Labbé, Alejandro; Elguero, José

2013-09-07

The mechanism of the S(N)2 model glycosylation reaction between ethanol, 1,2-ethanediol and methoxymethanol has been studied theoretically at the B3LYP/6-311+G(d,p) computational level. Three different types of reactions have been explored: (i) the exchange of hydroxyl groups between these model systems; (ii) the basic catalysis reactions by combination of the substrates as glycosyl donors (neutral species) and acceptors (enolate species); and (iii) the effect on the reaction profile of an explicit H2O molecule in the reactions considered in (ii). The reaction force, the electronic chemical potential and the reaction electronic flux have been characterized for the reaction path in each case. Energy calculations show that methoxymethanol is the worst glycosyl donor model among the ones studied here, while 1,2-ethanediol is the best, having the lowest activation barrier of 74.7 kJ mol(-1) for the reaction between this one and the ethanolate as the glycosyl acceptor model. In general, the presence of direct interactions between the atoms involved in the penta-coordinated TS increases the activation energies of the processes.

N-Glycosylation of Lipocalin 2 Is Not Required for Secretion or Exosome Targeting

Directory of Open Access Journals (Sweden)

Erawan Borkham-Kamphorst

2018-04-01

Full Text Available Lipocalin 2 (LCN2 is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.

Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

Science.gov (United States)

Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

2017-10-31

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular cloning and characterization of an alpha-amylase from Pichia burtonii 15-1.

Science.gov (United States)

Kato, Saemi; Shimizu-Ibuka, Akiko; Mura, Kiyoshi; Takeuchi, Akiko; Tokue, Chiyoko; Arai, Soichi

2007-12-01

An alpha-amylase secreted by Pichia burtonii 15-1 isolated from a traditional starter murcha of Nepal, named Pichia burtonii alpha-amylase (PBA), was studied. The gene was cloned and its nucleotide sequence was determined. PBA was deduced to consist of 494 amino acid residues. It shared certain degrees of amino acid sequence identity with other homologous proteins: 60% with Schwanniomyces occidentalis alpha-amylase, 58% with Saccharomycopsis sp. alpha-amylase, and 47% with Taka-amylase A from Aspergillus oryzae. A three-dimensional structural model of PBA generated using the known three-dimensional structure of Taka-amylase A as a template suggested high structural similarity between them. Kinetic analysis revealed that the K(m) values of PBA were lower than those of Taka-amylase A for the oligosaccharides. Although the k(cat) values of PBA were lower than those of Taka-amylase A for the oligosaccharide substrates, the k(cat)/K(m) values of PBA were higher.

Measurement of Glycosylated Alpha-Fetoprotein Improves Diagnostic Power over the Native Form in Hepatocellular Carcinoma

Science.gov (United States)

Jin, Jonghwa; Park, Jiyoung; Yu, Su Jong; Yoon, Jung-Hwan; Kim, Youngsoo

2014-01-01

Serum alpha-fetoprotein (AFP) has long been used as a diagnostic marker for hepatocellular carcinoma (HCC), albeit controversially. Although it remains widely used in clinics, the value of AFP in HCC diagnosis has recently been challenged due to its significant rates of false positive and false negative findings. To improve the efficacy of AFP as HCC diagnostic marker, we developed a method of measuring total and glycosylated AFP by multiple reaction monitoring (MRM)-MS. In this study, we verified the total amount of AFP (nonglycopeptide levels) and the degree of glycosylated AFP (deglycopeptide levels) in 60 normal (41 men and 19 women; mean age 53 years; range 32–74 years), 35 LC (23 men and 12 women; mean age 56 years; range 43–78 years; HBV-related), and 60 HCC subjects (42 men and 18 women; mean age 58 years; range 38–76 years; HBV-related; 30 stage I, 15 stage II, and 10 stage III). By MRM-MS analysis, the nonglycopeptide had 56.7% sensitivity, 68.3% specificity, and an AUC of 0.687 [cutoff value: ≥0.02 (light/heavy ratio)], comparing the normal and HCC group, whereas the deglycopeptide had 93.3% sensitivity, 68.3% specificity, and an AUC of 0.859 [cutoff value: ≥0.02 (light/heavy ratio)]. In comparing the stage I HCC subgroup with the LC group, the nonglycopeptide had a sensitivity of 66.7%, specificity of 80.0%, and an AUC of 0.712 [cutoff value: ≥0.02 (light/heavy ratio)], whereas the deglycopeptide had a sensitivity of 96.7%, specificity of 80.0%, and an AUC of 0.918 [cutoff value: ≥0.02 (light/heavy ratio)]. These data demonstrate that the discriminatory power of the deglycopeptide is greater than that of the nonglycopeptide. We conclude that deglycopeptide can distinguish cancer status between normal subjects and HCC patients better than nonglycopeptide. PMID:25310463

Proceedings, High-Precision $\\alpha_s$ Measurements from LHC to FCC-ee

Energy Technology Data Exchange (ETDEWEB)

d' Enterria, David [CERN; Skands, Peter Z. [Monash U.

2015-01-01

This document provides a writeup of all contributions to the workshop on "High precision measurements of $\\alpha_s$: From LHC to FCC-ee" held at CERN, Oct. 12--13, 2015. The workshop explored in depth the latest developments on the determination of the QCD coupling $\\alpha_s$ from 15 methods where high precision measurements are (or will be) available. Those include low-energy observables: (i) lattice QCD, (ii) pion decay factor, (iii) quarkonia and (iv) $\\tau$ decays, (v) soft parton-to-hadron fragmentation functions, as well as high-energy observables: (vi) global fits of parton distribution functions, (vii) hard parton-to-hadron fragmentation functions, (viii) jets in $e^\\pm$p DIS and $\\gamma$-p photoproduction, (ix) photon structure function in $\\gamma$-$\\gamma$, (x) event shapes and (xi) jet cross sections in $e^+e^-$ collisions, (xii) W boson and (xiii) Z boson decays, and (xiv) jets and (xv) top-quark cross sections in proton-(anti)proton collisions. The current status of the theoretical and experimental uncertainties associated to each extraction method, the improvements expected from LHC data in the coming years, and future perspectives achievable in $e^+e^-$ collisions at the Future Circular Collider (FCC-ee) with $\\cal{O}$(1--100 ab$^{-1}$) integrated luminosities yielding 10$^{12}$ Z bosons and jets, and 10$^{8}$ W bosons and $\\tau$ leptons, are thoroughly reviewed. The current uncertainty of the (preliminary) 2015 strong coupling world-average value, $\\alpha_s(m_Z)$ = 0.1177 $\\pm$ 0.0013, is about 1\\%. Some participants believed this may be reduced by a factor of three in the near future by including novel high-precision observables, although this opinion was not universally shared. At the FCC-ee facility, a factor of ten reduction in the $\\alpha_s$ uncertainty should be possible, mostly thanks to the huge Z and W data samples available.

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

Science.gov (United States)

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Functional Analysis of Glycosylation of Zika Virus Envelope Protein

Directory of Open Access Journals (Sweden)

Camila R. Fontes-Garfias

2017-10-01

Full Text Available Summary: Zika virus (ZIKV infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. : Zika virus (ZIKV causes devastating congenital abnormities and Guillain-Barré syndrome. Fontes-Garfias et al. showed that the glycosylation of ZIKV envelope protein plays an important role in infecting mosquito vectors and pathogenesis in mouse. Keywords: Zika virus, glycosylation, flavivirus entry, mosquito transmission, vaccine

Cyclohex-1-ene carboxylic acids: synthesis and biological evaluation of novel inhibitors of human 5 alpha reductase.

Science.gov (United States)

Baston, Eckhard; Salem, Ola I A; Hartmann, Rolf W

2003-03-01

In search of novel nonsteroidal mimics of steroidal inhibitors of 5 alpha reductase, 4-(2-phenylethyl)cyclohex-1-ene carboxylic acids 1-5 were synthesized with different substituents in para position of the phenyl ring (1: N, N-diisopropylcarbamoyl, 2: phenyl, 3: phenoxy, 4: benzoyl, and 5: benzyl). The principal synthetic approach for the desired compounds consisted of a Wittig olefination between 1, 4-dioxaspiro [4.5]-decane-8-carbaldehyde (4g and the appropriate phosphonium salts. The compounds were tested for inhibition of human 5 alpha reductase isozymes 1 and 2 using DU 145 cells and preparations from prostatic tissue, respectively. They turned out to be good inhibitors of the prostatic isozyme 2 with compound 1 being the most potent one (IC(50) = 760 nM). Isozyme 1 was only slightly inhibited. It is concluded that the novel structures are appropriate for being further optimized, aiming at the development of a novel drug for the treatment of benign prostatic hyperplasia.

Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein.

Science.gov (United States)

Kim, Unyong; Oh, Myung Jin; Seo, Youngsuk; Jeon, Yinae; Eom, Joon-Ho; An, Hyun Joo

2017-09-01

Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.

The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase

DEFF Research Database (Denmark)

Juge, N.; Nøhr, J.; Le Gal-Coëffet, M.-F.

2006-01-01

High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley alpha......-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg x L(-1) and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms...... in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed...

Quantifying risk of penile prosthesis infection with elevated glycosylated hemoglobin.

Science.gov (United States)

Wilson, S K; Carson, C C; Cleves, M A; Delk, J R

1998-05-01

Elevation of glycosylated hemoglobin above levels of 11.5 mg.% has been considered a contraindication to penile prosthesis implantation in diabetic patients. We determine the predictive value of glycosylated hemoglobin A1C in penile prosthesis infections in diabetic and nondiabetic patients to confirm or deny this prevalent opinion. We conducted a 2-year prospective study of 389 patients, including 114 diabetics, who underwent 3-piece penile prosthesis implantation. All patients had similar preoperative preparation without regard to diabetic status, control or glycosylated hemoglobin A1C level. Risk of infection was statistically analyzed for diabetics versus nondiabetics, glycosylated hemoglobin A1C values above and below 11.5 mg.%, insulin dependent versus oral medication diabetics, and fasting blood sugars above and below 180 mg.%. Prosthesis infections developed in 10 diabetics (8.7%) and 11 nondiabetics (4.0%). No increased infection rate was observed in diabetics with high fasting sugars or diabetics on insulin. There was no statistically significant increased infection risk with increased levels of glycosylated hemoglobin A1C among all patients or among only the diabetics. In fact, there was no meaningful difference in the median or mean level of glycosylated hemoglobin A1C in the infected and noninfected patients regardless of diabetes. Use of glycosylated hemoglobin A1C values to identify and exclude surgical candidates with increased risk of infections is not proved by this study. Elevation of fasting sugar or insulin dependence also does not increase risk of infection in diabetics undergoing prosthesis implantation.

Glycosylated hemoglobin as a forecast factor of progressing of diabetic nephropathy in patients with diabetes type 1

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Pertseva N.O.

2017-12-01

Full Text Available The aim of the study was to propose a mathematical model for prediction of development of diabetic nephropathy in patients with diabetes mellitus by determining the level of glycosylated hemoglobin - as a factor in the development and progression of diabetic nephropathy. A survey of 136 patients with type 1 diabetes was performed in the endocrinology department of the OSH «Clinic of the Medical Academy», Dnipro in 2016-2017. Clinical laboratory examination included: determination of the level of glycosylated hemoglobin (HbA1c, level of blood creatinine, level of albuminuria. The GFR was calculated by the formula CKD-EPI. The obtained results of the study, using methods of correlation and regression analysis, show a clear correlation between the GFR score in patients with diabetes mellitus and the level of glycosylated hemoglobin. Statistical methods of analysis have shown that the level of glycosylated hemoglobin can be considered as an early predictor of development of diabetic nephropathy. The mathematical equation of prognosis for the onset of diabetic nephropathy can be used to determine the prognosis for the development of diabetic nephropathy in diabetes mellitus patients in clinical practice for the timely inclusion of patients with a high prognostic risk in a group requiring more stringent glycemic control.

Inhibition of steroid 5 alpha-reductase by specific aliphatic unsaturated fatty acids.

Science.gov (United States)

Liang, T; Liao, S

1992-01-01

Human or rat microsomal 5 alpha-reductase activity, as measured by enzymic conversion of testosterone into 5 alpha-dihydrotestosterone or by binding of a competitive inhibitor, [3H]17 beta-NN-diethulcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA) to the reductase, is inhibited by low concentrations (less than 10 microM) of certain polyunsaturated fatty acids. The relative inhibitory potencies of unsaturated fatty acids are, in decreasing order: gamma-linolenic acid greater than cis-4,7,10,13,16,19-docosahexaenoic acid = cis-6,9,12,15-octatetraenoic acid = arachidonic acid = alpha-linolenic acid greater than linoleic acid greater than palmitoleic acid greater than oleic acid greater than myristoleic acid. Other unsaturated fatty acids such as undecylenic acid, erucic acid and nervonic acid, are inactive. The methyl esters and alcohol analogues of these compounds, glycerols, phospholipids, saturated fatty acids, retinoids and carotenes were inactive even at 0.2 mM. The results of the binding assay and the enzymic assay correlated well except for elaidic acid and linolelaidic acid, the trans isomers of oleic acid and linoleic acid respectively, which were much less active than their cis isomers in the binding assay but were as potent in the enzymic assay. gamma-Linolenic acid had no effect on the activities of two other rat liver microsomal enzymes: NADH:menadione reductase and glucuronosyl transferase. gamma-Linolenic acid, the most potent inhibitor tested, decreased the Vmax. and increased Km values of substrates, NADPH and testosterone, and promoted dissociation of [3H]4-MA from the microsomal reductase. gamma-Linolenic acid, but not the corresponding saturated fatty acid (stearic acid), inhibited the 5 alpha-reductase activity, but not the 17 beta-dehydrogenase activity, of human prostate cancer cells in culture. These results suggest that unsaturated fatty acids may play an important role in regulating androgen action in target cells. PMID:1637346

Simultaneous improvement in production of microalgal biodiesel and high-value alpha-linolenic acid by a single regulator acetylcholine

OpenAIRE

Parsaeimehr, Ali; Sun, Zhilan; Dou, Xiao; Chen, Yi-Feng

2015-01-01

Background Photoautotrophic microalgae are a promising avenue for sustained biodiesel production, but are compromised by low yields of biomass and lipids at present. We are developing a chemical approach to improve microalgal accumulation of feedstock lipids as well as high-value alpha-linolenic acid which in turn might provide a driving force for biodiesel production. Results We demonstrate the effectiveness of the small bioactive molecule ?acetylcholine? on accumulation of biomass, total li...

Towards control of aggregational behaviour of alpha-lactalbumin at acidic pH.

Science.gov (United States)

Pedersen, Jane B; Fojan, Peter; Sorensen, John; Petersen, Steffen B

2006-07-01

alpha-Lactalbumin (alpha-La) undergoes considerable structural changes upon loss of bound Ca2+ at acidic pH, leaving alpha-La in a molten globule structure. Using fluorescence the present work provides more insight into the structural transition of alpha-La at acidic pH leading to protein aggregation, most likely caused by a combination of hydrophobic and electrostatic interactions. The rate of aggregation is determined by the protein concentration and temperature applied. Availability of Ca2+ stabilises the protein, and thus prevent aggregation at pH values as low as pH 2.9. In contrast, presence of Cu2+ induces a destabilisation of the protein, which can be explained by a binding to the Zn2+ binding site in alpha-La, possibly resulting in structural alterations of the protein. In general, presence of anions destabilize alpha-La at pH values below pI, with SO4(2-) exhibiting the strongest effect on the protein stability, thus correlating well with the Hofmeister series. At more acidic pH values far from pI, alpha-La becomes more stable towards ion induced aggregation, since higher ion activity is required to efficiently screen the charges on the protein surface. The results presented in this paper provide detailed knowledge on the external parameters leading to aggregation of alpha-La at acidic pH, thus permitting rational design of the aggregation process.

Effect of alpha 2-adrenoceptor agonists on gastric pepsin and acid secretion in the rat.

Science.gov (United States)

Tazi-Saad, K.; Chariot, J.; Del Tacca, M.; Rozé, C.

1992-01-01

1. The purpose of the present study was to analyze the effects of the alpha 2-adrenoceptor agonists clonidine, guanabenz, detomidine and medetomidine on pepsin secretion in conscious rats provided with gastric chronic fistula and to compare this with acid secretion. 2. Basal interdigestive gastric secretion, which is mainly neurally driven in the rat, and the secretion directly stimulated by the two main stimulants of chief cells, cholecystokinin octapeptide (CCK8) and methacholine, were studied. 3. Basal secretion of pepsin and acid was inhibited by all four drugs with comparable EC50S. 4. CCK-stimulated pepsin and acid secretion was less sensitive than basal pepsin and acid secretion to alpha 2-adrenoceptor inhibition. 5. Methacholine-stimulated pepsin and acid secretion was not changed by clonidine and guanabenz; methacholine-stimulated acid was even marginally increased by clonidine. 6. These results do not favour the presence of alpha 2-receptors on chief cells in the rat stomach. They rather suggest that pepsin inhibition by alpha 2-adrenoceptor agonists is indirect and due to central or peripheral inhibition of the discharge of nerve fibres activating pepsin secretion. PMID:1356566

Electrospray Ionization Mass Spectrometric Analysis of Highly Reactive Glycosyl Halides

Directory of Open Access Journals (Sweden)

Lajos Kovács

2012-07-01

Full Text Available Highly reactive glycosyl chlorides and bromides have been analysed by a routine mass spectrometric method using electrospray ionization and lithium salt adduct-forming agents in anhydrous acetonitrile solution, providing salient lithiated molecular ions [M+Li]⁺, [2M+Li]⁺ etc. The role of other adduct-forming salts has also been evaluated. The lithium salt method is useful for accurate mass determination of these highly sensitive compounds.

An efficient method to control high mannose and core fucose levels in glycosylated antibody production using deoxymannojirimycin.

Science.gov (United States)

Shalel Levanon, Sagit; Aharonovitz, Orit; Maor-Shoshani, Ayelet; Abraham, Gita; Kenett, Dan; Aloni, Yehoshua

2018-06-20

Glycosylation on the Fc region of recombinant Immunoglobulin G (IgG) therapeutic antibodies is a critical protein quality attribute which may affect the efficacy and safety of the molecule. During the development of biosimilar therapeutics, adjustment of the glycosylation profile is required in order to match the reference innovator profile. Deoxymannojirimycin (DMJ), a known inhibitor of mannosidase, was used in this study to modulate the glycosylation pattern of antibodies. The effect of DMJ, at concentrations of 5 μM - 500 μM, on non-fucosylated glycoform levels was tested in the biosynthesis processes of two different IgG1 (IgG1 #A and IgG1 #B) using two Chinese hamster ovary (CHO) cell lines (CHO-DXB-11 and CHOK1SV, respectively) in Erlenmeyer flasks and in lab scale bioreactors. DMJ affected glycan forms in a dose response manner. At the highest concentration tested, DMJ reduced N-linked complex glycoform and core fucose levels by 15 and 14 fold, respectively, and increased high mannose level by 21 fold. 10 μM DMJ decreased IgG1 #A core fucose level in CHO-DXB-11 from 92% to 73% and increased high mannose level from 4% to 22% in Erlenmeyer flasks. Furthermore, in lab scale bioreactors, 15 μM DMJ decreased IgG1 #A core fucose level from 95% to 84% and increased high mannose level from 3% to 13%. Core fucose level of IgG1 #B in CHOK1SV was decreased from 81% to 73% using 10 μM DMJ in lab scale bioreactors while high mannose was increased from 6% to 15%. While affecting core fucose and high mannose levels, DMJ decreased maximum viable cell concentration by 16% and did not significantly affect cell productivity (less than 10%). This study demonstrated that DMJ can enable the control of core fucosylated and high mannose levels of IgG1 antibodies in a defined range. Copyright © 2018 Elsevier B.V. All rights reserved.

Scanning the available Dictyostelium discoideum proteome for O-linked GlcNAc glycosylation sitesusing neural networks

DEFF Research Database (Denmark)

Gupta, Ramneek; Jung, Eva; Gooley, Andrew A

1999-01-01

Dictyostelium discoideum has been suggested as a eukaryotic model organism for glycobiology studies. Presently, the characteristics of acceptor sites for the N-acetylglucosaminyl-transferases in Dictyostelium discoideum, which link GlcNAc in an alpha linkage to hydroxyl residues, are largely...... unknown. This motivates the development of a species specific method for prediction of O-linked GlcNAc glycosylation sites in secreted and membrane proteins of D. discoideum. The method presented here employs a jury of artificial neural networks. These networks were trained to recognize the sequence...... context and protein surface accessibility in 39 experimentally determined O-alpha-GlcNAc sites found in D. discoideum glycoproteins expressed in vivo. Cross-validation of the data revealed a correlation in which 97% of the glycosylated and nonglycosylated sites were correctly identified. Based...

Synthesis and evaluation of phytotoxic activity of {alpha}-Santonin derivatives; Sintese e avaliacao da atividade fitotoxica de derivados da {alpha}-Santonina

Energy Technology Data Exchange (ETDEWEB)

Alvarenga, Elson S.; Barbosa, Luiz C.A.; Saliba, William A.; Arantes, Francisco F.P.; Demuner, Antonio J. [Universidade Federal de Vicosa (UFV), MG (Brazil). Dept. de Quimica]. E-mail: elson@ufv.br; Silva, Antonio A. [Universidade Federal de Vicosa (UFV), MG (Brazil). Dept. de Fitotecnia

2009-07-01

Mixtures of {alpha}-Santonin and various solvents were irradiated by either high or low pressure mercury lamps. The photochemical reactions afforded lumisantonin (11) (76% in acetonitrile), (3 S,3a S,9{beta}S)-3,6,6-trimethyl-3,3a,4,5-tetrahydronafto[1,2-b]furan-2,7({eta}6,9{beta}{eta}) dione (12) (100% in acetonitrile), 10{alpha}-acetoxy-3-oxo-1,7{alpha}H{eta},6,11{alpha}a{eta}-guaia-4-en-6,12-oli= de (8) (26% in acetic acid), 10{alpha}-hydroxy-3-oxo-1,7{alpha}a{eta},6,11{alpha}{eta}-guaia-4-en-6,12-olid= e (10) (32%) and (E)-3-((3 S,3a S,7{alpha}S)-3-methyl-2-oxo-6-(propan-2-ylidene)hexahydrobenzofuran- 7 - (7{alpha}{eta})-ylidene)propanoic acid (9) (44%) (in water/ acetic acid 1:1, v/v). Lactone 12 was also prepared by irradiation of lumisantonin in diethyl ether. Lactones 8 and 10 were converted, respectively, into the 10 {alpha}-acetoxy-3{alpha}-hydroxy-1,7{alpha}H,6,11{alpha}H-guaia-4-en-6,12-olid= e (13) (87%) and 3a,10a-dihydroxy-1,7{alpha}H,6,11{alpha}H-guaia-4-en-6,12-olide (14) (75%) by sodium borohydride reduction. The effects of the compounds on the development of radicle of Sorghum bicolor and Cucumis sativus were evaluated. (author)

Distribution of N-glycosylation sequons in proteins: how apart are they?

DEFF Research Database (Denmark)

Rao, Shyama Prasad; Buus, Ole Thomsen; Wollenweber, Bernd

2011-01-01

of experimentally confirmed eukaryotic N-glycoproteins we analyzed the relative position and distribution of sequons. N-Glycosylation probability was found to be lower in the termini of protein sequences compared to the mid region. N-glycosylated sequons were found much farther from C terminus compared to the N......N-glycosylation is a common protein modification process, which affects a number of properties of proteins. Little is known about the distribution of N-glycosylation sequons, for example, the distance between glycosylated sites and their position in the protein primary sequence. Using a large set......-terminus of the protein sequence and this effect was more pronounced for NXS sequons. The distribution of sequons, modeled based on balls-in-boxes classical occupancy, showed a near-maximum probability. Considerable proportion of sequons was found within a distance of ten amino acids, indicating that the steric hindrance...

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2

DEFF Research Database (Denmark)

Elster, L; Kristiansen, U; Pickering, D S

2001-01-01

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma......, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important...... for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed...

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

International Nuclear Information System (INIS)

Kalra, Rajkumar S.; Wadhwa, Renu

2015-01-01

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

Energy Technology Data Exchange (ETDEWEB)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp [Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST Central 4), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

Determination of radium-226 by high-resolution alpha spectrometry

International Nuclear Information System (INIS)

Sill, C.W.

1983-01-01

The determination of radium-226 by alpha spectrometry has been investigated critically to determine experimental conditions under which high resolution and accurate and reliable results can be obtained. Refractory solids such as soils, ores, and tailings from uranium mills are dissolved completely by fusion with potassium fluoride in the presence of barium-133 tracer. The fluoride cake is then transposed with sulfuric acid to a pyrosulfate fusion with simultaneous volatilization of all silica and fluoride. Radium is precipitated with barium already present in the sample by addition of lead perchlorate to a dilute hydrochloric acid solution of the pyrosulfate cake. The resulting insoluble sulfates are dissolved in an alkaline solution of diethylenetriaminepentaacetic acid, and the radium and barium sulfates are reprecipitated with acetic acid. The precipitate is mounted on a membrane filter and analyzed by alpha spectrometry. Water samples are partially evaporated and treated similarly. Resolution of the subsequent alpha spectra is much better than has been achieved previously from barium sulfate, and is almost as good as is obtainable with actinides electrodeposited on polished steel plates. The resolution is about 60 keV full-width-half-maximum with 100 μg of barium on a 1-inch filter with a 450 mm 2 detector at 20% counting efficiency. Recovery is about 97% and accuracy is generally as good as the counting statistics obtained will permit. Grossly inaccurate results can be obtained under certain conditions when barium-133 tracer is used to determine the chemical yield of radium-226. Severe contamination of the surface-barrier detector by polonium-210 and by recoil products of the radium isotopes being counted is demonstrated, amd methods for virtual elimination of both problems are discussed

Synthesis of tritiated 1-alpha-methadol and 1-alpha-acetylmethadol

Energy Technology Data Exchange (ETDEWEB)

Thang, D.C.; Nam, N.H.; Pontikis, R. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital Fernand Widal, 75 - Paris (France)); Pichat, L. (CEA Centre d' Etudes Nucleaires de Saclay, 91 - Gif-sur-Yvette (France). Service des Molecules Marquees)

1982-04-01

dl-Methadone was resolved by crystallization of its ammonium d- ..cap alpha.. -bromocamphor-..pi..-sulfonate salt to give d-methadone. The latter in ethyl acetate solution was reduced with tritium gas to 1-..cap alpha..-methadol /sup 3/H in presence of Adams platinum oxide at normal temperature and pressure. Acetylation of 1-..cap alpha..-carbinol hydrochloride by means of acetyl chloride afforded 1-..cap alpha..-acetylmethadol /sup 3/H, specific activity: 20 Ci/mMole. The positions and extent of tritium labelling were determined by /sup 3/H NMR spectroscopy.

ADD1/SREBP1c activates the PGC1-alpha promoter in brown adipocytes

DEFF Research Database (Denmark)

Hao, Qin; Hansen, Jacob B; Petersen, Rasmus K

2010-01-01

Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and beta-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor alpha (LXRalpha), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol...

Fc-Glycosylation in Human IgG1 and IgG3 Is Similar for Both Total and Anti-Red-Blood Cell Anti-K Antibodies

Directory of Open Access Journals (Sweden)

Myrthe E. Sonneveld

2018-01-01

Full Text Available After albumin, immunoglobulin G (IgG are the most abundant proteins in human serum, with IgG1 and IgG3 being the most abundant subclasses directed against protein antigens. The quality of the IgG-Fc-glycosylation has important functional consequences, which have been found to be skewed toward low fucosylation in some antigen-specific immune responses. This increases the affinity to IgG1-Fc-receptor (FcγRIIIa/b and thereby directly affects downstream effector functions and disease severity. To date, antigen-specific IgG-glycosylation have not been analyzed for IgG3. Here, we analyzed 30 pregnant women with anti-K alloantibodies from a prospective screening cohort and compared the type of Fc-tail glycosylation of total serum- and antigen-specific IgG1 and IgG3 using mass spectrometry. Total serum IgG1 and IgG3 Fc-glycoprofiles were highly similar. Fc glycosylation of antigen-specific IgG varied greatly between individuals, but correlated significantly with each other for IgG1 and IgG3, except for bisection. However, although the magnitude of changes in fucosylation and galactosylation were similar for both subclasses, this was not the case for sialylation levels, which were significantly higher for both total and anti-K IgG3. We found that the combination of relative IgG1 and IgG3 Fc-glycosylation levels did not improve the prediction of anti-K mediated disease over IgG1 alone. In conclusion, Fc-glycosylation profiles of serum- and antigen-specific IgG1 and IgG3 are highly similar.

Condensation reactions catalyzed by α-N-acetylgalactosaminidase from Aspergillus niger yielding a-N-acetylgalactosaminides

Czech Academy of Sciences Publication Activity Database

Weignerová, Lenka; Pelantová, Helena; Manglová, Dana; Michálková, Klára; Křen, Vladimír

2010-01-01

Roč. 28, č. 2 (2010), s. 150-155 ISSN 1024-2422 R&D Projects: GA MŠk OC 136 Keywords : alpha-N-Acetylgalactosaminidase * amino acid glycosylation * enzymatic glycosylation Subject RIV: EE - Microbiology, Virology Impact factor: 1.275, year: 2010

Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

Science.gov (United States)

Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

1997-06-25

Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

Non-CpG methylation of the PGC-1alpha promoter through DNMT3B controls mitochondrial density

DEFF Research Database (Denmark)

Barres, Romain; Osler, Megan E; Yan, Jie

2009-01-01

-CpG nucleotides. Non-CpG methylation was acutely increased in human myotubes by exposure to tumor necrosis factor-alpha (TNF-alpha) or free fatty acids, but not insulin or glucose. Selective silencing of the DNA methyltransferase 3B (DNMT3B), but not DNMT1 or DNMT3A, prevented palmitate-induced non......-CpG methylation of PGC-1alpha and decreased mtDNA and PGC-1alpha mRNA. We provide evidence for PGC-1alpha hypermethylation, concomitant with reduced mitochondrial content in type 2 diabetic patients, and link DNMT3B to the acute fatty-acid-induced non-CpG methylation of PGC-1alpha promoter....

Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins.

Science.gov (United States)

Liu, Liming

2015-06-01

Understanding the impact of glycosylation and keeping a close control on glycosylation of product candidates are required for both novel and biosimilar monoclonal antibodies (mAbs) and Fc-fusion protein development to ensure proper safety and efficacy profiles. Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 and, in some cases, in the Fab region. For Fc-fusion proteins, glycosylation also frequently occurs in the fusion partners. Depending on the expression host, glycosylation patterns in mAb or Fc-fusions can be significantly different, thus significantly impacting the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs. Glycans that have a major impact on PK and PD of mAb or Fc-fusion proteins include mannose, sialic acids, fucose (Fuc), and galactose (Gal). Mannosylated glycans can impact the PK of the molecule, leading to reduced exposure and potentially lower efficacy. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of Fc-fusion molecules. Core Fuc in the glycan structure reduces IgG antibody binding to IgG Fc receptor IIIa relative to IgG lacking Fuc, resulting in decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activities. Glycoengineered Chinese hamster ovary (CHO) expression systems can produce afucosylated mAbs that have increased ADCC activities. Terminal Gal in a mAb is important in the complement-dependent cytotoxicity (CDC) in that lower levels of Gal reduce CDC activity. Glycans can also have impacts on the safety of mAb. mAbs produced in murine myeloma cells such as NS0 and SP2/0 contain glycans such as Galα1-3Galβ1-4N-acetylglucosamine-R and N-glycolylneuraminic acid (NGNA) that are not naturally present in humans and can be immunogenic when used as therapeutics. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Role of protein glycosylation on the expression of muscarinic receptors of N4TG1 neuroblastoma cells

International Nuclear Information System (INIS)

Ahmad, A.; Chiang, P.K.

1986-01-01

Muscarinic acetylcholine receptors (mAChR) are glycoproteins. Experiments were conducted to determine whether active glycosylation of proteins in N4TG1 neuroblastoma cells could affect the expression of muscarinic receptors on the cell surface. The binding of radioactive N-methylscopolamine, a membrane impermeable ligand, to intact cells was used as a measure of mAChR. In the presence of the inhibitors of glycosylation, such as tunicamycin, monensin and amphomycin, N-linked glycosylation of proteins in the N4TG1 cells was inhibited, as measured by the incorporation of radioactive glucosamine or mannose in proteins. At the concentrations of tunicamycin and monensin used, the glycosylation of proteins after 3 hours were drastically reduced, but the number of mAChR in the cells was not altered. The apparent lack of effect within a short incubation period could be attributed to the presence of preformed oligosaccharide dolichol readily available for N-glycosylation. However, after 24 hours, tunicamycin (0.05 μg/ml) caused a decrease in the number of mAChR by 17% without having any effect on protein synthesis. Therefore, de novo glycosylation of proteins may be required for the expression of mAChR receptors in the N4TG1 neuroblastoma cell surface

Effects of glycosylation on the conformation and dynamics of O-linked glycoproteins: Carbon-13 NMR studies of ovine submaxillary mucin

International Nuclear Information System (INIS)

Gerken, T.A.; Butenhof, K.J.; Shogren, R.

1989-01-01

Carbon-13 NMR spectroscopic studies of native and sequentially deglycosylated ovine submaxillary mucin (OSM) have been performed to examine the effects of glycosylation on the conformation and dynamics of the peptide core of O-linked glycoproteins. OSM is a large nonglobular glycoprotein in which nearly one-third of the amino acid residues are Ser and Thr which are glycosylated by the α-Neu-NAc(2-6)α-Ga1NAc- disaccharide. The β-carbon resonances of glycosylated Ser and Thr residues in intact and asialo mucin display considerable chemical shift heterogeneity which, upon the complete removal of carbohydrate, coalesces to single sharp resonances. This chemical shift heterogeneity is due to peptide sequence variability and is proposed to reflect the presence of sequence-dependent conformations of the peptide core. These different conformations are thought to be determined by steric interactions of the Ga1NAc residue with adjacent peptide residues. The absence of chemical shift heterogeneity in apo mucin is taken to indicate a loss in the peptide-carbohydrate steric interactions, consistent with a more relaxed random coiled structure. On the basis of the 13 C relaxation behavior the dynamics of the α-carbons appear to be unique to each amino acid type and glycosylation state. These results are consistent with the changes in molecular dimensions determined by light-scattering techniques for the same series of modified mucins. Taken together, these results further demonstrate that mucins possess a highly expanded conformation that is dominated by steric interactions between the peptide core and the O-linked Ga1NAc residue

Palladium-Catalyzed alpha-Arylation of Tetramic Acids

DEFF Research Database (Denmark)

Storgaard, Morten; Dorwald, F. Z.; Peschke, B.

2009-01-01

A mild, racemization-free, palladium-Catalyzed alpha-arylation of tetramic acids (2,4-pyrrolidinediones) has been developed. Various amino acid-derived tetramic acids were cleanly arylated by treatment with 2 mol % of Pd(OAc)(2), 4 mol % of a sterically demanding biaryl phosphine, 2.3 equiv of K2CO...... no effect on their reactivity: both electron-rich and electron-poor aryl chlorides and bromides or triflates led to good yields. Ortho-substituted aryl halides and heteroaryl halides, however, did not undergo the title reaction....

PROCESS FOR HYDROGENOLYSIS OF ALPHA-HYDROXY ESTERS OR ACIDS USING A HETEROGENEOUS CATALYST

DEFF Research Database (Denmark)

2017-01-01

The present invention relates to a method for hydrogenolysis of alpha-hydroxy esters or acids, comprising reacting the alpha-hydroxy ester or acid in the presence of a heterogeneous catalyst. The present invention also relates to a method for producing propionic acid ester, and the use of any...

Synthesis of omega-hydroxy carboxylic acids and alpha,omega-dimethyl ketones using alpha,omega-diols as alkylating agents.

Science.gov (United States)

Iuchi, Yosuke; Hyotanishi, Megumi; Miller, Brittany E; Maeda, Kensaku; Obora, Yasushi; Ishii, Yasutaka

2010-03-05

Synthesis of omega-hydroxy carboxylic acids and alpha,omega-dimethyl diketones was successfully achieved by using alpha,omega-diols as alkylating agents under the influence of an iridium catalyst. For example, the alkylation of butyl cyanoacetate with 1,13-tridecanediol in the presence of [IrCl(cod)](2) or [IrCl(coe)(2)](2) gave rise to butyl 2-cyano-15-hydroxypentadecanoate in good yield which is easily converted to cyclopentadecanolide (CPDL). In addition, the alkylation of acetone with 1,10-decanediol in the presence of [IrCl(cod)](2) and KOH resulted in an important muscone precursor, 2,15-hexadecanedione (HDDO), in good yield.

Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

Directory of Open Access Journals (Sweden)

Erica E Rosenbaum

2014-05-01

Full Text Available As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II, α-mannosidase-IIb (α-Man-IIb, a β-N-acetylglucosaminidase called fused lobes (Fdl, and hexosaminidase 1 (Hexo1. We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights

Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

Science.gov (United States)

Rosenbaum, Erica E.; Vasiljevic, Eva; Brehm, Kimberley S.; Colley, Nansi Jo

2014-01-01

As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the

Actions of alpha2 adrenoceptor ligands at alpha2A and 5-HT1A receptors: the antagonist, atipamezole, and the agonist, dexmedetomidine, are highly selective for alpha2A adrenoceptors.

Science.gov (United States)

Newman-Tancredi, A; Nicolas, J P; Audinot, V; Gavaudan, S; Verrièle, L; Touzard, M; Chaput, C; Richard, N; Millan, M J

1998-08-01

This study examined the activity of chemically diverse alpha2 adrenoceptor ligands at recombinant human (h) and native rat (r) alpha2A adrenoceptors compared with 5-HT1A receptors. First, in competition binding experiments at h alpha2A and h5-HT1A receptors expressed in CHO cells, several compounds, including the antagonists 1-(2-pyrimidinyl)piperazine (1-PP), (+/-)-idazoxan, benalfocin (SKF 86466), yohimbine and RX 821,002, displayed preference for h alpha2A versus h5-HT1A receptors of only 1.4-, 3.6-, 4-, 10- and 11-fold, respectively (based on differences in pKi values). Clonidine, brimonidine (UK 14304), the benzopyrrolidine fluparoxan and the guanidines guanfacine and guanabenz exhibited intermediate selectivity (22- to 31-fold) for h alpha2A receptors. Only the antagonist atipamezole and the agonist dexmedetomidine (DMT) displayed high preference for alpha2 adrenoceptors (1290- and 91-fold, respectively). Second, the compounds were tested for their ability to induce h5-HT1A receptor-mediated G-protein activation, as indicated by the stimulation of [35S]GTPgammaS binding. All except atipamezole and RX 821,002 exhibited agonist activity, with potencies which correlated with their affinity for h5-HT1A receptors. Relative efficacies (Emax values) were 25-35% for guanabenz, guanfacine, WB 4101 and benalfocin, 50-65% for 1-PP, (+/-)-idazoxan and clonidine, and over 70% for fluparoxan, oxymetazoline and yohimbine (relative to 5-HT = 100%). Yohimbine-induced [35S]GTPgammaS binding was inhibited by the selective 5-HT1A receptor antagonist WAY 100,635. In contrast, RX 821,002 was the only ligand which exhibited antagonist activity at h5-HT1A receptors, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Atipamezole, which exhibited negligeable affinity for 5-HT1A receptors, was inactive. Third, the affinities for r alpha2A differed considerably from the affinities for h alpha2A receptors whereas the affinities for r5-HT1A differed much less from the affinities for h5-HT

An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

DEFF Research Database (Denmark)

Hägglund, Per; Matthiesen, R.; Elortza, F.

2007-01-01

and N-acetyl-β-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides......, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide- N-glycosidase (PNGase) digestion, with concomitant deamidation...... of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β- N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond...

Stability of HAMLET--a kinetically trapped alpha-lactalbumin oleic acid complex.

Science.gov (United States)

Fast, Jonas; Mossberg, Ann-Kristin; Svanborg, Catharina; Linse, Sara

2005-02-01

The stability toward thermal and urea denaturation was measured for HAMLET (human alpha-lactalbumin made lethal to tumor cells) and alpha-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than alpha-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15 degrees C lower for apo HAMLET than for apo alpha-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.

Main metabolites of 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea and 1-(2-chloroethyl)-3-(2',3', 4'-tri-O-acetyl-alpha, beta-D-ribopyranosyl)-1-nitrosourea in rats

International Nuclear Information System (INIS)

Madelmont, J.C.; Moreau, M.F.; Godeneche, D.; Duprat, J.; Plagne, R.; Meyniel, G.

1982-01-01

The metabolism of two glycosylnitrosoureas, 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea (RFCNU) and 1-(2-chloroethyl)-3-(2',3',4'-tri-O-acetyl-alpha, beta-D-ribopyranosyl)-1-nitrosourea (RPCNU), has been investigated in the rat. With the label on the carboxyl moiety of RFCNU, we have shown that hydrolysis of the 4-nitrobenzoyl ester occurred to a large extent in vivo; 4-nitrobenzoic acid and its glucuronide were the major urinary metabolites. Two other minor metabolites and their glucuronides were identified as 4-aminobenzoic acid and 4-acetamidobenzoic acid. With the label on the chloroethyl moieties of RFCNU and RPCNU, we have shown that chloroethanol was a major degradation product of this alkylating part of the molecule. The concentration of chloroethanol in plasma vs. time has been determined. In urine, four metabolites derived from alkylated glutathione, namely thiodiacetic acid and its sulfoxide, N-acetylcarboxymethylcysteine, and N-acetylhydroxyethylcysteine, have been identified

UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation.

Science.gov (United States)

Wen, Chao; Huang, Wei; Zhu, Xue-Lin; Li, Xiao-San; Zhang, Fan; Jiang, Ren-Wang

2018-02-02

A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C 21 steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

Chemoenzymatic synthesis of alpha-L-rhamnosides using recombinant alpha-L-rhamnosidase from Aspergillus terreus

Czech Academy of Sciences Publication Activity Database

De Winter, K.; Šimčíková, Daniela; Schlack, B.; Weignerová, Lenka; Pelantová, Helena; Soetaert, W.; Desmet, T.; Křen, Vladimír

2013-01-01

Roč. 147, NOV 2013 (2013), s. 640-644 ISSN 0960-8524 R&D Projects: GA MŠk(CZ) LD13042; GA MŠk(CZ) 7E11011 Institutional support: RVO:61388971 Keywords : alpha-L-Rhamnosidase * Aspergillus terreus * Glycosylation Subject RIV: CE - Biochemistry Impact factor: 5.039, year: 2013

N-glycosylation of Colorectal Cancer Tissues

Science.gov (United States)

Balog, Crina I. A.; Stavenhagen, Kathrin; Fung, Wesley L. J.; Koeleman, Carolien A.; McDonnell, Liam A.; Verhoeven, Aswin; Mesker, Wilma E.; Tollenaar, Rob A. E. M.; Deelder, André M.; Wuhrer, Manfred

2012-01-01

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ∼1 million cases and an annual mortality rate of ∼655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers. PMID:22573871

[Effect of high-intensity interval training on the reduction of glycosylated hemoglobin in type-2 diabetic adult patients].

Science.gov (United States)

Aguilera Eguía, Raúl Alberto; Russell Guzmán, Javier Antonio; Soto Muñoz, Marcelo Enrique; Villegas González, Bastián Eduardo; Poblete Aro, Carlos Emilio; Ibacache Palma, Alejandro

2015-03-05

Type 2 diabetes mellitus is one of the major non-communicable chronic diseases in the world. Its prevalence in Chile is significant, and complications associated with this disease involve great costs, which is why prevention and treatment of this condition are essential. Physical exercise is an effective means for prevention and treatment of type 2 diabetes mellitus. The emergence of new forms of physical training, such as "high intensity interval training", presents novel therapeutic alternatives for patients and health care professionals. To assess the validity and applicability of the results regarding the effectiveness of high intensity interval training in reducing glycosylated hemoglobin in adult patients with type 2 diabetes mellitus and answer the following question: In subjects with type 2 diabetes, can the method of high intensity interval training compared to moderate intensity exercise decrease glycosylated hemoglobin? We performed a critical analysis of the article "Feasibility and preliminary effectiveness of high intensity interval training in type 2 diabetes". We found no significant differences in the amount of glycosylated hemoglobin between groups of high intensity interval training and moderate-intensity exercise upon completion of the study (p>0.05). In adult patients with type 2 diabetes mellitus, high intensity interval training does not significantly improve glycosylated hemoglobin levels. Despite this, the high intensity interval training method shows as much improvement in body composition and physical condition as the moderate intensity exercise program.

Effect of astaxanthin in combination with alpha-tocopherol or ascorbic acid against oxidative damage in diabetic ODS rats.

Science.gov (United States)

Nakano, Masako; Onodera, Aya; Saito, Emi; Tanabe, Miyako; Yajima, Kazue; Takahashi, Jiro; Nguyen, Van Chuyen

2008-08-01

The present study was performed to investigate the effect of astaxanthin in combination with other antioxidants against oxidative damage in streptozotocin (STZ)-induced diabetic Osteogenic Disorder Shionogi (ODS) rats. Diabetic-ODS rats were divided into five groups: control, astaxanthin, ascorbic acid, alpha-tocopherol, and tocotrienol. Each of the four experimental groups was administered a diet containing astaxanthin (0.1 g/kg), in combination with ascorbic acid (3.0 g/kg), alpha-tocopherol (0.1 g/kg), or tocotrienol (0.1 g/kg) for 20 wk. The effects of astaxanthin with other antioxidants on lipid peroxidation, urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) excretion, serum creatinine (Cr) level, creatinine clearance (Ccr), and urinary protein content were assessed. The serum lipid peroxide levels and chemiluminescent (CL) intensity in the liver of the alpha-tocopherol and tocotrienol groups were significantly reduced in comparison to that of the control group. In the alpha-tocopherol group, urinary 8-OHdG excretion, serum Cr level, Ccr, urinary albumin excretion, and urinary protein concentration were significantly decreased as compared with those in the control group. Additionally, the CL intensity in the kidney of the alpha-tocopherol group was significantly lower, but that of the ascorbic acid group was significantly higher than that in the control group. These results indicate that dietary astaxanthin in combination with alpha-tocopherol has an inhibitory effect on oxidative stress. On the other hand, our study suggests that excessive ascorbic acid intake increases lipid peroxidation in diabetic rats.

Phytanic acid alpha-oxidation: decarboxylation of 2-hydroxyphytanoyl-CoA to pristanic acid in human liver

NARCIS (Netherlands)

Verhoeven, N. M.; Wanders, R. J.; Schor, D. S.; Jansen, G. A.; Jakobs, C.

1997-01-01

The degradation of the first intermediate in the alpha-oxidation of phytanic acid, 2-hydroxyphytanoyl-CoA, was investigated. Human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA or 2-hydroxyphytanic acid, after which formation of 2-ketophytanic acid and pristanic acid were studied.

Effects of omega-3 fatty acid plus alpha-tocopherol supplementation on malnutrition-inflammation score, biomarkers of inflammation and oxidative stress in chronic hemodialysis patients.

Science.gov (United States)

Asemi, Zatollah; Soleimani, Alireza; Shakeri, Hossein; Mazroii, Navid; Esmaillzadeh, Ahmad

2016-11-01

The current study was carried out to assess the effects of omega-3 fatty acid and alpha-tocopherol co-supplementation on malnutrition-inflammation score (MIS), biomarkers of inflammation and oxidative stress in chronic hemodialysis (HD) patients. In a randomized double-blind placebo-controlled clinical trial, 120 patients with chronic HD were included. Patients were randomly allocated into four groups to receive: (1) 1250 mg/day omega-3 fatty acid containing 600 mg EPA and 300 mg DHA + alpha-tocopherol placebo (n = 30); (2) 400 IU/day alpha-tocopherol + omega-3 fatty acids placebo (n = 30); (3) 1250 mg omega-3 fatty acids/day + 400 IU/day alpha-tocopherol (n = 30); and (4) omega-3 fatty acids placebo + alpha-tocopherol placebo (n = 30) for 12 weeks. After 12 weeks of intervention, all three groups of alpha-tocopherol only, individual omega-3 fatty acids, and combined omega-3 fatty acids and alpha-tocopherol experienced a significant improvements in MIS compared with the placebo group; however, improvements were much greater in the individual omega-3 fats (-1.4 ± 1.4) and combined omega-3 fats and alpha-tocopherol (-1.1 ± 2.3) groups compared with alpha-tocopherol group alone (-0.5 ± 1.7, P = 0.004). Furthermore, both individual and combined intervention with omega-3 fats and alpha-tocopherol led to a significant increase in plasma nitric oxide (NO) (combined group: +17.6 ± 29.3; alpha-tocopherol: +43.1 ± 36.3; omega-3 fats: +31.0 ± 40.0; and placebo: -0.5 ± 18.5 µmol/L, respectively, P acids and alpha-tocopherol co-supplementation for 12 weeks among HD patients improved MIS, plasma NO and TAC levels. Future studies with longer duration of the intervention are needed to confirm the validity of our findings. CLINICAL REGISTRATION: www.irct.ir as IRCT201410245623N28.

Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits

International Nuclear Information System (INIS)

Gesundheit, N.; Gyves, P.W.; DeCherney, G.S.; Stannard, B.S.; Winston, R.L.; Weintraub, B.D.

1989-01-01

Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with [35S]sulfate and [3H]mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharide species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of [3H]mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned

Mirrors in the PDB: left-handed alpha-turns guide design with D-amino acids.

Science.gov (United States)

Annavarapu, Srinivas; Nanda, Vikas

2009-09-22

Incorporating variable amino acid stereochemistry in molecular design has the potential to improve existing protein stability and create new topologies inaccessible to homochiral molecules. The Protein Data Bank has been a reliable, rich source of information on molecular interactions and their role in protein stability and structure. D-amino acids rarely occur naturally, making it difficult to infer general rules for how they would be tolerated in proteins through an analysis of existing protein structures. However, protein elements containing short left-handed turns and helices turn out to contain useful information. Molecular mechanisms used in proteins to stabilize left-handed elements by L-amino acids are structurally enantiomeric to potential synthetic strategies for stabilizing right-handed elements with D-amino acids. Propensities for amino acids to occur in contiguous alpha(L) helices correlate with published thermodynamic scales for incorporation of D-amino acids into alpha(R) helices. Two backbone rules for terminating a left-handed helix are found: an alpha(R) conformation is disfavored at the amino terminus, and a beta(R) conformation is disfavored at the carboxy terminus. Helix capping sidechain-backbone interactions are found which are unique to alpha(L) helices including an elevated propensity for L-Asn, and L-Thr at the amino terminus and L-Gln, L-Thr and L-Ser at the carboxy terminus. By examining left-handed alpha-turns containing L-amino acids, new interaction motifs for incorporating D-amino acids into right-handed alpha-helices are identified. These will provide a basis for de novo design of novel heterochiral protein folds.

Effect of ascorbic acid and alpha-tocopherol supplementations on serum leptin, tumor necrosis factor alpha, and serum amyloid A levels in individuals with type 2 diabetes mellitus

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Mostafa Jamalan

2015-10-01

Full Text Available Objective: Diabetes mellitus Type 2 is one of the most widespread chronic metabolic diseases. In most cases, this type of diabetes is associated with alterations in levels of some inflammatory cytokines and hormones. Considering anti-inflammatory properties of plant extracts rich in ascorbic acid (vitamin C and alpha-tocopherol (vitamin E, anti-diabetic properties of these two well-known antioxidant vitamins were investigated through measurement of serum levels of high-sensitivity C-reactive protein (hs-CRP, insulin, leptin, tumor necrosis factor alpha (TNF-α, and serum amyloid A (SAA in patients with diabetes mellitus type 2. Methods: Male patients (n=80 were randomly divided into two groups each consisted of 40 subjects. Test groups were supplemented with ascorbic acid (1000 mg/day or alpha-tocopherol (300 mg/day orally during four weeks. Before and after treatment, serum biochemical factors of subjects were measured and compared. Results: Our results showed that both ascorbic acid and alpha-tocopherol could induce significant anti-inflammatory effects by decreasing the level of inflammatory factors such as TNF-α, SAA, and hs-CRP in diabetes mellitus type 2 patients. Effects of alpha-tocopherol and ascorbic acid in decreasing serum leptin level were similar. Ascorbic acid in contrast to alpha-tocopherol diminished fasting insulin and HOMA index but had no effect on LDL serum level. Conclusion: Concerning the obtained results, it is concluded that consumption of supplementary vitamins C and E could decrease induced inflammatory response in patients with diabetes mellitus type 2.  It is also possible that vitamin C and vitamin E supplementation can attenuate incidence of some proposed pathological effects of diabetes mellitus.

Studies of iso-alpha-acids : analysis, purification, and stability.

NARCIS (Netherlands)

Khatib, Alfi

2006-01-01

The female cones of hop (Humulus lupulus L.) are added to beer, providing taste and flavour and contributing to the stability of foam. The main constituents of hop related to these properties are generically known as alpha-acids. During the brewing process, these acids are isomerized, resulting in

HEK293T cell lines defective for O-linked glycosylation.

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James M Termini

Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

cDNA cloning and characterization of Type I procollagen alpha1 chain in the skate Raja kenojei.

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Hwang, Jae-Ho; Yokoyama, Yoshihiro; Mizuta, Shoshi; Yoshinaka, Reiji

2006-05-01

A full-length cDNA of the Type I procollagen alpha1 [pro-alpha1(I)] chain (4388 bp), coding for 1463 amino acid residues in the total length, was determined by RACE PCR using a cDNA library constructed from 4-week embryo of the skate Raja kenojei. The helical region of the skate pro-alpha1(I) chain consisted of 1014 amino acid residues - the same as other fibrillar collagen alpha chains from higher vertebrates. Comparison on denaturation temperatures of Type I collagens from the skate, rainbow trout (Oncorhynchus mykiss) and rat (Rattus norvegicus) revealed that the number of Gly-Pro-Pro and Gly-Gly in the alpha1(I) chains could be directly related to the thermal stability of the helix. The expression property of the skate pro-alpha1(I) chain mRNA and phylogenetic analysis with other vertebrate pro-alpha1(I) chains suggested that skate pro-alpha1(I) chain could be a precursor form of the skate Type I collagen alpha1 chain. The present study is the first evidence for the primary structure of full-length pro-alpha1(I) chain in an elasmobranch.

Macrocyclic bis-thioureas catalyze stereospecific glycosylation reactions.

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Park, Yongho; Harper, Kaid C; Kuhl, Nadine; Kwan, Eugene E; Liu, Richard Y; Jacobsen, Eric N

2017-01-13

Carbohydrates are involved in nearly all aspects of biochemistry, but their complex chemical structures present long-standing practical challenges to their synthesis. In particular, stereochemical outcomes in glycosylation reactions are highly dependent on the steric and electronic properties of coupling partners; thus, carbohydrate synthesis is not easily predictable. Here we report the discovery of a macrocyclic bis-thiourea derivative that catalyzes stereospecific invertive substitution pathways of glycosyl chlorides. The utility of the catalyst is demonstrated in the synthesis of trans-1,2-, cis-1,2-, and 2-deoxy-β-glycosides. Mechanistic studies are consistent with a cooperative mechanism in which an electrophile and a nucleophile are simultaneously activated to effect a stereospecific substitution reaction. Copyright © 2017, American Association for the Advancement of Science.

High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Lausen, Thomas Frederik

2017-01-01

A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play...... led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy...

Chemical reactivity of {alpha}-isosaccharinic acid in heterogeneous alkaline systems

Energy Technology Data Exchange (ETDEWEB)

Glaus, M. A.; Loon, L. R. Van

2009-05-15

Cellulose degradation under alkaline conditions is of relevance for the mobility of many radionuclides in the near-field of a cementitious repository for radioactive waste, because metal-binding degradation products may be formed. Among these, {alpha}- isosaccharinic acid ({alpha}-ISA) is the strongest complexant. The prediction of the equilibrium concentration of {alpha}-ISA in cement pore water is therefore an important step in the assessment of the influence of cellulose degradation products on the speciation of radionuclides in such environments. The present report focuses on possible chemical transformation reactions of {alpha}-ISA in heterogeneous alkaline model systems containing either Ca(OH){sub 2} or crushed hardened cement paste. The transformation reactions were monitored by measuring the concentration of {alpha}-ISA by high performance anion exchange chromatography and the formation of reaction products by high performance ion exclusion chromatography. The overall loss of organic species from solution was monitored by measuring the concentration of non-purgeable organic carbon. The reactions were examined in diluted and compacted suspensions, at either 25 {sup o}C or 90 {sup o}C, and under anaerobic atmospheres obtained by various methods. It was found that {alpha}-ISA was transformed under all conditions tested to some extent. Reaction products, such as glycolate, formate, lactate and acetate, all compounds with less complexing strength than {alpha}-ISA, were detected. The amount of reaction products identified by the chromatographic technique applied was {approx} 50 % of the amount of {alpha}-ISA reacted. Sorption of {alpha}-ISA to Ca(OH){sub 2} contributed only to a minor extent to the loss of {alpha}-ISA from the solution phase. As the most important conclusion of the present work it was demonstrated that the presence of oxidising agents had a distinctive influence on the turnover of {alpha}-ISA. Under aerobic conditions {alpha}-ISA was

Glycosylation intermediates studied using low temperature ¹H- and ¹⁹F-DOSY NMR

DEFF Research Database (Denmark)

Qiao, Yan; Ge, Wenzhi; Jia, Lingyu

2016-01-01

Low temperature 1H- and 19F-DOSY have been used for analyzing reactive intermediates in glycosylation reactions, where a glycosyl trichloroacetimidate donor has been activated using different catalysts. The DOSY protocols have been optimized for low temperature experiments and provided new insight...

Trim24 (Tif1 alpha): an essential 'brake' for retinoic acid-induced transcription to prevent liver cancer.

Science.gov (United States)

Khetchoumian, Konstantin; Teletin, Marius; Tisserand, Johan; Herquel, Benjamin; Ouararhni, Khalid; Losson, Régine

2008-12-01

Retinoic acid (RA), the active derivative of vitamin A, is an important signaling molecule that controls various developmental processes and influence the proliferation and differentiation of a variety of cell types. RA exerts its biological functions primarily through binding to and activating nuclear RA receptors (RARs, which include the RAR alpha, beta and gamma isotypes RARA, RARB and RARC). Aberrant expression or impaired function of these nuclear receptors has been linked to diverse types of cancer. RARs are RA-dependent transcription factors that regulate gene expression through the recruitment of different co-regulators (co-activators and co-repressors). TRIM24 (formerly known as TIF1 alpha) was among the first co-regulators identified as interacting with RARs in a ligand-dependent fashion, and it was recently shown to function in mice as a potent liver-specific tumor suppressor by attenuating Rara-mediated transcription. The fact that Trim24(-/-), but not Trim24(-/-)Rara(+/-), mutant mice are highly predisposed to the development of hepatocellular carcinoma (HCC) has significant implications in cancer research. This result, along with the observation that in response to pharmacological inhibition of the RA signaling, hepatocytes lacking Trim24 loose their ability to proliferate, strongly implicates Rara as a proto-oncogene in hepatocytes and demonstrates that overactivated RA signaling is deleterious to liver homeostasis.

Detection of site specific glycosylation in proteins using flow cytometry†

Science.gov (United States)

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Heparin Binding Ability

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Chiung-Fang Chang

2016-06-01

Full Text Available R-spondin 1 (Rspo1 plays an essential role in stem cell biology by potentiating Wnt signaling activity. Despite the fact that Rspo1 holds therapeutic potential for a number of diseases, its biogenesis is not fully elucidated. All Rspo proteins feature two amino-terminal furin-like repeats, which are responsible for Wnt signal potentiation, and a thrombospondin type 1 (TSR1 domain that can provide affinity towards heparan sulfate proteoglycans. Using chemical inhibitors, deglycosylase and site-directed mutagenesis, we found that human Rspo1 and Rspo3 are both N-glycosylated at N137, a site near the C-terminus of the furin repeat 2 domain, and Rspo2 is N-glycosylated at N160, a position near the N-terminus of TSR1 domain. Elimination of N-glycosylation at these sites affects their accumulation in media but have no effect on the ability towards heparin. Introduction of the N-glycosylation site to Rspo2 mutant at the position homologous to N137 in Rspo1 restored full glycosylation and rescued the accumulation defect of nonglycosylated Rspo2 mutant in media. Similar effect can be observed in the N137 Rspo1 or Rspo3 mutant engineered with Rspo2 N-glycosylation site. The results highlight the importance of N-glycosylation at these two positions in efficient folding and secretion of Rspo family. Finally, we further showed that human Rspo1 is subjected to endoplasmic reticulum (ER quality control in N-glycan-dependent manner. While N-glycan of Rspo1 plays a role in its intracellular stability, it had little effect on secreted Rspo1. Our findings provide evidence for the critical role of N-glycosylation in the biogenesis of Rspo1.

Importance of N-Glycosylation on CD147 for Its Biological Functions

Science.gov (United States)

Bai, Yang; Huang, Wan; Ma, Li-Tian; Jiang, Jian-Li; Chen, Zhi-Nan

2014-01-01

Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation. PMID:24739808

Importance of N-Glycosylation on CD147 for Its Biological Functions

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Yang Bai

2014-04-01

Full Text Available Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation.

Characterisation of a thiamine diphosphate-dependent alpha-keto acid decarboxylase from Proteus mirabilis JN458.

Science.gov (United States)

Wang, Biying; Bai, Yajun; Fan, Taiping; Zheng, Xiaohui; Cai, Yujie

2017-10-01

Alpha-keto acid decarboxylases can convert keto acids to their corresponding aldehydes, which are often volatile aroma compounds. The gene encoding α-keto acid decarboxylase in Proteus mirabilis JN458 was cloned, and the enzyme overexpressed in Escherichia coli BL21 (DE3), purified in high yield, and characterised. The molecular weight is 62.291kDa by MALDI-TOF MS, and optimum activity at pH 6.0 and 40-50°C. The enzyme is a typical decarboxylase, dependent on thiamine diphosphate and Mg 2+ as cofactors. For the decarboxylation reaction, the enzyme displayed a broad substrate range. Kinetic parameters were determined using 4-methyl-2-oxopentanoic acid, phenyl pyruvate and 3-methyl-2-oxopentanoic acid as substrates. K m and k cat values for phenyl pyruvate were 0.62mM and 77.38s -1 , respectively, and the k cat /K m value was 124.81mM -1 s -1 . The enzyme properties suggest it may act effectively under cheese ripening conditions. Copyright © 2017. Published by Elsevier Ltd.

Engineering Mammalian Mucin-type O-Glycosylation in Plants

DEFF Research Database (Denmark)

Yang, Zhang; Drew, Damian P; Jørgensen, Bodil

2012-01-01

-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity...... was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon a2b. In plants, prolines in certain...... classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host...

Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

Science.gov (United States)

Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

2009-01-01

Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

Glycation and transglutaminase mediated glycosylation of fish gelatin peptides with glucosamine enhance bioactivity.

Science.gov (United States)

Hong, Pui Khoon; Gottardi, Davide; Ndagijimana, Maurice; Betti, Mirko

2014-01-01

A mixture of novel glycopeptides from glycosylation between cold water fish skin gelatin hydrolysates and glucosamine (GlcN) via transglutaminase (TGase), as well as glycation between fish gelatin hydrolysate and GlcN were identified by their pattern of molecular distribution using MALDI-TOF-MS. Glycated/glycosylated hydrolysates showed superior bioactivity to their original hydrolysates. Alcalase-derived fish skin gelatin hydrolysate glycosylated with GlcN in the presence of TGase at 25°C (FAT25) possessed antioxidant activity when tested in a linoleic acid oxidation system, when measured according to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and when tested at the cellular level with human hepatocarcinoma (HepG2) cells as target cells. In addition, Alcalase-derived glycosylated hydrolysates showed specificity toward the inhibition of Escherichia coli (E. coli). The Flavourzyme-derived glycopeptides prepared at 37°C (FFC37 and FFT37) showed better DPPH scavenging activity than their native hydrolysates. The glycated Flavourzyme-derived hydrolysates were found to act as potential antimicrobial agents when incubated with E. coli and Bacillus subtilis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN) on monocytes/macrophages.

Science.gov (United States)

Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

2015-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN on monocytes/macrophages.

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Heng Ge

Full Text Available Extracellular matrix metalloproteinase inducer (EMMPRIN is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages.The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway.1 It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN that increased after being exposed to inflammatory signals (PMA and H2O2. 2 Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG the simple type. 3 Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression.Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Preparation of. alpha. -deuterated L-amino acids using E. coli cells containing tryptophanase. Poluchenie. alpha. -dejterirovannykh L-aminokislot s ispol'zovaniem kletok E. coli, soderzhashchikh triptofanazy

Energy Technology Data Exchange (ETDEWEB)

Faleev, N G; Ruvinov, S B; Saporovskaya, M B; Belikov, V M; Zakomyrdina, L N; Sakharova, I S; Torchinskij, Yu M [AN SSSR, Moscow (USSR). Inst. Ehlementoorganicheskikh Soedinenij AN SSSR, Moscow (USSR). Inst. Molekulyarnoj Biologii AN SSSR, Moscow (USSR)

1989-10-01

Method for preparation of a series of {alpha}-deuterated L-amino acids of high optical purity with quantitative chemica yield, suing stereospecific isotopic exchange in D{sub 2}O under the effect of E.coli cells with high tryptophanase activity was developed.

VITAMIN C, VITAMIN A AND ALPHA HYDROXY ACID IN BENGKOANG (Pachyrhizus Erosus

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Ardian Widyatmoko

2016-04-01

Full Text Available Bengkoang or Pachyrhizus erosus has been used as a traditional cosmetic material since many years ago. Until now, many cosmetic preparations using bengkoang as a main material have been produced by cosmetic industries, mainly for skin care, whitening and sunscreen preparation. However, content of vitamin A, vitamin C and alpha hydroxyl acid has not been discovered yet. These compounds are essential in skin care process because of their activities in cell regeneration, antioxidant as well as peeling of dead skin cell layer. The aim of this research is to provide information about the content of three compounds in bengkoang. Vitamin A has been analysed by spectrophotometer, vitamin C analysed using titration method and alpha hydroxyl acid analysed using gas chromatography. The result showed that concentrations of vitamin A and vitamin C are 179.21 ± 8.19 ppm and 0.31 ± 0.06 %, respectively. Meanwhile the content of alpha hydroxyl acid was 0.80 ± 0.01% (measured as glycolic acid.

Oxidative stability of dark chicken meat through frozen storage: influence of dietary fat and alpha-tocopherol and ascorbic acid supplementation.

Science.gov (United States)

Grau, A; Guardiola, F; Grimpa, S; Barroeta, A C; Codony, R

2001-11-01

We used factorial design to ascertain the influence of dietary fat source (linseed, sunflower and oxidized sunflower oils, and beef tallow) and the dietary supplementation with alpha-tocopheryl acetate (alpha-TA) (225 mg/kg of feed) and ascorbic acid (AA) (110 mg/kg) on dark chicken meat oxidation (lipid hydroperoxide and TBA values and cholesterol oxidation product content). alpha-TA greatly protected ground and vacuum-packaged raw or cooked meat from fatty acid and cholesterol oxidation after 0, 3.5, or 7 mo of storage at -20 C. In contrast, AA provided no protection, and no synergism between alpha-TA and AA was observed. Polyunsaturated fatty acid-enriched diets (those containing linseed, sunflower, or oxidized sunflower oils) increased meat susceptibility to oxidation. Cooking always involved more oxidation, especially in samples from linseed oil diets. The values of all the oxidative parameters showed a highly significant negative correlation with the alpha-tocopherol content of meat.

Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation

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Olgun eGuvench

2015-06-01

Full Text Available The extracellular N-terminal hyaluronan binding domain (HABD of CD44 is a small globular domain that confers hyaluronan (HA binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA binding site from a low affinity to a high affinity state; in the partially-disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently-attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD.

Deorphanization of GPRC6A: a promiscuous L-alpha-amino acid receptor with preference for basic amino acids

DEFF Research Database (Denmark)

Wellendorph, Petrine; Hansen, Kasper B; Balsgaard, Anders

2005-01-01

with the signal transducing transmembrane and C terminus of the homologous goldfish 5.24 receptor allowed us to overcome these obstacles. Homology modeling of the hGPRC6A ATD based on the crystal structure of the metabotropic glutamate receptor subtype 1 predicted interaction with alpha-amino acids...

Kinetics of alpha-amylase secretion in Aspergillus oryzae

DEFF Research Database (Denmark)

Henriksen, Anne Laurence Santerre; Carlsen, Morten; Bang de, H.

1999-01-01

-chase experiments were carried out to investigate the alpha-amylase secretion kinetics in A. oryzae. No unglycosylated alpha-amylase was detected neither intracellularly nor extracellularly demonstrating that glycosylation was not the rate controlling step in the secretory pathway. The pulse chase experiments...... indicated that there are two pools of intracellular alpha-amylase: a fast secreted and a slow secreted. The secretion of those two pools were described with a kinetic model, which was fitted to the pulse chase experiments. (C) 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 76-82, 1999....

[Cholesterol metabolism and lipid peroxidation processes in hypodynamia. Effect of using ascorbic acid and alpha-tocopherol].

Science.gov (United States)

Elikov, A V; Tsapok, P I

2010-01-01

Study status of cholesterol metabolism, processes of lipid peroxidation and antioxidant protection in blood plasma, erythrocytes and homogenates of the, heart, liver, muscle femors of rats attached to movement active. Establishment effects application of ascorbic acid and alpha-tocopherol. Ascorbic acid and alpha-tocopherol were infused daily. The daily dosage was 2 and 1 mg respectively. Characteristic shift changes of cholesterol metabolism in conditions of limited muscular activity were revealed. It was shown that vitamin antioxidants play a role in correction of metabolic disorders in case of immobile distress syndrome.

Insufficient intake of alpha-linolenic fatty acid (18:3n-3 during pregnancy and associated factors

Directory of Open Access Journals (Sweden)

Letícia Garcia VASCONCELOS

Full Text Available ABSTRACT Objective: To analyze alpha-linolenic fatty acid intake in two cohorts of pregnant women, and to identify factors associated with alpha-linolenic acid intake. Methods: This is a cohort study involving pregnant women with low obstetric risk (N=353 in public health system from a municipality of São Paulo state, Brazil. In each trimester, two 24-hour food recalls were collected. Descriptive analyses of dietary lipid profiles were performed, followed by a multiple comparison test. According to the trimester of pregnancy, differences were assessed using the mean difference test. To evaluate the adequacy of linoleic fatty acid and alpha-linolenic acid intake, the adequate intake test was used. The association between alpha-linolenic acid intake adequacy and maternal characteristics was investigated using a binary logistic regression model. Results: Total lipids intake and the percentage contribution to dietary energy met recommended levels. One-third of the diets demonstrated a lower than daily recommended intake of alpha-linolenic acid. Overweight pregnant women were twice as likely to have inadequate alpha-linolenic acid intake. Pregnant women from a more disadvantaged socioeconomic situation had greater risks of inadequate intake. Conclusion: Over-intake of lipids is not problematic, but quality is an issue, with one third of the pregnant women and their fetuses exposed to adverse effects due to low intake of omega-3 fatty acids, indicating important nutritional vulnerability in this population.

“CLEAR BRANDY” PRODUCTION USING ALPHA ACID EXTRACT FROM HOPS IN THE BIOCIDAL CONTROL OF THE FERMENTATION PROCESS

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Tassiana Amélia de Oliveira e Silva

2016-01-01

Full Text Available The major problem in the production of “clear brandy” is the contamination of the must by bacteria of the species Lactobacillus. To overcome this problem, this work intends to evaluate the usage of the alpha acids from hop extract (Humulus lupulus as antibacterial agent during brandy production. In addition, the maximum cell recycling was evaluated during the clear brandy production. Preliminary experiments using the Sabourand synthetic medium added with 40 ppm of the alpha acids reduced the cell viability of L. casei and L. plantarum bacteria from 108 CFU.mL-1 to 105 CFU.mL-1 and showed no effect in the growth of Saccharomyces cerevisiae. These experiments were conducted at 120 rpm and 25°C. The clear brandy production (200 L using the same alpha acids concentration (40 ppm and Saccharomyces cerevisiae cell recycling (14 times showed no contamination by bacteria. Then, the clear brandy was distilled in a 160 L cooper distiller. The clear brandy produced with and without alpha acids, was analyzed in the Laboratory of Alcohol and Beverages as well as in the Laboratory of Spectroscopy of the Cuban Research Institute of the Sugarcane Derivatives (ICIDCA in Spanish in Havana and showed similarities in their composition and sensorial chemical analysis.

Comparative Glycoproteome Analysis: Dynamics of Protein Glycosylation during Metamorphic Transition from Pelagic to Benthic Life Stages in Three Invertebrates

KAUST Repository

Chandramouli, Kondethimmanahalli

2012-02-03

The life cycle of most benthic marine invertebrates has two distinct stages: the pelagic larval stage and the sessile juvenile stage. The transition between the larval stage and the juvenile stage is often abrupt and may be triggered by post-translational modification of proteins. Glycosylation, a very important post-translational modification, influences the biological activity of proteins. We used two-dimensional gel electrophoresis (2-DE) followed by glycoprotein-specific fluorescence staining and mass spectrometry with the goal of identifying glycosylation pattern changes during larval settlement and metamorphosis in barnacles, bryozoans, and polychaetes. Our results revealed substantial changes in the protein glycosylation patterns from larval to juvenile stages. Before metamorphosis, the degree of protein glycosylation was high in the barnacle Balanus (=Amphibalanus) amphitrite and the spionid polychaete Pseudopolydora vexillosa, whereas it increased after metamorphosis in the bryozoan Bugula neritina. We identified 19 abundant and differentially glycosylated proteins in these three species. Among the proteins, cellular stress- and metabolism-related proteins exhibited distinct glycosylation in B. amphitrite and B. neritina, whereas fatty acid metabolism-related proteins were abundantly glycosylated in P. vexillosa. Furthermore, the protein and gene expression analysis of some selected glycoproteins revealed that the degree of protein glycosylation did not always complement with transcriptional and translational changes associated with the larval-juvenile transition. The current study provides preliminary information on protein glycosylation in marine invertebrates that will serve as a solid basis for future comprehensive analysis of glycobiology during larval settlement and metamorphosis. © 2011 American Chemical Society.

Beta-alanine/alpha-ketoglutarate aminotransferase for 3-hydroxypropionic acid production

Science.gov (United States)

Jessen, Holly Jean [Chanhassen, MN; Liao, Hans H [Eden Prairie, MN; Gort, Steven John [Apple Valley, MN; Selifonova, Olga V [Plymouth, MN

2011-10-04

The present disclosure provides novel beta-alanine/alpha ketoglutarate aminotransferase nucleic acid and protein sequences having increased biological activity. Also provided are cells containing such enzymes, as well as methods of their use, for example to produce malonyl semialdehyde and downstream products thereof, such as 3-hydroxypropionic acid and derivatives thereof.

Evaluation on a radioimmunoassay of. alpha. /sub 1/ microglobulin (. alpha. /sub 1/-m) with simplified procedures

Energy Technology Data Exchange (ETDEWEB)

Matsui, Kazuyo; Moriuma, Hatsuko; Mishima, Chiho; Honda, Minoru; Tomonobu, Masahiro; Kanao, Keisuke; Fushimi, Hisako (Sumitomo Hospital, Osaka (Japan))

1989-06-01

A newly established double antibody radioimmunoassay (RIA) was fundamentally and clinically evaluated. Original procedures were partially modified as follows: Sample volume for serum and urine was changed to 25{mu}l, and thus 200 mg/l of {alpha}/sub 1/-m standard was prepared using 50 {mu}l of original standard solution (100 mg/l). The results were satisfactory in sensitivity (0.3 mg/l obtained from -2SD method), intraassay precision with its coefficient variation (CV) ranging from 3.0 to 7.4%, interassay precision with its CV ranging from 3.0 to 10.7%, and recovery with the mean value of 102.4% in serum and 108.2% in urine respectively. There were no changes about {alpha}/sub 1/-m value between diluted (2 times) and undiluted with high concentration samples. Normal levels of {alpha}/sub 1/-m were less than 25 mg/l is serum and less than 10 mg/l in urine. The present results indicate that the determination of {alpha}/sub 1/-m could be very simple and useful for the most sensitive screening test for the evaluation of renal function. (author).

PANP is a novel O-glycosylated PILR{alpha} ligand expressed in neural tissues

Energy Technology Data Exchange (ETDEWEB)

Kogure, Amane [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); Shiratori, Ikuo [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Wang, Jing [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); Lanier, Lewis L. [Department of Microbiology and Immunology and the Cancer Research Institute, University of California San Francisco, San Francisco, CA 94143 (United States); Arase, Hisashi, E-mail: arase@biken.osaka-u.ac.jp [Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871 (Japan); Laboratory of Immunochemistry, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871 (Japan); JST CREST, Saitama 332-0012 (Japan)

2011-02-18

Research highlights: {yields} A Novel molecule, PANP, was identified to be a PILR{alpha} ligand. {yields} Sialylated O-glycan structures on PANP were required for PILR{alpha} recognition. {yields} Transcription of PANP was mainly observed in neural tissues. {yields} PANP seems to be involved in immune regulation as a ligand for PILR{alpha}. -- Abstract: PILR{alpha} is an immune inhibitory receptor possessing an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain enabling it to deliver inhibitory signals. Binding of PILR{alpha} to its ligand CD99 is involved in immune regulation; however, whether there are other PILR{alpha} ligands in addition to CD99 is not known. Here, we report that a novel molecule, PILR-associating neural protein (PANP), acts as an additional ligand for PILR{alpha}. Transcription of PANP was mainly observed in neural tissues. PILR{alpha}-Ig fusion protein bound cells transfected with PANP and the transfectants stimulated PILR{alpha} reporter cells. Specific O-glycan structures on PANP were found to be required for PILR recognition of this ligand. These results suggest that PANP is involved in immune regulation as a ligand of the PILR{alpha}.

A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, Jakob; Elortza, Felix

2004-01-01

remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach...

Lower critical solution temperature behavior of alpha-substituted poly(acrylic acids)s, cyclopolymerization of N-vinylformamido-methylacrylates, and use of the World-Wide Web in polymer science education

Science.gov (United States)

Michalovic, Mark Stephen

A series of alpha-substituted poly(acrylic acid)s was synthesized and characterized. Their aqueous solution properties were investigated with respect to lower critical solution temperature (LCST) behavior. Poly(alpha-methoxymethylacrylic acid) was found to have a lower critical solution temperature (LCST) of 46°C, poly(alpha-methoxyethoxymethylacrylic acid) showed an LCST of 26.5°C and poly(alpha-methoxyethoxyethoxymethylacrylic acid) showed an LCST of 66°C. The cloud points of the solutions of these polymers were found to be sensitive to pH, and to concentrations of additives such as urea, salts, and surfactants. Because of low molecular weight due to chain transfer, high molecular weight analogs of the ether-linked polymers were synthesized in which ester linkages joined the oligo-oxyethylene segment to the acrylate moiety. Poly(alpha-methoxyethoxyacetoxymethylacrylic acid) was the only one of this series to give an LCST with a value of 52.5°C. Copolymers of t-butyl alpha-methoxymethylacrylate (tBMMA) with alpha-(1H,1H- perfluorooctyloxymethyl)acrylic acid (PFOMA) were synthesized, deprotected and their lower critical solution temperatures (LCSTs) evaluated. At PFOMA feed ratios of 0.25 mol % or less, no observable change in the LCST was observed, while at PFOMA feed ratios of above 0.25 mol % to 1.125 mol %, a large linear decrease in the LCST was observed with increasing fluorocarbon content. t-Butyl alpha-(N-vinylformamidomethyl)acrylate (tBVFA) and ethyl alpha-(N-vinylformamidomethyl)acrylate (EVFA) were synthesized from t-butyl alpha-bromomethylacrylate and ethyl alpha-chloromethylacrylate, respectively. tBVFA was found to cyclopolymerize at 120°C in DMF, DMSO, and 1,2-dichlorobenzene at solvent:monomer ratios of 10:1 vol:wt. Molecular weights for poly(tBVFA) ranged from 10,000 to 13,000 as estimated by size-exclusion chromatography. At lower solvent monomer ratio (1:1), and at lower temperature (71°C), crosslinking occurred. EVFA was found to

Effect of alpha linolenic acid supplementation on serum prostate specific antigen (PSA: results from the alpha omega trial.

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Ingeborg A Brouwer

Full Text Available Alpha linolenic acid (ALA is the major omega-3 fatty acid in the diet. Evidence on health effects of ALA is not conclusive, but some observational studies found an increased risk of prostate cancer with higher intake of ALA. We examined the effect of ALA supplementation on serum concentrations of prostate-specific antigen (PSA, a biomarker for prostate cancer.The Alpha Omega Trial (ClinicalTrials.gov Identifier: NCT00127452 was a double-blind, placebo-controlled trial of ALA and the fish fatty acids eicosapentanoic acid (EPA and docosahexanoic acid (DHA on the recurrence of cardiovascular disease, using a 2×2 factorial design. Blood was collected at the start and the end of the intervention period. The present analysis included 1622 patients with a history of a myocardial infarction, aged 60-80 years with an initial PSA concentration 4 ng/mL.Mean serum PSA increased by 0.42 ng/mL on placebo (n = 815 and by 0.52 ng/mL on ALA (n = 807, a difference of 0.10 (95% confidence interval: -0.02 to 0.22 ng/mL (P = 0·12. The odds ratio for PSA rising above 4 ng/mL on ALA versus placebo was 1.15 (95% CI: 0.84-1.58.An additional amount of 2 g of ALA per day increased PSA by 0.10 ng/mL, but the confidence interval ranged from -0.02 to 0.22 ng/mL and included no effect. Therefore, more studies are needed to establish whether or not ALA intake has a clinically significant effect on PSA or prostate cancer.ClinicalTrials.gov; Identifier: NCT00127452. URL: http://www.clinicaltrials.gov/ct2/show/NCT00127452.

Secretion of alpha 2-plasmin inhibitor is impaired by amino acid deletion in a small region of the molecule.

Science.gov (United States)

Toyota, S; Hirosawa, S; Aoki, N

1994-02-01

Alpha 2-plasmin inhibitor (alpha 2PI) deficiency Okinawa results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of the deletion of Glu137 in the amino acid sequence of alpha 2PI. To examine the effects of replacing the amino acid occupying position 137 or deleting its neighboring amino acid on alpha 2PI secretion, we used oligonucleotide-directed mutagenesis of alpha 2PI cDNA to change the codon specifying Glu137 or delete a codon specifying its neighboring amino acid. The effects were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of media from transiently transfected COS-7 cells. Replacement of Glu137 with an amino acid other than Cys had little effect on alpha 2PI secretion. In contrast, deletion of an amino acid in a region spanning a sequence of less than 30 amino acids including positions 127 and 137 severely impaired the secretion. The results suggest that structural integrity of the region, rather than its component amino acids, is important for the intracellular transport and secretion of alpha 2PI.

Synthesis and biological evaluation of 2-heteroarylthioalkanoic acid analogues of clofibric acid as peroxisome proliferator-activated receptor alpha agonists.

Science.gov (United States)

Giampietro, Letizia; Ammazzalorso, Alessandra; Giancristofaro, Antonella; Lannutti, Fabio; Bettoni, Giancarlo; De Filippis, Barbara; Fantacuzzi, Marialuigia; Maccallini, Cristina; Petruzzelli, Michele; Morgano, Annalisa; Moschetta, Antonio; Amoroso, Rosa

2009-10-22

A series of 2-heteroarylthioalkanoic acids were synthesized through systematic structural modifications of clofibric acid and evaluated for human peroxisome proliferator-activated receptor alpha (PPARalpha) transactivation activity, with the aim of obtaining new hypolipidemic compounds. Some thiophene and benzothiazole derivatives showing a good activation of the receptor alpha were screened for activity against the PPARgamma isoform. The gene induction of selected compounds was also investigated in the human hepatoma cell line.

Halide-mediated regioselective 6-O-glycosylation of unprotected hexopyranosides with perbenzylated glycosyl bromide donors

DEFF Research Database (Denmark)

Niedbal, Dominika Alina; Madsen, Robert

2016-01-01

The regio- and stereoselective glycosylation at the 6-position in 2,3,4,6-unprotected hexopyranosides has been investigated with dibutyltin oxide as the directing agent. Perbenzylated hexopyranosyl bromides were employed as the donors and the glycosylations were promoted by tetrabutylammonium...... bromide. The couplings were completely selective for both glucose and galactose donors and acceptors as long as the stannylene acetal of the acceptor was soluble in dichloromethane. This gave rise to a number of 1,2-cis-linked disaccharides in reasonable yields. Mannose donors and acceptors, on the other...

Synthesis and antimicrobial evaluation of new 3-alkyl/aryl-2-[((alpha,alpha-diphenyl-alpha-hydroxy)acetyl)hydrazono]-5-methyl-4-thiazolidinones.

Science.gov (United States)

Güzeldemirci, Nuray Ulusoy; Ilhan, Eser; Küçükbasmaci, Omer; Satana, Dilek

2010-01-01

New 4-thiazolidinone derivatives of benzilic acid (alpha,alpha-diphenyl-alpha-hydroxyacetic acid) have been synthesized and evaluated for antibacterial and antifungal activities. The reaction of 1- (alpha,alpha-diphenyl-alpha-hydroxy)acetyl-4-alkyl/arylthiosemicarbazides with ethyl 2-bromopropionate gave 3-alkyl/aryl-2-[((alpha,alpha-diphenyl-alpha-hydroxy)acetyl)hydrazono]-5-methyl-4-thiazolidinone derivatives. Their antibacterial and antifungal activities were evaluated against S. aureus ATCC 29213, P. aeruginosa ATCC 27853, E. coli ATCC 25922, C. albicans ATCC 10231, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, T. mentagrophytes var. erinacei NCPF 375, M. gypseum NCPF 580 and T. tonsurans NCPF 245. 3e, 3f, 3g and 3h showed the highest antibacterial activity. Particularly 3a and 3e showed the highest antifungal activities against C. parapsilosis ATCC 22019, T. tonsurans NCPF 245 and M. gypseum NCPF 580.

Mechanisms and regulation of TSH glycosylation

International Nuclear Information System (INIS)

Gesundheit, N.; Weintraub, B.D.

1986-01-01

Some differences in carbohydrate composition of secreted TSH-alpha, TSH-beta, and free alpha-subunits are examined. In order to explore whether each of the secreted TSH subunits has a distinctive carbohydrate composition, the authors incubated hypothyroid mouse tritiated sugar precursors: tritium-glucosamine; tritium-mannose; tritium-l-fucose; and tritium-N-acetylmannosamine, a specific precursor of the sialic acid tritium-N-acetylneuraminic acid. It was shown that although the two TSH subunits, alpha and beta, combine soon after synthesis and are secreted as a dimeric protein, there are notable differences in the carbohydrate processing of the two subunits

Amino acid sequences mediating vascular cell adhesion molecule 1 binding to integrin alpha 4: homologous DSP sequence found for JC polyoma VP1 coat protein

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Michael Andrew Meyer

2013-07-01

Full Text Available The JC polyoma viral coat protein VP1 was analyzed for amino acid sequences homologies to the IDSP sequence which mediates binding of VLA-4 (integrin alpha 4 to vascular cell adhesion molecule 1. Although the full sequence was not found, a DSP sequence was located near the critical arginine residue linked to infectivity of the virus and binding to sialic acid containing molecules such as integrins (3. For the JC polyoma virus, a DSP sequence was found at residues 70, 71 and 72 with homology also noted for the mouse polyoma virus and SV40 virus. Three dimensional modeling of the VP1 molecule suggests that the DSP loop has an accessible site for interaction from the external side of the assembled viral capsid pentamer.

Tolerability in the elderly population of high-dose alpha lipoic acid: a potential antioxidant therapy for the eye

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Sarezky D

2016-09-01

Full Text Available Daniel Sarezky, Aaishah R Raquib, Joshua L Dunaief, Benjamin J Kim Scheie Eye Institute, Department of Ophthalmology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Purpose: Alpha lipoic acid (ALA is an antioxidant and iron-chelating supplement that has potential benefits for geographic atrophy in dry age-related macular degeneration as well as other eye diseases. The purpose of this study was to determine the tolerability of ALA in the elderly population. Patients and methods: Fifteen subjects, age ≥65 years, took sequential ALA doses of 600, 800, and 1,200 mg. Each dose was taken once daily with a meal for 5 days. After each dose was taken by the subjects for 5 days, the subjects were contacted by phone, a review of systems was performed, and they were asked if they thought they could tolerate taking that dose of ALA for an extended period of time. Results: The 600 mg dose was well tolerated. At the 800 mg dose, one subject had an intolerable flushing sensation. At the 1,200 mg dose, two subjects had intolerable upper gastrointestinal side effects and one subject had an intolerable flushing sensation. Subjects taking gastrointestinal prophylaxis medications had no upper gastrointestinal side effects. Conclusion: High-dose ALA is not completely tolerated by the elderly. These preliminary data suggest that gastrointestinal prophylaxis may improve tolerability. (ClinicalTrials.gov, NCT02613572. Keywords: age-related macular degeneration, geographic atrophy, antioxidant, gastrointestinal, dietary supplements, lipoic acid

An NMR and ab initio quantum chemical study of acid-base equilibria for conformationally constrained acidic alpha-amino acids in aqueous solution

DEFF Research Database (Denmark)

Nielsen, Peter Aadal; Jaroszewski, Jerzy W.; Norrby, Per-Ola

2001-01-01

The protonation states of a series of piperidinedicarboxylic acids (PDAs), which are conformationally constrained acidic alpha -amino acids, have been studied by C-13 NMR titration in water. The resulting data have been correlated with theoretical results obtained by HF/6-31+G* calculations using...

Effect of alpha linolenic acid supplementation on serum prostate specific antigen (PSA): results from the alpha omega trial

NARCIS (Netherlands)

Brouwer, Ingeborg A.; Geleijnse, Johanna M.; Klaasen, Veronique M.; Smit, Liesbeth A.; Giltay, Erik J.; de Goede, Janette; Heijboer, Annemieke C.; Kromhout, Daan; Katan, Martijn B.

2013-01-01

Alpha linolenic acid (ALA) is the major omega-3 fatty acid in the diet. Evidence on health effects of ALA is not conclusive, but some observational studies found an increased risk of prostate cancer with higher intake of ALA. We examined the effect of ALA supplementation on serum concentrations of

Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

DEFF Research Database (Denmark)

Scott, N. E.; Kinsella, R. L.; Edwards, A. V. G.

2014-01-01

nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison......-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans...

Method Development in the Regioselective Glycosylation of Unprotected Carbohydrates

DEFF Research Database (Denmark)

Niedbal, Dominika Alina

and the glycosylations were promoted by tetrabutylammonium bromide. The couplings were completely selective and gave rise to a number of 1,6-linked disaccharides with 1,2- cis-linked orientation. Project 2: Boron-mediated glycosylation of unprotected carbohydrates Boron-mediated regioselective Koenigs...

The remarkable stability of chimeric, sialic acid-derived alpha/delta-peptides in human blood plasma.

Science.gov (United States)

Saludes, Jonel P; Natarajan, Arutselvan; DeNardo, Sally J; Gervay-Hague, Jacquelyn

2010-05-01

Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-alpha-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric alpha/delta-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en alpha/delta-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with (111)In, incubated in human blood plasma at 37 degrees C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural alpha-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.

Classification of alpha 1-adrenoceptor subtypes

NARCIS (Netherlands)

Michel, M. C.; Kenny, B.; Schwinn, D. A.

1995-01-01

Two alpha 1-adrenoceptor subtypes (alpha 1A and alpha 1B) have been detected in various tissues by pharmacological techniques, and three distinct cDNAs encoding alpha 1-adrenoceptor subtypes have been cloned. The profile of an increasing number of subtype-selective compounds at cloned and endogenous

Anti-pp,. cap alpha cap alpha. and p. cap alpha. elastic scattering at high energies and Chou-Yang conjecture

Energy Technology Data Exchange (ETDEWEB)

Saleem, M.; Fazal-e-Aleem; Rifique, M.

1987-03-01

The recent experimental measurements for anti-pp and ..cap alpha cap alpha.. elastic scattering at high energies have shown that the Chou-Yang conjecture regarding the relationship between the electromagnetic and the hadronic form factor of a particle is only an approximation. A new ansatz has been proposed to obtain hadronic form factors of proton and the ..cap alpha..-particle. These form factors have been used to explain the various characteristics of anti-pp, ..cap alpha cap alpha.. and p..cap alpha.. elastic scattering at high energies.

The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes.

Science.gov (United States)

Fushitani, K; Higashiyama, K; Moriyama, E N; Imai, K; Hosokawa, K

1996-09-01

To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.

Thermophilic and thermoacidophilic glycosylation genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

Science.gov (United States)

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2016-01-12

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for glycosylating and/or post-translationally modifying proteins using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Alpha lipoic acid attenuates high-fructose-induced pancreatic toxicity.

Science.gov (United States)

Topsakal, Senay; Ozmen, Ozlem; Cankara, Fatma Nihan; Yesilot, Sukriye; Bayram, Dilek; Genç Özdamar, Nilüfer; Kayan, Sümeyra

2016-01-01

Chronic consumption of high-fructose corn syrup (HFCS) causes several problems such as insulin resistance. The goal of the study was to investigate pancreatic damage induced by chronic HFCS consumption and the protective effects of alpha lipoic acid (ALA) on pancreatic cells. Wistar Albino, 4-month-old, female rats weighing 250-300 g were randomly distributed into three groups, each containing eight rats. The study included an HFCS group, an HFCS + ALA-administered group and a control group (CON). The prepared 30% solution of HFCS (F30) (24% fructose, 28% dextrose) was added to the drinking water for 10 weeks. ALA treatment was begun 4 weeks after the first HFCS administration (100 mg/kg/oral, last 6 weeks). Rats were anaesthetised and euthanised by cervical dislocation 24 h after the last ALA administration. Blood samples for biochemical tests (amylase, lipase, malondialdehyde (MDA) and catalase (CAT)) and tissue samples for histopathological and immunohistochemical examinations (caspase-3, insulin and glucagon) were collected. Comparing the control and HFCS groups, serum glucose (150.92 ± 39.77 and 236.50 ± 18.28, respectively, p < 0.05), amylase (2165.00 ± 150.76 and 3027.66 ± 729.19, respectively, p < 0.01), lipase (5.58 ± 2.22 and 11.51 ± 2.74, respectively, p < 0.01) and pancreatic tissue MDA (0.0167 ± 0.004 and 0.0193 ± 0.006, respectively, p < 0.05) levels were increased, whereas tissue CAT (0.0924 ± 0.029 and 0.0359 ± 0.023, respectively, p < 0.05) activity decreased in the HFCS group significantly. Histopathological examination revealed degenerative and necrotic changes in Langerhans islet cells and slight inflammatory cell infiltration in pancreatic tissue in the HFCS group. Immunohistochemically there was a significant decrease in insulin (2.85 ± 0.37 and 0.87 ± 0.64, respectively, p < 0.001) and glucagon (2.71 ± 0.48 and 1.00 ± 0.75, respectively, p < 0.001) secreting cell scores, whereas a

Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: role of cysteine alkylation during sample processing.

Science.gov (United States)

Franc, Vojtěch; Řehulka, Pavel; Raus, Martin; Stulík, Jiří; Novak, Jan; Renfrow, Matthew B; Šebela, Marek

2013-10-30

electrophoresis under denaturing conditions, the heavy chain of IgA1 was subjected to in-gel digestion by trypsin. O-glycopeptides were separated from the digest on capillary columns using a microgradient chromatographic device (replacing commonly used liquid chromatographs) and subjected to MALDI-TOF/TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS) involving post-source decay fragmentation. We show that the complete modification of cysteines by iodoacetamide prior to electrophoresis is critical for successful MS/MS analyses on the way to deciphering the microheterogeneity of O-glycosylation in IgA1. Similarly, the removal of the excess of the reagent is equally important. The acquired MS/MS allowed assigning up to six O-glycosylation sites and identification of isomeric O-glycoforms. We show that our simplified approach is efficient and has a high potential to provide a method for the rapid assessment of IgA1 heterogeneity that is a less expensive and yet corroborating alternative to LC-(high-resolution)-MS protocols. The novelty and biological significance reside in the demonstration, for the first time, of the distribution of the most abundant isoforms of HR O-glycopeptides of IgA1. As another new feature, we introduce a software solution for the interpretation of MS/MS data of O-glycopeptide isoforms, which provides the possibility of fast and easier data processing. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. © 2013 Elsevier B.V. All rights reserved.

Capillary zone electrophoresis method for a highly glycosylated and sialylated recombinant protein: development, characterization and application for process development.

Science.gov (United States)

Zhang, Le; Lawson, Ken; Yeung, Bernice; Wypych, Jette

2015-01-06

A purity method based on capillary zone electrophoresis (CZE) has been developed for the separation of isoforms of a highly glycosylated protein. The separation was found to be driven by the number of sialic acids attached to each isoform. The method has been characterized using orthogonal assays and shown to have excellent specificity, precision and accuracy. We have demonstrated the CZE method is a useful in-process assay to support cell culture and purification development of this glycoprotein. Compared to isoelectric focusing (IEF), the CZE method provides more quantitative results and higher sample throughput with excellent accuracy, qualities that are required for process development. In addition, the CZE method has been applied in the stability testing of purified glycoprotein samples.

GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

Energy Technology Data Exchange (ETDEWEB)

Yoo, Jiho; Mashalidis, Ellene H.; Kuk, Alvin C. Y.; Yamamoto, Kazuki; Kaeser, Benjamin; Ichikawa, Satoshi; Lee, Seok-Yong

2018-02-19

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.

Prediction of glycosylation sites using random forests

Directory of Open Access Journals (Sweden)

Hirst Jonathan D

2008-11-01

Full Text Available Abstract Background Post translational modifications (PTMs occur in the vast majority of proteins and are essential for function. Prediction of the sequence location of PTMs enhances the functional characterisation of proteins. Glycosylation is one type of PTM, and is implicated in protein folding, transport and function. Results We use the random forest algorithm and pairwise patterns to predict glycosylation sites. We identify pairwise patterns surrounding glycosylation sites and use an odds ratio to weight their propensity of association with modified residues. Our prediction program, GPP (glycosylation prediction program, predicts glycosylation sites with an accuracy of 90.8% for Ser sites, 92.0% for Thr sites and 92.8% for Asn sites. This is significantly better than current glycosylation predictors. We use the trepan algorithm to extract a set of comprehensible rules from GPP, which provide biological insight into all three major glycosylation types. Conclusion We have created an accurate predictor of glycosylation sites and used this to extract comprehensible rules about the glycosylation process. GPP is available online at http://comp.chem.nottingham.ac.uk/glyco/.

Expression and functional importance of collagen-binding integrins, alpha 1 beta 1 and alpha 2 beta 1, on virus-activated T cells

DEFF Research Database (Denmark)

Andreasen, Susanne Ø; Thomsen, Allan R; Koteliansky, Victor E

2003-01-01

decreased responses were seen upon transfer of alpha(1)-deficient activated/memory T cells. Thus, expression of alpha(1)beta(1) and alpha(2)beta(1) integrins on activated T cells is directly functionally important for generation of inflammatory responses within tissues. Finally, the inhibitory effect......Adhesive interactions are crucial to cell migration into inflammatory sites. Using murine lymphocytic choriomeningitis virus as an Ag model system, we have investigated expression and function of collagen-binding integrins, alpha(1)beta(1) and alpha(2)beta(1), on activated and memory T cells. Using...... this system and MHC tetramers to define Ag-specific T cells, we demonstrate that contrary to being VLAs, expression of alpha(1)beta(1) and alpha(2)beta(1) can be rapidly induced on acutely activated T cells, that expression of alpha(1)beta(1) remains elevated on memory T cells, and that expression of alpha(1...

Patterns of glycemic control using glycosylated hemoglobin in diabetics.

Science.gov (United States)

Kahlon, Arunpreet Singh; Pathak, Rambha

2011-07-01

Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS) is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3%) had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came out to be .311. This correlation was found to be statistically

Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

Energy Technology Data Exchange (ETDEWEB)

Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

1998-06-29

Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

Characterization of the N-linked glycosylation site of recombinant pectate lyase

NARCIS (Netherlands)

Colangelo, J.; Licon, V.; Benen, J.A.E.; Visser, J.; Bergmann, C.; Orlando, R.

1999-01-01

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for

Hallmarks of glycosylation in cancer.

Science.gov (United States)

Munkley, Jennifer; Elliott, David J

2016-06-07

Aberrant glycosylation plays a fundamental role in key pathological steps of tumour development and progression. Glycans have roles in cancer cell signalling, tumour cell dissociation and invasion, cell-matrix interactions, angiogenesis, metastasis and immune modulation. Aberrant glycosylation is often cited as a 'hallmark of cancer' but is notably absent from both the original hallmarks of cancer and from the next generation of emerging hallmarks. This review discusses how glycosylation is clearly an enabling characteristic that is causally associated with the acquisition of all the hallmark capabilities. Rather than aberrant glycosylation being itself a hallmark of cancer, another perspective is that glycans play a role in every recognised cancer hallmark.

Catabolism of 6-ketoprostaglandin F1alpha by the rat kidney cortex.

Science.gov (United States)

Pace-Asciak, C R; Domazet, Z; Carrara, M

1977-05-25

Homogenates of the rat kidney cortex converted 5,8,9,11,12,14,15-hepta-tritiated 6-ketoprostaglandin F 1alpha into one major product identified by gas chromatography-mass spectrometry of the methoxime-methyl ester trimethylsilyl ether derivative as 6,15-diketo-9,11-dihydroxyprost-13-enoic acid. The sequence of derivatisation i.e. methoximation prior to methylation, was crucial as methylation of 15-keto catabolites of the E, F and 6-keto-F series affords degradation products. The corresponding 15-keto-13,14-dihydro catabolite was formed in much smaller quantities. Time course studies indicated that 6-keto-prostaglandin F1alpha was catabolised at a slower rate (about 2-5 fold) than prostaglandin F1alpha. The catabolic activity was blocked by NADH.

N-Glycosylation of Carnosinase Influences Protein Secretion and Enzyme Activity Implications for Hyperglycemia

NARCIS (Netherlands)

Riedl, Eva; Koeppel, Hannes; Pfister, Frederick; Peters, Verena; Sauerhoefer, Sibylle; Sternik, Paula; Brinkkoetter, Paul; Zentgraf, Hanswalter; Navis, Gerjan; Henning, Robert H.; Van Den Born, Jacob; Bakker, Stephan J. L.; Janssen, Bart; van der Woude, Fokko J.; Yard, Benito A.

OBJECTIVE-The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1

Uptake of neutral alpha- and beta-amino acids by human proximal tubular cells

DEFF Research Database (Denmark)

Jessen, H; Røigaard, H; Jacobsen, Christian

1996-01-01

experiments revealed that all the neutral amino acids tested reduced the uptake of AIB, whereas there was no effect of taurine, L-aspartic acid, and L-arginine. By contrast, the influx of beta-alanine was only drastically reduced by beta-amino acids, whereas the inhibition by neutral alpha-amino acids...

Alpha 1A and alpha 1B-adrenoceptors enhance inositol phosphate generation in rat renal cortex

NARCIS (Netherlands)

Michel, M. C.; Büscher, R.; Philipp, T.; Brodde, O. E.

1993-01-01

We have studied the role of alpha 1A- and alpha 1B-adrenoceptors in noradrenaline- and methoxamine-stimulated inositol phosphate accumulation in rat renal cortical slices. [3H]Prazosin binding studies with and without inactivation of alpha 1B-adrenoceptors by chloroethylclonidine treatment suggested

Action of ornithine alpha-ketoglutarate, ornithine hydrochloride, and calcium alpha-ketoglutarate on plasma amino acid and hormonal patterns in healthy subjects.

Science.gov (United States)

Cynober, L; Coudray-Lucas, C; de Bandt, J P; Guéchot, J; Aussel, C; Salvucci, M; Giboudeau, J

1990-02-01

Ornithine alpha-ketoglutarate (OKG) has been useful as an adjuvant of enteral and parenteral nutrition. However, its metabolism and mechanism of action remain unclear although it is known that alpha-ketoglutarate (alpha KG) and ornithine (ORN) follow, in part, common metabolic pathways. Six fasting healthy male subjects underwent three separate oral load tests: (i) they received 10 g of OKG (i.e., 3.6 g of alpha KG and 6.4 g of ORN); (ii) 6.4 g of ORN as ornithine hydrochloride, and (iii) 3.6 g of alpha KG as calcium alpha-ketoglutarate. Blood was drawn 15 times over a five-hour period for measurements of plasma amino acids, alpha KG, insulin, and glucagon. After OKG and ORN administration, plasma ORN peaked at 60-75 min (494 +/- 91 and 541 +/- 85 mumol/L). The increase in plasma alpha KG was very small. OKG, alpha KG, and ORN all increased glutamate concentrations at 60 min (mean: +43%, +68%, +68%, respectively, p less than 0.05 compared to basal values). However, only OKG increased proline and arginine levels at 60 min (mean: +35%, p less than 0.01 and mean: +41%, p less than 0.05). Furthermore, glutamate, proline, and arginine concentrations correlated linearly with ornithine levels at 60 min. Finally, OKG increased insulinemia and glucagonemia (mean: +24% at 15 min, p less than 0.05 and +30% at 60 min, p less than 0.01, respectively). These data provide evidence that the combination of ORN and alpha KG modifies amino acid metabolism in a way which is not observed when they are administered separately. In addition, the OKG-mediated increase in insulin levels probably does not appear to result from a direct action of ORN on pancreatic secretion.

Charged amino acid variability related to N-glyco -sylation and epitopes in A/H3N2 influenza: Hem -agglutinin and neuraminidase.

Directory of Open Access Journals (Sweden)

Zhong-Zhou Huang

Full Text Available The A/H3N2 influenza viruses circulated in humans have been shown to undergo antigenic drift, a process in which amino acid mutations result from nucleotide substitutions. There are few reports regarding the charged amino acid mutations. The purpose of this paper is to explore the relations between charged amino acids, N-glycosylation and epitopes in hemagglutinin (HA and neuraminidase (NA.A total of 700 HA genes (691 NA genes of A/H3N2 viruses were chronologically analyzed for the mutational variants in amino acid features, N-glycosylation sites and epitopes since its emergence in 1968.It was found that both the number of HA N-glycosylation sites and the electric charge of HA increased gradually up to 2016. The charges of HA and HA1 increased respectively 1.54-fold (+7.0 /+17.8 and 1.08-fold (+8.0/+16.6 and the number of NGS in nearly doubled (7/12. As great diversities occurred in 1990s, involving Epitope A, B and D mutations, the charged amino acids in Epitopes A, B, C and D in HA1 mutated at a high frequency in global circulating strains last decade. The charged amino acid mutations in Epitopes A (T135K has shown high mutability in strains near years, resulting in a decrease of NGT135-135. Both K158N and K160T not only involved mutations charged in epitope B, but also caused a gain of NYT158-160. Epitope B and its adjacent N-glycosylation site NYT158-160 mutated more frequently, which might be under greater immune pressure than the rest.The charged amino acid mutations in A/H3N2 Influenza play a significant role in virus evolution, which might cause an important public health issue. Variability related to both the epitopes (A and B and N-glycosylation is beneficial for understanding the evolutionary mechanisms, disease pathogenesis and vaccine research.

Influence of glucoregulation quality on C-reactive protein, interleukin-6 and tumor necrosis factor-alpha level in patients with diabetes type 1.

Science.gov (United States)

Mitrović, Milena; Ilić, Tatjana; Stokić, Edita; Paro, Jovanka Novaković; Naglić, Dragana Tomić; Bajkin, Ivana; Icin, Tijana

2011-09-01

Results of studies which have proved an increased inflammatory activity in diabetes type 1, have been published over recent years. One of possible mechanisms that are used to explain chronic inflammation in diabetes is the state of hyperglycemia leading to the enhanced synthesis of glycosylation end products (AGEs) which activate macrophages, increase the oxidative stress and affect the synthesis of interleukins (IL-1, IL-6), tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein (CRP). The aim of the study was to determine the inflammatory markers (CRP, IL-6, TNF-alpha) in patients with diabetes type 1 and to establish their correlation with glucoregulation parameters and other cardiovascular risk factors as well as to compare them with the healthy controls. The study included 76 patients with diabetes type 1 and 30 healthy controls. We determined values of inflammatory markers (CRP, IL-6, TNF-alpha) and glucoregulation parameters (fasting glucose HbA(1c)). The values of CRP (p = 0.014), IL-6 (p = 0.020) and TNF-alpha (p = 0.037) were statistically significantly higher in the diabetic patients than in the healthy controls. There was a positive correlation between CRP with postprandial glycemia (p = 0.004); the multivariate regression analysis revealed a statistically significant correlation between CRP and age (p = 0.001), smoking (p = 0.055), fasting glucose (p = 0.021) and triglycerides (p = 0.048) as well as between IL-6 and LDL-cholesterol (p = 0.009). No statistically significant correlations were found between glycosilated hemoglobin (HbA(1c)) and the inflammatory markers (CRP, IL-6 and TNF-alpha). The patients with type 1 diabetes were found to have a low level of inflammatory activity manifested by the increased values of CRP, IL-6 and TNF-alpha.

DNA methylation in Cosmc promoter region and aberrantly glycosylated IgA1 associated with pediatric IgA nephropathy.

Directory of Open Access Journals (Sweden)

Qiang Sun

Full Text Available IgA nephropathy (IgAN is one of the most common glomerular diseases leading to end-stage renal failure. Elevation of aberrantly glycosylated IgA1 is a key feature of it. The expression of the specific molecular chaperone of core1ß1, 3galactosyl transferase (Cosmc is known to be reduced in IgAN. We aimed to investigate whether the methylation of CpG islands of Cosmc gene promoter region could act as a possible mechanism responsible for down-regulation of Cosmc and related higher secretion of aberrantly glycosylated IgA1in lymphocytes from children with IgA nephropathy. Three groups were included: IgAN children (n = 26, other renal diseases (n = 11 and healthy children (n = 13. B-lymphocytes were isolated and cultured, treated or not with IL-4 or 5-Aza-2'-deoxycytidine (AZA. The levels of DNA methylation of Cosmc promotor region were not significantly different between the lymphocytes of the three children populations (P = 0.113, but there were significant differences between IgAN lymphocytes and lymphocytes of the other two children populations after IL-4 (P<0.0001 or AZA (P<0.0001. Cosmc mRNA expression was low in IgAN lymphocytes compared to the other two groups (P<0.0001. The level of aberrantly glycosylated IgA1 was markedly higher in IgAN group compared to the other groups (P<0.0001. After treatment with IL-4, the levels of Cosmc DNA methylation and aberrantly glycosylated IgA1 in IgAN lymphocytes were remarkably higher than the other two groups (P<0.0001 with more markedly decreased Cosmc mRNA content (P<0.0001. After treatment with AZA, the levels in IgAN lymphocytes were decreased, but was still remarkably higher than the other two groups (P<0.0001, while Cosmc mRNA content in IgAN lymphocytes were more markedly increased than the other two groups (P<0.0001. The alteration of DNA methylation by IL-4 or AZA specifically correlates in IgAN lymphocytes with alterations in Cosmc mRNA expression and with the level of aberrantly glycosylated

A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B.; Goth, Christoffer K.

2011-01-01

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition...... and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc......-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model...

Determination of radium-226 by high-resolution alpha spectrometry

International Nuclear Information System (INIS)

Sill, C.W.

1983-01-01

Condition were determined under which high resolution and accurate and reliable results can be obtained. Refractory solids are dissolved completely by fusion with KF and Ba-133 tracer. The fluoride cake is then transposed with sulfuric acid to a pyrosulfate fusion. Radium is precipitated with barium by addition of lead perchlorate to a dilute HCl solution of the pyrosulfate cake. The resulting insoluble sulfates are dissolved in an alkaline solution of DTPA and the Ra and Ba sulfates are reprecipitated with acetic acid to produce very small crystals. The precipitate is mounted on 0.1-μm membrane filter and analyzed by alpha spectrometry. Water samples are partially evaporated and treated similarly. Resolution, almost as good as with actinides electrodeposited on polished steel plates, is about 60 keV full-width-half-maximum with 100 μg of barium on a 1-inch filter with a 450 mm 2 detector at 20% counting efficiency. Recovery is about 97%. One solid sample can be prepared for counting in less than 2 hours. Methods are discussed for ensuring reliability of the results. Severe contamination of the surface-barrier detector by polonium-210 and recoil products is discussed

Alpha-eleostearic acid (9Z11E13E-18:3) is quickly converted to conjugated linoleic acid (9Z11E-18:2) in rats.

Science.gov (United States)

Tsuzuki, Tsuyoshi; Tokuyama, Yoshiko; Igarashi, Miki; Nakagawa, Kiyotaka; Ohsaki, Yusuke; Komai, Michio; Miyazawa, Teruo

2004-10-01

We previously showed that alpha-eleostearic acid (alpha-ESA; 9Z11E13E-18:3) is converted to conjugated linoleic acid (CLA; 9,11-18:2) in the liver and plasma of rats that were given diets including 1% alpha-ESA for 4 wk. In this study, we investigated this phenomenon in detail. First, the chemical structure of CLA produced by alpha-ESA administration was determined. After alpha-ESA was orally administered to rats, CLA in rat liver was isolated by HPLC. The positional and geometric isomerism was determined using GC-EI/MS and (13)C-NMR, respectively, and the CLA generated in rats after alpha-ESA feeding was confirmed to be 9Z11E-CLA. Next, the concentrations of alpha-ESA and CLA were determined 0, 3, 6, and 24 h after oral administration of alpha-ESA to rats. Moreover, we also investigated whether enteric bacteria are involved in the conversion of alpha-ESA to CLA using germ-free rats. alpha-ESA was orally administered to germ-free and normal rats and alpha-ESA and CLA were detected in the organs of both groups. In addition, to confirm that this reaction was enzyme-mediated, alpha-ESA was reacted with tissue homogenates (liver, kidney, and small intestine mucous) and coenzymes (NADH, NAD(+), NADPH, and NADP(+)), and the enzyme activities were estimated from the amount of CLA produced. CLA was detected when alpha-ESA was reacted with liver, kidney, and small intestine mucous homogenates and a coenzyme (NADPH). These results indicated that alpha-ESA is converted to 9Z11E-CLA in rats by a Delta13-saturation reaction carried out by an NADPH-dependent enzyme.

Decreased levels of genuine large free hCG alpha in men presenting with abnormal semen analysis

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Plas Eugen

2011-08-01

Full Text Available Abstract Background The pregnancy hormone human chorionic gonadotropin (hCG and its free subunits (hCG alpha, hCG beta are produced in the male reproductive tract and found in high concentrations in seminal fluid, in particular hCG alpha. This study aimed to elucidate changes in peptide hormone profiles in patients showing abnormal semen analyses and to determine the genuineness of the highly abundant hCG alpha. Methods Seminal plasma was obtained from 45 male patients undergoing semen analysis during infertility workups. Comprehensive peptide hormone profiles were established by a panel of immunofluorometric assays for hCG, hCG alpha, hCG beta and its metabolite hCG beta core fragment, placental lactogen, growth hormone and prolactin in seminal plasma of patients with abnormal semen analysis results (n = 29 versus normozoospermic men (n = 16. The molecular identity of large hyperglycosylated hCG alpha was analyzed by mass-spectrometry and selective deglycosylation. Results hCG alpha levels were found to be significantly lower in men with impaired semen quality (1346 +/- 191 vs. 2753 +/- 533 ng/ml, P = 0.022. Moreover, patients with reduced sperm count had reduced intact hCG levels compared with normozoospermic men (0.097 +/- 0.022 vs. 0.203 +/- 0.040 ng/ml, P = 0.028. Using mass-spectrometry, the biochemical identity of hCG alpha purified from seminal plasma was verified. Under non-reducing conditions in SDS-PAGE, hCG alpha isolated from seminal plasma migrated in a manner comparable with large free hCG alpha with an apparent molecular mass (Mr, app of 24 kDa, while hCG alpha dissociated from pregnancy-derived holo-hCG migrated at approximately 22 kDa. After deglycosylation with PNGase F under denaturing conditions, all hCG alpha variants showed an Mr, app of 15 kDa, indicating identical amino acid backbones. Conclusions The findings indicate a pathophysiological relevance of hCG, particularly its free alpha subunit, in spermatogenesis. The

Splitting of α-Helical Structure as Molecular Basis for Abolishing an Amyloid Formation by Multiple Glycosylation: A Molecular Dynamics Simulation Study

Energy Technology Data Exchange (ETDEWEB)

Choi, Youngjin [Hoseo University, Asan (Korea, Republic of); Cho, Eunae; Jung, Seunho [Konkuk University, Seoul (Korea, Republic of)

2016-07-15

Molecular details played by glycosylation are complicated by the subtle nature of variations in the glycan structure, and this complexity is one of the research barriers to establish structure-function relationship on the protein modification. This is particularly true for understanding the exact structural consequence of the glycosylation of the biological proteins. The present MD simulation revealed molecular-level mechanism of the glycosylation effect on the peptide to understand the experimentally observed phenomenon for inhibiting amyloid formation in the model peptide. The galactose residue on the Ser17 undermined the helical integrity of main protein region by enhancing sugar–amino acid interaction and perturbing natural interactions between amino acid residues.

Salivary tissue inhibitor of metalloproteinases-1 localization and glycosylation profile analysis

DEFF Research Database (Denmark)

Holten-Andersen, Lars; Thaysen-Andersen, Morten; Jensen, Siri Beier

2011-01-01

tissue samples (four parotid gland and four submandibular gland biopsies) were analysed for the presence of TIMP-1 mRNA and protein expression. To assess TIMP-1 glycosylation profiles in blood and saliva, the protein was isolated from plasma and unstimulated and stimulated whole saliva as well...... as stimulated parotid and submandibular saliva and analysed by MALDI-TOF mass spectrometry. TIMP-1 protein was demonstrated in mucous acinar cells of the submandibular gland and in ductal cells of both the parotid and submandibular gland. However, no TIMP-1 mRNA was detected in any of these cells...

Evolutionary Pattern of N-Glycosylation Sequon Numbers  in Eukaryotic ABC Protein Superfamilies

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R. Shyama Prasad Rao

2010-02-01

Full Text Available Many proteins contain a large number of NXS/T sequences (where X is any amino acid except proline which are the potential sites of asparagine (N linked glycosylation. However, the patterns of occurrence of these N-glycosylation sequons in related proteins or groups of proteins and their underlying causes have largely been unexplored. We computed the actual and probabilistic occurrence of NXS/T sequons in ABC protein superfamilies from eight diverse eukaryotic organisms. The ABC proteins contained significantly higher NXS/T sequon numbers compared to respective genome-wide average, but the sequon density was significantly lower owing to the increase in protein size and decrease in sequon specific amino acids. However, mammalian ABC proteins have significantly higher sequon density, and both serine and threonine containing sequons (NXS and NXT have been positively selected—against the recent findings of only threonine specific Darwinian selection of sequons in proteins. The occurrence of sequons was positively correlated with the frequency of sequon specific amino acids and negatively correlated with proline and the NPS/T sequences. Further, the NPS/T sequences were significantly higher than expected in plant ABC proteins which have the lowest number of NXS/T sequons. Accord- ingly, compared to overall proteins, N-glycosylation sequons in ABC protein superfamilies have a distinct pattern of occurrence, and the results are discussed in an evolutionary perspective.

Microheterogeneity of alpha 1-acid glycoprotein in healthy elderly subjects: patterns obtained by crossed affino-immunoelectrophoresis.

Science.gov (United States)

Kawerk, N; Succari-Aderschlag, M; Foglietti, M J

1991-10-14

Total serum alpha 1-acid glycoprotein (AGP) concentration and concanavalin A-dependent microheterogeneity were studied in 31 healthy elderly subjects (18 men, 13 women, 71 to 76 yr old). Crossed affino-immunoelectrophoresis (CAIE) revealed three microheterogeneity variants of AGP: non-reactive, weakly reactive and strongly reactive with ConA. Two patterns were found in both elderly men and women, i.e. a normal pattern and one with an increase in the non-reactive form. Mean serum AGP levels in the elderly subjects with slightly higher than in a reference group of younger subjects. The Con A non-reactive form of AGP was increased in 42% of the elderly population. An increase in the non-reactive form of AGP in CAIE should be considered as general expression of chronic inflammation which is of no clinical relevance.

Increased humoral immunity by DNA vaccination using an alpha-tocopherol-based adjuvant

DEFF Research Database (Denmark)

Karlsson, Ingrid; Borggren, Marie; Nielsen, Jens

2017-01-01

approaches. We tested whether the emulsion-based and alpha-tocopherol containing adjuvant Diluvac Forte® has the ability to enhance the immunogenicity of a naked DNA vaccine (i.e., plasmid DNA). As a model vaccine, we used plasmids encoding both a surface-exposed viral glycoprotein (hemagglutinin......) and an internal non-glycosylated nucleoprotein in the Th1/Th2 balanced CB6F1 mouse model. The naked DNA (50 µg) was premixed at a 1:1 volume/volume ratio with Diluvac Forte®, an emulsion containing different concentrations of alpha-tocopherol, the emulsion alone or endotoxin-free phosphate-buffered saline (PBS......). The animals received two intracutaneous immunizations spaced 3 weeks apart. When combined with Diluvac Forte® or the emulsion containing alpha-tocopherol, the DNA vaccine induced a more potent and balanced immunoglobulin G (IgG)1 and IgG2c response, and both IgG subclass responses were significantly enhanced...

New entry for synthesis of N-acylhydrazones, pyridazinones, and 1,3,4-oxadiazin-6-ones from alpha-amino acid esters.

Science.gov (United States)

Yasui, Eiko; Wada, Masao; Takamura, Norio

2007-11-01

Versatile electrophiles N-acylhydrazones are synthesized via diazotization, reduction, and acylation of alpha-amino acid esters. Reduction of diazo esters with L-selectride or tributylphosphine affords the corresponding hydrazones in good yields. Both reducing agents give anti-hydrazones as the major product although the reactivity of each reductant is slightly different. The resulting hydrazones are acylated to give N-acylhydrazones, which are subjected to further reactions to give 1,3,4-oxadiazin-6-ones that serve as useful synthetic intermediates for the Diels-Alder reaction.

Glycosyltransferase glycosylating flavokermesic acid and/or kermesic acid

DEFF Research Database (Denmark)

2016-01-01

An isolated glycosyltransferase (GT) polypeptide capable of: (I) : conjugating glucose to flavokermesic acid (FK); and/or (II) : conjugating glucose to kermesic acid (KA) and use of this GT to e.g. make Carminic acid.......An isolated glycosyltransferase (GT) polypeptide capable of: (I) : conjugating glucose to flavokermesic acid (FK); and/or (II) : conjugating glucose to kermesic acid (KA) and use of this GT to e.g. make Carminic acid....

GLYCOSYLTRANSFERASE GLYCOSYLATING FLAVOKERMESIC ACID AND/OR KERMESIC ACID

DEFF Research Database (Denmark)

2015-01-01

An isolated glycosyltransferase (GT) polypeptide capable of: (I): conjugating glucose to flavokermesic acid (FK); and/or (II): conjugating glucose to kermesic acid (KA) and use of this GT to e.g. make Carminic acid.......An isolated glycosyltransferase (GT) polypeptide capable of: (I): conjugating glucose to flavokermesic acid (FK); and/or (II): conjugating glucose to kermesic acid (KA) and use of this GT to e.g. make Carminic acid....

Lithium abundances in high- and low-alpha halo stars

DEFF Research Database (Denmark)

Nissen, P. E.; Schuster, W. J.

2012-01-01

A previous study of F and G main-sequence stars in the solar neighborhood has revealed the existence of two distinct halo populations with a clear separation in [alpha /Fe] for the metallicity range -1.4 < [Fe/H] < -0.7. The kinematics of the stars and models of galaxy formation suggest that the ......A previous study of F and G main-sequence stars in the solar neighborhood has revealed the existence of two distinct halo populations with a clear separation in [alpha /Fe] for the metallicity range -1.4 ... that the ``high-alpha '' stars were formed in situ in the inner parts of the Galaxy, whereas the ``low-alpha '' ones have been accreted from satellite galaxies. In order to see if there is any systematic difference in the lithium abundances of high- and low-alpha stars, equivalent widths of the iLi 6707.8 Å line...... have been measured from VLT/UVES and NOT/FIES spectra and used to derive Li abundances. Furthermore, stellar masses are determined from evolutionary tracks in the log T_eff - log g diagram. For stars with masses 0.7 lithium abundance...

Alpha-lipoic acid reduces body weight and regulates triglycerides in obese patients with diabetes mellitus.

Science.gov (United States)

Okanović, Azra; Prnjavorac, Besim; Jusufović, Edin; Sejdinović, Rifat

2015-08-01

To determine an influence of alpha-lipoic acid to reduction of body weight and regulation of total cholesterol concentration, triglycerides and glucose serum levels in obese patients with diabetes mellitus type 2. A prospective study includes two groups of obese patients with diabetes mellitus and signs of peripheral polyneuropathia: examined group (30 patients; 15 females and 15 males), and control group (30 patients; 12 females and 18 males). All were treated with metformin (850-1700 mg/day). Examined patients were additionally treated with alpha-lipoic acid 600 mg/day during 20 weeks. Body mass index and concentrations of total cholesterol, triglycerides and glucose in serum were compared before and after the treatment. The group treated with 600 mg alpha-lipoic acid lost significantly more weight, and had lower triglyceride level than the control group. There were no significant differences in total cholesterol and glucose serum levels between the groups. Alpha-lipoic acid of 600 mg/day treatment have influenced weight and triglycerides loss in obese patients with diabetes mellitus type 2. It should be considered as an important additive therapy in obese patients with diabetes mellitus type 2. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

The implications of particle energy and acidic media on gross alpha and gross beta determination using liquid scintillation

Energy Technology Data Exchange (ETDEWEB)

Zapata-Garcia, D. [Laboratori de Radiologia Ambiental (LRA), Departament de Quimica Analitica, Universitat de Barcelona, Marti i Franques, 1-11 Planta 3, E-08028 Barcelona (Spain); Llaurado, M., E-mail: montse.llaurado@ub.edu [Laboratori de Radiologia Ambiental (LRA), Departament de Quimica Analitica, Universitat de Barcelona, Marti i Franques, 1-11 Planta 3, E-08028 Barcelona (Spain); Rauret, G. [Laboratori de Radiologia Ambiental (LRA), Departament de Quimica Analitica, Universitat de Barcelona, Marti i Franques, 1-11 Planta 3, E-08028 Barcelona (Spain)

2012-04-15

The interaction of humans with radioactivity present in the environment from natural and artificial sources necessitates an evaluation of its risk on human health. Gross alpha and gross beta activities can provide a rapid evaluation of the radioactive content of a sample and can be simultaneously determined by using liquid scintillation counters. However, calibration of the liquid scintillation counter is required and is affected by many factors, such as particle energy and the acidity of the media. This study investigates what effect the particle energy used for calibration has on misclassification and how to account for this misclassification in routine measurements. The variability in measurement produced by the final pH, as well as any acids used in sample treatment, was also studied. These results showed that the most commonly used acid for these types of analyses, HNO{sub 3}, produced a high amount of misclassifications at very low pH. The results improved when HCl was used to adjust the sample to low pH. - Highlights: Black-Right-Pointing-Pointer We study the effect of alpha and beta energies on PSA optimisation. Black-Right-Pointing-Pointer The optimum PSA shifts to higher values as the alpha energy increases. Beta energies do not affect it. Black-Right-Pointing-Pointer We study the effect of pH on the simultaneous determination of gross alpha/beta activities. Black-Right-Pointing-Pointer HNO{sub 3} produces a high amount of misclassification at very low pH. Black-Right-Pointing-Pointer The results improve when HCl is used to adjust the sample to low pH.

The implications of particle energy and acidic media on gross alpha and gross beta determination using liquid scintillation

International Nuclear Information System (INIS)

Zapata-García, D.; Llauradó, M.; Rauret, G.

2012-01-01

The interaction of humans with radioactivity present in the environment from natural and artificial sources necessitates an evaluation of its risk on human health. Gross alpha and gross beta activities can provide a rapid evaluation of the radioactive content of a sample and can be simultaneously determined by using liquid scintillation counters. However, calibration of the liquid scintillation counter is required and is affected by many factors, such as particle energy and the acidity of the media. This study investigates what effect the particle energy used for calibration has on misclassification and how to account for this misclassification in routine measurements. The variability in measurement produced by the final pH, as well as any acids used in sample treatment, was also studied. These results showed that the most commonly used acid for these types of analyses, HNO 3 , produced a high amount of misclassifications at very low pH. The results improved when HCl was used to adjust the sample to low pH. - Highlights: ► We study the effect of alpha and beta energies on PSA optimisation. ► The optimum PSA shifts to higher values as the alpha energy increases. Beta energies do not affect it. ► We study the effect of pH on the simultaneous determination of gross alpha/beta activities. ► HNO 3 produces a high amount of misclassification at very low pH. ► The results improve when HCl is used to adjust the sample to low pH.

Glycosylation in HIV-1 envelope glycoprotein and its biological implications

KAUST Repository

Ho, Yung Shwen; Saksena, Nitin K.

2013-01-01

architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may

Bile acid metabolism in cirrhosis. VIII. Quantitative evaluation of bile acid synthesis from [7 beta-3H]7 alpha-hydroxycholesterol and [G-3H]26-hydroxycholesterol

International Nuclear Information System (INIS)

Goldman, M.; Vlahcevic, Z.R.; Schwartz, C.C.; Gustafsson, J.; Swell, L.

1982-01-01

In order to evaluate more definitively the observed aberrations in the synthesis of cholic and chenodeoxycholic acids in patients with advanced cirrhosis, two bile acid biosynthesis pathways were examined by determining the efficiency of conversion of [ 3 H]7 alpha-hydroxycholesterol and [ 3 H] 26-hydroxycholesterol to primary bile acids. Bile acid kinetics were determined by administration of [ 14 C]cholic and [ 14 C]chenodeoxycholic acids. Cholic acid synthesis in cirrhotic patients was markedly depressed (170 vs 927 μmoles per day)( while chenodeoxycholic acid synthesis was reduced to a much lesser degree (227 vs 550 μmoles per day). The administration of [ 3 H]7 alpha-hydroxycholesterol allowed for an evaluation of the major pathway of bile acid synthesis via the 7 alpha-hydroxylation of cholesterol. This compound was efficiently incorporated into primary bile acids by the two normal subjects (88 and 100%) and two cirrhotic patients (77 and 91%). However, the recovery of the label in cholic acid was slightly less in cirrhotic patients than in normal subjects. [ 3 H]26-hydroxycholesterol was administered to ascertain the contribution of the 26-hydroxylation pathway to bile acid synthesis. All study subjects showed poor conversion (9 to 22%) of this intermediate into bile acids. The results of this study suggest that a major block in the bile acid synthesis pathway in cirrhosis is at the level of 7 alpha-hydroxylation of cholesterol (impairment of 7 alpha-hydroxylase) and/or in the feedback triggering mechanism regulating bile acid synthesis. The data also suggest that the 26-hydroxylation pathway in normal subjects and patients with cirrhosis is a minor contributor to synthesis of the primary bile acids. Therefore, the relative sparing of chenodeoxycholic acid synthesis observed in cirrhotic patients is not due to preferential synthesis of this bile acid via the 26-hydroxylation pathway

Are Ichthyosporea animals or fungi? Bayesian phylogenetic analysis of elongation factor 1alpha of Ichthyophonus irregularis.

Science.gov (United States)

Ragan, Mark A; Murphy, Colleen A; Rand, Thomas G

2003-12-01

Ichthyosporea is a recently recognized group of morphologically simple eukaryotes, many of which cause disease in aquatic organisms. Ribosomal RNA sequence analyses place Ichthyosporea near the divergence of the animal and fungal lineages, but do not allow resolution of its exact phylogenetic position. Some of the best evidence for a specific grouping of animals and fungi (Opisthokonta) has come from elongation factor 1alpha, not only phylogenetic analysis of sequences but also the presence or absence of short insertions and deletions. We sequenced the EF-1alpha gene from the ichthyosporean parasite Ichthyophonus irregularis and determined its phylogenetic position using neighbor-joining, parsimony and Bayesian methods. We also sequenced EF-1alpha genes from four chytrids to provide broader representation within fungi. Sequence analyses and the presence of a characteristic 12 amino acid insertion strongly indicate that I. irregularis is a member of Opisthokonta, but do not resolve whether I. irregularis is a specific relative of animals or of fungi. However, the EF-1alpha of I. irregularis exhibits a two amino acid deletion heretofore reported only among fungi.

Optimization of a colorimetric assay for glycosylated human serum albumin

International Nuclear Information System (INIS)

Bohney, J.P.; Feldhoff, R.C.

1986-01-01

The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100 0 C. A NaBH 4 reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with [ 3 H]glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation

Operation of a high-purity silicon diode alpha particle detector at 1.4 K

International Nuclear Information System (INIS)

Martoff, C.J.; Kaczanowicz, E.; Neuhauser, B.J.; Lopez, E.; Zhang, Y.; Ziemba, F.P.

1991-01-01

Detection of alpha particles at temperatures as low as 1.4 K was demonstrated using a specially fabricated Si diode. The diode was 475 mm 2 by 0.280 mm thick, fabricated from high-purity silicon with degenerately doped contacts. This is an important step toward development of dual-mode (ionization plus phonon) silicon detectors for low energy radiation. (orig.)

Operation of a high-purity silicon diode alpha particle detector at 1. 4 K

Energy Technology Data Exchange (ETDEWEB)

Martoff, C.J.; Kaczanowicz, E. (Temple Univ., Philadelphia, PA (USA)); Neuhauser, B.J.; Lopez, E.; Zhang, Y. (San Francisco State Univ., CA (USA)); Ziemba, F.P. (Quantrad Corp. (USA))

1991-03-01

Detection of alpha particles at temperatures as low as 1.4 K was demonstrated using a specially fabricated Si diode. The diode was 475 mm{sup 2} by 0.280 mm thick, fabricated from high-purity silicon with degenerately doped contacts. This is an important step toward development of dual-mode (ionization plus phonon) silicon detectors for low energy radiation. (orig.).

A Comparative Study of Effects of Omega‑3 Fatty Acids, Alpha Lipoic ...

African Journals Online (AJOL)

Background: Diabetes Mellitus is a metabolic disorder characterized by abnormal lipid and glucose metabolism. Various modes of adjuvant therapy have been advocated to ameliorate insulin resistance. Aim: This study was intended to assess the effects of antioxidants; alpha lipoic acid (ALA), omega 3 fatty acid and vitamin ...

Patterns of glycemic control using glycosylated hemoglobin in diabetics

Directory of Open Access Journals (Sweden)

Arunpreet Singh Kahlon

2011-01-01

Full Text Available Aim : Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. Objective : To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. Materials and Methods : A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Results : Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3% had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came

Dietary enrichment with alpha-linolenic acid during pregnancy attenuates insulin resistance in adult offspring in mice.

Science.gov (United States)

Hollander, K S; Tempel Brami, C; Konikoff, F M; Fainaru, M; Leikin-Frenkel, A

2014-07-01

Our objective was to test the contribution of dietary enrichment in essential or saturated fatty acids, in normocaloric diets, on the lipid accumulation and insulin resistance in the adult offspring in a C57Bl6/J mice model. Pregnant mothers were fed normocaloric diets containing 6% fat enriched in essential fatty acids (EFA): alpha-linolenic (ALA-18:3, n-3), linoleic (LA-18:2, n-6), or saturated fatty acids (SFA). After a washing-out period with regular diet, the offspring received a high-fat diet before euthanization. Adult mice fed maternal ALA showed lower body weight gain and lower liver fat accumulation, lower HOMA index and lower stearoyl-CoA desaturase (SCD1) activity than those fed maternal SFA. The results observed using this novel model suggest that ALA in maternal diet may have the potential to inhibit insulin resistance in adult offspring.

Diversity and functions of protein glycosylation in insects.

Science.gov (United States)

Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

2017-04-01

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

Energy Technology Data Exchange (ETDEWEB)

Garrison, W. M.

1981-12-01

An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

Alpha1-antitrypsin deficiency

DEFF Research Database (Denmark)

Stolk, Jan; Seersholm, Niels; Kalsheker, Noor

2006-01-01

The Alpha One International Registry (AIR), a multinational research program focused on alpha1-antitrypsin (AAT) deficiency, was formed in response to a World Health Organization recommendation. Each of the nearly 20 participating countries maintains a national registry of patients with AAT defic...

A new highly selective metabotropic excitatory amino acid agonist: 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid

DEFF Research Database (Denmark)

Bräuner-Osborne, Hans; Sløk, F A; Skjaerbaek, N

1996-01-01

The homologous series of acidic amino acids, ranging from aspartic acid (1) to 2-aminosuberic acid (5), and the corresponding series of 3-isoxazolol bioisosteres of these amino acids, ranging from (RS)-2-amino-2-(3-hydroxy-5-methylisoxazol-4-yl)acetic acid (AMAA, 6) to (RS)-2-amino-6-(3-hydroxy-5......-methylisoxazol-4-yl)hexanoic acid (10), were tested as ligands for metabotropic excitatory amino acid receptors (mGlu1 alpha, mGlu2, mGlu4a, and mGlu6). Whereas AMAA (6) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinoic acid (AMPA, 7) are potent and highly selective agonists at N......-methyl-D-aspartic acid (NMDA) and AMPA receptors, respectively, the higher homologue of AMPA (7), (RS)-2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid (homo-AMPA, 8), is inactive at ionotropic excitatory amino acid receptors. Homo-AMPA (8), which is a 3-isoxazolol bioisostere of 2-aminoadipic acid (3), was...

Recombinant human acid alpha-glucosidase: high level production in mouse milk, biochemical characteristics, correction of enzyme deficiency in GSDII KO mice

NARCIS (Netherlands)

A.G.A. Bijvoet (Agnes); M.A. Kroos (Marian); F.R. Pieper (Frank); M. Van der Vliet (Martin); H.A. de Boer (Herman); A.T. van der Ploeg (Ans); M.Ph. Verbeet (Martin); A.J.J. Reuser (Arnold)

1998-01-01

textabstractGlycogen storage disease type II (GSDII) is caused by lysosomal acid alpha-glucosidase deficiency. Patients have a rapidly fatal or slowly progressive impairment of muscle function. Enzyme replacement therapy is under investigation. For large-scale, cost-effective

Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

NARCIS (Netherlands)

Wang, Guanbo; de Jong, Rob N; van den Bremer, Ewald T J; Parren, Paul W H I; Heck, Albert J R

2017-01-01

The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

HPLC Analysis of [Alpha]- and [Beta]-Acids in Hops

Science.gov (United States)

Danenhower, Travis M.; Force, Leyna J.; Petersen, Kenneth J.; Betts, Thomas A.; Baker, Gary A.

2008-01-01

Hops have been used for centuries to impart aroma and bitterness to beer. The cones of the female hop plant contain both essential oils, which include many of the fragrant components of hops, and a collection of compounds known as [alpha]- and [beta]-acids that are the precursors to bittering agents. In order for brewers to predict the ultimate…

Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger.

Science.gov (United States)

Sauer, J; Christensen, T; Frandsen, T P; Mirgorodskaya, E; McGuire, K A; Driguez, H; Roepstorff, P; Sigurskjold, B W; Svensson, B

2001-08-07

Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.

No need to be HAMLET or BAMLET to interact with histones: binding of monomeric alpha-lactalbumin to histones and basic poly-amino acids.

Science.gov (United States)

Permyakov, Serge E; Pershikova, Irina V; Khokhlova, Tatyana I; Uversky, Vladimir N; Permyakov, Eugene A

2004-05-18

The ability of a specific complex of human alpha-lactalbumin with oleic acid (HAMLET) to induce cell death with selectivity for tumor and undifferentiated cells was shown recently to be mediated by interaction of HAMLET with histone proteins irreversibly disrupting chromatin structure [Duringer, C., et al. (2003) J. Biol. Chem. 278, 42131-42135]. Here we show that monomeric alpha-lactalbumin (alpha-LA) in the absence of fatty acids is also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of Ca(2+) content. Thus, the modification of alpha-LA by oleic acid is not required for binding to histones. We suggest that interaction of negatively charged alpha-LA with the basic histone stabilizes apo-alpha-LA and destabilizes the Ca(2+)-bound protein due to compensation for excess negative charge of alpha-LA's Ca(2+)-binding loop by positively charged residues of the histone. Spectrofluorimetric curves of titration of alpha-LA by histone H3 were well approximated by a scheme of cooperative binding of four alpha-LA molecules per molecule of histone, with an equilibrium dissociation constant of 1.0 microM. Such a stoichiometry of binding implies that the binding process is not site-specific with respect to histone and likely is driven by just electrostatic interactions. Co-incubation of positively charged poly-amino acids (poly-Lys and poly-Arg) with alpha-LA resulted in effects which were similar to those caused by histone HIII, confirming the electrostatic nature of the alpha-LA-histone interaction. In all cases that were studied, the binding was accompanied by aggregation. The data indicate that alpha-lactalbumin can be used as a basis for the design of antitumor agents, acting through disorganization of chromatin structure due to interaction between alpha-LA and histone proteins.

Direct chemical synthesis of 1 alpha,25-dihydroxy[26,27-3H] vitamin D3 with high specific activity: its use in receptor studies

International Nuclear Information System (INIS)

Napoli, J.L.; Mellon, W.S.; Fivizzani, M.A.; Schnoes, H.K.; DeLuca, H.F.

1980-01-01

The first direct chemical synthesis of radiolabeled 1 alpha, 25-dihydroxyvitamin D3 is reported. Unlike all previous syntheses, the new approach does not rely on enzymatic 1 alpha-hydroxylation of radiolabeled precursors. Rather, isotope is introduced in the last synthetic step by reaction of [3H] -methylmagnesium bromide with methyl 1 alpha-hydroxy-26,27-dinorvitamin D3-25-carboxylate to give 1 alpha,25-dihydroxy-[26,27-3H] vitamin D3 with a specific activity of 160 Ci/mmol. Mass spectroscopy confirmed that the radiohormone consists of a single isomer with six tritium atoms bound to carbons 26 and 27. Synthetically produced 1 alpha,25-dihydroxy [26,27-3H] vitamin D3 is indistinguishable from 1 alpha,25-dihydroxy-[26,27-3H] vitamin D3 obtained from the enzymatic 1 alpha-hydroxylation of 25-hydroxy[26,27-3H] vitamin D3 (160 Ci/mmol) by high-pressure liquid chromatography analysis and in the competitive binding assay using chick intestinal cytosol as the receptor source. Equilibrium dissociation constant measurements with the high specific activity radiohormone indicate a Kd of 8.2 x 10(-11) M for the chick intestinal cytosol 1 alpha,25-dihydroxyvitamin D3 receptor--a value considerably lower than the constants in the range of (1-5) x 10(-9) M previously reported

In vitro studies on the translocation of acid phosphatase into the endoplasmic reticulum of the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Krebs, H O; Hoffschulte, H K; Müller, M

1989-05-01

We demonstrate here the in vitro translocation of yeast acid phosphatase into rough endoplasmic reticulum. The precursor of the repressible acid phosphatase from Saccharomyces cerevisiae encoded by the PHO5 gene, was synthesized in a yeast lysate programmed with in vitro transcribed PHO5 mRNA. In the presence of yeast rough microsomes up to 16% of the acid phosphatase synthesized was found to be translocated into the microsomes, as judged by proteinase resistance, and fully core-glycosylated. The translocation efficiency however, decreased to 3% if yeast rough microsomes were added after synthesis of acid phosphatase had been terminated. When a wheat-germ extract was used for in vitro synthesis, the precursor of acid phosphatase was translocated into canine pancreatic rough microsomes and thereby core-glycosylated in a signal-recognition-particle-dependent manner. Replacing canine with yeast rough microsomes in the wheat-germ translation system, however, resulted in a significant decrease in the ability to translocate and glycosylate the precursor. Translocation and glycosylation were partially restored by a high-salt extract prepared from yeast ribosomes. The results presented here suggest that yeast-specific factors are needed to translocate and glycosylate acid phosphatase efficiently in vitro.

Is glycosylated haemoglobin a marker of fertility?

DEFF Research Database (Denmark)

Hjollund, N H; Jensen, T K; Bonde, J P

1999-01-01

We performed a follow-up study of time to pregnancy in a population of first-time pregnancy planners without previous reproductive experience. The objective of this paper is to report and discuss a finding of a strong relationship between glycosylated haemoglobin (HbA1C) and fertility. A total...... concentration of inhibin A. No association was found between HbA1C and psychosocial distress. The reduced fertility among women with high HbA1C may be due to an association with subclinical polycystic ovaries as indicated by the hormonal profile....

Importance of glycosylation on function of a potassium channel in neuroblastoma cells.

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M K Hall

Full Text Available The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type, partially glycosylated (N220Q and N229Q, and unglycosylated (N220Q/N229Q Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.

Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

International Nuclear Information System (INIS)

Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

1990-01-01

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

Reduced apolipoprotein glycosylation in patients with the metabolic syndrome.

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Olga V Savinova

Full Text Available The purpose of this study was to compare the apolipoprotein composition of the three major lipoprotein classes in patients with metabolic syndrome to healthy controls.Very low density (VLDL, intermediate/low density (IDL/LDL, hereafter LDL, and high density lipoproteins (HDL fractions were isolated from plasma of 56 metabolic syndrome subjects and from 14 age-sex matched healthy volunteers. The apolipoprotein content of fractions was analyzed by one-dimensional (1D gel electrophoresis with confirmation by a combination of mass spectrometry and biochemical assays.Metabolic syndrome patients differed from healthy controls in the following ways: (1 total plasma--apoA1 was lower, whereas apoB, apoC2, apoC3, and apoE were higher; (2 VLDL--apoB, apoC3, and apoE were increased; (3 LDL--apoC3 was increased, (4 HDL--associated constitutive serum amyloid A protein (SAA4 was reduced (p<0.05 vs. controls for all. In patients with metabolic syndrome, the most extensively glycosylated (di-sialylated isoform of apoC3 was reduced in VLDL, LDL, and HDL fractions by 17%, 30%, and 25%, respectively (p<0.01 vs. controls for all. Similarly, the glycosylated isoform of apoE was reduced in VLDL, LDL, and HDL fractions by 15%, 26%, and 37% (p<0.01 vs. controls for all. Finally, glycosylated isoform of SAA4 in HDL fraction was 42% lower in patients with metabolic syndrome compared with controls (p<0.001.Patients with metabolic syndrome displayed several changes in plasma apolipoprotein composition consistent with hypertriglyceridemia and low HDL cholesterol levels. Reduced glycosylation of apoC3, apoE and SAA4 are novel findings, the pathophysiological consequences of which remain to be determined.

The role of the active site Zn in the catalytic mechanism of the GH38 Golgi alpha-mannosidase II: Implications from noeuromycin inhibition

DEFF Research Database (Denmark)

Bols, Mikael; Kuntz, Douglas A.; Rose, David R.

2006-01-01

Golgi alpha-mannosidase II (GMII) is a Family 38 glycosyl hydrolase involved in the eukaryotic N-glycosylation pathway in protein synthesis. Understanding of its catalytic mechanism has been of interest for the development of specific inhibitors that could lead to novel anti-metastatic or anti-in...

The group B streptococcal alpha C protein binds alpha1beta1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells.

Science.gov (United States)

Bolduc, Gilles R; Madoff, Lawrence C

2007-12-01

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR', bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with alpha(1)beta(1)-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds alpha(1)beta(1)-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind alpha(1)beta(1)-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.

The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells.

Science.gov (United States)

Fujita, Naonobu; Tamura, Ayako; Higashidani, Aya; Tonozuka, Takashi; Freeze, Hudson H; Nishikawa, Atsushi

2008-02-01

Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.

[Effects of low-protein diet plus alpha-keto acid on micro-inflammation and the relationship between micro-inflammation and nutritional status in patients performing continuous ambulatory peritoneal dialysis: a randomized controlled trial].

Science.gov (United States)

Chen, Wei; Guo, Zhi-Yong; Wu, Hao; Sun, Li-Jing; Cai, Li-Li; Xu, Hai-Yan

2008-05-01

To investigate the effects of the combination of alpha-keto acid and low-protein diet on the levels of serum cytokines in patients performing continuous ambulatory peritoneal dialysis (CAPD) and to explore the relationship between inflammation and malnutrition in CAPD patients. Eighty-nine CAPD patients were randomized into three groups, and 78 cases completed a one-year follow-up and with complete data. There were 31 cases in low-protein diet plus alpha-keto acid group, 26 cases in low-protein diet group and 21 cases in routine-protein diet group. The levels of serum albumin (Alb), prealbumin (PA), retinol-binding protein (RBP), transferrin (TRF), cholesterol (TC), triglycerides (TG), leptin, and triceps skinfold thickness (TSF), mid-arm muscle circumference (MAMC), body mass index (BMI) were measured. The changes of serum interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein (CRP) were also detected. Compared with low-protein diet group, serum levels of PA, RBP and TRF were significantly increased both in low-protein diet plus alpha-keto acid and routine-protein diet groups ( Pdiet plus alpha-keto acid group and routine-protein diet group. There was an increased tendency in the content of Alb, TC, TG, BMI, TSF and MAMC, but there were no significant differences. The plasma levels of IL-1alpha, IL-6 and TNF-alpha in low-protein diet plus alpha-keto acid group were decreased as compared with the routine-protein diet group, but there were no significant differences. The plasma level of CRP in low-protein diet plus alpha-keto acid group was lower than that in the routine-protein diet group ( Pketo acid and low-protein diet can ameliorate malnutrition and micro-inflammation in CAPD patients.

Dual Effects of Alpha-Hydroxy Acids on the Skin

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Sheau-Chung Tang

2018-04-01

Full Text Available AHAs are organic acids with one hydroxyl group attached to the alpha position of the acid. AHAs including glycolic acid, lactic acid, malic acid, tartaric acid, and citric acid are often used extensively in cosmetic formulations. AHAs have been used as superficial peeling agents as well as to ameliorate the appearance of keratoses and acne in dermatology. However, caution should be exercised in relation to certain adverse reactions among patients using products with AHAs, including swelling, burning, and pruritus. Whether AHAs enhance or decrease photo damage of the skin remains unclear, compelling us to ask the question, is AHA a friend or a foe of the skin? The aim of this manuscript is to review the various biological effects and mechanisms of AHAs on human keratinocytes and in an animal model. We conclude that whether AHA is a friend or foe of human skin depends on its concentration. These mechanisms of AHAs are currently well understood, aiding the development of novel approaches for the prevention of UV-induced skin damage.

The Emerging Importance of IgG Fab Glycosylation in Immunity.

Science.gov (United States)

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

Alpha-mangostin attenuates diabetic nephropathy in association with suppression of acid sphingomyelianse and endoplasmic reticulum stress.

Science.gov (United States)

Liu, Tingting; Duan, Wang; Nizigiyimana, Paul; Gao, Lin; Liao, Zhouning; Xu, Boya; Liu, Lerong; Lei, Minxiang

2018-02-05

Diabetic nephropathy is a common complication of diabetes, but there are currently few treatment options. The aim of this study was to gain insight into the effect of alpha-mangostin on diabetic nephropathy and possible related mechanisms. Goto-Kakizaki rats were used as a diabetic model and received alpha-mangostin or desipramine treatment with normal saline as a control. Ten age-matched Sprague Dawley rats were used as normal controls and treated with normal saline. At week 12, blood glucose, albuminuria, apoptosis and renal pathologic changes were assessed. Protein levels for acid sphingomyelinase, glucose-regulated protein 78, phosphorylated PKR-like ER-resident kinase, activated transcription factor 4, CCAAT/enhancer-binding protein, homologous protein), and cleaved-caspase12 were measured. The level of acid sphingomyelinase was significantly increased, and ER stress was activated in diabetic rat kidneys when compared to the control animals. When acid sphingomyelinase was inhibited by alpha-mangostin, the expression of ER stress-related proteins was down-regulated in association with decreased levels of diabetic kidney injury. Alpha-mangostin, an acid sphingomyelinase inhibitor plays a protective role in diabetic neuropathy by relieving ER stress induced-renal cell apoptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Differentiation of the mRNA transcripts originating from the alpha 1- and alpha 2-globin loci in normals and alpha-thalassemics.

OpenAIRE

Liebhaber, S A; Kan, Y W

1981-01-01

The alpha-globin polypeptide is encoded by two adjacent genes, alpha 1 and alpha 2. In the normal diploid state (alpha alpha/alpha alpha) all four alpha-globin genes are expressed. Loss or dysfunction of one or more of these genes leads to deficient alpha-globin production and results in alpha-thalassemia. We present a technique to differentially assess the steady-state levels of the alpha 1- and alpha-2-globin messenger RNA (mRNA) transcripts and thus delineate the relative level of expressi...

Optimization of a colorimetric assay for glycosylated human serum albumin

Energy Technology Data Exchange (ETDEWEB)

Bohney, J.P.; Feldhoff, R.C.

1986-05-01

The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.

3 alpha-Hydroxylated bile acid profiles in clinically normal cats, cats with severe hepatic lipidosis, and cats with complete extrahepatic bile duct occlusion.

Science.gov (United States)

Center, S A; Thompson, M; Guida, L

1993-05-01

Concentrations of 3 alpha-hydroxylated bile acids were measured in serum and urine of clinically normal (healthy) cats (n = 6), cats with severe hepatic lipidosis (n = 9), and cats with complete bile duct occlusion (n = 4). Bile acid concentrations were measured by use of a gradient flow high-performance liquid chromatography procedure with an acetonitrile and ammonium phosphate mobile phase and an in-line postanalytic column containing 3 alpha-hydroxy-steroid dehydrogenase and a fluorescence detector. Specific identification of all bile acid peaks was not completed; unidentified moieties were represented in terms of their elution time (in minutes). Significant differences in serum and urine bile acid concentrations, quantitative and proportional, were determined among groups of cats. Cats with hepatic lipidosis and bile duct occlusion had significantly (P > or = 0.05) greater total serum and urine bile acids concentrations than did healthy cats. The proportion of hydrophobic bile acids in serum, those eluting at > or = 400 minutes, was 1.9% for healthy cats, 3.3% for cats with lipidosis, and 5.4% for bile duct-obstructed cats. Both groups of ill cats had a broader spectrum of unidentified late-eluting serum bile acids than did healthy cats; the largest spectrum developed in bile duct-occluded cats.(ABSTRACT TRUNCATED AT 250 WORDS)

LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031(T).

Science.gov (United States)

Song, Qinghao; Wang, Yan; Yin, Chong; Zhang, Xiao-Hua

2016-08-01

Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031(T) and expressed in Escherichia coli BL21. The gene has a length of 1428bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45°C, retaining high activity even at low temperatures (almost 38% residual activity at 10°C). In addition, 1mM Na(+), K(+), and Mn(2+) enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry. Copyright © 2016 Elsevier Inc. All rights reserved.

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

Science.gov (United States)

Lee, Dae-Hee; Lee, Yong J

2008-10-01

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

Alternate method of source preparation for alpha spectrometry: No electrodeposition, no hydrofluoric acid

International Nuclear Information System (INIS)

Kurosaki, Hiromu; Mueller, Rebecca J.; Lambert, Susan B.; Rao, Govind R.

2016-01-01

An alternate method of preparing actinide alpha counting sources was developed in place of electrodeposition or lanthanide fluoride micro-precipitation. The method uses lanthanide hydroxide micro-precipitation to avoid the use of hazardous hydrofluoric acid. Lastly, it provides a quicker, simpler, and safer way of preparing actinide alpha counting sources in routine, production-type laboratories that process many samples daily.

Alternate method of source preparation for alpha spectrometry: no electrodeposition, no hydrofluoric acid

International Nuclear Information System (INIS)

Hiromu Kurosaki; Lambert, S.B.; Rao, G.R.; Mueller, R.J.

2017-01-01

An alternate method of preparing actinide alpha counting sources was developed in place of electrodeposition or lanthanide fluoride micro-precipitation. The method uses lanthanide hydroxide micro-precipitation to avoid the use of hazardous hydrofluoric acid. It provides a quicker, simpler, and safer way of preparing actinide alpha counting sources in routine, production-type laboratories that process many samples daily. (author)

The role of glycosylation in breast cancer metastasis and cancer control

Directory of Open Access Journals (Sweden)

Alexandra eKölbl

2015-10-01

Full Text Available AbstractGlycosylation and its correlation to the formation of remote metastasis in breast cancer had been an important scientific topic in the last 25 years. With the development of new analytical techniques new insights were gained on the mechanisms underlying metastasis formation and the role of aberrant glycosylation within. Mucin-1 and Galectin were recognized as key players in glycosylation. Interestingly, aberrant carbohydrate structures seem to support the development of brain metastasis in breast cancer patients, as changes in glycosylation structures facilitate an overcoming of blood-brain barrier. Changes in the gene expression of glycosyltransferases are the leading cause for a modification of carbohydrate chains, so that also altered gene expression plays a role for glycosylation. In consequence, glycosylation and changes within can be useful for cancer diagnosis, determination of tumour stage and prognosis, but can as well be targets for therapeutic strategies. Thus, further research on this topic would worth wile for cancer combating.

Amphiphilic glycosylated block copolypeptides as macromolecular surfactants in the emulsion polymerization of styrene

NARCIS (Netherlands)

Jacobs, Jaco; Gathergood, N.; Heuts, J.P.A.; Heise, A.

2015-01-01

Diblock copolymers consisting of poly(L-phenyl alanine) and poly(benzyl-L-glutamate) or poly(CBZ-L-lysine), respectively, were synthesized via sequential NCA polymerization. After deprotection, subsequent partial glycosylation of the glutamic acid and lysine units with galactosamine hydrochloride or

Effects of L-ascorbic acid and alpha-tocopherol on biochemical ...

African Journals Online (AJOL)

Background: The purpose of this study is to determine the effect of L-ascorbic acid and alpha-tocopherol as well as combination of these vitamins with or without exposure to physical exercise on intensity of lipid peroxidation, activity of xanthine oxidase, activity of total antioxidative system, concentration of glutathione, and ...

Does Glycosylation as a modifier of Original Antigenic Sin explain the case age distribution and unusual toxicity in pandemic novel H1N1 influenza?

Directory of Open Access Journals (Sweden)

Nishiura Hiroshi

2010-01-01

Full Text Available Abstract Background A pandemic novel H1N1 swine-origin influenza virus has emerged. Most recently the World Health Organization has announced that in a country-dependent fashion, up to 15% of cases may require hospitalization, often including respiratory support. It is now clear that healthy children and young adults are disproportionately affected, most unusually among those with severe respiratory disease without underlying conditions. One possible explanation for this case age distribution is the doctrine of Original Antigenic Sin, i.e., novel H1N1 may be antigenically similar to H1N1 viruses that circulated at an earlier time. Persons whose first exposure to influenza viruses was to such similar viruses would be relatively immune. However, this principle is not sufficient to explain the graded susceptibility between ages 20 and 60, the reduced susceptibility in children below age 10, and the unusual toxicity observed. Methods We collected case data from 11 countries, about 60% of all cases reported through mid-July 2009. We compared sequence data for the hemagglutinin of novel H1N1 with sequences of H1N1 viruses from 1918 to the present. We searched for sequence differences that imply loss of antigenicity either directly through amino acid substitution or by the appearance of sites for potential glycosylation proximal to sites known to be antigenic in humans. We also considered T-cell epitopes. Results In our composite, over 75% of confirmed cases of novel H1N1 occurred in persons ≤ 30 years old, with peak incidence in the age range 10-19 years. Less than 3% of cases occurred in persons over 65, with a gradation in incidence between ages 20 and 60 years. The sequence data indicates that novel H1N1 is most similar to H1N1 viruses that circulated before 1943. Novel H1N1 lacks glycosylation sites on the globular head of hemagglutinin (HA1 near antigenic regions, a pattern shared with the 1918 pandemic strain and H1N1 viruses that circulated

Darbepoetin alpha for the treatment of anaemia in low-intermediate risk myelodysplastic syndromes

DEFF Research Database (Denmark)

Musto, Pellegrino; Lanza, Francesco; Balleari, Enrico

2005-01-01

Thirty-seven anaemic subjects with low-to-intermediate risk myelodysplastic syndrome (MDS) received the highly glycosylated, long-acting erythropoiesis-stimulating molecule darbepoetin-alpha (DPO) at the single, weekly dose of 150 microg s.c. for at least 12 weeks. Fifteen patients (40.5%) achieved......, no excess of blasts and hypoplastic bone marrow. This study suggests that DPO, at the dose and schedule used, can be safely given in low-intermediate risk MDS and may be effective in a significant proportion of these patients....

The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

Energy Technology Data Exchange (ETDEWEB)

Klapper, Maja [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Boehme, Mike [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Nitz, Inke [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany); Doering, Frank [Molecular Nutrition, Institute of Human Nutrition and Food Science, Christian-Albrechts-University of Kiel, Heinrich-Hecht-Platz 10, D-24118 Kiel (Germany)

2007-04-27

The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

Determination of branched chain amino acids, methionine, phenylalanine, tyrosine and alpha-keto acids in plasma and dried blood samples using HPLC with fluorescence detection.

Science.gov (United States)

Kand'ár, Roman; Záková, Pavla; Jirosová, Jana; Sladká, Michaela

2009-01-01

The determination of branched chain amino acids [BCAA; valine (Val), leucine (Leu), isoleucine (Ile)], alpha-keto acids derived from BCAA [BCKA; alpha-ketoisovaleric acid (KIV), alpha-ketoisocaproic acid (KIC), alpha-ketomethylvaleric acid (KMV)], methionine (Met), phenylalanine (Phe) and tyrosine (Tyr) is currently the most reliable approach for the diagnosis of maple syrup urine disease (MSUD), hypermethioninemia, phenylketonuria (PKU) and tyrosinemia. The aim of this study was to develop rapid and simple HPLC methods for measurement of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples. Samples of peripheral venous blood with EDTA as anticoagulant were obtained from a group of healthy blood donors (n=70, 35 females, 27-41 years of age and 35 males, 28-43 years of age). Blood-spot samples from a group of newborns (n=80, 40 girls and 40 boys 3-5 days of age) were collected onto #903 Specimen Collection Paper and allowed to dry for at least 24 h before analysis. Prior to separation, the amino acids (AA) were derivatized with o-phthaldialdehyde (OPA) and BCKA with o-phenylenediamine (OPD). Reverse phase column chromatography (LiChroCart 125-4 Purospher RP-18e, 5 microm) was used for separation and fluorescence detection used to monitoring of effluent. For AA analysis, 25 mmol/L sodium hydrogenphosphate-methanol (90:10, v/v), pH 6.5+/-0.1 was used as mobile phase A and 100% methanol was used as mobile phase B. Measurement of BCKA used a mixture of methanol and deionized water (55:45, v/v) as mobile phase A and mobile phase B consisted of 100% methanol. Analytical performance of these methods was satisfactory for the determination of all AA and BCKA. The intra-assay and inter-assay coefficients of variation were below 10% and recovery ranged from 90%-110%. We have developed simple, rapid and selective HPLC methods with fluorescence detection for the determination of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples.

Effects of cell culture conditions on antibody N-linked glycosylation--what affects high mannose 5 glycoform.

Science.gov (United States)

Pacis, Efren; Yu, Marcella; Autsen, Jennifer; Bayer, Robert; Li, Feng

2011-10-01

The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process. Copyright © 2011 Wiley Periodicals, Inc.

Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.

Science.gov (United States)

Biswas, Himadri; Chattopadhyaya, Rajagopal

2016-04-01

Curcuma longa rhizome lectin, a mannose-binding protein of non-seed portions of turmeric, is known to have antifungal, antibacterial and α-glucosidase inhibitory activities. We studied the role of complex-type glycans attached to asparagine (Asn) 66 and Asn 110 to elucidate the role of carbohydrates in lectin activity and stability. Apart from the native lectin, the characteristics of a deglycosylated Escherichia coli expressed lectin, high-mannose oligosaccharides at both asparagines and its glycosylation mutants N66Q and N110Q expressed in Pichia pastoris, were compared to understand the relationship between glycosylation and activity. Far UV circular dichroism (CD) spectra, fluorescence emission maximum, hemagglutination assay show no change in secondary or tertiary structures or sugar-binding properties between wild-type and aforementioned recombinant lectins under physiological pH. But reduced agglutination activity and loss of tertiary structure are observed in the acidic pH range for the deglycosylated and the N110Q protein. In thermal and guanidine hydrochloride (GdnCl)-induced unfolding, the wild-type and high-mannose lectins possess higher stability compared with the deglycosylated recombinant lectin and both mutants, as measured by a higher Tm of denaturation or a greater free energy change, respectively. Reversibility experiments after thermal denaturation reveal that deglycosylated proteins tend to aggregate during thermal inactivation but the wild type shows a much greater recovery to the native state upon refolding. These results suggest that N-glycosylation in turmeric lectin is important for the maintenance of its proper folding upon changes in pH, and that the oligosaccharides help in maintaining the active conformation and prevent aggregation in unfolded or partially folded molecules. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

High specific activity N-Acetyl-3{sup H}-{alpha}-Aspartyl- L-Glutamic at micro mole scale; Sintesis de N-Acetil-3{sup H}- {alpha} -Aspartil-Glutamico a escala de Micromoles

Energy Technology Data Exchange (ETDEWEB)

Suarez, C

1984-07-01

High specific activity N-Acetyl-3{sup H}- {alpha} -Aspartyl-I-Glutamic acid at micro mole scale in prepared acetylating L- {alpha} -Aspartyl-L-glutamic with 3{sup H}-acetic anhydride in re distilled toluene. The product le purified through cationic and anionic columns. The radiochemical purity as determined by thin-layer chromatography is greater then 99% at the time preparation. (Author) 5 refs.

Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells.

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Nicolas Rochereau

2013-09-01

Full Text Available Intestinal microfold (M cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell-mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower......Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...

Alternative splicing of T cell receptor (TCR) alpha chain transcripts containing V alpha 1 or V alpha 14 elements.

Science.gov (United States)

Mahotka, C; Hansen-Hagge, T E; Bartram, C R

1995-10-01

Human acute lymphoblastic leukemia cell lines represent valuable tools to investigate distinct steps of the complex regulatory pathways underlying T cell receptor recombination and expression. A case in point are V delta 2D delta 3 and subsequent V delta 2D delta 3J alpha rearrangements observed in human leukemic pre-B cells as well as in normal lymphopoiesis. The functional expression of these unusual (VD) delta (JC) alpha hybrids is almost exclusively prevented by alternative splicing events. In this report we show that alternative splicing at cryptic splice donor sites within V elements is not a unique feature of hybrid TCR delta/alpha transcripts. Among seven V alpha families analyzed by RT-PCR, alternatively spliced products were observed in TCR alpha recombinations containing V alpha 1 or V alpha 14 elements. In contrast to normal peripheral blood cells and thymocytes, the leukemia cell line JM expressing functional V alpha 1J alpha 3C alpha transcripts lacked evidence of aberrant TCR alpha RNA species.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

Energy Technology Data Exchange (ETDEWEB)

Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai; Lee, Kyung-Dall; Amidon, Gordon L. (Michigan)

2008-07-08

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Mining the Virgin Land of Neurotoxicology: A Novel Paradigm of Neurotoxic Peptides Action on Glycosylated Voltage-Gated Sodium Channels

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Zhirui Liu

2012-01-01

Full Text Available Voltage-gated sodium channels (VGSCs are important membrane protein carrying on the molecular basis for action potentials (AP in neuronal firings. Even though the structure-function studies were the most pursued spots, the posttranslation modification processes, such as glycosylation, phosphorylation, and alternative splicing associating with channel functions captured less eyesights. The accumulative research suggested an interaction between the sialic acids chains and ion-permeable pores, giving rise to subtle but significant impacts on channel gating. Sodium channel-specific neurotoxic toxins, a family of long-chain polypeptides originated from venomous animals, are found to potentially share the binding sites adjacent to glycosylated region on VGSCs. Thus, an interaction between toxin and glycosylated VGSC might hopefully join the campaign to approach the role of glycosylation in modulating VGSCs-involved neuronal network activity. This paper will cover the state-of-the-art advances of researches on glycosylation-mediated VGSCs function and the possible underlying mechanisms of interactions between toxin and glycosylated VGSCs, which may therefore, fulfill the knowledge in identifying the pharmacological targets and therapeutic values of VGSCs.

[Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins].

Science.gov (United States)

Avanov, A Ia; Lipkind, G M

1990-03-01

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.

Catalpic acid decreases abdominal fat deposition, improves glucose homeostasis and upregulates PPAR alpha expression in adipose tissue.

Science.gov (United States)

Hontecillas, Raquel; Diguardo, Maggie; Duran, Elisa; Orpi, Marcel; Bassaganya-Riera, Josep

2008-10-01

Catalpic acid (CAT) is a conjugated linolenic acid (CLN) isomer containing trans-9, trans-11, cis-13 double bonds in an 18-carbon chain and it is found primarily in the seed oil of ornamental and medicinal trees and shrubs of the family Bignoniaceae. The objective of this study was to investigate whether CAT decreases obesity and ameliorates insulin sensitivity and glucose tolerance in mice fed high-fat diets. To test the efficacy of CAT in decreasing obesity and diabetes we used both a model of diet-induced obesity (DIO) and a genetic model of obesity (i.e., mice lacking the leptin receptor). Blood was collected on days 0, 7, 14, 21 and 28 for determining fasting glucose and insulin concentrations in plasma. In addition, a glucose tolerance test was administered on day 28. We found that dietary CAT (1g/100g) decreased fasting plasma glucose and insulin concentrations, ameliorated the glucose normalizing ability following glucose challenge and decreased abdominal white adipose tissue accumulation. In white adipose tissue (WAT), CAT upregulated peroxisome proliferator-activated receptor (PPAR) alpha and its responsive genes [i.e., stearoyl-coenzyme A desaturase (SCD1) and enoyl-coenzyme A hydratase (ECH)], increased concentrations of high-density lipoprotein (HDL) cholesterol and decreased plasma triglyceride (TG) levels. CAT decreased abdominal fat deposition, increased HDL cholesterol, decreased TG concentrations, decreased glucose and insulin homeostasis and modulated WAT gene expression in a manner reminiscent of the actions of the PPAR alpha-activating fibrate class of lipid-lowering drugs.

Glycosylated yellow laccases of the basidiomycete Stropharia aeruginosa.

Science.gov (United States)

Daroch, Maurycy; Houghton, Catharine A; Moore, Jonathan K; Wilkinson, Mark C; Carnell, Andrew J; Bates, Andrew D; Iwanejko, Lesley A

2014-05-10

Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases. Copyright © 2014 Elsevier Inc. All rights reserved.

Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns.

Science.gov (United States)

Yang, Ting; Stoopen, Geert; Yalpani, Nasser; Vervoort, Jacques; de Vos, Ric; Voster, Alessandra; Verstappen, Francel W A; Bouwmeester, Harro J; Jongsma, Maarten A

2011-07-01

Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize (Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MICLippia dulcis under the control of a ubiquitin promoter. The volatile and non-volatile metabolite profiles of leaves from transgenic and control lines were compared. The headspaces collected from intact seedlings of transgenic and control plants were not significantly different, although detached leaves of transgenic plants emitted 5-fold more geranyl acetate compared to control plants. Non-targeted LC-MS profiling and LC-MS-MS identification of extracts from maize leaves revealed that the major significantly different non-volatile compounds were 2 geranic acid derivatives, a geraniol dihexose and 4 different types of hydroxyl-geranic acid-hexoses. A geranic acid glycoside was the most abundant, and identified by NMR as geranoyl-6-O-malonyl-β-d-glucopyranoside with an average concentration of 45μM. Fungal bioassays with C. graminicola and F. graminearum did not reveal an effect of these changes in secondary metabolite composition on plant resistance to either fungus. The results demonstrate that metabolic engineering of geraniol into geranic acid can rely on the existing default pathway, but branching glycosylation pathways must be controlled to achieve accumulation of the aglycones. Copyright © 2011 Elsevier Inc. All rights reserved.

A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype

NARCIS (Netherlands)

Iqbal, Z.; Shahzad, M.; Vissers, L.E.L.M.; Scherpenzeel, M. van; Gilissen, C.; Razzaq, A.; Zahoor, M.Y.; Khan, S.N.; Kleefstra, T.; Veltman, J.A.; Brouwer, A.P.M. de; Lefeber, D.J.; Bokhoven, H. van; Riazuddin, S.

2013-01-01

Congenital disorders of glycosylation (CDG) are a large group of recessive multisystem disorders caused by impaired protein or lipid glycosylation. The CDG-I subgroup is characterized by protein N-glycosylation defects originating in the endoplasmic reticulum. The genetic defect is known for 17

The Protective Effects of Alpha-Lipoic Acid and Coenzyme Q10 Combination on Ovarian Ischemia-Reperfusion Injury: An Experimental Study

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Ahmet Ali Tuncer

2016-01-01

Full Text Available Objective. This study aims to evaluate whether alpha-lipoic acid and/or coenzyme Q10 can protect the prepubertal ovarian tissue from ischemia-reperfusion injury in an experimental rat model of ovarian torsion. Materials and Methods. Forty-two female preadolescent Wistar-Albino rats were divided into 6 equal groups randomly. The sham group had laparotomy without torsion; the other groups had torsion/detorsion procedure. After undergoing torsion, group 2 received saline, group 3 received olive oil, group 4 received alpha-lipoic acid, group 5 received coenzyme Q10, and group 6 received both alpha-lipoic acid and coenzyme Q10 orally. The oxidant-antioxidant statuses of these groups were compared using biochemical measurement of oxidized/reduced glutathione, glutathione peroxidase and malondialdehyde, pathological evaluation of damage and apoptosis within the ovarian tissue, and immunohistochemical assessment of nitric oxide synthase. Results. The left ovaries of the alpha-lipoic acid + coenzyme Q10 group had significantly lower apoptosis scores and significantly higher nitric oxide synthase content than the left ovaries of the control groups. The alpha-lipoic acid + coenzyme Q10 group had significantly higher glutathione peroxidase levels and serum malondialdehyde concentrations than the sham group. Conclusions. The combination of alpha-lipoic acid and coenzyme Q10 has beneficial effects on oxidative stress induced by ischemia-reperfusion injury related to ovarian torsion.

An isozyme of acid alpha-glucosidase with reduced catalytic activity for glycogen.

Science.gov (United States)

Beratis, N G; LaBadie, G U; Hirschhorn, K

1980-03-01

Both the common and a variant isozyme of acid alpha-glucosidase have been purified from a heterozygous placenta with CM-Sephadex, ammonium sulfate precipitation, dialysis, Amicon filtration, affinity chromatography by Sephadex G-100, and DEAE-cellulose chromatography. Three and two activity peaks, from the common and variant isozymes, respectively, were obtained by DEAE-cellulose chromatography using a linear NaCl gradient. The three peaks of activity of the common isozyme were eluted with 0.08, 0.12, and 0.17 M NaCl, whereas the two peaks of the variant, with 0.01 and 0.06 M NaCl. The pH optimum and thermal denaturation at 57 degrees C were the same in all enzyme peaks of both isozymes. Rabbit antiacid alpha-glucosidase antibodies produced against the common isozyme were found to cross-react with both peaks of the variant isozyme. The two isozymes shared antigenic identity and had similar Km's with maltose as substrate. Normal substrate saturation kinetics were observed with the common isozyme when glycogen was the substrate, but the variant produced an S-shaped saturation curve indicating a phase of negative and positive cooperativity at low and high glycogen concentrations, respectively. The activity of the variant was only 8.6% and 19.2% of the common isozyme when assayed with nonsaturating and saturating concentrations of glycogen, respectively. A similar rate of hydrolysis of isomaltose by both isozymes was found indicating that the reduced catalytic activity of the variant isozyme toward glycogen is not the result of a reduced ability of this enzyme to cleave the alpha-1,6 linkages of glycogen.

A randomized phase II chemoprevention trial of 13-CIS retinoic acid with or without alpha tocopherol or observation in subjects at high risk for lung cancer.

Science.gov (United States)

Kelly, Karen; Kittelson, John; Franklin, Wilbur A; Kennedy, Timothy C; Klein, Catherine E; Keith, Robert L; Dempsey, Edward C; Lewis, Marina; Jackson, Mary K; Hirsch, Fred R; Bunn, Paul A; Miller, York E

2009-05-01

No chemoprevention strategies have been proven effective for lung cancer. We evaluated the effect of 13-cis retinoic acid (13-cis RA), with or without alpha tocopherol, as a lung cancer chemoprevention agent in a phase II randomized controlled clinical trial of adult subjects at high risk for lung cancer as defined by the presence of sputum atypia, history of smoking, and airflow obstruction, or a prior surgically cured nonsmall cell lung cancer (disease free, >3 years). Subjects were randomly assigned to receive either 13-cis RA, 13-cis RA plus alpha tocopherol (13-cis RA/alpha toco) or observation for 12 months. Outcome measures are derived from histologic evaluation of bronchial biopsy specimens obtained by bronchoscopy at baseline and follow-up. The primary outcome measure is treatment "failure" defined as histologic progression (any increase in the maximum histologic score) or failure to return for follow-up bronchoscopy. Seventy-five subjects were randomized (27/22/26 to observations/13-cis RA/13-cis RA/alpha toco); 59 completed the trial; 55 had both baseline and follow-up bronchoscopy. The risk of treatment failure was 55.6% (15 of 27) and 50% (24 of 48) in the observation and combined (13 cis RA plus 13 cis RA/alpha toco) treatment arms, respectively (odds ratio adjusted for baseline histology, 0.97; 95% confidence interval, 0.36-2.66; P = 0.95). Among subjects with complete histology data, maximum histology score in the observation arm increased by 0.37 units and by 0.03 units in the treated arms (difference adjusted for baseline, -0.18; 95% confidence interval, -1.16 to 0.81; P = 0.72). Similar (nonsignificant) results were observed for treatment effects on endobronchial proliferation as assessed by Ki-67 immunolabeling. Twelve-month treatment with 13-cis RA produced nonsignificant changes in bronchial histology, consistent with results in other trials. Agents advancing to phase III randomized trials should produce greater histologic changes. The

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide.

Science.gov (United States)

Daubenspeck, James M; Jordan, David S; Simmons, Warren; Renfrow, Matthew B; Dybvig, Kevin

2015-01-01

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.

Kinetic studies of acid inactivation of alpha-amylase from Aspergillus oryzae

DEFF Research Database (Denmark)

Carlsen, Morten; Nielsen, Jens Bredal; Villadsen, John

1996-01-01

The stability of alpha-amylase from Aspergillus oryzae has been studied at different pH. The enzyme is extremely stable at neutral pH (pH 5-8), whereas outside this pH-range a substantial loss of activity is observed. The acid-inactivation of alpha-amylase from A. oryzae was monitored on...... regains part of its activity, and the reactivation process also follows first-order kinetics. The irreversible loss of activity is found not to result from a protease contamination of the protein samples. A proposed model, where irreversibly inactivated a-amylase is formed both directly from the active...

Molecular pharmacology of 4-substituted glutamic acid analogues at ionotropic and metabotropic excitatory amino acid receptors

DEFF Research Database (Denmark)

Bräuner-Osborne, Hans; Nielsen, B; Stensbøl, T B

1997-01-01

(subtypes 1alpha and 2), respectively, whereas (S)-4-methyleneglutamic acid showed high but rather non-selective affinity for the (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), kainic acid, NMDA and mGlu receptors (subtypes 1alpha and 2). Although none of the compounds were specific......The pharmacology of (2S,4R)-4-methylglutamic acid, (2S,4S)-4-methylglutamic acid and (S)- and (R)-4-methyleneglutamic acids (obtained in high chemical and enantiomeric purity from racemic 4-methyleneglutamic acid by chiral HPLC using a Crownpak CR(+) column), was examined in binding experiments...... using rat brain ionotropic glutamate receptors, and in functional assays using cloned metabotropic glutamate (mGlu) receptors. As a notable result of these studies, (2S,4R)-4-methylglutamic acid and (2S,4S)-4-methylglutamic acid were shown to be selective for kainic acid receptors and mGlu receptors...

Synthesis of 4-O-glycosylated 1,5-anhydro-D-fructose and of 1,5-anhydro-D-tagatose from a common intermediate 2,3-O-isopropylidene -D-fructose

DEFF Research Database (Denmark)

Agoston, Karoly; Dekany, Gyula; Lundt, Inge

2009-01-01

Four novel disaccharides of glycosylated 1,5-anhydro-D-ketoses have been prepared: 1,5-anhydro-4-O-b-D-glucopyranosyl-D-fructose, 1,5-anhydro-4-O-b-D-galactopyranosyl-D-fructose, 1,5-anhydro-4-O-b-D-glucopyranosyl-D-tagatose and 1,5-anhydro-4-O-b-D-galactopyranosyl-D-tagatose. The common...... intermediate, 1,5-anhydro-2,3-O-isopropylidene-b-D-fructopyranose, was prepared from D-fructose and converted into the D-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The prepared anhydro-ketoses were glycosylated and deprotected to the disaccharides....

Synthesis of 4-O-glycosylated 1,5-anhydro-D-fructose and of 1,5-anhydro-D-tagatose from a common intermediate 2,3-O-isopropylidene-D-fructose.

Science.gov (United States)

Agoston, Károly; Dékány, Gyula; Lundt, Inge

2009-05-26

Four novel disaccharides of glycosylated 1,5-anhydro-D-ketoses have been prepared: 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-fructose, 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-fructose, 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-tagatose, and 1,5-anhydro-4-O-beta-D-galactopyranosyl-D-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-beta-D-fructopyranose, was prepared from D-fructose and was converted into the D-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.

Alpha-lipoic acid induces sodium iodide symporter expression in TPC-1 thyroid cancer cell line

International Nuclear Information System (INIS)

Choi, Hyun-Jeung; Kim, Tae Yong; Ruiz-Llorente, Sergio; Jeon, Min Ji; Han, Ji Min; Kim, Won Gu; Shong, Young Kee; Kim, Won Bae

2012-01-01

Introduction: Patients with metastatic thyroid cancers that do not uptake iodine need effective therapeutic option. Differentiation-inducing agents have been tried to restore functional expression of sodium iodide symporter (NIS) without success. Our objective was to assess the effect of alpha-lipoic acid (ALA), known as potential antioxidant, on expression of sodium iodide symporter in thyroid cancer cells. Methods: Human thyroid cancer-derived cell lines, TPC-1, were treated with ALA, and changes in NIS mRNA and protein expression were measured. ALA's effect on NIS gene promoter was evaluated, and functional NIS expression was assessed by iodide uptake assay. Results: Treatment with ALA increased NIS mRNA expression up to ten folds of control dose-dependently after 24 h of exposure. ALA increased NIS promoter activity, and increased iodide uptake by 1.6 fold. ALA induced expression of NIS protein, but had no significant effect on the plasma membrane trafficking. ALA increased phosphorylation of CREB and nuclear translocation of pCREB, and co-treatment of ALA and trichostatin A increased iodide uptake by three folds in TPC-1 cells. Conclusions: ALA is a potential agent to increase NIS transcription in TPC-1. It could be used as an adjunctive agent to increase efficacy of radioiodine therapy if combined with a strategy to increase NIS protein trafficking to cell membrane.

Thermometric titrimetry Studies of the cerium(IV) oxidation of alpha-mercaptocarboxylic acids.

Science.gov (United States)

Alexander, W A; Mash, C J; McAuley, A

1969-04-01

The cerium(IV) oxidation of thioglycollic, thiolactic and thiomalic acids has been examined by thermometric titration. The titration curves indicate stoichiometries of more than 1 mole of cerium(IV) per mole of alpha-thiol, suggesting possible side-reactions. In the presence of methyl acrylate, however, the expected ratio is observed. The overall heat of each reaction has been derived. Only with a titration method of this kind where allowance can be made for side-reactions can the heats of reaction for these systems be measured.

Hypolipidaemic and antiplatelet activity of phenoxyacetic acid derivatives related to alpha-asarone.

Science.gov (United States)

Pérez-Pastén, Ricardo; García, Rosa Virginia; Garduño, Leticia; Reyes, Elba; Labarrios, Fernando; Tamariz, Joaquín; Chamorro, Germán

2006-10-01

The phenoxyacetic acid derivatives 1-6 [2-methoxy-4-(2-propenyl)phenoxyacetic acid (1); 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetic acid (2); methyl 2-methoxy-4-(2-propenyl)phenoxyacetate (3); ethyl 2-methoxy-4-(2-propenyl)phenoxyacetate (4); methyl 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetate (5); ethyl 2-methoxy-5-nitro-4-(2-propenyl)phenoxyacetate (6)] related to alpha-asarone have been reported previously as hypolipidaemic agents in diet-induced hyperlipidaemic mice. We have aimed to expand the pharmacological profile of these derivatives by investigating their hypolipidaemic activity in rats and mice under different experimental conditions. The antiplatelet activity was tested also in-vitro from blood derived from consenting healthy volunteers. In normolipidaemic rats, compounds 2, 3 and 5 at oral doses of 40 and 80 mg kg(-1) significantly decreased total cholesterol and LDL-cholesterol levels. Moreover, analogues 3 and 5 administered to hypercholesterolaemic rats at the same doses for seven days also produced a reduction in the content of these same lipoproteins. In neither case were the high-density lipoprotein cholesterol and triglyceride concentrations affected. However, practically all tested compounds were found to be hypocholesterolaemic agents, and were shown to effectively lower low-density lipoprotein cholesterol and triglyceride levels in Triton-induced hyperlipidaemic mice at oral doses of 50 and 100 mg kg(-1). In all tests, all animals appeared to be healthy throughout the experimental period in their therapeutic ranges. Triton-induced hypercholesterolaemic mice appeared to be a desirable model for this class of hypolipidaemic drugs. On the other hand, compounds 1, 2, 4 and 5 significantly inhibited ADP-induced aggregation in-vitro. These findings indicated that all of these compounds appeared to be promising for the treatment of human hyperlipidaemia and thrombotic diseases.

Syntheses of {gamma}-aminobutyric-1-{sup 14}C and of {alpha}-aminoadipic-6-{sup 14}C acid from methoxy-3 chloropropyl-magnesium and marked carbon dioxide; Syntheses de l'acide {gamma}-aminobutyrique{sup 14}C-1 et de l'acide {alpha}-aminoadipique {sup 14}C-6 a partir de methoxy-3 chloropropylmagnesium et d'anhydride carbonique marque

Energy Technology Data Exchange (ETDEWEB)

Liem, Phung Nhu [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires, Departement des radioelements, Service des molecules marquees

1967-04-01

Carbonation of {gamma}-methoxypropyl-magnesium chloride by CO{sub 2} gives {gamma}-methoxy-butyric carboxylic-{sup 14}C acid with a yield of about 95 per cent. When the latter is treated successively with anhydrous HBr and with diazomethane, methyl carboxylic {gamma}-bromobutyrate-{sup 14}C is formed. This in turn gives {gamma}-amino-butyric carboxylic-{sup 14}C acid with an overall yield of 66 per cent with respect to Ba{sup 14}CO{sub 3}, when it is condensed with potassium phthalimide and hydrolyzed by acid. By reacting methyl-{gamma}-bromobutyrate-{sup 14}C with the sodium derivative of ethyl cyanacetamido-acetate in ethanol, followed by an acid hydrolysis, {alpha}-aminoadipic-6-{sup 14}C acid is obtained with an overall yield of 46 per cent with respect to Ba{sup 14}CO{sub 3}. (author) [French] La carbonatation du chlorure de {gamma}-methoxypropylmagnesium par {sup 14}CO{sub 2} donne l'acide {gamma}-methoxybutyrique carboxyle {sup 14}C avec un rendement d'environ 95 pour cent. Ce dernier traite successivement par HBr anhydre et par le diazomethane conduit au {gamma}-bromobutyrate de methyle carboxyle {sup 14}C. Celui-ci condense avec le phtalimide de potassium suivi d'une hydrolyse acide fournit l'acide {gamma}-aminobutyrique carboxyle {sup 14}C avec un rendement global de 66 pour cent par rapport a Ba{sup 14}CO{sub 3}. L'action du {gamma}-bromobutyrate de methyle {sup 14}C sur le derive sode du cyanacetamidoacetate d'ethyle dans l'ethanol suivie d'hydrolyse acide donne l'acide {alpha}-aminoadipique {sup 14}C-6 avec un rendement global de 46 pour cent par rapport a Ba{sup 14}CO{sub 3}. (auteur)

Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen.

Science.gov (United States)

Schulmeister, Ulrike; Hochwallner, Heidrun; Swoboda, Ines; Focke-Tejkl, Margarete; Geller, Beate; Nystrand, Mats; Härlin, Annika; Thalhamer, Josef; Scheiblhofer, Sandra; Keller, Walter; Niggemann, Bodo; Quirce, Santiago; Ebner, Christoph; Mari, Adriano; Pauli, Gabrielle; Herz, Udo; Valenta, Rudolf; Spitzauer, Susanne

2009-06-01

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow's milk allergen, alphaS1-casein, from a bovine mammary gland cDNA library with allergic patients' IgE Abs. Recombinant alphaS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of alphaS1-casein were determined with recombinant fragments and synthetic peptides spanning the alphaS1-casein sequence using microarrayed components and sera from 66 cow's milk-sensitized patients. The allergenic activity of ralphaS1-casein and the alphaS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcepsilonRI, which had been loaded with the patients' serum IgE. Our results demonstrate that ralphaS1-casein as well as alphaS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact ralphaS1-casein induced strong basophil degranulation. These results suggest that primarily intact alphaS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant alphaS1-casein as well as alphaS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

OpenAIRE

Kuhn, H; Fietzek, P P; Lampen, J O

1982-01-01

The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.

Glycosylation: a hallmark of cancer?

Science.gov (United States)

Vajaria, Bhairavi N; Patel, Prabhudas S

2017-04-01

The hallmarks of cancer are characterized by functional capabilities that allow cancer cells to survive, proliferate and disseminate during the multistep tumorigenesis. Cancer being a cellular disease, changes in cellular glycoproteins play an important role in malignant transformation and cancer progression. The present review summarizes various studies that depicted correlation of glycosylation with tumor initiation, progression and metastasis, which are helpful in early diagnosis, disease monitoring and prognosis. The results are further strengthened by our reports, which depicted alterations in sialylation and fucosylation in different cancers. Alterations in glycosyltransferases are also involved in formation of various tumor antigens (e.g. Sialyl Lewis x) which serves as ligand for the cell adhesion molecule, selectin which is involved in adhesion of cancer cells to vascular endothelium and thus contributes to hematogenous metastasis. Increased glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC)s are also involved in loss of E-cadherin, a key molecule implicated in metastatic dissemination of cells. The present review also summarizes the correlation of glycosylation with all the hallmarks of cancer. The enormous progress in the design of novel inhibitors of pathway intermediates of sialylation and fucosylation can prove wonders in combating the dreadful disease. The results provide the evidence that altered glycosylation is linked to tumor initiation, progression and metastasis. Hence, it can be considered as a new hallmark of cancer development and strategies to develop novel glycosylation targeted molecules should be strengthened.

Urinary alpha 1-microglobulin as an indicator protein of renal tubular dysfunction caused by environmental cadmium exposure

Energy Technology Data Exchange (ETDEWEB)

Tohyama, C.; Kobayashi, E.; Saito, H.; Sugihara, N.; Nakano, A.; Mitane, Y.

1986-06-01

An epidemiologic investigation was carried out to clarify the significance of the urinary excretion of alpha 1-microglobulin (alpha 1-MG) in people aged 50 years and over living in a Cd-polluted area in Japan. Approximately 80% of the population participated in the health examination. The urinary and serum levels and the relative clearance of alpha 1-MG to creatinine clearance were compared with various parameters (age, urinary beta 2-microglobulin (beta 2-MG), total protein, Cd, Cu and Zn, serum beta 2-MG, creatinine and blood urea nitrogen and relative clearances of alpha 1-MG, beta 2-MG, inorganic phosphate and uric acid). It was found that the urinary excretion of alpha 1-MG is closely associated with the urinary Cd and Cu and with the indices of renal dysfunction listed above. These results suggest that the urinary alpha 1-MG level markedly reflects a degree of proximal tubular dysfunction and that it may be useful as one of the screening measures for proximal tubular dysfunction caused by environmental Cd exposure.

Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods. [106 references

Energy Technology Data Exchange (ETDEWEB)

Garrison, W.M.

1979-03-01

An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and pathogenic toxins. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

The interaction between the first transmembrane domain and the thumb of ASIC1a is critical for its N-glycosylation and trafficking.

Directory of Open Access Journals (Sweden)

Lan Jing

Full Text Available Acid-sensing ion channel-1a (ASIC1a, the primary proton receptor in the brain, contributes to multiple diseases including stroke, epilepsy and multiple sclerosis. Thus, a better understanding of its biogenesis will provide important insights into the regulation of ASIC1a in diseases. Interestingly, ASIC1a contains a large, yet well organized ectodomain, which suggests the hypothesis that correct formation of domain-domain interactions at the extracellular side is a key regulatory step for ASIC1a maturation and trafficking. We tested this hypothesis here by focusing on the interaction between the first transmembrane domain (TM1 and the thumb of ASIC1a, an interaction known to be critical in channel gating. We mutated Tyr71 and Trp287, two key residues involved in the TM1-thumb interaction in mouse ASIC1a, and found that both Y71G and W287G decreased synaptic targeting and surface expression of ASIC1a. These defects were likely due to altered folding; both mutants showed increased resistance to tryptic cleavage, suggesting a change in conformation. Moreover, both mutants lacked the maturation of N-linked glycans through mid to late Golgi. These data suggest that disrupting the interaction between TM1 and thumb alters ASIC1a folding, impedes its glycosylation and reduces its trafficking. Moreover, reducing the culture temperature, an approach commonly used to facilitate protein folding, increased ASIC1a glycosylation, surface expression, current density and slowed the rate of desensitization. These results suggest that correct folding of extracellular ectodomain plays a critical role in ASIC1a biogenesis and function.

Dietary alpha-Lipoic Acid Alters Piglet Neurodevelopment

Directory of Open Access Journals (Sweden)

Austin T Mudd

2016-05-01

Full Text Available Introduction: Alpha-lipoic acid (a-LA is an antioxidant shown to ameliorate age-associated impairments of brain and cardiovascular function. Human milk is known to have high antioxidant capacity, however the role of antioxidants in the developing brain is largely uncharacterized. This exploratory study aimed to examine the dose response effects of a-LA on piglet growth and neurodevelopment. Methods: Beginning at 2 d of age, 31 male pigs received one of three diets: control (CONT [0 mg a-LA/100g], low a-LA (LOW [120 mg a-LA/100g], or high a-LA (HIGH [240 mg a-LA/100g]. From 14 to 28 d of age, pigs were subjected to spatial T-maze assessment and macrostructural and microstructural neuroimaging procedures were performed at 31 d of age.Results: No differences due to diet were observed for bodyweight gain or intestinal weight and length. Spatial T-maze assessment did not reveal learning differences due to diet in proportion of correct choices or latency to choice measures. Diffusion tensor imaging revealed decreased (P = 0.01 fractional anisotropy (FA in the internal capsule of HIGH fed pigs compared with both the CONT (P < 0.01 and LOW (P = 0.03 fed pigs, which were not different from one another. Analysis of axial diffusivity (AD within the internal capsule revealed a main effect of diet (P < 0.01 in which HIGH fed piglets exhibited smaller (P < 0.01 rates of diffusion compared with CONT piglets, but HIGH fed piglets were not different (P = 0.12 than LOW fed piglets. Tract-based spatial statistics, a comparison of FA values along white matter tracts, revealed 1,650 voxels where CONT piglets exhibited higher (P < 0.05 values compared with HIGH fed piglets. Conclusion: The lack of differences in intestinal and bodyweight measures among piglets indicate a-LA supplementation does not impact overall growth, regardless of concentration. Additionally, no observed differences between CONT and LOW fed piglets in behavior and neuroimaging measures indicate a

Nonenzymatic glycosylation of bovine myelin basic protein

International Nuclear Information System (INIS)

Hitz, J.B.

1987-01-01

In the CNS myelin sheath the nonenzymatic glycosylation reaction (at the early stage of the Amadori product) occurs only with the myelin basic protein and not with the other myelin proteins. This was observed in isolated bovine myelin by in vitro incubation with [ 14 C]-galactose and [ 14 C]-glucose. The respective in-vitro incorporation rates for purified bovine myelin basic protein with D-galactose, D-glucose and D-mannose were 7.2, 2.4 and 2.4 mmoles/mole myelin basic protein per day at 37 0 C. A more rapid, HPLC method was devised and characterized to specifically analyze for the Amadori product. The HPLC method was correlated to the [ 14 C]-sugar incorporation method for myelin basic protein under a set of standard reaction conditions using [ 14 C]-glucose and [ 14 C]-mannose with HPLC values at 1/6 and 1/5 of the [ 14 C]-sugar incorporation method. A novel myelin basic protein purification step has been developed that yields a relativity proteolytic free preparation that is easy to work with, being totally soluble at a neutral pH. Nine new spots appear for a trypsinized glycosylated MBP in the paper peptide map of which eight correspond to positions of the [ 3 H]-labeled Amadori product in affinity isolated peptides. These studies provide a general characterization of and a structural basis for investigations on nonenzymatically glycosylated MBP as well as identifying MBP as the only nonenzymatically glycosylated protein in the CNS myelin sheath which may accumulate during aging, diabetes, and demyelinating diseases in general

Localization of an O-glycosylated site in the recombinant barley alpha-amylase 1 produced in yeast and correction of the amino acid sequence using matrix-assisted laser desorption/ionization mass spectrometry of peptide mixtures

DEFF Research Database (Denmark)

Andersen, Jens S.; Søgaard, M; Svensson, B

1994-01-01

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of peptide mixtures was used to characterize recombinant barley alpha-amylase 1, produced in yeast. Three peptide mixtures were generated by cleavage with CNBr, digestion with endoproteinase Lys-C and Asp-N, respectively...

Competition between folding and glycosylation in the endoplasmic reticulum

DEFF Research Database (Denmark)

Holst, B; Bruun, A W; Kielland-Brandt, Morten

1996-01-01

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...

Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

DEFF Research Database (Denmark)

Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

2012-01-01

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing......, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo...

Evaluation of [1-11C]-α-aminoisobutyric acid for tumor detection and amino acid transport measurement: Spontaneous canine tumor studies

International Nuclear Information System (INIS)

Bigler, R.E.; Zanzonico, P.B.; Schmall, B.; Conti, P.S.; Dahl, J.R.; Rothman, L.; Sgouros, G.

1985-01-01

Alpha-aminoisobutyric acid (AIB) or α-methyl alanine, is a nonmetabolized amino acid treansported into cells particularly malignant cells, predominantly by the ''A'' amino acid transport system. Since it is not metabolized, [1- 11 C]-AIB can be used to quantify A-type amino acid transport into cells using a relatively simple compartmental model and quantitative imaging procedures (e.g. positron tomography). The tissue distribution of [1- 11 C]-AIB was determined in six dogs bearing spontaneous tumors, including lymphosarcoma, osteogenic sarcoma, mammary carcinoma, and adenocarcinoma. Quantitative imaging with tissue radioassay confirmation at necropsy showed poor to excellent tumor localization. However, in all cases the concentrations achieved appear adequate for amino acid transport measurement at known tumor locations. The observed low normal brain (due to blood-brain barrier exclusion) and high (relative to brain) tumor concentrations of [1- 11 C]-AIB suggest that this agent may prove effective for the early detection of human brain tumors. (orig.)

Tumor necrosis factor alpha is associated with insulin-mediated suppression of free fatty acids and net lipid oxidation in HIV-infected patients with lipodystrophy

DEFF Research Database (Denmark)

Haugaard, SB; Andersen, O; Pedersen, Steen Bønløkke

2006-01-01

Tumor necrosis factor alpha (TNF-alpha) stimulates lipolysis in man. We examined whether plasma TNF-alpha is associated with the degree by which insulin suppresses markers of lipolysis, for example, plasma free fatty acid (FFA) and net lipid oxidation (LIPOX) rate in HIV-infected patients...... with lipodystrophy (LIPO) and those without (controls). LIPOX was estimated by indirect calorimetry during fasting and steady state of a hyperinsulinemic euglycemic clamp in 36 (18 LIPO and 18 controls) normoglycemic HIV-infected men on highly active antiretroviral therapy. In LIPO, TNF-alpha correlated with clamp...... were significant in controls. In all patients, TNF-alpha correlated with clamp FFA (r = 0.61, P

N-glycosylation by N-acetylglucosaminyltransferase V enhances the interaction of CD147/basigin with integrin β1 and promotes HCC metastasis.

Science.gov (United States)

Cui, Jian; Huang, Wan; Wu, Bo; Jin, Jin; Jing, Lin; Shi, Wen-Pu; Liu, Zhen-Yu; Yuan, Lin; Luo, Dan; Li, Ling; Chen, Zhi-Nan; Jiang, Jian-Li

2018-05-01

While the importance of protein N-glycosylation in cancer cell migration is well appreciated, the precise mechanisms by which N-acetylglucosaminyltransferase V (GnT-V) regulates cancer processes remain largely unknown. In the current study, we report that GnT-V-mediated N-glycosylation of CD147/basigin, a tumor-associated glycoprotein that carries β1,6-N-acetylglucosamine (β1,6-GlcNAc) glycans, is upregulated during TGF-β1-induced epithelial-to-mesenchymal transition (EMT), which correlates with tumor metastasis in patients with hepatocellular carcinoma (HCC). Interruption of β1,6-GlcNAc glycan modification of CD147/basigin decreased matrix metalloproteinase (MMP) expression in HCC cell lines and affected the interaction of CD147/basigin with integrin β1. These results reveal that β1,6-branched glycans modulate the biological function of CD147/basigin in HCC metastasis. Moreover, we showed that the PI3K/Akt pathway regulates GnT-V expression and that inhibition of GnT-V-mediated N-glycosylation suppressed PI3K signaling. In summary, β1,6-branched N-glycosylation affects the biological function of CD147/basigin and these findings provide a novel approach for the development of therapeutic strategies targeting metastasis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.