N-Glycosylation analysis of yeast Carboxypeptidase Y reveals the ultimate removal of phosphate from glycans at Asn368.

PubMed

B S, Gnanesh Kumar; Surolia, Avadhesha

2017-05-01

Carboxypeptidase Y from Saccharomyces cerivisiae was characterized for its site specific N-glycosylation through mass spectrometry. The N-glycopeptides were derived using non specific proteases and are analysed directly on liquid chromatography coupled to ion trap mass spectrometer in tandem mode. The evaluation of glycan fragment ions and the Y 1 ions (peptide+HexNAc) +n revealed the glycan sequence and the corresponding site of attachment. We observed the microheterogeneity in N-glycans such as Man 11-15 GlcNAc 2 at Asn 13 , Man 8-12 GlcNAc 2 at Asn 87 , Man 9-14 GlcNAc 2 at Asn 168 and phosphorylated Man 12-17 GlcNAc 2 as well as Man 11-16 GlcNAc 2 at Asn 368 . The presence of N-glycans with Man <18 GlcNAc 2 indicated that in vacuoles the steady release of mannose/phospho mannose residues from glycans occurs initially at Asn 13 or Asn 168 followed by at Asn 368 . However, glycans at Asn 87 which comprises Man 8-12 residues as reported earlier remain intact suggesting its inaccessibility for a similar processing. This in turn indicates the interaction of the glycan at Asn 87 with the polypeptide chain implicating it in the folding of the protein. Copyright © 2017 Elsevier B.V. All rights reserved.

Oxidative Addition of Disulfides, Alkyl Sulfides, and Diphosphides to an Aluminum(I) Center.

PubMed

Chu, Terry; Boyko, Yaroslav; Korobkov, Ilia; Kuzmina, Lyudmila G; Howard, Judith A K; Nikonov, Georgii I

2016-09-06

The aluminum(I) compound NacNacAl (1) reacts with diphenyl disulfide and diethyl sulfide to form the respective four-coordinate bis(phenyl sulfide) complex NacNacAl(SPh)2 (2) and alkyl thiolate aluminum complex NacNacAlEt(SEt) (3). As well, reaction of 1 with tetraphenyl diphosphine furnishes the bis(diphenyl phosphido) complex NacNacAl(PPh2)2 (4). Production of 3 and 4 are the first examples of C(sp(3))-S and R2P-PR2 activation by a main-group element complex. All three complexes were characterized by multinuclear NMR spectroscopy and X-ray crystal structure analysis. Furthermore, a variable-temperature NMR spectroscopic study was undertaken on 4 to study its dynamic behavior in solution.

Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER.

PubMed

Sobering, Andrew K; Watanabe, Reika; Romeo, Martin J; Yan, Benjamin C; Specht, Charles A; Orlean, Peter; Riezman, Howard; Levin, David E

2004-05-28

The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner. We showed previously that loss of Eri1 function results in hyperactive Ras phenotypes. Here, we demonstrate that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins. We also show that GTP bound Ras2 associates with the GPI-GnT complex in vivo and inhibits its activity, indicating that yeast Ras uses the ER as a signaling platform from which to negatively regulate the GPI-GnT. We propose that diminished GPI-anchor protein production contributes to hyperactive Ras phenotypes.

Modeling the conformation of polyphenols and their complexation with polypeptides: self-association of catechin and its complexation with L-proline glycine oligomers.

Treesearch

Fred L. Tobiason; Richard W. Hemingway; Gerard Vergoten

1999-01-01

Over the past 10 years, several scientific thrusts have come together in the study of flavanoids that make it possible to move forward into the study of complexation between polyphenols and polypeptides. Enhanced understanding of the conformational properties of flavanoid monomers and polyflavanoids through...

A novel enhancer of the Apaf1 apoptosome involved in cytochrome c-dependent caspase activation and apoptosis.

PubMed

Chu, Z L; Pio, F; Xie, Z; Welsh, K; Krajewska, M; Krajewski, S; Godzik, A; Reed, J C

2001-03-23

Apaf1/CED4 family members play central roles in apoptosis regulation as activators of caspase family cell death proteases. These proteins contain a nucleotide-binding (NB) self-oligomerization domain and a caspase recruitment domain (CARD). A novel human protein was identified, NAC, that contains an NB domain and CARD. The CARD of NAC interacts selectively with the CARD domain of Apaf1, a caspase-activating protein that couples mitochondria-released cytochrome c (cyt-c) to activation of cytosolic caspases. Cyt-c-mediated activation of caspases in cytosolic extracts and in cells is enhanced by overexpressing NAC and inhibited by reducing NAC using antisense/DNAzymes. Furthermore, association of NAC with Apaf1 is cyt c-inducible, resulting in a mega-complex (>1 MDa) containing both NAC and Apaf1 and correlating with enhanced recruitment and proteolytic processing of pro-caspase-9. NAC also collaborates with Apaf1 in inducing caspase activation and apoptosis in intact cells, whereas fragments of NAC representing only the CARD or NB domain suppress Apaf1-dependent apoptosis induction. NAC expression in vivo is associated with terminal differentiation of short lived cells in epithelia and some other tissues. The ability of NAC to enhance Apaf1-apoptosome function reveals a novel paradigm for apoptosis regulation.

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

PubMed

Horynová, Milada; Takahashi, Kazuo; Hall, Stacy; Renfrow, Matthew B; Novak, Jan; Raška, Milan

2012-02-01

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system. Copyright © 2011 Elsevier Inc. All rights reserved.

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

PubMed Central

Avezov, Edward; Ron, Efrat; Izenshtein, Yana; Adan, Yosef; Lederkremer, Gerardo Z.

2010-01-01

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation. PMID:20424595

Pulse-chase analysis of N-linked sugar chains from glycoproteins in mammalian cells.

PubMed

Avezov, Edward; Ron, Efrat; Izenshtein, Yana; Adan, Yosef; Lederkremer, Gerardo Z

2010-04-27

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-(3)H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

Making Home Sweet and Sturdy: Toxoplasma gondii ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity

PubMed Central

Tomita, Tadakimi; Sugi, Tatsuki; Yakubu, Rama; Tu, Vincent; Ma, Yanfen

2017-01-01

ABSTRACT The protozoan intracellular parasite Toxoplasma gondii forms latent cysts in the central nervous system (CNS) and persists for the lifetime of the host. This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O-GalNAc glycosylated. A family of enzymes termed polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism. PMID:28074022

Surface active complexes formed between keratin polypeptides and ionic surfactants.

PubMed

Pan, Fang; Lu, Zhiming; Tucker, Ian; Hosking, Sarah; Petkov, Jordan; Lu, Jian R

2016-12-15

Keratins are a group of important proteins in skin and hair and as biomaterials they can provide desirable properties such as strength, biocompatibility, and moisture regaining and retaining. The aim of this work is to develop water-soluble keratin polypeptides from sheep wool and then explore how their surface adsorption behaves with and without surfactants. Successful preparation of keratin samples was demonstrated by identification of the key components from gel electrophoresis and the reproducible production of gram scale samples with and without SDS (sodium dodecylsulphate) during wool fibre dissolution. SDS micelles could reduce the formation of disulphide bonds between keratins during extraction, reducing inter-molecular crosslinking and improving keratin polypeptide solubility. However, Zeta potential measurements of the two polypeptide batches demonstrated almost identical pH dependent surface charge distributions with isoelectric points around pH 3.5, showing complete removal of SDS during purification by dialysis. In spite of different solubility from the two batches of keratin samples prepared, very similar adsorption and aggregation behavior was revealed from surface tension measurements and dynamic light scattering. Mixing of keratin polypeptides with SDS and C 12 TAB (dodecyltrimethylammonium bromide) led to the formation of keratin-surfactant complexes that were substantially more effective at reducing surface tension than the polypeptides alone, showing great promise in the delivery of keratin polypeptides via the surface active complexes. Neutron reflection measurements revealed the coexistence of surfactant and keratin polypeptides at the interface, thus providing the structural support to the observed surface tension changes associated with the formation of the surface active complexes. Copyright © 2016. Published by Elsevier Inc.

Identification of a major polypeptide of the nuclear pore complex

PubMed Central

1982-01-01

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes. PMID:7153248

Studies on the synthesis and processing of the asparagine-linked carbohydrate units of glycoproteins.

PubMed

Spiro, R G; Spiro, M J

1982-12-24

It has become apparent in recent years from the work of a number of laboratories that the N-glycosylation of both membrane and secretory glycoproteins is effected by the transfer en bloc to nascent polypeptides of a glucose-containing oligosaccharide (Glc3Man9GlcNAc2) from a dolichyl pyrophosphoryl carrier; this is followed by a series of modifying reactions to yield the mature polymannose and complex asparagine-linked carbohydrate units. The enzymic steps involved in the assembly of the precursor oligosaccharide, its transfer to protein and its subsequent processing represent potential sites for the regulation of glycoprotein synthesis. Studies performed in our laboratory have dealt primarily with thyroid slices and particulate enzymes with special regard to the role of glucose in these events. Thyroglobulin, the major secretory glycoprotein of this tissue, has well defined complex and polymannose saccharide units, and indeed the most complete form of the latter (Man9GlcNAc2) has the same structure as the lipid-linked oligosaccharide without the glucose. Our studies indicate that effective N-glycosylation requires a complete glucose chain (Glc3) and that the glucose sequence is assembled from dolichol-P-glucose in a stepwise manner through the concerted action of at least two transferases in a fashion complementary to the subsequent excision of this sugar by glucosidases. Pulse-chase studies indicate that, after the transfer to protein, the removal of all three glucose residues as well as of the first mannose takes place in the endoplasmic reticulum and three additional mannoses are excised in the Golgi complex, because in the presence of an inhibitor of intracellular transport, carbonyl cyanide m-chlorophenylhydrazone (CCCP), there is a pronounced accumulation of protein-linked Man8GlcNAc2. Studies with metabolic inhibitors (CCCP, antimycin, N2) indicate that, under conditions of energy depletion, glucosylation of oligosaccharide-lipid is selectively impaired, resulting in an accumulation, as measured chemically or metabolically, of high-mannose-containing (Man9GlcNAc2 and Man8GlcNAc2) lipid-linked saccharides. Further evidence that the glucosylation reaction is very sensitive to the metabolic state is suggested by the observation that tissues not rapidly frozen after removal from the animal show a similar depletion of the glucose-containing oligosaccharide lipids. Another important aspect for the regulation of N-glycosylation of proteins is the availability of dolichyl phosphate for the formation of the lipid-linked mono- and oligosaccharides. Our studies with puromycin suggest that there is a limited supply of the lipid carrier, because in the presence of this inhibitor there is no accumulation of any of the oligosaccharide-lipid species.

Primary light-harvesting system: phycobilisomes and associated membranes. Progress report, January 1, 1981-December 31, 1981

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gantt, E.

1981-01-01

Phycobilisomes, serving as primary light harvesting complexes in cyanobacteria and red algae, were investigated. Structurally the phycobilisomes of both groups have the same fundamental phycobiliprotein arrangement. Allophycocyanin is in the center near the thylakoid. Stacked rods composed of phycocyanin, or phycocyanin-phycoerythrin radiate peripherally from the allophycocyanin core. Phycobilisomes of Nostoc sp. and Fremyella diplosiphon, after separation into separate allophycocyanin and phycoerythrin-phycocyanin fractions have been associated in vitro. Hybrid phycobilisomes, derived from mixtures of phycobiliprotein from these species were also obtained. The interaction is specific since reassociation was not obtained with phycobiliprotein complexes of some other algae. Phycobilisomes, whether native, ormore » associated in vitro, were similar in their sedimentation, absorption, fluorescence excitation, fluorescence emission, and by electron microscopy. Furthermore, many of the colorless polypeptides were also highly similar between Nostoc and Fremyella. The similarity formed may reflect an evolutionary relationship between the two species. The polypeptide composition of Porphyridium cruentum phycobilisomes is the most complex of any thus far examined. The phycobiliprotein containing polypeptides comprised 84% of the total stainable protein, while the remaining were colorless. Most of the colorless polypeptides occurred in a pelletable fraction, which was enriched in allophycocyanin and phycocyanin, it is probable that some are involved in the linking of these phycobiliproteins.« less

Making Home Sweet and Sturdy: Toxoplasma gondii ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity.

PubMed

Tomita, Tadakimi; Sugi, Tatsuki; Yakubu, Rama; Tu, Vincent; Ma, Yanfen; Weiss, Louis M

2017-01-10

The protozoan intracellular parasite Toxoplasma gondii forms latent cysts in the central nervous system (CNS) and persists for the lifetime of the host. This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O-GalNAc glycosylated. A family of enzymes termed polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism. Toxoplasma gondii is an obligate intracellular parasite that infects a third of the world's population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the brain cyst. Our studies provide new insights into the mechanisms of O-GalNAc glycosylation in T. gondii. Copyright © 2017 Tomita et al.

Evaluations of alcohol consequences moderate social anxiety risk for problematic drinking.

PubMed

Nitka, Danit; O'Connor, Roisin M

2017-02-01

The link between social anxiety (SA) and problematic drinking is complex; this seems predominantly true among young adults. Individuals high on SA are thought to be particularly sensitive to the negative effects of alcohol, which should deter them from drinking. Yet, some evidence suggests that those high on SA continue to drink despite experiencing negative alcohol-related consequences (NACs) (Morris, Stewart, & Ham, 2005). Although traditionally, researchers assume NACs are perceived as averse, emerging evidence suggests these are not categorically viewed as negative by undergraduates. The study goal was to test whether evaluations of NACs moderate the effect of SA on problematic drinking. It was hypothesized that high SA would predict elevated alcohol use and number of NACs experienced, but only for those who evaluate NACs as less negative. Undergraduate drinkers (N=130, 80 women) completed self-reports of social anxiety, NAC evaluations (ratings of how 'bad' experiencing each alcohol-related consequence would be), alcohol use, and NACs experienced. Regression analyses revealed that NAC evaluations moderated the effect of SA on number of NACs experienced, but not the effect of SA on weekly alcohol use. Simple slopes analyses showed that high SA was associated with elevated NACs experienced for those with weak negative NAC evaluations, controlling for alcohol use. These findings help explain the mixed SA-problematic drinking literature by identifying perceptions of NACs as an important moderator of SA risk for experiencing NACs. Moreover, clinical interventions aimed at reducing SA risk for undergraduate problematic drinking may benefit from targeting NAC evaluations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chirality-selected phase behaviour in ionic polypeptide complexes

DOE PAGES

Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; ...

2015-01-14

In this study, polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with amore » β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.« less

Sindbis virus glycoproteins are abnormally glycosylated in Chinese hamster ovary cells deprived of glucose.

PubMed

Davidson, S K; Hunt, L A

1985-07-01

We have previously demonstrated that Sindbis virus infection of Chinese hamster ovary (CHO) cells altered the protein glycosylation machinery of the cell, so that both normal, full-size (nine mannose-containing) oligosaccharides and abnormal, "truncated' (five mannose-containing) oligosaccharides are transferred from lipid-linked precursors to newly synthesized viral membrane glycoproteins. In the present studies, we have examined the precursor oligosaccharides on viral glycoproteins that were pulse-labelled with [3H]mannose in the presence or absence of glucose, since glucose starvation of uninfected CHO cells has been reported to induce synthesis of truncated precursor oligosaccharides. Pulse-labelling in the absence of glucose led to a greater than 10-fold increase in the relative amount of the truncated precursor oligosaccharides being transferred to the newly synthesized viral glycoproteins and to an apparent underglycosylation of some precursor viral polypeptides, with some asparaginyl sites not acquiring covalently linked oligosaccharides. The mature virion glycoproteins from CHO cells which were pulse-labelled in the absence of glucose and then 'chased' in the presence of glucose contained proportionately more unusual Man3GlcNAc2-size oligosaccharides. These small neutral-type oligosaccharides were apparently not as good a substrate for further processing into complex acidic-type oligosaccharides as the normal Man5GlcNAc2 intermediate that results from the full-size precursor oligosaccharides.

Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA).

PubMed

Kumari, Amrita; Koyama, Tetsuo; Hatano, Ken; Matsuoka, Koji

2016-10-01

A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C6 linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λex 295nm and it was found to be very high Ka∼1.39×10(7) M(-1) in case of ABG complex as compared to GlcNAc only Ka∼1.01×10(4) M(-1) with the phenomenon proven to be due to glycocluster effect. Copyright © 2016 Elsevier Inc. All rights reserved.

Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

PubMed

Chandrasekaran, E V; Xue, Jun; Xia, Jie; Khaja, Siraj D; Piskorz, Conrad F; Locke, Robert D; Neelamegham, Sriram; Matta, Khushi L

2016-10-01

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucβ1-3 GalNAc and Fucα-1-2 D-Fucβ-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc β-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Formation of hybrid phycobilisomes by association of phycobiliproteins from Nostoc and Fremyella

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canaani, O.; Gantt, E.

1982-09-01

Formation of phycobilisomes has been accomplished in vitro from isolated phycobiliprotein fraction obtained from the same blue-green alga (intrageneric) and from different blue-green algae (intergeneric). Phycobilisomes, which are supramolecular complexes of phycobiliproteins, serve as major light-harvesting antennae for photosynthesis in blue-green and red algae. Intrageneric association into energetically functional phycobilisomes, previously reported to occur with Nostoc sp. allophycocyanin and phycoerythrin-phycocyanin complexes has been obtained with Fremyella diplosiphon. By their spectral propeties (absorption, fluorescence excitation, and emission) and electron microscopic images, the native and in vitro-associated phycobilisomes were virtually indistinguishable. Intergeneic phycobilisomes have been produced from allophycocyanin of Nostoc sp. strainmore » Mac, and phycoerythrin-phycocyanin of F. diplosiphon, as well as from the reverse mixtures. Phycobilisomes of Nostoc and Fremyella, analyzed by NaDodSO/sub 4//polyacrylamide gel electrophoresis, possessed a number of polypeptides having similar molecular weights: the usual ..cap alpha..- and ..beta..-phycobilin-containing polypeptides of M/sub r/ 15,000-22,000, a faint band at M/sub r/ ca. 95,000, and a prominent band at M/sub r/ ca. 31,000. The M/sub r/ 31,000 polypeptide is assumed to provide the recognition site for attachment of the phycoerythrin-phycocyanin complexes with the allophycocyanin core. In vitro association was not obtained between allophycocyanin from Nostoc and phycoerythrin-phycocyanin complexes from Phormidium persicinum or Porphyridium sordidum.« less

Membrane fractions active in poliovirus RNA replication contain VPg precursor polypeptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takegami, T.; Semler, B.L.; Anderson, C.W.

1983-01-01

The poliovirus specific polypeptide P3-9 is of special interest for studies of viral RNA replication because it contains a hydrophobic region and, separated by only seven amino acids from that region, the amino acid sequence of the genome-linked protein VPg. Membraneous complexes of poliovirus-infected HeLa cells that contain poliovirus RNA replicating proteins have been analyzed for the presence of P3-9 by immunoprecipitation. Incubation of a membrane fraction rich in P3-9 with proteinase leaves the C-terminal 69 amino acids of P3-9 intact, an observation suggesting that this portion is protected by its association with the cellular membrane. These studies have alsomore » revealed two hitherto undescribed viral polypeptides consisting of amino acid sequences of the P2 andf P3 regions of the polyprotein. Sequence analysis by stepwise Edman degradation show that these proteins are 3b/9 (M/sub r/77,000) and X/9 (M/sub r/50,000). 3b/9 and X/9 are membrane bound and are turned over rapidly and may be direct precursors to proteins P2-X and P3-9 of the RNA replication complex. P2-X, a polypeptide void of hydrophobic amino acid sequences but also found associated with membranes, is rapidly degraded when the membraneous complex is treated with trypsin. It is speculated that P2-X is associated with membranes by its affinity to the N-terminus of P3-9.« less

Immediate effects of a single exercise bout on protein O-GlcNAcylation and chromatin regulation of cardiac hypertrophy

PubMed Central

Medford, Heidi M.; Porter, Karen

2013-01-01

Cardiac hypertrophy induced by pathological stimuli is regulated by a complex formed by the repressor element 1-silencing transcription factor (REST) and its corepressor mSin3A. We previously reported that hypertrophic signaling is blunted by O-linked attachment of β-N-acetylglucosamine (O-GlcNAc) to proteins. Regular exercise induces a physiological hypertrophic phenotype in the heart that is associated with decreased O-GlcNAc levels, but a link between O-GlcNAc, the REST complex, and initiation of exercise-induced cardiac hypertrophy is not known. Therefore, mice underwent a single 15- or 30-min bout of moderate- to high-intensity treadmill running, and hearts were harvested immediately and compared with sedentary controls. Cytosolic O-GlcNAc was lower (P < 0.05) following 15 min exercise with no differences in nuclear levels (P > 0.05). There were no differences in cytosolic or nuclear O-GlcNAc levels in hearts after 30 min exercise (P > 0.05). Cellular compartment levels of O-GlcNAc transferase (OGT, the enzyme that removes the O-GlcNAc moiety from proteins), REST, mSin3A, and histone deacetylases (HDACs) 1, 2, 3, 4, and 5 were not changed with exercise. Immunoprecipitation revealed O-GlcNAcylation of OGT and HDACs 1, 2, 4, and 5 that was not changed with acute exercise; however, exercised hearts did exhibit lower interactions between OGT and REST (P < 0.05) but not between OGT and mSin3A. These data suggest that hypertrophic signaling in the heart may be initiated by as little as 15 min of exercise via intracellular changes in protein O-GlcNAcylation distribution and reduced interactions between OGT and the REST chromatin repressor. PMID:23624624

Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli.

PubMed

Lauber, Jennifer; Handrick, René; Leptihn, Sebastian; Dürre, Peter; Gaisser, Sabine

2015-01-13

Recombinant protein-based therapeutics have become indispensable for the treatment of many diseases. They are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. The majority of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. The development of an engineered Escherichia coli-based expression system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs. This work describes the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. For expression, a codon-optimised gene encoding amino acids 52-571 of GalNAcT2 lacking the transmembrane N-terminal domain was inserted into a pET-23 derived vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase was produced in E. coli using a recently published expression system. Soluble HisDapGalNAcT2 produced in SHuffle® T7 host cells was purified using nickel affinity chromatography and was subsequently analysed by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and circular dichroism spectroscopy to determine molecular mass, folding state and thermal transitions of the protein. The activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay based on the release of phosphate during transfer of glycosyl residues to a model acceptor peptide or, alternatively, to the granulocyte-colony stimulating growth factor (G-CSF). Modifications were assessed by Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry analysis (MALDI-TOF-MS) and Electrospray Mass Spectrometry analysis (ESI-MS). The results clearly indicate the glycosylation of the acceptor peptide and of G-CSF. In the present work, we isolated a human-derived glycosyltransferase by expressing soluble HisDapGalNAcT2 in E. coli. The functional activity of the enzyme was shown in vitro. Further investigations are needed to assess the potential of in vivo glycosylation in E. coli.

The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A carbohydrate in Streptococcus pyogenes.

PubMed

Rush, Jeffrey S; Edgar, Rebecca J; Deng, Pan; Chen, Jing; Zhu, Haining; van Sorge, Nina M; Morris, Andrew J; Korotkov, Konstantin V; Korotkova, Natalia

2017-11-24

In many Lactobacillales species ( i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes , synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N -acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry, and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltransferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Nuclear Involvement in the Appearance of a Chloroplast-Encoded 32,000 Dalton Thylakoid Membrane Polypeptide Integral to the Photosystem II Complex 1

PubMed Central

Leto, Kenneth J.; Keresztes, Aron; Arntzen, Charles J.

1982-01-01

The genetic locus for the high chlorophyll fluorescent photosystem II-deficient maize mutant hcf*-3 has been definitively located to the nuclear genome. Fluorography of lamellar polypeptides labeled with [35S]methionine in vivo revealed the specific loss of a heavily labeled 32,000 dalton thylakoid membrane polypeptide as well as its chloroplast encoded precursor species at 34,000 daltons. Examination of freeze-fractured mesophyll and bundle sheath thylakoids from hcf*-3 revealed that both plastid types lacked the large EFs particles believed to consist of the photosystem II reaction center-core complex and associated light harvesting chlorophyll-proteins. The present evidence suggests that the synthesis or turnover/integration of the chloroplast-encoded 34,000 to 32,000 dalton polypeptide is under nuclear control, and that these polyipeptides are integral components of photosystem II which may be required for the assembly or structural stabilization of newly formed photosystem II reaction centers in both mesophyll and bundle sheath chloroplasts. Images PMID:16662421

A recombinant fungal lectin for labeling truncated glycans on human cancer cells.

PubMed

Audfray, Aymeric; Beldjoudi, Mona; Breiman, Adrien; Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

2015-01-01

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.

A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

PubMed Central

Hurbin, Amandine; Boos, Irene; Unverzagt, Carlo; Bouras, Mourad; Lantuejoul, Sylvie; Coll, Jean-Luc; Varrot, Annabelle; Le Pendu, Jacques; Busser, Benoit; Imberty, Anne

2015-01-01

Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc) residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL) has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac). Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases. PMID:26042789

Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

PubMed

Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

2016-03-01

Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome reaction, whereas the IZUMO1 and lactadherin polypeptides remain associated to the particulate fraction. Almost entire population of bovine sperm IZUMO1 relocates to the equatorial segment during the LPC-induced acrosome reaction. We propose that the interaction of OMC32 matrix polypeptide with detergent-soluble acrosomal proteins regulates the release of hydrolases/other acrosomal protein(s) during the acrosome reaction.

Bivalent Carbohydrate Binding Is Required for Biological Activity of Clitocybe nebularis Lectin (CNL), the N,N′-Diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc)-specific Lectin from Basidiomycete C. nebularis*

PubMed Central

Pohleven, Jure; Renko, Miha; Magister, Špela; Smith, David F.; Künzler, Markus; Štrukelj, Borut; Turk, Dušan; Kos, Janko; Sabotič, Jerica

2012-01-01

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N′-diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency. PMID:22298779

Patient Satisfaction and Nipple-Areola Sensitivity After Bilateral Prophylactic Mastectomy and Immediate Implant Breast Reconstruction in a High Breast Cancer Risk Population: Nipple-Sparing Mastectomy Versus Skin-Sparing Mastectomy.

PubMed

van Verschuer, Victorien M T; Mureau, Marc A M; Gopie, Jessica P; Vos, Elvira L; Verhoef, Cornelis; Menke-Pluijmers, Marian B E; Koppert, Linetta B

2016-08-01

Prophylactic skin-sparing mastectomy (SSM) and nipple-sparing mastectomy (NSM) both are associated with major risk reduction in women with high breast cancer risk. Skin-sparing mastectomy followed by nipple-areola complex (NAC) reconstruction is standard of care, but NSM is increasingly being performed. Preservation of the NAC in NSM may increase patient satisfaction. Therefore, we measured NAC sensitivity after NSM and compared patient satisfaction as well as body image after SSM with NSM. Women who underwent prophylactic bilateral SSM or NSM and immediate implant breast reconstruction between 2002 and 2012 were eligible. Patient satisfaction was assessed using the Breast-Q reconstruction questionnaire, body image using Hopwood's body image scale (BIS), and satisfaction with the (reconstructed) NAC using a study-specific questionnaire. In the NSM group, NAC sensitivity was assessed using Semmes Weinstein monofilaments with a 5-point scale and compared with NAC sensitivity in a nonoperated control group. The SSM group comprised 25 women (50 SSMs) and the NSM group 20 women (39 NSMs). Median follow-up was 65 months in the SSM group compared with 27 months in the NSM group (P < 0.01). In univariable analyses, Breast-Q scores were favorable in the SSM group compared with the NSM group with trends for higher "satisfaction with breasts" (66.2 vs 56.6; P = 0.06) and "satisfaction with outcome" (76.1 vs 61.5; P = 0.09). Mean BIS score of 7.1/30 in the SSM group and 9.3/30 in the NSM group (P = 0.35). Adjusted for follow-up, there were no significant differences in Breast-Q scores, nor in BIS scores. Interestingly, satisfaction with the (reconstructed) NAC was similar after SSM and NSM. Nipple-areola complex sensitivity was lower in the NSM group (mean score, 1.9; 95% confidence interval, 1.5-2.3) compared with the control group (mean score, 4.7; 95% confidence interval, 4.6-4.9; P < 0.01). Breast-Q scores regarding satisfaction with breasts and overall outcome were in favor of the SSM group. Residual NAC sensitivity after NSM was low. This suggests that SSM followed by NAC reconstruction is a balanced alternative to NSM. We observed no significant differences in body image and NAC-specific satisfaction between the NSM and SSM groups.

Stepwise Catalytic Mechanism via Short-Lived Intermediate Inferred from Combined QM/MM MERP and PES Calculations on Retaining Glycosyltransferase ppGalNAcT2

PubMed Central

Trnka, Tomáš; Kozmon, Stanislav; Tvaroška, Igor; Koča, Jaroslav

2015-01-01

The glycosylation of cell surface proteins plays a crucial role in a multitude of biological processes, such as cell adhesion and recognition. To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals. PMID:25849117

Stepwise catalytic mechanism via short-lived intermediate inferred from combined QM/MM MERP and PES calculations on retaining glycosyltransferase ppGalNAcT2.

PubMed

Trnka, Tomáš; Kozmon, Stanislav; Tvaroška, Igor; Koča, Jaroslav

2015-04-01

The glycosylation of cell surface proteins plays a crucial role in a multitude of biological processes, such as cell adhesion and recognition. To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals.

The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities.

PubMed

Zhang, Y; LeRoy, G; Seelig, H P; Lane, W S; Reinberg, D

1998-10-16

Histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. ATP-dependent nucleosome remodeling, mediated by different multisubunit complexes, was thought to be involved only in transcription activation. Here we report the isolation of a protein complex that contains both histone deacetylation and ATP-dependent nucleosome remodeling activities. The complex contains the histone deacetylases HDAC1/2, histone-binding proteins, the dermatomyositis-specific autoantigen Mi2beta, a polypeptide related to the metastasis-associated protein 1, and a novel polypeptide of 32 kDa. Patients with dermatomyositis have a high rate of malignancy. The finding that Mi2beta exists in a complex containing histone deacetylase and nucleosome remodeling activities suggests a role for chromatin reorganization in cancer metastasis.

O-GlcNAc Transferase/Host Cell Factor C1 Complex Regulates Gluconeogenesis by Modulating PGC-1α Stability

PubMed Central

Ruan, Hai-Bin; Han, Xuemei; Li, Min-Dian; Singh, Jay Prakash; Qian, Kevin; Azarhoush, Sascha; Zhao, Lin; Bennett, Anton M.; Samuel, Varman T.; Wu, Jing; Yates, John R.; Yang, Xiaoyong

2012-01-01

SUMMARY A major cause of hyperglycemia in diabetic patients is inappropriate hepatic gluconeogenesis. PGC-1α is a master regulator of gluconeogenesis, and its activity is controlled by various post-translational modifications. A small portion of glucose metabolizes through the hexosamine biosynthetic pathway, which leads to O-linked β-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins. Using a proteomic approach, we identified a broad variety of proteins associated with O-GlcNAc transferase (OGT), among which host cell factor C1 (HCF-1) is highly abundant. HCF-1 recruits OGT to O-GlcNAcylate PGC-1α and O-GlcNAcylation facilitates the binding of the deubiquitinase BAP1, thus protecting PGC-1α from degradation and promoting gluconeogenesis. Glucose availability modulates gluconeogenesis through the regulation of PGC-1α O-GlcNAcylation and stability by the OGT/HCF1 complex. Hepatic knockdown of OGT and HCF-1 improves glucose homeostasis in diabetic mice. These findings define the OGT/HCF-1 complex as a glucose sensor and key regulator of gluconeogenesis, shedding light on new strategies for treating diabetes. PMID:22883232

Managing Necrosis of the Nipple Areolar Complex Following Reduction Mammaplasty and Mastopexy.

PubMed

Handel, Neal; Yegiyants, Sara

2016-04-01

The objectives of this article are to explain the mechanisms of injury that result in ischemia of the nipple areolar complex (NAC) after reduction mammaplasty or mastopexy, to offer recommendations about the management of this complication, and to illustrate reconstructive techniques that can be used to correct deformities arising from necrosis of the NAC. With these goals in mind, the article is divided into 3 sections: prevention of ischemia of the NAC, management of the ischemic nipple, and reconstruction after ischemic necrosis of the nipple and areola. Necrosis of all or part of the NAC is a devastating complication after breast surgery. However, with properly timed and well-executed reconstructive procedures, it is possible in most cases to restore a natural-appearing NAC. Copyright © 2016 Elsevier Inc. All rights reserved.

Peptide Regulation of Cells Renewal Processes in Kidney Tissue Cultures from Young and Old Animals.

PubMed

Chalisova, N I; Lin'kova, N S; Nichik, T E; Ryzhak, A P; Dudkov, A V; Ryzhak, G A

2015-05-01

Polypeptide complex isolated from calf kidneys stimulates the processes of cell renewal in organotypic kidney tissue cultures from young and old rats. The polypeptide complex enhances expression of proliferation marker Ki-67 and reduces expression of proapoptotic peptide p53 in kidney explants obtained from young and old animals. Short peptides T-31 (AED) and T-35 (EDL) also stimulate proliferation and reduce apoptosis of the kidney cells, but to a lesser degree than the polypeptide complex. The results provide the basis for further investigation of the polypeptide complex as a preparation for the therapy of kidney diseases, including age-related pathologies.

The abundance and organization of polypeptides associated with antigens of the Rh blood group system.

PubMed

Gardner, B; Anstee, D J; Mawby, W J; Tanner, M J; von dem Borne, A E

1991-06-01

Twelve murine monoclonal antibodies, which react with human red cells of common Rh phenotype but give weak or negative reactions with Rh null erythrocytes, were used in quantitative binding assays and competitive binding assays to investigate the abundance and organization of polypeptides involved in the expression of antigens of the Rh blood group system. Antibodies of the R6A-type (R6A, BRIC-69, BRIC-207) and the 2D10-type (MB-2D10, LA18.18, LA23.40) recognize related structures and 100,000-200,000 molecules of each antibody bind maximally to erythrocytes of common Rh phenotype. Antibodies of the BRIC-125 type (BRICs 32, 122, 125, 126, 168, 211) recognize structures that are unrelated to those recognized by R6A-type and 2D10-type antibodies and between 10,000 and 50,000 antibody molecules bind maximally to erythrocytes of the common Rh phenotype. The binding of antibodies of the R6A-type and the 2D10-type, but not of antibodies of the BRIC-125-type could be partially inhibited by human anti-D antibodies (polyclonal and monoclonal) and a murine anti-e-like antibody. These results are consistent with evidence (Moore & Green 1987; Avent et al., 1988b) that the Rh blood group antigens are associated with a complex that comprises two groups of related polypeptides of M(r) 30,000 and M(r) 35,000-100,000, respectively, and suggest that there are 1-2 x 10(5) copies of this complex per erythrocyte. The polypeptide recognized by antibodies of the BRIC-125 type is likely to be associated with this complex.

β4-Integrin/PI3K Signaling Promotes Tumor Progression through the Galectin-3-N-Glycan Complex.

PubMed

Kariya, Yukiko; Oyama, Midori; Hashimoto, Yasuhiro; Gu, Jianguo; Kariya, Yoshinobu

2018-06-01

Malignant transformation is associated with aberrant N -glycosylation, but the role of protein N -glycosylation in cancer progression remains poorly defined. β4-integrin is a major carrier of N -glycans and is associated with poor prognosis, tumorigenesis, and metastasis. Here, N -glycosylation of β4-integrin contributes to the activation of signaling pathways that promote β4-dependent tumor development and progression. Increased expression of β1,6GlcNAc-branched N -glycans was found to be colocalized with β4-integrin in human cutaneous squamous cell carcinoma tissues, and that the β1,6GlcNAc residue was abundant on β4-integrin in transformed keratinocytes. Interruption of β1,6GlcNAc-branching formation on β4-integrin with the introduction of bisecting GlcNAc by N -acetylglucosaminyltransferase III overexpression was correlated with suppression of cancer cell migration and tumorigenesis. N -Glycan deletion on β4-integrin impaired β4-dependent cancer cell migration, invasion, and growth in vitro and diminished tumorigenesis and proliferation in vivo The reduced abilities of β4-integrin were accompanied with decreased phosphoinositol-3 kinase (PI3K)/Akt signals and were restored by the overexpression of the constitutively active p110 PI3K subunit. Binding of galectin-3 to β4-integrin via β1,6GlcNAc-branched N -glycans promoted β4-integrin-mediated cancer cell adhesion and migration. In contrast, a neutralizing antibody against galectin-3 attenuated β4-integrin N -glycan-mediated PI3K activation and inhibited the ability of β4-integrin to promote cell motility. Furthermore, galectin-3 knockdown by shRNA suppressed β4-integrin N -glycan-mediated tumorigenesis. These findings provide a novel role for N -glycosylation of β4-integrin in tumor development and progression, and the regulatory mechanism for β4-integrin/PI3K signaling via the galectin-3- N -glycan complex. Implications: N -Glycosylation of β4-integrin plays a functional role in promoting tumor development and progression through PI3K activation via the galectin-3- N -glycan complex. Mol Cancer Res; 16(6); 1024-34. ©2018 AACR . ©2018 American Association for Cancer Research.

Citrus CitNAC62 cooperates with CitWRKY1 to participate in citric acid degradation via up-regulation of CitAco3.

PubMed

Li, Shao-Jia; Yin, Xue-Ren; Wang, Wen-Li; Liu, Xiao-Fen; Zhang, Bo; Chen, Kun-Song

2017-06-15

Citric acid is the predominant organic acid of citrus fruit. Degradation of citric acid occurs during fruit development, influencing fruit acidity. Associations of CitAco3 transcripts and citric acid degradation have been reported for citrus fruit. Here, transient overexpression of CitAco3 significantly reduced the citric acid content of citrus leaves and fruits. Using dual luciferase assays, it was shown that CitNAC62 and CitWRKY1 could transactivate the promoter of CitAco3. Subcellular localization results showed that CitWRKY1 was located in the nucleus and CitNAC62 was not. Yeast two-hybrid analysis and bimolecular fluorescence complementation (BiFC) assays indicated that the two differently located transcription factors could interact with each other. Furthermore, BiFC showed that the protein-protein interaction occurred only in the nucleus, indicating the potential mobility of CitNAC62 in plant cells. A synergistic effect on citrate content was observed between CitNAC62 and CitWRKY1. Transient overexpression of CitNAC62 or CitWRKY1 led to significantly lower citrate content in citrus fruit. The combined expression of CitNAC62 and CitWRKY1 resulted in lower citrate content compared with the expression of CitNAC62 or CitWRKY1 alone. The transcript abundance of CitAco3 was consistent with the citrate content. Thus, we propose that a complex of CitWRKY1 and CitNAC62 contributes to citric acid degradation in citrus fruit, potentially via modulation of CitAco3. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

PubMed Central

Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

1999-01-01

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

Niacinamide mitigated the acute lung injury induced by phorbol myristate acetate in isolated rat's lungs

PubMed Central

2012-01-01

Background Phorbol myristate acetate (PMA) is a strong neutrophil activator and has been used to induce acute lung injury (ALI). Niacinamide (NAC) is a compound of B complex. It exerts protective effects on the ALI caused by various challenges. The purpose was to evaluate the protective effects of niacinamide (NAC) on the PMA-induced ALI and associated changes. Methods The rat's lungs were isolated in situ and perfused with constant flow. A total of 60 isolated lungs were randomized into 6 groups to received Vehicle (DMSO 100 μg/g), PMA 4 μg/g (lung weight), cotreated with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. We measured the lung weight and parameters related to ALI. The pulmonary arterial pressure and capillary filtration coefficient (Kfc) were determined in isolated lungs. ATP (adenotriphosphate) and PARP [poly(adenosine diphophate-ribose) polymerase] contents in lung tissues were detected. Real-time PCR was employed to display the expression of inducible and endothelial NO synthases (iNOS and eNOS). The neutrophil-derived mediators in lung perfusate were determined. Results PMA caused increases in lung weight parameters. This agent produced pulmonary hypertension and increased microvascular permeability. It resulted in decrease in ATP and increase in PARP. The expression of iNOS and eNOS was upregulated following PMA. PMA increased the neutrophil-derived mediators. Pathological examination revealed lung edema and hemorrhage with inflammatory cell infiltration. Immunohistochemical stain disclosed the presence of iNOS-positive cells in macrophages and endothelial cells. These pathophysiological and biochemical changes were diminished by NAC treatment. The NAC effects were dose-dependent. Conclusions Our results suggest that neutrophil activation and release of neutrophil-derived mediators by PMA cause ALI and associated changes. NO production through the iNOS-producing cells plays a detrimental role in the PMA-induced lung injury. ATP is beneficial, while PARP plays a deteriorative effect on the PMA-induced ALI. NAC exerts protective effects on the inflammatory cascade leading to pulmonary injury. This B complex compound may be applied for clinical usage and therapeutic regimen. PMID:22375599

Niacinamide mitigated the acute lung injury induced by phorbol myristate acetate in isolated rat's lungs.

PubMed

Lin, Chia-Chih; Hsieh, Nan-Kuang; Liou, Huey Ling; Chen, Hsing I

2012-03-01

Phorbol myristate acetate (PMA) is a strong neutrophil activator and has been used to induce acute lung injury (ALI). Niacinamide (NAC) is a compound of B complex. It exerts protective effects on the ALI caused by various challenges. The purpose was to evaluate the protective effects of niacinamide (NAC) on the PMA-induced ALI and associated changes. The rat's lungs were isolated in situ and perfused with constant flow. A total of 60 isolated lungs were randomized into 6 groups to received Vehicle (DMSO 100 μg/g), PMA 4 μg/g (lung weight), cotreated with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. We measured the lung weight and parameters related to ALI. The pulmonary arterial pressure and capillary filtration coefficient (Kfc) were determined in isolated lungs. ATP (adenotriphosphate) and PARP [poly(adenosine diphophate-ribose) polymerase] contents in lung tissues were detected. Real-time PCR was employed to display the expression of inducible and endothelial NO synthases (iNOS and eNOS). The neutrophil-derived mediators in lung perfusate were determined. PMA caused increases in lung weight parameters. This agent produced pulmonary hypertension and increased microvascular permeability. It resulted in decrease in ATP and increase in PARP. The expression of iNOS and eNOS was upregulated following PMA. PMA increased the neutrophil-derived mediators. Pathological examination revealed lung edema and hemorrhage with inflammatory cell infiltration. Immunohistochemical stain disclosed the presence of iNOS-positive cells in macrophages and endothelial cells. These pathophysiological and biochemical changes were diminished by NAC treatment. The NAC effects were dose-dependent. Our results suggest that neutrophil activation and release of neutrophil-derived mediators by PMA cause ALI and associated changes. NO production through the iNOS-producing cells plays a detrimental role in the PMA-induced lung injury. ATP is beneficial, while PARP plays a deteriorative effect on the PMA-induced ALI. NAC exerts protective effects on the inflammatory cascade leading to pulmonary injury. This B complex compound may be applied for clinical usage and therapeutic regimen.

Genetic architecture of wood properties based on association analysis and co-expression networks in white spruce.

PubMed

Lamara, Mebarek; Raherison, Elie; Lenz, Patrick; Beaulieu, Jean; Bousquet, Jean; MacKay, John

2016-04-01

Association studies are widely utilized to analyze complex traits but their ability to disclose genetic architectures is often limited by statistical constraints, and functional insights are usually minimal in nonmodel organisms like forest trees. We developed an approach to integrate association mapping results with co-expression networks. We tested single nucleotide polymorphisms (SNPs) in 2652 candidate genes for statistical associations with wood density, stiffness, microfibril angle and ring width in a population of 1694 white spruce trees (Picea glauca). Associations mapping identified 229-292 genes per wood trait using a statistical significance level of P < 0.05 to maximize discovery. Over-representation of genes associated for nearly all traits was found in a xylem preferential co-expression group developed in independent experiments. A xylem co-expression network was reconstructed with 180 wood associated genes and several known MYB and NAC regulators were identified as network hubs. The network revealed a link between the gene PgNAC8, wood stiffness and microfibril angle, as well as considerable within-season variation for both genetic control of wood traits and gene expression. Trait associations were distributed throughout the network suggesting complex interactions and pleiotropic effects. Our findings indicate that integration of association mapping and co-expression networks enhances our understanding of complex wood traits. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Robust and Porous β-Diketiminate-Functionalized Metal–Organic Frameworks for Earth-Abundant-Metal-Catalyzed C–H Amination and Hydrogenation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thacker, Nathan C.; Lin, Zekai; Zhang, Teng

We have designed a strategy for postsynthesis installation of the β-diketiminate (NacNac) functionality in a metal–organic framework (MOF) of UiO-topology. Metalation of the NacNac-MOF (I) with earth-abundant metal salts afforded the desired MOF-supported NacNac-M complexes (M = Fe, Cu, and Co) with coordination environments established by detailed EXAFS studies. The NacNac-Fe-MOF catalyst, I•Fe(Me), efficiently catalyzed the challenging intramolecular sp 3 C–H amination of a series of alkyl azides to afford α-substituted pyrrolidines. The NacNac-Cu-MOF catalyst, I•Cu(THF), was effective in promoting the intermolecular sp 3 C–H amination of cyclohexene using unprotected anilines to provide access to secondary amines in excellent selectivity.more » Finally, the NacNac-Co-MOF catalyst, I•Co(H), was used to catalyze alkene hydrogenation with turnover numbers (TONs) as high as 700 000. All of the NacNac-M-MOF catalysts were more effective than their analogous homogeneous catalysts and could be recycled and reused without a noticeable decrease in yield. The NacNac-MOFs thus provide a novel platform for engineering recyclable earth-abundant-element-based single-site solid catalysts for many important organic transformations.« less

21 CFR 314.70 - Supplements and other changes to an approved application.

Code of Federal Regulations, 2014 CFR

2014-04-01

... derived from such studies; (vi) For a natural product, a recombinant DNA-derived protein/polypeptide, or a...) Changes solely affecting a natural protein, a recombinant DNA-derived protein/polypeptide or a complex or..., recombinant DNA-derived protein/polypeptide, complex or conjugate of a drug substance with a monoclonal...

Sequential Desorption of Nitroaromatic Compounds (NAC) from Soils

DTIC Science & Technology

2005-03-01

the soil solution . Weissmahr et al. [20] suggest an electron donor acceptor (EDA) complex between oxygens of the siloxane surface of the clays and the...release of NACs into the soil solution . At high pH values desorption is superimposed by NACs hydrolysis. Therefore, in- creasing pH values impedes the...presented demonstrate that both the cation present in the soil solution and its concentration may affect the desorption behavior of NACs in contaminated soils

Characterization of auxin-binding proteins from zucchini plasma membrane

NASA Technical Reports Server (NTRS)

Hicks, G. R.; Rice, M. S.; Lomax, T. L.

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Characterization of auxin-binding proteins from zucchini plasma membrane.

PubMed

Hicks, G R; Rice, M S; Lomax, T L

1993-01-01

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

Helicobacter pylori β1,3-N-acetylglucosaminyltransferase for versatile synthesis of type 1 and type 2 poly-LacNAcs on N-linked, O-linked and I-antigen glycans

PubMed Central

Peng, Wenjie; Pranskevich, Jennifer; Nycholat, Corwin; Gilbert, Michel; Wakarchuk, Warren; Paulson, James C; Razi, Nahid

2012-01-01

Poly-N-acetyllactosamine extensions on N- and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial β3-N-acetylglucosaminyltransferase (β3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1, -2, -3 and -4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galβ1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in β1-3 linkage to type-1 (Galβ1-3GlcNAc) or type-3/4 (Galβ1-3GalNAcα/β) sequences. Thus, by alternating the use of the H. pylori β3GlcNAcT with galactosyltransferases that make the β1-4 or β1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the β3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori β3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans. PMID:22786570

Survey of O-GlcNAc level variations in Xenopus laevis from oogenesis to early development.

PubMed

Dehennaut, Vanessa; Lefebvre, Tony; Leroy, Yves; Vilain, Jean-Pierre; Michalski, Jean-Claude; Bodart, Jean-François

2009-04-01

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I-V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.

Tumor infiltrating lymphocytes in triple negative breast cancer receiving neoadjuvant chemotherapy

PubMed Central

Castaneda, Carlos A; Mittendorf, Elizabeth; Casavilca, Sandro; Wu, Yun; Castillo, Miluska; Arboleda, Patricia; Nunez, Teresa; Guerra, Henry; Barrionuevo, Carlos; Dolores-Cerna, Ketty; Belmar-Lopez, Carolina; Abugattas, Julio; Calderon, Gabriela; De La Cruz, Miguel; Cotrina, Manuel; Dunstan, Jorge; Gomez, Henry L; Vidaurre, Tatiana

2016-01-01

AIM To determine influence of neoadjuvant-chemotherapy (NAC) over tumor-infiltrating-lymphocytes (TIL) in triple-negative-breast-cancer (TNBC). METHODS TILs were evaluated in 98 TNBC cases who came to Instituto Nacional de Enfermedades Neoplasicas from 2005 to 2010. Immunohistochemistry staining for CD3, CD4, CD8 and FOXP3 was performed in tissue microarrays (TMA) sections. Evaluation of H/E in full-face and immunohistochemistry in TMA sections was performed in pre and post-NAC samples. STATA software was used and P value < 0.05 was considered statistically significant. RESULTS Higher TIL evaluated in full-face sections from pre-NAC tumors was associated to pathologic-complete-response (pCR) (P = 0.0251) and outcome (P = 0.0334). TIL evaluated in TMA sections showed low level of agreement with full-face sections (ICC = 0.017-0.20) and was not associated to pCR or outcome. TIL in post-NAC samples were not associated to response or outcome. Post-NAC lesions with pCR had similar TIL levels than those without pCR (P = 0.6331). NAC produced a TIL decrease in full-face sections (P < 0.0001). Percentage of TIL subpopulations was correlated with their absolute counts. Higher counts of CD3, CD4, CD8 and FOXP3 in pre-NAC samples had longer disease-free-survival (DFS). Higher counts of CD3 in pre-NAC samples had longer overall-survival. Higher ratio of CD8/CD4 counts in pre-NAC was associated with pCR. Higher ratio of CD4/FOXP3 counts in pre-NAC was associated with longer DFS. Higher counts of CD4 in post-NAC samples were associated with pCR. CONCLUSION TIL in pre-NAC full-face sections in TNBC are correlated to longer survival. TIL in full-face differ from TMA sections, absolute count and percentage analysis of TIL subpopulation closely related. PMID:27777881

N-Glycosylation Profiling of Porcine Reproductive and Respiratory Syndrome Virus Envelope Glycoprotein 5

PubMed Central

Li, Juan; Tao, Shujuan; Orlando, Ron; Murtaugh, Michael P.

2015-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV. PMID:25726973

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

PubMed Central

1986-01-01

A collection of 17 monoclonal antibodies elicited against the light- harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC- II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b. PMID:3528171

Negative feedback regulation of ABA biosynthesis in peanut (Arachis hypogaea): a transcription factor complex inhibits AhNCED1 expression during water stress

PubMed Central

Liu, Shuai; Li, Meijuan; Su, Liangchen; Ge, Kui; Li, Limei; Li, Xiaoyun; Liu, Xu; Li, Ling

2016-01-01

Abscisic acid (ABA), a key plant stress-signaling hormone, is produced in response to drought and counteracts the effects of this stress. The accumulation of ABA is controlled by the enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). In Arabidopsis, NCED3 is regulated by a positive feedback mechanism by ABA. In this study in peanut (Arachis hypogaea), we demonstrate that ABA biosynthesis is also controlled by negative feedback regulation, mediated by the inhibitory effect on AhNCED1 transcription of a protein complex between transcription factors AhNAC2 and AhAREB1. AhNCED1 was significantly down-regulated after PEG treatment for 10 h, at which time ABA content reached a peak. A ChIP-qPCR assay confirmed AhAREB1 and AhNAC2 binding to the AhNCED1 promoter in response to ABA. Moreover, the interaction between AhAREB1 and AhNAC2, and a transient expression assay showed that the protein complex could negatively regulate the expression of AhNCED1. The results also demonstrated that AhAREB1 was the key factor in AhNCED1 feedback regulation, while AhNAC2 played a subsidiary role. ABA reduced the rate of AhAREB1 degradation and enhanced both the synthesis and degradation rate of the AhNAC2 protein. In summary, the AhAREB1/AhNAC2 protein complex functions as a negative feedback regulator of drought-induced ABA biosynthesis in peanut. PMID:27892506

Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity

PubMed Central

1984-01-01

The processing of asparagine-linked oligosaccharides on the alpha- chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N- acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin. PMID:6420419

Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity.

PubMed

Hickman, S; Theodorakis, J L; Greco, J M; Brown, P H

1984-02-01

The processing of asparagine-linked oligosaccharides on the alpha-chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N-acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.

Implementation of a nutrition assessment, counseling and support program and its association with body mass index among people living with HIV in Accra, Ghana.

PubMed

Sackey, Joachim; Zhang, Fang Fang; Rogers, Beatrice; Aryeetey, Richmond; Wanke, Christine

2018-05-01

In spite of the important role nutrition plays in the management of HIV, access to nutrition services is inadequate, especially in resource limited settings. In addition, nutrition programs for people living with HIV (PLWH) have not been sufficiently evaluated for efficacy and this study was conducted to address this gap. This study aimed to evaluate the implementation of the nutrition assessment, counseling and support (NACS) program in Accra, Ghana, and to assess whether the level of implementation of NACS was associated with the body mass index (BMI) of PLWH. A cross-sectional study was conducted in six HIV clinics (3 NACS designated and 3 non-NACS). Study participants were 152 adult PLWH at least 6 months on antiretroviral therapy and not pregnant or breastfeeding. Using a NACS implementation scale developed for this study ranging from 0 to 8 (a higher score indicating better NACS implementation), median NACS implementation score was not different between NACS-designated, and non-NACS HIV clinics (5 vs 4, p = 0.14). Almost half (47%) of the respondents were overweight or obese. A higher score on the NACS implementation scale was not significantly associated with overweight or obesity (BMI >24.9 kg/m 2 ) after adjusting for other covariates. It was concluded that, there was poor implementation of NACS in the NACS designated HIV clinics surveyed with no nutrition counseling offered nor food support available to those who might need it.

MRI to predict nipple-areola complex (NAC) involvement: An automatic method to compute the 3D distance between the NAC and tumor.

PubMed

Giannini, Valentina; Bianchi, Veronica; Carabalona, Silvia; Mazzetti, Simone; Maggiorotto, Furio; Kubatzki, Franziska; Regge, Daniele; Ponzone, Riccardo; Martincich, Laura

2017-12-01

To assess the role in predicting nipple-areola complex (NAC) involvement of a newly developed automatic method which computes the 3D tumor-NAC distance. Ninety-nine patients scheduled to nipple sparing mastectomy (NSM) underwent magnetic resonance (MR) examination at 1.5 T, including sagittal T2w and dynamic contrast enhanced (DCE)-MR imaging. An automatic method was developed to segment the NAC and the tumor and to compute the 3D distance between them. The automatic measurement was compared with manual axial and sagittal 2D measurements. NAC involvement was defined by the presence of invasive ductal or lobular carcinoma and/or ductal carcinoma in situ or ductal intraepithelial neoplasia (DIN1c - DIN3). Tumor-NAC distance was computed on 95/99 patients (25 NAC+), as three tumors were not correctly segmented (sensitivity = 97%), and 1 NAC was not detected (sensitivity = 99%). The automatic 3D distance reached the highest area under the receiver operating characteristic (ROC) curve (0.830) with respect to the manual axial (0.676), sagittal (0.664), and minimum distances (0.664). At the best cut-off point of 21 mm, the 3D distance obtained sensitivity = 72%, specificity = 80%, positive predictive value = 56%, and negative predictive value = 89%. This method could provide a reproducible biomarker to preoperatively select breast cancer patients candidates to NSM, thus helping surgical planning and intraoperative management of patients. © 2017 Wiley Periodicals, Inc.

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

PubMed

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.

Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product.

PubMed

Hadfield, A T; Harvey, D J; Archer, D B; MacKenzie, D A; Jeenes, D J; Radford, S E; Lowe, G; Dobson, C M; Johnson, L N

1994-11-11

The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6) geometry of the sofa is stabilised by hydrogen bonds from the O-6 hydroxyl group to the main-chain N of Val109 and main-chain O of Ala107. The sugar in site D adopts the alpha configuration, seemingly in conflict with the observation that the hydrolysis of beta (1-4) glycosidie linkage by HEWL proceeds with 99.9% retention of beta-configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

Cation-π interaction of the univalent sodium cation with [2.2.2]paracyclophane: Experimental and theoretical study

NASA Astrophysics Data System (ADS)

Makrlík, Emanuel; Sýkora, David; Böhm, Stanislav; Vaňura, Petr

2018-02-01

By employing electrospray ionization mass spectrometry (ESI-MS), it was proven experimentally that the univalent sodium cation (Na+) forms with [2.2.2]paracyclophane (C24H24) the cationic complex [Na(C24H24)]+. Further, applying quantum chemical DFT calculations, the most probable structure of the [Na(C24H24)]+ complex was derived. In the resulting complex with a symmetry very close to C3, the "central" cation Na+, fully located in the cavity of the parent [2.2.2]paracyclophane ligand, is bound to all three benzene rings of [2.2.2]paracyclophane via cation-π interaction. Finally, the interaction energy, E(int), of the considered cation-π complex [Na(C24H24)]+ was found to be -267.3 kJ/mol, confirming the formation of this fascinating complex species as well.

Association of phycoerythrin and phycocyanin: in vitro formation of a functional energy transferring phycobilisome complex of Porphyridium sordidum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lipschultz, C.A.; Gantt, E.

1981-01-01

Functional in vitro association and dissociation of a phycobiliprotein complex, isolated from phycobilisomes of the red alga Porphyridium sordidum, were studied. The complex contained large bangiophyceaen phycoerythrin and cyanophytan phycocyanin in an equimolar ratio and had absorption maxima at 625, 567, and 550 nm and a shoulder at 495 nm. Emission at 655 nm (with excitation at 545 nm) from phycocyanin indicated functional coupling. The complex was stable over a wide buffer concentration range, and, notably, it was maximally stable in low phosphate, <0.01 M, unlike the phycobilisomes, which dissociate at this concentration. Its molecular weight was estimated to bemore » ca. 510 000, and by electron microscopy it was seen to consist of two units of similar size. The complex in 0.1 M phosphate was separated on a sucrose gradient into a homogeneous phycoerythrin band and a spectrally heterogeneous phycocyanin band. In vitro association of phycoerythrin and phycocyanin resulted in a complex with the same absorbance, emission, sedimentation, and molar pigment ratio as those of the native complex. The spectrally heterogeneous phycocyanin fractions from the dissociation gradient varied in the degree of association with phycoerythrin. Phycocyanin fractions absorbing from 622 to 633 nm exhibited high associability (>70%), whereas those with maxima at 617-620 nm had low associability (<30%). The presence of a 30 000 molecular weight polypeptide accompanied high associability, where it was ca. 2-fold more prominent. It is suggested that this polypeptide is involved in complex formation and could serve either in the stabilization of the conformational state of cyanophytan phycocyanin or as a direct linker between phycobiliproteins.« less

Mucin-type O-glycans in Tears of Normal Subjects and Patients with Non-Sjögren’s Dry Eye

PubMed Central

Guzman-Aranguez, Ana; Mantelli, Flavio; Argüeso, Pablo

2009-01-01

Purpose O-linked carbohydrates (O-glycans) contribute to the hydrophilic character of mucins in mucosal tissues. This study aimed to identify the repertoire of O-glycans in the tear film, and the glycosyltransferases associated with their biosynthesis, in normal subjects and patients with non-Sjögren’s dry eye. Methods Human tear fluid was collected from the inferior conjunctival fornix. O-glycans were released by hydrazinolysis, labeled with 2-aminobenzamide, and analyzed by fluorometric, high-performance liquid chromatography (HPLC) coupled with exoglycosidase digestions. O-glycan structures identified in tears were related to potential biosynthetic pathways in human conjunctival epithelium using a glycogene microarray database. Lectin-binding analyses were performed using agglutinins from Arachis hypogaea, Maackia amurensis, and Sambucus nigra. Results The O-glycan profile of human tears consisted primarily of core 1 (Galβ1-3GalNAcα1-Ser/Thr)-based structures. Mono-sialyl O-glycans represented approximately 66% of the glycan pool, being α2-6-sialyl core 1 the predominant O-glycan structure in human tears (48%). Four families of glycosyltranferases potentially related to the biosynthesis of these structures were identified in human conjunctiva. These included thirteen polypeptide-GalNAc-transferases (GALNT), the core 1 β-3-galactosyltransferase (T-synthase), three α2-6-sialyltransferases (ST6GalNAc), and two α2-3-sialyltransferases (ST3Gal). No significant differences in total amount of O-glycans were detected between tears of normal subjects and dry eye patients, by HPLC and lectin blot. Likewise, no differences in glycosyltransferase expression were found by glycogene microarray. Conclusions This study identifies the most common mucin-type O-glycans in human tears and their expected biosynthetic pathways in ocular surface epithelia. Patients with non-Sjögren’s dry eye show no alterations in composition and amount of O-glycans in the tear fluid. PMID:19407012

A split motor domain in a cytoplasmic dynein

PubMed Central

Straube, Anne; Enard, Wolfgang; Berner, Alexandra; Wedlich-Söldner, Roland; Kahmann, Regine; Steinberg, Gero

2001-01-01

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP-cleavage region and the microtubule-binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule-binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co-immunoprecipitated and co-localized within growing cells, and Dyn1–Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1–Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1–Dyn2 complex serves multiple cellular functions. PMID:11566874

An antibody to the GM1/GalNAc-GD1a complex correlates with development of pure motor Guillain-Barré syndrome with reversible conduction failure.

PubMed

Ogawa, Go; Kaida, Ken-ichi; Kuwahara, Motoi; Kimura, Fumihiko; Kamakura, Keiko; Kusunoki, Susumu

2013-01-15

Antibodies to a ganglioside complex consisting of GM1 and GalNAc-GD1a (GM1/GalNAc-GD1a) are found in sera from patients with Guillain-Barré syndrome (GBS). To elucidate the clinical significance of anti-GM1/GalNAc-GD1a antibodies in GBS, clinical features of 58 GBS patients with IgG anti-GM1/GalNAc-GD1a antibodies confirmed by enzyme-linked immunosorbent assay and thin layer chromatography immunostaining were analyzed. Compared to GBS patients without anti-GM1/GalNAc-GD1a antibodies, anti-GM1/GalNAc-GD1a-positive patients more frequently had a preceding respiratory infection (n=38, 66%, p<0.01) and were characterized by infrequency of cranial nerve deficits (n=9, 16%, p<0.01) and sensory disturbances (n=26, 45%, p<0.01). Of the 28 anti-GM1/GalNAc-GD1a-positive patients for whom electrophysiological data were available, 14 had conduction blocks (CBs) at intermediate segments of motor nerves, which were not followed by evident remyelination. Eight of 10 bedridden cases were able to walk independently within one month after the nadir. These results show that the presence of anti-GM1/GalNAc-GD1a antibodies correlated with pure motor GBS characterized by antecedent respiratory infection, fewer cranial nerve deficits, and CBs at intermediate sites of motor nerves. The CB may be generated through alteration of the regulatory function of sodium channels in the nodal axolemma. Copyright © 2012 Elsevier B.V. All rights reserved.

Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway.

PubMed

Geisler, Christoph; Jarvis, Donald L

2012-03-02

Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2) is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man(5)GlcNAc(2), Man(3)GlcNAc(2), and GlcNAc(β1-2)Man(α1-6)[Man(α1-3)]ManGlcNAc(2), demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2), demonstrating that it initiates complex N-glycan production, but cannot use Man(3)GlcNAc(2) to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the net outcome of the insect cell N-glycosylation pathway.

Loss of gastric gland mucin-specific O-glycan is associated with progression of differentiated-type adenocarcinoma of the stomach.

PubMed

Shiratsu, Kazuo; Higuchi, Kayoko; Nakayama, Jun

2014-01-01

Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides having terminal α1,4-linked N-acetylglucosamine (αGlcNAc) residues largely attached to a MUC6 scaffold. Previously, we generated A4gnt-deficient mice, which totally lack αGlcNAc, and showed that αGlcNAc functions as a tumor suppressor for gastric cancer. Here, to determine the clinicopathological significance of αGlcNAc in gastric carcinomas, we examined immunohistochemical expression of αGlcNAc and mucin phenotypic markers including MUC5AC, MUC6, MUC2, and CD10 in 214 gastric adenocarcinomas and compared those expression patterns with clinicopathological parameters and cancer-specific survival. The αGlcNAc loss was evaluated in MUC6-positive gastric carcinoma. Thirty-three (61.1%) of 54 differentiated-type gastric adenocarcinomas exhibiting MUC6 in cancer cells lacked αGlcNAc expression. Loss of αGlcNAc was significantly correlated with depth of invasion, stage, and venous invasion by differentiated-type adenocarcinoma. Loss of αGlcNAc was also significantly associated with poorer patient prognosis in MUC6-positive differentiated-type adenocarcinoma. By contrast, no significant correlation between αGlcNAc loss and any clinicopathologic variable was observed in undifferentiated-type adenocarcinoma. Expression of MUC6 was also significantly correlated with several clinicopathological variables in differentiated-type adenocarcinoma. However, unlike the case with αGlcNAc, its expression showed no correlation with cancer-specific survival in patients. In undifferentiated-type adenocarcinoma, we observed no significant correlation between mucin phenotypic marker expression, including MUC6, and any clinicopathologic variable. These results together indicate that loss of αGlcNAc in MUC6-positive cancer cells is associated with progression and poor prognosis in differentiated, but not undifferentiated, types of gastric adenocarcinoma. © 2013 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Biological Activities and Potential Oral Applications of N-Acetylcysteine: Progress and Prospects

PubMed Central

Pei, Yanping; Liu, Huan; Yang, Yi; Yang, Yanwei

2018-01-01

N-Acetylcysteine (NAC), a cysteine prodrug and glutathione (GSH) precursor, has been used for several decades in clinical therapeutic practices as a mucolytic agent and for the treatment of disorders associated with GSH deficiency. Other therapeutic activities of NAC include inhibition of inflammation/NF-κB signaling and expression of proinflammatory cytokines. N-Acetylcysteine is also a nonantibiotic compound possessing antimicrobial property and exerts anticarcinogenic and antimutagenic effects against certain types of cancer. Recently, studies describing potentially important biological and pharmacological activities of NAC have stimulated interests in using NAC-based therapeutics for oral health care. The present review focused on the biological activities of NAC and its potential oral applications. The potential side effects of NAC and formulations for drug delivery were also discussed, with the intent of advancing NAC-associated treatment modalities in oral medicine. PMID:29849877

Effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pan, Y.T.; Elbein, A.D.

1985-11-01

Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with (2-/sup 3/H)mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps bymore » inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with (/sup 3/H)mannose or (/sup 3/H)galactose.« less

Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation.

PubMed

Spiro, R G

2004-05-01

Misfolded or incompletely assembled multisubunit glycoproteins undergo endoplasmic reticulum-associated degradation (ERAD) regulated in large measure by their N-linked polymannose oligosaccharides. In this quality control system lectin interaction with Glc(3)Man(9)GlcNAc(2) glycans after trimming with endoplasmic reticulum (ER) alpha-glucosidases and alpha-mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and proteolytic degradation. Monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins take part in the calnexin/calreticulin glucosylation-deglucosylation cycle, while the Man(8)GlcNAc(2) isomer B product of ER mannosidase I interacts with EDEM. Proteasomal degradation requires retrotranslocation into the cytosol through a Sec61 channel and deglycosylation by peptide: N-glycosidase (PNGase); in alternate models both PNGase and proteasomes may be either free in the cytosol or ER membrane-imbedded/attached. Numerous proteins appear to undergo nonproteasomal degradation in which deglycosylation and proteolysis take place in the ER lumen. The released free oligosaccharides (OS) are transported to the cytosol as OS-GlcNAc(2) along with similar components produced by the hydrolytic action of the oligosaccharyltransferase, where they together with OS from the proteasomal pathway are trimmed to Man(5)GlcNAc(1) by the action of cytosolic endo-beta- N-acetylglucosaminidase and alpha-mannosidase before entering the lysosomes. Some misfolded glycoproteins can recycle between the ER, intermediate and Golgi compartments, where they are further processed before ERAD. Moreover, properly folded glycoproteins with mannose-trimmed glycans can be deglucosylated in the Golgi by endomannosidase, thereby releasing calreticulin and permitting formation of complex OS. A number of regulatory controls have been described, including the glucosidase-glucosyltransferase shuttle, which controls the level of Glc(3)Man(9)GlcNAc(2)-P-P-Dol, and the unfolded protein response, which enhances synthesis of components of the quality control system.

Differential expression and localization of glycosidic residues in in vitro- and in vivo-matured cumulus-oocyte complexes in equine and porcine species.

PubMed

Accogli, Gianluca; Douet, Cécile; Ambruosi, Barbara; Martino, Nicola Antonio; Uranio, Manuel Filioli; Deleuze, Stefan; Dell'Aquila, Maria Elena; Desantis, Salvatore; Goudet, Ghylène

2014-12-01

Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and βN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models. © 2014 Wiley Periodicals, Inc.

Application of novel Ni(II) complex and ZrO2 nanoparticle as mediators for electrocatalytic determination of N-acetylcysteine in drug samples.

PubMed

Karimi-Maleh, Hassan; Salehi, Mehdi; Faghani, Fatemeh

2017-10-01

The electrooxidation of N-acetylcysteine (N-AC) was studied by a novel Ni(II) complex modified ZrO 2 nanoparticle carbon paste electrode [Ni(II)/ZrO 2 /NPs/CPE] using voltammetric methods. The results showed that Ni(II)/ZrO 2 /NPs/CPE had high electrocatalytic activity for the electrooxidation of N-AC in aqueous buffer solution (pH = 7.0). The electrocatalytic oxidation peak currents increase linearly with N-AC concentrations over the concentration ranges of 0.05-600μM using square wave voltammetric methods. The detection limit for N-AC was equal to 0.009μM. The catalytic reaction rate constant, k h , was calculated (7.01 × 10 2  M -1  s -1 ) using the chronoamperometry method. Finally, Ni(II)/ZrO 2 /NPs/CPE was also examined as an ultrasensitive electrochemical sensor for the determination of N-AC in real samples such as tablet and urine. Copyright © 2017. Published by Elsevier B.V.

A little sugar goes a long way: The cell biology of O-GlcNAc

PubMed Central

2015-01-01

Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport. PMID:25825515

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

PubMed Central

Kapuria, Vaibhav; Röhrig, Ute F.; Bhuiyan, Tanja; Borodkin, Vladimir S.; van Aalten, Daan M.F.; Zoete, Vincent; Herr, Winship

2016-01-01

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc), O-linked-GlcNAc transferase (OGT) catalyzes Ser/Thr O-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase–protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase–protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT. PMID:27056667

Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein.

PubMed

Ohta, M; Hamako, J; Yamamoto, S; Hatta, H; Kim, M; Yamamoto, T; Oka, S; Mizuochi, T; Matsuura, F

1991-10-01

Asparagine-linked oligosaccharides present on hen egg-yolk immunoglobulin, termed IgY, were liberated from the protein by hydrazinolysis. After N-acetylation, the oligosaccharides were labelled with a UV-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-derivatized oligosaccharides were fractionated by anion exchange, normal phase and reversed phase HPLC, and their structures were determined by a combination of sugar composition analysis, methylation analysis, negative ion FAB-MS, 500 MHz 1H-NMR and sequential exoglycosidase digestions. IgY contained monoglucosylated oligomannose type oligosaccharides with structures of Glc alpha 1-3Man7-9-GlcNAc-GlcNAc, oligomannose type oligosaccharides with the size range of Man5-9GlcNAc-GlcNAc, and biantennary complex type oligosaccharides with core region structure of Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc. The glucosylated oligosaccharides, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, have not previously been reported in mature glycoproteins from any source.

The active site of O-GlcNAc transferase imposes constraints on substrate sequence

PubMed Central

Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.

2016-01-01

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509

Computerized N-acetylcysteine physician order entry by template protocol for acetaminophen toxicity.

PubMed

Thompson, Trevonne M; Lu, Jenny J; Blackwood, Louisa; Leikin, Jerrold B

2011-01-01

Some medication dosing protocols are logistically complex for traditional physician ordering. The use of computerized physician order entry (CPOE) with templates, or order sets, may be useful to reduce medication administration errors. This study evaluated the rate of medication administration errors using CPOE order sets for N-acetylcysteine (NAC) use in treating acetaminophen poisoning. An 18-month retrospective review of computerized inpatient pharmacy records for NAC use was performed. All patients who received NAC for the treatment of acetaminophen poisoning were included. Each record was analyzed to determine the form of NAC given and whether an administration error occurred. In the 82 cases of acetaminophen poisoning in which NAC was given, no medication administration errors were identified. Oral NAC was given in 31 (38%) cases; intravenous NAC was given in 51 (62%) cases. In this retrospective analysis of N-acetylcysteine administration using computerized physician order entry and order sets, no medication administration errors occurred. CPOE is an effective tool in safely executing complicated protocols in an inpatient setting.

Caffeine-water-polypeptide interaction in aqueous solution

NASA Astrophysics Data System (ADS)

Ghabi, Habib; Dhahbi, Mahmoud

1999-04-01

The interaction of caffeine monomer with the synthetic polypeptides polyasparagine (pAg) and polyaspartic acid (pAsp) was studied by UV spectrophotometry. The results show that different types of interactions are possible depending on the nature of polypeptide. The form of the complex was discussed.

Assessing Contributions of Nucleus Accumbens Shell Subregions to Reward-Seeking Behavior

PubMed Central

Reed, Michael D.; Hildebrand, David G. C.; Santangelo, Gabrielle; Moffa, Anthony; Pira, Ashley S.; Rycyna, Lisa; Radic, Mia; Price, Katherine; Archbold, Jonathan; McConnell, Kristi; Girard, Lauren; Morin, Kristen; Tang, Anna; Febo, Marcelo; Stellar, James R.

2015-01-01

Background The nucleus accumbens (NAc) plays a key role in brain reward processes including drug seeking and reinstatement. Several anatomical, behavioral, and neurochemical studies discriminate between the limbic-associated shell and the motor-associated core regions. Less studied is the fact that the shell can be further subdivided into a dorsomedial shell (NAcDMS) and an intermediate zone (NAcINT) based on differential expression of transient c-Fos and long-acting immediate-early gene ΔFosB upon cocaine sensitization. These disparate expression patterns suggest that NAc shell subregions may play distinct roles in reward-seeking behavior. In this study, we examined potential differences in the contributions of the NAcDMS and the NAcINT to reinstatement of reward-seeking behavior after extinction. Methods Rats were trained to intravenously self-administer cocaine, extinguished, and subjected to a reinstatement test session consisting of either an intracranial microinfusion of amphetamine or vehicle targeted to the NAcDMS or the NAcINT. Results Small amphetamine microinfusions targeted to the NAcDMS resulted in statistically significant reinstatement of lever pressing, whereas no statistical difference was observed for microinfusions targeted to the NAcINT. No significant difference was found for vehicle microinfusions in either case. Conclusion These results suggest heterogeneity in the behavioral relevance of NAc shell subregions, a possibility that can be tested in specific neuronal populations in the future with recently developed techniques including optogenetics. PMID:26048642

Tuning the conformational properties of the prion peptide.

PubMed

Ho, Chai-Chi; Lee, Lily Y-L; Huang, Kuo-Ting; Lin, Chun-Cheng; Ku, Mei-Yun; Yang, Chien-Chih; Chan, Sunney I; Hsu, Ruei-Lin; Chen, Rita P-Y

2009-07-01

Previously, we disclosed that O-linked glycosylation of Ser-132 or Ser-135 could dramatically change the amyloidogenic property of the hamster prion peptide (sequence 108-144). This peptide, which corresponds to the flexible loop and the first beta-strand in the structure of the prion protein, is a random coil when it is initially dissolved in buffer, but amyloid fibrils are formed with time. Thus, it offers a convenient model system to observe and compare how different chemical modifications and sequence mutations alter the amyloidogenic property of the peptide within a reasonable experimental time frame. In our earlier study, aside from uncovering a site-specificity of the glycosylation on the fibrillogenesis, different effects of alpha-GalNAc and beta-GlcNAc were observed. In this work, we explore further how different sugar configurations affect the conformational property of the polypeptide chain. We compare the effects of O-linked glycosylation by the common sugars alpha-GalNAc, beta-GlcNAc with their non-native analogs beta-GalNAc, alpha-GlcNAc in an effort to uncover the origin of the sugar-specificity on the fibril formation. We find that the anomeric configuration of the sugar is the most important factor affecting the fibrillogenesis. Sugars with the glycosidic bond in the alpha-configuration at Ser-135 have a dramatic inhibitory effect on the structural conversion of the glycosylated peptide. Because O-glycosylation of Ser-135 with alpha-linked sugars also promote the formation of three slowly converting conformations at the site of glycosylation, we surmise that the amyloidogenic property of the peptide is related to its conformational flexibility, and the proclivity of this region of the peptide to undergo the structural conversion from the random coil to form the beta-structure. Upon O-glycosylation with an alpha-linked sugar, this conversion is inhibited and the nucleation of fibril formation is largely retarded. Consistent with this scenario, Arg-136 is the residue most affected in the TOCSY NMR spectra of the glycosylated peptides, other than the serine site modified. In addition, when Arg-136 is substituted by Gly, a mutation that should provide higher structural flexibility in this part of the peptide, the amyloidogenic property of the peptide is greatly enhanced, and the inhibition effect of glycosylation is largely diminished. These results are consistent with Ser-135 and Arg-136 being part of the kink region involved in the structural conversion.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein.

PubMed

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncations of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T][T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein.

Electrophoretic mobility shift assay reveals a novel recognition sequence for Setaria italica NAC protein

PubMed Central

Puranik, Swati; Kumar, Karunesh; Srivastava, Prem S

2011-01-01

The NAC (NAM/ATAF1,2/CUC2) proteins are among the largest family of plant transcription factors. Its members have been associated with diverse plant processes and intricately regulate the expression of several genes. Inspite of this immense progress, knowledge of their DNA-binding properties are still limited. In our recent publication,1 we reported isolation of a membrane-associated NAC domain protein from Setaria italica (SiNAC). Transactivation analysis revealed that it was a functionally active transcription factor as it could stimulate expression of reporter genes in vivo. Truncation of the transmembrane region of the protein lead to its nuclear localization. Here we describe expression and purification of SiNAC DNA-binding domain. We further report identification of a novel DNA-binding site, [C/G][A/T] [T/A][G/C]TC[C/G][A/T][C/G][G/C] for SiNAC by electrophoretic mobility shift assay. The SiNAC-GST protein could bind to the NAC recognition sequence in vitro as well as to sequences where some bases had been reshuffled. The results presented here contribute to our understanding of the DNA-binding specificity of SiNAC protein. PMID:21918373

Molecular cloning and characterization of a membrane associated NAC family gene, SiNAC from foxtail millet [Setaria italica (L.) P. Beauv].

PubMed

Puranik, Swati; Bahadur, Ranjit Prasad; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280-287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and in silico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC(1-158):GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.

Designing healthy communities: creating evidence on metrics for built environment features associated with walkable neighbourhood activity centres.

PubMed

Gunn, Lucy Dubrelle; Mavoa, Suzanne; Boulangé, Claire; Hooper, Paula; Kavanagh, Anne; Giles-Corti, Billie

2017-12-04

Evidence-based metrics are needed to inform urban policy to create healthy walkable communities. Most active living research has developed metrics of the environment around residential addresses, ignoring other important walking locations. Therefore, this study examined: metrics for built environment features surrounding local shopping centres, (known in Melbourne, Australia as neighbourhood activity centres (NACs) which are typically anchored by a supermarket); the association between NACs and transport walking; and, policy compliance for supermarket provision. In this observational study, cluster analysis was used to categorize 534 NACs in Melbourne, Australia by their built environment features. The NACS were linked to eligible Victorian Integrated Survey of Travel Activity 2009-2010 (VISTA) survey participants (n=19,984). Adjusted multilevel logistic regressions estimated associations between each cluster typology and two outcomes of daily walking: any transport walking; and, any 'neighbourhood' transport walking. Distance between residential dwellings and closest NAC was assessed to evaluate compliance with local planning policy on supermarket locations. Metrics for 19 built environment features were estimated and three NAC clusters associated with walkability were identified. NACs with significantly higher street connectivity (mean:161, SD:20), destination diversity (mean:16, SD:0.4); and net residential density (mean:77, SD:65) were interpreted as being 'highly walkable' when compared with 'low walkable' NACs, which had lower street connectivity (mean:57, SD:15); destination diversity (mean:11, SD:3); and net residential density (mean:10, SD:3). The odds of any daily transport walking was 5.85 times higher (95% CI: 4.22, 8.11), and for any 'neighborhood' transport walking 8.66 (95% CI: 5.89, 12.72) times higher, for residents whose closest NAC was highly walkable compared with those living near low walkable NACs. Only highly walkable NACs met the policy requirement that residents live within 1km of a local supermarket. Built environment features surrounding NACs must reach certain levels to encourage walking and deliver walkable communities. Research and metrics about the type and quantity of built environment features around both walking trip origins and destinations is needed to inform urban planning policies and urban design guidelines.

A transposable element in a NAC gene is associated with drought tolerance in maize seedlings

PubMed Central

Mao, Hude; Wang, Hongwei; Liu, Shengxue; Li, Zhigang; Yang, Xiaohong; Yan, Jianbing; Li, Jiansheng; Tran, Lam-Son Phan; Qin, Feng

2015-01-01

Drought represents a major constraint on maize production worldwide. Understanding the genetic basis for natural variation in drought tolerance of maize may facilitate efforts to improve this trait in cultivated germplasm. Here, using a genome-wide association study, we show that a miniature inverted-repeat transposable element (MITE) inserted in the promoter of a NAC gene (ZmNAC111) is significantly associated with natural variation in maize drought tolerance. The 82-bp MITE represses ZmNAC111 expression via RNA-directed DNA methylation and H3K9 dimethylation when heterologously expressed in Arabidopsis. Increasing ZmNAC111 expression in transgenic maize enhances drought tolerance at the seedling stage, improves water-use efficiency and induces upregulation of drought-responsive genes under water stress. The MITE insertion in the ZmNAC111 promoter appears to have occurred after maize domestication and spread among temperate germplasm. The identification of this MITE insertion provides insight into the genetic basis for natural variation in maize drought tolerance. PMID:26387805

Solution NMR Analyses of the C-type Carbohydrate Recognition Domain of DC-SIGNR Protein Reveal Different Binding Modes for HIV-derived Oligosaccharides and Smaller Glycan Fragments

PubMed Central

Probert, Fay; Whittaker, Sara B.-M.; Crispin, Max; Mitchell, Daniel A.; Dixon, Ann M.

2013-01-01

The C-type lectin DC-SIGNR (dendritic cell-specific ICAM-3-grabbing non-integrin-related; also known as L-SIGN or CD299) is a promising drug target due to its ability to promote infection and/or within-host survival of several dangerous pathogens (e.g. HIV and severe acute respiratory syndrome coronavirus (SARS)) via interactions with their surface glycans. Crystallography has provided excellent insight into the mechanism by which DC-SIGNR interacts with small glycans, such as (GlcNAc)2Man3; however, direct observation of complexes with larger, physiological oligosaccharides, such as Man9GlcNAc2, remains elusive. We have utilized solution-state nuclear magnetic resonance spectroscopy to investigate DC-SIGNR binding and herein report the first backbone assignment of its active, calcium-bound carbohydrate recognition domain. Direct interactions with the small sugar fragments Man3, Man5, and (GlcNAc)2Man3 were investigated alongside Man9GlcNAc derived from recombinant gp120 (present on the HIV viral envelope), providing the first structural data for DC-SIGNR in complex with a virus-associated ligand, and unique binding modes were observed for each glycan. In particular, our data show that DC-SIGNR has a different binding mode for glycans on the HIV viral envelope compared with the smaller glycans previously observed in the crystalline state. This suggests that using the binding mode of Man9GlcNAc, instead of those of small glycans, may provide a platform for the design of DC-SIGNR inhibitors selective for high mannose glycans (like those on HIV). 15N relaxation measurements provided the first information on the dynamics of the carbohydrate recognition domain, demonstrating that it is a highly flexible domain that undergoes ligand-induced conformational and dynamic changes that may explain the ability of DC-SIGNR to accommodate a range of glycans on viral surfaces. PMID:23788638

Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing.

PubMed

Brown, P H; Hickman, S

1986-02-25

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.

Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos

PubMed Central

Famiglietti, Amber L.; Wei, Zheng; Beres, Thomas M.; Milac, Adina L.; Tran, Duy T.; Patel, Divya; Angerer, Robert C.; Angerer, Lynne M.

2017-01-01

Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus. PMID:28448610

Reagent Anions for Charge Inversion of Polypeptide/Protein Cations in the Gas Phase

PubMed Central

He, Min; Emory, Joshua F.; McLuckey, Scott A.

2005-01-01

Various reagent anions capable of converting polypeptide cations to anions via ion/ion reactions have been investigated. The major charge inversion reaction channels include multiple proton transfer and adduct formation. Dianions composed of sulfonate groups as the negative charge carriers show essentially exclusive adduct formation in converting protonated peptides and proteins to anions. Dianions composed of carboxylate groups, on the other hand, show far more charge inversion via multiple proton transfer, with the degree of adduct formation dependent upon both the size of the polypeptide and the spacings between carboxylate groups in the dianion. More highly charged carboxylate-containing anions, such as those derived from carboxylate-terminated polyamidoamine half-generation dendrimers show charge inversion to give anion charges as high in magnitude as −4, with the degree of adduct formation being inversely related to dendrimer generation. All observations can be interpreted on the basis of charge inversion taking place via a long-lived chemical complex. The lifetime of this complex is related to the strengths and numbers of the interactions of the reactants in the complex. Calculations with model systems are fully consistent with sulfonate groups giving rise to more stable complexes. The kinetic stability of the complex can also be affected by the presence of electrostatic repulsion if it is multiply charged. In general, this situation destabilizes the complex and reduces the likelihood for observation of adducts. The findings highlight the characteristics of multiply charged anions that play roles in determining the nature of charge inversion products associated with protonated peptides and proteins. PMID:15889906

Structure of the N-linked oligosaccharides of MHC class I molecules from cells deficient in the antigenic peptide transporter. Implications for the site of peptide association.

PubMed

Hayes, B K; Esquivel, F; Bennink, J R; Yewdell, J W; Varki, A

1995-10-15

Class I molecules are N-linked glycoproteins encoded by the MHC. They carry cytosolic protein-derived peptides to the cell surface, displaying them to enable immune surveillance of cellular processes. Peptides are delivered to class I molecules by the transporter associated with Ag processing (TAP). Peptide association is known to occur before exposure of class I molecules to the medial Golgi-processing enzyme alpha-mannosidase II, but there is limited information regarding the location or timing of peptide binding within the earlier regions of the endoplasmic reticulum (ER)-Golgi pathway. A reported association of newly synthesized class I molecules with the ER chaperonin calnexin raises the possibility of persistence of the monoglycosylated N-linked oligosaccharide (NLO) Glc1Man8GlcNAc2, known to be recognized by this lectin. To explore these matters, we determined the structure of the NLOs on the subset of newly synthesized class I molecules awaiting the loading of peptide. We pulse-labeled murine MHC H-2Db class I molecules in RMA/S cells, which lack one of the TAP subunits, causing the great majority of the molecules to be retained for prolonged periods in an early secretory compartment, awaiting peptide binding. MHC molecules pulse-labeled with [3H]glucosamine were isolated, the NLOs specifically released and structurally analyzed by a variety of techniques. Within the chosen window of biosynthetic time, most Db molecules from parental RMA cells carried mature NLOs of the biantennary complex-type, with one to two sialic acid residues. In RMA/S cells, such chains were in the minority, the majority consisting of the precursor forms Man8GlcNAc2 and Man9GlcNAc2. No glucosylated forms were detected, nor were the later processing intermediates Man5-7GlcNAc2 or GlcNAc1Man4-5GlcNAc2. Thus, most Db molecules in TAP-deficient cells are retained in an early compartment of the secretory pathway, before the point of first access to the Golgi alpha-mannosidase I, which trims alpha 1-2 linked mannose residues, but beyond the point where the alpha 1-3-linked glucose residue is finally removed by the ER glucosidase II. Thus, structural analysis of NLOs on class I molecules within a defined biosynthetic window has established a biochemical measure of the timing of peptide association.

The diagnostic utility of preoperative breast magnetic resonance imaging (MRI) and/or intraoperative sub-nipple biopsy in nipple-sparing mastectomy.

PubMed

Chan, S-E; Liao, C-Y; Wang, T-Y; Chen, S-T; Chen, D-R; Lin, Y-J; Chen, C-J; Wu, H-K; Chen, S-L; Kuo, S-J; Lee, C-W; Lai, H-W

2017-01-01

The necessity of routine sub-nipple biopsy was uncertain, and the role of preoperative magnetic resonance imaging (MRI) in detecting nipple invasion in patients who have been selected for nipple sparing mastectomy (NSM) has not been adequately evaluated. We retrospectively collected and analyzed the medical and surgical records of 434 patients with primary operable breast cancer who met the criteria for NSM and underwent breast surgery during the period January 2011 to December 2015. Patients were stratified into three risk groups (low, intermediate, and high) according to tumor size and tumor-to-nipple distance. Among the 434 patients in this study, 29 (6.7%) had occult invasion of the nipple-areola complex (NAC). Sub-nipple biopsy had a sensitivity of 84.6%, a specificity of 100%, a false negative rate of 1.2%, a false positive rate of 0%, and an overall accuracy rate of 98.8% in confirming NAC invasion. The NAC invasion rate was 0% in the low-risk group, 5.1% in the intermediate-risk group, and 19.7% in the high-risk group (P < 0.01). The overall NPV of preoperative MRI for predicting NAC invasion was 94.8%. Cost analysis revealed that the cost of NSM with sub-nipple biopsy was significantly higher than that of NSM alone, with a mean difference in cost of USD 238.5 (P < 0.01). The high negative predictive value of MRI for NAC invasion is useful for selection of patients receiving NSM. Sub-nipple biopsy is a reliable procedure to detect occult NAC invasion, however, routine use is not cost-effect for low risk patients. Copyright © 2016 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

Similarities between N-acetylcysteine and Glutathione in Binding to Lead(II) Ions

PubMed Central

Sisombath, Natalie S.; Jalilehvand, Farideh

2015-01-01

N -acetylcysteine is a natural thiol-containing antioxidant, a precursor for cysteine and glutathione, and a potential detoxifying agent for heavy metal ions. However, previous accounts of the efficiency of N-acetylcysteine (H2NAC) in excretion of lead are few and contradicting. Here we report results on the nature of lead(II) complexes formed with N-acetylcysteine in aqueous solution, which were obtained by combining information from several spectroscopic methods, including 207Pb, 13C and 1H NMR, Pb LIII-edge X-ray absorption, Ultraviolet-visible (UV-vis.) spectroscopy and electro-spray ionization mass spectrometry (ESI-MS). Two series of solutions were used containing CPb(II) = 10 and 100 mM, respectively, varying the H2NAC / Pb(II) mole ratios from 2.1 to 10.0 at pH = 9.1 – 9.4. The coordination environments obtained resemble those previously found for the Pb(II) glutathione system: at a ligand-to-lead mole ratio of 2.1 dimeric or oligomeric Pb(II) N-acetylcysteine complexes are formed, while a tri-thiolate [Pb(NAC)3]4− complex dominates in solutions with H2NAC/Pb(II) mole ratios > 3.0. PMID:26624959

Assessing contributions of nucleus accumbens shell subregions to reward-seeking behavior.

PubMed

Reed, Michael D; Hildebrand, David G C; Santangelo, Gabrielle; Moffa, Anthony; Pira, Ashley S; Rycyna, Lisa; Radic, Mia; Price, Katherine; Archbold, Jonathan; McConnell, Kristi; Girard, Lauren; Morin, Kristen; Tang, Anna; Febo, Marcelo; Stellar, James R

2015-08-01

The nucleus accumbens (NAc) plays a key role in brain reward processes including drug seeking and reinstatement. Several anatomical, behavioral, and neurochemical studies discriminate between the limbic-associated shell and the motor-associated core regions. Less studied is the fact that the shell can be further subdivided into a dorsomedial shell (NAcDMS) and an intermediate zone (NAcINT) based on differential expression of transient c-Fos and long-acting immediate-early gene ΔFosB upon cocaine sensitization. These disparate expression patterns suggest that NAc shell subregions may play distinct roles in reward-seeking behavior. In this study, we examined potential differences in the contributions of the NAcDMS and the NAcINT to reinstatement of reward-seeking behavior after extinction. Rats were trained to intravenously self-administer cocaine, extinguished, and subjected to a reinstatement test session consisting of an intracranial microinfusion of either amphetamine or vehicle targeted to the NAcDMS or the NAcINT. Small amphetamine microinfusions targeted to the NAcDMS resulted in statistically significant reinstatement of lever pressing, whereas no significant difference was observed for microinfusions targeted to the NAcINT. No significant difference was found for vehicle microinfusions in either case. These results suggest heterogeneity in the behavioral relevance of NAc shell subregions, a possibility that can be tested in specific neuronal populations in the future with recently developed techniques including optogenetics. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

O-GlcNAc cycling in the developing, adult and geriatric brain.

PubMed

Lagerlöf, Olof

2018-06-01

Hundreds of proteins in the nervous system are modified by the monosaccharide O-GlcNAc. A single protein is often O-GlcNAcylated on several amino acids and the modification of a single site can play a crucial role for the function of the protein. Despite its complexity, only two enzymes add and remove O-GlcNAc from proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Global and local regulation of these enzymes make it possible for O-GlcNAc to coordinate multiple cellular functions at the same time as regulating specific pathways independently from each other. If O-GlcNAcylation is disrupted, metabolic disorder or intellectual disability may ensue, depending on what neurons are affected. O-GlcNAc's promise as a clinical target for developing drugs against neurodegenerative diseases has been recognized for many years. Recent literature puts O-GlcNAc in the forefront among mechanisms that can help us better understand how neuronal circuits integrate diverse incoming stimuli such as fluctuations in nutrient supply, metabolic hormones, neuronal activity and cellular stress. Here the functions of O-GlcNAc in the nervous system are reviewed.

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harada, Y.; Li, H.; Li, Hua

2009-04-28

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of {approx}1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings,more » we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.« less

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

PubMed Central

Harada, Yoichiro; Li, Hua; Li, Huilin; Lennarz, William J.

2009-01-01

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of ≈1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes. PMID:19365066

Region-specific role of Rac in nucleus accumbens core and basolateral amygdala in consolidation and reconsolidation of cocaine-associated cue memory in rats.

PubMed

Ding, Zeng-Bo; Wu, Ping; Luo, Yi-Xiao; Shi, Hai-Shui; Shen, Hao-Wei; Wang, Shen-Jun; Lu, Lin

2013-08-01

Drug reinforcement and the reinstatement of drug seeking are associated with the pathological processing of drug-associated cue memories that can be disrupted by manipulating memory consolidation and reconsolidation. Ras-related C3 botulinum toxin substrate (Rac) is involved in memory processing by regulating actin dynamics and neural structure plasticity. The nucleus accumbens (NAc) and amygdala have been implicated in the consolidation and reconsolidation of emotional memories. Therefore, we hypothesized that Rac in the NAc and amygdala plays a role in the consolidation and reconsolidation of cocaine-associated cue memory. Conditioned place preference (CPP) and microinjection of Rac inhibitor NSC23766 were used to determine the role of Rac in the NAc and amygdala in the consolidation and reconsolidation of cocaine-associated cue memory in rats. Microinjections of NSC23766 into the NAc core but not shell, basolateral (BLA), or central amygdala (CeA) after each cocaine-conditioning session inhibited the consolidation of cocaine-induced CPP. A microinjection of NSC23766 into the BLA but not CeA, NAc core, or NAc shell immediately after memory reactivation induced by exposure to a previously cocaine-paired context disrupted the reconsolidation of cocaine-induced CPP. The effect of memory disruption on cocaine reconsolidation was specific to reactivated memory, persisted at least 2 weeks, and was not reinstated by a cocaine-priming injection. Our findings indicate that Rac in the NAc core and BLA are required for the consolidation and reconsolidation of cocaine-associated cue memory, respectively.

Immunological tumor status may predict response to neoadjuvant chemotherapy and outcome after radical cystectomy in bladder cancer.

PubMed

Tervahartiala, Minna; Taimen, Pekka; Mirtti, Tuomas; Koskinen, Ilmari; Ecke, Thorsten; Jalkanen, Sirpa; Boström, Peter J

2017-10-04

Bladder cancer (BC) is the ninth most common cancer worldwide. Radical cystectomy (RC) with neoadjuvant chemotherapy (NAC) is recommended for muscle-invasive BC. The challenge of the neoadjuvant approach relates to challenges in selection of patients to chemotherapy that are likely to respond to the treatment. To date, there are no validated molecular markers or baseline clinical characteristics to identify these patients. Different inflammatory markers, including tumor associated macrophages with their plastic pro-tumorigenic and anti-tumorigenic functions, have extensively been under interests as potential prognostic and predictive biomarkers in different cancer types. In this immunohistochemical study we evaluated the predictive roles of three immunological markers, CD68, MAC387, and CLEVER-1, in response to NAC and outcome of BC. 41% of the patients had a complete response (pT0N0) to NAC. Basic clinicopathological variables did not predict response to NAC. In contrast, MAC387 + cells and CLEVER-1 + macrophages associated with poor NAC response, while CLEVER-1 + vessels associated with more favourable response to NAC. Higher counts of CLEVER-1 + macrophages associated with poorer overall survival and CD68 + macrophages seem to have an independent prognostic value in BC patients treated with NAC. Our findings point out that CD68, MAC387, and CLEVER-1 may be useful prognostic and predictive markers in BC.

Rod outer segment-associated N-acetylgalactosaminylphosphotransferase.

PubMed

Sweatt, A J; Balsamo, J; Lilien, J

1995-01-01

To determine the exact location of a cell surface glycosyltransferase (N-acetylgalactosaminylphosphotransferase, (GalNAcPTase) immunochemically identified in mammalian rod outer segments (ROS), to determine whether anti-GalNAcPTase antibody recognizes retinal molecules that possess transferase activity and to characterize ROS transferase enzyme activity and acceptors. The GalNAcPTase is known to be associated with the adhesion molecule N-cadherin in embryonic avian retinas and with E-cadherin in mammalian pancreatic islet cells. Purified, fixed ROS were reacted with anti-chick GalNAcPTase antibody followed by secondary antibody conjugated to colloidal gold and were examined by electron microscopy. Fractions of retinal and ROS proteins enriched in the transferase were obtained through batch adsorption on Sepharose, separated by gel electrophoresis, transferred to nitrocellulose, and either reacted with anti-GalNAcPTase antibody or assayed for transferase activity. Interphotoreceptor matrix (IPM) was examined for the presence of immunoreactive GalNAcPTase by gel electrophoresis and immunoblot. The kinetics and endogenous acceptors of the cow ROS transferase were characterized. ROS are specifically labeled by anti-GalNAcPTase antibody at the cell surface. The immunogold label was associated with the cell surface and with flocculent material adherent to the cell surface. In addition, soluble and particulate fractions of the IPM showed GalNAcPTase-like immunoreactivity. The transferase appears as single immunoreactive band at or near 220 kd. Transferase enzyme activity was present at this position on Western transfers of retinal and ROS proteins. In whole ROS, transferase activity was directed toward endogenous acceptors of very high molecular mass. The GalNAcPTase is localized on ROS in association with the cell surface and with components of the IPM. The molecule recognized by the anti-GalNAcPTase antibody possesses transferase activity toward itself and a few other proteins, but mostly toward very large molecules that may be IPM proteoglycans. It is not yet known whether the enzyme of the adult retina specifically transfers sugar or sugar-phosphate groups to its acceptors. It is proposed that the ROS GalNAcPTase is involved in the modulation of adhesive phenomena between or within photoreceptors or between photoreceptors and the interphotoreceptor matrix.

N-acetylglucosamine affects Cryptococcus neoformans cell-wall composition and melanin architecture.

PubMed

Camacho, Emma; Chrissian, Christine; Cordero, Radames J B; Liporagi-Lopes, Livia; Stark, Ruth E; Casadevall, Arturo

2017-11-01

Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15 N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother-daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In summary, GlcNAc supplementation had pleiotropic effects on cell-wall and melanin architectures, and thus established its capacity to perturb these structures, a property that could prove useful for metabolic tracking studies.

N-acetylglucosamine affects Cryptococcus neoformans cell-wall composition and melanin architecture

PubMed Central

Camacho, Emma; Chrissian, Christine; Cordero, Radames J. B.; Liporagi-Lopes, Livia; Stark, Ruth E.; Casadevall, Arturo

2017-01-01

Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother–daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In summary, GlcNAc supplementation had pleiotropic effects on cell-wall and melanin architectures, and thus established its capacity to perturb these structures, a property that could prove useful for metabolic tracking studies. PMID:29043954

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

Membrane-associated precursor to poliovirus VPg identified by immunoprecipitation with antibodies directed against a synthetic heptapeptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Semelr, B.L.; Anderson, C.W.; Hanecak, R.

1982-02-01

A synthetic heptapeptide corresponding to the C-terminal sequence of the poliovirus genome protein (VPg) has been linked to bovine serum albumin and used to raise antibodies in rabbits. These antibodies precipitate not only VPg but also at least two more virus-specific polypeptides. The smaller polypeptide, denoted P3-9 (12,000 daltons), has been mapped by Edman degradation and by fragmentation with cyanogen bromide and determined to be the N-terminal cleavage product of polypeptide P3-1b, a precursor to the RNA polymerase. P3-9 contains the sequence of the basic protein VPg (22 amino acids) at its C terminus. As predicted by the known RNAmore » sequence of poliovirus, P3-9 also contains a hydrophobic region of 22 amino acids preceding VPg, an observation suggesting that P3-9 may be membrane-associated. This was confirmed by fractionation of infected cells in the presence or absence of detergent. We speculate that P3-9 may be the donor of VPg to RNA chains in the membrane-bound RNA replication complex.« less

Structural and functional diversity in Listeria cell wall teichoic acids.

PubMed

Shen, Yang; Boulos, Samy; Sumrall, Eric; Gerber, Benjamin; Julian-Rodero, Alicia; Eugster, Marcel R; Fieseler, Lars; Nyström, Laura; Ebert, Marc-Olivier; Loessner, Martin J

2017-10-27

Wall teichoic acids (WTAs) are the most abundant glycopolymers found on the cell wall of many Gram-positive bacteria, whose diverse surface structures play key roles in multiple biological processes. Despite recent technological advances in glycan analysis, structural elucidation of WTAs remains challenging due to their complex nature. Here, we employed a combination of ultra-performance liquid chromatography-coupled electrospray ionization tandem-MS/MS and NMR to determine the structural complexity of WTAs from Listeria species. We unveiled more than 10 different types of WTA polymers that vary in their linkage and repeating units. Disparity in GlcNAc to ribitol connectivity, as well as variable O -acetylation and glycosylation of GlcNAc contribute to the structural diversity of WTAs. Notably, SPR analysis indicated that constitution of WTA determines the recognition by bacteriophage endolysins. Collectively, these findings provide detailed insight into Listeria cell wall-associated carbohydrates, and will guide further studies on the structure-function relationship of WTAs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of laser-assisted lipolysis on nipple-areola complex.

PubMed

Sönmez Ergün, Selma; Kayan, Reşit Burak; Güleş, Mustafa Ekrem; Kuzu, İsmail Melih

2017-08-01

Gynecomastia, as a most common benign condition, represents itself as the enlargement of the male breast and also nipple-areola complex as the severity of the condition increases. With this study, we aimed to clarify the effects of 980-nm diode laser on nipple-areola complex (NAC). Although numerous open techniques have been described to correct gynecomastia, nowadays trends have shifted to minimally invasive techniques such as laser-assisted lipolysis (LAL). A total of 25 patients with bilateral gynecomastia treated with LAL by using a 980-nm diode laser. The resultant contour and reduced size of the complex were satisfactory. LAL leads to significant reduction of the size of NAC.

The number of cycles of neoadjuvant chemotherapy is associated with prognosis of stage IIIc-IV high-grade serous ovarian cancer.

PubMed

Xu, Xia; Deng, Fei; Lv, Mengmeng; Chen, Xiaoxiang

2017-02-01

No consensus exists on the number of chemotherapy cycles to be administered before and after interval debulking surgery (IDS) in patients with advanced stage epithelial ovarian cancer. The present study aims to explore the optimal number of cycles of neoadjuvant chemotherapy (NAC) and post-operation chemotherapy to treat the International Federation of Gynecology and Obstetrics stage IIIc-IV high-grade serous ovarian cancer (HG-SOC). A total of 129 IIIc-IV stage HG-SOC cases were retrospectively analyzed. Cases were comprised of patients who underwent NAC followed by IDS and who achieved clinical complete response (CCR) at the end of primary therapy. Patients were recruited from the Jiangsu Institute of Cancer Research between 1993 and 2013. Optimal IDS-associated factors were explored with logistic regression. The association between progression-free survival (PFS), overall survival (OS) duration, and covariates was assessed by Cox proportional hazards model and log-rank test. The median number of NAC cycle was 3 (range 1-8). CA-125 decreasing kinetics (p = 0.01) was independently associated with optimal IDS. CA-125 decreasing kinetics, optimal IDS, and NAC cycles was independently associated with OS (p < 0.01, p < 0.01, p = 0.03, respectively) and PFS (p < 0.01, p < 0.01, p = 0.04, respectively). The PFS of patients who underwent ≥5 NAC cycles was shorter than those of patients who underwent <5 NAC cycles (12.3 versus 17.2 months). The PFS and OS of patients who underwent <5 cycles of adjuvant chemotherapy post-IDS were shorter than those of patients who underwent ≥5 cycles (14.2 and 20.3 versus 21.2 and 28.8 months). NAC cycles, CA-125 decreasing kinetics, and optimal debulking are independently associated with the prognosis of patients with advanced stage HG-SOC who underwent NAC/IDS and achieved CCR. The number of administered NAC cycles should not exceed 4.

CBP70, a glycosylated nuclear lectin.

PubMed

Rousseau, C; Felin, M; Doyennette-Moyne, M A; Sève, A P

1997-09-01

Some years ago, a lectin designated CBP70 that recognized glucose (Glc) but had a stronger affinity for N-acetylglucosamine (GlcNAc), was first isolated from HL60 cell nuclei. Recently, a cytoplasmic form of this lectin was described, and one 82 kDa nuclear ligand was characterized for the nuclear CBP70. In the present study, the use of Pronase digestion and the trifluoromethanesulphonic acid (TFMS) procedure strongly suggest that the nuclear and the cytoplasmic CBP70 have a same 23 kDa polypeptide backbone and, consequently, could be the same protein. In order to know the protein better and to obtain the best recombinant possible in the future, the post-translational modification of the nuclear and cytoplasmic CBP70 was analyzed in terms of glycosylation. Severals lines of evidence indicate that both forms of CBP70 are N- and O-glycosylated. Surprisingly, this glycosylation pattern differs between the two forms, as revealed by beta-elimination, hydrazinolysis, peptide-N-glycosydase F (PNGase F), and TFMS reactions. The two preparations were analyzed by affinity chromatography on immobilized lectins [Ricinus communis-l agglutinin (RCA-I), Arachis hypogaea agglutinin (PNA), Galanthus nivalis agglutinin (GNA), and wheat germ agglutinin (WGA)] and by lectin-blotting analysis Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), Lotus tetragonolobus (Lotus), succinylated-WGA, and Psathyrella velutina agglutinin (PVA)]. Both forms of CBP70 have the following sugar moities: terminal beta Gal residues, Gal beta 1-3 GalNAc, Man alpha 1-3 Man, sialic acid alpha 2-6 linked to Gal or GalNAc; and sialic acid alpha 2-3 linked to Gal. However, only nuclear CBP70 have terminal GlcNAc and alpha-L-fucose residues. All these data are consistent with the fact that different glycosylation pattern found for each form of CBP70 might act as a complementary signal for cellular targeting.

Biomimetic assembly of polypeptide-stabilized CaCO(3) nanoparticles.

PubMed

Zhang, Zhongping; Gao, Daming; Zhao, Hui; Xie, Chenggen; Guan, Guijian; Wang, Dapeng; Yu, Shu-Hong

2006-05-04

In this paper, we report a simple polypeptide-directed strategy for fabricating large spherical assembly of CaCO(3) nanoparticles. Stepwise growth and assembly of a large number of nanoparticles have been observed, from the formation of an amorphous liquidlike CaCO(3)-polypeptide precursor, to the crystallization and stabilization of polypeptide-capped nanoparticles, and eventually, the spherical assembly of nanoparticles. The "soft" poly(aspartate)-capping layer binding on a nanoparticle surface resulted in the unusual soft nature of nanoparticle assembly, providing a reservoir of primary nanoparticles with a moderate mobility, which is the basis of a new strategy for reconstructing nanoparticle assembly into complex nanoparticle architectures. Moreover, the findings of the secondary assembly of nanoparticle microspheres and the morphology transformation of nanoparticle assembly demonstrate a flexible and controllable pathway for manipulating the shapes and structures of nanoparticle assembly. In addition, the combination of the polypeptide with a double hydrophilic block copolymer (DHBC) allows it to possibly further control the shape and complexity of the nanoparticle assembly. A clear perspective is shown here that more complex nanoparticle materials could be created by using "soft" biological proteins or peptides as a mediating template at the organic-inorganic interface.

Crystal structure of product-bound complex of UDP-N-acetyl-D-mannosamine dehydrogenase from Pyrococcus horikoshii OT3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pampa, K.J., E-mail: sagarikakj@gmail.com; Lokanath, N.K.; Girish, T.U.

Highlights: • Determined the structure of UDP-D-ManNAcADH to a resolution of 1.55 Å. • First complex structure of PhUDP-D-ManNAcADH with UDP-D-ManMAcA. • The monomeric structure consists of three distinct domains. • Cys258 acting as catalytic nucleophilic and Lys204 acts as acid/base catalyst. • Oligomeric state plays an important role for the catalytic function. - Abstract: UDP-N-acetyl-D-mannosamine dehydrogenase (UDP-D-ManNAcDH) belongs to UDP-glucose/GDP-mannose dehydrogenase family and catalyzes Uridine-diphospho-N-acetyl-D-mannosamine (UDP-D-ManNAc) to Uridine-diphospho-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA) through twofold oxidation of NAD{sup +}. In order to reveal the structural features of the Pyrococcus horikoshii UDP-D-ManNAcADH, we have determined the crystal structure of the product-bound enzyme bymore » X-ray diffraction to resolution of 1.55 Å. The protomer folds into three distinct domains; nucleotide binding domain (NBD), substrate binding domain (SBD) and oligomerization domain (OD, involved in the dimerization). The clear electron density of the UDP-D-ManNAcA is observed and the residues binding are identified for the first time. Crystal structures reveal a tight dimeric polymer chains with product-bound in all the structures. The catalytic residues Cys258 and Lys204 are conserved. The Cys258 acts as catalytic nucleophile and Lys204 as acid/base catalyst. The product is directly interacts with residues Arg211, Thr249, Arg244, Gly255, Arg289, Lys319 and Arg398. In addition, the structural parameters responsible for thermostability and oligomerization of the three dimensional structure are analyzed.« less

Defining Normal Parameters for the Male Nipple-Areola Complex: A Prospective Observational Study and Recommendations for Placement on the Chest Wall.

PubMed

Yue, Dominic; Cooper, Lilli R L; Kerstein, Ryan; Charman, Susan C; Kang, Norbert V

2018-01-10

The nipple-areola complex (NAC) is important aesthetically and functionally for both sexes. Methods for positioning the NAC in males are less well established in the literature compared to females but are just as important. This study aims to determine the normal parameters for the male NAC, to review literature, and to present a reliable method for preoperative placement. Normal male patients, with no prior chest wall conditions, were prospectively recruited to participate. General demographics and chest wall dimensions were recorded-sternal notch to nipple (SNND), internipple (IND), anterior axillary folds distances (AFD), NAC, and chest circumference were measured. Comparisons were made using t test and ANOVA. One hundred and fifty-eight patients were recruited (age range, 18-90 years); mostly (86.7%) with normal or overweight BMI. The IND averaged 249.4 mm, the SNND averaged 204.2 mm, and the AFD averaged 383.8 mm. Areola diameter averaged 26.6 mm and for the nipple, 6.9 mm. The IND:AFD ratio was 0.65. There was no statistical difference in the IND:AFD ratio, SNND, or NAC parameters comparing different ethnic groups. The SNND increased with greater BMI (P ≤ 0.001). Using these data, we suggest ideal NAC dimensions and devised a simple method for positioning of the NAC on the male chest wall. This is the largest study, with the widest range in age and BMI, to date on this topic. Although fewer men than women undergo surgery to the breast, there is a growing awareness for enhancing the appearance of the male chest wall. © 2018 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

Differential expression of isoproterenol-induced salivary polypeptides in two mouse strains that are congenic for the H-2 histocompatibility gene complex.

PubMed

López Solís, Remigio O; Weis, Ulrike Kemmerling; Ceballos, Alicia Ramos; Salas, Gustavo Hoecker

2003-12-01

Two inbred mouse strains, A/Snell and A.Swiss, which were produced as congenic with regard to the H-2 histocompatibility gene complex, are homozygous for two different groups of isoproterenol-induced salivary polypeptides (IISP). These polypeptides, which have been considered as markers of the hypertrophic growth of the parotid acinar cells, are members of the complex family of salivary proline-rich proteins (PRP) on the basis of both their massive accumulation in the parotid acinar cells in response to chronic isoproterenol, secretory character, high solubility in trichloroacetic acid and metachromatic staining by Coomassie blue. IISP expressed in both mouse strains were identified by unidimensional SDS-polyacrylamide electrophoresis and Coomassie blue staining both in parotid gland homogenates and in whole salivas obtained from mice repeatedly stimulated at 24-h intervals with isoproterenol. Parotid glands from 40 mice (20 A/Snell and 20 A.Swiss) and salivas from 270 mice (200 A/Snell and 70 A.Swiss) were analyzed. One of the congenic strains (A/Snell) expressed five IISP (Mr 65, 61, 51.5, 38, and 37 kDa) and the other strain (A.Swiss) expressed six IISP (Mr 59, 57, 54.5, 46, 36, and 34 kDa). No inter-individual intra-strain variations were observed, thus defining strain-associated patterns of IISP (PRP). Copyright 2003 Wiley-Liss, Inc.

Circulating blood and platelets supply glycosyltransferases that enable extrinsic extracellular glycosylation.

PubMed

Lee-Sundlov, Melissa M; Ashline, David J; Hanneman, Andrew J; Grozovsky, Renata; Reinhold, Vernon N; Hoffmeister, Karin M; Lau, Joseph Ty

2017-01-01

Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

PubMed

Gradišar, Helena; Božič, Sabina; Doles, Tibor; Vengust, Damjan; Hafner-Bratkovič, Iva; Mertelj, Alenka; Webb, Ben; Šali, Andrej; Klavžar, Sandi; Jerala, Roman

2013-06-01

Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

Predictors of candidaemia caused by non-albicans Candida species: results of a population-based surveillance in Barcelona, Spain.

PubMed

Rodríguez, D; Almirante, B; Cuenca-Estrella, M; Rodríguez-Tudela, J L; Mensa, J; Ayats, J; Sanchez, F; Pahissa, A

2010-11-01

Although Candida albicans (CA) is the most common cause of Candida bloodstream infections (BSIs), recent studies have observed an increasing percentage of candidaemias caused by non-albicans Candida species (NAC). In the present study, we attempted to identify the predictors of candidaemia due to NAC compared to CA. We analyzed data from an active population-based surveillance in Barcelona (Spain) from January 2002 to December 2003. Factors associated with NAC fungaemia were determined by multivariate analysis. A total of 339 episodes of Candida BSI, in 336 patients (median age 63 years, interquartile range: 41-72 years), were included. CA was the most commonly isolated (52%), followed by Candida parapsilosis (23%), Candida tropicalis (10%), Candida glabrata (8.6%), Candida krusei (3.4%) and other NAC spp. (3%).Overall, 48% of cases were due to NAC spp. Multivariate logistic regression analysis identified factors associated with a risk of BSI due to NAC spp.: having received a haematologic transplant (OR 10.8; 95% CI 1.31-90.01; p 0.027), previous fluconazole exposure (OR 4.47; 95% CI 2.12-9.43; p <0.001) and neonatal age (OR 4.42; 95% CI 1.63-12.04; p 0.004). Conversely, previous CA colonization (OR 0.33; 95% CI 0.19-0.57; p 0.001) and previous antibiotic use (OR 0.42; 95% CI 0.21-0.85; p 0.017) were associated with CA fungaemia compared to NAC. In conclusion, NAC candidaemia comprised 48% of cases in our series. Predictors of NAC include having received a haematologic transplant, neonatal age and previous fluconazole use. © 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.

The nucleus accumbens 5-HTR₄-CART pathway ties anorexia to hyperactivity.

PubMed

Jean, A; Laurent, L; Bockaert, J; Charnay, Y; Dusticier, N; Nieoullon, A; Barrot, M; Neve, R; Compan, V

2012-12-11

In mental diseases, the brain does not systematically adjust motor activity to feeding. Probably, the most outlined example is the association between hyperactivity and anorexia in Anorexia nervosa. The neural underpinnings of this 'paradox', however, are poorly elucidated. Although anorexia and hyperactivity prevail over self-preservation, both symptoms rarely exist independently, suggesting commonalities in neural pathways, most likely in the reward system. We previously discovered an addictive molecular facet of anorexia, involving production, in the nucleus accumbens (NAc), of the same transcripts stimulated in response to cocaine and amphetamine (CART) upon stimulation of the 5-HT(4) receptors (5-HTR(4)) or MDMA (ecstasy). Here, we tested whether this pathway predisposes not only to anorexia but also to hyperactivity. Following food restriction, mice are expected to overeat. However, selecting hyperactive and addiction-related animal models, we observed that mice lacking 5-HTR(1B) self-imposed food restriction after deprivation and still displayed anorexia and hyperactivity after ecstasy. Decryption of the mechanisms showed a gain-of-function of 5-HTR(4) in the absence of 5-HTR(1B), associated with CART surplus in the NAc and not in other brain areas. NAc-5-HTR(4) overexpression upregulated NAc-CART, provoked anorexia and hyperactivity. NAc-5-HTR(4) knockdown or blockade reduced ecstasy-induced hyperactivity. Finally, NAc-CART knockdown suppressed hyperactivity upon stimulation of the NAc-5-HTR(4). Additionally, inactivating NAc-5-HTR(4) suppressed ecstasy's preference, strengthening the rewarding facet of anorexia. In conclusion, the NAc-5-HTR(4)/CART pathway establishes a 'tight-junction' between anorexia and hyperactivity, suggesting the existence of a primary functional unit susceptible to limit overeating associated with resting following homeostasis rules.

The nucleus accumbens 5-HTR4-CART pathway ties anorexia to hyperactivity

PubMed Central

Jean, A; Laurent, L; Bockaert, J; Charnay, Y; Dusticier, N; Nieoullon, A; Barrot, M; Neve, R; Compan, V

2012-01-01

In mental diseases, the brain does not systematically adjust motor activity to feeding. Probably, the most outlined example is the association between hyperactivity and anorexia in Anorexia nervosa. The neural underpinnings of this ‘paradox', however, are poorly elucidated. Although anorexia and hyperactivity prevail over self-preservation, both symptoms rarely exist independently, suggesting commonalities in neural pathways, most likely in the reward system. We previously discovered an addictive molecular facet of anorexia, involving production, in the nucleus accumbens (NAc), of the same transcripts stimulated in response to cocaine and amphetamine (CART) upon stimulation of the 5-HT4 receptors (5-HTR4) or MDMA (ecstasy). Here, we tested whether this pathway predisposes not only to anorexia but also to hyperactivity. Following food restriction, mice are expected to overeat. However, selecting hyperactive and addiction-related animal models, we observed that mice lacking 5-HTR1B self-imposed food restriction after deprivation and still displayed anorexia and hyperactivity after ecstasy. Decryption of the mechanisms showed a gain-of-function of 5-HTR4 in the absence of 5-HTR1B, associated with CART surplus in the NAc and not in other brain areas. NAc-5-HTR4 overexpression upregulated NAc-CART, provoked anorexia and hyperactivity. NAc-5-HTR4 knockdown or blockade reduced ecstasy-induced hyperactivity. Finally, NAc-CART knockdown suppressed hyperactivity upon stimulation of the NAc-5-HTR4. Additionally, inactivating NAc-5-HTR4 suppressed ecstasy's preference, strengthening the rewarding facet of anorexia. In conclusion, the NAc-5-HTR4/CART pathway establishes a ‘tight-junction' between anorexia and hyperactivity, suggesting the existence of a primary functional unit susceptible to limit overeating associated with resting following homeostasis rules. PMID:23233022

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis

PubMed Central

1978-01-01

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome- tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate- sized filaments is discussed. PMID:569157

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.

PubMed

Drochmans, P; Freudenstein, C; Wanson, J C; Laurent, L; Keenan, T W; Stadler, J; Leloup, R; Franke, W W

1978-11-01

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate-sized filaments is discussed.

Mechanisms and kinetics of alkaline hydrolysis of the energetic nitroaromatic compounds 2,4,6-trinitrotoluene (TNT) and 2,4-dinitroanisole (DNAN).

PubMed

Salter-Blanc, Alexandra J; Bylaska, Eric J; Ritchie, Julia J; Tratnyek, Paul G

2013-07-02

The environmental impacts of energetic compounds can be minimized through the design and selection of new energetic materials with favorable fate properties. Building predictive models to inform this process, however, is difficult because of uncertainties and complexities in some major fate-determining transformation reactions such as the alkaline hydrolysis of energetic nitroaromatic compounds (NACs). Prior work on the mechanisms of the reaction between NACs and OH(-) has yielded inconsistent results. In this study, the alkaline hydrolysis of 2,4,6-trinitrotoluene (TNT) and 2,4-dinitroanisole (DNAN) was investigated with coordinated experimental kinetic measurements and molecular modeling calculations. For TNT, the results suggest reversible formation of an initial product, which is likely either a Meisenheimer complex or a TNT anion formed by abstraction of a methyl proton by OH(-). For DNAN, the results suggest that a Meisenheimer complex is an intermediate in the formation of 2,4-dinitrophenolate. Despite these advances, the remaining uncertainties in the mechanisms of these reactions-and potential variability between the hydrolysis mechanisms for different NACs-mean that it is not yet possible to generalize the results into predictive models (e.g., quantitative structure-activity relationships, QSARs) for hydrolysis of other NACs.

Shape, Position and Dimension of the Nipple Areola Complex in the Ideal Male Chest: A Quick and Simple Operating Room Technique.

PubMed

Tanini, Sara; Lo Russo, Giulia

2018-04-11

The anatomical features of the chest identify an individual as male or female and even the smallest details of these features determine the appropriate appearance for each gender. In female-to-male patients, the creation of an aesthetically pleasing male chest is the most important step. Incorrect positioning of the nipple areola complex (NAC) on the chest wall and suboptimal shaping and sizing of the NAC are common pitfalls in male NAC creation. We have analyzed the anatomical chest features of 26 water polo players, to verify our hypothesis of the relationship between the pectoralis major muscle and NAC and to create a method for repositioning the NAC that is applicable in the operating room, is easy, practical and reproducible without the use of formulas and based on an easily identifiable landmark. In our reference group, the NAC has a constant relationship with the pectoralis major muscle, positioned on average 3 cm medial to the lateral border of the pectoralis muscle and 2.5 cm above the inferior pectoralis major insertion. This supports our hypothesis and our surgical technique. We use the index finger to find a vertical axis and a line 2.5 cm above the inferior pectoralis shadow to find the horizontal axis. We also introduce a modification to the receiving site to recreate an oval areola more similar to that of an ideal male chest. Our anatomical study and statistical analysis support a consistent relationship between the position and shape of the NAC and the borders of the pectoral muscle. We have used this relationship to develop our "trick," which is easily applicable in the operating room to find the NAC position without using formulas and numbers. This method allowed us to place the NAC in a position very close to that of a typical male subject, and it permitted us to reduce the surgery time. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Heat-induced formation of a specific binding site for self-assembled Congo Red in the V domain of immunoglobulin L chain lambda.

PubMed

Piekarska, B; Konieczny, L; Rybarska, J; Stopa, B; Zemanek, G; Szneler, E; Król, M; Nowak, M; Roterman, I

2001-11-01

Moderate heating (40-50 degrees C) of immunoglobulins makes them accessible for binding with Congo Red and some related highly associated dyes. The binding is specific and involves supramolecular dye ligands presenting ribbon-like micellar bodies. The L chain lambda dimer, which upon heating disclosed the same binding requirement with respect to supramolecular dye ligands, was used in this work to identify the site of their attachment. Two clearly defined dye-protein (L lambda chain) complexes arise upon heating, here called complex I and complex II. The first is formed at low temperatures (up to 40-45 degrees C) and hence by a still native protein, while the formation of the second one is associated with domain melting above 55 degrees C. They contain 4 and 8 dye molecules bound per L chain monomer, respectively. Complex I also forms efficiently at high dye concentration even at ambient temperature. Complex I and its formation was the object of the present studies. Three structural events that could make the protein accessible to penetration by the large dye ligand were considered to occur in L chains upon heating: local polypeptide chain destabilization, VL-VL domain incoherence, and protein melting. Of these three possibilities, local low-energy structural alteration was found to correlate best with the formation of complex I. It was identified as decreased packing stability of the N-terminal polypeptide chain fragment, which as a result made the V domain accessible for dye penetration. The 19-amino acid N-terminal fragment becomes susceptible to proteolytic cleavage after being replaced by the dye at its packing locus. Its splitting from the dye-protein complex was proved by amino acid sequence analysis. The emptied packing locus, which becomes the site that holds the dye, is bordered by strands of amino acids numbered 74-80 and 105-110, as shown by model analysis. The character of the temperature-induced local polypeptide chain destabilization and its possible role in intramolecular antibody signaling is discussed. Copyright 2001 John Wiley & Sons, Inc.

Structure-Guided Functional Characterization of DUF1460 Reveals a Highly Specific NlpC/P60 Amidase Family

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Patin, Delphine; ...

2014-11-20

GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a novel, highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo-structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predictedmore » in silico based on structural and bioinformatics data, and were subsequently characterized experimentally. Ultimately, further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines a new amidase family.« less

skNAC, a Smyd1-interacting transcription factor, is involved in cardiac development and skeletal muscle growth and regeneration.

PubMed

Park, Chong Yon; Pierce, Stephanie A; von Drehle, Morgan; Ivey, Kathryn N; Morgan, Jayson A; Blau, Helen M; Srivastava, Deepak

2010-11-30

Cardiac and skeletal muscle development and maintenance require complex interactions between DNA-binding proteins and chromatin remodeling factors. We previously reported that Smyd1, a muscle-restricted histone methyltransferase, is essential for cardiogenesis and functions with a network of cardiac regulatory proteins. Here we show that the muscle-specific transcription factor skNAC is the major binding partner for Smyd1 in the developing heart. Targeted deletion of skNAC in mice resulted in partial embryonic lethality by embryonic day 12.5, with ventricular hypoplasia and decreased cardiomyocyte proliferation that were similar but less severe than in Smyd1 mutants. Expression of Irx4, a ventricle-specific transcription factor down-regulated in hearts lacking Smyd1, also depended on the presence of skNAC. Viable skNAC(-/-) adult mice had reduced postnatal skeletal muscle growth and impaired regenerative capacity after cardiotoxin-induced injury. Satellite cells isolated from skNAC(-/-) mice had impaired survival compared with wild-type littermate satellite cells. Our results indicate that skNAC plays a critical role in ventricular cardiomyocyte expansion and regulates postnatal skeletal muscle growth and regeneration in mice.

Structure-Guided Functional Characterization of DUF1460 Reveals a Highly Specific NlpC/P60 Amidase Family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Patin, Delphine

GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a novel, highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo-structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predictedmore » in silico based on structural and bioinformatics data, and were subsequently characterized experimentally. Ultimately, further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines a new amidase family.« less

Structure-guided functional characterization of DUF1460 reveals a highly specific NlpC/P60 amidase family.

PubMed

Xu, Qingping; Mengin-Lecreulx, Dominique; Patin, Delphine; Grant, Joanna C; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W; Godzik, Adam; Lesley, Scott A; Elsliger, Marc-André; Deacon, Ashley M; Wilson, Ian A

2014-12-02

GlcNAc-1,6-anhydro-MurNAc-tetrapeptide is a major peptidoglycan degradation intermediate and a cytotoxin. It is generated by lytic transglycosylases and further degraded and recycled by various enzymes. We have identified and characterized a highly specific N-acetylmuramoyl-L-alanine amidase (AmiA) from Bacteroides uniformis, a member of the DUF1460 protein family, that hydrolyzes GlcNAc-1,6-anhydro-MurNAc-peptide into disaccharide and stem peptide. The high-resolution apo structure at 1.15 Å resolution shows that AmiA is related to NlpC/P60 γ-D-Glu-meso-diaminopimelic acid amidases and shares a common catalytic core and cysteine peptidase-like active site. AmiA has evolved structural adaptations that reconfigure the substrate recognition site. The preferred substrates for AmiA were predicted in silico based on structural and bioinformatics data, and subsequently were characterized experimentally. Further crystal structures of AmiA in complexes with GlcNAc-1,6-anhydro-MurNAc and GlcNAc have enabled us to elucidate substrate recognition and specificity. DUF1460 is highly conserved in structure and defines another amidase family. Copyright © 2014 Elsevier Ltd. All rights reserved.

You are what you eat: O-linked N-acetylglucosamine in disease, development and epigenetics.

PubMed

Olivier-Van Stichelen, Stéphanie; Hanover, John A

2015-07-01

The O-linked N-acetylglucosamine (O-GlcNAc) modification is both responsive to nutrient availability and capable of altering intracellular cellular signalling. We summarize data defining a role for O-GlcNAcylation in metabolic homeostasis and epigenetic regulation of development in the intrauterine environment. O-GlcNAc transferase (OGT) catalyzes nutrient-driven O-GlcNAc addition and is subject to random X-inactivation. OGT plays key roles in growth factor signalling, stem cell biology, epigenetics and possibly imprinting. The O-GlcNAcase, which removes O-GlcNAc, is subject to tight regulation by higher order chromatin structure. O-GlcNAc cycling plays an important role in the intrauterine environment wherein OGT expression is an important biomarker of placental stress. Regulation of O-GlcNAc cycling by X-inactivation, epigenetic regulation and nutrient-driven processes makes it an ideal candidate for a nutrient-dependent epigenetic regulator of human disease. In addition, O-GlcNAc cycling influences chromatin modifiers critical to the regulation and timing of normal development including the polycomb repression complex and the ten-eleven translocation proteins mediating DNA methyl cytosine demethylation. The pathway also impacts the hypothalamic-pituitary-adrenal axis critical to intrauterine programming influencing disease susceptibility in later life.

NAC transcription factor genes: genome-wide identification, phylogenetic, motif and cis-regulatory element analysis in pigeonpea (Cajanus cajan (L.) Millsp.).

PubMed

Satheesh, Viswanathan; Jagannadham, P Tej Kumar; Chidambaranathan, Parameswaran; Jain, P K; Srinivasan, R

2014-12-01

The NAC (NAM, ATAF and CUC) proteins are plant-specific transcription factors implicated in development and stress responses. In the present study 88 pigeonpea NAC genes were identified from the recently published draft genome of pigeonpea by using homology based and de novo prediction programmes. These sequences were further subjected to phylogenetic, motif and promoter analyses. In motif analysis, highly conserved motifs were identified in the NAC domain and also in the C-terminal region of the NAC proteins. A phylogenetic reconstruction using pigeonpea, Arabidopsis and soybean NAC genes revealed 33 putative stress-responsive pigeonpea NAC genes. Several stress-responsive cis-elements were identified through in silico analysis of the promoters of these putative stress-responsive genes. This analysis is the first report of NAC gene family in pigeonpea and will be useful for the identification and selection of candidate genes associated with stress tolerance.

Influence of lectins, hexoses, and neuraminidase on the association of purified elementary bodies of Chlamydia trachomatis UW-31 with HeLa cells.

PubMed Central

Bose, S K; Smith, G B; Paul, R G

1983-01-01

Using highly purified elementary bodies of Chlamydia trachomatis UW-31 (serotype K), we found that HeLa 229 monolayer cultures bound more 32P-labeled chlamydiae after pretreatment with the lectin wheat germ agglutinin. The lectin, on the other hand, inhibited competitively when chlamydial association was assayed in the presence of polycations. The two effects of wheat germ agglutinin were abolished when N-acetylneuraminic acid (NeuNAc)- or N-acetylglucosamine (GlcNAc)-preincubated wheat germ agglutinin was used. Brief exposure of HeLa cells to neuraminidase abolished the ability to bind the elementary bodies, whether or not polycations were present. Furthermore, at 5 degrees C but not at 37 degrees C, NeuNAc, GlcNAc and N-acetylgalactosamine inhibited chlamydial association only in the absence of the polycation DEAE-dextran. The results suggest that NeuNAc residues on the plasma membrane are the principal, but not the only, receptors for this strain of C. trachomatis. PMID:6687878

Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases.

PubMed

Foddy, L; Hughes, R C

1988-08-01

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.

Decreased background parenchymal enhancement of the contralateral breast after two cycles of neoadjuvant chemotherapy is associated with tumor response in HER2-positive breast cancer.

PubMed

You, Chao; Gu, Yajia; Peng, Wen; Li, Jianwei; Shen, Xuxia; Liu, Guangyu; Peng, Weijun

2018-07-01

Background Several recent studies have focused on the association between background parenchymal enhancement (BPE) and tumor response to neoadjuvant chemotherapy (NAC), but early prediction of tumor response based on BPE has yet not been investigated. Purpose To retrospectively investigate whether changes in the BPE of the contralateral breast following NAC could help predict tumor response in early stage HER2-positive breast cancer. Material and Methods Data from 71 patients who were diagnosed with unilateral HER2 positive breast cancer and then underwent NAC with trastuzumab before surgery were analyzed retrospectively. Two experienced radiologists independently categorized the patients' levels of BPE of the contralateral breast into four categories (1 = minimal, 2 = mild, 3 = moderate, 4 = marked) at baseline and after the second cycle of NAC. After undergoing surgery, 34 patients achieved pathologic complete response (pCR) and 37 patients had residual disease (non-pCR). The association between BPE and histopathologic tumor response was analyzed. Result The level of BPE was higher in premenopausal than post-menopausal women both at baseline and after the second cycle of NAC ( P < 0.005). A significant reduction in BPE ( P < 0.001) was observed after the second NAC cycle; however, a more obvious decrease in BPE was identified in premenopausal relative to post-menopausal women ( P = 0.041). No significant association was identified between pCR and baseline BPE ( P = 0.287). However, after the second NAC cycle, decreased BPE was significantly associated with pCR ( P = 0.003). Conclusion For HER2-positive patients, changes in BPE may serve as an additional imaging biomarker of treatment response at an early stage.

Induction of salivary polypeptides associated with parotid hypertrophy by gallotannins administered topically into the mouse mouth.

PubMed

Gho, Francesca; Peña-Neira, Alvaro; López-Solís, Remigio O

2007-02-01

Isoproterenol-induced salivary polypeptides (IISP), a group of proline-rich proteins synthesized by mouse parotids, have been considered as markers for isoproterenol-induced parotid hypertrophy. Rodents fed diets containing high-tannin cereals (sorghum), also develop parotid hypertrophy. To test whether tannins are directly involved in provoking sialotrophic growth, we studied the effect of intraperitoneal and topical oral administrations of tannic acid (TA) on the induction of IISP polypeptides in endogamic mice (A/Snell). TA was characterized by HPLC chromatography and spectral analysis and shown to be composed solely of gallotannins, a complex family of glucose and gallic acid esters. IISP polypeptides were monitored in saliva by SDS-polyacrylamide gel electrophoresis during 36 h after ending TA stimulation. Single daily intraperitoneal administrations of TA for 3 consecutive days (0.033 mg/g bw/day), at variance of parallel administrations of isoproterenol (0.042 mg/g bw/day) failed to induce IISP polypeptides. However, repeated topical applications of TA into the mouse mouths (1.21 mg/g bw divided into three equal doses given at 4-h intervals within a single day) resulted in unequivocal induction of IISP polypeptides. That response was clearly intensified by increasing the stimulation frequency to eight equivalent doses given at 1.5-h intervals within a single day (corresponding to 3.23 mg/g bw) and even further by repeating this protocol for 3 days. Under these productive schemes of stimulations by TA, electrophoretic fractionation of parotid homogenates showed new polypeptide bands migrating in parallel to salivary IISP. These results suggest that topically administered gallotannins are effective inducers of trophic growth in mouse parotids.

Dopamine D1 receptor agonist treatment attenuates extinction of morphine conditioned place preference while increasing dendritic complexity in the nucleus accumbens core.

PubMed

Kobrin, Kendra L; Arena, Danielle T; Heinrichs, Stephen C; Nguyen, Olivia H; Kaplan, Gary B

2017-03-30

The dopamine D1 receptor (D1R) has a role in opioid reward and conditioned place preference (CPP), but its role in CPP extinction is undetermined. We examined the effect of D1R agonist SKF81297 on the extinction of opioid CPP and associated dendritic morphology in the nucleus accumbens (NAc), a region involved with reward integration and its extinction. During the acquisition of morphine CPP, mice received morphine and saline on alternate days; injections were given immediately before each of eight daily conditioning sessions. Mice subsequently underwent six days of extinction training designed to diminish the previously learned association. Mice were treated with either 0.5mg/kg SKF81297, 0.8mg/kg SKF81297, or saline immediately after each extinction session. There was a dose-dependent effect, with the highest dose of SKF81297 attenuating extinction, as mice treated with this dose had significantly higher CPP scores than controls. Analysis of medium spiny neuron morphology revealed that in the NAc core, but not in the shell, dendritic arbors were significantly more complex in the morphine conditioned, SKF81297-treated mice compared to controls. In separate experiments using mice conditioned with only saline, SKF81297 administration after extinction sessions had no effect on CPP and produced differing effects on dendritic morphology. At the doses used in our experiments, SKF81297 appears to maintain previously learned opioid conditioned behavior, even in the face of new information. The D1R agonist's differential, rather than unidirectional, effects on dendritic morphology in the NAc core suggests that it may be involved in encoding reward information depending on previously learned behavior. Published by Elsevier B.V.

Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains.

PubMed

Li, Shan; Zhang, Li; Yao, Qing; Li, Lin; Dong, Na; Rong, Jie; Gao, Wenqing; Ding, Xiaojun; Sun, Liming; Chen, Xing; Chen, She; Shao, Feng

2013-09-12

The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors. Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD). Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB (NF-κB) signalling. NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg 235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.

Patterns of Drug Use and Serum Sodium Concentrations in Older Hospitalized Patients: A Latent Class Analysis Approach.

PubMed

Woodman, Richard J; Wood, Karen M; Kunnel, Aline; Dedigama, Maneesha; Pegoli, Matthew A; Soiza, Roy L; Mangoni, Arduino A

2016-12-01

Several drugs may lower serum sodium concentrations (NaC) in older patients. However, distinguishing their individual effects is particularly difficult in this population because of the high prevalence of polypharmacy and disease states that are per se associated with hyponatremia. Our objective was to identify specific patterns of medication use in older hospitalized patients and determine whether these patterns were associated with serum NaC. We collected clinical and demographic data, pre-admission drugs, Drug Burden Index (DBI) score, and average NaC during hospitalization in a consecutive series of older medical patients (n = 101, mean ± standard deviation [SD] age 87 ± 6 years). We used latent class analysis (LCA) to identify specific patterns of drug use and multivariate regression to determine the associations between 14 separate drug classes, identified patterns of drug use, and NaC. LCA revealed three patterns: lower overall drug use (class 1), anticoagulant use and higher drug use (class 2), and antiplatelet use (class 3). Mean (±SD) DBI score in each class was 2.7 ± 1.3, 3.3 ± 1.6, and 2.4 ± 1.5, respectively (p = 0.04). Mean (± SD) NaC in classes 1, 2, and 3 were 140.6 ± 6.8, 138.7 ± 5.3, and 136.5 ± 4.7 mmol/l, respectively (p = 0.006). After adjustment for age, sex, Charlson Comorbidity Index score, estimated glomerular filtration rate (eGFR), DBI score, and digoxin use, mean NaC in class 2 and class 3 was significantly lower than in class 1 (-3.9 mmol/l; 95% confidence interval [CI] -7.1 to -0.8, p = 0.01 and -5.2 mmol/l; 95% CI -7.9 to -2.5, p < 0.001, respectively). Mean serum NaC was not significantly associated with any of the 14 individually assessed drug classes. In addition to latent class, increasing age and higher eGFR were also independently associated with lower serum NaC (p = 0.002 and p = 0.03, respectively). LCA enabled us to identify patterns of drug use associated with lower serum NaC in older inpatients. Our results suggest that older patients using antiplatelets or anticoagulants are especially at risk of lower serum NaC.

LROC Advances in Lunar Science

NASA Astrophysics Data System (ADS)

Robinson, M. S.

2012-12-01

Since entering orbit in 2009 the Lunar Reconnaissance Orbiter Camera (LROC) has acquired over 700,000 Wide Angle Camera (WAC) and Narrow Angle Camera (NAC) images of the Moon. This new image collection is fueling research into the origin and evolution of the Moon. NAC images revealed a volcanic complex 35 x 25 km (60N, 100E), between Compton and Belkovich craters (CB). The CB terrain sports volcanic domes and irregular depressed areas (caldera-like collapses). The volcanic complex corresponds to an area of high-silica content (Diviner) and high Th (Lunar Prospector). A low density of impact craters on the CB complex indicates a relatively young age. The LROC team mapped over 150 volcanic domes and 90 volcanic cones in the Marius Hills (MH), many of which were not previously identified. Morphology and compositional estimates (Diviner) indicate that MH domes are silica poor, and are products of low-effusion mare lavas. Impact melt deposits are observed with Copernican impact craters (>10 km) on exterior ejecta, the rim, inner wall, and crater floors. Preserved impact melt flow deposits are observed around small craters (25 km diam.), and estimated melt volumes exceed predictions. At these diameters the amount of melt predicted is small, and melt that is produced is expected to be ejected from the crater. However, we observe well-defined impact melt deposits on the floor of highland craters down to 200 m diameter. A globally distributed population of previously undetected contractional structures were discovered. Their crisp appearance and associated impact crater populations show that they are young landforms (<1 Ga). NAC images also revealed small extensional troughs. Crosscutting relations with small-diameter craters and depths as shallow as 1 m indicate ages <50 Ma. These features place bounds on the amount of global radial contraction and the level of compressional stress in the crust. WAC temporal coverage of the poles allowed quantification of highly illuminated regions, including one site that remains lit for 94% of a year (longest eclipse period of 43 hours). Targeted NAC images provide higher resolution characterization of key sites with permanent shadow and extended illumination. Repeat WAC coverage provides an unparalleled photometric dataset allowing spatially resolved solutions (currently 1 degree) to Hapke's photometric equation - data invaluable for photometric normalization and interpreting physical properties of the regolith. The WAC color also provides the means to solve for titanium, and distinguish subtle age differences within Copernican aged materials. The longevity of the LRO mission allows follow up NAC and WAC observations of previously known and newly discovered targets over a range of illumination and viewing geometries. Of particular merit is the acquisition of NAC stereo pairs and oblique sequences. With the extended SMD phase, the LROC team is working towards imaging the whole Moon with pixel scales of 50 to 200 cm.

Citrullus colocynthis NAC transcription factors CcNAC1 and CcNAC2 are involved in light and auxin signaling.

PubMed

Wang, Zhuoyu; Rashotte, Aaron M; Dane, Fenny

2014-10-01

Two novel NAC transcription factors from C itrullus colocynthis implicated in light and auxin signaling pathway. NAC transcription factors (NAM, ATAF1, 2, CUC2) have multiple functions in plant growth and development. Two NACs, CcNAC1 and CcNAC2, were recently identified in the highly drought-tolerant cucurbit species, Citrullus colocynthis. This study examines the functional role of these genes under different qualities of light based on the in silico analysis of the CcNAC1 and CcNAC2 promoters that revealed the presence of several light-associated motifs. The impact of both light and auxin on CcNAC1 and CcNAC2 expression was examined in C. colocynthis leaves, and using reporter (pCcNAC1, 2::GUS) lines in Arabidopsis. Furthermore, the effects of constitutive overexpression (OE-CcNAC1, 2) in Arabidopsis were also examined under a range of conditions to confirm reporter line linkages. White, blue, red, and far-red light treatments resulted in similar patterns of quantitative changes in CcNAC1and CcNAC2 expression in both species, with the highest transcript increases following red light. Photomorphogenic changes in Arabidopsis hypocotyls were correlated with gene transcript levels. In the absence of light, hypocotyls of OE-CcNAC1/CcNAC2 lines were significantly longer as compared to WT. The addition of exogenous auxin (+IAA) to growth medium also resulted in changes to the hypocotyl lengths of overexpression lines and spatiotemporal reporter line changes in seedlings. Our data suggest that CcNAC1, 2 might be functionally important in the light signaling pathway, and appear connected to the hormone auxin. This is the first study to indicate that NAC genes might play a role in both light and auxin signaling pathways.

Electron transfer dissociation (ETD): The mass spectrometric breakthrough essential for O-GlcNAc protein site assignments – A study of the O-GlcNAcylated protein Host Cell Factor C1

PubMed Central

Myers, Samuel A.; Daou, Salima; Affar, El Bachir; Burlingame, AL

2014-01-01

The development of electron-based, unimolecular dissociation mass spectrometric methods, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile post-translational modification (PTM) site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD mass spectrometry. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cells and subcellular structures. Our ability to identify and unambiguously assign the site of O-GlcNAc modifications using ETD is rapidly increasing our understanding of this regulatory glycosylation and its potential interaction with other PTMs. Here, we discuss the advantages of using ETD, complimented with collisional-activation mass spectrometry (CID/CAD), in a study of O-GlcNAc modified peptides of the extensively O-GlcNAcylated protein Host Cell Factor C1 (HCF-1). HCF-1 is a transcriptional co-regulator, forms a stable complex with O-GlcNAc transferase and is involved in control of cell cycle progression. ETD, along with higher energy collisional dissociation (HCD) mass spectrometry, was employed to assign the PTMs of the HCF-1 protein isolated from HEK293T cells. These include nineteen sites of O-GlcNAcylation, two sites of phosphorylation and two sites bearing dimethylarginine, and showcase the residue-specific, PTM complexity of this regulator of cell proliferation. PMID:23335398

Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response

PubMed Central

McKibbin, Craig; Mares, Alina; Piacenti, Michela; Williams, Helen; Roboti, Peristera; Puumalainen, Marjo; Callan, Anna C.; Lesiak-Mieczkowska, Karolina; Linder, Stig; Harant, Hanna; High, Stephen; Flitsch, Sabine L.; Whitehead, Roger C.; Swanton, Eileithyia

2011-01-01

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ESI (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ESI causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation. PMID:22145777

The bark of Robinia pseudoacacia contains a complex mixture of lectins.Characterization of the proteins and the cDNA clones.

PubMed Central

Van Damme, E J; Barre, A; Smeets, K; Torrekens, S; Van Leuven, F; Rougé, P; Peumans, W J

1995-01-01

Two lectins were isolated from the inner bark of Robinia pseudoacacia (black locust). The first (and major) lectin (called RPbAI) is composed of five isolectins that originate from the association of 31.5- and 29-kD polypeptides into tetramers. In contrast, the second (minor) lectin (called RPbAII) is a hometetramer composed of 26-kD subunits. The cDNA clones encoding the polypeptides of RPbAI and RPbAII were isolated and their sequences determined. Apparently all three polypeptides are translated from mRNAs of approximately 1.2 kb. Alignment of the deduced amino acid sequences of the different clones indicates that the 31.5- and 29-kD RPbAI polypeptides show approximately 80% sequence identity and are homologous to the previously reported legume seed lectins, whereas the 26-kD RPbAII polypeptide shows only 33% sequence identity to the previously described legume lectins. Modeling the 31.5-kD subunit of RPbAI predicts that its three-dimensional structure is strongly related to the three-dimensional models that have been determined thus far for a few legume lectins. Southern blot analysis of genomic DNA isolated from Robinia has revealed that the Robinia bark lectins are the result of the expression of a small family of lectin genes. PMID:7716244

The National Astronomy Consortium (NAC)

NASA Astrophysics Data System (ADS)

Von Schill, Lyndele; Ivory, Joyce

2017-01-01

The National Astronomy Consortium (NAC) program is designed to increase the number of underrepresented minority students into STEM and STEM careers by providing unique summer research experiences followed by long-term mentoring and cohort support. Hallmarks of the NAC program include: research or internship opportunities at one of the NAC partner sites, a framework to continue research over the academic year, peer and faculty mentoring, monthly virtual hangouts, and much more. NAC students also participate in two professional travel opportunities each year: the annual NAC conference at Howard University and poster presentation at the annual AAS winter meeting following their summer internship.The National Astronomy Consortium (NAC) is a program led by the National Radio Astronomy Consortium (NRAO) and Associated Universities, Inc. (AUI), in partnership with the National Society of Black Physicist (NSBP), along with a number of minority and majority universities.

Characterization of mutants expressing thermostable D1 and D2 polypeptides of photosystem II in the cyanobacterium Synechococcus elongatus PCC 7942.

PubMed

Haraguchi, Norihisa; Kaseda, Jun; Nakayama, Yasumune; Nagahama, Kazuhiro; Ogawa, Takahira; Matsuoka, Masayoshi

2018-06-08

Photosystem II complex embedded in thylakoid membrane performs oxygenic photosynthesis where the reaction center D1/D2 heterodimer accommodates all components of the electron transport chain. To express thermostable D1/D2 heterodimer in a cyanobacterium Synechococcus elongatus PCC 7942, we constructed a series of mutant strains whose psbA1 and psbD1 genes encoding, respectively, the most highly expressed D1 and D2 polypeptides were replaced with those of a thermophilic strain, Thermosynechococcus vulcanus. Because the C-terminal 16 amino acid sequences of D1 polypeptides should be processed prior to maturation but diverge from each other, we also constructed the psbA1ΔC-replaced strain expressing a thermostable D1 polypeptide devoid of the C-terminal extension. The psbA1/psbD1-replaced strain showed decreased growth rate and oxygen evolution rate, suggesting inefficient photosystem II. Immunoblot analyses for thermostable D1, D2 polypeptides revealed that the heterologous D1 protein was absent in thylakoid membrane from any mutant strains with psbA1, psbA1ΔC, and psbA1/psbD1-replacements, whereas the heterologous D2 protein was present in thylakoid membrane as well as purified photosystem II complex from the psbA1/psbD1-replaced strain. In the latter strain, the compensatory expression of psbA3 and psbD2 genes was elevated. These data suggest that heterologous D2 polypeptide could be combined with the host D1 polypeptide to form chimeric D1/D2 heterodimer, whereas heterologous D1 polypeptide even without the C-terminal extension was unable to make complex with the host D2 polypeptide. Since the heterologous D1 could not be detected even in the whole cells of psbA1/psbD1-replaced strain, the rapid degradation of unprocessed or unassembled heterologous D1 was implicated. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, D.J.; Lidstrom, M.E.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome c/sub L/. In this study, four polypeptides of M/sub r/, 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement ofmore » the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the M/sub r/-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the M/sub r/-20,000 polypeptide, was identified as mature cytochrome c/sub L/, and the product of moxI, the M/sub r/-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the M/sub r/-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the M/sub r/-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the M/sub r/-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the M/sub r/-60,000 MeDH polypeptide. Our data suggest that both the M/sub r/-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.« less

Nipple sparing mastectomy for breast cancer is associated with high patient satisfaction and safe oncological outcomes.

PubMed

Mesdag, V; Régis, C; Tresch, E; Chauvet, M-P; Boulanger, L; Collinet, P; Giard, S

2017-10-01

The preservation of the nipple areolar complex (NAC) for cancer treatment is still a matter of debate because of suspected increase of local recurrence and surgery-specific complications. The aim of the study was to investigate both the relapse risk associated with nipple sparing mastectomy (NSM) for breast cancer and women's satisfaction with preservation of the NAC. We included retrospectively all patients who had skin-sparing mastectomy (SSM) or NSM from 2007 to 2012 for breast cancer or ductal carcinoma in situ (DCIS). We compared NSM and SSM group for oncological and surgical outcomes. Patients' satisfaction and quality of life has been evaluated by a specifically designed questionnaire. We included 63NSM (41.5%) and 89SM (58.5%). Eighty-nine (58.6%) patients had DCIS, and the other had small invasive disease. Median follow-up was 42 (IQR: 18-58) months. Local recurrence rate was 1.7% (n=1) in the NSM group and 0% in the SSM group without recurrence in the preserved nipple. After NSM, one patient had complete NAC necrosis, and three patients suffered partial necrosis. Satisfaction with the NAC was higher in the NSM group compared to the SSM group with delayed reconstruction of the nipple (75% vs. 59%, P=0.14). Patients with NSM required less psychological support before (P=0.028) and immediately after surgery (P=0.14) than patients in the SSM group. NSM can successfully and safely be performed for pre-invasive and small invasive breast cancer. Besides esthetic aspects, preserving the nipple may ease the acceptance of these radical form of surgery. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

cerebral Markers of the Serotonergic System in Rat Models of Obesity and After Roux-en-Y Gastric Bypass

PubMed Central

Ratner, Cecilia; Ettrup, Anders; Bueter, Marco; Haahr, Mette E.; Compan, Valérie; le Roux, Carel W.; Levin, Barry; Hansen, Henrik H.; Knudsen, Gitte M.

2013-01-01

Food intake and body weight are regulated by a complex system of neural and hormonal signals, of which the anorexigenic neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) is central. In this study, rat models of obesity and weight loss intervention were compared with regard to several 5-HT markers. Using receptor autoradiography, brain regional-densities of the serotonin transporter (SERT) and the 5-HT2A and 5-HT4 receptors were measured in (i) selectively bred polygenic diet-induced obese (pgDIO) rats, (ii) outbred DIO rats, and (iii) Roux-en-Y gastric bypass (RYGB)-operated rats. pgDIO rats had higher 5-HT4 and 5-HT2A receptor binding and lower SERT binding when compared to polygenic diet-resistant (pgDR) rats. The most pronounced difference between pgDIO and pgDR rats was observed in the nucleus accumbens shell (NAcS), a brain region regulating reward aspects of feeding. No differences were found in the 5-HT markers between DIO rats, chow-fed control rats, and DIO rats experiencing a weight loss. The 5-HT markers were also similar in RYGB and sham-operated rats except for a downregulation of 5-HT2A receptors in the NAcS. The higher receptor and lower SERT binding in pgDIO as compared to pgDR rats corresponds to what is reported in overweight humans and suggests that the dysfunctions of the 5-HT system associated with overeating or propensity to become overweight are polygenically determined. Our results support that the obesity-prone rat model has high translational value and suggests that susceptibility to develop obesity is associated with changed 5-HT tone in the brain that may also regulate hedonic aspects of feeding. PMID:22450706

Targeted polypeptide degradation

DOEpatents

Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

2008-05-13

This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

Synthesis of N-acetyl-L-cysteine capped Mn:doped CdS quantum dots for quantitative detection of copper ions

NASA Astrophysics Data System (ADS)

Yang, Xiupei; Jia, Zhihui; Cheng, Xiumei; Luo, Na; Choi, Martin M. F.

2018-06-01

In this work, a new assembled copper ions sensor based on the Mn metal-enhanced fluorescence of N-acetyl-L-cysteine protected CdS quantum dots (NAC-Mn:CdS QDs) was developed. The NAC and Mn:CdS QDs nanoparticles were assembled into NAC-Mn:CdS QDs complexes through the formation of Cdsbnd S and Mnsbnd S bonds. As compared to NAC capped CdS QDs, higher fluorescence quantum yields of NAC-Mn:CdS QDs was observed, which is attributed to the surface plasmon resonance of Mn metal. In addition, the fluorescence intensity of as-formed complexes weakened in the presence of copper ions. The decrease in fluorescence intensity presented a linear relationship with copper ions concentration in the range from 0.16-3.36 μM with a detection limit of 0.041 μM . The characterization of as-formed QDs was analyzed by photoluminescence (PL), ultra violet-visible (UV-vis), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and energy dispersive spectroscopy (EDS) respectively. Furthermore, the recoveries and relative standard deviations of Cu2+ spiked in real water samples for the intra-day and inter-day analyses were 88.20-117.90, 95.20-109.90, 0.80-5.80 and 1.20-3.20%, respectively. Such a metal-enhanced QDs fluorescence system may have promising application in chemical and biological sensors.

Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

NASA Astrophysics Data System (ADS)

Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

2017-02-01

The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides.

PubMed

Kostova, Kamena K; Hickey, Kelsey L; Osuna, Beatriz A; Hussmann, Jeffrey A; Frost, Adam; Weinberg, David E; Weissman, Jonathan S

2017-07-28

Ribosome stalling leads to recruitment of the ribosome quality control complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a carboxyl-terminal alanine and threonine (CAT) tail through a noncanonical elongation reaction. Here we examined the role of CAT-tailing in nascent-chain degradation in budding yeast. We found that Ltn1p efficiently accessed only nascent-chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enabled degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription

PubMed Central

Waast, Laetitia; Kuo, Mei-Shiue; Mangeney, Marianne; Martella, Christophe; Souidi, Mouloud; Issad, Tarik

2017-01-01

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5’LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex. PMID:28742148

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription.

PubMed

Groussaud, Damien; Khair, Mostafa; Tollenaere, Armelle I; Waast, Laetitia; Kuo, Mei-Shiue; Mangeney, Marianne; Martella, Christophe; Fardini, Yann; Coste, Solène; Souidi, Mouloud; Benit, Laurence; Pique, Claudine; Issad, Tarik

2017-07-01

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5'LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.

Structural studies on molecular interactions between camel peptidoglycan recognition protein, CPGRP-S, and peptidoglycan moieties N-acetylglucosamine and N-acetylmuramic acid.

PubMed

Sharma, Pradeep; Yamini, Shavait; Dube, Divya; Singh, Amar; Sinha, Mau; Dey, Sharmistha; Mitra, Dipendra K; Kaur, Punit; Sharma, Sujata; Singh, Tej P

2012-06-22

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

Identification of the triazine receptor protein as a chloroplast gene product

PubMed Central

Steinback, Katherine E.; McIntosh, Lee; Bogorad, Lawrence; Arntzen, Charles J.

1981-01-01

The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem II. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed “B.” The polypeptide that is believed to be a component of the B complex has recently been identified as a 32- to 34-kilo-dalton polypeptide by using a photoaffinity labeling probe, azido-[14C]atrazine. A 34-kilodalton polypeptide of pea chloroplasts rapidly incorporates [35S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. Trypsin treatment of membranes tagged with azido-[14C]atrazine, [35S]methionine in vivo, or [35S]methionine in isolated intact chloroplasts results in identical, sequential alterations of the 34-kilo-dalton polypeptide to species of 32, then 18 and 16 kilodaltons. From the identical pattern of susceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product of chloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein of photosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide. Images PMID:16593133

Determinants of short and long term outcomes in patients undergoing immediate breast reconstruction following neoadjuvant chemotherapy.

PubMed

Wengler, Craig A; Valente, Stephanie A; Al-Hilli, Zahraa; Woody, Neil M; Muntean, Julia H; Abraham, Jame; Tendulkar, Rahul D; Djohan, Risal; O'Rourke, Colin; Crowe, Joseph P; Grobmyer, Stephen R

2017-12-01

We evaluated oncologic outcomes and complications of skin-sparing mastectomy (SSM) and nipple-sparing mastectomy (NSM) with immediate reconstruction (IR) after neoadjuvant chemotherapy (NAC) in patients with early-stage and locally advanced breast cancer (BC). BC patients from 2000 to 2014 treated with NAC followed by SSM/NSM and IR were reviewed. Patient demographics, tumor characteristics, NAC response, complications, and recurrence were analyzed. Two hundred sixty-nine patients with 280 BCs were treated with NAC followed by SSM (94%) or NSM (6%) with IR. Median age was 47 (26-72) years with a median follow-up of 45 months. Pathologic complete response (pCR) was noted in 49 (17.5%) cases. Overall 30-day complication rate was 13.2%. Variables associated with complications included BMI (P < 0.0001), tobacco use (P = 0.015), and adjuvant radiation (P = 0.025). Local-regional recurrence was 3.2% and metastatic recurrence was 13.2%. Variables predicting recurrence risk were pre-NAC tumor size (P < 0.001), residual tumor size (P = 0.002), Grade III (P = 0.002), HER-2 negative (P = 0.025), pre-NAC nodal disease (P = 0.05), and lack of pCR (P = 0.045). Following NAC, risk factors for complications in patients undergoing SSM/NSM with IR are high BMI, smoking, and adjuvant XRT. SSM/NSM following NAC is associated with excellent local control. These data support expanding the indications for NSM/SSM to include patients receiving NAC. © 2017 Wiley Periodicals, Inc.

Contemporary use and effectiveness of N-acetylcysteine in preventing contrast-induced nephropathy among patients undergoing percutaneous coronary intervention.

PubMed

Gurm, Hitinder S; Smith, Dean E; Berwanger, Otavio; Share, David; Schreiber, Theodore; Moscucci, Mauro; Nallamothu, Brahmajee K

2012-01-01

The aim of this study was to examine the use of and outcomes associated with use of N-acetylcysteine (NAC) in real-world practice. The role of NAC in the prevention of contrast-induced nephropathy (CIN) is controversial, leading to widely varying recommendations for its use. Use of NAC was assessed in consecutive patients undergoing nonemergent percutaneous coronary intervention from 2006 to 2009 in the Blue Cross Blue Shield of Michigan Cardiovascular Consortium, a large multicenter quality improvement collaborative. We examined the overall prevalence of NAC use in these patients and then used propensity matching to link its use with clinical outcomes, including CIN, nephropathy-requiring dialysis, and death. Of the 90,578 percutaneous coronary interventions performed during the study period, NAC was used in 10,574 (11.6%) procedures, with its use steadily increasing over the study period. Patients treated with NAC were slightly older and more likely to have baseline renal insufficiency and other comorbidities. In propensity-matched, risk-adjusted models, we found no differences in outcomes between patients treated with NAC and those not receiving NAC for CIN (5.5% vs. 5.5%, p = 0.99), nephropathy-requiring dialysis (0.6% vs. 0.6%, p = 0.69), or death (0.6% vs. 0.8%, p = 0.15). These findings were consistent across many prespecified subgroups. Use of NAC is common and has steadily increased over the study period but does not seem to be associated with improved clinical outcomes in real-world practice. Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Does nipple preservation in mastectomy improve satisfaction with cosmetic results, psychological adjustment, body image and sexuality?

PubMed

Didier, F; Radice, D; Gandini, S; Bedolis, R; Rotmensz, N; Maldifassi, A; Santillo, B; Luini, A; Galimberti, V; Scaffidi, E; Lupo, F; Martella, S; Petit, J Y

2009-12-01

We investigated the influence of nipple areolar complex (NAC) sparing in mastectomy, on patient satisfaction with cosmetic results, body-image, sexuality and psychological well-being. We developed a specific questionnaire and compared two groups of women who underwent radical mastectomy with immediate breast reconstruction (IBR). Between 2004 and 2006, 310 women with NAC preservation and 143 patients with successive NAC reconstruction were mailed the questionnaire at follow-up 1 year after definitive complete breast reconstruction surgery. 256 questionnaires was available. Our results showed significant differences in favour of the NAC sparing group regarding body image (difficulty in looking at themselves naked and being seen naked by their partners after surgery, P = 0.001 and P = 0.003, respectively); regarding satisfaction with the appearance of the nipple (P < .0001) and with the sensitivity of the nipple (P = 0.001); regarding the feeling of mutilation (P = 0.003). NAC sparing in mastectomy has a positive impact on patient satisfaction, body image and psychological adjustment.

N-Glycan Modification in Aspergillus Species▿

PubMed Central

Kainz, Elke; Gallmetzer, Andreas; Hatzl, Christian; Nett, Juergen H.; Li, Huijuan; Schinko, Thorsten; Pachlinger, Robert; Berger, Harald; Reyes-Dominguez, Yazmid; Bernreiter, Andreas; Gerngross, Tillmann; Wildt, Stefan; Strauss, Joseph

2008-01-01

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi. PMID:18083888

2.2 A resolution structure analysis of two refined N-acetylneuraminyl-lactose--wheat germ agglutinin isolectin complexes.

PubMed

Wright, C S

1990-10-20

The crystal structures of complexes of isolectins 1 and 2 of wheat germ agglutinin (WGA1 and WGA2) with N-acetylneuraminyl-lactose (NeuNAc-alpha(2-3)-Gal-beta(1-4)-Glc) have been refined on the basis of data in the 8 to 2.2 A resolution range to final crystallographic R-factors of 17.2% and 15.3% (Fo greater than 1 sigma), respectively. Specific binding interactions and water association, as well as changes in conformation and mobility of the structure upon ligand binding, were compared in the two complexes. The temperature factors (B = 16.3 A2 and 18.4 A2) were found to be much lower compared with those of their respective native structures (19 to 22 A2). Residues involved in sugar binding, dimerization and in lattice contacts exhibit the largest decreases in B-value, suggesting that sugar binding reduces the overall mobility of the protein molecules in the crystal lattice. The binding mode of this sialyl-trisaccharide, an important cell receptor analogue, has been compared in the two isolectins. Only one of the two unique binding sites (4 per dimer), located in the subunit/subunit interface, is occupied in the crystals. This site, termed the "primary" binding site, contains one of the five amino acid substitutions that differentiate WGA1 and WGA2. Superposition of the refined models in each of the independent crystallographic environments indicates a close match only of the terminal non-reducing NeuNAc residue (root-mean-square delta r of 0.5 to 0.6 A). The Gal-Glc portion was found to superimpose poorly, lack electron density, and possess high atomic thermal factors. In both complexes NeuNAc is stabilized through contact with six amino acid side-chains (Ser114 and Glu115 of subunit 1 and Ser62, Tyr64, Tyr(His)66 and Tyr73 of subunit 2), involving all NeuNAc ring substituents. Refinement has allowed accurate assessment of the contact distances for four hydrogen bonds, a strong buried non-polar contact with the acetamido CH3 group and a large number of van der Waals' interactions with the three aromatic side-chains. The higher affinity of N-acetylneuraminyl-lactose observed by nuclear magnetic resonance studies for WGA1 can be explained by the more favorable binding interactions that occur when residue 66 is a Tyr. The tyrosyl side-chain provides a larger surface for van der Waals' stacking against the NeuNAc pyranose ring than His66 and a hydrogen bond contact with Gal (C2-OH), not possible in WGA2.(ABSTRACT TRUNCATED AT 400 WORDS)

Association Between Reward Reactivity and Drug Use Severity is Substance Dependent: Preliminary Evidence From the Human Connectome Project.

PubMed

Peechatka, Alyssa L; Janes, Amy C

2017-06-01

Blunted nucleus accumbens (NAc) reactivity to reward is common across drug users. One theory is that individuals abuse substances due to this reward deficit. However, whether there is a relationship between the amount an individual uses and the severity of NAc dysfunction is unclear. It also is possible that such a relationship is substance specific, as nicotine transiently increases reward system sensitivity while alcohol, another commonly used substance, does not. As smokers may use nicotine to bolster NAc reward function, we hypothesize that NAc reactivity to reward will be related to volume of cigarette use, but not volume of alcohol use. A functional magnetic resonance imaging incentive-processing task collected by the Human Connectome Project was assessed in a cohort of tobacco smokers who reported smoking between 5-20 cigarettes/day and a cohort of alcohol users who reported drinking 7-25 drinks/wk. Number of cigarettes/day and drinks/wk were correlated with right and left NAc reactivity to the receipt of a monetary reward relative to baseline. Individuals who smoke greater numbers of cigarettes/day showed lower right NAc reactivity to reward (r = 0.853, p ≤ .001). Left NAc reactivity was not correlated with cigarettes/day. No association was found with drinks/wk. A negative association was found between NAc reactivity to reward and cigarettes/day, but not alcohol drinks/wk. Given nicotine's unique ability to increase sensitivity to rewards, these findings suggest that individuals who smoke more cigarettes/day may be compensating for more dysfunctional NAc reward reactivity. The present study demonstrates that a relationship between NAc reactivity to nondrug reward and volume of substance use is present in nicotine but not alcohol use. While prior work has implicated dysfunctional reward processing in addictions, these findings clarify a substance-specific role that blunted reward function has in determining patterns of use among chronic users. © The Author 2017. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Breast irradiation causes pallor in the nipple-areolar complex in women with Celtic skin type (result from the St. George and Wollongong randomised breast boost trial).

PubMed

Lee, Yoo Young Dominique; Hau, Eric; Browne, Lois H; Chin, Yaw; Lee, Jessica; Szwajcer, Alison; Cail, Stacy; Nolan, David N; Graham, Peter H

2014-04-01

The nipple-areolar complex (NAC) has special histological properties with higher melanocyte concentration than breast skin. To date, there are no data describing the late effects on the NAC following breast-conserving therapy (BCT). This study evaluated colour changes in the NAC in patients treated with breast-conserving surgery and adjuvant radiotherapy after 5 years. Digital photographs obtained at 5 years following breast irradiation from the St. George and Wollongong (SGW) trial (NCT00138814) were evaluated by five experts using an iPad® (Apple Inc., Cupertino, CA, USA) application specifically created for this study. The SGW trial randomised 688 patients with Tis-2, N0-1, M0 carcinoma to the control arm of 50 Gy in 25 fractions and boost arm of 45 Gy in 25 fractions and 16 Gy in 8 fractions electron boost. A total of 141/372 (38%) patients had altered NAC (86% lighter, 10% darker). Patients with Celtic skin type had increased likelihood of having an altered NAC (odds ratio (OR), 1.75 (CI 1.1-2.7, P = 0.011)). On subgroup analysis, those with Celtic skin type receiving biologically equivalent dose (BED) Gy3 ≥ 80 Gy had OR of 3.03 (95% CI 1.2-7.5, P = 0.016) for having altered colour. There was a dose response with more profound changes seen in the NAC compared with irradiated breast skin if BED Gy3 ≥ 80 Gy with OR of 2.42 (95% CI 1.1-5.6, P = 0.036). In this Caucasian BCT population, over 30% of patients developed lighter NAC and more commonly in women with Celtic skin type. The degree of this effect increased with higher radiation dose. © 2014 The Royal Australian and New Zealand College of Radiologists.

Specific Binding of Protoporphyrin IX to a Membrane-Bound 63 Kilodalton Polypeptide in Cucumber Cotyledons Treated with Diphenyl Ether-Type Herbicides.

PubMed

Sato, R; Oshio, H; Koike, H; Inoue, Y; Yoshida, S; Takahashi, N

1991-06-01

Porphyrin accumulation in excised cucumber cotyledons (Cucumis sativus L.) treated with a N-phenylimide S-23142 (N-[4-chloro-2-fluoro-5-propargyloxyphenyl]-3,4,5,6- tetrahydrophthalimide) and a diphenylether acifluorfen-ethyl (ethyl-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro benzoic acid) was studied. Most of the accumulated porphyrins were found in the membrane fractions of 6,000g and 30,000g pellets, forming a complex with a membrane polypeptide. The complex was solubilized with 1% n-dodecyl beta-d-maltoside and its molecular mass was estimated to be 63,000 and 66,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation high performance liquid chromatography (HPLC), respectively. The polypeptide also existed in untreated cotyledons but had little protoporphyrin IX. The complex was also formed in vitro by mixing the 30,000g pellets from untreated cotyledons and authentic protoporphyrin IX. However, protoporphyrin IX formed the complex specifically with the 63,000 dalton polypeptide and not with the other proteins both in vivo and in vitro. At least four fluorescent porphyrins, including protoporphyrin IX, were found in the acetone extract of the cotyledons by HPLC using a reversed phase column. Protoporphyrin IX was one of the two porphyrins that formed the complex. These results suggest that S-23142 and acifluorfenethyl enhance the accumulation of protoporphyrin IX, which forms the complex with the membrane protein.

Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases

PubMed Central

Castillo, Virginia; Ventura, Salvador

2009-01-01

Protein aggregation underlies a wide range of human disorders. The polypeptides involved in these pathologies might be intrinsically unstructured or display a defined 3D-structure. Little is known about how globular proteins aggregate into toxic assemblies under physiological conditions, where they display an initially folded conformation. Protein aggregation is, however, always initiated by the establishment of anomalous protein-protein interactions. Therefore, in the present work, we have explored the extent to which protein interaction surfaces and aggregation-prone regions overlap in globular proteins associated with conformational diseases. Computational analysis of the native complexes formed by these proteins shows that aggregation-prone regions do frequently overlap with protein interfaces. The spatial coincidence of interaction sites and aggregating regions suggests that the formation of functional complexes and the aggregation of their individual subunits might compete in the cell. Accordingly, single mutations affecting complex interface or stability usually result in the formation of toxic aggregates. It is suggested that the stabilization of existing interfaces in multimeric proteins or the formation of new complexes in monomeric polypeptides might become effective strategies to prevent disease-linked aggregation of globular proteins. PMID:19696882

Effects of brefeldin A on oligosaccharide processing. Evidence for decreased branching of complex-type glycans and increased formation of hybrid-type glycans.

PubMed

Chawla, D; Hughes, R C

1991-10-01

Brefeldin A (BFA), a drug that induces redistribution of Golgi-apparatus proteins into the endoplasmic reticulum, was used to determine the role of subcellular compartmentalization in the processing of asparagine-linked oligosaccharides. Baby-hamster kidney cells were pulse-labelled with [3H]mannose for 30-60 min and chased for up to several hours in the presence or in the absence of BFA or labelled continuously for several hours with and without the drug. Cellular glycoproteins were digested to glycopeptides with Pronase and either fractionated into glycan classes by lectin affinity chromatography or digested further by endoglycosidase H and endoglycosidase D. Released oligosaccharides obtained in the latter procedure were then separated from each other and from endoglycosidase-resistant glycopeptides by paper chromatography. The results show that BFA induces a very fast processing of protein-linked Glc3Man9GlcNAc2 oligosaccharide down to man5GlcNAc2 and conversion into complex-type and hybrid-type glycans. The major difference between untreated and BFA-treated cells is a large increase in bi-antennary and hybrid-type glycans in the latter cells. These results indicate that galactosylation of a mono-antennary GlcNAcMan5GlcNAc2 hybrid blocks subsequent action by mannosidase II and N-acetylglucosaminyl transferase II, producing galactosylated hybrid-type glycans. Similarly, galactosylation of the product of N-acetylglucosaminyltransferases I and II, i.e. a Man3GlcNAc2 core substituted with GlcNAc beta 1----2 on both alpha 1----3- and alpha 1----6-linked mannose residues, blocks branching N-acetylglucosaminyltransferases IV and V, thereby causing an increase in bi-antennary glycans and a decrease in tri- and tetra-antennary glycans.

N-acetylcysteine Protects Mice from High Fat Diet-induced Metabolic Disorders.

PubMed

Ma, Yongjie; Gao, Mingming; Liu, Dexi

2016-08-01

To study the effects of N-acetylcysteine (NAC, C5H9NO3S) on diet-induced obesity and obesity-related metabolic disorders. Six-week-old male C57BL/6 mice fed a chow or high-fat diet (HFD) were treated with NAC (2 g/L) in drinking water for 11 weeks. Its influences on body weight and food intake were manually measured, and influence on body composition were analyzed by magnetic residence imaging. Glucose meter and ELISA were used to determine serum glucose and insulin levels, as well as lipid content in the liver. The effects of NAC treatment on mRNA levels of genes involved in inflammation, thermogenesis, and lipid metabolism in various tissues were determined by real time PCR. NAC supplementation inhibited the increase of fat mass and the development of obesity when mice were fed an HFD. NAC treatment significantly lowered HFD-induced macrophage infiltration, and enhanced adiponectin gene expression, resulting in reduced hyperglycemia and hyperinsulinemia, and improvement of insulin resistance. NAC oral administration suppressed hepatic lipid accumulation, as evidenced by lower levels of triglyceride and cholesterol in the liver. The beneficial effects are associated with a decrease of hepatic Pparγ and its target gene expression, and an increase in the expression of genes responsible for lipid oxidation and activation of farnesoid X receptor. Furthermore, NAC treatment also stimulates expression of thermogenic genes. These results provide direct proof of the protective potential of NAC against HFD-induced obesity and obesity-associated metabolic disorders.

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum.

PubMed

Carey, Anne-Marie; Hacking, Kirsty; Picken, Nichola; Honkanen, Suvi; Kelly, Sharon; Niedzwiedzki, Dariusz M; Blankenship, Robert E; Shimizu, Yuuki; Wang-Otomo, Zheng-Yu; Cogdell, Richard J

2014-11-01

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila. Copyright © 2014. Published by Elsevier B.V.

Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

PubMed Central

Yu, Seok-Ho; Boyce, Michael; Wands, Amberlyn M.; Bond, Michelle R.; Bertozzi, Carolyn R.; Kohler, Jennifer J.

2012-01-01

O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation. PMID:22411826

Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence

PubMed Central

Christiansen, Michael W.; Matthewman, Colette; Podzimska-Sroka, Dagmara; O’Shea, Charlotte; Lindemose, Søren; Møllegaard, Niels Erik; Holme, Inger B.; Hebelstrup, Kim; Skriver, Karen; Gregersen, Per L.

2016-01-01

The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence. It was significantly up-regulated following ABA treatment, supported by ABA-responsive elements in its promoter, but it was not up-regulated during dark-induced senescence. The C-termini of proteins closely related to HvNAC005 showed overall high divergence but also contained conserved short motifs. A serine- and leucine-containing central motif was essential for transcriptional activity of the HvNAC005 C-terminus in yeast. Over-expression of HvNAC005 in barley resulted in a strong phenotype with delayed development combined with precocious senescence. The over-expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling pathways. Binding of HvNAC005 to promoter sequences of putative target genes containing the T[G/A]CGT core motif was shown by direct protein–DNA interactions of HvNAC005 with promoters for two of the up-regulated genes. In conclusion, HvNAC005 was shown to be a strong positive regulator of senescence and so is an obvious target for the fine-tuning of gene expression in future attempts to improve nutrient remobilization related to the senescence process in barley. PMID:27436280

Plant development. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation.

PubMed

Furuta, Kaori Miyashima; Yadav, Shri Ram; Lehesranta, Satu; Belevich, Ilya; Miyashima, Shunsuke; Heo, Jung-ok; Vatén, Anne; Lindgren, Ove; De Rybel, Bert; Van Isterdael, Gert; Somervuo, Panu; Lichtenberger, Raffael; Rocha, Raquel; Thitamadee, Siripong; Tähtiharju, Sari; Auvinen, Petri; Beeckman, Tom; Jokitalo, Eija; Helariutta, Ykä

2014-08-22

Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain-containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism. Copyright © 2014, American Association for the Advancement of Science.

Appetitive Pavlovian conditioned stimuli increase CREB phosphorylation in the nucleus accumbens.

PubMed

Shiflett, Michael W; Mauna, Jocelyn C; Chipman, Amanda M; Peet, Eloise; Thiels, Edda

2009-10-01

The transcription factor cAMP response element-binding protein (CREB) in the nucleus accumbens (NAc) has been shown to regulate an animal's behavioral responsiveness to emotionally salient stimuli, and an increase in CREB phosphorylation in the NAc has been observed during exposure to rewarding stimuli, such as drugs of abuse. Here we show that CREB phosphorylation increases in the NAc also during exposure to cues that an animal has associated with delivery of natural rewards. Adult male Sprague-Dawley rats (rattus norvegicus) were trained to associate an auditory stimulus with delivery of food pellets, and CREB phosphorylation was examined in the striatum following training. We found that repeated tone-food pairings resulted in an increase in CREB phosphorylation in the NAc but not in the adjacent dorsal striatum or in the NAc 3h after the final training session. We further found that the cue itself, as opposed to the food pellets, the training context, or tone-food pairings, was sufficient to increase CREB phosphorylation in the NAc. These results suggest that the processing of primary rewarding stimuli and of environmental cues that predict them triggers similar accumbal signaling mechanisms.

Effect of double-tailed surfactant architecture on the conformation, self-assembly, and processing in polypeptide-surfactant complexes.

PubMed

Junnila, Susanna; Hanski, Sirkku; Oakley, Richard J; Nummelin, Sami; Ruokolainen, Janne; Faul, Charl F J; Ikkala, Olli

2009-10-12

This work describes the solid-state conformational and structural properties of self-assembled polypeptide-surfactant complexes with double-tailed surfactants. Poly(L-lysine) was complexed with three dialkyl esters of phosphoric acid (i.e., phosphodiester surfactants), where the surfactant tail branching and length was varied to tune the supramolecular architecture in a facile way. After complexation with the branched surfactant bis(2-ethylhexyl) phosphate in an aqueous solution, the polypeptide chains adopted an alpha-helical conformation. These rod-like helices self-assembled into cylindrical phases with the amorphous alkyl tails pointing outward. In complexes with dioctyl phosphate and didodecyl phosphate, which have two linear n-octyl or n-dodecyl tails, respectively, the polypeptide formed antiparallel beta-sheets separated by alkyl layers, resulting in well-ordered lamellar self-assemblies. By heating, it was possible to trigger a partial opening of the beta-sheets and disruption of the lamellar phase. After repeated heating/cooling, all of these complexes also showed a glass transition between 37 and 50 degrees C. Organic solvent treatment and plasticization by overstoichiometric amount of surfactant led to structure modification in poly(L-lysine)-dioctyl phosphate complexes, PLL(diC8)(x) (x = 1.0-3.0). Here, the alpha-helical PLL is surrounded by the surfactants and these bottle-brush-like chains self-assemble in a hexagonal cylindrical morphology. As x is increased, the materials are clearly plasticized and the degree of ordering is improved: The stiff alpha-helical backbones in a softened surfactant matrix give rise to thermotropic liquid-crystalline phases. The complexes were examined by Fourier transform infrared spectroscopy, small- and wide-angle X-ray scattering, transmission electron microscopy, differential scanning calorimetry, polarized optical microscopy, and circular dichroism.

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin.

PubMed

Olden, K; Hunter, V A; Yamada, K M

1980-10-15

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose of [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-beta-N-acetylgluosaminidase H, which specifically cleaves the 'high-mannose' class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing. This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a 'high-mannose' oligosaccharide precursor and a 'complex' carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-beta-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistant to endo-beta-N-acetylglucosaminidase H cleavage. The final 'complex' or processed oligosaccharide structure contained approximately two-thirds less [3H]mannose, was insensitive to endo-beta-N-acetylglucosaminidase H and had an apparent Mr of 2300 as estimated by gel filtration. We conclude that the carbohydrate portion of fibronectin is synthesized as a 'high-mannose' intermediate and is subsequently processed to give the characteristic 'complex' oligosaccharide chains of fibronectin.

Update on the pathological processes, molecular biology, and clinical utility of N-acetylcysteine in chronic obstructive pulmonary disease

PubMed Central

Tse, Hoi Nam; Tseng, Cee Zhung Steven

2014-01-01

Chronic obstructive pulmonary disease (COPD) is a common and morbid disease characterized by high oxidative stress. Its pathogenesis is complex, and involves excessive oxidative stress (redox imbalance), protease/antiprotease imbalance, inflammation, apoptosis, and autoimmunity. Among these, oxidative stress has a pivotal role in the pathogenesis of COPD by initiating and mediating various redox-sensitive signal transduction pathways and gene expression. The protective physiological mechanisms of the redox balance in the human body, their role in the pathogenesis of COPD, and the clinical correlation between oxidative stress and COPD are reviewed in this paper. N-acetylcysteine (NAC) is a mucolytic agent with both antioxidant and anti-inflammatory properties. This paper also reviews the use of NAC in patients with COPD, especially the dose-dependent properties of NAC, eg, its effects on lung function and the exacerbation rate in patients with the disease. Earlier data from BRONCUS (the Bronchitis Randomized on NAC Cost-Utility Study) did not suggest that NAC was beneficial in patients with COPD, only indicating that it reduced exacerbation in an “inhaled steroid-naïve” subgroup. With regard to the dose-dependent properties of NAC, two recent randomized controlled Chinese trials suggested that high-dose NAC (1,200 mg daily) can reduce exacerbations in patients with COPD, especially in those with an earlier (moderately severe) stage of disease, and also in those who are at high risk of exacerbations. However, there was no significant effect on symptoms or quality of life in patients receiving NAC. Further studies are warranted to investigate the effect of NAC at higher doses in non-Chinese patients with COPD. PMID:25125976

Glucose-induced expression of MIP-1 genes requires O-GlcNAc transferase in monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chikanishi, Toshihiro; ERATO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012; Fujiki, Ryoji

2010-04-16

O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1{alpha} and -1{beta}more » genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1{alpha} gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1{alpha} gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.« less

Induction, immunochemical identity and immunofluorescence localization of an 80 000-molecular-weight peroxisome-proliferation-associated polypeptide (polypeptide PPA-80) and peroxisomal enoyl-CoA hydratase of mouse liver and renal cortex.

PubMed

Lalwani, N D; Reddy, M K; Mangkornkanok-Mark, M; Reddy, J K

1981-07-15

The hypolipidaemic drugs methyl clofenapate, BR-931, Wy-14643 and procetofen induced a marked proliferation of peroxisomes in the parenchymal cells of liver and the proximal-convoluted-tubular epithelium of mouse kidney. The proliferation of peroxisomes was associated with 6-12-fold increase in the peroxisomal palmitoyl-CoA oxidizing capacity of the mouse liver. Enhanced activity of the peroxisomal palmitoyl-CoA oxidation system was also found in the renal-cortical homogenates of hypolipidaemic-drug-treated mice. The activity of enoyl-CoA hydratase in the mouse liver increased 30-50-fold and in the kidney cortex 3-5-fold with hypolipidaemic-drug-induced peroxisome proliferation in these tissues, and over 95% of this induced activity was found to be heat-labile peroxisomal enzyme in both organs. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of large-particle and microsomal fractions obtained from the liver and kidney cortex of mice treated with hypolipidaemic peroxisome proliferators demonstrated a substantial increase in the quantity of an 80000-mol.wt. peroxisome-proliferation-associated polypeptide (polypeptide PPA-80). The heat-labile peroxisomal enoyl-CoA hydratase was purified from the livers of mice treated with the hypolipidaemic drug methyl clofenapate; the antibodies raised against this electrophoretically homogeneous protein yielded a single immunoprecipitin band with purified mouse liver enoyl-CoA hydratase and with liver and kidney cortical extracts of normal and hypolipidaemic-drug-treated mice. These anti-(mouse liver enoyl-CoA hydratase) antibodies also cross-reacted with purified rat liver enoyl-CoA hydratase and with the polypeptide PPA-80 obtained from rat and mouse liver. Immunofluorescence studies with anti-(polypeptide PPA-80) and anti-(peroxisomal enoyl-CoA hydratase) provided visual evidence for the localization and induction of polypeptide PPA-80 and peroxisomal enoyl-CoA hydratase in the liver and kidney respectively of normal and hypolipidaemic-drug-treated mice. In the kidney, the distribution of these two proteins is identical and limited exclusively to the cytoplasm of proximal-convoluted-tubular epithelium. The immunofluorescence studies clearly complement the biochemical and ultrastructural observations of peroxisome induction in the liver and kidney cortex of mice fed on hypolipidaemic drugs. In addition, preliminary ultrastructural studies with the protein-A-gold-complex technique demonstrate that the heat-labile hepatic enoyl-CoA hydratase is localized in the peroxisome matrix.

A new tomato NAC (NAM/ATAF1/2/CUC2) transcription factor, SlNAC4, functions as a positive regulator of fruit ripening and carotenoid accumulation.

PubMed

Zhu, Mingku; Chen, Guoping; Zhou, Shuang; Tu, Yun; Wang, Yi; Dong, Tingting; Hu, Zongli

2014-01-01

Fruit ripening in tomato (Solanum lycopersicum) is a complicated development process affected by both endogenous hormonal and genetic regulators and external signals. Although the role of NOR, a member of the NAC domain family, in mediating tomato fruit ripening has been established, its underlying molecular mechanisms remain unclear. To explore further the role of NAC transcription factors in fruit ripening, we characterized a new tomato NAC domain protein, named SlNAC4, which shows high accumulation in sepal and at the onset of fruit ripening. Various stress treatments including wounding, NaCl, dehydration and low temperature significantly increased the expression of SlNAC4. Reduced expression of SlNAC4 by RNA interference (RNAi) in tomato resulted in delayed fruit ripening, suppressed Chl breakdown and decreased ethylene synthesis mediated mainly through reduced expression of ethylene biosynthesis genes of system-2, and reduced carotenoids by alteration of the carotenoid pathway flux. Transgenic tomato fruits also displayed significant down-regulation of multiple ripening-associated genes, indicating that SlNAC4 functions as a positive regulator of fruit ripening by affecting ethylene synthesis and carotenoid accumulation. Moreover, we also noted that SlNAC4 could not be induced by ethylene and may function upstream of the ripening regulator RIN and positively regulate its expression. Yeast two-hybrid assay further revealed that SlNAC4 could interact with both RIN and NOR protein. These results suggested that ethylene-dependent and -independent processes are regulated by SlNAC4 in the fruit ripening regulatory network.

Banana NAC transcription factor MusaNAC042 is positively associated with drought and salinity tolerance.

PubMed

Tak, Himanshu; Negi, Sanjana; Ganapathi, T R

2017-03-01

Banana is an important fruit crop and its yield is hampered by multiple abiotic stress conditions encountered during its growth. The NAC (NAM, ATAF, and CUC) transcription factors are involved in plant response to biotic and abiotic stresses. In the present study, we studied the induction of banana NAC042 transcription factor in drought and high salinity conditions and its overexpression in transgenic banana to improve drought and salinity tolerance. MusaNAC042 expression was positively associated with stress conditions like salinity and drought and it encoded a nuclear localized protein. Transgenic lines of banana cultivar Rasthali overexpressing MusaNAC042 were generated by Agrobacterium-mediated transformation of banana embryogenic cells and T-DNA insertion was confirmed by PCR and Southern blot analysis. Our results using leaf disc assay indicated that transgenic banana lines were able to tolerate drought and high salinity stress better than the control plants and retained higher level of total chlorophyll and lower level of MDA content (malondialdehyde). Transgenic lines analyzed for salinity (250 mM NaCl) and drought (Soil gravimetric water content 0.15) tolerance showed higher proline content, better Fv/Fm ratio, and lower levels of MDA content than control suggesting that MusaNAC042 may be involved in responses to higher salinity and drought stresses in banana. Expression of several abiotic stress-related genes like those coding for CBF/DREB, LEA, and WRKY factors was altered in transgenic lines indicating that MusaNAC042 is an efficient modulator of abiotic stress response in banana.

Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murray, M.T.; Krohne, G.; Franke, W.W.

1991-01-01

To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occurmore » in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.« less

Structural and immunochemical relatedness suggests a conserved pathogenicity motif for secondary cell wall polysaccharides in Bacillus anthracis and infection-associated Bacillus cereus

PubMed Central

Saile, Elke; Klee, Silke R.; Hoffmaster, Alex; Kannenberg, Elmar L.

2017-01-01

Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying “anthrax-like” symptoms in Cameroon (Bc CA) and Côte d’Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10–20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains. PMID:28832613

Structural and immunochemical relatedness suggests a conserved pathogenicity motif for secondary cell wall polysaccharides in Bacillus anthracis and infection-associated Bacillus cereus.

PubMed

Kamal, Nazia; Ganguly, Jhuma; Saile, Elke; Klee, Silke R; Hoffmaster, Alex; Carlson, Russell W; Forsberg, Lennart S; Kannenberg, Elmar L; Quinn, Conrad P

2017-01-01

Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying "anthrax-like" symptoms in Cameroon (Bc CA) and Côte d'Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684 (a unique strain producing an SCWP lacking all Gal substitutions) and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10-20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains.

The molecular architecture of human N-acetylgalactosamine kinase.

PubMed

Thoden, James B; Holden, Hazel M

2005-09-23

Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.

Genomic analysis of NAC transcription factors in banana (Musa acuminata) and definition of NAC orthologous groups for monocots and dicots.

PubMed

Cenci, Albero; Guignon, Valentin; Roux, Nicolas; Rouard, Mathieu

2014-05-01

Identifying the molecular mechanisms underlying tolerance to abiotic stresses is important in crop breeding. A comprehensive understanding of the gene families associated with drought tolerance is therefore highly relevant. NAC transcription factors form a large plant-specific gene family involved in the regulation of tissue development and responses to biotic and abiotic stresses. The main goal of this study was to set up a framework of orthologous groups determined by an expert sequence comparison of NAC genes from both monocots and dicots. In order to clarify the orthologous relationships among NAC genes of different species, we performed an in-depth comparative study of four divergent taxa, in dicots and monocots, whose genomes have already been completely sequenced: Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa. Due to independent evolution, NAC copy number is highly variable in these plant genomes. Based on an expert NAC sequence comparison, we propose forty orthologous groups of NAC sequences that were probably derived from an ancestor gene present in the most recent common ancestor of dicots and monocots. These orthologous groups provide a curated resource for large-scale protein sequence annotation of NAC transcription factors. The established orthology relationships also provide a useful reference for NAC function studies in newly sequenced genomes such as M. acuminata and other plant species.

[Cloning, subcellular localization, and heterologous expression of ApNAC1 gene from Andrographis paniculata].

PubMed

Wang, Jian; Qi, Meng-Die; Guo, Juan; Shen, Ye; Lin, Hui-Xin; Huang, Lu-Qi

2017-03-01

Andrographis paniculata is widely used as medicinal herb in China for a long time and andrographolide is its main medicinal constituent. To investigate the underlying andrographolide biosynthesis mechanisms, RNA-seq for A. paniculata leaves with MeJA treatment was performed. In A. paniculata transcriptomic data, the expression pattern of one member of NAC transcription factor family (ApNAC1) matched with andrographolide accumulation. The coding sequence of ApNAC1 was cloned by RT-PCR, and GenBank accession number was KY196416. The analysis of bioinformatics showed that the gene encodes a peptide of 323 amino acids, with a predicted relative molecular weight of 35.9 kDa and isoelectric point of 6.14. To confirm the subcellular localization, ApNAC1-GFP was transiently expressed in A. paniculata protoplast. The results indicated that ApNAC1 is a nucleus-localized protein. The analysis of real-time quantitative PCR revealed that ApNAC1 gene predominantly expresses in leaves. Compared with control sample, its expression abundance sharply increased with methyl jasmonate treatment. Based on its expression pattern, ApNAC1 gene might involve in andrographolide biosynthesis. ApNAC1 was heterologously expressed in Escherichia coli and recombinant protein was purified by Ni-NTA agarose. Further study will help us to understand the function of ApNAC1 in andrographolide biosynthesis. Copyright© by the Chinese Pharmaceutical Association.

Applications of site-specific labeling to study HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

PubMed

Mercer, Natalia; Ramakrishnan, Boopathy; Boeggeman, Elizabeth; Qasba, Pradman K

2011-01-01

Alpha-lactalbumin (α-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of β1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milk's main carbohydrate. When calcium is removed from α-LA, it adopts a molten globule form, and this form, interestingly, when complexed with oleic acid (OA) acquires tumoricidal activity. Such a complex made from human α-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established. In the present work, we have used site-specific labeling, a technique previously developed in our laboratory, to label HAMLET with biotin, or a fluoroprobe for confocal microscopy studies. In addition to full length hLA, the α-domain of hLA (αD-hLA) alone is also included in the present study. We have engineered these proteins with a 17-amino acid C-terminal extension (hLA-ext and αD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose) using polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules. We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity.

The Rose (Rosa hybrida) NAC Transcription Factor 3 Gene, RhNAC3, Involved in ABA Signaling Pathway Both in Rose and Arabidopsis

PubMed Central

Lü, Peitao; Liu, Jitao; Gao, Junping; Zhang, Changqing

2014-01-01

Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (Rosa hybrida), RhNAC3, was previously found to increase dehydration tolerance in both rose and Arabidopsis. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of RhNAC3 and found many stress-related cis-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of Arabidopsis thaliana plants transformed with the putative RhNAC3 promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that RhNAC3 is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the RhNAC3 promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of RhNAC3 in A. thaliana resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing RhNAC3 than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding cis-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 A. thaliana genes previously shown by microarray analysis to be regulated by heterologous overexpression RhNAC3, 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in RhNAC3-silenced petals. Taken together, we propose that the rose RhNAC3 protein could mediate ABA signaling both in rose and in A. thaliana. PMID:25290154

The rose (Rosa hybrida) NAC transcription factor 3 gene, RhNAC3, involved in ABA signaling pathway both in rose and Arabidopsis.

PubMed

Jiang, Guimei; Jiang, Xinqiang; Lü, Peitao; Liu, Jitao; Gao, Junping; Zhang, Changqing

2014-01-01

Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (Rosa hybrida), RhNAC3, was previously found to increase dehydration tolerance in both rose and Arabidopsis. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of RhNAC3 and found many stress-related cis-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of Arabidopsis thaliana plants transformed with the putative RhNAC3 promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that RhNAC3 is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the RhNAC3 promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of RhNAC3 in A. thaliana resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing RhNAC3 than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding cis-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 A. thaliana genes previously shown by microarray analysis to be regulated by heterologous overexpression RhNAC3, 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in RhNAC3-silenced petals. Taken together, we propose that the rose RhNAC3 protein could mediate ABA signaling both in rose and in A. thaliana.

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

PubMed

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-12-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

Optimal Positioning of the Nipple-Areola Complex in Men Using the Mohrenheim-Estimated-Tangential-Tracking-Line (METT-Line): An Intuitive Approach.

PubMed

Mett, Tobias R; Krezdorn, Nicco; Luketina, Rosalia; Boyce, Maria K; Henseler, Helga; Ipaktchi, Ramin; Vogt, Peter M

2017-12-01

The reconstruction of the body shape after post-bariatric surgery or high-grade gynecomastia involves, besides skin tightening, the repositioning of anatomical, apparent landmarks. The surgeon usually defines these during the preoperative planning. In particular, the positions of the nipple-areola complexes (NAC) should contribute to the gender-appropriate appearance. While in the female breast numerous methods have been developed to determine the optimal position of the NACs, there are only a few, metric and often impractical algorithms for positioning the nipples and areoles in the male. With this study, we show the accuracy of the intuitive positioning of the nipple-areola complex in men. From a pre-examined and measured quantity of 10 young and healthy men, six subjects were selected, which corresponded, on the basis of their chest and trunk dimensions, to the average of known data from the literature. The photographed frontal views were retouched in two steps. Initially, only the NACs were removed and the chest contours were left. In a second step, all contours and the navel were blurred. These pictures were submitted to resident and consultant plastic surgeons, who were asked to draw the missing NACs without any tools. The original positions of the nipples were compared with the inscriptions. Furthermore, the results were compared between the contoured and completely retouched pictures and between the residents and consultants. A total of 8 consultants and 7 residents were included. In the contoured and completely retouched images, a significant deviation of the marked positions of the missing features was found. The height of the NAC was determined somewhat more precisely than the vertical position. There was no significant difference between the contoured and completely retouched images, with a discretely more accurate tendency on the contoured images. In comparison with the professional experience, the consultants were tangentially more precise, but without a statistically significant impact. The intuitive determination of the NACs is a challenge for the plastic surgeon. In this study, a statistically significant deviation was seen in almost all dimensions, although the clinical relevance cannot be conclusively assessed. We found a positional relationship of the NAC to the infraclavicular groove ("Mohrenheim pit") in the vertical and 4-4.5 cm above the submammary fold. The position of the NAC can be satisfactorily determined by a combination of plastic surgical intuition, patient wishes and practical metric methods using the Mohrenheim-Estimated-Tangential-Tracking-Line (METT-Line). This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to Table of Contents or the online Instructions to Authors www.springer.com/00266 .

N-acetylcysteine inhibits in vivo oxidation of native low-density lipoprotein

PubMed Central

Cui, Yuqi; Narasimhulu, Chandrakala A.; Liu, Lingjuan; Zhang, Qingbin; Liu, Patrick Z.; Li, Xin; Xiao, Yuan; Zhang, Jia; Hao, Hong; Xie, Xiaoyun; He, Guanglong; Cui, Lianqun; Parthasarathy, Sampath; Liu, Zhenguo

2015-01-01

Low-density lipoprotein (LDL) is non-atherogenic, while oxidized LDL (ox-LDL) is critical to atherosclerosis. N-acetylcysteine (NAC) has anti-atherosclerotic effect with largely unknown mechanisms. The present study aimed to determine if NAC could attenuate in vivo LDL oxidation and inhibit atherosclerosis. A single dose of human native LDL was injected intravenously into male C57BL/6 mice with and without NAC treatment. Serum human ox-LDL was detected 30 min after injection, reached the peak in 3 hours, and became undetectable in 12 hours. NAC treatment significantly reduced serum ox-LDL level without detectable serum ox-LDL 6 hours after LDL injection. No difference in ox-LDL clearance was observed in NAC-treated animals. NAC treatment also significantly decreased serum ox-LDL level in patients with coronary artery diseases and hyperlipidemia without effect on LDL level. Intracellular and extracellular reactive oxidative species (ROS) production was significantly increased in the animals treated with native LDL, or ox-LDL and in hyperlipidemic LDL receptor knockout (LDLR−/−) mice that was effectively prevented with NAC treatment. NAC also significantly reduced atherosclerotic plaque formation in hyperlipidemic LDLR−/− mice. NAC attenuated in vivo oxidation of native LDL and ROS formation from ox-LDL associated with decreased atherosclerotic plaque formation in hyperlipidemia. PMID:26536834

A Developmental Study of Photosystem I Peripheral Chlorophyll Proteins 1

PubMed Central

Mullet, John E.; Burke, John J.; Arntzen, Charles J.

1980-01-01

An isolated “native” photosystem I (PSI complex) contains three spectral populations of chlorophyll a antennae (Mullet, Burke, Arntzen 1980 Plant Physiol 65: 814-822). It was hypothesized that nearly one-half of these antennae (≃45 Chl/P700) are associated with polypeptides of 21,500 to 24,500 daltons. The present study utilizes two developmental systems to verify this association. Chloroplasts were isolated from a Chl b-less barley mutant and from partially-developed cucumber cotyledons (greened under intermittent illumination [ImL] chloroplasts) and were compared to control chloroplasts isolated from wild-type barley and mature cucumber. Both the mutant and ImL chloroplasts exhibited a long wavelength fluorescence maximum at 724 nanometers at 77 K as compared to 735 to 738 nanometers emission maximum in the respective controls. Both the mutant and ImL chloroplasts were deficient in polypeptides of 21,500 to 24,500 daltons which were present in control membranes and in PSI fractions isolated from control membranes. In light-induced maturation of the ImL cucumbers, the synthesis of polypeptides in the 21,500 to 24,500 molecular weight range paralleled the appearance of PSI Chl species fluorescing at long wavelength (≃735 nm). The PSI spectral properties of the control membranes were retained in isolated PSI particles containing 100 to 120 Chl/P700 (PSI-110). Detergent extraction of PSI-110 removed polypeptides of 21,500 to 24,500 daltons plus ≃ 45 Chl/P700. The antennae-depleted PSI particle mimics PSI properties exhibited by incompletely differentiated mutant or ImL chloroplasts. Images PMID:16661289

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beattie, Ross J.; Sutton, Andrew D.; Scott, Brian L.

The sterically encumbered NacNac ligand, [HC(MeCNAr) 2] – (Ar = 2,6- i-Pr 2C 6H 3), was investigated as a platform for supporting Lu-halide complexes, sought for their potential capability of being further converted into hydrocarbyl derivatives via metathetical chemistries with alkali metal alkyls. As a result, these substituted analogs were targeted as potentially viable candidates for alkane elimination chemistries, with an eye towards the formation of an isolable Lu-alkylidene fragment.

Effect of bisecting GlcNAc and core fucosylation on conformational properties of biantennary complex-type N-glycans in solution.

PubMed

Nishima, Wataru; Miyashita, Naoyuki; Yamaguchi, Yoshiki; Sugita, Yuji; Re, Suyong

2012-07-26

The introduction of bisecting GlcNAc and core fucosylation in N-glycans is essential for fine functional regulation of glycoproteins. In this paper, the effect of these modifications on the conformational properties of N-glycans is examined at the atomic level by performing replica-exchange molecular dynamics (REMD) simulations. We simulate four biantennary complex-type N-glycans, namely, unmodified, two single-substituted with either bisecting GlcNAc or core fucose, and disubstituted forms. By using REMD as an enhanced sampling technique, five distinct conformers in solution, each of which is characterized by its local orientation of the Manα1-6Man glycosidic linkage, are observed for all four N-glycans. The chemical modifications significantly change their conformational equilibria. The number of major conformers is reduced from five to two and from five to four upon the introduction of bisecting GlcNAc and core fucosylation, respectively. The population change is attributed to specific inter-residue hydrogen bonds, including water-mediated ones. The experimental NMR data, including nuclear Overhauser enhancement and scalar J-coupling constants, are well reproduced taking the multiple conformers into account. Our structural model supports the concept of "conformer selection", which emphasizes the conformational flexibility of N-glycans in protein-glycan interactions.

The NACS Education Program

ERIC Educational Resources Information Center

Thodt, Charles A.

1978-01-01

The National Association of College Stores has for 31 years offered intensive formal training in bookstore management. The NACS Management Survey/Management Seminar, Booksellers School, and Advanced Seminars on Personnel Supervision and Financial Management are described. (LBH)

A Novel RNA Polymerase I Transcription Initiation Factor, TIF-IE, Commits rRNA Genes by Interaction with TIF-IB, Not by DNA Binding

PubMed Central

Al-Khouri, Anna Maria; Paule, Marvin R.

2002-01-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE. PMID:11784852

A novel RNA polymerase I transcription initiation factor, TIF-IE, commits rRNA genes by interaction with TIF-IB, not by DNA binding.

PubMed

Al-Khouri, Anna Maria; Paule, Marvin R

2002-02-01

In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.

Radiotherapy and nipple-areolar complex necrosis after nipple-sparing mastectomy: a systematic review and meta-analysis.

PubMed

Zheng, Yajuan; Zhong, Miaochun; Ni, Chao; Yuan, Hongjun; Zhang, Jingxia

2017-03-01

To perform a meta-analysis to determine the effect of radiotherapy (RT) on nipple-areolar complex (NAC) and skin flap necrosis, and local recurrence in women who undergo nipple-sparing mastectomy (NSM) and immediate breast reconstruction. Medline, PubMed, Cochrane, and Google Scholar databases were searched until October 16, 2015. Randomized-controlled-trials, prospective, retrospective, and cohort studies were included. The primary outcome was the NAC necrosis rate, and the secondary outcomes were the skin flap necrosis and local recurrence rates. Of 186 studies identified, 2 prospective and 5 retrospective studies including a total of 3692 patients were included in the meta-analysis. Five, 3, and 2 studies reported data of NAC necrosis (3461 breasts), skin flap necrosis (2490 breasts), and local recurrence (988 breasts), respectively. Pooled results showed no difference in the odds of NAC necrosis [odds ratio (OR) = 1.250, 95% confidence interval (CI) 0.481-3.247, P = 0.647], or local recurrence (OR = 0.564, 95% CI 0.056-5.710, P = 0.627) between patients who received and did not receive RT. Patients treated with RT had a higher likelihood of skin flap necrosis (OR = 2.534, 95% CI 1.720-3.735, P < 0.001). Significant heterogeneity, however, was noted in the analysis of NAC and local recurrence. Because of the limitations of the small number of studies and heterogeneity in the analysis, this study does not allow drawing any definitive conclusions and highlights the need of well-controlled trials to determine the effect of RT in patients undergoing NSM.

The arterial supply of the nipple areola complex (NAC) and its relations: an analysis of angiographic CT imaging for breast pedicle design.

PubMed

Stirling, Aaron D; Murray, Conor P; Lee, Mark A

2017-10-01

To investigate the blood supply to the nipple areola complex (NAC) on thoracic CT angiograms (CTA) to improve breast pedicle design in reduction mammoplasty. In a single centre, CT scans of the thorax were retrospectively reviewed for suitability by a cardiothoracic radiologist. Suitable scans had one or both breasts visible in extended fields, with contrast enhancement of breast vasculature in a female patient. The arterial sources, intercostal space perforated, glandular/subcutaneous course, vessel entry point, and the presence of periareolar anastomoses were recorded for the NAC of each breast. From 69 patients, 132 breasts were suitable for inclusion. The most reproducible arterial contribution to the NAC was perforating branches arising from the internal thoracic artery (ITA) (n = 108, 81.8%), followed by the long thoracic artery (LTA) (n = 31, 23.5%) and anterior intercostal arteries (AI) (n = 21, 15.9%). Blood supply was superficial versus deep in (n = 86, 79.6%) of ITA sources, (n = 28, 90.3%) of LTA sources, and 10 (47.6%) of AI sources. The most vascularly reliable breast pedicle would be asymmetrical in 7.9% as a conservative estimate. We suggest that breast CT angiography can provide valuable information about NAC blood supply to aid customised pedicle design, especially in high-risk, large-volume breast reductions where the risk of vascular-dependent complications is the greatest and asymmetrical dominant vasculature may be present. Superficial ITA perforator supplies are predominant in a majority of women, followed by LTA- and AIA-based sources, respectively.

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

PubMed

Fan, Qiong; Moen, Anders; Anonsen, Jan Haug; Bindesbøll, Christian; Sæther, Thomas; Carlson, Cathrine Rein; Grønning-Wang, Line M

2018-05-05

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20 LWKPGAQDASSQAQGGSSCILRE 42 . However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT. Copyright © 2018 Elsevier Inc. All rights reserved.

PpNAC1, a main regulator of phenylalanine biosynthesis and utilization in maritime pine.

PubMed

Pascual, María Belén; Llebrés, María-Teresa; Craven-Bartle, Blanca; Cañas, Rafael A; Cánovas, Francisco M; Ávila, Concepción

2018-05-01

The transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long-lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the down-regulation of several genes associated with cell wall biogenesis and secondary metabolism. Furthermore, transactivation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the ArabidopsisSND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and paves the way for applications in conifer biotechnology. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

BEND3 mediates transcriptional repression and heterochromatin organization

PubMed Central

Khan, Abid; Prasanth, Supriya G

2015-01-01

Transcription repression plays a central role in gene regulation. Transcription repressors utilize diverse strategies to mediate transcriptional repression. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein represses rDNA transcription by stabilizing a NoRC component. We discuss the role of BEND3 as a global regulator of gene expression and propose a model whereby BEND3 associates with chromatin remodeling complexes to modulate gene expression and heterochromatin organization. PMID:26507581

BEND3 mediates transcriptional repression and heterochromatin organization.

PubMed

Khan, Abid; Prasanth, Supriya G

2015-01-01

Transcription repression plays a central role in gene regulation. Transcription repressors utilize diverse strategies to mediate transcriptional repression. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein represses rDNA transcription by stabilizing a NoRC component. We discuss the role of BEND3 as a global regulator of gene expression and propose a model whereby BEND3 associates with chromatin remodeling complexes to modulate gene expression and heterochromatin organization.

Antioxidants reverse the changes in energy metabolism of rat brain after chronic administration of L.-tyrosine.

PubMed

Teodorak, Brena P; Scaini, Giselli; Carvalho-Silva, Milena; Gomes, Lara M; Teixeira, Letícia J; Rebelo, Joyce; De Prá, Samira D T; Zeni, Neila; Schuck, Patrícia F; Ferreira, Gustavo C; Streck, Emilio L

2017-04-01

Tyrosinemia type II is a rare autosomal recessive disease caused by deficiency of hepatic tyrosine aminotransferase and is associated with neurologic and development difficulties in numerous patients. Considering that the mechanisms underlying the neurological dysfunction in hypertyrosinemic patients are poorly known and that high concentrations of tyrosine provoke mitochondrial dysfunction and oxidative stress, in the present study we investigated the in vivo influence of antioxidants (N-acetylcysteine, NAC; and deferoxamine, DFX) administration on the inhibitory effects on parameters of energy metabolism in cerebral cortex, hippocampus and striatum of rats, provoked by chronic administration of L.-tyrosine. Our results showed that chronic administration of L.-tyrosine results in a marked decrease in the activity of citrate synthase in all the analyzed structures and succinate dehydrogenase activities in hippocampus and striatum, and that antioxidants administration can prevent this inhibition in hippocampus and striatum. Moreover, chronic administration of L.-tyrosine inhibited the activity of complex I, II-III and IV in the striatum, which can be prevented by antioxidant treatment. However, the co-administration of NAC plus DFX could not prevent the inhibition of creatine kinase activity in the striatum. In conclusion, the present study demonstrates that the administration of antioxidants NAC and DFX attenuates the L.-tyrosine effects on enzymes of the Krebs cycle and the mitochondrial respiratory chain, suggesting that impairment of energy metabolism can be involved with oxidative stress. These results also indicate a possible neuroprotective role for NAC and DFX as a potential adjuvant therapy to the patients with Tyrosinemia type II.

ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors.

PubMed

Halasi, Marianna; Wang, Ming; Chavan, Tanmay S; Gaponenko, Vadim; Hay, Nissim; Gartel, Andrei L

2013-09-01

NAC (N-acetyl-L-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H₂O₂. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis.

Label-Free Neuroproteomics of the Hippocampal-Accumbal Circuit Reveals Deficits in Neurotransmitter and Neuropeptide Signaling in Mice Lacking Ethanol-Sensitive Adenosine Transporter.

PubMed

Oliveros, Alfredo; Starski, Phillip; Lindberg, Daniel; Choi, Sun; Heppelmann, Carrie J; Dasari, Surendra; Choi, Doo-Sup

2017-04-07

The neural circuit of the dorsal hippocampus (dHip) and nucleus accumbens (NAc) contributes to cue-induced learning and addictive behaviors, as demonstrated by the escalation of ethanol-seeking behaviors observed following deletion of the adenosine equilibrative nucleoside transporter 1 (ENT1 -/- ) in mice. Here we perform quantitative LC-MS/MS neuroproteomics in the dHip and NAc of ENT1 -/- mice. Using Ingenuity Pathway Analysis, we identified proteins associated with increased long-term potentiation, ARP2/3-mediated actin cytoskeleton signaling and protein expression patterns suggesting deficits in glutamate degradation, GABAergic signaling, as well as significant changes in bioenergetics and energy homeostasis (oxidative phosphorylation, TCA cycle, and glycolysis). These pathways are consistent with previously reported behavioral and biochemical phenotypes that typify mice lacking ENT1. Moreover, we validated decreased expression of the SNARE complex protein VAMP1 (synaptobrevin-1) in the dHip as well as decreased expression of pro-dynorphin (PDYN), neuroendocrine convertase (PCSK1), and Leu-Enkephalin (dynorphin-A) in the NAc. Taken together, our proteomic approach provides novel pathways indicating that ENT1-regulated signaling is essential for neurotransmitter release and neuropeptide processing, both of which underlie learning and reward-seeking behaviors.

Superoxide scavenging effects of N-acetylcysteine and vitamin C in subjects with essential hypertension.

PubMed

Schneider, Markus P; Delles, Christian; Schmidt, Bernhard M W; Oehmer, Sebastian; Schwarz, Thomas K; Schmieder, Roland E; John, Stefan

2005-08-01

It is not known whether the beneficial effects of N-acetylcysteine (NAC) in conditions associated with increased oxidative stress are caused by direct superoxide scavenging. We therefore compared the acute superoxide scavenging efficacy of NAC against vitamin C (VITC) on impaired endothelium-dependent vasodilation in subjects with essential hypertension. In a cross-over randomized study, the effects of intra-arterial administration of either NAC (48 mg/min) or VITC (18 mg/min) were examined in 15 subjects with essential hypertension and in 15 normotensive control subjects. Both endothelium-dependent and endothelium-independent vasodilation were determined as forearm blood flow (FBF) response to the intra-arterial administration of acetylcholine (Ach) and sodium nitroprusside (NP) in doses of 12 and 48 mug/min and 3.2 and 12.8 mug/min, respectively. Subjects with essential hypertension had impaired responses to both doses of Ach (Delta% FBF to higher dose of Ach: 325 +/- 146 in subjects with essential hypertension v 540 +/- 199 in control subjects; P = .02) and an impaired response to the higher dose of NP (330 +/- 108 v 500 +/- 199; P = .03). The intra-arterial administration of NAC had no effect on these responses (higher dose of Ach: 325 +/- 146 without v 338 +/- 112 with NAC, NS). In contrast, intra-arterial VITC improved both the response to Ach (320 +/- 132 without v 400 +/- 185 with VITC, P = .05) and to NP (383 +/- 162 v 447 +/- 170, P = .05). We found that NAC showed no statistically significant effect on either endothelium-dependent or endothelium-independent vasodilation in hypertensive subjects, whereas VITC did. We conclude that NAC is therefore not an effective superoxide scavenger in vivo. Other, nonimmediate effects such as the generation of glutathione may explain the beneficial effects of NAC in conditions associated with oxidative stress.

Two cofactors and cytoplasmic chaperonin are required for the folding of alpha- and beta-tubulin.

PubMed Central

Gao, Y; Vainberg, I E; Chow, R L; Cowan, N J

1993-01-01

Though the chaperonins that mediate folding in prokaryotes, mitochondria, and chloroplasts have been relatively well characterized, the folding of proteins in the eukaryotic cytosol is much less well understood. We recently identified a cytoplasmic chaperonin as an 800-kDa multisubunit toroid which forms a binary complex with unfolded actin; the correctly folded polypeptide is released upon incubation with Mg-ATP (Y. Gao, J. O. Thomas, R. L. Chow, G.-H. Lee, and N. J. Cowan, Cell 69:1043-1050, 1992). Here we show that the same chaperonin also forms a binary complex with unfolded alpha- or beta-tubulin; however, there is no detectable release of the correctly folded product, irrespective of the concentration of added Mg-ATP and Mg-GTP or the presence of added carrier tubulin heterodimers with which newly folded alpha- or beta-tubulin polypeptides might exchange. Rather, two additional protein cofactors are required for the generation of properly folded alpha- or beta-tubulin, which is then competent for exchange into preexisting alpha/beta-tubulin heterodimers. We show that actin and tubulins compete efficiently with one another for association with cytoplasmic chaperonin complexes. These data imply that actin and alpha- and beta-tubulin interact with the same site(s) on chaperonin complexes. Images PMID:8096061

[Proteins and saponins in the lipid preparation obtained by extraction of soybean flour].

PubMed

Baukova, N A; Alekseeva, S G; Sorokoumova, G M; Selishcheva, A A; Martynova, O M; Rogozhkina, E A; Shvets, V I

2002-01-01

A complex lipid preparation was obtained by extraction of soybean flour with organic solvents. This preparation was shown to include not only phospholipids (major components), but also up to 30% saponins. These compounds were identified by TLC, HPLC, and 1H-NMR spectroscopy. Minor components of the lipid extract were represented by polypeptides associated with phospholipids via electrostatic or hydrophobic forces.

Divergent Evolutionary Patterns of NAC Transcription Factors Are Associated with Diversification and Gene Duplications in Angiosperm

PubMed Central

Jin, Xiaoli; Ren, Jing; Nevo, Eviatar; Yin, Xuegui; Sun, Dongfa; Peng, Junhua

2017-01-01

NAC (NAM/ATAF/CUC) proteins constitute one of the biggest plant-specific transcription factor (TF) families and have crucial roles in diverse developmental programs during plant growth. Phylogenetic analyses have revealed both conserved and lineage-specific NAC subfamilies, among which various origins and distinct features were observed. It is reasonable to hypothesize that there should be divergent evolutionary patterns of NAC TFs both between dicots and monocots, and among NAC subfamilies. In this study, we compared the gene duplication and loss, evolutionary rate, and selective pattern among non-lineage specific NAC subfamilies, as well as those between dicots and monocots, through genome-wide analyses of sequence and functional data in six dicot and five grass lineages. The number of genes gained in the dicot lineages was much larger than that in the grass lineages, while fewer gene losses were observed in the grass than that in the dicots. We revealed (1) uneven constitution of Clusters of Orthologous Groups (COGs) and contrasting birth/death rates among subfamilies, and (2) two distinct evolutionary scenarios of NAC TFs between dicots and grasses. Our results demonstrated that relaxed selection, resulting from concerted gene duplications, may have permitted substitutions responsible for functional divergence of NAC genes into new lineages. The underlying mechanism of distinct evolutionary fates of NAC TFs shed lights on how evolutionary divergence contributes to differences in establishing NAC gene subfamilies and thus impacts the distinct features between dicots and grasses. PMID:28713414

Purification and chemical characterisation of a cell wall-associated β-galactosidase from mature sweet cherry (Prunus avium L.) fruit.

PubMed

Gerardi, Carmela; Blando, Federica; Santino, Angelo

2012-12-01

Using four different chromatographic steps, β-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium β-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and β-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing β-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry β-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple β-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry β-galactosidase exhibited a strict specificity towards p-nitrophenyl β-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc.

PubMed

Chandrasekaran, E V; Jain, R K; Larsen, R D; Wlasichuk, K; Matta, K L

1996-07-09

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an alpha 1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha 1,6-linked Fuo.

Regulated binding of PTP1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of beta-catenin

PubMed Central

1996-01-01

Cadherins are a family of cell-cell adhesion molecules which play a central role in controlling morphogenetic movements during development. Cadherin function is regulated by its association with the actin containing cytoskeleton, an association mediated by a complex of cytoplasmic proteins, the catenins: alpha, beta, and gamma. Phosphorylated tyrosine residues on beta-catenin are correlated with loss of cadherin function. Consistent with this, we find that only nontyrosine phosphorylated beta-catenin is associated with N-cadherin in E10 chick retina tissue. Moreover, we demonstrate that a PTP1B-like tyrosine phosphatase associates with N-cadherin and may function as a regulatory switch controlling cadherin function by dephosphorylating beta-catenin, thereby maintaining cells in an adhesion-competent state. The PTP1B-like phosphatase is itself tyrosine phosphorylated. Moreover, both direct binding experiments performed with phosphorylated and dephosphorylated molecules, and treatment of cells with tyrosine kinase inhibitors indicate that the interaction of the PTP1B-like phosphatase with N-cadherin depends on its tyrosine phosphorylation. Concomitant with the tyrosine kinase inhibitor-induced loss of the PTP1B-like phosphatase from its association with N-cadherin, phosphorylated tyrosine residues are retained on beta-catenin, the association of N- cadherin with the actin containing cytoskeleton is lost and N-cadherin- mediated cell adhesion is prevented. Tyrosine phosphatase inhibitors also result in the accumulation of phosphorylated tyrosine residues on beta-catenin, loss of the association of N-cadherin with the actin- containing cytoskeleton, and prevent N-cadherin mediated adhesion, presumably by directly blocking the function of the PTP1B-like phosphatase. We previously showed that the binding of two ligands to the cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase), the monoclonal antibody 1B11 and a proteoglycan with a 250-kD core protein, results in the accumulation of phosphorylated tyrosine residues on beta-catenin, uncoupling of N-cadherin from its association with the actin containing cytoskeleton, and loss of N- cadherin function. We now report that binding of these ligands to the GalNAcPTase results in the absence of the PTP1B-like phosphatase from its association with N-cadherin as well as the loss of the tyrosine kinase and tyrosine phosphatase activities that otherwise co- precipitate with N-cadherin. Control antibodies and proteoglycans have no such effect. This effect is similar to that observed with tyrosine kinase inhibitors, suggesting that the GalNAcPTase/proteoglycan interaction inhibits a tyrosine kinase, thereby preventing the phosphorylation of the PTP1B-like phosphatase, and its association with N-cadherin. Taken together these data indicate that a PTP1B-like tyrosine phosphatase can regulate N-cadherin function through its ability to dephosphorylate beta-catenin and that the association of the phosphatase with N-cadherin is regulated via the interaction of the GalNAcPTase with its proteoglycan ligand. In this manner the GalNAcPTase-proteoglycan interaction may play a major role in morphogenetic cell and tissue interactions during development. PMID:8707857

Catfish (Clarias batrachus) serum lectin recognizes polyvalent Tn [alpha-D-GalpNAc1-Ser/Thr], Talpha [beta-D-Galp-(1-->3)-alpha-D-GalpNAc1-Ser/Thr], and II [beta-D-Galp(1-->4)-beta-D-GlcpNAc1-] mammalian glycotopes.

PubMed

Singha, Biswajit; Adhya, Mausumi; Chatterjee, Bishnu P

2008-09-22

A new calcium dependent GalNAc/Gal specific lectin was isolated from the serum of Indian catfish, Clarias batrachus and designated as C. batrachus lectin (CBL). It is a disulfide-linked homodecameric lectin of 74.65kDa subunits and the oligomeric form is essential for its activity. Binding specificity of CBL was investigated by enzyme-linked lectin-sorbent assay using a series of simple sugars, polysaccharides, and glycoproteins. GalNAc was more potent inhibitor than Gal; and alpha glycosides of both were more inhibitory than their beta counterparts. CBL showed maximum affinity for human tumor-associated Tn-antigens (GalNAcalpha1-Ser/Thr) at the molecular level and was 3.5 times higher than GalNAc. CBL interacted strongly with polyvalent Tn and Talpha (Galbeta1,3GalNAcalpha1-) as well as multivalent-II (Galbeta1,4GlcNAcbeta1-) antigens containing glycoproteins and intensity of inhibition was 10(3)-10(5) times more than monovalent ones. The overall specificity of CBL lies in the order of polyvalent Tn, Talpha and II>monovalent Tn > or = Me-alphaGalNAc>monovalent Talpha> Me-betaGalNAc>Me-alphaGal>monovalent T>GalNAc>monovalent F>monovalent II>Me-betaGal>Gal.

Evaluation of the antioxidant properties of N-acetylcysteine in human platelets: prerequisite for bioconversion to glutathione for antioxidant and antiplatelet activity.

PubMed

Gibson, Kyle R; Neilson, Ilene L; Barrett, Fiona; Winterburn, Tim J; Sharma, Sushma; MacRury, Sandra M; Megson, Ian L

2009-10-01

N-Acetylcysteine (NAC) is a frequently used "antioxidant" in vitro, but the concentrations applied rarely correlate with those encountered with oral dosing in vivo. Here, we investigated the in vitro antioxidant and antiplatelet properties of NAC at concentrations (10-100 microM) that are achievable in plasma with tolerable oral dosing. The impact of NAC pretreatment (2 hours) on aggregation of platelets from healthy volunteers in response to thrombin and adenosine diphosphate and on platelet-derived nitric oxide (NO) was examined. NAC was found to be a weak reducing agent and a poor antioxidant compared with glutathione (reduced form) (GSH). However, platelets treated with NAC showed enhanced antioxidant activity and depression of reactive oxygen species generation associated with increases in intraplatelet GSH levels. An approximately 2-fold increase in NO synthase-derived nitrite was observed with 10 microM NAC treatment, but the effect was not concentration dependent. Finally, NAC significantly reduced both thrombin-induced and adenosine diphosphate-induced platelet aggregation. NAC should be considered a weak antioxidant that requires prior conversion to GSH to convey antioxidant and antithrombotic benefit at therapeutically relevant concentrations. Our results suggest that NAC might be an effective antiplatelet agent in conditions where increased oxidative stress contributes to heightened risk of thrombosis but only if the intraplatelet machinery to convert it to GSH is functional.

Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

PubMed

McElduff, A; Watkinson, A; Hedo, J A; Gorden, P

1986-11-01

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits.

Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

PubMed Central

McElduff, A; Watkinson, A; Hedo, J A; Gorden, P

1986-01-01

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits. Images Fig. 1. PMID:3827820

Evidence from in vivo manipulations of lipid composition in mutants that the delta 3-trans-hexadecenoic acid-containing phosphatidylglycerol is involved in the biogenesis of the light-harvesting chlorophyll a/b-protein complex of Chlamydomonas reinhardtii.

PubMed

Dubertret, G; Mirshahi, A; Mirshahi, M; Gerard-Hirne, C; Tremolieres, A

1994-12-01

The phosphatidylglycerol containing the unusual delta 3-trans hexadecenoic fatty acid is specifically found in photosynthetic membranes of eukaryotic organisms. Its involvement in the biogenesis and the structure of the light-harvesting chlorophyll a/b-protein complex has been evidenced by in vivo targeting this lipid to photosynthetic membranes of Chlamydomonas reinhardtii mutants lacking this lipid. In the mf1 and mf2 mutants, this deficiency results in (a) the absence of the oligomeric light-harvesting complex of photosystem 2; (b) an extensive destacking of thylakoid membranes; (c) a very low 77-K fluorescence emission in the photosystem-2 region. We show in this paper that these deficiencies result from modifications in the pigment and polypeptide compositions of the photosystem-2 light-harvesting complex; it contains less chlorophyll b and some of its constitutive polypeptides are absent or reduced in amount, while immunologically related polypeptides of lower molecular mass accumulate. The direct involvement of the lack of trans-C16: 1-phosphatidylglycerol in these deficiencies is evidenced by the partial restoration of normal characteristics of the light-harvesting complex (pigment and polypeptide composition, oligomerization) after liposome-mediated, in vivo incorporation of this lipid into the photosynthetic membranes of the mf2 mutant. Trans-C16:1-phosphatidylglycerol, therefore, is involved in the biogenesis of the photosystem-2 light-harvesting chlorophyll a/b-protein complex through a mechanism that may prevent degradation processes. Its contribution to the structural conformation of neosynthesized monomers and to their organization into stable oligomeric form is discussed.

Extinction of opiate reward reduces dendritic arborization and c-Fos expression in the nucleus accumbens core.

PubMed

Leite-Morris, Kimberly A; Kobrin, Kendra L; Guy, Marsha D; Young, Angela J; Heinrichs, Stephen C; Kaplan, Gary B

2014-04-15

Recurrent opiate use combined with environmental cues, in which the drug was administered, provokes cue-induced drug craving and conditioned drug reward. Drug abuse craving is frequently linked with stimuli from a prior drug-taking environment via classical conditioning and associative learning. We modeled the conditioned morphine reward process by using acquisition and extinction of conditioned place preference (CPP) in C57BL/6 mice. Mice were trained to associate a morphine injection with a drug context using a classical conditioning paradigm. In morphine conditioning (0, 0.25, 0.5, 1, 5, or 10 mg/kg) experimental mice acquired a morphine CPP dose response with 10mg/kg as most effective. During morphine CPP extinction experiments, mice were divided into three test groups: morphine CPP followed by extinction training, morphine CPP followed by sham extinction, and saline controls. Extinction of morphine CPP developed within one extinction experiment (4 days) that lasted over two more trials (another 8 days). However, the morphine CPP/sham extinction group retained a place preference that endured through all three extinction trials. Brains were harvested following CPP extinction and processed using Golgi-Cox impregnation. Changes in dendritic morphology and spine quantity were examined in the nucleus accumbens (NAc) Core and Shell neurons. In the NAcCore only, morphine CPP/extinguished mice produced less dendritic arborization, and a decrease in neuronal activity marker c-Fos compared to the morphine CPP/sham extinction group. Extinction of morphine CPP is associated with decreased structural complexity of dendrites in the NAcCore and may represent a substrate for learning induced structural plasticity relevant to addiction. Published by Elsevier B.V.

Sodium-potassium-activated adenosine triphosphatase of electrophorus electric organ. X. Immunochemical properties of the Lubrol-solubilized enzume and its constituent polypeptides.

PubMed

Jean, D H; Albers, R W; Koval, G J

1975-02-10

Detergent (Lubrol WX)-solubilized sodium-potassium-activated adenosine triphosphatase ((Na+ + K+)-ATPase) of electrophorus electric organ contains two major constituent polypeptides with molecular weights of 96,000 and 58,000 which can be readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These two polypeptides can be clearly separated and can be obtained in milligram quantities by preparative sodium dodecyl sulfate gel electrophoresis. The separated polypeptides, after removal of sodium dodecyl sulfate, and Lubrol-solubilized (Na+ + K+)-ATPase activity to some degree. Moreover, the degree of inhibition is directly proportional to the increasing amounts of antisera. The inhibition is maximal 4 weeks after the first injection. Immunodiffusion in 1% agar gel indicated that only Lubrol-solubilized enzyme antiserum, but not 58,000-dalton or 96,00-dalton polypeptide antiserum, gives one major precipitin band. However, specific complex formation between each polypeptide antiserum and Lubrol-solubilized enzyme occurs. This was demonstrated indirectly. After incubating Lubrol-solubilized enzyme with increasing amounts of polypeptide antisera at 37 degrees for 15 min, they were placed in the side wells of an immunodiffusion plate with antiserum against Lubrol-solubilized enzyme in the central well. The intensity of the precipitin band decreased with increasing amounts of polypeptide antisera. Thus, the results indicate that both 96,000-dalton and 58,000-dalton polypeptides are integral subunits of (Na+ + K+)-ATPase.

Molecular structure of the pyruvate dehydrogenase complex from Escherichia coli K-12.

PubMed

Vogel, O; Hoehn, B; Henning, U

1972-06-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 x 10(6). All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This "excess" component is bound differently than are the eight dimers in the core complex.

Overexpression of a Novel Apple NAC Transcription Factor Gene, MdNAC1, Confers the Dwarf Phenotype in Transgenic Apple (Malus domestica)

PubMed Central

Jia, Dongfeng; Gong, Xiaoqing; Li, Mingjun; Li, Chao; Sun, Tingting

2018-01-01

Plant height is an important trait for fruit trees. The dwarf characteristic is commonly associated with highly efficient fruit production, a major objective when breeding for apple (Malus domestica). We studied the function of MdNAC1, a novel NAC transcription factor (TF) gene in apple related to plant dwarfing. Localized primarily to the nucleus, MdNAC1 has transcriptional activity in yeast cells. Overexpression of the gene results in a dwarf phenotype in transgenic apple plants. Their reduction in size is manifested by shorter, thinner stems and roots, and a smaller leaf area. The transgenics also have shorter internodes and fewer cells in the stems. Levels of endogenous abscisic acid (ABA) and brassinosteroid (BR) are lower in the transgenic plants, and expression is decreased for genes involved in the biosynthesis of those phytohormones. All of these findings demonstrate that MdNAC1 has a role in plants dwarfism, probably by regulating ABA and BR production. PMID:29702625

Pre-clinical evaluation of N-acetylcysteine reveals side effects in the mdx mouse model of Duchenne muscular dystrophy.

PubMed

Pinniger, Gavin J; Terrill, Jessica R; Assan, Evanna B; Grounds, Miranda D; Arthur, Peter G

2017-12-01

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a therapeutic intervention for DMD boys, but potential adverse effects of NAC have not been widely investigated. We used young (6 weeks old) growing mdx mice to investigate the capacity of NAC supplementation (2% in drinking water for 6 weeks) to improve dystrophic muscle function and to explore broader systemic effects of NAC treatment. NAC treatment improved normalised measures of muscle function, and decreased inflammation and oxidative stress, but significantly reduced body weight gain, muscle weight and liver weight. Unexpected significant adverse effects of NAC on body and muscle weights indicate that interpretation of muscle function based on normalised force measures should be made with caution and careful consideration is needed when proposing the use of NAC as a therapeutic treatment for young DMD boys. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disease characterised by severe muscle weakness, necrosis, inflammation and oxidative stress. The antioxidant N-acetylcysteine (NAC) has been proposed as a potential therapeutic intervention for DMD boys. We investigated the capacity of NAC to improve dystrophic muscle function in the mdx mouse model of DMD. Young (6 weeks old) mdx and non-dystrophic C57 mice receiving 2% NAC in drinking water for 6 weeks were compared with untreated mice. Grip strength and body weight were measured weekly, before the 12 week old mice were anaesthetised and extensor digitorum longus (EDL) muscles were excised for functional analysis and tissues were sampled for biochemical analyses. Compared to untreated mice, the mean (SD) normalised grip strength was significantly greater in NAC-treated mdx [3.13 (0.58) vs 4.87 (0.78) g body weight (bw) -1 ; P < 0.001] and C57 mice [3.90 (0.32) vs 5.32 (0.60) g bw -1 ; P < 0.001]. Maximum specific force was significantly greater in NAC-treated mdx muscles [9.80 (2.27) vs 13.07 (3.37) N cm -2 ; P = 0.038]. Increased force in mdx mice was associated with reduced thiol oxidation and inflammation in fast muscles, and increased citrate synthase activity in slow muscle. Importantly, NAC significantly impaired body weight gain in both strains of young growing mice, and reduced liver weight in C57 mice and muscle weight in mdx mice. These potentially adverse effects of NAC emphasise the need for caution when interpreting improvements in muscle function based on normalised force measures, and that careful consideration be given to these effects when proposing NAC as a potential treatment for young DMD boys. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Intravenous Administration of Stable-Labeled N-Acetylcysteine Demonstrates an Indirect Mechanism for Boosting Glutathione and Improving Redox Status.

PubMed

Zhou, Jie; Coles, Lisa D; Kartha, Reena V; Nash, Nardina; Mishra, Usha; Lund, Troy C; Cloyd, James C

2015-08-01

There is an increasing interest in using N-acetylcysteine (NAC) as a treatment for neurodegenerative disorders to increase glutathione (GSH) levels and its redox status. The purpose of this study was to characterize the biosynthesis of NAC to GSH using a novel stable isotope-labeled technique, and investigate the pharmacodynamics of NAC in vivo. Female wild-type mice were given a single intravenous bolus dose of 150 mg kg(-1) stable-labeled NAC. Plasma, red blood cells (RBC), and brain tissues were collected at predesignated time points. Stable-labeled NAC and its metabolite GSH (both labeled and unlabeled forms) were quantified in blood and brain samples. Molar ratios of the reduced and oxidized forms of GSH (GSH divided by glutathione disulfide, redox ratio) were also determined. The elimination phase half-life of NAC was approximately 34 min. Both labeled and unlabeled GSH in RBC were found to increase; however, the area under the curve above baseline (AUCb0-280 ) of labeled GSH was only 1% of the unlabeled form. These data indicate that NAC is not a direct precursor of GSH. In addition, NAC has prolonged effects in brain even when the drug has been eliminated from systemic circulation. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Prognostic significance of the prognostic nutritional index in esophageal cancer patients undergoing neoadjuvant chemotherapy.

PubMed

Nakatani, M; Migita, K; Matsumoto, S; Wakatsuki, K; Ito, M; Nakade, H; Kunishige, T; Kitano, M; Kanehiro, H

2017-08-01

Nutritional status is one of the most important issues faced by cancer patients. Several studies have shown that a low preoperative nutritional status is associated with a worse prognosis in patients with various types of cancer, including esophageal cancer (EC). Recently, neoadjuvant chemotherapy (NAC) and/or radiotherapy have been accepted as the standard treatment for resectable advanced EC. However, NAC has the potential to deteriorate the nutritional status of a patient. This study aimed to evaluate the prognostic significance of the nutritional status for EC patients who underwent NAC. We retrospectively reviewed 66 squamous cell EC patients who underwent NAC consisting of docetaxel, cisplatin, and 5-fluorouracil followed by subtotal esophagectomy at Nara Medical University Hospital between January 2009 and August 2015. To assess the patients' nutritional status, the prognostic nutritional index (PNI) before commencing NAC and prior to the operation was calculated as 10 × serum albumin (g/dl) + 0.005 × total lymphocyte count in the peripheral blood (per mm3). The cutoff value of the PNI was set at 45. A multivariable analysis was performed to identify prognostic factors for overall survival (OS) and relapse-free survival (RFS). The mean pre-NAC and preoperative PNI were 50.2 ± 5.7 and 48.1 ± 4.7, respectively (P = 0.005). The PNI decreased following NAC in 44 (66.7%) patients. Before initiating NAC, 9 (13.6%) patients had a low PNI, and 12 (18.2%) patients had a low PNI prior to the operation. The pre-NAC PNI and preoperative PNI were significantly associated with the OS (P = 0.013 and P = 0.004, respectively) and RFS (P = 0.036 and P = 0.005, respectively) rates. The multivariable analysis identified the preoperative PNI as an independent prognostic factor for poor OS and RFS, although the pre-NAC PNI was not an independent predictor. Our results suggest that the preoperative PNI is a useful marker for predicting the long-term outcomes of EC patients undergoing NAC and subsequent subtotal esophagectomy. Therefore, patients with a low preoperative nutritional status may be at a higher risk of EC recurrence. © The Authors 2017. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

N-Acetyl-Cysteine Alleviates Gut Dysbiosis and Glucose Metabolic Disorder in High-Fat Diet-Induced Mice.

PubMed

Zheng, Junping; Yuan, Xubing; Zhang, Chen; Jia, Peiyuan; Jiao, Siming; Zhao, Xiaoming; Yin, Heng; Du, Yuguang; Liu, Hongtao

2018-05-30

N-acetyl cysteine (NAC), an anti-oxidative reagent for clinical diseases, shows potential application to diabetes and other metabolic diseases. However, it is unknown how NAC modulates the gut microbiota of mice with metabolic syndrome. In present study, we aim to demonstrate the preventive effect of NAC on intestinal dysbiosis and glucose metabolic disorder. C57BL/6J mice were fed with normal chow diet (NCD), NCD plus NAC, high-fat diet (HFD) or HFD plus NAC for five months. After the treatment, the glucose level, circulating endotoxin and metabolism-related key proteins were determined. The fecal samples were analyzed by 16S rRNA sequencing. A novel analysis was carried out to predict the functional changes of gut microbiota. In addition, Spearman's correlation between metabolic biomarkers and bacterial abundance was also assayed. The results show that NAC treatment significantly reversed the glucose intolerance, fasting glucose level, body weight and plasma endotoxin in HFD-fed mice. Further, NAC upregulated the levels of Occludin protein and mucin glycoproteins in proximal colons of HFD-treated mice. Noticeably, NAC promoted the growth of beneficial bacteria such as Akkermansia, Bifidobacterium, Lactobacillus and Allobaculum, and hampered the population of diabetes-related genera including Desulfovibrio and Blautia. Also, NAC may influence the metabolic pathways of intestinal bacteria including lipopolysaccharide biosynthesis, oxidative stress and bacterial motility. Finally, the modified gut microbiota showed close association with the metabolic changes of the NAC treated HFD-fed mice. In summary, NAC may be a potential drug to prevent glucose metabolic disturbance by reshaping the structure of gut microbiota. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

PubMed

Dhandhukia, Jugal P; Brill, Dab A; Kouhi, Aida; Pastuszka, Martha K; MacKay, J Andrew

2017-09-01

Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent K d of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates. © 2017 The Protein Society.

Molecular mechanism and structure of Trigger Factor bound to the translating ribosome

PubMed Central

Merz, Frieder; Boehringer, Daniel; Schaffitzel, Christiane; Preissler, Steffen; Hoffmann, Anja; Maier, Timm; Rutkowska, Anna; Lozza, Jasmin; Ban, Nenad; Bukau, Bernd; Deuerling, Elke

2008-01-01

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome–nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors. PMID:18497744

Identification of O-linked β-d-N-acetylglucosamine-Modified Proteins from Arabidopsis

PubMed Central

Xu, Shou-Ling; Chalkley, Robert J.; Wang, Zhi-Yong; Burlingame, Alma L.

2013-01-01

The posttranslational modification of proteins with O-linked β-d-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues occurs in all animals and plants. This modification is dynamic and ubiquitous, and regulates many cellular processes, including transcription, signaling and cytokinesis and is associated with several diseases. Cycling of O-GlcNAc is tightly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Plants have two OGTs, SPINDLY (SPY) and SECRET AGENT (SEC); disruption of both causes embryo lethality. Despite O-GlcNAc modification of proteins being discovered more than 20-years ago, identification and mapping of protein GlcNAcylation is still a challenging task. Here we describe the use of lectin affinity chromatography combined with electron transfer dissociation mass spectrometry to enrich and to detect O-GlcNAc modified peptides from Arabidopsis. PMID:22576084

Nonlinear association criterion, nonlinear Granger causality and related issues with applications to neuroimage studies.

PubMed

Tao, Chenyang; Feng, Jianfeng

2016-03-15

Quantifying associations in neuroscience (and many other scientific disciplines) is often challenged by high-dimensionality, nonlinearity and noisy observations. Many classic methods have either poor power or poor scalability on data sets of the same or different scales such as genetical, physiological and image data. Based on the framework of reproducing kernel Hilbert spaces we proposed a new nonlinear association criteria (NAC) with an efficient numerical algorithm and p-value approximation scheme. We also presented mathematical justification that links the proposed method to related methods such as kernel generalized variance, kernel canonical correlation analysis and Hilbert-Schmidt independence criteria. NAC allows the detection of association between arbitrary input domain as long as a characteristic kernel is defined. A MATLAB package was provided to facilitate applications. Extensive simulation examples and four real world neuroscience examples including functional MRI causality, Calcium imaging and imaging genetic studies on autism [Brain, 138(5):13821393 (2015)] and alcohol addiction [PNAS, 112(30):E4085-E4093 (2015)] are used to benchmark NAC. It demonstrates the superior performance over the existing procedures we tested and also yields biologically significant results for the real world examples. NAC beats its linear counterparts when nonlinearity is presented in the data. It also shows more robustness against different experimental setups compared with its nonlinear counterparts. In this work we presented a new and robust statistical approach NAC for measuring associations. It could serve as an interesting alternative to the existing methods for datasets where nonlinearity and other confounding factors are present. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcriptional control in Alicyclobacillus acidocaldarius and associated genes, proteins, and methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Brady D.; Thompson, David N.; Apel, William A.

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of modulating transcription or transcription or transcriptional control using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Transcriptional control in Alicyclobacillus acidocaldarius and associated genes, proteins, and methods

DOEpatents

Lee, Brady Deneys; Thompson, David N; Apel, William A.; Thompson, Vicki Slavchev; Reed, David W; Lacey, Jeffrey A

2014-05-06

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of modulating transcription or transcription or transcriptional control using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Transcriptional control in Alicyclobacillus acidocaldarius and associated genes, proteins, and methods

DOEpatents

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

2015-11-17

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of modulating transcription or transcription or transcriptional control using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Transcriptional control in alicyclobacillus acidocaldarius and associated genes, proteins, and methods

DOEpatents

Lee, Brady D; Thompson, David N; Apel, William A; Thompson, Vicki S; Reed, David W; Lacey, Jeffrey A

2016-11-22

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods of modulating transcription or transcription or transcriptional control using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

New Small Polypeptides Associated with DNA-Dependent RNA Polymerase of Escherichia coli after Infection with Bacteriophage T4

PubMed Central

Stevens, Audrey

1972-01-01

Four new small polypeptides are associated with DNA-dependent RNA polymerase from E. coli after infection with T4 phage. The new polypeptides are easily detected in RNA polymerase from E. coli cells labeled with amino acids after phage infection. Their molecular weights range from 10,000 to 22,000, as detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All four polypeptides are found after infection with either wild-type T4 phage or T4 early amber mutants in genes 44, 42, 47, and 46. None of the polypeptides is labeled significantly before 5 min after infection at 30°. When two maturation-defective amber mutants in gene 55 of T4 phage are used for infection, a polypeptide with a molecular weight of 22,000 is absent. When a maturation-defective amber mutant in gene 33 of T4 phage is used, another small protein is absent. PMID:4551978

Protecting the proteome: Eukaryotic cotranslational quality control pathways

PubMed Central

2014-01-01

The correct decoding of messenger RNAs (mRNAs) into proteins is an essential cellular task. The translational process is monitored by several quality control (QC) mechanisms that recognize defective translation complexes in which ribosomes are stalled on substrate mRNAs. Stalled translation complexes occur when defects in the mRNA template, the translation machinery, or the nascent polypeptide arrest the ribosome during translation elongation or termination. These QC events promote the disassembly of the stalled translation complex and the recycling and/or degradation of the individual mRNA, ribosomal, and/or nascent polypeptide components, thereby clearing the cell of improper translation products and defective components of the translation machinery. PMID:24535822

[Anatomical study of men's nipple areola complex].

PubMed

Vaucher, R; Dast, S; Assaf, N; Sinna, R

2016-06-01

The surgical approach of gynecomastia, sexual reassignment surgery in female-to-male transsexuals and the increase of number of obese wishing to turn to plastic surgery led us to deepen the anatomical knowledge of the nipple areola complex (NAC) in men, poorly retailed in the literature. By inspiring us of the methodology of a Japanese study, we studied 50 healthy volunteers male, from 18 to 55 years old, from July till August 2015. We measured various distances relative to the NAC to define its vertical and horizontal position, as well as the internipple distance according to the size, to the weight and to the body mass index (BMI). At the end of the analysis, we were able to underline a lower vertical thoracic position of the NAC in the tall category of person, a more side horizontal position to the subject presenting a high BMI and a linear relation between the BMI and the internipple (Em) defined by (Em)=8.96×BMI. The surgeon's judgment and the desires of the patient are essentials basis of therapeutics decisions that could be lean on this anatomical study, which allowed to establish an idea of the cartography of the NAC in man. It will be interesting and necessary to confront it with other studies with larger scale. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Structural Basis of Specific Recognition of Non-Reducing Terminal N-Acetylglucosamine by an Agrocybe aegerita Lectin

PubMed Central

Ren, Xiao-Ming; Li, De-Feng; Jiang, Shuai; Lan, Xian-Qing; Hu, Yonglin; Sun, Hui; Wang, Da-Cheng

2015-01-01

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently. PMID:26114302

The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs.

PubMed

O'Leary, Brendan; Park, Joonho; Plaxton, William C

2011-05-15

PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled enzyme located at the core of plant C-metabolism that catalyses the irreversible β-carboxylation of PEP to form oxaloacetate and Pi. The critical role of PEPC in assimilating atmospheric CO(2) during C(4) and Crassulacean acid metabolism photosynthesis has been studied extensively. PEPC also fulfils a broad spectrum of non-photosynthetic functions, particularly the anaplerotic replenishment of tricarboxylic acid cycle intermediates consumed during biosynthesis and nitrogen assimilation. An impressive array of strategies has evolved to co-ordinate in vivo PEPC activity with cellular demands for C(4)-C(6) carboxylic acids. To achieve its diverse roles and complex regulation, PEPC belongs to a small multigene family encoding several closely related PTPCs (plant-type PEPCs), along with a distantly related BTPC (bacterial-type PEPC). PTPC genes encode ~110-kDa polypeptides containing conserved serine-phosphorylation and lysine-mono-ubiquitination sites, and typically exist as homotetrameric Class-1 PEPCs. In contrast, BTPC genes encode larger ~117-kDa polypeptides owing to a unique intrinsically disordered domain that mediates BTPC's tight interaction with co-expressed PTPC subunits. This association results in the formation of unusual ~900-kDa Class-2 PEPC hetero-octameric complexes that are desensitized to allosteric effectors. BTPC is a catalytic and regulatory subunit of Class-2 PEPC that is subject to multi-site regulatory phosphorylation in vivo. The interaction between divergent PEPC polypeptides within Class-2 PEPCs adds another layer of complexity to the evolution, physiological functions and metabolic control of this essential CO(2)-fixing plant enzyme. The present review summarizes exciting developments concerning the functions, post-translational controls and subcellular location of plant PTPC and BTPC isoenzymes.

O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease

NASA Astrophysics Data System (ADS)

Marotta, Nicholas P.; Lin, Yu Hsuan; Lewis, Yuka E.; Ambroso, Mark R.; Zaro, Balyn W.; Roth, Maxwell T.; Arnold, Don B.; Langen, Ralf; Pratt, Matthew R.

2015-11-01

Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

Antenna complexes protect Photosystem I from Photoinhibition

PubMed Central

Alboresi, Alessandro; Ballottari, Matteo; Hienerwadel, Rainer; Giacometti, Giorgio M; Morosinotto, Tomas

2009-01-01

Background Photosystems are composed of two moieties, a reaction center and a peripheral antenna system. In photosynthetic eukaryotes the latter system is composed of proteins belonging to Lhc family. An increasing set of evidences demonstrated how these polypeptides play a relevant physiological function in both light harvesting and photoprotection. Despite the sequence similarity between antenna proteins associated with the two Photosystems, present knowledge on their physiological role is mostly limited to complexes associated to Photosystem II. Results In this work we analyzed the physiological role of Photosystem I antenna system in Arabidopsis thaliana both in vivo and in vitro. Plants depleted in individual antenna polypeptides showed a reduced capacity for photoprotection and an increased production of reactive oxygen species upon high light exposure. In vitro experiments on isolated complexes confirmed that depletion of antenna proteins reduced the resistance of isolated Photosystem I particles to high light and that the antenna is effective in photoprotection only upon the interaction with the core complex. Conclusion We show that antenna proteins play a dual role in Arabidopsis thaliana Photosystem I photoprotection: first, a Photosystem I with an intact antenna system is more resistant to high light because of a reduced production of reactive oxygen species and, second, antenna chlorophyll-proteins are the first target of high light damages. When photoprotection mechanisms become insufficient, the antenna chlorophyll proteins act as fuses: LHCI chlorophylls are degraded while the reaction center photochemical activity is maintained. Differences with respect to photoprotection strategy in Photosystem II, where the reaction center is the first target of photoinhibition, are discussed. PMID:19508723

Preventive Strategies for Contrast-Induced Acute Kidney Injury in Patients Undergoing Percutaneous Coronary Procedures: Evidence From a Hierarchical Bayesian Network Meta-Analysis of 124 Trials and 28 240 Patients.

PubMed

Giacoppo, Daniele; Gargiulo, Giuseppe; Buccheri, Sergio; Aruta, Patrizia; Byrne, Robert A; Cassese, Salvatore; Dangas, George; Kastrati, Adnan; Mehran, Roxana; Tamburino, Corrado; Capodanno, Davide

2017-05-01

The effectiveness of currently available effective preventive strategies for contrast-induced acute kidney injury (CIAKI) is a matter of debate. We performed a Bayesian random-effects network meta-analysis of 124 trials (28 240 patients) comparing a total of 10 strategies: saline, statin, N-acetylcysteine (NAC), sodium bicarbonate (NaHCO 3 ), NAC+NaHCO 3 , ascorbic acid, xanthine, dopaminergic agent, peripheral ischemic preconditioning, and natriuretic peptide. Compared with saline, the risk of CIAKI was reduced by using statin (odds ratio [OR], 0.42; 95% credible interval [CrI], 0.26-0.67), xanthine (OR, 0.32; 95% CrI, 0.17-0.57), ischemic preconditioning (OR, 0.48; 95% CrI, 0.26-0.87), NAC+NaHCO 3 (OR, 0.50; 95% CrI, 0.33-0.76), NAC (OR, 0.68; 95% CrI, 0.55-0.84), and NaHCO 3 (OR, 0.66; 95% CrI, 0.47-0.90). The benefit of statin therapy was consistent across multiple sensitivity analyses, whereas the efficacy of all the other strategies was questioned by restricting the analysis to high-quality trials. Overall, high heterogeneity was observed for comparisons involving xanthine and ischemic preconditioning, although the impact of NAC and xanthine was probably influenced by publication bias/small-study effect. Hydration alone was the least effective preventive strategy for CIAKI. Meta-regressions did not reveal significant associations with baseline creatinine and contrast volume. In patients with diabetes mellitus, no strategy was found to reduce the incidence of CIAKI. In patients undergoing percutaneous coronary procedures, statin administration is associated with a marked and consistent reduction in the risk of CIAKI compared with saline. Although xanthine, NAC, NaHCO 3 , NAC+NaHCO 3 , ischemic preconditioning, and natriuretic peptide may have nephroprotective effects, these results were not consistent across multiple sensitivity analyses. © 2017 American Heart Association, Inc.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

PubMed Central

Munkley, Jennifer; Oltean, Sebastian; Vodák, Daniel; Wilson, Brian T.; Livermore, Karen E.; Zhou, Yan; Star, Eleanor; Floros, Vasileios I.; Johannessen, Bjarne; Knight, Bridget; McCullagh, Paul; McGrath, John; Crundwell, Malcolm; Skotheim, Rolf I.; Robson, Craig N.; Leung, Hing Y.; Harries, Lorna W.; Rajan, Prabhakar; Mills, Ian G.; Elliott, David J.

2015-01-01

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, being significantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression. PMID:26452038

Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRζ fragments

PubMed Central

Kim, Walter M.; Sigalov, Alexander B.; Stern, Lawrence J.

2010-01-01

HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-­cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (R work = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (R work = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning. PMID:20124696

Expression of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) in the nucleus accumbens is critical for the acquisition, expression and reinstatement of morphine-induced conditioned place preference.

PubMed

Lv, Xiu-Fang; Xu, Ya; Han, Ji-Sheng; Cui, Cai-Lian

2011-09-30

Activity-regulated cytoskeleton-associated protein (Arc), also known as activity-regulated gene 3.1 (Arg3.1), is an immediate early gene whose mRNA is selectively targeted to recently activated synaptic sites, where it is translated and enriched. This unique feature suggests a role for Arc/Arg3.1 in coupling synaptic activity to protein synthesis, leading to synaptic plasticity. Although the Arc/Arg3.1 gene has been shown to be induced by a variety of abused drugs and its protein has been implicated in diverse forms of long-term memory, relatively little is known about its role in drug-induced reward memory. In this study, we investigated the potential role of Arc/Arg3.1 protein expression in reward-related associative learning and memory using morphine-induced conditioned place preference (CPP) in rats. We found that (1) intraperitoneal (i.p.) injection of morphine (10mg/kg) increased Arc/Arg3.1 protein levels after 2h in the NAc core but not in the NAc shell. (2) In CPP experiments, Arc/Arg3.1 protein was increased in the NAc shell of rats following both morphine conditioning and the CPP expression test compared to rats that received the conditioning without the test or those that did not receive morphine conditioning. (3) Microinjection of Arc/Arg3.1 antisense oligodeoxynucleotide (AS) into the NAc core inhibited the acquisition, expression and reinstatement of morphine CPP; however, intra-NAc shell infusions of the AS only blocked the expression of CPP. These findings suggest that expression of the Arc/Arg3.1 protein in the NAc core is required for the acquisition, context-induced retrieval and reinstatement of morphine-associated reward memory, whereas Arc/Arg3.1 protein expression in the NAc shell is only critical for the context-induced retrieval of memory. As a result, Arc/Arg3.1 may be a potential therapeutic target for the prevention of drug abuse or the relapse of drug use. Copyright © 2011 Elsevier B.V. All rights reserved.

N-acetylcysteine inhibits endothelial cell invasion and angiogenesis.

PubMed

Cai, T; Fassina, G; Morini, M; Aluigi, M G; Masiello, L; Fontanini, G; D'Agostini, F; De Flora, S; Noonan, D M; Albini, A

1999-09-01

The thiol N-acetylcysteine (NAC) is a chemopreventive agent that acts through a variety of mechanisms and can prevent in vivo carcinogenesis. We have previously shown that NAC inhibits invasion and metastasis of malignant cells as well as tumor take. Neovascularization is critical for tumor mass expansion and metastasis formation. We investigated whether a target of the anti-cancer activity of NAC could be the inhibition of the tumor angiogenesis-associated phenotype in vitro and in vivo using the potent angiogenic mixture of Kaposi's sarcoma cell products as a stimulus. Two endothelial (EAhy926 and human umbilical vein endothelial [HUVE]) cell lines were utilized in a panel of assays to test NAC ability in inhibiting chemotaxis, invasion, and gelatinolytic activity in vitro. NAC treatment of EAhy926 and HUVE cells in vitro dose-dependently reduced their ability to invade a reconstituted basement membrane, an indicator of endothelial cell activation. Invasion of HUVE cells was inhibited with an ID50 of 0.24 mM NAC, whereas inhibition of chemotaxis required a 10 fold higher doses, indicating that invasion is a preferential target. NAC inhibited the enzymatic activity and conversion to active forms of the gelatinase produced by endothelial cells. The matrigel in vivo assay was used for the evaluation of angiogenesis; NAC strongly inhibited neovascularization of the matrigel sponges in response to Kaposi's sarcoma cell products. NAC prevented angiogenesis while preserving endothelial cells, implying that it could be safely used as an anti-angiogenic treatment.

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

PubMed

Winton, Alexander J; Baptiste, Janae L; Allen, Mark A

2018-09-01

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. Copyright © 2018. Published by Elsevier Inc.

Mechanisms and Kinetics of Alkaline Hydrolysis of the Energetic Nitroaromatic Compounds 2,4,6-Trinitrotoluene (TNT) and 2,4-Dinitroanisole (DNAN)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salter-Blanc, Alexandra J.; Bylaska, Eric J.; Ritchie, Julia J.

2013-07-02

The environmental impacts of energetic compounds can be minimized through the design and selection of new energetic materials with favorable fate properties. Building predictive models to inform this process, however, is difficult because of uncertainties and complexities in some major fate-determining transformation reactions such as the alkaline hydrolysis of energetic nitroaromatic compounds (NACs). Prior work on the mechanisms of the reaction between NACs and OH– has yielded inconsistent results. In this study, the alkaline hydrolysis of 2,4,6-trinitrotoluene (TNT) and 2,4-dinitroanisole (DNAN) was investigated with coordinated experimental kinetic measurements and molecular modeling calculations. For TNT, the results suggest reversible formation ofmore » an initial product, which is likely either a Meisenheimer complex or a TNT anion formed by abstraction of a methyl proton by OH–. For DNAN, the results suggest that a Meisenheimer complex is an intermediate in the formation of 2,4-dinitrophenolate. Despite these advances, the remaining uncertainties in the mechanisms of these reactions—and potential variability between the hydrolysis mechanisms for different NACs—mean that it is not yet possible to generalize the results into predictive models (e.g., quantitative structure–activity relationships, QSARs) for hydrolysis of other NACs.« less

Structure of human O-GlcNAc transferase and its complex with a peptide substrate

PubMed Central

Lazarus, Michael B.; Nam, Yunsun; Jiang, Jiaoyang; Sliz, Piotr; Walker, Suzanne

2010-01-01

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that couples metabolic status to the regulation of a wide variety of cellular signaling pathways by acting as a nutrient sensor1. OGT catalyzes the transfer of N-acetyl-glucosamine from UDP-GlcNAc to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins2,3, including numerous transcription factors4, tumor suppressors, kinases5, phosphatases1, and histone-modifying proteins6. Aberrant O-GlcNAcylation by OGT has been linked to insulin resistance7, diabetic complications8, cancer9 and neurodegenerative diseases including Alzheimer’s10. Despite the importance of OGT, the details of how it recognizes and glycosylates its protein substrates are largely unknown. We report here two crystal structures of human OGT, as a binary complex with UDP (2.8 A) and a ternary complex with UDP and a peptide substrate (1.95 A). The structures provide clues to the enzyme mechanism, show how OGT recognizes target peptide sequences, and reveal the fold of the unique domain between the two halves of the catalytic region. This information will accelerate the rational design of biological experiments to investigate OGT’s functions and the design of inhibitors for use as cellular probes and to assess its potential as a therapeutic target. PMID:21240259

Molecular Structure of the Pyruvate Dehydrogenase Complex from Escherichia coli K-12

PubMed Central

Vogel, Otto; Hoehn, Barbara; Henning, Ulf

1972-01-01

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex that can be obtained with a unique polypeptide chain composition, has a molecular weight of 3.75 × 106. All results obtained agree with the following numerology. The core complex consists of 48 polypeptide chains. There are 16 chains (molecular weight = 100,000) of the pyruvate dehydrogenase component, 16 chains (molecular weight = 80,000) of the dihydrolipoamide dehydrogenase component, and 16 chains (molecular weight = 56,000) of the dihydrolipoamide dehydrogenase component. Usually, but not always, pyruvate dehydrogenase complex is produced in vivo containing at least 2-3 mol more of dimers of the pyruvate dehydrogenase component than the stoichiometric ratio with respect to the core complex. This “excess” component is bound differently than are the eight dimers in the core complex. Images PMID:4556465

O-GlcNAcylation: A New Cancer Hallmark?

PubMed

Fardini, Yann; Dehennaut, Vanessa; Lefebvre, Tony; Issad, Tarik

2013-01-01

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression.

O-GlcNAcylation: A New Cancer Hallmark?

PubMed Central

Fardini, Yann; Dehennaut, Vanessa; Lefebvre, Tony; Issad, Tarik

2013-01-01

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression. PMID:23964270

Effects of hypo-O-GlcNAcylation on Drosophila development.

PubMed

Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

2018-05-11

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc ( O -GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila , null mutants of the Polycomb gene O -GlcNAc transferase ( OGT ; also known as super sex combs ( sxc )) display homeotic phenotypes. To dissect the requirement for O -GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxc K872M , was recessive lethal, whereas a second mutant, the hypomorphic sxc H537A , was homozygous viable. We observed that reduced total protein O -GlcNAcylation in the sxc H537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc H537A and a null allele of Drosophila host cell factor ( dHcf ), encoding an extensively O -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxc H537A flies lacking a copy of skuld ( skd ), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxc H537A enabled us to identify pleiotropic effects of globally reduced protein O -GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development. © 2018 Mariappa et al.

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein-protein interactions.

PubMed

Lambert, Matthias; Richard, Elodie; Duban-Deweer, Sophie; Krzewinski, Frederic; Deracinois, Barbara; Dupont, Erwan; Bastide, Bruno; Cieniewski-Bernard, Caroline

2016-09-01

The sarcomere structure of skeletal muscle is determined through multiple protein-protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization. C2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level. Our data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin. For the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure. In the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation. Copyright © 2016 Elsevier B.V. All rights reserved.

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

PubMed

Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

2017-07-05

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Role of projections from ventral subiculum to nucleus accumbens shell in context-induced reinstatement of heroin seeking in rats.

PubMed

Bossert, Jennifer M; Adhikary, Sweta; St Laurent, Robyn; Marchant, Nathan J; Wang, Hui-Ling; Morales, Marisela; Shaham, Yavin

2016-05-01

In humans, exposure to contexts previously associated with heroin use can provoke relapse. In rats, exposure to heroin-paired contexts after extinction of drug-reinforced responding in different contexts reinstates heroin seeking. We previously demonstrated that the projections from ventral medial prefrontal cortex (vmPFC) to nucleus accumbens (NAc) shell play a role in this reinstatement. The ventral subiculum (vSub) sends glutamate projections to NAc shell and vmPFC. Here, we determined whether these projections contribute to context-induced reinstatement. We trained rats to self-administer heroin (0.05-0.1 mg/kg/infusion) for 3 h per day for 12 days; drug infusions were paired with a discrete tone-light cue. Lever pressing in the presence of the discrete cue was subsequently extinguished in a different context. We then tested the rats for reinstatement in the heroin- and extinction-associated contexts under extinction conditions. We combined Fos with the retrograde tracer Fluoro-Gold (FG) to determine projection-specific activation during the context-induced reinstatement tests. We also used anatomical disconnection procedures to determine whether the vSub → NAc shell and vSub → vmPFC projections are functionally involved in this reinstatement. Exposure to the heroin but not the extinction context reinstated lever pressing. Context-induced reinstatement of heroin seeking was associated with increased Fos expression in vSub neurons, including those projecting to NAc shell and vmPFC. Anatomical disconnection of the vSub → NAc shell projection, but not the vSub → vmPFC projection, decreased this reinstatement. Our data indicate that the vSub → NAc shell glutamatergic projection, but not the vSub → vmPFC projection, contributes to context-induced reinstatement of heroin seeking.

C-Phycocyanin protects against acute tributyltin chloride neurotoxicity by modulating glial cell activity along with its anti-oxidant and anti-inflammatory property: A comparative efficacy evaluation with N-acetyl cysteine in adult rat brain.

PubMed

Mitra, Sumonto; Siddiqui, Waseem A; Khandelwal, Shashi

2015-08-05

Spirulina is a widely used health supplement and is a dietary source of C-Phycocyanin (CPC), a potent anti-oxidant. We have previously reported the neurotoxic potential of tributyltin chloride (TBTC), an environmental pollutant and potent biocide. In this study, we have evaluated the protective efficacy of CPC against TBTC induced neurotoxicity. To evaluate the extent of neuroprotection offered by CPC, its efficacy was compared with the degree of protection offered by N-acetylcysteine (NAC) (a well known neuroprotective drug, taken as a positive control). Male Wistar rats (28 day old) were administered with 20mg/kg TBTC (oral) and 50mg/kg CPC or 50mg/kg NAC (i.p.), alone or in combination, and various parameters were evaluated. These include blood-brain barrier (BBB) damage; redox parameters (ROS, GSH, redox pathway associated enzymes, oxidative stress markers); inflammatory, cellular, and stress markers; apoptotic proteins and in situ cell death assay (TUNEL). We observed increased CPC availability in cortical tissue following its administration. Although BBB associated proteins like claudin-5, p-glycoprotein and ZO-1 were restored, CPC/NAC failed to protect against TBTC induced overall BBB permeability (Evans blue extravasation). Both CPC and NAC remarkably reduced oxidative stress and inflammation. NAC effectively modulated redox pathway associated enzymes whereas CPC countered ROS levels efficiently. Interestingly, CPC and NAC were equivalently capable of reducing apoptotic markers, astroglial activation and cell death. This study illustrates the various pathways involved in CPC mediated neuroprotection against this environmental neurotoxicant and highlights its capability to modulate glial cell activity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Gene transcripts selectively down-regulated in the shell of the nucleus accumbens long after heroin self-administration are up-regulated in the core independent of response contingency.

PubMed

Jacobs, Edwin H; de Vries, Taco J; Smit, August B; Schoffelmeer, Anton N M

2004-01-01

Long-term drug-induced alterations in neurotransmission within the nucleus accumbens (NAc) shell and core may underlie relapse to drug-seeking behavior and drug-taking upon re-exposure to drugs and drug-associated stimuli (cues) during abstinence. Using an open screening strategy, we recently identified 25 gene transcripts, encoding for proteins involved in neuronal functioning and structure that are down-regulated in rat NAc shell after contingent (active), but not after non-contingent (passive), heroin administration. Studying the expression of the same transcripts in the NAc core by means of quantitative PCR, we now demonstrate that most of these transcripts are up-regulated in that NAc subregion long (3 weeks) after heroin self-administration in rats. A similar up-regulation in gene expression was also apparent in the NAc core of animals with a history of non-contingent heroin administration (yoked controls). These data indicate that heroin self-administration differentially regulates genes in the NAc core as compared with the shell. Moreover, whereas cognitive processes involved in active drug self-administration (e.g., instrumental learning) seems to direct gene expression in the NAc shell, neuroplasticity in the NAc core may be due to the pharmacological effects of heroin (including Pavlovian conditioning), as expressed in rats upon contingent as well as non-contingent administration of heroin.

Effects of oral N-acetylcysteine on fatigue, critical power, and W' in exercising humans.

PubMed

Corn, Sarah D; Barstow, Thomas J

2011-09-15

The accumulation of reactive oxygen species (ROS) is associated with muscular fatigue. The antioxidant N-acetylcysteine (NAC) can extend time to fatigue (TTF), but the effect appears to be exercise intensity dependent. The purpose of this study was to determine the effects of an acute oral dose of NAC on time to fatigue (TTF), critical power (CP), W' (curvature constant), V(O2) kinetics and muscle EMG during cycling exercise. Male (n=7) subjects performed four tests at power outputs corresponding to 80, 90, 100, and 110% of the peak power output achieved during the incremental test (Pmax) under NAC and placebo (PLA) conditions. TTF was increased only in the 80% Pmax trial (p=0.033). CP was higher with NAC (NAC: 232±28 W versus PLA: 226±31 W; p=0.032), but W' tended to decrease (NAC: 15.5±3.8 kJ versus W': 16.4±4.5 kJ; p=0.10). The change in W' was negatively related to the change in CP (r = -0.96). MdPF and RMS of EMG tended to change less with NAC. There were no significant differences in V(O2) kinetics. These results demonstrate that oral NAC was successful in extending time to fatigue at 80% Pmax but not at higher work rates. Copyright © 2011 Elsevier B.V. All rights reserved.

New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro.

PubMed

Qi, Jieqiong; Wang, Ruihong; Zeng, Yazhen; Yu, Wengong; Gu, Yuchao

2017-08-09

O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3',5,5'-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.

Mitochondrial Based Treatments that Prevent Post-Traumatic Osteoarthritis in a Translational Large Animal Intraarticular Fracture Survival Model

DTIC Science & Technology

2013-09-01

included an oxidant scavenger, (N- Acetylcysteine ), a drug that reduces mitochondrial superoxide production by blocking electron flow through complex I...selection of compounds included an oxidant scavenger, (N- Acetylcysteine ), a drug that reduces mitochondrial superoxide production by blocking...2mM N- acetylcysteine 5 3. 5mM NAC 5 4. 20mM NAC 5 5. 20µM Cytochalasin B 5 6. 10µM Nocodazole 4 7. 2.5mM Amobarbital 5 Table. Dose

Lutetium functionalities supported by a sterically encumbered β-diketiminate ligand

DOE PAGES

Beattie, Ross J.; Sutton, Andrew D.; Scott, Brian L.; ...

2018-01-06

The sterically encumbered NacNac ligand, [HC(MeCNAr) 2] – (Ar = 2,6- i-Pr 2C 6H 3), was investigated as a platform for supporting Lu-halide complexes, sought for their potential capability of being further converted into hydrocarbyl derivatives via metathetical chemistries with alkali metal alkyls. As a result, these substituted analogs were targeted as potentially viable candidates for alkane elimination chemistries, with an eye towards the formation of an isolable Lu-alkylidene fragment.

A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3)

PubMed Central

van Wijk, Xander M.; Lawrence, Roger; Thijssen, Victor L.; van den Broek, Sebastiaan A.; Troost, Ran; van Scherpenzeel, Monique; Naidu, Natasha; Oosterhof, Arie; Griffioen, Arjan W.; Lefeber, Dirk J.; van Delft, Floris L.; van Kuppevelt, Toin H.

2015-01-01

Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 μM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50–60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5′-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.—Van Wijk, X. M., Lawrence, R., Thijssen, V. L., van den Broek, S. A., Troost, R., van Scherpenzeel, M., Naidu, N., Oosterhof, A., Griffioen, A. W., Lefeber, D. J., van Delft, F. L., van Kuppevelt, T. H. A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3). PMID:25868729

N-Acetylcysteine improves mitochondrial function and ameliorates behavioral deficits in the R6/1 mouse model of Huntington's disease

PubMed Central

Wright, D J; Renoir, T; Smith, Z M; Frazier, A E; Francis, P S; Thorburn, D R; McGee, S L; Hannan, A J; Gray, L J

2015-01-01

Huntington's disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington's disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy. PMID:25562842

Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

PubMed

Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

2015-01-01

Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and revealed that the glycoform influenced ADCC activity.

The role of protein O-linked beta-N-acetylglucosamine in mediating cardiac stress responses.

PubMed

Chatham, John C; Marchase, Richard B

2010-02-01

The modification of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) has emerged as a highly dynamic post-translational modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of its adverse effects in mediating a range of chronic disease processes, including diabetes, cancer and neurodegenerative diseases. However, at the cellular level it has been shown that O-GlcNAc levels are increased in response to stress; augmentation of this response improved cell survival while attenuation decreased cell viability. Thus, it has become apparent that strategies that augment O-GlcNAc levels are pro-survival, whereas those that reduce O-GlcNAc levels decrease cell survival. There is a long history demonstrating the effectiveness of acute glucose-insulin-potassium (GIK) treatment and to a lesser extent glutamine in protecting against a range of stresses, including myocardial ischemia. A common feature of these approaches for metabolic cardioprotection is that they both have the potential to stimulate O-GlcNAc synthesis. Consequently, here we examine the links between metabolic cardioprotection with the ischemic cardioprotection associated with acute increases in O-GlcNAc levels. Some of the protective mechanisms associated with activation of O-GlcNAcylation appear to be transcriptionally mediated; however, there is also strong evidence to suggest that transcriptionally independent mechanisms also play a critical role. In this context we discuss the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis including the role of non-voltage gated, capacitative calcium entry as a potential mechanism contributing to this protection. Copyright 2009 Elsevier B.V. All rights reserved.

Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels.

PubMed

Liu, Jia; Marchase, Richard B; Chatham, John C

2007-01-01

It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identified. Glutamine:fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP), and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5 mM glucose, and global, no-flow ischemia was induced for 20 min followed by 60 min of reperfusion. Thirty-minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6+/-5.7% vs. 59.4+/-6.1%; p<0.05) and decreased cardiac troponin I release (25.4+/-3.0 vs. 4.7+/-1.9 ng/ml; p<0.05) during reperfusion. This protection was associated with a significant increase in the levels of protein O-GlcNAc and ATP. Pre-treatment with 80 muM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in protein O-GlcNAc. O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc, and inhibition of OGT with 5 mM alloxan also reversed the protection associated with glutamine. These data support the hypothesis that in the ex vivo perfused heart glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels.

Nucleus Accumbens Core and Shell Differentially Encode Reward-Associated Cues after Reinforcer Devaluation

PubMed Central

West, Elizabeth A.

2016-01-01

Nucleus accumbens (NAc) neurons encode features of stimulus learning and action selection associated with rewards. The NAc is necessary for using information about expected outcome values to guide behavior after reinforcer devaluation. Evidence suggests that core and shell subregions may play dissociable roles in guiding motivated behavior. Here, we recorded neural activity in the NAc core and shell during training and performance of a reinforcer devaluation task. Long–Evans male rats were trained that presses on a lever under an illuminated cue light delivered a flavored sucrose reward. On subsequent test days, each rat was given free access to one of two distinctly flavored foods to consume to satiation and were then immediately tested on the lever pressing task under extinction conditions. Rats decreased pressing on the test day when the reinforcer earned during training was the sated flavor (devalued) compared with the test day when the reinforcer was not the sated flavor (nondevalued), demonstrating evidence of outcome-selective devaluation. Cue-selective encoding during training by NAc core (but not shell) neurons reliably predicted subsequent behavioral performance; that is, the greater the percentage of neurons that responded to the cue, the better the rats suppressed responding after devaluation. In contrast, NAc shell (but not core) neurons significantly decreased cue-selective encoding in the devalued condition compared with the nondevalued condition. These data reveal that NAc core and shell neurons encode information differentially about outcome-specific cues after reinforcer devaluation that are related to behavioral performance and outcome value, respectively. SIGNIFICANCE STATEMENT Many neuropsychiatric disorders are marked by impairments in behavioral flexibility. Although the nucleus accumbens (NAc) is required for behavioral flexibility, it is not known how NAc neurons encode this information. Here, we recorded NAc neurons during a training session in which rats learned that a cue predicted a specific reward and during a test session when that reward value was changed. Although encoding in the core during training predicted the ability of rats to change behavior after the reward value was altered, the NAc shell encoded information about the change in reward value during the test session. These findings suggest differential roles of the core and shell in behavioral flexibility. PMID:26818502

Studies on a lipopolysaccharide-protein complex from Yersinia pseudotuberculosis. 1 isolation and characterization.

PubMed

Solov'eva, T F; Yermak, I M; Bondarenko, O D; Frolova, G M; Ovodov, Y S

1979-01-01

A comparative study of various procedures of a lipopolysaccharide-protein complex (LPPC) from Yersinia pseudotuberculosis was carried out. The materials obtained were fractionated by molecular-sieve chromatography on Sepharose 2B resulting in highly aggregated complexes with antigen activity. LPPC aggregates dissociated in the presence of sodium dodecylsulphate (SDS) and urea. The chemical composition and serologic properties of fractions obtained are under consideration. The protein component of the complex consists of two major polypeptides (molecular weights--45,000 and 20,000) and some minor ones. The LPS component appeared to give 2--3 narrow bands in gel under conditions of SDS-polyacrylamide gel electrophoresis. It is suggested that such fractionation is caused by LPS association-dissociation in the course of electrophoresis.

Regulation of Alcohol Extinction and Cue-Induced Reinstatement by Specific Projections among Medial Prefrontal Cortex, Nucleus Accumbens, and Basolateral Amygdala.

PubMed

Keistler, Colby R; Hammarlund, Emma; Barker, Jacqueline M; Bond, Colin W; DiLeone, Ralph J; Pittenger, Christopher; Taylor, Jane R

2017-04-26

The ability to inhibit drinking is a significant challenge for recovering alcoholics, especially in the presence of alcohol-associated cues. Previous studies have demonstrated that the regulation of cue-guided alcohol seeking is mediated by the basolateral amygdala (BLA), nucleus accumbens (NAc), and medial prefrontal cortex (mPFC). However, given the high interconnectivity between these structures, it is unclear how mPFC projections to each subcortical structure, as well as projections between BLA and NAc, mediate alcohol-seeking behaviors. Here, we evaluate how cortico-striatal, cortico-amygdalar, and amygdalo-striatal projections control extinction and relapse in a rat model of alcohol seeking. Specifically, we used a combinatorial viral technique to express diphtheria toxin receptors in specific neuron populations based on their projection targets. We then used this strategy to create directionally selective ablations of three distinct pathways after acquisition of ethanol self-administration but before extinction and reinstatement. We demonstrate that ablation of mPFC neurons projecting to NAc, but not BLA, blocks cue-induced reinstatement of alcohol seeking and neither pathway is necessary for extinction of responding. Further, we show that ablating BLA neurons that project to NAc disrupts extinction of alcohol approach behaviors and attenuates reinstatement. Together, these data provide evidence that the mPFC→NAc pathway is necessary for cue-induced reinstatement of alcohol seeking, expand our understanding of how the BLA→NAc pathway regulates alcohol behavior, and introduce a new methodology for the manipulation of target-specific neural projections. SIGNIFICANCE STATEMENT The vast majority of recovering alcoholics will relapse at least once and understanding how the brain regulates relapse will be key to developing more effective behavior and pharmacological therapies for alcoholism. Given the high interconnectivity of cortical, striatal, and limbic structures that regulate alcohol intake, it has been difficult to disentangle how separate projections between them may control different aspects of these complex behaviors. Here, we demonstrate a new approach for noninvasively ablating each of these pathways and testing their necessity for both extinction and relapse. We show that inputs to the nucleus accumbens from medial prefrontal cortex and amygdala regulate alcohol-seeking behaviors differentially, adding to our understanding of the neural control of alcoholism. Copyright © 2017 the authors 0270-6474/17/374462-10$15.00/0.

Regulation of Alcohol Extinction and Cue-Induced Reinstatement by Specific Projections among Medial Prefrontal Cortex, Nucleus Accumbens, and Basolateral Amygdala

PubMed Central

Bond, Colin W.; DiLeone, Ralph J.

2017-01-01

The ability to inhibit drinking is a significant challenge for recovering alcoholics, especially in the presence of alcohol-associated cues. Previous studies have demonstrated that the regulation of cue-guided alcohol seeking is mediated by the basolateral amygdala (BLA), nucleus accumbens (NAc), and medial prefrontal cortex (mPFC). However, given the high interconnectivity between these structures, it is unclear how mPFC projections to each subcortical structure, as well as projections between BLA and NAc, mediate alcohol-seeking behaviors. Here, we evaluate how cortico-striatal, cortico-amygdalar, and amygdalo-striatal projections control extinction and relapse in a rat model of alcohol seeking. Specifically, we used a combinatorial viral technique to express diphtheria toxin receptors in specific neuron populations based on their projection targets. We then used this strategy to create directionally selective ablations of three distinct pathways after acquisition of ethanol self-administration but before extinction and reinstatement. We demonstrate that ablation of mPFC neurons projecting to NAc, but not BLA, blocks cue-induced reinstatement of alcohol seeking and neither pathway is necessary for extinction of responding. Further, we show that ablating BLA neurons that project to NAc disrupts extinction of alcohol approach behaviors and attenuates reinstatement. Together, these data provide evidence that the mPFC→NAc pathway is necessary for cue-induced reinstatement of alcohol seeking, expand our understanding of how the BLA→NAc pathway regulates alcohol behavior, and introduce a new methodology for the manipulation of target-specific neural projections. SIGNIFICANCE STATEMENT The vast majority of recovering alcoholics will relapse at least once and understanding how the brain regulates relapse will be key to developing more effective behavior and pharmacological therapies for alcoholism. Given the high interconnectivity of cortical, striatal, and limbic structures that regulate alcohol intake, it has been difficult to disentangle how separate projections between them may control different aspects of these complex behaviors. Here, we demonstrate a new approach for noninvasively ablating each of these pathways and testing their necessity for both extinction and relapse. We show that inputs to the nucleus accumbens from medial prefrontal cortex and amygdala regulate alcohol-seeking behaviors differentially, adding to our understanding of the neural control of alcoholism. PMID:28336571

N-acetylcysteine prevents stress-induced anxiety behavior in zebrafish.

PubMed

Mocelin, Ricieri; Herrmann, Ana P; Marcon, Matheus; Rambo, Cassiano L; Rohden, Aline; Bevilaqua, Fernanda; de Abreu, Murilo Sander; Zanatta, Leila; Elisabetsky, Elaine; Barcellos, Leonardo J G; Lara, Diogo R; Piato, Angelo L

2015-12-01

Despite the recent advances in understanding the pathophysiology of anxiety disorders, the pharmacological treatments currently available are limited in efficacy and induce serious side effects. A possible strategy to achieve clinical benefits is drug repurposing, i.e., discovery of novel applications for old drugs, bringing new treatment options to the market and to the patients who need them. N-acetylcysteine (NAC), a commonly used mucolytic and paracetamol antidote, has emerged as a promising molecule for the treatment of several neuropsychiatric disorders. The mechanism of action of this drug is complex, and involves modulation of antioxidant, inflammatory, neurotrophic and glutamate pathways. Here we evaluated the effects of NAC on behavioral parameters relevant to anxiety in zebrafish. NAC did not alter behavioral parameters in the novel tank test, prevented the anxiety-like behaviors induced by an acute stressor (net chasing), and increased the time zebrafish spent in the lit side in the light/dark test. These data may indicate that NAC presents an anti-stress effect, with the potential to prevent stress-induced psychiatric disorders such as anxiety and depression. The considerable homology between mammalian and zebrafish genomes invests the current data with translational validity for the further clinical trials needed to substantiate the use of NAC in anxiety disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

O-GlcNAc reports ambient temperature and confers heat resistance on ectotherm development.

PubMed

Radermacher, Pablo T; Myachina, Faina; Bosshardt, Fritz; Pandey, Rahul; Mariappa, Daniel; Müller, H-Arno J; Lehner, Christian F

2014-04-15

Effects of temperature on biological processes are complex. Diffusion is less affected than the diverse enzymatic reactions that have distinct individual temperature profiles. Hence thermal fluctuations pose a formidable challenge to ectothermic organisms in which body temperature is largely dictated by the ambient temperature. How cells in ectotherms cope with the myriad disruptive effects of temperature variation is poorly understood at the molecular level. Here we show that nucleocytoplasmic posttranslational modification of proteins with O-linked GlcNAc (O-GlcNAc) is closely correlated with ambient temperature during development of distantly related ectotherms ranging from the insect Drosophila melanogaster to the nematode Caenorhabditis elegans to the fish Danio rerio. Regulation seems to occur at the level of activity of the only two enzymes, O-GlcNAc transferase and O-GlcNAcase, that add and remove, respectively, this posttranslational modification in nucleus and cytoplasm. With genetic approaches in D. melanogaster and C. elegans, we demonstrate the importance of high levels of this posttranslational modification for successful development at elevated temperatures. Because many cytoplasmic and nuclear proteins in diverse pathways are O-GlcNAc targets, temperature-dependent regulation of this modification might contribute to an efficient coordinate adjustment of cellular processes in response to thermal change.

[The application of N-acetylcysteine in optimization of specific pharmacological therapies].

PubMed

Hołyńska-Iwan, Iga; Wróblewski, Marcin; Olszewska-Słonina, Dorota; Tyrakowski, Tomasz

2017-09-29

Based on the analysis of data from clinical trials it could be postulated that N-acetylcysteine has a positive impact on the treatment of various diseases. However, less is known about specific molecular and physiological mechanisms underlying the reported therapeutic effects. N-acetylcysteine (NAC, N-acetyl-L-cysteine) is an amino acid derivative containing a thiol group. It is a precursor of L-cysteine and glutathione. NAC is well absorbed and safe for the body at doses up to 300 mg per kg of body weight. Side effects are relatively rare. NAC is used as an mucolytic agent in adjunctive therapy of respiratory diseases causing the retention of secretions, as well as an antidote in the treatment of paracetamol poisoning. Moreover, NAC protects against the toxic effects of reactive oxygen species and their active metabolites. NAC is involved in free radical scavenging processes via several independent mechanisms, including a direct reduction of free radicals, providing substrates for oxidation-reduction reactions and activation of antioxidant enzymes. In the blood, NAC decreases the level of low density lipoprotein peroxidation. In various tissues, NAC may increase the levels of glutathione and cysteine and stimulate the superoxide dismutase action. NAC is used as a supplement in the treatment of various diseases associated with impaired exterior and intracellular oxidative balance. NAC increases the concentrations of amino acids and their derivatives, including cysteine, cystine, and glutathione. It also stabilizes the antioxidant status of the cells and the intercellular spaces. NAC changes the levels of transcription factors, modifying the transcription of selected genes and acting on the protein translation. It works on the activation of several enzymes in the cells and outside the cells. Based on the analysis of data from clinical trials it can be concluded, that an administration of NAC may be beneficial for these groups of patients, in whom the reversible accumulation and the negative action of free radicals was observed.

N-Acetylcysteine in Agriculture, a Novel Use for an Old Molecule: Focus on Controlling the Plant–Pathogen Xylella fastidiosa

PubMed Central

Muranaka, Lígia S.; Giorgiano, Thais E.; Takita, Marco A.; Forim, Moacir R.; Silva, Luis F. C.; Coletta-Filho, Helvécio D.; Machado, Marcos A.; de Souza, Alessandra A.

2013-01-01

Xylella fastidiosa is a plant pathogen bacterium that causes diseases in many different crops. In citrus, it causes Citrus Variegated Chlorosis (CVC). The mechanism of pathogenicity of this bacterium is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants, and there is not yet any method to directly reduce populations of this pathogen in the field. In this study, we investigated the inhibitory effect of N-Acetylcysteine (NAC), a cysteine analogue used mainly to treat human diseases, on X. fastidiosa in different experimental conditions. Concentrations of NAC over 1 mg/mL reduced bacterial adhesion to glass surfaces, biofilm formation and the amount of exopolysaccharides (EPS). The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to X. fastidiosa-infected plants in hydroponics, fertigation, and adsorbed to organic fertilizer (NAC-Fertilizer). HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL but not at 6 mg/mL. Sweet orange plants with CVC symptoms treated with NAC (0.48 and 2.4 mg/mL) in hydroponics showed clear symptom remission and reduction in bacterial population, as analyzed by quantitative PCR and bacterial isolation. Experiments using fertigation and NAC-Fertilizer were done to simulate a condition closer to that normally is used in the field. For both, significant symptom remission and a reduced bacterial growth rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment increased to around eight months. This is the first report of the anti-bacterial effect of NAC against a phytopathogenic bacterium. The results obtained in this work together with the characteristics of this molecule indicate that the use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogenic bacteria. PMID:24009716

N-acetylcysteine in agriculture, a novel use for an old molecule: focus on controlling the plant-pathogen Xylella fastidiosa.

PubMed

Muranaka, Lígia S; Giorgiano, Thais E; Takita, Marco A; Forim, Moacir R; Silva, Luis F C; Coletta-Filho, Helvécio D; Machado, Marcos A; de Souza, Alessandra A

2013-01-01

Xylella fastidiosa is a plant pathogen bacterium that causes diseases in many different crops. In citrus, it causes Citrus Variegated Chlorosis (CVC). The mechanism of pathogenicity of this bacterium is associated with its capacity to colonize and form a biofilm in the xylem vessels of host plants, and there is not yet any method to directly reduce populations of this pathogen in the field. In this study, we investigated the inhibitory effect of N-Acetylcysteine (NAC), a cysteine analogue used mainly to treat human diseases, on X. fastidiosa in different experimental conditions. Concentrations of NAC over 1 mg/mL reduced bacterial adhesion to glass surfaces, biofilm formation and the amount of exopolysaccharides (EPS). The minimal inhibitory concentration of NAC was 6 mg/mL. NAC was supplied to X. fastidiosa-infected plants in hydroponics, fertigation, and adsorbed to organic fertilizer (NAC-Fertilizer). HPLC analysis indicated that plants absorbed NAC at concentrations of 0.48 and 2.4 mg/mL but not at 6 mg/mL. Sweet orange plants with CVC symptoms treated with NAC (0.48 and 2.4 mg/mL) in hydroponics showed clear symptom remission and reduction in bacterial population, as analyzed by quantitative PCR and bacterial isolation. Experiments using fertigation and NAC-Fertilizer were done to simulate a condition closer to that normally is used in the field. For both, significant symptom remission and a reduced bacterial growth rate were observed. Using NAC-Fertilizer the lag for resurgence of symptoms on leaves after interruption of the treatment increased to around eight months. This is the first report of the anti-bacterial effect of NAC against a phytopathogenic bacterium. The results obtained in this work together with the characteristics of this molecule indicate that the use of NAC in agriculture might be a new and sustainable strategy for controlling plant pathogenic bacteria.

Nutrient-driven O-GlcNAc in proteostasis and neurodegeneration.

PubMed

Akan, Ilhan; Olivier-Van Stichelen, Stephanie; Bond, Michelle R; Hanover, John A

2018-01-01

Proteostasis is essential in the mammalian brain where post-mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient-sensitive nucleocytoplasmic post-translational modification O-linked N-acetylglucosamine (O-GlcNAc) regulates protein homeostasis. The O-GlcNAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O-GlcNAc transferase- and O-GlcNAcase-knockout alleles have helped define the role O-GlcNAc plays in development as well as age-associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O-GlcNAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O-GlcNAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O-GlcNAc cycling. Like the quality control system in the endoplasmic reticulum which uses a 'mannose timer' to monitor protein folding, we propose that cytoplasmic proteostasis relies on an 'O-GlcNAc timer' to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O-GlcNAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain-selective knockout mouse models will be an important tool for understanding the role of O-GlcNAc in the physiology of the brain and its susceptibility to neurodegenerative injury. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step

PubMed Central

Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Kamiya, Yukiko; Kato, Koichi; Horimoto, Satoshi; Ishikawa, Tokiro; Takeda, Shunichi; Sakuma, Tetsushi; Yamamoto, Takashi

2014-01-01

Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease–mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2. PMID:25092655

Interactive effects of N-acetylcysteine and antidepressants.

PubMed

Costa-Campos, Luciane; Herrmann, Ana P; Pilz, Luísa K; Michels, Marcus; Noetzold, Guilherme; Elisabetsky, Elaine

2013-07-01

N-acetylcysteine (NAC), a glutathione precursor and glutamate modulator, has been shown to possess various clinically relevant psychopharmacological properties. Considering the role of glutamate and oxidative stress in depressive states, the poor effectiveness of antidepressant drugs (ADs) and the benefits of drug combination for treating depression, the aim of this study was to explore the possible benefit of NAC as an add on drug to treat major depression. For that matter we investigated the combination of subeffective and effective doses of NAC with subeffective and effective doses of several ADs in the mice tail suspension test. The key finding of this study is that a subeffective dose of NAC reduced the minimum effective doses of imipramine and escitalopram, but not those of desipramine and bupropion. Moreover, the same subeffective dose of NAC increased the minimum effective dose of fluoxetine in the same model. In view of the advantages associated with using the lowest effective dose of antidepressant, the results of this study suggest the potential of a clinically useful interaction of NAC with imipramine and escitalopram. Further studies are necessary to better characterize the molecular basis of such interactions, as well as to typify the particular drug combinations that would optimize NAC as an alternative for treating depression. Copyright © 2013 Elsevier Inc. All rights reserved.

Influence of Sulfolane on ESI-MS Measurements of Protein-Ligand Affinities

NASA Astrophysics Data System (ADS)

Yao, Yuyu; Richards, Michele R.; Kitova, Elena N.; Klassen, John S.

2016-03-01

The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand affinities are described. Binding measurements carried out on four protein-carbohydrate complexes, lysozyme with β- d-GlcNAc-(1→4)-β- d-GlcNAc-(1→4)-β- d-GlcNAc-(1→4)- d-GlcNAc, a single chain variable fragment and α- d-Gal-(1→2)-[α- d-Abe-(1→3)]-α- d-Man-OCH3, cholera toxin B subunit homopentamer with β- d-Gal-(1→3)-β- d-GalNAc-(1→4)[α- d-Neu5Ac-(2→3)]-β- d-Gal-(1→4)-β- d-Glc, and a fragment of galectin 3 and α- l-Fuc-(1→2)-β- d-Gal-(1→3)-β- d-GlcNAc-(1→3)-β- d-Gal-(1→4)-β- d-Glc, revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein-ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme-tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.

Influence of Sulfolane on ESI-MS Measurements of Protein-Ligand Affinities.

PubMed

Yao, Yuyu; Richards, Michele R; Kitova, Elena N; Klassen, John S

2016-03-01

The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand affinities are described. Binding measurements carried out on four protein-carbohydrate complexes, lysozyme with β-D-GlcNAc-(1→4)-β-D-GlcNAc-(1→4)-β-D-GlcNAc-(1→4)-D-GlcNAc, a single chain variable fragment and α-D-Gal-(1→2)-[α-D-Abe-(1→3)]-α-D-Man-OCH3, cholera toxin B subunit homopentamer with β-D-Gal-(1→3)-β-D-GalNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β-D-Gal-(1→4)-β-D-Glc, and a fragment of galectin 3 and α-L-Fuc-(1→2)-β-D-Gal-(1→3)-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-β-D-Glc, revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein-ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme-tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.

Arabidopsis thaliana alpha1,2-glucosyltransferase (ALG10) is required for efficient N-glycosylation and leaf growth

PubMed Central

Farid, Akhlaq; Pabst, Martin; Schoberer, Jennifer; Altmann, Friedrich; Glössl, Josef; Strasser, Richard

2011-01-01

Assembly of the dolichol-linked oligosaccharide precursor (Glc3Man9GlcNAc2) is highly conserved among eukaryotes. In contrast to yeast and mammals, little is known about the biosynthesis of dolichol-linked oligosaccharides and the transfer to asparagine residues of nascent polypeptides in plants. To understand the biological function of these processes in plants we characterized the Arabidopsis thaliana homolog of yeast ALG10, the α1,2-glucosyltransferase that transfers the terminal glucose residue to the lipid-linked precursor. Expression of an Arabidopsis ALG10–GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed a reticular distribution pattern resembling endoplasmic reticulum (ER) localization. Analysis of lipid-linked oligosaccharides showed that Arabidopsis ALG10 can complement the yeast Δalg10 mutant strain. A homozygous Arabidopsis T-DNA insertion mutant (alg10-1) accumulated mainly lipid-linked Glc2Man9GlcNAc2 and displayed a severe protein underglycosylation defect. Phenotypic analysis of alg10-1 showed that mutant plants have altered leaf size when grown in soil. Moreover, the inactivation of ALG10 in Arabidopsis resulted in the activation of the unfolded protein response, increased salt sensitivity and suppression of the phenotype of α-glucosidase I-deficient plants. In summary, these data show that Arabidopsis ALG10 is an ER-resident α1,2-glucosyltransferase that is required for lipid-linked oligosaccharide biosynthesis and subsequently for normal leaf development and abiotic stress response. PMID:21707802

Antimicrobial activity of alexidine alone and associated with N-acetylcysteine against Enterococcus faecalis biofilm

PubMed Central

Silveira, Luiz Fernando Machado; Baca, Pilar; Arias-Moliz, María Teresa; Rodríguez-Archilla, Alberto; Ferrer-Luque, Carmen María

2013-01-01

The purpose of this study was to assess the efficacy of alexidine (ALX), alone and combined with N-acetylcysteine (NAC), in eradicating two Enterococcus faecalis strain biofilms. The biofilms of E. faecalis ATCC 29212 and the clinical isolate E. faecalis D1 were grown in the MBEC-high-throughput device for 24 h and were exposed to five twofold dilutions of ALX (2%–0.007 8%) alone and combined with 100 mg⋅mL−1 NAC, for 1 and 5 min. Eradication was defined as 100% kill of biofilm bacteria. The Student's t-test was used to compare the efficacy of the associations of the two irrigants. After 1-min contact time, ALX eradicated the biofilms at all concentrations except for 0.007 8% and 0.015 6%–0.007 8% with E. faecalis ATCC 29212 and E. faecalis D1, respectively. Similar results for eradication and concentration were obtained when it was combined with 100 mg⋅mL−1 NAC. After 5 min of contact time, ALX alone and combined with NAC eradicated all enterococci biofilms. ALX showed antimicrobial properties against the two E. faecalis strain biofilms tested at very low concentrations, and its combined use with NAC was not seen to enhance its activity. PMID:23970139

Epigenetic activation of MGAT3 and corresponding bisecting GlcNAc shortens the survival of cancer patients.

PubMed

Kohler, Reto S; Anugraham, Merrina; López, Mónica Núñez; Xiao, Christina; Schoetzau, Andreas; Hettich, Timm; Schlotterbeck, Goetz; Fedier, André; Jacob, Francis; Heinzelmann-Schwarz, Viola

2016-08-09

Bisecting GlcNAc on N-glycoproteins is described in E-cadherin-, EGF-, Wnt- and integrin- cancer-associated signaling pathways. However, the mechanisms regulating bisecting GlcNAc expression are not clear. Bisecting GlcNAc is attached to N-glycans through beta 1-4 N-acetylglucosaminyl transferase III (MGAT3), which is encoded by two exons flanked by high-density CpG islands. Despite a recently described correlation of MGAT3 and bisecting GlcNAc in ovarian cancer cells, it remains unknown whether DNA methylation is causative for the presence of bisecting GlcNAc. Here, we narrow down the regulatory genomic region and show that reconstitution of MGAT3 expression with 5-Aza coincides with reduced DNA methylation at the MGAT3 transcription start site. The presence of bisecting GlcNAc on released N-glycans was detected by mass spectrometry (LC-ESI-qTOF-MS/MS) in serous ovarian cancer cells upon DNA methyltransferase inhibition. The regulatory impact of DNA methylation on MGAT3 was further evaluated in 18 TCGA cancer types (n = 6118 samples) and the results indicate an improved overall survival in patients with reduced MGAT3 expression, thereby identifying long-term survivors of high-grade serous ovarian cancers (HGSOC). Epigenetic activation of MGAT3 was also confirmed in basal-like breast cancers sharing similar molecular and genetic features with HGSOC. These results provide novel insights into the epigenetic regulation of MGAT3/bisecting GlcNAc and demonstrate the importance of N-glycosylation in cancer progression.

Cytochrome bc1 complexes of microorganisms.

PubMed Central

Trumpower, B L

1990-01-01

The cytochrome bc1 complex is the most widely occurring electron transfer complex capable of energy transduction. Cytochrome bc1 complexes are found in the plasma membranes of phylogenetically diverse photosynthetic and respiring bacteria, and in the inner mitochondrial membrane of all eucaryotic cells. In all of these species the bc1 complex transfers electrons from a low-potential quinol to a higher-potential c-type cytochrome and links this electron transfer to proton translocation. Most bacteria also possess alternative pathways of quinol oxidation capable of circumventing the bc1 complex, but these pathways generally lack the energy-transducing, protontranslocating activity of the bc1 complex. All cytochrome bc1 complexes contain three electron transfer proteins which contain four redox prosthetic groups. These are cytochrome b, which contains two b heme groups that differ in their optical and thermodynamic properties; cytochrome c1, which contains a covalently bound c-type heme; and a 2Fe-2S iron-sulfur protein. The mechanism which links proton translocation to electron transfer through these proteins is the proton motive Q cycle, and this mechanism appears to be universal to all bc1 complexes. Experimentation is currently focused on understanding selected structure-function relationships prerequisite for these redox proteins to participate in the Q-cycle mechanism. The cytochrome bc1 complexes of mitochondria differ from those of bacteria, in that the former contain six to eight supernumerary polypeptides, in addition to the three redox proteins common to bacteria and mitochondria. These extra polypeptides are encoded in the nucleus and do not contain redox prosthetic groups. The functions of the supernumerary polypeptides of the mitochondrial bc1 complexes are generally not known and are being actively explored by genetically manipulating these proteins in Saccharomyces cerevisiae. Images PMID:2163487

Low Pretreatment Impulsivity and High Medication Adherence Increase the Odds of Abstinence in a Trial of N-Acetylcysteine in Adolescents with Cannabis Use Disorder

PubMed Central

Bentzley, Jessica P.; Tomko, Rachel L.; Gray, Kevin M.

2016-01-01

Background In light of recent progress toward pharmacologic interventions to treat adolescent cannabis use disorder, it is important to consider which adolescent characteristics may be associated with a favorable response to treatment. This study presents secondary analyses from a parent randomized controlled trial of N-acetylcysteine (NAC) in adolescents with cannabis use disorder. We hypothesized high pretreatment impulsivity and medication non-adherence would be associated with reduced abstinence rates. Methods Participants were treatment-seeking adolescents (N = 115) who met criteria for cannabis use disorder and were assessed for pretreatment impulsivity. They received 1200 mg NAC or placebo orally twice daily for 8 weeks. An intent-to-treat analysis using a repeated-measures logistic regression model was used to relate pretreatment impulsivity (Barratt Impulsiveness Scale) and treatment group to abstinence rates, measured by urine cannabinoid tests. To explore mechanisms by which NAC may reduce cannabis use, relationships between impulsivity, adherence, and abstinence were assessed in a second statistical model using data from participants with recorded adherence and urine cannabinoid test results (n = 54). Results In the intent-to-treat analysis, low pretreatment impulsivity, NAC treatment, and negative baseline urine cannabinoid test results independently increased the odds of having negative urine cannabinoid tests during treatment (OR = 2.1, 2.3, 5.3 respectively). In the sample of participants with adherence data (n = 54), adherence tripled the odds of abstinence. Notably, the effect of adherence on abstinence was only observed in the NAC treatment group. Lastly, although the highly impulsive participants had reduced rates of abstinence, highly impulsive individuals adherent to NAC treatment had increased abstinence rates compared to non-adherent individuals. Conclusion Low impulsivity, NAC treatment, medication adherence, and baseline negative cannabinoid testing were associated with increased rates of abstinence in adolescents seeking treatment for cannabis use disorder. Efforts to optimize pharmacotherapy adherence may be particularly crucial for highly impulsive individuals. Understanding and addressing factors, such as impulsivity and adherence, which may affect outcomes, may aid in the successful evaluation and development of potentially promising pharmacotherapies. PMID:26827257

SANS with contrast variation study of the bacteriorhodopsin-octyl glucoside complex

NASA Astrophysics Data System (ADS)

Mo, Yiming; Heller, William T.

2010-11-01

Membrane proteins (MPs), which play vital roles in trans-membrane trafficking and signalling between cells and their external environment, comprise a major fraction of the expressed proteomes of many organisms. MP production for biophysical characterization requires detergents for extracting MPs from their native membrane and to solubilize the MP in solution for purification and study. In a proper detergent solution, the detergent-associated MPs retain their native fold and oligomerization state, key requirements for biophysical characterization and crystallization. SANS with contrast variation was performed to characterize BR in complex with OG to better understand the MP-detergent complex. Contrast variation makes it possible to not only probe the conformation of the entire structure but also investigate the conformation of the polypeptide chain within the BR-OG complex. The BR-OG SANS contrast variation series is not consistent with a compact structure, such as a trimeric BR complex surrounded by a belt of detergent. The data strongly suggest that the protein is partially unfolded through its association with the detergent micelles.

Survival benefit of neoadjuvant chemotherapy for resectable breast cancer: A meta-analysis.

PubMed

Chen, Yan; Shi, Xiu-E; Tian, Jin-Hui; Yang, Xu-Juan; Wang, Yong-Feng; Yang, Ke-Hu

2018-05-01

Neoadjuvant chemotherapy (NAC) increases breast conservation rates in patients with resectable breast cancer at the associated cost of higher locoregional recurrence rates; however, the magnitude of the survival benefits of NAC for these patients remains undefined. Therefore, we aimed to clarify the survival benefit of NAC versus postoperative chemotherapy by conducting an updated meta-analysis of randomized clinical trials (RCTs). The authors searched the Cochrane Library, PubMed, Embase, Web of Science, Chinese biomedical literature database, and Chinese Scientific Journals full-text database from their inception to December 2016. The authors identified relevant RCTs that compared NAC with postoperative chemotherapy in the treatment of operable breast cancer. The main endpoints were overall survival (OS) and recurrence-free survival (RFS). A total of 21 citations representing 16 unique studies were eligible. There were 787 deaths among 2794 patients assigned to NAC groups and 816 deaths among 2799 patients assigned to adjuvant chemotherapy groups. A meta-analysis of data indicated that there was no significant benefit in terms of OS ([hazard ratio [HR] = 1.03, 95% confidence interval [CI]: 0.94-1.13, P = .51) and RFS (HR = 1.01, 95% CI: 0.93-1.10, P = .80) between the NAC and postoperative chemotherapy groups. The pooled HR estimate for OS was not influenced by NAC cycles, the total number of chemotherapy cycles, administration of tamoxifen, administration of adjuvant chemotherapy, or type of NAC regimen. Subgroup analysis showed that the pooled HR estimate for RFS was influenced by anthracycline-containing regimens. Patients with a pathological complete response had superior survival outcomes compared with patients who had residual disease. The survival benefits for patients with operable breast cancer who received either NAC or adjuvant chemotherapy based on anthracycline regimens were comparable.

N-acetylcysteine in a Double-Blind Randomized Placebo-Controlled Trial: Toward Biomarker-Guided Treatment in Early Psychosis

PubMed Central

Conus, Philippe; Seidman, Larry J; Fournier, Margot; Xin, Lijing; Cleusix, Martine; Baumann, Philipp S; Ferrari, Carina; Cousins, Ann; Alameda, Luis; Gholam-Rezaee, Mehdi; Golay, Philippe; Jenni, Raoul; Woo, T -U Wilson; Keshavan, Matcheri S; Eap, Chin B; Wojcik, Joanne; Cuenod, Michel; Buclin, Thierry; Gruetter, Rolf

2018-01-01

Abstract Biomarker-guided treatments are needed in psychiatry, and previous data suggest oxidative stress may be a target in schizophrenia. A previous add-on trial with the antioxidant N-acetylcysteine (NAC) led to negative symptom reductions in chronic patients. We aim to study NAC’s impact on symptoms and neurocognition in early psychosis (EP) and to explore whether glutathione (GSH)/redox markers could represent valid biomarkers to guide treatment. In a double-blind, randomized, placebo-controlled trial in 63 EP patients, we assessed the effect of NAC supplementation (2700 mg/day, 6 months) on PANSS, neurocognition, and redox markers (brain GSH [GSHmPFC], blood cells GSH levels [GSHBC], GSH peroxidase activity [GPxBC]). No changes in negative or positive symptoms or functional outcome were observed with NAC, but significant improvements were found in favor of NAC on neurocognition (processing speed). NAC also led to increases of GSHmPFC by 23% (P = .005) and GSHBC by 19% (P = .05). In patients with high-baseline GPxBC compared to low-baseline GPxBC, subgroup explorations revealed a link between changes of positive symptoms and changes of redox status with NAC. In conclusion, NAC supplementation in a limited sample of EP patients did not improve negative symptoms, which were at modest baseline levels. However, NAC led to some neurocognitive improvements and an increase in brain GSH levels, indicating good target engagement. Blood GPx activity, a redox peripheral index associated with brain GSH levels, could help identify a subgroup of patients who improve their positive symptoms with NAC. Thus, future trials with antioxidants in EP should consider biomarker-guided treatment. PMID:29462456

Mobilization of Cd from human serum albumin by small molecular weight thiols.

PubMed

Morris, Thomas T; Keir, Jennifer L A; Boshart, Steven J; Lobanov, Victor P; Ruhland, Anthony M A; Bahl, Nishita; Gailer, Jürgen

2014-05-01

Although the toxic metal Cd is an established human nephrotoxin, little is known about the role that interactions with plasma constitutents play in determining its mammalian target organs. To gain insight, a Cd-human serum albumin (HSA) complex was analyzed on a system consisting of size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using phosphate buffered saline (pH 7.4) as the mobile phase, we investigated the effect of 1-10mM oxidized glutathione (GSSG), l-cysteine (Cys), l-glutathione (GSH), or N-acetyl-l-cysteine (NAC) on the elution of Cd. As expected, GSSG did not mobilize Cd from the Cd-HSA complex up to a concentration of 4mM. With 1.0mM NAC, ∼30% of the injected Cd-HSA complex eluted as such, while the mobilized Cd was lost on the column. With 1.0mM of Cys or GSH, no parent Cd-HSA complex was detected and 88% and 82% of the protein bound Cd eluted close to the elution volume, likely in form of Cd(Cys)2 and a Cd-GSH 1:1 complex. Interestingly, with GSH and NAC concentrations >4.0mM, a Cd double peak was detected, which was rationalized in terms of the elution of a polynuclear Cd complex baseline-separated from a mononuclear Cd complex. In contrast, mobile phases which contained Cys concentrations ≥2mM resulted in the detection of only a single Cd peak, probably Cd(Cys)4. Our results establish SEC-FAAS as a viable tool to probe the mobilization of Cd from binding sites on plasma proteins at near physiological conditions. The detected complexes between Cd and Cys or GSH may be involved in the translocation of Cd to mammalian target organs. Copyright © 2014 Elsevier B.V. All rights reserved.

An Ab Initio Study of Alkali-C60 Complexes

NASA Astrophysics Data System (ADS)

Frick, Nathan; Hira, A. S.; Ray, A. K.

2003-03-01

We extend our previous work on fullerene-alkali complexes1-2 by presenting the results of an ab initio theoretical study of the alkali LiC60+, LiC60, NaC60+, NaC60, KC60+, and KC60 complexes. In the endohedral complexes for Li and Na, there is displacement of the adatom from the center. Of the ions, exohedral Li+ will sit closest to the cage, and among the neutrals, exohedral K remains closest. Bond lengths are consistently longer for the fivefold and threefold approaches. Adsorbates inside the fullerene donate negative charge to the carbons, but ions outside obtain a small amount, resulting in a polarization of the molecule. In the ion complexes, there is lowering of the orbital energy levels by 3 to 4 eV, resulting in an increase in the number of bound, but unoccupied, electronic orbitals. The HOMO-LUMO gap, of interest in superconductivity studies, is reduced by about 50 1. A.S. Hira and A.K. Ray, Phys. Rev. A 52, 141(1995); A 54, 2205(1996). 2. Ajit Hira and A. K. Ray, "An Initio Modeling of the Endohedral and Exohedral Complexes of C60Na2+ Complexes", Bull. Am. Phys. Soc. 47 (March 2002).

Adjunctive N-acetylcysteine in depression: exploration of interleukin-6, C-reactive protein and brain-derived neurotrophic factor.

PubMed

Hasebe, Kyoko; Gray, Laura; Bortolasci, Chiara; Panizzutti, Bruna; Mohebbi, Mohammadreza; Kidnapillai, Srisaiyini; Spolding, Briana; Walder, Ken; Berk, Michael; Malhi, Gin; Dodd, Seetal; Dean, Olivia M

2017-12-01

This study aimed to explore effects of adjunctive N-acetylcysteine (NAC) treatment on inflammatory and neurogenesis markers in unipolar depression. We embarked on a 12-week clinical trial of NAC (2000 mg/day compared with placebo) as an adjunctive treatment for unipolar depression. A follow-up visit was conducted 4 weeks following the completion of treatment. We collected serum samples at baseline and the end of the treatment phase (week 12) to determine changes in interleukin-6 (IL6), C-reactive protein (CRP) and brain-derived neurotrophic factor (BDNF) following NAC treatment. NAC treatment significantly improved depressive symptoms on the Montgomery-Asberg Depression Rating Scale (MADRS) over 16 weeks of the trial. Serum levels of IL6 were associated with reductions of MADRS scores independent of treatment response. However, we found no significant changes in IL6, CRP and BDNF levels following NAC treatment. Overall, this suggests that our results failed to support the hypothesis that IL6, CRP and BDNF are directly involved in the therapeutic mechanism of NAC in depression. IL6 may be a useful marker for future exploration of treatment response.

Current evidence for the use of N-acetylcysteine following liver resection.

PubMed

Kemp, Richard; Mole, Jonathan; Gomez, Dhanny

2018-06-01

N-acetylcysteine (NAC) has many uses in medicine; notable in the management of paracetamol toxicity, acute liver failure and liver surgery. The aim of this review was to critically appraise the published literature for the routine use of NAC in liver resection surgery. An electronic search was performed of EBSCOhost (Medline and CINAHL database), PubMed and the Cochrane Library for the period 1990-2016. MeSH headings: 'acetyl-cysteine', 'liver resection' and 'hepatectomy' were used to identify all relevant articles published in English. Following the search criteria used, three articles were included. Two of these studies were randomized controlled trials. All the studies collated data on morbidity and mortality. All three studies did not show a significant difference in overall complications rates in patients that underwent hepatic resection that had NAC infusion compared with patients that did not. In one study, NAC administration was associated with a higher frequency of grade A post-hepatectomy liver failure. In another study, a significantly higher incidence of delirium was observed in the NAC group, which led to the trial to be terminated early. The current published data do not support the routine use of NAC following liver resection. © 2017 Royal Australasian College of Surgeons.

Evaluation of N-acetylcysteine and methylprednisolone as therapies for oxygen and acrolein-induced lung damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Critchley, J.A.J.H.; Beeley, J.M.; Clark, R.J.

1990-04-01

Reactive oxidizing species are implicated in the etiology of a range of inhalational pulmonary injuries. Consequently, various free radical scavengers have been tested as potential prophylactic agents. The sulfydryl compound, N-acetylcysteine (NAC) is the only such compound clinically available for use in realistic dosages, and it is well established as an effective antidote for the hepatic and renal toxicity of paracetamol. Another approach in pulmonary injury prophylaxis is methylprednisolone therapy. The authors evaluated NAC and methylprednisolone in two rats models of inhalation injury: 40-hr exposure to >97% oxygen at 1.1 bar and 15-min exposure to acrolein vapor (210 ppm). Themore » increases in lung wet/dry weight ratios, seen with both oxygen and acrolein toxicity were reduced with both treatments. However, with oxygen, NAC therapy was associated with considerably increased mortality and histological changes. Furthermore, IP NAC administration resulted in large volumes of ascitic fluid. With acrolein, IV, NAC had no significant effect on mortality or pulmonary histological damage. Methylprednisolone had no beneficial effects on either the mortality or histological damage observed in either toxicity model. They caution against the ad hoc use of NAC in the management of inhalational pulmonary injury.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woodson, W.R.; Handa, A.K.

Changes in proteins associated with senescence of the flowers of Hibiscus rosa-sinensis was studied using SDS-PAGE. Total extractable protein from petals decreased with senescence. Changes were noted in patterns of proteins from aging petals. Flower opening and senescence was associated with appearance and disappearance of several polypeptides. One new polypeptide with an apparent mw of 41 kd was first seen the day of flower opening and increased to over 9% of the total protein content of senescent petal tissue. Protein synthesis during aging was investigated by following uptake and incorporation of /sup 3/H-leucine into TCA-insoluble fraction of petal discs. Proteinmore » synthesis, as evidenced by the percent of label incorporated into the TCA-insoluble fraction, was greatest (32%) the day before flower opening. Senescent petal tissue incorporated 4% of label taken up into protein. Proteins were separated by SDS-PAGE and labelled polypeptides identified by fluorography. In presenescent petal tissue, radioactivity was distributed among several major polypeptides. In senescent tissue, much of the radioactivity was concentrated in the 41 kd polypeptide.« less

Caudal Nucleus Accumbens Core Is Critical in the Regulation of Cue-Elicited Approach-Avoidance Decisions

PubMed Central

Hamel, Laurie; Thangarasa, Tharshika; Samadi, Osai

2017-01-01

The nucleus accumbens (NAc) is thought to be a site of integration of positively and negatively valenced information and action selection. Functional differentiation in valence processing has previously been found along the rostrocaudal axis of the shell region of the NAc in assessments of unconditioned motivation. Given that the core region of the NAc has been implicated in the elicitation of motivated behavior in response to conditioned cues, we sought to assess the role of caudal, intermediate, and rostral sites within this subregion in cue-elicited approach-avoidance decisions. Rats were trained to associate visuo-tactile cues with appetitive, aversive, and neutral outcomes. Following the successful acquisition of the cue-outcome associations, rats received microinfusions of GABAA and GABAB receptor agonists (muscimol/baclofen) or saline into the caudal, intermediate, or rostral NAc core and were then exposed to a superimposition of appetitively and aversively valenced cues versus neutral cues in a “conflict test,” as well as to the appetitive versus neutral cues, and aversive cues versus neutral cues, in separate conditioned preference/avoidance tests. Disruption of activity in the intermediate to caudal parts of the NAc core resulted in a robust avoidance bias in response to motivationally conflicting cues, as well as a potentiated avoidance of aversive cues as compared with control animals, coupled with an attenuated conditioned preference for the appetitive cue. These results suggest that the caudal NAc core may have the capacity to exert bidirectional control over appetitively and aversively motivated responses to valence signals. PMID:28275709

Multidrug Resistance-Associated Protein 1 (MRP1) mediated vincristine resistance: effects of N-acetylcysteine and Buthionine Sulfoximine

PubMed Central

Akan, Ilhan; Akan, Selma; Akca, Hakan; Savas, Burhan; Ozben, Tomris

2005-01-01

Background Multidrug resistance mediated by the multidrug resistance-associated protein 1 (MRP1) decreases cellular drug accumulation. The exact mechanism of MRP1 involved multidrug resistance has not been clarified yet, though glutathione (GSH) is likely to have a role for the resistance to occur. N-acetylcysteine (NAC) is a pro-glutathione drug. DL-Buthionine (S,R)-sulfoximine (BSO) is an inhibitor of GSH synthesis. The aim of our study was to investigate the effect of NAC and BSO on MRP1-mediated vincristine resistance in Human Embryonic Kidney (HEK293) and its MRP1 transfected 293MRP cells. Human Embryonic Kidney (HEK293) cells were transfected with a plasmid encoding whole MRP1 gene. Both cells were incubated with vincristine in the presence or absence of NAC and/or BSO. The viability of both cells was determined under different incubation conditions. GSH, Glutathione S-Transferase (GST) and glutathione peroxidase (GPx) levels were measured in the cell extracts obtained from both cells incubated with different drugs. Results N-acetylcysteine increased the resistance of both cells against vincristine and BSO decreased NAC-enhanced MRP1-mediated vincristine resistance, indicating that induction of MRP1-mediated vincristine resistance depends on GSH. Vincristine decreased cellular GSH concentration and increased GPx activity. Glutathione S-Transferase activity was decreased by NAC. Conclusion Our results demonstrate that NAC and BSO have opposite effects in MRP1 mediated vincristine resistance and BSO seems a promising chemotherapy improving agent in MRP1 overexpressing tumor cells. PMID:16042792

Secretion of pancreatic polypeptide in patients with pancreatic endocrine tumors.

PubMed

Adrian, T E; Uttenthal, L O; Williams, S J; Bloom, S R

1986-07-31

Pancreatic polypeptide is often secreted by pancreatic endocrine tumors and is considered a marker for such tumors. To investigate the diagnostic value of this marker, we studied 323 patients with proved pancreatic endocrine tumors. We found plasma concentrations of pancreatic polypeptide to be elevated (more than 300 pmol per liter) in 144 patients (diagnostic sensitivity, 45 percent). However, plasma levels of pancreatic polypeptide can also be elevated in the absence of a pancreatic tumor. To ascertain whether the administration of atropine could distinguish between normal and tumor-associated polypeptide secretion, we studied 30 patients with pancreatic tumors and high plasma levels of pancreatic polypeptide, 18 patients without tumors who had elevated levels of pancreatic polypeptide, and eight normal controls. Polypeptide levels in the 18 patients without tumors were substantially lower than in the 30 patients with tumors. Atropine (1 mg intramuscularly) did not suppress polypeptide levels in patients with tumors, but did suppress plasma levels by more than 50 percent in all subjects without tumors. Thus, although its diagnostic sensitivity is low, pancreatic polypeptide appears to be a useful adjunctive marker of many pancreatic endocrine tumors, and the atropine suppression test can be used to distinguish normal from tumor-related secretion of the polypeptide. Identification of the type of pancreatic endocrine tumor still requires measurement of the hormone that is specific for the tumor.

Analysis of the internal nuclear matrix. Oligomers of a 38 kD nucleolar polypeptide stabilized by disulfide bonds.

PubMed

Fields, A P; Kaufmann, S H; Shaper, J H

1986-05-01

When rat liver nuclei are treated with the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) prior to nuclease treatment and extraction with 1.6 M NaCl, residual nucleoli and an extensive non-chromatin intranuclear network remain associated with the nuclear envelope. Subsequent treatment of this structure with 1 M NaCl containing 20 mM dithiothreitol (DTT) solubilizes the intranuclear material, while the nuclear envelope remains structurally intact. We have isolated and partially characterized a major polypeptide of the disulfide-stabilized internal nuclear matrix. The polypeptide, which has an apparent molecular mass 38 kD and isoelectric point 5.3, has been localized to the nucleolus of rat liver nuclei by indirect immunofluorescence using a specific polyclonal chicken antiserum. Based on its molecular mass, isoelectric point, intracellular localization and amino acid composition, the 38 kD polypeptide appears to be analogous to the nucleolar phosphoprotein B23 described by Prestayko et al. (Biochemistry 13 (1974) 1945) [20]. Immunologically related polypeptides have likewise been localized to the nucleoli of both hamster and human tissue culture cell lines as well as the cellular slime mold Physarum polycephalum. By immunoblotting, a single 38 kD polypeptide is recognized by the antiserum in rat, mouse, hamster and human cell lines. The antiserum has been utilized to investigate the oligomeric structure of the 38 kD polypeptide and the nature of its association with the rat liver nuclear matrix. By introducing varying numbers of disulfide bonds, we have found that the 38 kD polypeptide becomes incorporated into the internal nuclear matrix in a two-step process. Soluble disulfide-bonded homodimers of the polypeptide are first formed and then are rendered salt-insoluble by more extensive disulfide cross-linking.

O-GlcNAc cycling: Emerging Roles in Development and Epigenetics

PubMed Central

Love, Dona C.; Krause, Michael W.; Hanover, John A.

2010-01-01

The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These ‘evo-devo’ relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the ‘vicious cycle’ observed in children of mothers with type-2 diabetes and metabolic disease. PMID:20488252

Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens.

PubMed

Volgers, Charlotte; Benedikter, Birke J; Grauls, Gert E; Hellebrand, Pauline H M; Savelkoul, Paul H M; Stassen, Frank R M

2017-05-01

Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

OsNAC2 positively affects salt-induced cell death and binds to the OsAP37 and OsCOX11 promoters.

PubMed

Mao, Chanjuan; Ding, Jialin; Zhang, Bin; Xi, Dandan; Ming, Feng

2018-05-01

Plant development and adaptation to environmental stresses are intimately associated with programmed cell death (PCD). Although some of the mechanisms regulating PCD [e.g., accumulation of reactive oxygen species (ROS)] are common among responses to different abiotic stresses, the pathways mediating salt-induced PCD remain largely uncharacterized. Here we report that overexpression of OsNAC2, which encodes a plant-specific transcription factor, promotes salt-induced cell death accompanied by the loss of plasma membrane integrity, nuclear DNA fragmentation, and changes to caspase-like activity. In OsNAC2-knockdown lines, cell death was markedly decreased in response to severe salt stress. Additionally, OsNAC2 expression was enhanced in rice seedlings exposed to a high NaCl concentration. Moreover, the results of quantitative real-time PCR, chromatin immunoprecipitation, dual-luciferase, and yeast one-hybrid assays indicated that OsNAC2 targeted genes that encoded an ROS scavenger (OsCOX11) and a caspase-like protease (OsAP37). Furthermore, K + -efflux channels (OsGORK and OsSKOR) were clearly activated by OsNAC2. Overall, our results suggested that OsNAC2 accelerates NaCl-induced PCD and provide new insights into the mechanisms that affect ROS accumulation, plant caspase-like activity, and K + efflux. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

The National Association of College Stores College Store Personnel Placement Service Is a Valuable Tool for Members.

ERIC Educational Resources Information Center

College Store Journal, 1980

1980-01-01

The NACS Placement Service, which consists of two activities--resume referral and advertisements in a special "positions wanted/available" supplement sheet distributed with the weekly NACS College Stores Confidential Bulletin--is described. The position available listing, position wanted listing, and the resume are discussed. (MLW)

Can Tech Transcend the Textbook?

ERIC Educational Resources Information Center

Waters, John K.

2011-01-01

After traveling a long, tortuous road, the much-anticipated e-book revolution has finally arrived. So where is the revolution in the "e-textbook" market? According to the National Association of College Stores (NACS), digital books currently account for less than 3 percent of textbook sales. NACS expects that percentage to reach 10 to 15 percent…

Structural and functional determination of homologs of the Mycobacterium tuberculosisN-acetylglucosamine-6-phosphate deacetylase (NagA).

PubMed

Ahangar, Mohd Syed; Furze, Christopher M; Guy, Collette S; Cooper, Charlotte; Maskew, Kathryn S; Graham, Ben; Cameron, Alexander D; Fullam, Elizabeth

2018-05-04

The Mycobacterium tuberculosis (Mtb) pathogen encodes an N -acetylglucosamine-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of N -acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential anti-tubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereo-selective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 Å and 2.0 Å resolutions, respectively. The GlcNAc6P-complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of Candidate Genes Associated with Leaf Senescence in Cultivated Sunflower (Helianthus annuus L.)

PubMed Central

Moschen, Sebastian; Bengoa Luoni, Sofia; Paniego, Norma B.; Hopp, H. Esteban; Dosio, Guillermo A. A.

2014-01-01

Cultivated sunflower (Helianthus annuus L.), an important source of edible vegetable oil, shows rapid onset of senescence, which limits production by reducing photosynthetic capacity under specific growing conditions. Carbon for grain filling depends strongly on light interception by green leaf area, which diminishes during grain filling due to leaf senescence. Transcription factors (TFs) regulate the progression of leaf senescence in plants and have been well explored in model systems, but information for many agronomic crops remains limited. Here, we characterize the expression profiles of a set of putative senescence associated genes (SAGs) identified by a candidate gene approach and sunflower microarray expression studies. We examined a time course of sunflower leaves undergoing natural senescence and used quantitative PCR (qPCR) to measure the expression of 11 candidate genes representing the NAC, WRKY, MYB and NF-Y TF families. In addition, we measured physiological parameters such as chlorophyll, total soluble sugars and nitrogen content. The expression of Ha-NAC01, Ha-NAC03, Ha-NAC04, Ha-NAC05 and Ha-MYB01 TFs increased before the remobilization rate increased and therefore, before the appearance of the first physiological symptoms of senescence, whereas Ha-NAC02 expression decreased. In addition, we also examined the trifurcate feed-forward pathway (involving ORE1, miR164, and ETHYLENE INSENSITIVE 2) previously reported for Arabidopsis. We measured transcription of Ha-NAC01 (the sunflower homolog of ORE1) and Ha-EIN2, along with the levels of miR164, in two leaves from different stem positions, and identified differences in transcription between basal and upper leaves. Interestingly, Ha-NAC01 and Ha-EIN2 transcription profiles showed an earlier up-regulation in upper leaves of plants close to maturity, compared with basal leaves of plants at pre-anthesis stages. These results suggest that the H. annuus TFs characterized in this work could play important roles as potential triggers of leaf senescence and thus can be considered putative candidate genes for senescence in sunflower. PMID:25110882

Early Use of N-acetylcysteine With Nitrate Therapy in Patients Undergoing Primary Percutaneous Coronary Intervention for ST-Segment-Elevation Myocardial Infarction Reduces Myocardial Infarct Size (the NACIAM Trial [N-acetylcysteine in Acute Myocardial Infarction]).

PubMed

Pasupathy, Sivabaskari; Tavella, Rosanna; Grover, Suchi; Raman, Betty; Procter, Nathan E K; Du, Yang Timothy; Mahadavan, Gnanadevan; Stafford, Irene; Heresztyn, Tamila; Holmes, Andrew; Zeitz, Christopher; Arstall, Margaret; Selvanayagam, Joseph; Horowitz, John D; Beltrame, John F

2017-09-05

Contemporary ST-segment-elevation myocardial infarction management involves primary percutaneous coronary intervention, with ongoing studies focusing on infarct size reduction using ancillary therapies. N-acetylcysteine (NAC) is an antioxidant with reactive oxygen species scavenging properties that also potentiates the effects of nitroglycerin and thus represents a potentially beneficial ancillary therapy in primary percutaneous coronary intervention. The NACIAM trial (N-acetylcysteine in Acute Myocardial Infarction) examined the effects of NAC on infarct size in patients with ST-segment-elevation myocardial infarction undergoing percutaneous coronary intervention. This randomized, double-blind, placebo-controlled, multicenter study evaluated the effects of intravenous high-dose NAC (29 g over 2 days) with background low-dose nitroglycerin (7.2 mg over 2 days) on early cardiac magnetic resonance imaging-assessed infarct size. Secondary end points included cardiac magnetic resonance-determined myocardial salvage and creatine kinase kinetics. Of 112 randomized patients with ST-segment-elevation myocardial infarction, 75 (37 in NAC group, 38 in placebo group) underwent early cardiac magnetic resonance imaging. Median duration of ischemia pretreatment was 2.4 hours. With background nitroglycerin infusion administered to all patients, those randomized to NAC exhibited an absolute 5.5% reduction in cardiac magnetic resonance-assessed infarct size relative to placebo (median, 11.0%; [interquartile range 4.1, 16.3] versus 16.5%; [interquartile range 10.7, 24.2]; P =0.02). Myocardial salvage was approximately doubled in the NAC group (60%; interquartile range, 37-79) compared with placebo (27%; interquartile range, 14-42; P <0.01) and median creatine kinase areas under the curve were 22 000 and 38 000 IU·h in the NAC and placebo groups, respectively ( P =0.08). High-dose intravenous NAC administered with low-dose intravenous nitroglycerin is associated with reduced infarct size in patients with ST-segment-elevation myocardial infarction undergoing percutaneous coronary intervention. A larger study is required to assess the impact of this therapy on clinical cardiac outcomes. Australian New Zealand Clinical Trials Registry. URL: http://www.anzctr.org.au/. Unique identifier: 12610000280000. © 2017 American Heart Association, Inc.

A double-blind, randomized, controlled trial on N-acetylcysteine for the prevention of acute kidney injury in patients undergoing allogeneic hematopoietic stem cell transplantation.

PubMed

Ataei, Sara; Hadjibabaie, Molouk; Moslehi, Amirhossein; Taghizadeh-Ghehi, Maryam; Ashouri, Asieh; Amini, Elham; Gholami, Kheirollah; Hayatshahi, Alireza; Vaezi, Mohammad; Ghavamzadeh, Ardeshir

2015-06-01

Acute kidney injury (AKI) is one of the complications of hematopoietic stem cell transplantation and is associated with increased mortality. N-acetylcysteine (NAC) is a thiol compound with antioxidant and vasodilatory properties that has been investigated for the prevention of AKI in several clinical settings. In the present study, we evaluated the effects of intravenous NAC on the prevention of AKI in allogeneic hematopoietic stem cell transplantation patients. A double-blind randomized placebo-controlled trial was conducted, and 80 patients were recruited to receive 100 mg/kg/day NAC or placebo as intermittent intravenous infusion from day -6 to day +15. AKI was determined on the basis of the Risk-Injury-Failure-Loss-End-stage renal disease and AKI Network criteria as the primary outcome. We assessed urine neutrophil gelatinase-associated lipocalin (uNGAL) on days -6, -3, +3, +9 and +15 as the secondary outcome. Moreover, transplant-related outcomes and NAC adverse reactions were evaluated during the study period. Statistical analysis was performed using appropriate parametric and non-parametric methods including Kaplan-Meier for AKI and generalized estimating equation for uNGAL. At the end of the trial, data from 72 patients were analysed (NAC: 33 patients and placebo: 39 patients). Participants of each group were not different considering baseline characteristics. AKI was observed in 18% of NAC recipients and 15% of placebo group patients, and the occurrence pattern was not significantly different (p = 0.73). Moreover, no significant difference was observed between groups for uNGAL measures (p = 0.10). Transplant-related outcomes were similar for both groups, and all patients had successful engraftment. Three patients did not tolerate NAC because of abdominal pain, shortness of breath and rash with pruritus and were dropped from the intervention group before transplantation. However, the frequency of adverse reactions was not significantly different between groups. In conclusion, our findings could not show any clinical benefits from high-dose NAC particularly for AKI prevention in allogeneic hematopoietic stem cell transplantation patients. Copyright © 2014 John Wiley & Sons, Ltd.

Identification of candidate genes associated with leaf senescence in cultivated sunflower (Helianthus annuus L.).

PubMed

Moschen, Sebastian; Bengoa Luoni, Sofia; Paniego, Norma B; Hopp, H Esteban; Dosio, Guillermo A A; Fernandez, Paula; Heinz, Ruth A

2014-01-01

Cultivated sunflower (Helianthus annuus L.), an important source of edible vegetable oil, shows rapid onset of senescence, which limits production by reducing photosynthetic capacity under specific growing conditions. Carbon for grain filling depends strongly on light interception by green leaf area, which diminishes during grain filling due to leaf senescence. Transcription factors (TFs) regulate the progression of leaf senescence in plants and have been well explored in model systems, but information for many agronomic crops remains limited. Here, we characterize the expression profiles of a set of putative senescence associated genes (SAGs) identified by a candidate gene approach and sunflower microarray expression studies. We examined a time course of sunflower leaves undergoing natural senescence and used quantitative PCR (qPCR) to measure the expression of 11 candidate genes representing the NAC, WRKY, MYB and NF-Y TF families. In addition, we measured physiological parameters such as chlorophyll, total soluble sugars and nitrogen content. The expression of Ha-NAC01, Ha-NAC03, Ha-NAC04, Ha-NAC05 and Ha-MYB01 TFs increased before the remobilization rate increased and therefore, before the appearance of the first physiological symptoms of senescence, whereas Ha-NAC02 expression decreased. In addition, we also examined the trifurcate feed-forward pathway (involving ORE1, miR164, and ethylene insensitive 2) previously reported for Arabidopsis. We measured transcription of Ha-NAC01 (the sunflower homolog of ORE1) and Ha-EIN2, along with the levels of miR164, in two leaves from different stem positions, and identified differences in transcription between basal and upper leaves. Interestingly, Ha-NAC01 and Ha-EIN2 transcription profiles showed an earlier up-regulation in upper leaves of plants close to maturity, compared with basal leaves of plants at pre-anthesis stages. These results suggest that the H. annuus TFs characterized in this work could play important roles as potential triggers of leaf senescence and thus can be considered putative candidate genes for senescence in sunflower.

Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

NASA Astrophysics Data System (ADS)

Du, Yi; May, Kimberly; Xu, Wei; Liu, Hongcheng

2012-07-01

The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

Protective effect of N-acetylcysteine against ethanol-induced gastric ulcer: A pharmacological assessment in mice

PubMed Central

Jaccob, Ausama Ayoob

2015-01-01

Aim: Since there is an increasing need for gastric ulcer therapies with optimum benefit-risk profile. This study was conducted to investigate gastro-protective effects of N-acetylcysteine (NAC) against ethanol-induced gastric ulcer models in mice. Materials and Methods: A total of 41 mice were allocated into six groups consisted of 7 mice each. Groups 1 (normal control) and 2 (ulcer control) received distilled water at a dose of 10 ml/kg, groups 3, 4 and 5 were given NAC at doses 100, 300 and 500 mg/kg, respectively, and the 6th group received ranitidine (50 mg/kg). All drugs administered orally once daily for 7 days, on the 8th day absolute ethanol (7 ml/kg) was administrated orally to all mice to induce the acute ulcer except normal control group. Then 3 h after, all animals were sacrificed then consequently the stomachs were excised for examination. Results: NAC administration at the tested doses showed a dose-related potent gastro-protective effect with significant increase in curative ratio, PH of gastric juice and mucus content viscosity seen with the highest dose of NAC and it is comparable with that observed in ranitidine group. Conclusion: The present findings demonstrate that, oral NAC shows significant gastro-protective effects comparable to ranitidine confirmed by anti-secretory, cytoprotective, histological and biochemical data, but the molecular mechanisms behind such protection are complex. PMID:26401392

N-acetylcysteine improves coronary and peripheral vascular function.

PubMed

Andrews, N P; Prasad, A; Quyyumi, A A

2001-01-01

We investigated whether N-acetylcysteine (NAC), a reduced thiol that modulates redox state and forms adducts of nitric oxide (NO), improves endothelium-dependent vasomotion. Coronary atherosclerosis is associated with endothelial dysfunction and reduced NO activity. In 16 patients undergoing cardiac catheterization, seven with and nine without atherosclerosis, we assessed endothelium-dependent vasodilation with acetylcholine (ACH) and endothelium-independent vasodilation with nitroglycerin (NTG) and sodium nitroprusside (SNP) before and after intracoronary NAC. In 14 patients femoral vascular responses to ACH, NTG and SNP were measured before and after NAC. Intraarterial NAC did not change resting coronary or peripheral vascular tone. N-acetylcysteine potentiated ACH-mediated coronary vasodilation; coronary blood flow was 36 +/- 11% higher (p < 0.02), and epicardial diameter changed from -1.2 +/- 2% constriction to 4.7 +/- 2% dilation after NAC (p = 0.03). Acetylcholine-mediated femoral vasodilation was similarly potentiated by NAC (p = 0.001). Augmentation of the ACH response was similar in patients with or without atherosclerosis. N-acetylcysteine did not affect NTG-mediated vasodilation in either the femoral or coronary circulations and did not alter SNP responses in the femoral circulation. In contrast, coronary vasodilation with SNP was significantly greater after NAC (p < 0.05). Thiol supplementation with NAC improves human coronary and peripheral endothelium-dependent vasodilation. Nitroglycerin responses are not enhanced, but SNP-mediated responses are potentiated only in the coronary circulation. These NO-enhancing effects of thiols reflect the importance of the redox state in the control of vascular function and may be of therapeutic benefit in treating acute and chronic manifestations of atherosclerosis.

Evolution of heliobacteria: implications for photosynthetic reaction center complexes

NASA Technical Reports Server (NTRS)

Vermaas, W. F.; Blankenship, R. E. (Principal Investigator)

1994-01-01

The evolutionary position of the heliobacteria, a group of green photosynthetic bacteria with a photosynthetic apparatus functionally resembling Photosystem I of plants and cyanobacteria, has been investigated with respect to the evolutionary relationship to Gram-positive bacteria and cyanobacteria. On the basis of 16S rRNA sequence analysis, the heliobacteria appear to be most closely related to Gram-positive bacteria, but also an evolutionary link to cyanobacteria is evident. Interestingly, a 46-residue domain including the putative sixth membrane-spanning region of the heliobacterial reaction center protein show rather strong similarity (33% identity and 72% similarity) to a region including the sixth membrane-spanning region of the CP47 protein, a chlorophyll-binding core antenna polypeptide of Photosystem II. The N-terminal half of the heliobacterial reaction center polypeptide shows a moderate sequence similarity (22% identity over 232 residues) with the CP47 protein, which is significantly more than the similarity with the Photosystem I core polypeptides in this region. An evolutionary model for photosynthetic reaction center complexes is discussed, in which an ancestral homodimeric reaction center protein (possibly resembling the heliobacterial reaction center protein) with 11 membrane-spanning regions per polypeptide has diverged to give rise to the core of Photosystem I, Photosystem II, and of the photosynthetic apparatus in green, purple, and heliobacteria.

Peptide Beacons: A New Design for Polypeptide-Based Optical Biosensors

PubMed Central

Oh, Kenneth J.; Cash, Kevin J.; Hugenberg, Verena; Plaxco, Kevin W.

2008-01-01

Phage display and other in vitro selection techniques produce short polypeptides that tightly and specifically bind to any of a wide range of macromolecular targets. Here we demonstrate a potentially general means of converting such polypeptides into optical biosensors. The sensing architecture we have developed, termed peptide beacons, is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed to their target. Using this effect to segregate a long-lived fluorophore from an electron transfer-based contact quencher, both covalently attached to the peptide, we have produced a robust optical sensor for anti-HIV antibodies. The binding-induced segregation of the fluorophore-quencher pair produces a six-fold increase in sensor emission, thus allowing us to readily detect as low as ∼250 pM of the target antibody. Because the sensor is based on binding-induced folding and a visible-light fluorophore, it is sufficiently selective to work directly in complex, contaminant-ridden samples such as saliva and blood. PMID:17461545

F44. AN ADD-ON TRIAL WITH N-ACETYL-CYSTEINE (NAC) IN EARLY PSYCHOSIS PATIENTS: TOWARDS BIOMARKER GUIDED TREATMENT

PubMed Central

Conus, Philippe; Fournier, Margot; Xin, Lijing; Cleusix, Martine; Baumann, Philipp S; Ferrari, Carina; Cousins, Ann; Alameda, Luis; Gholam-Razaee, Mehdi; Golay, Philippe; Jenni, Raoul; Woo, Tsung-Ung Wilson; Keshavan, Matcheri; Eap, Chin B; Wojcik, Joanne; Cuenod, Michel; Buclin, Thierry; Gruetter, Rolf; Seidman, Larry; Do, Kim

2018-01-01

Abstract Background Oxidative stress, coupled with dysregulation of inflammation, NMDAR and dopamine, is involved in schizophrenia (SZ) pathophysiology. Earlier add-on clinical trials showed in chronic SZ patients that NAC, a precursor of glutathione (GSH), an important cerebral antioxidant, improved negative symptoms, mismatch negativity and local synchronization. We hypothesized that NAC at an earlier stage of illness would have a greater impact. Methods Early psychosis patients (EP, less than 5 years of illness, N=63; NAC=32, placebo=31) were supplemented with NAC (2.7g/day, 6 months) in a double-blind randomized placebo-controlled trial. Outcome measures: PANSS and neurocognition (MATRICS Consensus Cognitive Battery; n=36); quantification of medial prefronfal cortex glutathione (GSHmPFC) by 1H-magnetic-resonance-spectroscopy, of white matter diffusion properties estimated by generalized fractional anisotropy (gFA) computed from diffusion spectrum imaging (DSI), of blood cells GSH (GSHBC) and GSH peroxidase activity (GPxBC) at start and end of trial Results While PANSS negative and positive were not affected by NAC, NAC improved Processing Speed (NAC > Placebo; F(1, 30)=5.849, p=.022), favoring 2 of 3 processing speed tasks (Trail Making A, F(1, 30)=4.279, p=.048 & Verbal Fluency, F(1, 30)=5.749, p=.023). GSHmPFC (+23%, p=0.005) and GSHBC (+19%, p=0.05) were increased following NAC treatment. In patients with high-baseline GPxBC (>22.3U/gHb), subgroup explorations revealed an improvement of PANSS positive compared to placebo (p=0.02). The change of PANSS positive correlated negatively with that of GPxBC activity, showing that the improvement paralleled the restoration of redox status. NAC group showed 11% increase in fornix white matter integrity as measured by gFA, correlating with an increase in GSHmPFC over the 6-months period. Discussion This is the first clinical trial assessing the impact of NAC treatment in a sample of EP and the potential predictive role of peripheral biomarkers of redox dysregulation. The hypothesis that NAC would be beneficial to negative symptoms in EP was not confirmed in this small sample, most likely in reason of their very low level at baseline. The NAC induced GSHmPFC increase demonstrates its target engagement. NAC improved Processing Speed showing a therapeutic enhancement of cognitive functions. Most importantly, NAC improved fornix integrity, in association with brain GSH elevation, demonstrating for the first time that a redox regulator can enhance structural connectivity. Peripheral redox status allows identifying a subgroup of patients with improved positive symptoms. Future biomarker guided antioxidant interventions in larger EP samples should replicate these findings.

Oxytocin receptors modulate a social salience neural network in male prairie voles.

PubMed

Johnson, Zachary V; Walum, Hasse; Xiao, Yao; Riefkohl, Paula C; Young, Larry J

2017-01-01

Social behavior is regulated by conserved neural networks across vertebrates. Variation in the organization of neuropeptide systems across these networks is thought to contribute to individual and species diversity in network function during social contexts. For example, oxytocin (OT) is an ancient neuropeptide that binds to OT receptors (OTRs) in the brain and modulates social and reproductive behavior across vertebrate species, including humans. Central OTRs exhibit extraordinarily diverse expression patterns that are associated with individual and species differences in social behavior. In voles, OTR density in the nucleus accumbens (NAc)-a region important for social and reward learning-is associated with individual and species variation in social attachment behavior. Here we test whether OTRs in the NAc modulate a social salience network (SSN)-a network of interconnected brain nuclei thought to encode valence and incentive salience of sociosensory cues-during a social context in the socially monogamous male prairie vole. Using a selective OTR antagonist, we test whether activation of OTRs in the NAc during sociosexual interaction and mating modulates expression of the immediate early gene product Fos across nuclei of the SSN. We show that blockade of endogenous OTR signaling in the NAc during sociosexual interaction and mating does not strongly modulate levels of Fos expression in individual nodes of the network, but strongly modulates patterns of correlated Fos expression between the NAc and other SSN nuclei. Published by Elsevier Inc.

Silencing of the potato StNAC103 gene enhances the accumulation of suberin polyester and associated wax in tuber skin

PubMed Central

Verdaguer, Roger; Soler, Marçal; Serra, Olga; Garrote, Aïda; Fernández, Sandra; Company-Arumí, Dolors; Anticó, Enriqueta; Molinas, Marisa; Figueras, Mercè

2016-01-01

Suberin and wax deposited in the cork (phellem) layer of the periderm form the lipophilic barrier that protects mature plant organs. Periderm lipids have been widely studied for their protective function with regards to dehydration and for how they respond to environmental stresses and wounding. However, despite advances in the biosynthetic pathways of suberin and associated wax, little is known about the regulation of their deposition. Here, we report on a potato NAC transcription factor gene, StNAC103, induced in the tuber phellem (skin). The StNAC103 promoter is active in cells undergoing suberization such as in the basal layer of the phellem, but also in the root apical meristem. Gene silencing in potato periderm correlates with an increase in the suberin and wax load, and specifically in alkanes, ω-hydroxyacids, diacids, ferulic acid, and primary alcohols. Concomitantly, silenced lines also showed up-regulation of key genes related to the biosynthesis and transport of suberin and wax in the tuber periderm. Taken together, our results suggest that StNAC103 has a role in the tight regulation of the formation of apoplastic barriers and is, to the best of our knowledge, the first candidate gene to be identified as being involved in the repression of suberin and wax deposition. PMID:27520790

Eotaxin, but not IL-8, is increased in upper and lower airways of allergic rhinitis subjects after nasal allergen challenge.

PubMed

Semik-Orzech, Aleksandra; Barczyk, Adam; Wiaderkiewicz, Ryszard; Pierzchała, Władysław

2011-01-01

The aim of this study was to assess the impact of a single nasal allergen challenge (NAC) on levels of eotaxin and IL-8 and the inflammatory cells in upper and lower airways of allergic rhinitis (AR) patients. Twenty-four AR patients and 12 control subjects entered a sequential nasal placebo challenge and NAC study, out of the pollen season. Nasal lavage fluid (NLF) was obtained at baseline, 15 minutes, and 1, 5, and 24 hours postchallenge. Before and 24 hours after placebo/allergen challenge induced sputum was performed. NLF and induced sputum were evaluated for total cell count (TCC) and differential cell count and analyzed for concentrations of eotaxin and IL-8 using ELISA method. NAC in AR subjects was associated with significantly increased sputum (p = 0.008) and NLF (p < 0.001) eotaxin levels. Post-NAC IL-8 levels were significantly increased in NLF (p < 00001) but not in sputum (p = 0.080) of AR subjects. Increased eotaxin levels in NLF positively correlated with the increased TCC and eosinophils. Positive correlations were also found between NLF increased eotaxin level and sputum TCC, eosinophils, and macrophages. NAC is associated with the increased levels of eotaxin in lower airways of AR subjects. Allergen-induced secretion of eotaxin in nasal mucosa of AR subjects is involved in determining the cellular character of both upper and lower airway inflammation.

Expression of N-acetyl-D-galactosamine associated epitope in synovium: a potential marker of glycoprotein production.

PubMed

El-Gabalawy, H; King, R; Bernstein, C; Ma, G; Mou, Y; Alguacil-Garcia, A; Fritzler, M; Wilkins, J

1997-07-01

To investigate synovial glycoprotein production in situ, a novel monoclonal antibody (Mab), A13D8, was used to evaluate the expression of an epitope containing N-acetyl-D-galactosamine (GalNAc) in normal and pathological synovium. Immunohistological and cytochemical analysis of synovial tissue samples was undertaken with single and double staining techniques using the A13D8 Mab, anti-CD68, vascular cell adhesion molecule-1 (VCAM-1), the hyaluronan associated enzyme uridine diphosphoglucose dehydrogenase (UDPGD), and the anti-Golgi Mab SSN/HR-1992. The specificity of the A13D8 Mab was established through blocking studies using carbohydrate residues, including GalNAc and N-acetylglucosamine (GlcNAc). A13D8 is expressed intensely in the cytoplasm of normal type B lining cells, which coexpress VCAM-1 and UDPGD, and is not expressed by CD68+ type A lining cells. In the lining layer of RA synovium, there is a negative correlation between A13D8 expression and the level of lymphocytic infiltration in the sublining areas (r = -0.43, p < 0.001). The endothelium of a subset of venules, typically in lymphocyte-rich aggregates, also stains intensely for A13D8. Pretreatment of the Mab with GalNAc completely eliminates the tissue staining, as well as the 110 kDa band seen on immunoblot, whereas pretreatment of A13D8 with GlcNAc and lactose has no effect. Double staining of HEp-2 cells with A13D8 and the anti-Golgi Mab SSN/HR-1992 reveals co-localization of the A13D8 epitope to the Golgi apparatus. Type B synovial lining cells and selected synovial endothelium express GalNAc containing epitope identified by Mab A13D8. Marked reduction in the expression of this epitope in the lining layer of inflamed RA synovium suggests that the synovial production of GalNAc containing glycoproteins, such as mucins, may be altered in this disorder.

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

PubMed

Serafini-Cessi, F; Dall'Olio, F; Pereira, L; Campadelli-Fiume, G

1984-09-01

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).

Characterization of an Extremely Basic Protein Derived from Granulosis Virus Nucleocapsids †

PubMed Central

Tweeten, Kathleen A.; Bulla, Lee A.; Consigli, Richard A.

1980-01-01

Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex. Images PMID:16789190

Developmental Loss of Photosystem II Activity and Structure in a Chloroplast-Encoded Tobacco Mutant, Lutescens-11

PubMed Central

Chia, Catherine P.; Duesing, John H.; Arntzen, Charles J.

1986-01-01

Lutescens-1, a tobacco mutant with a maternally inherited dysfunction, displayed an unusual developmental phenotype. In vivo measurement of chlorophyll fluorescence revealed deterioration in photosystem II (PSII) function as leaves expanded. Analysis of thylakoid membrane proteins by polyacrylamide gel electrophoresis indicated the physical loss of nuclear- and chloroplast-encoded polypeptides comprising the PSII core complex concomitant with loss of activity. Freeze fracture electron micrographs of mutant thylakoids showed a reduced density, compared to wild type, of the EFs particles which have been shown previously to be the structural entity containing PSII core complexes and associated pigment-proteins. The selective loss of PSII cores from thylakoids resulted in a higher ratio of antenna chlorophyll to reaction centers and an altered 77 K chlorophyll fluorescence emission spectra; these data are interpreted to indicate functional isolation of light-harvesting chlorophyll a/b complexes in the absence of PSII centers. Examination of PSII reaction centers (which were present at lower levels in mutant membranes) by monitoring the light-dependent phosphorylation of PSII polypeptides and flash-induced O2 evolution patterns demonstrated that the PSII cores which were assembled in mutant thylakoids were functionally identical to those of wild type. We conclude that the lutescens-1 mutation affected the correct stoichiometry of PSII centers, in relation to other membrane constituents, by disrupting the proper assembly and maintenance of PSII complexes in lutescens-1 thylakoid membranes. Images Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16664990

Identification of a new membrane-associated polypeptide specified by the coronavirus infectious bronchitis virus.

PubMed

Smith, A R; Boursnell, M E; Binns, M M; Brown, T D; Inglis, S C

1990-01-01

Nucleotide sequences from the third open reading frame of mRNA D (D3) of infectious bronchitis virus (IBV) were expressed in bacteria as part of a fusion protein with beta-galactosidase. Antiserum raised in rabbits against this fusion protein immunoprecipitated from IBV-infected chick kidney or Vero cells a polypeptide of 12.4K, the size expected for a D3-encoded product. The D3 polypeptide is apparently non-glycosylated, and appears to be associated with the membrane fraction of infected cells, as judged by cell fractionation and immunofluorescence.

Tunable drug loading and release from polypeptide multilayer nanofilms

PubMed Central

Jiang, Bingbing; Li, Bingyun

2009-01-01

Polypeptide multilayer nanofilms were prepared using electrostatic layer-by-layer self-assembly nanotechnology. Small charged drug molecules (eg, cefazolin, gentamicin, and methylene blue) were loaded in polypeptide multilayer nanofilms. Their loading and release were found to be pH-dependent and could also be controlled by changing the number of film layers and drug incubation time, and applying heat-treatment after film formation. Antibioticloaded polypeptide multilayer nanofilms showed controllable antibacterial properties against Staphylococcus aureus. The developed biodegradable polypeptide multilayer nanofilms are capable of loading both positively- and negatively-charged drug molecules and promise to serve as drug delivery systems on biomedical devices for preventing biomedical device-associated infection, which is a significant clinical complication for both civilian and military patients. PMID:19421369

Thio-Linked UDP–Peptide Conjugates as O-GlcNAc Transferase Inhibitors

PubMed Central

2018-01-01

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay. PMID:29723473

Cocaine self-administration abolishes associative neural encoding in the nucleus accumbens necessary for higher-order learning.

PubMed

Saddoris, Michael P; Carelli, Regina M

2014-01-15

Cocaine use is often associated with diminished cognitive function, persisting even after abstinence from the drug. Likely targets for these changes are the core and shell of the nucleus accumbens (NAc), which are critical for mediating the rewarding aspects of drugs of abuse as well as supporting associative learning. To understand this deficit, we recorded neural activity in the NAc of rats with a history of cocaine self-administration or control subjects while they learned Pavlovian first- and second-order associations. Rats were trained for 2 weeks to self-administer intravenous cocaine or water. Later, rats learned a first-order Pavlovian discrimination where a conditioned stimulus (CS)+ predicted food, and a control (CS-) did not. Rats then learned a second-order association where, absent any food reinforcement, a novel cued (SOC+) predicted the CS+ and another (SOC-) predicted the CS-. Electrophysiological recordings were taken during performance of these tasks in the NAc core and shell. Both control subjects and cocaine-experienced rats learned the first-order association, but only control subjects learned the second-order association. Neural recordings indicated that core and shell neurons encoded task-relevant information that correlated with behavioral performance, whereas this type of encoding was abolished in cocaine-experienced rats. The NAc core and shell perform complementary roles in supporting normal associative learning, functions that are impaired after cocaine experience. This impoverished encoding of motivational behavior, even after abstinence from the drug, might provide a key mechanism to understand why addiction remains a chronically relapsing disorder despite repeated attempts at sobriety. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.

PubMed

Moore, S E; Spiro, R G

1992-04-25

Studies on N-linked oligosaccharide processing in the mouse lymphoma glucosidase II-deficient mutant cell line (PHAR2.7) as well as the parent BW5147 cells indicated that the former maintain their capacity to synthesize complex carbohydrate units through the use of the deglucosylation mechanism provided by endomannosidase. The in vivo activity of this enzyme was evident in the mutant cells from their production of substantial amounts of glucosylated mannose saccharides, predominantly Glc2Man; moreover, in the presence of 1-deoxymannojirimycin or kifunensine to prevent processing by mannosidase I, N-linked Man8GlcNAc2 was observed entirely in the form of the characteristic isomer in which the terminal mannose of the alpha 1,3-linked branch is missing (isomer A). In contrast, parent lymphoma cells, as well as HepG2 cells in the presence of 1-deoxymannojirimycin accumulated Man9GlcNAc2 as the primary deglucosylated N-linked oligosaccharide and contained only about 16% of their Man8GlcNAc2 as isomer A. In the presence of the glucosidase inhibitor castanospermine the mutant released Glc3Man instead of Glc2Man, and the parent cells converted their deglucosylation machinery to the endomannosidase route. Despite the mutant's capacity to accommodate a large traffic through this pathway no increase in the in vitro determined endomannosidase activity was evident. The exclusive utilization of endomannosidase by the mutant for the deglucosylation of its predominant N-linked Glc2Man9GlcNAc2 permitted an exploration of the in vivo site of this enzyme's action. Pulse-chase studies utilizing sucrose-D2O density gradient centrifugation indicated that the Glc2Man9GlcNAc2 to Man8GlcNAc2 conversion is a relatively late event that is temporally separated from the endoplasmic reticulum-situated processing of Glc3Man9GlcNAc2 to Glc2Man9GlcNAc2 and in contrast to the latter takes place in the Golgi compartment.

N-acetylcysteine and vitamin E rescue animal longevity and cellular oxidative stress in pre-clinical models of mitochondrial complex I disease.

PubMed

Polyak, Erzsebet; Ostrovsky, Julian; Peng, Min; Dingley, Stephen D; Tsukikawa, Mai; Kwon, Young Joon; McCormack, Shana E; Bennett, Michael; Xiao, Rui; Seiler, Christoph; Zhang, Zhe; Falk, Marni J

2018-04-01

Oxidative stress is a known contributing factor in mitochondrial respiratory chain (RC) disease pathogenesis. Yet, no efficient means exists to objectively evaluate the comparative therapeutic efficacy or toxicity of different antioxidant compounds empirically used in human RC disease. We postulated that pre-clinical comparative analysis of diverse antioxidant drugs having suggested utility in primary RC disease using animal and cellular models of RC dysfunction may improve understanding of their integrated effects and physiologic mechanisms, and enable prioritization of lead antioxidant molecules to pursue in human clinical trials. Here, lifespan effects of N-acetylcysteine (NAC), vitamin E, vitamin C, coenzyme Q10 (CoQ10), mitochondrial-targeted CoQ10 (MS010), lipoate, and orotate were evaluated as the primary outcome in a well-established, short-lived C. elegans gas-1(fc21) animal model of RC complex I disease. Healthspan effects were interrogated to assess potential reversal of their globally disrupted in vivo mitochondrial physiology, transcriptome profiles, and intermediary metabolic flux. NAC or vitamin E fully rescued, and coenzyme Q, lipoic acid, orotic acid, and vitamin C partially rescued gas-1(fc21) lifespan toward that of wild-type N2 Bristol worms. MS010 and CoQ10 largely reversed biochemical pathway expression changes in gas-1(fc21) worms. While nearly all drugs normalized the upregulated expression of the "cellular antioxidant pathway", they failed to rescue the mutant worms' increased in vivo mitochondrial oxidant burden. NAC and vitamin E therapeutic efficacy were validated in human fibroblast and/or zebrafish complex I disease models. Remarkably, rotenone-induced zebrafish brain death was preventable partially with NAC and fully with vitamin E. Overall, these pre-clinical model animal data demonstrate that several classical antioxidant drugs do yield significant benefit on viability and survival in primary mitochondrial disease, where their major therapeutic benefit appears to result from targeting global cellular, rather than intramitochondria-specific, oxidative stress. Clinical trials are needed to evaluate whether the two antioxidants, NAC and vitamin E, that show greatest efficacy in translational model animals significantly improve the survival, function, and feeling of human subjects with primary mitochondrial RC disease. Copyright © 2018 Elsevier Inc. All rights reserved.

Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.

PubMed

Hosokawa, Nobuko; Tremblay, Linda O; You, Zhipeng; Herscovics, Annette; Wada, Ikuo; Nagata, Kazuhiro

2003-07-11

Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant-null Hong Kong alpha1-antitrypsin is enhanced by overexpression of the ER processing alpha1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded alpha1-antitrypsin and accelerates its degradation (Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415-422). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded alpha1-antitrypsin. We also show that misfolded alpha1-antitrypsin NHK contains labeled Glc1Man9GlcNAc and Man5-9GlcNAc released by endo-beta-N-acetylglucosaminidase H in pulse-chase experiments with [2-3H]mannose. Overexpression of ER ManI greatly increases the formation of Man8GlcNAc, induces the formation of Glc1Man8GlcNAc and increases trimming to Man5-7GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man8GlcNAc2 isomer B as previous studies have suggested.

Evidence for an asialoglycoprotein receptor on nonparenchymal cells for O-linked glycoproteins.

PubMed

Stefanich, Eric G; Ren, Song; Danilenko, Dimitry M; Lim, Amy; Song, An; Iyer, Suhasini; Fielder, Paul J

2008-11-01

B cell-activating factor receptor 3 (BR3)-Fc is an IgG1-receptor dimeric fusion protein that has multiple O-linked glycosylation sites and sialylation levels that can vary in the manufacturing process. Increased sialic acid levels resulted from increased site occupancy with the O-linked N-acetylgalactosamine (GalNAc-Gal), but because the ratio of sialic acid per mole of oligosaccharide remained approximately 1, this led to increased asialo terminal GalNAc. Previous studies have demonstrated an effect of terminal asialo Gal or GalNAc on the clearance of glycoproteins due to uptake and degradation by lectin receptors in the liver. However, the previous studies examined N-linked oligosaccharides, and there are less data regarding O-linked oligosaccharides. The objective of these studies was to determine the effects on the pharmacokinetics and distribution of the asialo terminal GalNAc and varying amounts of sialic acid residues on BR3-Fc. The results of the data presented here suggest that exposed Gal on the desialylated BR3-Fc led to rapid clearance due to uptake and degradation in the liver that was associated with nonparenchymal cells. It is interesting to note that the data indicated a decreased clearance and increased exposure of BR3-Fc as the sialic acid levels increased, even though increased sialic acid was associated with increased asialo GalNAc. Therefore, the exposed GalNAc did not seem to play a role in the clearance of BR3-Fc; although the Gal linked to the hydroxyl group at position 3 may have prevented an interaction. Because we did not see uptake of desialylated BR3-Fc in hepatocytes where the asialoglycoprotein receptor is localized, this nonparenchymal cell lectin may have preference for O-linked glycoproteins.

Synthesis of aryl azide derivatives of UDP-GlcNAc and UDP-GalNAc and their use for the affinity labeling of glycosyltransferases and the UDP-HexNAc pyrophosphorylase.

PubMed

Zeng, Y; Shabalin, Y; Szumilo, T; Pastuszak, I; Drake, R R; Elbein, A D

1996-07-15

The chemical synthesis and utilization of two photoaffinity analogs, 125I-labeled 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc and -UDP-GalNAc, is described. Starting with either UDP-GlcNAc or UDP-GalNAc, the synthesis involved the preparation of the 5-mercuri-UDP-HexNAc and then attachment of an allylamine to the 5 position to give 5-(3-amino)allyl-UDP-HexNAc. This was followed by acylation with N-hydroxysuccinimide p-aminosalicylic acid to form the final product, i.e., 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc or UDP-GalNAc. These products could then be iodinated with chloramine T to give the 125I-derivatives. Both the UDP-GlcNAc and the UDP-GalNAc derivatives reacted in a concentration-dependent manner with a highly purified UDP-HexNAc pyrophosphorylase, and both specifically labeled the subunit(s) of this protein. The labeling of the protein by the UDP-GlcNAc derivative was inhibited in dose-dependent fashion by either unlabeled UDP-GlcNAc or unlabeled UDP-GalNAc. Likewise, labeling with the UDP-GalNAc probe was blocked by either UDP-GlcNAc or UDP-GalNAc. The UDP-GlcNAc probe also specifically labeled a partially purified preparation of GlcNAc transferase I.

Escherichia coli cellular responses to exposure to atmospheric-pressure dielectric barrier discharge plasma-treated N-acetylcysteine solution.

PubMed

Ercan, U K; Sen, B; Brooks, A D; Joshi, S G

2018-04-06

To understand the underlying cellular mechanisms during inactivation of Escherichia coli in response to antimicrobial solution of nonthermal plasma-activated N-acetylcysteine (NAC). The recommended techniques were used to demonstrate E. coli cellular and transcriptomic changes caused associated with peroxynitrite and compared with plasma-treated NAC solution. The findings demonstrate that E. coli cells respond to plasma-treated NAC and undergo severe oxidative and nitrosative stress, and leading to stress-induced damages to different components of bacterial cells, which includes loss of membrane potential, formation of oxidized glutathione (GSSG), formation of nitrotyrosine (a known marker of nitrosative stress), DNA damage, and generated a prominent pool of peroxynitrite. Reverse-transcriptase (RT)-polymerase chain reaction analysis of reactive nitrogen species (RNS) responsive genes indicated their differential expressions. For the first time, we report that the plasma-treated NAC solution activates predominantly nitrosative stress-responsive genes in E. coli and is responsible for cell death. The reactive species generated in solutions by nonthermal plasma treatment depends on the type of solution or solvent used. The plasma-treated NAC solution rapidly inactivates E. coli, mostly involving highly RNS generated in NAC solution, and has high potential as disinfectant. © 2018 The Society for Applied Microbiology.

Monitored Natural Attenuation and Enhanced Attenuation for Chlorinated Solvent Plumes - It’s All About Balance

DOE Office of Scientific and Technical Information (OSTI.GOV)

VANGELAS, KAREN

2005-05-19

Nature's inherent ability to cleanse itself is at the heart of Monitored Natural Attenuation (MNA). The complexity comes when one attempts to measure and calculate this inherent ability, called the Natural Attenuation Capacity (NAC), and determine if it is sufficient to cleanse the system to agreed upon criteria. An approach that is simple in concept for determining whether the NAC is sufficient for MNA to work is the concept of a mass balance. Mass balance is a robust framework upon which all decisions can be made. The inflows to and outflows from the system are balanced against the NAC ofmore » the subsurface system. For MNA to be acceptable, the NAC is balanced against the contaminant loading to the subsurface system with the resulting outflow from the system being in a range that is acceptable to the regulating and decision-making parties. When the system is such that the resulting outflow is not within an acceptable range, the idea of taking actions that are sustainable and that will bring the system within the acceptable range of outflows is evaluated. These sustainable enhancements are being developed under the Enhanced Attenuation (EA) concept.« less

Monitored natural attenuation and enhanced attenuation for chlorinated solvent plumes - It's all about balance

USGS Publications Warehouse

Adams, K.A.; Vangelas, K.M.; Looney, B.B.; Chapelle, F.; Early, T.; Gilmore, T.; Sink, C.H.

2005-01-01

Nature's inherent ability to cleanse itself is at the heart of Monitored Natural Attenuation (MNA). The complexity comes when one attempts to measure and calculate this inherent ability, called the Natural Attenuation Capacity (NAC), and determine if it is sufficient to cleanse the system to agreed upon criteria. An approach that is simple in concept for determining whether the NAC is sufficient for MNA to work is the concept of a mass balance. Mass balance is a robust framework upon which all decisions can be made. The inflows to and outflows from the system are balanced against the NAC of the subsurface system. For MNA to be acceptable, the NAC is balanced against the contaminant loading to the subsurface system with the resulting outflow from the system being in a range that is acceptable to the regulating and decision-making parties. When the system is such that the resulting outflow is not within an acceptable range, the idea of taking actions that are sustainable and that will bring the system within the acceptable range of outflows is evaluated. These sustainable enhancements are being developed under the Enhanced Attenuation (EA) concept. Copyright ASCE 2005.

Pancreatic polypeptide and calcitonin secretion from a pancreatic tumour-clinical improvement after hepatic artery embolization.

PubMed Central

Manche, A.; Wood, S. M.; Adrian, T. E.; Welbourn, R. B.; Bloom, S. R.

1983-01-01

We present a case in which plasma pancreatic polypeptide and calcitonin were found to be raised in association with an islet cell tumour of the pancreas and its hepatic metastases. In this patient, no specific endocrine syndrome was found. Therapeutic hepatic artery embolization improved the general health of the patient with no change in plasma pancreatic polypeptide, but a fall in calcitonin. PMID:6308585

Pancreatic polypeptide and calcitonin secretion from a pancreatic tumour-clinical improvement after hepatic artery embolization.

PubMed

Manche, A; Wood, S M; Adrian, T E; Welbourn, R B; Bloom, S R

1983-05-01

We present a case in which plasma pancreatic polypeptide and calcitonin were found to be raised in association with an islet cell tumour of the pancreas and its hepatic metastases. In this patient, no specific endocrine syndrome was found. Therapeutic hepatic artery embolization improved the general health of the patient with no change in plasma pancreatic polypeptide, but a fall in calcitonin.

Chitin-induced activation of immune signaling by the rice receptor CEBiP relies on a unique sandwich-type dimerization

PubMed Central

Hayafune, Masahiro; Berisio, Rita; Marchetti, Roberta; Silipo, Alba; Kayama, Miyu; Desaki, Yoshitake; Arima, Sakiko; Squeglia, Flavia; Ruggiero, Alessia; Tokuyasu, Ken; Molinaro, Antonio; Kaku, Hanae; Shibuya, Naoto

2014-01-01

Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile122 in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc)8 induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc)8-induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcNβ1,4GlcNAc)4, which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitin-elicitor receptor kinase-1. PMID:24395781

Environmental and Biofilm-dependent Changes in a Bacillus cereus Secondary Cell Wall Polysaccharide*

PubMed Central

Candela, Thomas; Maes, Emmanuel; Garénaux, Estelle; Rombouts, Yoann; Krzewinski, Frédéric; Gohar, Michel; Guérardel, Yann

2011-01-01

Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(β1–3)]GlcNAc(β1–6)[Glc(β1-3)][ManNAc(α1–4)]GalNAc(α1–4)ManNAc(β1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1–2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(β1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm. PMID:21784857

Regulation of neuraminidase expression in Streptococcus pneumoniae

PubMed Central

2012-01-01

Background Sialic acid (N-acetylneuraminic acid; NeuNAc) is one of the most important carbohydrates for Streptococcus pneumoniae due of its role as a carbon and energy source, receptor for adhesion and invasion and molecular signal for promotion of biofilm formation, nasopharyngeal carriage and invasion of the lung. Results In this work, NeuNAc and its metabolic derivative N-acetyl mannosamine (ManNAc) were used to analyze regulatory mechanisms of the neuraminidase locus expression. Genomic and metabolic comparison to Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii and Streptococcus sanguinis elucidates the metabolic association of the two amino sugars to different parts of the locus coding for the two main pneumococcal neuraminidases and confirms the substrate specificity of the respective ABC transporters. Quantitative gene expression analysis shows repression of the locus by glucose and induction of all predicted transcriptional units by ManNAc and NeuNAc, each inducing with higher efficiency the operon encoding for the transporter with higher specificity for the respective amino sugar. Cytofluorimetric analysis demonstrated enhanced surface exposure of NanA on pneumococci grown in NeuNAc and ManNAc and an activity assay allowed to quantify approximately twelve times as much neuraminidase activity on induced cells as opposed to glucose grown cells. Conclusions The present data increase the understanding of metabolic regulation of the nanAB locus and indicate that experiments aimed at the elucidation of the relevance of neuraminidases in pneumococcal virulence should possibly not be carried out on bacteria grown in glucose containing media. PMID:22963456

Effects of N-acetylcysteine on semen parameters and oxidative/antioxidant status.

PubMed

Ciftci, Halil; Verit, Ayhan; Savas, Murat; Yeni, Ercan; Erel, Ozcan

2009-07-01

To examine whether a beneficial effect of N-acetylcysteine (NAC) on semen parameters and oxidative/antioxidant status in idiopathic male infertility exists. The production of reactive oxygen species is a normal physiologic event in various organs. However, overproduction of reactive oxygen species can be detrimental to sperm and has been associated with male infertility. Our study included 120 patients who had attended our clinic and were diagnosed with idiopathic infertility according to medical history and physical and seminal examination findings, as initial evaluations. The patients were divided randomly into 2 groups. Those in the study group (60 men) were given NAC (600 mg/d orally) for 3 months; the control group (60 men) received a placebo. The oxidative status was determined by measuring the total antioxidant capacity, total peroxide and oxidative stress index in plasma samples. The sperm parameters were evaluated after NAC treatment and were compared with those in the control group. NAC had significant improving effects on the volume, motility, and viscosity of semen. After NAC treatment, the serum total antioxidant capacity was greater and the total peroxide and oxidative stress index were lower in the NAC-treated group compared with the control group. These beneficial effects resulted from reduced reactive oxygen species in the serum and reduced viscosity of the semen. No significant differences were found in the number or morphology of the sperm between the 2 groups. We believe that NAC could improve some semen parameters and the oxidative/antioxidant status in patients with male infertility.

The Combination of N-Acetyl Cysteine, Alpha-Lipoic Acid, and Bromelain Shows High Anti-Inflammatory Properties in Novel In Vivo and In Vitro Models of Endometriosis

PubMed Central

Agostinis, C.; Zorzet, S.; De Leo, R.; Zauli, G.; De Seta, F.; Bulla, R.

2015-01-01

To evaluate the efficacy of an association of N-acetyl cystein, alpha-lipoic acid, and bromelain (NAC/LA/Br) in the treatment of endometriosis we set up a new in vivo murine model. We explored the anti-inflammatory and proapoptotic effect of this combination on human endometriotic endothelial cells (EECs) and on endothelial cells isolated from normal uterus (UtMECs). We implanted fragments of human endometriotic cysts intraperitoneally into SCID mice to evaluate the efficacy of NAC/LA/Br treatment. UtMECs and EECs, untreated or treated with NAC/LA/Br, were activated with the proinflammatory stimulus TNF-α and their response in terms of VCAM1 expression was evaluated. The proapoptotic effect of higher doses of NAC/LA/Br on UtMECs and EECs was measured with a fluorogenic substrate for activated caspases 3 and 7. The preincubation of EECs with NAC/LA/Br prior to cell stimulation with TNF-α prevents the upregulation of the expression of the inflammatory “marker” VCAM1. Furthermore NAC/LA/Br were able to induce EEC, but not UtMEC, apoptosis. Finally, the novel mouse model allowed us to demonstrate that mice treated with NAC/LA/Br presented a lower number of cysts, smaller in size, compared to untreated mice. Our findings suggest that these dietary supplements may have potential therapeutic uses in the treatment of chronic inflammatory diseases like endometriosis. PMID:25960622

The combination of N-acetyl cysteine, alpha-lipoic acid, and bromelain shows high anti-inflammatory properties in novel in vivo and in vitro models of endometriosis.

PubMed

Agostinis, C; Zorzet, S; De Leo, R; Zauli, G; De Seta, F; Bulla, R

2015-01-01

To evaluate the efficacy of an association of N-acetyl cystein, alpha-lipoic acid, and bromelain (NAC/LA/Br) in the treatment of endometriosis we set up a new in vivo murine model. We explored the anti-inflammatory and proapoptotic effect of this combination on human endometriotic endothelial cells (EECs) and on endothelial cells isolated from normal uterus (UtMECs). We implanted fragments of human endometriotic cysts intraperitoneally into SCID mice to evaluate the efficacy of NAC/LA/Br treatment. UtMECs and EECs, untreated or treated with NAC/LA/Br, were activated with the proinflammatory stimulus TNF-α and their response in terms of VCAM1 expression was evaluated. The proapoptotic effect of higher doses of NAC/LA/Br on UtMECs and EECs was measured with a fluorogenic substrate for activated caspases 3 and 7. The preincubation of EECs with NAC/LA/Br prior to cell stimulation with TNF-α prevents the upregulation of the expression of the inflammatory "marker" VCAM1. Furthermore NAC/LA/Br were able to induce EEC, but not UtMEC, apoptosis. Finally, the novel mouse model allowed us to demonstrate that mice treated with NAC/LA/Br presented a lower number of cysts, smaller in size, compared to untreated mice. Our findings suggest that these dietary supplements may have potential therapeutic uses in the treatment of chronic inflammatory diseases like endometriosis.

Clinical trials of N-acetylcysteine in psychiatry and neurology: A systematic review.

PubMed

Deepmala; Slattery, John; Kumar, Nihit; Delhey, Leanna; Berk, Michael; Dean, Olivia; Spielholz, Charles; Frye, Richard

2015-08-01

N-acetylcysteine (NAC) is recognized for its role in acetaminophen overdose and as a mucolytic. Over the past decade, there has been growing evidence for the use of NAC in treating psychiatric and neurological disorders, considering its role in attenuating pathophysiological processes associated with these disorders, including oxidative stress, apoptosis, mitochondrial dysfunction, neuroinflammation and glutamate and dopamine dysregulation. In this systematic review we find favorable evidence for the use of NAC in several psychiatric and neurological disorders, particularly autism, Alzheimer's disease, cocaine and cannabis addiction, bipolar disorder, depression, trichotillomania, nail biting, skin picking, obsessive-compulsive disorder, schizophrenia, drug-induced neuropathy and progressive myoclonic epilepsy. Disorders such as anxiety, attention deficit hyperactivity disorder and mild traumatic brain injury have preliminary evidence and require larger confirmatory studies while current evidence does not support the use of NAC in gambling, methamphetamine and nicotine addictions and amyotrophic lateral sclerosis. Overall, NAC treatment appears to be safe and tolerable. Further well designed, larger controlled trials are needed for specific psychiatric and neurological disorders where the evidence is favorable. Copyright © 2015 Elsevier Ltd. All rights reserved.

The National Astronomy Consortium - An Adaptable Model for OAD?

NASA Astrophysics Data System (ADS)

Sheth, Kartik

2015-08-01

The National Astronomy Consortium (NAC) is a program led by the National Radio Astronomy Observatory (NRAO) and Associated Universities Inc., (AUI) in partnership with the National Society of Black Physicists (NSBP), and a number of minority and majority universities to increase the numbers of students from underrepresented groups and those otherwise overlooked by the traditional academic pipeline into STEM or STEM-related careers. The seed for the NAC was a partnership between NRAO and Howard University which began with an exchange of a few summer students five years ago. Since then the NAC has grown tremendously. Today the NAC aims to host between 4 to 5 cohorts nationally in an innovative model in which the students are mentored throughout the year with multiple mentors and peer mentoring, continued engagement in research and professional development / career training throughout the academic year and throughout their careers.The NAC model has already shown success and is a very promising and innovative model for increasing participation of young people in STEM and STEM-related careers. I will discuss how this model could be adapted in various countries at all levels of education.

Nucleus Accumbens Deep Brain Stimulation in Patients with Substance Use Disorders and Delay Discounting.

PubMed

Peisker, Canan B; Schüller, Thomas; Peters, Jan; Wagner, Ben J; Schilbach, Leonhard; Müller, Ulf J; Visser-Vandewalle, Veerle; Kuhn, Jens

2018-01-27

Deep brain stimulation (DBS) of the nucleus accumbens (NAc) shows first promising results in patients with severe substance use disorder (SUD), a patient group known to have deficits in self-control. One facet of self-control is the ability to forego smaller sooner rewards in favor of larger later rewards (delay discounting, DD). The NAc has been suggested to integrate motivational information to guide behavior while the consequences of NAc-DBS on DD are unknown. To this end, nine patients with SUD performed a DD task with DBS on and after a 24 h DBS off period. Furthermore, 18 healthy controls were measured to assess possible alterations in DD in patients with SUD. Our findings implicate that DD was not significantly modulated by NAc-DBS and also that patients with SUD did not differ from healthy controls. While null results must be interpreted with caution, the commonly observed association of impaired DD in SUD might suggest a long-term effect of NAc-DBS that was not sufficiently modulated by a 24 h DBS off period.

Crystal Structure of Human [Beta]-Hexosaminidase B: Understanding the Molecular Basis of Sandhoff and Tay-Sachs Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mark, Brian L.; Mahuran, Don J.; Cherney, Maia M.

2010-12-01

In humans, two major {beta}-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits {alpha} and {beta} (60% identity), whereas Hex B is a homodimer of {beta}-subunits. Interest in human {beta}-hexosaminidase stems from its association with Tay-Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of G{sub M2}-ganglioside (G{sub M2}). Hex A degrades G{sub M2} by removing a terminal N-acetyl-D-galactosamine ({beta}-GalNAc) residue, and this activity requires the G{sub M2}-activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hexmore » B, alone (2.4 {angstrom}) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2 {angstrom}) or NAG-thiazoline (2.5 {angstrom}). From these, and the known X-ray structure of the G{sub M2}-activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how {alpha} and {beta}-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease ({beta}-subunit mutations) and Tay-Sachs disease ({alpha}-subunit mutations).« less

Transcriptomic analysis suggests a key role for SQUAMOSA PROMOTER BINDING PROTEIN LIKE, NAC and YUCCA genes in the heteroblastic development of the temperate rainforest tree Gevuina avellana (Proteaceae).

PubMed

Ostria-Gallardo, Enrique; Ranjan, Aashish; Chitwood, Daniel H; Kumar, Ravi; Townsley, Brad T; Ichihashi, Yasunori; Corcuera, Luis J; Sinha, Neelima R

2016-04-01

Heteroblasty, the temporal development of the meristem, can produce diverse leaf shapes within a plant. Gevuina avellana, a tree from the South American temperate rainforest shows strong heteroblasty affecting leaf shape, transitioning from juvenile simple leaves to highly pinnate adult leaves. Light availability within the forest canopy also modulates its leaf size and complexity. Here we studied how the interaction between the light environment and the heteroblastic progression of leaves is coordinated in this species. We used RNA-seq on the Illumina platform to compare the range of transcriptional responses in leaf primordia of G. avellana at different heteroblastic stages and growing under different light environments. We found a steady up-regulation of SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL), NAC, YUCCA and AGAMOUS-LIKE genes associated with increases in age, leaf complexity, and light availability. In contrast, expression of TCP, TPR and KNOTTED1 homeobox genes showed a sustained down-regulation. Additionally, genes involved in auxin synthesis/transport and jasmonate activity were differentially expressed, indicating an active regulation of processes controlled by these hormones. Our large-scale transcriptional analysis of the leaf primordia of G. avellana sheds light on the integration of internal and external cues during heteroblastic development in this species. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

PubMed

Endo, T; Ohbayashi, H; Kanazawa, K; Kochibe, N; Kobata, A

1992-01-15

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

PubMed

Wu, A M; Lin, S R; Chin, L K; Chow, L P; Lin, J Y

1992-09-25

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.

Methods for the Detection, Study, and Dynamic Profiling of O-GlcNAc Glycosylation.

PubMed

Thompson, John W; Griffin, Matthew E; Hsieh-Wilson, Linda C

2018-01-01

The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition "click" reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)-(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation. © 2018 Elsevier Inc. All rights reserved.

ETHQV6.3 is involved in melon climacteric fruit ripening and is encoded by a NAC domain transcription factor.

PubMed

Ríos, Pablo; Argyris, Jason; Vegas, Juan; Leida, Carmen; Kenigswald, Merav; Tzuri, Galil; Troadec, Christelle; Bendahmane, Abdelhafid; Katzir, Nurit; Picó, Belén; Monforte, Antonio J; Garcia-Mas, Jordi

2017-08-01

Fruit ripening is divided into climacteric and non-climacteric types depending on the presence or absence of a transient rise in respiration rate and the production of autocatalytic ethylene. Melon is ideal for the study of fruit ripening, as both climacteric and non-climacteric varieties exist. Two introgressions of the non-climacteric accession PI 161375, encompassed in the QTLs ETHQB3.5 and ETHQV6.3, into the non-climacteric 'Piel de Sapo' background are able to induce climacteric ripening independently. We report that the gene underlying ETHQV6.3 is MELO3C016540 (CmNAC-NOR), encoding a NAC (NAM, ATAF1,2, CUC2) transcription factor that is closely related to the tomato NOR (non-ripening) gene. CmNAC-NOR was functionally validated through the identification of two TILLING lines carrying non-synonymous mutations in the conserved NAC domain region. In an otherwise highly climacteric genetic background, both mutations provoked a significant delay in the onset of fruit ripening and in the biosynthesis of ethylene. The PI 161375 allele of ETHQV6.3 is similar to that of climacteric lines of the cantalupensis type and, when introgressed into the non-climacteric 'Piel de Sapo', partially restores its climacteric ripening capacity. CmNAC-NOR is expressed in fruit flesh of both climacteric and non-climacteric lines, suggesting that the causal mutation may not be acting at the transcriptional level. The use of a comparative genetic approach in a species with both climacteric and non-climacteric ripening is a powerful strategy to dissect the complex mechanisms regulating the onset of fruit ripening. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Eating 'Junk-Food' Produces Rapid and Long-Lasting Increases in NAc CP-AMPA Receptors: Implications for Enhanced Cue-Induced Motivation and Food Addiction.

PubMed

Oginsky, Max F; Goforth, Paulette B; Nobile, Cameron W; Lopez-Santiago, Luis F; Ferrario, Carrie R

2016-12-01

Urges to eat are influenced by stimuli in the environment that are associated with food (food cues). Obese people are more sensitive to food cues, reporting stronger craving and consuming larger portions after food cue exposure. The nucleus accumbens (NAc) mediates cue-triggered motivational responses, and activations in the NAc triggered by food cues are stronger in people who are susceptible to obesity. This has led to the idea that alterations in NAc function similar to those underlying drug addiction may contribute to obesity, particularly in obesity-susceptible individuals. Motivational responses are mediated in part by NAc AMPA receptor (AMPAR) transmission, and recent work shows that cue-triggered motivation is enhanced in obesity-susceptible rats after 'junk-food' diet consumption. Therefore, here we determined whether NAc AMPAR expression and function is increased by 'junk-food' diet consumption in obesity-susceptible vs -resistant populations using both outbred and selectively bred models of susceptibility. In addition, cocaine-induced locomotor activity was used as a general 'read out' of mesolimbic function after 'junk-food' consumption. We found a sensitized locomotor response to cocaine in rats that gained weight on a 'junk-food' diet, consistent with greater responsivity of mesolimbic circuits in obesity-susceptible groups. In addition, eating 'junk-food' increased NAc calcium-permeable-AMPAR (CP-AMPAR) function only in obesity-susceptible rats. This increase occurred rapidly, persisted for weeks after 'junk-food' consumption ceased, and preceded the development of obesity. These data are considered in light of enhanced cue-triggered motivation and striatal function in obesity-susceptible rats and the role of NAc CP-AMPARs in enhanced motivation and addiction.

Eating ‘Junk-Food' Produces Rapid and Long-Lasting Increases in NAc CP-AMPA Receptors: Implications for Enhanced Cue-Induced Motivation and Food Addiction

PubMed Central

Oginsky, Max F; Goforth, Paulette B; Nobile, Cameron W; Lopez-Santiago, Luis F; Ferrario, Carrie R

2016-01-01

Urges to eat are influenced by stimuli in the environment that are associated with food (food cues). Obese people are more sensitive to food cues, reporting stronger craving and consuming larger portions after food cue exposure. The nucleus accumbens (NAc) mediates cue-triggered motivational responses, and activations in the NAc triggered by food cues are stronger in people who are susceptible to obesity. This has led to the idea that alterations in NAc function similar to those underlying drug addiction may contribute to obesity, particularly in obesity-susceptible individuals. Motivational responses are mediated in part by NAc AMPA receptor (AMPAR) transmission, and recent work shows that cue-triggered motivation is enhanced in obesity-susceptible rats after ‘junk-food' diet consumption. Therefore, here we determined whether NAc AMPAR expression and function is increased by ‘junk-food' diet consumption in obesity-susceptible vs -resistant populations using both outbred and selectively bred models of susceptibility. In addition, cocaine-induced locomotor activity was used as a general ‘read out' of mesolimbic function after ‘junk-food' consumption. We found a sensitized locomotor response to cocaine in rats that gained weight on a ‘junk-food' diet, consistent with greater responsivity of mesolimbic circuits in obesity-susceptible groups. In addition, eating ‘junk-food' increased NAc calcium-permeable-AMPAR (CP-AMPAR) function only in obesity-susceptible rats. This increase occurred rapidly, persisted for weeks after ‘junk-food' consumption ceased, and preceded the development of obesity. These data are considered in light of enhanced cue-triggered motivation and striatal function in obesity-susceptible rats and the role of NAc CP-AMPARs in enhanced motivation and addiction. PMID:27383008

Osa-miR164a targets OsNAC60 and negatively regulates rice immunity against the blast fungus Magnaporthe oryzae.

PubMed

Wang, Zhaoyun; Xia, Yeqiang; Lin, Siyuan; Wang, Yanru; Guo, Baohuan; Song, Xiaoning; Ding, Shaochen; Zheng, Liyu; Feng, Ruiying; Chen, Shulin; Bao, Yalin; Sheng, Cong; Zhang, Xin; Wu, Jianguo; Niu, Dongdong; Jin, Hailing; Zhao, Hongwei

2018-05-18

Exploring the regulatory mechanism played by endogenous rice miRNAs in defense responses against the blast disease is of great significance in both resistant variety breeding and disease control management. We identified rice defense-related miRNAs by comparing rice miRNA expression patterns before and after Magnaporthe oryzae strain Guy11 infection. We discovered that osa-miR164a expression reduced upon Guy11 infection at both early and late stages, which was perfectly associated with the induced expression of its target gene, OsNAC60. OsNAC60 encodes a transcription factor, over-expression of which enhanced defense responses, such as increased programmed cell death, greater ion leakage, more ROS accumulation and callose deposition, and up-regulation of defense-related genes. By using transgenic rice over-expressing osa-miR164a, and a transposon insertion mutant of OsNAC60, we showed that when the miR164a/OsNAC60 regulatory module was dysfunctional, rice developed significant susceptibility to Guy11 infection. The co-expression of OsNAC60 and osa-miR164a abolished the OsNAC60 activity, but not its synonymous mutant. We further validated that this regulatory module is conserved in plant resistance to multiple plant diseases such as the rice sheath blight, tomato late blight, and soybean root and stem rot diseases. Our results demonstrate that the miR164a/OsNAC60 regulatory module manipulates rice defense responses to M. oryzae infection. This discovery is of great potential for resistant variety breeding and disease control to a broad spectrum of pathogens in the future. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Role of beta-catenin and endocannabinoids in the nucleus accumbens in extinction in rats exposed to shock and reminders.

PubMed

Korem, Nachshon; Lange, Rachel; Hillard, Cecilia J; Akirav, Irit

2017-08-15

The response to a traumatic experience may be rapid recovery or development of psychopathology such as post-traumatic stress disorder (PTSD). Impaired extinction of fear memories is thought to contribute to the development of the persistent trauma memories and avoidance. The Wnt/β-catenin pathway and the endocannabinoid system appear to play significant roles in anxiety and depressive symptoms. Here we examined the involvement of β-catenin in the nucleus accumbens (NAc) in extinction in rats exposed to the shock and reminders model of PTSD. We found that increased β-catenin levels in the NAc were correlated with facilitated extinction kinetics in rats exposed to shock and reminders, suggesting that increased levels of NAc β-catenin are associated with a resilient response to the stressor. Furthermore, downregulating β-catenin expression in the NAc in shocked rats using sulindac (0.0178, 0.178mg/side) impaired extinction whereas upregulating the Wnt/β-catenin pathway using LiCl (2µg/side) facilitated extinction. Exposure to shock and reminders resulted in attenuated levels of the endocannabinoid N-arachidonylethanolamine (AEA) in the NAc; the cannabinoid CB1/2 receptor agonist WIN55,212-2 (5µg/side) microinjected into the NAc facilitated extinction in shocked rats. Importantly, the facilitating effect of WIN55,212-2 on extinction was blocked by co-administration of sulindac in doses that downregulated β-catenin levels. Taken together, the results suggest that β-catenin in the NAc may serve as a protective buffer against the effects of severe stress, and that inhibiting this system in the NAc may prevent the therapeutic effects of cannabinoids against stress-related disorders. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

The effect of N-acetylcysteine on biofilms: Implications for the treatment of respiratory tract infections.

PubMed

Blasi, Francesco; Page, Clive; Rossolini, Gian Maria; Pallecchi, Lucia; Matera, Maria Gabriella; Rogliani, Paola; Cazzola, Mario

2016-08-01

In airway infections, biofilm formation has been demonstrated to be responsible for both acute and chronic events, and constitutes a genuine challenge in clinical practice. Difficulty in eradicating biofilms with systemic antibiotics has led clinicians to consider the possible role of non-antibiotic therapy. The aim of this review is to examine current evidence for the use of N-acetylcysteine (NAC) in the treatment of biofilm-related respiratory infections. Electronic searches of PUBMED up to September 2015 were conducted, searching for 'biofilm', 'respiratory tract infection', 'N-acetylcysteine', 'cystic fibrosis', 'COPD', 'bronchiectasis', 'otitis', and 'bronchitis' in titles and abstracts. Studies included for review were primarily in English, but a few in Italian were also selected. Biofilm formation may be involved in many infections, including ventilator-associated pneumonia, cystic fibrosis, bronchiectasis, bronchitis, and upper respiratory airway infections. Many in vitro studies have demonstrated that NAC is effective in inhibiting biofilm formation, disrupting preformed biofilms (both initial and mature), and reducing bacterial viability in biofilms. There are fewer clinical studies on the use of NAC in disruption of biofilm formation, although there is some evidence that NAC alone or in combination with antibiotics can decrease the risk of exacerbations of chronic bronchitis, chronic obstructive pulmonary disease, and rhinosinusitis. However, the usefulness of NAC in the treatment of cystic fibrosis and bronchiectasis is still matter of debate. Most of the studies published to date have used oral or intramuscular NAC formulations. Evidence from in vitro studies indicates that NAC has good antibacterial properties and the ability to interfere with biofilm formation and disrupt biofilms. Results from clinical studies have provided some encouraging findings that need to be confirmed and expanded using other routes of administration of NAC such as inhalation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Augmentation of Heroin Seeking Following Chronic Food Restriction in the Rat: Differential Role for Dopamine Transmission in the Nucleus Accumbens Shell and Core.

PubMed

D'Cunha, Tracey M; Daoud, Emilie; Rizzo, Damaris; Bishop, Audrey B; Russo, Melissa; Mourra, Gabrielle; Hamel, Laurie; Sedki, Firas; Shalev, Uri

2017-04-01

Caloric restriction during drug abstinence increases the risk for relapse in addicts. In rats, chronic food restriction during a period of withdrawal following heroin self-administration augments heroin seeking. The mechanisms underlying this effect are largely unknown. Here, we investigated the role of nucleus accumbens (NAc) shell and core dopamine (DA) in food restriction-induced augmentation of heroin seeking. Rats were trained to self-administer heroin (0.1 mg/kg/infusion) for 10 days. Next, rats were moved to the animal colony for a withdrawal period, during which rats were food restricted to 90% of their original body weight (FDR group) or given unrestricted access to food (sated group). On day 14 of food restriction, rats were returned to the operant conditioning chambers for a heroin-seeking test under extinction conditions. Extracellular DA levels were assessed using in vivo microdialysis. In separate experiments, the DA D1-like receptor antagonist SCH39166 (12.5, 25.0, or 50.0 ng/side) was administered into the NAc before the heroin-seeking test. In the NAc shell, pre-test exposure to the heroin-associated context increased DA only in FDR rats; but in the NAc core, DA increased regardless of feeding condition. Food restriction significantly augmented heroin seeking and increased DA in the NAc shell and core during the test. Intra-NAc shell administration of SCH39166 decreased heroin seeking in all rats. In contrast, in the NAc core, SCH39166 selectively decreased the augmentation of heroin-seeking induced by chronic food restriction. Taken together, these results suggest that activation of the DA D1-like receptor in the NAc core is important for food restriction-induced augmentation of heroin seeking.

Pretreatment with 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside Attenuates Cerebral Ischemia/Reperfusion-Induced Injury In Vitro and In Vivo

PubMed Central

Chen, Xia; Deng, Aiqing; Zhou, Tianqiu; Ding, Fei

2014-01-01

Salidroside, extracted from the root of Rhodiola rosea L, is known for its pharmacological properties, in particular its neuroprotective effects. 2-(4-Methoxyphenyl) ethyl-2-acetamido-2-deoxy-β-D- pyranoside (GlcNAc-Sal), an analog of salidroside, was recently synthesized and shown to possess neuroprotective properties. The purpose of the current study was to investigate the neuroprotective effects of GlcNAc-Sal against oxygen–glucose deprivation-reperfusion (OGD-R)-induced neurotoxicity in vitro and global cerebral ischemia-reperfusion (GCI-R) injury in vivo. Cell viability tests and Hoechst 33342 staining confirmed that GlcNAc-Sal pretreatment markedly attenuated OGD-R induced apoptotic cell death in immortalized mouse hippocampal HT22 cells. Western blot, immunofluorescence and PCR analyses revealed that GlcNAc-Sal pretreatment restored the balance of pro- and anti-apoptotic proteins and inhibited the activation of caspase-3 and PARP induced by OGD-R treatment. Further analyses showed that GlcNAc-Sal pretreatment antagonized reactive oxygen species (ROS) generation, iNOS-derived NO production and NO-related apoptotic cell death during OGD-R stimulation. GCI-R was induced by bilateral common carotid artery occlusion (BCCAO) and reperfusion in mice in vivo. Western blot analysis showed that GlcNAc-Sal pretreatment decreased the expression of caspase-3 and increased the expression of Bcl-2 (B-cell lymphoma 2)/Bax (Bcl-2-associated X protein) induced by GCI-R treatment. Our findings suggest that GlcNAc-Sal pretreatment prevents brain ischemia reperfusion injury by the direct or indirect suppression of cell apoptosis and GlcNAc-Sal could be developed as a broad-spectrum agent for the prevention and/or treatment of cerebral ischemic injury. PMID:24991917

N-acetylcysteine and endothelial cell injury by sulfur mustard.

PubMed

Atkins, K B; Lodhi, I J; Hurley, L L; Hinshaw, D B

2000-12-01

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells (Toxicol. Appl. Pharmacol. 1996; 141: 568-583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N-acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFkappaB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFkappaB following exposure to HD. The cells were treated with 0-10 mM GSH for 1 h prior to and during exposure to 0 or 500 microM HD for 5-6 h. Cells were also treated with 50 mM NAC or 200 microM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 microM HD for 5-6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFkappaB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 microM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFkappaB binding to its consensus sequence induced by 500 microM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFkappaB, although HD-induced activation of NFkappaB was partially suppressed by NAC at 5 h. Factor NFkappaB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFkappaB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFkappaB activity. The NFkappaB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines.

Molecular characterization of banana NAC transcription factors and their interactions with ethylene signalling component EIL during fruit ripening.

PubMed

Shan, Wei; Kuang, Jian-fei; Chen, Lei; Xie, Hui; Peng, Huan-huan; Xiao, Yun-yi; Li, Xue-ping; Chen, Wei-xin; He, Quan-guang; Chen, Jian-ye; Lu, Wang-jin

2012-09-01

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play important roles in plant growth, development, and stress responses. However, the precise role of NAC TFs in relation to fruit ripening is poorly understood. In this study, six NAC genes, designated MaNAC1-MaNAC6, were isolated and characterized from banana fruit. Subcellular localization showed that MaNAC1-MaNAC5 proteins localized preferentially to the nucleus, while MaNAC6 was distributed throughout the entire cell. A transactivation assay in yeast demonstrated that MaNAC4 and MaNAC6, as well as their C-terminal regions, possessed trans-activation activity. Gene expression profiles in fruit with four different ripening characteristics, including natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and a combination of 1-MCP with ethylene treatment, revealed that the MaNAC genes were differentially expressed in peel and pulp during post-harvest ripening. MaNAC1 and MaNAC2 were apparently upregulated by ethylene in peel and pulp, consistent with the increase in ethylene production. In contrast, MaNAC3 in peel and pulp and MaNAC5 in peel were constitutively expressed, and transcripts of MaNAC4 in peel and pulp and MaNAC6 in peel decreased, while MaNAC5 or MaNAC6 in pulp increased slightly during fruit ripening. Furthermore, the MaNAC2 promoter was activated after ethylene application, further enhancing the involvement of MaNAC2 in fruit ripening. More importantly, yeast two-hybrid and bimolecular fluorescence complementation analyses confirmed that MaNAC1/2 physically interacted with a downstream component of ethylene signalling, ethylene insensitive 3 (EIN3)-like protein, termed MaEIL5, which was downregulated during ripening. Taken together, these results suggest that MaNACs such as MaNAC1/MaNAC2, may be involved in banana fruit ripening via interaction with ethylene signalling components.

Molecular characterization of banana NAC transcription factors and their interactions with ethylene signalling component EIL during fruit ripening

PubMed Central

Shan, Wei; Kuang, Jian-fei; Chen, Jian-ye; Lu, Wang-jin

2012-01-01

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play important roles in plant growth, development, and stress responses. However, the precise role of NAC TFs in relation to fruit ripening is poorly understood. In this study, six NAC genes, designated MaNAC1–MaNAC6, were isolated and characterized from banana fruit. Subcellular localization showed that MaNAC1–MaNAC5 proteins localized preferentially to the nucleus, while MaNAC6 was distributed throughout the entire cell. A transactivation assay in yeast demonstrated that MaNAC4 and MaNAC6, as well as their C-terminal regions, possessed trans-activation activity. Gene expression profiles in fruit with four different ripening characteristics, including natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and a combination of 1-MCP with ethylene treatment, revealed that the MaNAC genes were differentially expressed in peel and pulp during post-harvest ripening. MaNAC1 and MaNAC2 were apparently upregulated by ethylene in peel and pulp, consistent with the increase in ethylene production. In contrast, MaNAC3 in peel and pulp and MaNAC5 in peel were constitutively expressed, and transcripts of MaNAC4 in peel and pulp and MaNAC6 in peel decreased, while MaNAC5 or MaNAC6 in pulp increased slightly during fruit ripening. Furthermore, the MaNAC2 promoter was activated after ethylene application, further enhancing the involvement of MaNAC2 in fruit ripening. More importantly, yeast two-hybrid and bimolecular fluorescence complementation analyses confirmed that MaNAC1/2 physically interacted with a downstream component of ethylene signalling, ethylene insensitive 3 (EIN3)-like protein, termed MaEIL5, which was downregulated during ripening. Taken together, these results suggest that MaNACs such as MaNAC1/MaNAC2, may be involved in banana fruit ripening via interaction with ethylene signalling components. PMID:22888129

N-Acetylcysteine-induced vasodilatation is modulated by KATP channels, Na+/K+-ATPase activity and intracellular calcium concentration: An in vitro study.

PubMed

Vezir, Özden; Çömelekoğlu, Ülkü; Sucu, Nehir; Yalın, Ali Erdinç; Yılmaz, Şakir Necat; Yalın, Serap; Söğüt, Fatma; Yaman, Selma; Kibar, Kezban; Akkapulu, Merih; Koç, Meryem İlkay; Seçer, Didem

2017-08-01

In this study, we aimed to investigate the role of ATP-sensitive potassium (K ATP ) channel, Na + /K + -ATPase activity, and intracellular calcium levels on the vasodilatory effect of N-acetylcysteine (NAC) in thoracic aorta by using electrophysiological and molecular techniques. Rat thoracic aorta ring preparations and cultured thoracic aorta cells were divided into four groups as control, 2mM NAC, 5mM NAC, and 10mM NAC. Thoracic aorta rings were isolated from rats for measurements of relaxation responses and Na + /K + -ATPase activity. In the cultured thoracic aorta cells, we measured the currents of K ATP channel, the concentration of intracellular calcium and mRNA expression level of K ATP channel subunits (KCNJ8, KCNJ11, ABCC8 and ABCC9). The relaxation rate significantly increased in all NAC groups compared to control. Similarly, Na + /K + - ATPase activity also significantly decreased in NAC groups. Outward K ATP channel current significantly increased in all NAC groups compared to the control group. Intracellular calcium concentration decreased significantly in all groups with compared control. mRNA expression level of ABCC8 subunit significantly increased in all NAC groups compared to the control group. Pearson correlation analysis showed that relaxation rate was significantly associated with K ATP current, intracellular calcium concentration, Na + /K + -ATPase activity and mRNA expression level of ABCC8 subunit. Our findings suggest that NAC relaxes vascular smooth muscle cells through a direct effect on K ATP channels, by increasing outward K+ flux, partly by increasing mRNA expression of K ATP subunit ABCC8, by decreasing in intracellular calcium and by decreasing in Na + /K + -ATPase activity. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

N-acetylcysteine improves established monocrotaline-induced pulmonary hypertension in rats

PubMed Central

2014-01-01

Background The outcome of patients suffering from pulmonary arterial hypertension (PAH) are predominantly determined by the response of the right ventricle to the increase afterload secondary to high vascular pulmonary resistance. However, little is known about the effects of the current available or experimental PAH treatments on the heart. Recently, inflammation has been implicated in the pathophysiology of PAH. N-acetylcysteine (NAC), a well-known safe anti-oxidant drug, has immuno-modulatory and cardioprotective properties. We therefore hypothesized that NAC could reduce the severity of pulmonary hypertension (PH) in rats exposed to monocrotaline (MCT), lowering inflammation and preserving pulmonary vascular system and right heart function. Methods Saline-treated control, MCT-exposed, MCT-exposed and NAC treated rats (day 14–28) were evaluated at day 28 following MCT for hemodynamic parameters (right ventricular systolic pressure, mean pulmonary arterial pressure and cardiac output), right ventricular hypertrophy, pulmonary vascular morphometry, lung inflammatory cells immunohistochemistry (monocyte/macrophages and dendritic cells), IL-6 expression, cardiomyocyte hypertrophy and cardiac fibrosis. Results The treatment with NAC significantly decreased pulmonary vascular remodeling, lung inflammation, and improved total pulmonary resistance (from 0.71 ± 0.05 for MCT group to 0.50 ± 0.06 for MCT + NAC group, p < 0.05). Right ventricular function was also improved with NAC treatment associated with a significant decrease in cardiomyocyte hypertrophy (625 ± 69 vs. 439 ± 21 μm2 for MCT and MCT + NAC group respectively, p < 0.001) and heart fibrosis (14.1 ± 0.8 vs. 8.8 ± 0.1% for MCT and MCT + NAC group respectively, p < 0.001). Conclusions Through its immuno-modulatory and cardioprotective properties, NAC has beneficial effect on pulmonary vascular and right heart function in experimental PH. PMID:24929652

Effects of N-acetyl-L-cysteine and hyaluronic acid on HBOC-201-induced systemic and cerebral vasoconstriction in the rat.

PubMed

Abutarboush, Rania; Scultetus, Anke; Pappas, Georgina; Arnaud, Francoise; Auker, Charles; McCarron, Richard; Moon-Massat, Paula F

2013-12-01

Hemoglobin-based oxygen carrier-201 (HBOC) was developed as a resuscitative fluid but concerns exist over potentially adverse vasoconstriction. This study evaluated whether concurrent IV (intra venous) N-acetyl-L-cysteine (NAC) or hyaluronic acid (HA) would attenuate HBOC-associated vasoconstriction, assessed by systemic blood pressures and cerebral pial microvasculature, when administered to healthy, anesthetized rats. Rats (8-9/group) received a 30 min infusion of 3 ml/kg HBOC, HBOC plus 600 mg/kg NAC (HBOC/NAC), HBOC plus 1.5 mg/kg HA (HBOC/HA) or 3 ml/kg Albumin. Mean (MAP) and systolic (SBP) blood pressures, blood chemistries and cerebral pial vessel diameters were measured at baseline, end of infusion, and intermittently for an additional 90 min. HBOC caused immediate and sustained increases in SBP and MAP (35.3 ± 3.6 and 29.1 ± 2.5 mm Hg peak increases above baseline, respectively; mean ± SEM) and immediate but progressive vasoconstriction (11 µm maximum reduction) in medium-sized (50-100 µm) pial arterioles. When NAC was co-administered, blood pressure changes were attenuated and vessel changes were abolished. Similar trends were noted with co-administration of HA but were not statistically different from HBOC-alone. Small-sized (< 50 µm) pial vessels and blood parameters showed no differences from baseline or among groups. No adverse clinical signs were observed. We demonstrated that it is possible for adjuvant drugs to reduce the vasoconstriction associated with HBOC-201. Coinfusion of the anti-oxidant NAC mitigated HBOC-201-associated increases in blood pressures and vasoconstriction in medium-sized cerebral pial vessels. The drag-reducing polymer HA may be more effective at a higher dose as a similar but non-significant trend was observed.

AMPA receptor plasticity in accumbens core contributes to incubation of methamphetamine craving

PubMed Central

Scheyer, Andrew F.; Loweth, Jessica A.; Christian, Daniel T.; Uejima, Jamie; Rabei, Rana; Le, Tuan; Dolubizno, Hubert; Stefanik, Michael T.; Murray, Connor H.; Sakas, Courtney; Wolf, Marina E.

2016-01-01

BACKGROUND The incubation of cue-induced drug craving in rodents provides a model of persistent vulnerability to craving and relapse in human addicts. After prolonged withdrawal, incubated cocaine craving depends on strengthening of nucleus accumbens (NAc) core synapses through incorporation of Ca2+-permeable AMPA receptors (CP-AMPARs). Through mGlu1-mediated synaptic depression, mGlu1 positive allosteric modulators (PAMs) remove CP-AMPARs from these synapses and thereby reduce cocaine craving. This study aimed to determine if similar plasticity accompanies incubation of methamphetamine craving. METHODS Rats self-administered saline or methamphetamine under extended-access conditions. Cue-induced seeking tests demonstrated incubation of methamphetamine craving. After withdrawal periods ranging from 1 to >40 days, rats underwent one of the following procedures: 1) whole-cell patch clamp recordings to characterize AMPAR transmission, 2) intra-NAc core injection of the CP-AMPAR antagonist 1-napthyl acetyl spermine (naspm) prior to a seeking test, or 3) systemic administration of an mGlu1 PAM prior to a seeking test. RESULTS Incubation of methamphetamine craving was associated with CP-AMPAR accumulation in NAc core, and both effects were maximal after ~1 week of withdrawal. Expression of incubated craving was decreased by intra-NAc naspm injection or systemic mGlu1 PAM administration. CONCLUSIONS These results are the first to demonstrate a role for the NAc in the incubation of methamphetamine craving and describe adaptations in synaptic transmission associated with this model. They establish that incubation of craving and associated CP-AMPAR plasticity occur much more rapidly during withdrawal from methamphetamine than cocaine. However, a common mGlu1-based therapeutic strategy may be helpful for recovering cocaine and methamphetamine addicts. PMID:27264310

Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer's disease: Brain protein O-GlcNAcylation in Alzheimer's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Sheng; Yang, Feng; Petyuk, Vladislav A.

Protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as an important factor in the pathogenesis of sporadic Alzheimer’s disease. Herein we report the most comprehensive, quantitative proteomics analysis for protein O-GlcNAcylation in post-mortem human brains with and without Alzheimer’s using isobaric tandem mass tags labeling, chemoenzymatic photocleavage enrichment and liquid chromatography coupled to mass spectrometry. A total of 1,850 O-GlcNAc peptides covering 1,094 O-GlcNAcylation sites were identified from 530 proteins in the human brain. 128 O-GlcNAc peptides covering 78 proteins were altered significantly in Alzheimer’s brain as compared to controls (q<0.05). Moreover, alteration of the O-GlcNAc peptide abundance could bemore » attributed more to O-GlcNAcylation level than to protein level changes. The altered O-GlcNAcylated proteins belong to several structural and functional categories, including synaptic proteins, cytoskeleton proteins, and memory-associated proteins. These findings suggest that dysregulation of O-GlcNAcylation of multiple brain proteins may be involved in the development of sporadic Alzheimer’s disease.« less

Peptide mediators of cholesterol efflux

DOEpatents

Bielicki, John K.; Johansson, Jan

2013-04-09

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Potent and selective mediators of cholesterol efflux

DOEpatents

Bielicki, John K; Johansson, Jan

2015-03-24

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Oligosaccharides in feces of breast- and formula-fed babies.

PubMed

Albrecht, Simone; Schols, Henk A; van Zoeren, Diny; van Lingen, Richard A; Groot Jebbink, Liesbeth J M; van den Heuvel, Ellen G H M; Voragen, Alphons G J; Gruppen, Harry

2011-10-18

So far, little is known on the fate of oligosaccharides in the colon of breast- and formula-fed babies. Using capillary electrophoresis with laser induced fluorescence detector coupled to a mass spectrometer (CE-LIF-MS(n)), we studied the fecal oligosaccharide profiles of 27 two-month-old breast-, formula- and mixed-fed preterm babies. The interpretation of the complex oligosaccharide profiles was facilitated by beforehand clustering the CE-LIF data points by agglomerative hierarchical clustering (AHC). In the feces of breast-fed babies, characteristic human milk oligosaccharide (HMO) profiles, showing genetic fingerprints known for human milk of secretors and non-secretors, were recognized. Alternatively, advanced degradation and bioconversion of HMOs, resulting in an accumulation of acidic HMOs or HMO bioconversion products was observed. Independent of the prebiotic supplementation of the formula with galactooligosaccharides (GOS) at the level used, similar oligosaccharide profiles of low peak abundance were obtained for formula-fed babies. Feeding influences the presence of diet-related oligosaccharides in baby feces and gastrointestinal adaptation plays an important role herein. Four fecal oligosaccharides, characterized as HexNAc-Hex-Hex, Hex-[Fuc]-HexNAc-Hex, HexNAc-[Fuc]-Hex-Hex and HexNAc-[Fuc]-Hex-HexNAc-Hex-Hex, highlighted an active gastrointestinal metabolization of the feeding-related oligosaccharides. Their presence was linked to the gastrointestinal mucus layer and the blood-group determinant oligosaccharides therein, which are characteristic for the host's genotype. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Differential Contribution of the Innate Immune System to a Good Pathological Response in the Breast and Axillary Lymph Nodes Induced by Neoadjuvant Chemotherapy in Women with Large and Locally Advanced Breast Cancers

PubMed Central

Verma, Chandan; Eremin, Jennifer M.; Cowley, Gerard; Ilyas, Mohammad; Satthaporn, Sukchai; Eremin, Oleg

2017-01-01

The tumour microenvironment consists of malignant cells, stroma, and immune cells. The role of adaptive immunity in inducing a pathological complete response (pCR) in breast cancer with neoadjuvant chemotherapy (NAC) is well studied. The contribution of innate immunity, however, is poorly documented. Breast tumours and axillary lymph nodes (ALNs) from 33 women with large and locally advanced breast cancers (LLABCs) undergoing NAC were immunohistochemically assessed for tumour-infiltrating macrophages (TIMs: M1 and M2), neutrophils (TINs), and dendritic cells (TIDCs) using labelled antibodies and semiquantitative methods. Patients' blood neutrophils (n = 108), DCs (mDC1 and pDC), and their costimulatory molecules (n = 30) were also studied. Pathological results were classified as pCR, good (GPR) or poor (PRR). In breast and metastatic ALNs, high levels of CD163+ TIMs were significantly associated with a pCR. In blood, high levels of neutrophils were significantly associated with pCR in metastatic ALNs, whilst the % of mDC1 and pDC and expression of HLA-DR, mDC1 CD40, and CD83 were significantly reduced. NAC significantly reduced tumour DCs but increased blood DCs. PPRs to NAC had significantly reduced HLA-DR, CD40, and CD86 expression. Our study demonstrated novel findings documenting the differential but important contributions of innate immunity to pCRs in patients with LLABCs undergoing NAC. PMID:28913366

Effects of cell penetrating Notch inhibitory peptide conjugated to elastin-like polypeptide on glioblastoma cells.

PubMed

Opačak-Bernardi, Teuta; Ryu, Jung Su; Raucher, Drazen

2017-07-01

Notch pathway was found to be activated in most glioblastomas (GBMs), underlining the importance of Notch in formation and recurrence of GBM. In this study, a Notch inhibitory peptide, dominant negative MAML (dnMAML), was conjugated to elastin-like polypeptide (ELP) for tumor targeted delivery. ELP is a thermally responsive polypeptide that can be actively and passively targeted to the tumor site by localized application of hyperthermia. This complex was further modified with the addition of a cell penetrating peptide, SynB1, for improved cellular uptake and blood-brain barrier penetration. The SynB1-ELP1-dnMAML was examined for its cellular uptake, cytotoxicity, apoptosis, cell cycle inhibition and the inhibition of target genes' expression. SynB1-ELP1-dnMAML inhibited the growth of D54 and U251 cells by inducing apoptosis and cell cycle arrest, especially in the presence of hyperthermia. Hyperthermia increased overall uptake of the polypeptide by the cells and enhanced the resulting pharmacological effects of dnMAML, showing the inhibition of targets of Notch pathway such as Hes-1 and Hey-L. These results confirm that dnMAML is an effective Notch inhibitor and combination with ELP may allow thermal targeting of the SynB1-ELP1-dnMAML complex in cancer cells while avoiding the dangers of systemic Notch inhibition.

Successful deep brain stimulation of the nucleus accumbens in severe alcohol dependence is associated with changed performance monitoring.

PubMed

Kuhn, Jens; Gründler, Theo O J; Bauer, Robert; Huff, Wolfgang; Fischer, Adrian G; Lenartz, Doris; Maarouf, Mohammad; Bührle, Christian; Klosterkötter, Joachim; Ullsperger, Markus; Sturm, Volker

2011-10-01

Following recent advances in neuromodulation therapy for mental disorders, we treated one patient with severe alcohol addiction with deep brain stimulation (DBS) of the nucleus accumbens (NAc). Before and one year following the surgery, we assessed the effects of DBS within the NAc on the addiction as well as on psychometric scores and electrophysiological measures of cognitive control. In our patient, DBS achieved normalization of addictive behavior and craving. An electrophysiological marker of error processing (the error-related negativity) linked to anterior mid-cingulate cortex (aMCC) functioning was altered through DBS, an effect that could be reversed by periods without stimulation. Thus, this case supports the hypothesis that DBS of the NAc could have a positive effect on addiction trough a normalization of craving associated with aMCC dysfunction. © 2011 The Authors, Addiction Biology © 2011 Society for the Study of Addiction.

The Post-Anaesthesia N-acetylcysteine Cognitive Evaluation (PANACEA) trial: study protocol for a randomised controlled trial.

PubMed

Skvarc, David R; Dean, Olivia M; Byrne, Linda K; Gray, Laura J; Ives, Kathryn; Lane, Stephen E; Lewis, Matthew; Osborne, Cameron; Page, Richard; Stupart, Douglas; Turner, Alyna; Berk, Michael; Marriott, Andrew J

2016-08-09

Some degree of cognitive decline after surgery occurs in as many as one quarter of elderly surgical patients, and this decline is associated with increased morbidity and mortality. Cognition may be affected across a range of domains, including memory, psychomotor skills, and executive function. Whilst the exact mechanisms of cognitive change after surgery are not precisely known, oxidative stress and subsequent neuroinflammation have been implicated. N-acetylcysteine (NAC) acts via multiple interrelated mechanisms to influence oxidative homeostasis, neuronal transmission, and inflammation. NAC has been shown to reduce oxidative stress and inflammation in both human and animal models. There is clinical evidence to suggest that NAC may be beneficial in preventing the cognitive decline associated with both acute physiological insults and dementia-related disorders. To date, no trials have examined perioperative NAC as a potential moderator of postoperative cognitive changes in the noncardiac surgery setting. This is a single-centre, randomised, double-blind, placebo-controlled clinical trial, with a between-group, repeated-measures, longitudinal design. The study will recruit 370 noncardiac surgical patients at the University Hospital Geelong, aged 60 years or older. Participants are randomly assigned to receive either NAC or placebo (1:1 ratio), and groups are stratified by age and surgery type. Participants undergo a series of neuropsychological tests prior to surgery, 7 days, 3 months, and 12 months post surgery. It is hypothesised that the perioperative administration of NAC will reduce the degree of postoperative cognitive changes at early and long-term follow-up, as measured by changes on individual measures of the neurocognitive battery, when compared with placebo. Serum samples are taken on the day of surgery and on day 2 post surgery to quantitate any changes in levels of biomarkers of inflammation and oxidative stress. The PANACEA trial aims to examine the potential efficacy of perioperative NAC to reduce the severity of postoperative cognitive dysfunction in an elderly, noncardiac surgery population. This is an entirely novel approach to the prevention of postoperative cognitive dysfunction and will have high impact and translatable outcomes if NAC is found to be beneficial. The PANACEA trial has been registered with the Therapeutic Goods Administration, and the Australian New Zealand Clinical Trials Registry: ACTRN12614000411640 ; registered on 15 April 2014.

Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thoden, James B.; Holden, Hazel M.

2010-09-08

2,3-Diacetamido-2,3-dideoxy-D-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3{prime} hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD{sup +} to NADH. This enzyme has been shown to usemore » {alpha}-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and {alpha}-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both {alpha}-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3{prime}, respectively) abutting the re face of the cofactor. They are positioned {approx}3 {angstrom} from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.« less

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

PubMed Central

Serafini-Cessi, F; Dall'Olio, F; Pereira, L; Campadelli-Fiume, G

1984-01-01

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983). Images PMID:6088806

Improvement of Learning and Memory Induced by Cordyceps Polypeptide Treatment and the Underlying Mechanism

PubMed Central

2018-01-01

Our previous research revealed that Cordyceps militaris can improve the learning and memory, and although the main active ingredient should be its polypeptide complexes, the underlying mechanism of its activity remains poorly understood. In this study, we explored the mechanisms by which Cordyceps militaris improves learning and memory in a mouse model. Mice were given scopolamine hydrobromide intraperitoneally to establish a mouse model of learning and memory impairment. The effects of Cordyceps polypeptide in this model were tested using the Morris water maze test; serum superoxide dismutase activity; serum malondialdehyde levels; activities of acetyl cholinesterase, Na+-k+-ATPase, and nitric oxide synthase; and gamma aminobutyric acid and glutamate contents in brain tissue. Moreover, differentially expressed genes and the related cellular signaling pathways were screened using an mRNA expression profile chip. The results showed that the genes Pik3r5, Il-1β, and Slc18a2 were involved in the effects of Cordyceps polypeptide on the nervous system of these mice. Our findings suggest that Cordyceps polypeptide may improve learning and memory in the scopolamine-induced mouse model of learning and memory impairment by scavenging oxygen free radicals, preventing oxidative damage, and protecting the nervous system. PMID:29736181

Reactive oxygen species scavenger N-acetyl cysteine reduces methamphetamine-induced hyperthermia without affecting motor activity in mice

PubMed Central

Sanchez-Alavez, Manuel; Bortell, Nikki; Galmozzi, Andrea; Conti, Bruno; Marcondes, Maria Cecilia G.

2014-01-01

Hyperthermia is a potentially lethal side effect of Methamphetamine (Meth) abuse, which involves the participation of peripheral thermogenic sites such as the Brown Adipose Tissue (BAT). In a previous study we found that the anti-oxidant N-acetyl cysteine (NAC) can prevent the high increase in temperature in a mouse model of Meth-hyperthermia. Here, we have further explored the ability of NAC to modulate Meth-induced hyperthermia in correlation with changes in BAT. We found that NAC treatment in controls causes hypothermia, and, when administered prior or upon the onset of Meth-induced hyperthermia, can ameliorate the temperature increase and preserve mitochondrial numbers and integrity, without affecting locomotor activity. This was different from Dantrolene, which decreased motor activity without affecting temperature. The effects of NAC were seen in spite of its inability to recover the decrease of mitochondrial superoxide induced in BAT by Meth. In addition, NAC did not prevent the Meth-induced decrease of BAT glutathione. Treatment with S-adenosyl-L-methionine, which improves glutathione activity, had an effect in ameliorating Meth-induced hyperthermia, but also modulated motor activity. This suggests a role for the remaining glutathione for controlling temperature. However, the mechanism by which NAC operates is independent of glutathione levels in BAT and specific to temperature. Our results show that, in spite of the absence of a clear mechanism of action, NAC is a pharmacological tool to examine the dissociation between Meth-induced hyperthermia and motor activity, and a drug of potential utility in treating the hyperthermia associated with Meth-abuse. PMID:26346736

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis.

PubMed

Zeng, Qinghua; Zhao, Rui-Xun; Chen, Jianfeng; Li, Yining; Li, Xiang-Dong; Liu, Xiao-Long; Zhang, Wei-Ming; Quan, Cheng-Shi; Wang, Yi-Shu; Zhai, Ying-Xian; Wang, Jian-Wei; Youssef, Mariam; Cui, Rutao; Liang, Jiyong; Genovese, Nicholas; Chow, Louise T; Li, Yu-Lin; Xu, Zhi-Xiang

2016-08-16

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18-transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis

PubMed Central

Zeng, Qinghua; Zhao, Rui-Xun; Chen, Jianfeng; Li, Yining; Li, Xiang-Dong; Liu, Xiao-Long; Zhang, Wei-Ming; Quan, Cheng-Shi; Wang, Yi-Shu; Zhai, Ying-Xian; Wang, Jian-Wei; Youssef, Mariam; Cui, Rutao; Liang, Jiyong; Genovese, Nicholas; Chow, Louise T.; Li, Yu-Lin; Xu, Zhi-Xiang

2016-01-01

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18–transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc. PMID:27482104

Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes

PubMed Central

Becker, Annemarie H.; Oh, Eugene; Weissman, Jonathan S.; Kramer, Günter; Bukau, Bernd

2014-01-01

A plethora of factors is involved in the maturation of newly synthesized proteins, including chaperones, membrane targeting factors, and enzymes. Many factors act cotranslationally through association with ribosome-nascent chain complexes (RNCs), but their target specificities and modes of action remain poorly understood. We developed selective ribosome profiling (SeRP) to identify substrate pools and points of RNC engagement of these factors. SeRP is based on sequencing mRNA fragments covered by translating ribosomes (general ribosome profiling, RP), combined with a procedure to selectively isolate RNCs whose nascent polypeptides are associated with the factor of interest. Factor–RNC interactions are stabilized by crosslinking, the resulting factor–RNC adducts are then nuclease-treated to generate monosomes, and affinity-purified. The ribosome-extracted mRNA footprints are converted to DNA libraries for deep sequencing. The protocol is specified for general RP and SeRP in bacteria. It was first applied to the chaperone trigger factor and is readily adaptable to other cotranslationally acting factors, including eukaryotic factors. Factor–RNC purification and sequencing library preparation takes 7–8 days, sequencing and data analysis can be completed in 5–6 days. PMID:24136347

Effect of N-acetylcysteine treatment on oxidative stress and inflammation after severe burn.

PubMed

Csontos, C; Rezman, B; Foldi, V; Bogar, L; Drenkovics, L; Röth, E; Weber, G; Lantos, J

2012-05-01

Oxidative stress and inflammation generate edema in burns. The aim of our study was to assess effect of N-acetylcysteine (NAC) on oxidative stress, inflammation, fluid requirement, multiple organ dysfunction (MOD) score and vasoactive drug requirement. In this study 15 patients were on standard therapy, whereas for other 15 patients NAC was supplemented. Blood samples were taken on admission and on the next five consecutive mornings. Levels of malondialdehyde, protein sulfhydril (PSH) groups, reduced gluthation (GSH), activity of myeloperoxidase, catalase and superoxide dismutase enzymes and induced free radical generating capacity were measured as well as concentrations of TNF-α, IL-6, IL-8, and IL-10. MOD score, use of vasopressor agents and fluid utilisation were recorded daily. NAC treatment increased GSH level on days 4-5 (p<0.05) and PSH level on days 2-6 (p<0.05) compared to controls. Plasma IL-6 was lower on days 4-5 (p<0.05), IL-8 on days 4-6 (p<0.05) and IL-10 on days 4-6 (p<0.05) in NAC group. NAC group received less catecholamines than controls (p<0.01) from day 4 without significant differences in MOD score. NAC treatment is associated with a diminished oxidative stress reflected in preserved antioxidant levels, lower inflammation mirrored in lower interleukin levels and less vasopressor requirement. Copyright © 2011 Elsevier Ltd and ISBI. All rights reserved.

Evaluation of antioxidant drugs for the treatment of ochronotic alkaptonuria in an in vitro human cell model.

PubMed

Tinti, Laura; Spreafico, Adriano; Braconi, Daniela; Millucci, Lia; Bernardini, Giulia; Chellini, Federico; Cavallo, Giovanni; Selvi, Enrico; Galeazzi, Mauro; Marcolongo, Roberto; Gallagher, James A; Santucci, Annalisa

2010-10-01

Alkaptonuria (AKU) is a rare autosomal recessive disease, associated with deficiency of homogentisate 1,2-dioxygenase activity in the liver. This leads to an accumulation of homogentisic acid (HGA) and its oxidized derivatives in polymerized form in connective tissues especially in joints. Currently, AKU lacks an appropriate therapy. Hence, we propose a new treatment for AKU using the antioxidant N-acetylcysteine (NAC) administered in combinations with ascorbic acid (ASC) since it has been proven that NAC counteracts the side-effects of ASC. We established an in vitro cell model using human articular primary chondrocytes challenged with an excess of HGA (0.33 mM). We used this experimental model to undertake pre-clinical testing of potential antioxidative therapies for AKU, evaluating apoptosis, viability, proliferation, and metabolism of chondrocytes exposed to HGA and treated with NAC and ASC administered alone or in combination addition of both. NAC decreased apoptosis induced in chondrocytes by HGA, increased chondrocyte growth reduced by HGA, and partially restored proteoglycan release inhibited by HGA. A significantly improvement in efficacy was found with combined addition of the two antioxidants in comparison with NAC and ASC alone. Our novel in vitro AKU model allowed us to demonstrate the efficacy of the co-administration of NAC and ASC to counteract the negative effects of HGA for the treatment of ochronotic arthropathy. (c) 2010 Wiley-Liss, Inc.

Essential role of gastric gland mucin in preventing gastric cancer in mice

PubMed Central

Karasawa, Fumitoshi; Shiota, Akira; Goso, Yukinobu; Kobayashi, Motohiro; Sato, Yoshiko; Masumoto, Junya; Fujiwara, Maiko; Yokosawa, Shuichi; Muraki, Takashi; Miyagawa, Shinichi; Ueda, Masatsugu; Fukuda, Michiko N.; Fukuda, Minoru; Ishihara, Kazuhiko; Nakayama, Jun

2012-01-01

Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycans) having terminal α1,4-linked N-acetylglucosamine residues (αGlcNAc). Previously, we identified human α1,4-N-acetylglucosaminyltransferase (α4GnT), which is responsible for the O-glycan biosynthesis and characterized αGlcNAc function in suppressing Helicobacter pylori in vitro. In the present study, we engineered A4gnt–/– mice to better understand its role in vivo. A4gnt–/– mice showed complete lack of αGlcNAc expression in gastric gland mucin. Surprisingly, all the mutant mice developed gastric adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absence of H. pylori infection. Microarray and quantitative RT-PCR analysis revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Il-11, and Hgf in the gastric mucosa of A4gnt–/– mice. Further supporting an important role for this O-glycan in cancer progression, we also observed significantly reduced αGlcNAc in human gastric adenocarcinoma and adenoma. Our results demonstrate that the absence of αGlcNAc triggers gastric tumorigenesis through inflammation-associated pathways in vivo. Thus, αGlcNAc-terminated gastric mucin plays dual roles in preventing gastric cancer by inhibiting H. pylori infection and also suppressing tumor-promoting inflammation. PMID:22307328

NAc Shell Arc/Arg3.1 Protein Mediates Reconsolidation of Morphine CPP by Increased GluR1 Cell Surface Expression: Activation of ERK-Coupled CREB is Required

PubMed Central

Lv, Xiu-Fang; Sun, Lin-Lin; Han, Ji-Sheng

2015-01-01

Background: Relapse into drug abuse evoked by reexposure to the drug-associated context has been a primary problem in the treatment of drug addiction. Disrupting the reconsolidation of drug-related context memory would therefore limit the relapse susceptibility. Methods: Morphine conditioned place preference (CPP) was used to assess activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and correlative molecule expression in the Nucleus accumbens (NAc) shell during the reconsolidation of morphine CPP. U0126 and Arc/Arg3.1 antisense oligodeoxynucleotide were adapted to evaluate the role and the underlying mechanism of Arc/Arg3.1 during the reconsolidation. Results: The retrieval of morphine CPP in rats specifically increased the Arc/Arg3.1 protein level in the NAc shell, accompanied simultaneously by increases in the phosphorylation of extracellular signal-regulated kinase1/2 (pERK1/2), the phosphorylation of Cyclic Adenosine monophosphate (cAMP) response element-binding (pCREB), and the up-regulation of the membrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors GluR1 subunit level. Intra-NAc shell infusion U0126, an inhibitor of the Mitogen-activated protein kinase kinase (MEK), prevented the retrieval-induced up-regulation of pERK1/2, pCREB, Arc/Arg3.1, and membrane GluR1 immediately after retrieval of morphine CPP. The effect of disrupting the reconsolidation of morphine CPP by U0126 could last for at least 14 days, and could not be evoked by a priming injection of morphine. Furthermore, the specific knockdown of Arc/Arg3.1 in the NAc shell decreased the membrane GluR1 level, and impaired both the reconsolidation and the reinstatement of morphine CPP. Conclusions: Arc/Arg3.1 in the NAc shell mediates the reconsolidation of morphine-associated context memory via up-regulating the level of membrane of GluR1, for which the local activation of the ERK-CREB signal pathway, as an upstream mechanism of Arc/Arg3.1, is required. PMID:25746394

Dysfunction of Nucleus Accumbens Is Associated With Psychiatric Problems in Patients With Chronic Low Back Pain: A Functional Magnetic Resonance Imaging Study.

PubMed

Kaneko, Hideaki; Zhang, Shuo; Sekiguchi, Miho; Nikaido, Takuya; Makita, Koshi; Kurata, Jiro; Konno, Shin-Ichi

2017-06-01

A cross-sectional study. The aim of this study was to evaluate activity of the nucleus accumbens (NAc) in response to lumbar mechanical stimulation in patients with chronic low back pain (cLBP) using functional magnetic resonance imaging (fMRI). Although a modified activity of the NAc was characterized in cLBP patients, its pathological significance has yet to be determined. We hypothesized that NAc activation in response to pain might differ depending on the extent of psychiatric problems, which might be associated with the affective/motivational background of chronic pain. Twenty-one patients with cLBP (four men, 17 women) were recruited. Subjects were divided into two groups on the basis of scores on the patient version of the Brief Scale for Psychiatric problems in Orthopaedic Patients (BS-POP) scores: ≥17 (High Score, HiS group) and <17 (non-High Score, non-HiS group). Each subject was placed in the prone position on a 3-Tesla magnetic resonance imaging (MRI) scanner and stimulated by mechanical stimulation on the left lower back. Three blocks of 30-second pain stimulus calibrated at either 3 or 5 on an 11-grade numerical rating scale (NRS) were applied with intervening 30-second rest conditions during whole-brain echo-planar imaging. Functional images were analyzed using a multisubject general linear model with Bonferroni multiple comparisons. Subjects in the HiS group had more intense daily pain and lower quality of life than those in the non-HiS group (P < 0.05). Catastrophic thinking in relation to pain experience did not differ between the groups. Activation at the NAc was smaller in the HiS group than in the non-HiS group (P < 0.001). The presence of psychiatric problems was associated with attenuated activity of the NAc in cLBP patients. Dysfunction of the NAc might potentially be involved in the affective/motivational factors in the chronification of LBP. N/A.

Sex Differences in Nucleus Accumbens Transcriptome Profiles Associated with Susceptibility versus Resilience to Subchronic Variable Stress.

PubMed

Hodes, Georgia E; Pfau, Madeline L; Purushothaman, Immanuel; Ahn, H Francisca; Golden, Sam A; Christoffel, Daniel J; Magida, Jane; Brancato, Anna; Takahashi, Aki; Flanigan, Meghan E; Ménard, Caroline; Aleyasin, Hossein; Koo, Ja Wook; Lorsch, Zachary S; Feng, Jian; Heshmati, Mitra; Wang, Minghui; Turecki, Gustavo; Neve, Rachel; Zhang, Bin; Shen, Li; Nestler, Eric J; Russo, Scott J

2015-12-16

Depression and anxiety disorders are more prevalent in females, but the majority of research in animal models, the first step in finding new treatments, has focused predominantly on males. Here we report that exposure to subchronic variable stress (SCVS) induces depression-associated behaviors in female mice, whereas males are resilient as they do not develop these behavioral abnormalities. In concert with these different behavioral responses, transcriptional analysis of nucleus accumbens (NAc), a major brain reward region, by use of RNA sequencing (RNA-seq) revealed markedly different patterns of stress regulation of gene expression between the sexes. Among the genes displaying sex differences was DNA methyltransferase 3a (Dnmt3a), which shows a greater induction in females after SCVS. Interestingly, Dnmt3a expression levels were increased in the NAc of depressed humans, an effect seen in both males and females. Local overexpression of Dnmt3a in NAc rendered male mice more susceptible to SCVS, whereas Dnmt3a knock-out in this region rendered females more resilient, directly implicating this gene in stress responses. Associated with this enhanced resilience of female mice upon NAc knock-out of Dnmt3a was a partial shift of the NAc female transcriptome toward the male pattern after SCVS. These data indicate that males and females undergo different patterns of transcriptional regulation in response to stress and that a DNA methyltransferase in NAc contributes to sex differences in stress vulnerability. Women have a higher incidence of depression than men. However, preclinical models, the first step in developing new diagnostics and therapeutics, have been performed mainly on male subjects. Using a stress-based animal model of depression that causes behavioral effects in females but not males, we demonstrate a sex-specific transcriptional profile in brain reward circuitry. This transcriptional profile can be altered by removal of an epigenetic mechanism, which normally suppresses DNA transcription, creating a hybrid male/female transcriptional pattern. Removal of this epigenetic mechanism also induces behavioral resilience to stress in females. These findings shed new light onto molecular factors controlling sex differences in stress response. Copyright © 2015 the authors 0270-6474/15/3516363-15$15.00/0.

Sex Differences in Nucleus Accumbens Transcriptome Profiles Associated with Susceptibility versus Resilience to Subchronic Variable Stress

PubMed Central

Hodes, Georgia E.; Pfau, Madeline L.; Purushothaman, Immanuel; Ahn, H. Francisca; Golden, Sam A.; Christoffel, Daniel J.; Magida, Jane; Brancato, Anna; Takahashi, Aki; Flanigan, Meghan E.; Ménard, Caroline; Aleyasin, Hossein; Koo, Ja Wook; Lorsch, Zachary S.; Feng, Jian; Heshmati, Mitra; Wang, Minghui; Turecki, Gustavo; Neve, Rachel; Zhang, Bin; Shen, Li; Nestler, Eric J.

2015-01-01

Depression and anxiety disorders are more prevalent in females, but the majority of research in animal models, the first step in finding new treatments, has focused predominantly on males. Here we report that exposure to subchronic variable stress (SCVS) induces depression-associated behaviors in female mice, whereas males are resilient as they do not develop these behavioral abnormalities. In concert with these different behavioral responses, transcriptional analysis of nucleus accumbens (NAc), a major brain reward region, by use of RNA sequencing (RNA-seq) revealed markedly different patterns of stress regulation of gene expression between the sexes. Among the genes displaying sex differences was DNA methyltransferase 3a (Dnmt3a), which shows a greater induction in females after SCVS. Interestingly, Dnmt3a expression levels were increased in the NAc of depressed humans, an effect seen in both males and females. Local overexpression of Dnmt3a in NAc rendered male mice more susceptible to SCVS, whereas Dnmt3a knock-out in this region rendered females more resilient, directly implicating this gene in stress responses. Associated with this enhanced resilience of female mice upon NAc knock-out of Dnmt3a was a partial shift of the NAc female transcriptome toward the male pattern after SCVS. These data indicate that males and females undergo different patterns of transcriptional regulation in response to stress and that a DNA methyltransferase in NAc contributes to sex differences in stress vulnerability. SIGNIFICANCE STATEMENT Women have a higher incidence of depression than men. However, preclinical models, the first step in developing new diagnostics and therapeutics, have been performed mainly on male subjects. Using a stress-based animal model of depression that causes behavioral effects in females but not males, we demonstrate a sex-specific transcriptional profile in brain reward circuitry. This transcriptional profile can be altered by removal of an epigenetic mechanism, which normally suppresses DNA transcription, creating a hybrid male/female transcriptional pattern. Removal of this epigenetic mechanism also induces behavioral resilience to stress in females. These findings shed new light onto molecular factors controlling sex differences in stress response. PMID:26674863

Targeting endogenous proteins for degradation through the affinity-directed protein missile system.

PubMed

Fulcher, Luke J; Hutchinson, Luke D; Macartney, Thomas J; Turnbull, Craig; Sapkota, Gopal P

2017-05-01

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel-Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. © 2017 The Authors.

Uncoordinated (UNC)119: coordinating the trafficking of myristoylated proteins.

PubMed

Constantine, Ryan; Zhang, Houbin; Gerstner, Cecilia D; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like β-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in Caenorhabditis elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking ofmyristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transport myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

Uncoordinated (UNC)119: Coordinating the Trafficking of Myristoylated Proteins

PubMed Central

Constantine, Ryan; Zhang, Houbin; Gerstner, Cecilia D.; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like β-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in C. elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking of myristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transpot myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins. PMID:23000199

Targeting endogenous proteins for degradation through the affinity-directed protein missile system

PubMed Central

Fulcher, Luke J.; Hutchinson, Luke D.; Macartney, Thomas J.; Turnbull, Craig

2017-01-01

Targeted proteolysis of endogenous proteins is desirable as a research toolkit and in therapeutics. CRISPR/Cas9-mediated gene knockouts are irreversible and often not feasible for many genes. Similarly, RNA interference approaches necessitate prolonged treatments, can lead to incomplete knockdowns and are often associated with off-target effects. Targeted proteolysis can overcome these limitations. In this report, we describe an affinity-directed protein missile (AdPROM) system that harbours the von Hippel–Lindau (VHL) protein, the substrate receptor of the Cullin2 (CUL2) E3 ligase complex, tethered to polypeptide binders that selectively bind and recruit endogenous target proteins to the CUL2-E3 ligase complex for ubiquitination and proteasomal degradation. By using synthetic monobodies that selectively bind the protein tyrosine phosphatase SHP2 and a camelid-derived VHH nanobody that selectively binds the human ASC protein, we demonstrate highly efficient AdPROM-mediated degradation of endogenous SHP2 and ASC in human cell lines. We show that AdPROM-mediated loss of SHP2 in cells impacts SHP2 biology. This study demonstrates for the first time that small polypeptide binders that selectively recognize endogenous target proteins can be exploited for AdPROM-mediated destruction of the target proteins. PMID:28490657

Ridges and Cliffs on Mercury Surface

NASA Image and Video Library

2008-01-20

A complex history of geological evolution is recorded in this frame from the Narrow Angle Camera NAC, part of the Mercury Dual Imaging System MDIS instrument, taken during NASA MESSENGER close flyby of Mercury on January 14, 2008.

Superomedial pedicle reduction mammaplasty: increased resection weight does not increase nipple necrosis.

PubMed

Brownlee, Pamela; Chesire, David; Crandall, Marie; Murray, John

2017-11-01

The superomedial pedicle (SMP) reduction mammaplasty offers several advantages over more traditional operative techniques, such as retained sensation to the nipple areola complex (NAC) and improved preservation of long-term breast shape. However, many surgeons believe that using the SMP can cause an increase in NAC necrosis rates up to 13.1%, especially in large volume reductions. The aims of this paper are to determine the rate of nipple necrosis when performing an SMP reduction mammaplasty at our institution and establish if there is a difference in those rates based on the weight of tissue removed. We then compared our overall rate of nipple necrosis to those found using a systematic review of literature involving other pedicles to determine if this technique is equivalent to more commonly used methods. We performed a retrospective chart review of patients who underwent breast reduction surgery using SMP at a single institution between May 1, 2013, and May 1, 2015. Each breast was counted separately, and the weight of the tissue removed was recorded. Six-mo follow-up data were analyzed using the chi-square analysis to compare rates of NAC necrosis among our patients and historical cohorts. One hundred thirty-five breasts (70 patients) met inclusion criteria. Patients had a mean resection weight per breast of 1016.7 g (±478.3). There was an NAC necrosis rate of 0% in the groups where 1200 g or less of tissue were removed and 2.3% (n = 1) in the group where greater than 1200 g of tissue were removed. We found no statistically significant difference in the rate of NAC necrosis with increased resection weights, (P = 0.32). The overall rate of NAC necrosis for this study was 0.7% per breast, which is equivalent to the rates found during the systematic review of literature. In this study, the SMP breast reduction technique shows a low rate of NAC. We found no statistically significant increase in NAC necrosis rates as the volume of tissue removed increases, and the SMP appears to have an overall rate of NAC necrosis similar to more commonly used pedicles. Although the results of this study may be related to patient selection or surgical technique, we believe that this pedicle preserves the breast's main blood supply so that less tissue ischemia occurs. Copyright © 2017 Elsevier Inc. All rights reserved.

N-acetylcysteine reduces the renal oxidative stress and apoptosis induced by hemorrhagic shock.

PubMed

Moreira, Miriam Aparecida; Irigoyen, Maria Claudia; Saad, Karen Ruggeri; Saad, Paulo Fernandes; Koike, Marcia Kiyomi; Montero, Edna Frasson de Souza; Martins, José Luiz

2016-06-01

Renal ischemia/reperfusion injury induced by hemorrhagic shock (HS) and subsequent fluid resuscitation is a common cause of acute renal failure. The objective of this study was to evaluate the effect of combining N-acetylcysteine (NAC) with fluid resuscitation on renal injury in rats that underwent HS. Two groups of male Wistar rats were induced to controlled HS at 35 mm Hg mean arterial pressure for 60 min. After this period, the HS and fluid resuscitation (HS/R) group was resuscitated with lactate containing 50% of the blood that was withdrawn. The HS/R + NAC group was resuscitated with Ringer's lactate combined with 150 mg/kg of NAC and blood. The sham group animals were catheterized but were not subjected to shock. All animals were kept under anesthesia and euthanized after 120 min of fluid resuscitation or observation. Animals treated with NAC presented attenuation of histologic lesions, reduced oxidative stress, and apoptosis markers when compared with animals from the HS/R group. The serum creatinine was similar in all the groups. NAC is a promising drug for combining with fluid resuscitation to attenuate the kidney injury associated with HS. Copyright © 2016 Elsevier Inc. All rights reserved.

High post-natal mortality associated with defects in lung maturation and reduced adiposity in mice with gestational exposure to high fat and N-acetylcysteine.

PubMed

Williams, Lyda; Charron, Maureen J; Sellers, Rani S

2017-10-01

Studies have demonstrated that maternal consumption of a high fat diet (HFD) increases offspring susceptibility to metabolic disease. This study was initiated to identify the mechanistic contribution of oxidative stress on this phenomenon. Two weeks prior to mating, dams were fed either HFD or Control diet with or without supplementation with the anti-oxidant N-acetylcysteine (NAC). Pups born to HFD dams had reduced crown rump length (CRL) at birth and higher neonatal mortality compared to pups from Control dams. Supplementation with NAC normalized CRL in pups from HFD dams, but notably increased mortality. Histological examination of the lungs postnatally and prenatally, revealed normal branching morphogenesis but delayed alveolarization in pups from dams fed HFD+NAC. Discontinuation of NAC at ED17.5 with re-introduction at PD3 improved offspring survival and lung maturation. Additionally, interscapular brown adipose tissue (BAT) was reduced in ED18.5 embryos from HFD dams. These findings suggest that increased mortality in offspring from dams fed HFD+NAC during pregnancy may in part be the result of delayed pulmonary alveolarization and decreased BAT. Copyright © 2017 Elsevier Ltd. All rights reserved.

Primary light-harvesting system: phycobilisomes and associated membranes. Progress report, December 1979-January 1983

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gantt, E.

1983-01-01

The structure of phycobilisomes, which serve as primary light-harvesting complexes for photosynthesis, were investigated in red and blue-green algae (cyanobacteria). Structurally the phycobilisomes have the same fundamental arrangement in the various species, with allophycocyanin being in the core from which peripheral rods composed of phycocyanin or phycocyanin-phycoerythyrin radiate. Such studies were extended to show that energetically functional phycobilisomes could be formed in vitro from separated fractions of allophycocyanin and phycoerythrin-phycocyanin within the same species (Nostoc) and between separate species (Fremyella and Nostoc). A large (95 kD) blue-colored polypeptide is being regarded as an anchor component between phycobilisomes and thylakoids. Themore » polypeptide, isolated from phycobilisomes, had two long wavelength-emitting types of chromophores, one similar to allophycocyanin and another to chlorophyll. Such a large (95 kD) polypeptide occurred in phycobilisomes of red and blue-green algae examined, with the exception of Anacystis where it was somewhat smaller (75 kD). The effect of light quality on phycobilisome structure varied among the blue-green species studied. The relationship of the photosystems and phycobilisomes was investigated in Anacystis wild type cells, and in mutants deficient in phycocyanin. Although the phycocyanin and chlorophyll content per cell changed with light quality the phycobilisome per Reaction Center 2 ratio remained at ca. 0.9 +- 0.3 suggesting that only the size of the antennae varied.« less

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes

PubMed Central

Liao, Shih-Fen; Liang, Chi-Hui; Hsu, Tsui-Ling; Tsai, Tsung-I; Hsieh, Yves S.-Y.; Tsai, Chih-Ming; Li, Shiou-Ting; Cheng, Yang-Yu; Tsao, Shu-Ming; Lin, Tung-Yi; Lin, Zong-Yan; Yang, Wen-Bin; Ren, Chien-Tai; Lin, Kuo-I; Khoo, Kay-Hooi; Lin, Chun-Hung; Hsu, Hsien-Yeh; Wu, Chung-Yi; Wong, Chi-Huey

2013-01-01

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galβ1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans. PMID:23908400

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

PubMed

Liao, Shih-Fen; Liang, Chi-Hui; Ho, Ming-Yi; Hsu, Tsui-Ling; Tsai, Tsung-I; Hsieh, Yves S-Y; Tsai, Chih-Ming; Li, Shiou-Ting; Cheng, Yang-Yu; Tsao, Shu-Ming; Lin, Tung-Yi; Lin, Zong-Yan; Yang, Wen-Bin; Ren, Chien-Tai; Lin, Kuo-I; Khoo, Kay-Hooi; Lin, Chun-Hung; Hsu, Hsien-Yeh; Wu, Chung-Yi; Wong, Chi-Huey

2013-08-20

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galβ1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Quantitative time-resolved chemoproteomics reveals that stable O-GlcNAc regulates box C/D snoRNP biogenesis

PubMed Central

Qin, Wei; Lv, Pinou; Fan, Xinqi; Quan, Baiyi; Zhu, Yuntao; Qin, Ke; Chen, Ying; Wang, Chu

2017-01-01

O-linked GlcNAcylation (O-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of O-GlcNAcylated proteins, we developed a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an O-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of O-GlcNAc or degradation of protein backbones. The stability of those hyperstable O-GlcNAcylated proteins was more sensitive to O-GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that O-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2′-O-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo. PMID:28760965

ABO (histo) blood group phenotype development and human reproduction as they relate to ancestral IgM formation: A hypothesis.

PubMed

Arend, Peter

2016-01-01

The formation of a histo (blood) group) ABO phenotype and the exclusion of an autoreactive IgM or isoagglutinin activity arise apparently in identical glycosylation of complementary domains on cell surfaces and plasma proteins. The fundamental O-glycan emptiness of the circulating IgM, which during the neonatal amino acid sequencing of the variable regions is exerting germline-specific O-GalNAc glycan-reactive serine/threonine residues that in the plasma of the adult human blood group O individuals apparently remain associated with the open glycosidic sites on the ABOH convertible red cell surface, must raise suggestions on a transient expression of developmental glycans, which have been "lost" over the course of maturation. In fact, while the mammalian non-somatic, embryogenic stem cell (ESC)- germ cell (GC) transformation is characterized by a transient and genetically as-yet-undefined trans-species-functional O-GalNAc glycan expression, in the C57BL/10 mouse such expression was potentially identified in growth-dependent, blood group A-like GalNAc glycan-bearing, ovarian glycolipids complementary with the syngeneic anti-A reactive IgM, which does not appear in early ovariectomized animals. This non-somatically encoded, polyreactive, ancestral IgM molecule has not undergone clonal selection and does primarily not differentiate between self and non-self and might, due to amino acid hydroxyl groups, highly suggest substrate competition with subsequent O-glycosylations in ongoing ESC-GC transformations and affecting GC maturation. However, the membrane-bound somatic N/O-glycotransferases, which initiate, after formation of the zygote, the complex construction of the human ABO phenotypes in the trans cisternae of the Golgi apparatus, are associated and/or completed with soluble enzyme versions exerting identical specificities in plasma and likely competing vice versa by glycosylation of neonatal IgM amino acids, where they suggest to accomplish the clearance of anti-A autoreactivity at germline serine and threonine residues. Sustaining the lineage-maintaining position of the classic A allele and the discovery of the OA hybrid alleles at the normal ABO locus and in heterozygous ESC lines have, together with clinical observations, raised discussions about a silent A-allelic support within blood group O reproduction. However, the question of whether a fictional "continued blood group O inbreeding" ultimately occurs without the A-allelic or somatic function remains unanswered because the genetic relationship between non-somatic O-GalNAc-glycosylations that operate before sperm-egg recognition and somatic O-GalNAc-glycosylations that arise after the formation of the zygote remains to be elucidated. Copyright © 2015 Elsevier GmbH. All rights reserved.

Evidence for core 2 to core 1 O-glycan remodeling during the recycling of MUC1

PubMed Central

Razawi, Hanieh; Kinlough, Carol L; Staubach, Simon; Poland, Paul A; Rbaibi, Youssef; Weisz, Ora A; Hughey, Rebecca P; Hanisch, Franz-Georg

2013-01-01

The apical transmembrane glycoprotein MUC1 is endocytosed to recycle through the trans-Golgi network (TGN) or Golgi complex to the plasma membrane. We followed the hypothesis that not only the known follow-up sialylation of MUC1 in the TGN is associated with this process, but also a remodeling of O-glycan core structures, which would explain the previously described differential core 2- vs core 1-based O-glycosylation of secreted, single Golgi passage and recycling membrane MUC1 isoforms (Engelmann K, Kinlough CL, Müller S, Razawi H, Baldus SE, Hughey RP, Hanisch F-G. 2005. Glycobiology. 15:1111–1124). Transmembrane and secreted MUC1 probes show trafficking-dependent changes in O-glycan core profiles. To address this novel observation, we used recombinant epitope-tagged MUC1 (MUC1-M) and mutant forms with abrogated clathrin-mediated endocytosis (MUC1-M-Y20,60N) or blocked recycling (palmitoylation-defective MUC1-M-CQC/AQA). We show that the CQC/AQA mutant transits the TGN at significantly lower levels, concomitant with a strongly reduced shedding from the plasma membrane and its accumulation in endosomal compartments. Intriguingly, the O-glycosylation of the shed MUC1 ectodomain subunit changes from preponderant sialylated core 1 (MUC1-M) to core 2 glycans on the non-recycling CQC/AQA mutant. The O-glycoprofile of the non-recycling CQC/AQA mutant resembles the core 2 glycoprofile on a secretory MUC1 probe that transits the Golgi complex only once. In contrast, the MUC1-M-Y20,60N mutant recycles via flotillin-dependent pathways and shows the wild-type phenotype with dominant core 1 expression. Differential radiolabeling of protein with [35S]Met/Cys or glycans with [3H]GlcNH2 in pulse-chase experiments of surface biotinylated MUC1 revealed a significantly shorter half-life of [3H]MUC1 when compared with [35S]MUC1, whereas the same ratio for the CQC/AQA mutant was close to one. This finding further supports the novel possibility of a recycling-associated O-glycan processing from Gal1-4GlcNAc1-6(Gal1-3)GalNAc (core 2) to Gal1-3GalNAc (core 1). PMID:23640779

O-Linked-N-Acetylglucosamine Cycling and Insulin Signaling Are Required for the Glucose Stress Response in Caenorhabditis elegans

PubMed Central

Mondoux, Michelle A.; Love, Dona C.; Ghosh, Salil K.; Fukushige, Tetsunari; Bond, Michelle; Weerasinghe, Gayani R.; Hanover, John A.; Krause, Michael W.

2011-01-01

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose–insulin homeostasis. PMID:21441213

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galβ1-4GlcNAc) and LacdiNAc (GalNAcβ1-4GlcNAc) motifs found on vertebrate and insect cells.

PubMed

Frederiksen, Rikki F; Yoshimura, Yayoi; Storgaard, Birgit G; Paspaliari, Dafni K; Petersen, Bent O; Chen, Kowa; Larsen, Tanja; Duus, Jens Ø; Ingmer, Hanne; Bovin, Nicolai V; Westerlind, Ulrika; Blixt, Ola; Palcic, Monica M; Leisner, Jørgen J

2015-02-27

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1-4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1-6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1-6 bond in LacNAcβ1-6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Plasma N-glycosylation as Analyzed by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance-MS Associates with Markers of Inflammation and Metabolic Health.

PubMed

Reiding, Karli R; Ruhaak, L Renee; Uh, Hae-Won; El Bouhaddani, Said; van den Akker, Erik B; Plomp, Rosina; McDonnell, Liam A; Houwing-Duistermaat, Jeanine J; Slagboom, P Eline; Beekman, Marian; Wuhrer, Manfred

2017-02-01

Glycosylation is an abundant co- and post-translational protein modification of importance to protein processing and activity. Although not template-defined, glycosylation does reflect the biological state of an organism and is a high-potential biomarker for disease and patient stratification. However, to interpret a complex but informative sample like the total plasma N-glycome, it is important to establish its baseline association with plasma protein levels and systemic processes. Thus far, large-scale studies (n >200) of the total plasma N-glycome have been performed with methods of chromatographic and electrophoretic separation, which, although being informative, are limited in resolving the structural complexity of plasma N-glycans. MS has the opportunity to contribute additional information on, among others, antennarity, sialylation, and the identity of high-mannose type species.Here, we have used matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR)-MS to study the total plasma N-glycome of 2144 healthy middle-aged individuals from the Leiden Longevity Study, to allow association analysis with markers of metabolic health and inflammation. To achieve this, N-glycans were enzymatically released from their protein backbones, labeled at the reducing end with 2-aminobenzoic acid, and following purification analyzed by negative ion mode intermediate pressure MALDI-FTICR-MS. In doing so, we achieved the relative quantification of 61 glycan compositions, ranging from Hex 4 HexNAc 2 to Hex 7 HexNAc 6 dHex 1 Neu5Ac 4 , as well as that of 39 glycosylation traits derived thereof. Next to confirming known associations of glycosylation with age and sex by MALDI-FTICR-MS, we report novel associations with C-reactive protein (CRP), interleukin 6 (IL-6), body mass index (BMI), leptin, adiponectin, HDL cholesterol, triglycerides (TG), insulin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and smoking. Overall, the bisection, galactosylation, and sialylation of diantennary species, the sialylation of tetraantennary species, and the size of high-mannose species proved to be important plasma characteristics associated with inflammation and metabolic health. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Plasma N-glycosylation as Analyzed by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance-MS Associates with Markers of Inflammation and Metabolic Health*

PubMed Central

Reiding, Karli R.; Ruhaak, L. Renee; Uh, Hae-Won; el Bouhaddani, Said; van den Akker, Erik B.; Plomp, Rosina; McDonnell, Liam A.; Houwing-Duistermaat, Jeanine J.; Slagboom, P. Eline; Beekman, Marian; Wuhrer, Manfred

2017-01-01

Glycosylation is an abundant co- and post-translational protein modification of importance to protein processing and activity. Although not template-defined, glycosylation does reflect the biological state of an organism and is a high-potential biomarker for disease and patient stratification. However, to interpret a complex but informative sample like the total plasma N-glycome, it is important to establish its baseline association with plasma protein levels and systemic processes. Thus far, large-scale studies (n >200) of the total plasma N-glycome have been performed with methods of chromatographic and electrophoretic separation, which, although being informative, are limited in resolving the structural complexity of plasma N-glycans. MS has the opportunity to contribute additional information on, among others, antennarity, sialylation, and the identity of high-mannose type species. Here, we have used matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR)-MS to study the total plasma N-glycome of 2144 healthy middle-aged individuals from the Leiden Longevity Study, to allow association analysis with markers of metabolic health and inflammation. To achieve this, N-glycans were enzymatically released from their protein backbones, labeled at the reducing end with 2-aminobenzoic acid, and following purification analyzed by negative ion mode intermediate pressure MALDI-FTICR-MS. In doing so, we achieved the relative quantification of 61 glycan compositions, ranging from Hex4HexNAc2 to Hex7HexNAc6dHex1Neu5Ac4, as well as that of 39 glycosylation traits derived thereof. Next to confirming known associations of glycosylation with age and sex by MALDI-FTICR-MS, we report novel associations with C-reactive protein (CRP), interleukin 6 (IL-6), body mass index (BMI), leptin, adiponectin, HDL cholesterol, triglycerides (TG), insulin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and smoking. Overall, the bisection, galactosylation, and sialylation of diantennary species, the sialylation of tetraantennary species, and the size of high-mannose species proved to be important plasma characteristics associated with inflammation and metabolic health. PMID:27932526

Systemic illness moderates the impact of N-acetyl cysteine in bipolar disorder.

PubMed

Magalhães, P V; Dean, O M; Bush, A I; Copolov, D L; Weisinger, D; Malhi, G S; Kohlmann, K; Jeavons, S; Schapkaitz, I; Anderson-Hunt, M; Berk, M

2012-04-27

Bipolar disorder (BD) is intricately associated with chronic clinical conditions. Medical comorbidity is not only more prevalent in mood disorders, but is associated with increased costs, cognitive impairment and, ultimately, premature mortality. Oxidative stress and inflammation may mediate part of this association. To further investigate the association between medical comorbidity status and clinical improvement with adjuvant N acetyl cysteine (NAC) in the context of a placebo-controlled trial. Placebo-controlled randomized clinical trial assessing the effect of NAC over 24 weeks. Symptomatic and functional outcomes were collected over the study period. Medical comorbidities were self-reported, and we took special interest in cardiovascular and endocrine conditions. We evaluated change from baseline to endpoint and the interaction between change and reported medical comorbidities. Fifty-one percent of patients reported have a cardiovascular or endocrine comorbidity. Although not found for depressive symptoms or quality of life, a significant interaction between medical comorbidity and change scores was consistently found for all functional outcomes. This indicated an advantage of NAC over placebo in those with a clinical comorbidity. Systemic illness moderated only the effect of NAC on functioning, not on depression. Demonstrating an improvement in functional outcomes with an agent that modulates redox and inflammatory pathways, this study lends empirical support to the idea that medical and psychiatric comorbidity are additive in contributing to allostatic states. One intriguing possibility is that comorbid clinical illness could be a marker for more severe oxidative stress states--and thus guide antioxidant use--in BD. Copyright © 2011 Elsevier Inc. All rights reserved.

A single nucleotide polymorphism in MGEA5 encoding O-GlcNAc-selective N-acetyl-beta-D glucosaminidase is associated with type 2 diabetes in Mexican Americans.

PubMed

Lehman, Donna M; Fu, Dong-Jing; Freeman, Angela B; Hunt, Kelly J; Leach, Robin J; Johnson-Pais, Teresa; Hamlington, Jeanette; Dyer, Thomas D; Arya, Rector; Abboud, Hanna; Göring, Harald H H; Duggirala, Ravindranath; Blangero, John; Konrad, Robert J; Stern, Michael P

2005-04-01

Excess O-glycosylation of proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) may be involved in the pathogenesis of type 2 diabetes. The enzyme O-GlcNAc-selective N-acetyl-beta-d glucosaminidase (O-GlcNAcase) encoded by MGEA5 on 10q24.1-q24.3 reverses this modification by catalyzing the removal of O-GlcNAc. We have previously reported the linkage of type 2 diabetes and age at diabetes onset to an overlapping region on chromosome 10q in the San Antonio Family Diabetes Study (SAFADS). In this study, we investigated menangioma-expressed antigen-5 (MGEA5) as a positional candidate gene. Twenty-four single nucleotide polymorphisms (SNPs), identified by sequencing 44 SAFADS subjects, were genotyped in 436 individuals from 27 families whose data were used in the original linkage report. Association tests indicated significant association of a novel SNP with the traits diabetes (P = 0.0128, relative risk = 2.77) and age at diabetes onset (P = 0.0017). The associated SNP is located in intron 10, which contains an alternate stop codon and may lead to decreased expression of the 130-kDa isoform, the isoform predicted to contain the O-GlcNAcase activity. We investigated whether this variant was responsible for the original linkage signal. The variance attributed to this SNP accounted for approximately 25% of the logarithm of odds. These results suggest that this variant within the MGEA5 gene may increase diabetes risk in Mexican Americans.

Regional Differences in Striatal Neuronal Ensemble Excitability Following Cocaine and Extinction Memory Retrieval in Fos-GFP Mice.

PubMed

Ziminski, Joseph J; Sieburg, Meike C; Margetts-Smith, Gabriella; Crombag, Hans S; Koya, Eisuke

2018-03-01

Learned associations between drugs of abuse and the drug administration environment have an important role in addiction. In rodents, exposure to a drug-associated environment elicits conditioned psychomotor activation, which may be weakened following extinction (EXT) learning. Although widespread drug-induced changes in neuronal excitability have been observed, little is known about specific changes within neuronal ensembles activated during the recall of drug-environment associations. Using a cocaine-conditioned locomotion (CL) procedure, the present study assessed the excitability of neuronal ensembles in the nucleus accumbens core and shell (NAc core and NAc shell ), and dorsal striatum (DS) following cocaine conditioning and EXT in Fos-GFP mice that express green fluorescent protein (GFP) in activated neurons (GFP+). During conditioning, mice received repeated cocaine injections (20 mg/kg) paired with a locomotor activity chamber (Paired) or home cage (Unpaired). Seven to 13 days later, both groups were re-exposed to the activity chamber under drug-free conditions and Paired, but not Unpaired, mice exhibited CL. In a separate group of mice, CL was extinguished by repeatedly exposing mice to the activity chamber under drug-free conditions. Following the expression and EXT of CL, GFP+ neurons in the NAc core (but not NAc shell and DS) displayed greater firing capacity compared to surrounding GFP- neurons. This difference in excitability was due to a generalized decrease in GFP- excitability following CL and a selective increase in GFP+ excitability following its EXT. These results suggest a role for both widespread and ensemble-specific changes in neuronal excitability following recall of drug-environment associations.

Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides. Polypeptide vesicles by conformation-specific assembly. Ordered chiral macroporous hybrid silica-polypeptide composites

NASA Astrophysics Data System (ADS)

Bellomo, Enrico Giuseppe

2005-07-01

Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides . The aqueous, lyotropic liquid-crystalline phase behavior of an alpha helical polypeptide, has been studied using optical microscopy and X-ray scattering. Solutions of optically pure polypeptide were found to form cholesteric liquid crystals at volume fractions that decreased with increasing average chain length. At very high volume fractions, the formation of a hexagonal mesophase was observed. The pitch of the cholesteric phase could be varied by a mixture of enantiomeric samples, where the pitch increased as the mixture approached equimolar. The cholesteric phases could be untwisted, using either magnetic field or shear flow, into nematic phases, which relaxed into cholesterics upon removal of field or shear. We have found that the phase diagram of this polypeptide in aqueous solution parallels that of poly(gamma-benzyl glutamate) in organic solvents, thus providing a useful system for liquid-crystal applications requiring water as solvent. Polypeptide vesicles by conformation-specific assembly. We have found that block copolymers composed of polypeptide segments provide significant advantages in controlling both the function and supramolecular structure of bioinspired self-assemblies. Incorporation of the stable chain conformations found in proteins into block copolymers was found to provide an additional element of control, beyond amphiphilicity and composition that defines self-assembled architecture. The abundance of functionality present in amino acids, and the ease by which they can be incorporated into these materials, also provides a powerful mechanism to impart block copolypeptides with function. This combination of structure and function work synergistically to enable significant advantages in the preparation of therapeutic agents as well as provide insight into design of self-assemblies beginning to approach the complexity of natural structures such as virus capsids. Ordered chiral macroporous hybrid silica-polypeptide composites. The mineralization of organic templates has been investigated as an effective way to control the size and structure of inorganic frameworks. Hybrid structures incorporating polypeptide with silica have been prepared and characterized using X-ray scattering, TGA, SEM and TEM. The results support the interaction between silica and polymer to form ordered chiral macroporous structures that can be easily controlled by polymer molecular weight and volume fraction.

Protein Complexation and pH Dependent Release Using Boronic Acid Containing PEG-Polypeptide Copolymers.

PubMed

Negri, Graciela E; Deming, Timothy J

2017-01-01

New poly(L-lysine)-b-poly(ethylene glycol) copolypeptides have been prepared, where the side-chain amine groups of lysine residues are modified to contain ortho-amine substituted phenylboronic acid, i.e., Wulff-type phenylboronic acid (WBA), groups to improve their pH responsive, carbohydrate binding properties. These block copolymers form nanoscale complexes with glycosylated proteins that are stable at physiological pH, yet dissociate and release the glycoproteins under acidic conditions, similar to those found in endosomal and lysosomal compartments within cells. These results suggest that WBA modified polypeptide copolymers are promising for further development as degradable carriers for intracellular protein delivery. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

N-Acetylmannosamine improves sleep-wake quality in middle-aged mice: relevance to autonomic nervous function.

PubMed

Kuwahara, Masayoshi; Ito, Koichi; Hayakawa, Koji; Yagi, Shintaro; Shiota, Kunio

2015-01-01

Aging is associated with a variety of physiological changes originating peripherally and centrally, including within the autonomic nervous system. Sleep-wake disturbances constitute reliable hallmarks of aging in several animal species and humans. Recent studies have been interested in N-acetylmannosamine (ManNAc) a potential therapeutic agent for improving quality of life, as well as preventing age-related cognitive decline. In this study, ManNAc (5.0 mg/ml) was administered in the drinking water of middle-aged male C57BL/6J mice (55 weeks old) for 7 days. Mice were housed under a 12:12 h light:dark cycle at 23-24 °C. We evaluated bio-behavioral activity using electrocardiogram, body temperature and locomotor activity recorded by an implanted telemetry transmitter. To estimate sleep-wake profile, surface electroencephalogram and electromyogram leads connected to a telemetry transmitter were also implanted in mice. Autonomic nervous activity was evaluated using power spectral analysis of heart rate variability. ManNAc-treated mice spent more time in a wakeful state and less time in slow wave sleep during the dark phase. Parasympathetic nervous activity was increased following ManNAc treatment, then the sympatho-vagal balance was shifted predominance of parasympathetic nervous system. Furthermore, improvement in sleep-wake pattern was associated with increased parasympathetic nervous activity. These results suggest that ManNAc treatment can improve bio-behavioral activity and sleep-wake quality in middle-aged mice. This may have implications for improving sleep patterns in elderly humans. Copyright © 2014 Elsevier B.V. All rights reserved.

Cloning and characterization of a novel NAC family gene CarNAC1 from chickpea (Cicer arietinum L.).

PubMed

Peng, Hui; Yu, Xingwang; Cheng, Huiying; Shi, Qinghua; Zhang, Hua; Li, Jiangui; Ma, Hao

2010-01-01

The plant-specific NAC (for NAM, ATAF1,2 and CUC2) proteins have been found to play important roles in plant development and stress responses. In this study, a NAC gene CarNAC1 (for Cicer arietinum L. NAC gene 1) was isolated from a cDNA library constructed with chickpea seedling leaves treated by polyethylene glycol. CarNAC1 encoded a putative protein with 239 amino acids and contained 3 exons and 2 introns within genomic DNA sequence. CarNAC1 had a conserved NAC domain in the N-terminus and the CarNAC1:GFP (green fluorescent protein) fusion protein was localized in the nucleus of onion epidermal cells. Additionally, CarNAC1 exhibited the trans-activation activity which was mapped to the C-terminus. The CarNAC1 transcript was detected in many chickpea organs including seedling leaves, stems, roots, flowers, and young pods, but less accumulated in young seeds. CarNAC1 was induced by leaf age and showed changes in expression during seed development and germination. Furthermore, the expression of CarNAC1 was strongly induced by drought, salt, cold, wounding, H(2)O(2), ethephon, salicylic acid, indole-3-acetic acid, and gibberellin. Our results suggest that CarNAC1 encodes a novel NAC-domain protein and may be a transcriptional activator involved in plant development and various stress responses.

Reduction of the Areolar Diameter After Ultrasound-Assisted Liposuction for Gynecomastia.

PubMed

Keskin, Mustafa; Sutcu, Mustafa; Hanci, Mustafa; Cigsar, Bulent

2017-08-01

One of the clinical aspects characterizing gynecomastia is the enlargement of the nipple-areolar complex (NAC) due to hypertrophic breast glands, and the excessive fatty tissue underneath. The purpose of this study was to quantify the reduction of the areolar diameter after ultrasound-assisted liposuction (UAL) of the male breast. The horizontal diameters of the NACs of 30 men who underwent UAL were measured before surgery, 1 month after surgery and 6 months after surgery in a standard fashion. Those patients with surgical gland removals of any kind were not included in this study. The mean age of the patients was 27.9 years, and all of the patients had bilateral grade I, II, or III gynecomastia. The mean diameter of the NACs before surgery was 35.36 mm (range, 26-55 mm), and after surgery, the mean diameter of the NACs was initially reduced to 28.8 mm (range, 23-44 mm) and later to 28.57 mm (range, 23-42 mm). The mean volume of breast tissue aspirated was 382 mL per breast, and the percentage of reduction was 17.3%. The reduction of areola diameter was statistically significant after first month. A significant positive correlation was identified between the liposuction volume and areolar diameter reduction. In cases of gynecomastia, the removal of the glandular and fatty tissue underneath the areola releases the expanding forces and pressure that enlarge it. In many cases of gynecomastia, UAL alone is effective in reducing the size of the NAC and allows the surgeon to avoid placing scars on the breast.

Central pedicled breast reduction technique in male patients after massive weight loss.

PubMed

Stoff, Alexander; Velasco-Laguardia, Fernando J; Richter, Dirk F

2012-03-01

Male patients after massive weight loss often suffer from redundant skin and soft tissue in the anterior and lateral chest region, causing various deformities of pseudogynecomastia. Techniques with free or pedicled nipple-areola complex (NAC) transposition are widely accepted. The authors present their approach to male breast reduction with preservation of the NAC on a central dermoglandular pedicle and a wide elliptical tissue excision of breast and lateral thorax tissue in combination with liposuction. Male breast reduction was performed on patients after moderate to massive weight loss due to diet or bariatric procedures. Former procedures included free nipple-areola grafts or inferior pedicled techniques for NAC preservation. As a modification, we performed a central pedicled breast reduction on nine male patients with excessive liposuction of the pedicle and a horizontal elliptical skin removal, allowing for sufficient tissue removal at the lateral thorax. From October 2010 until June 2011, nine male patients had central pedicled breast reconstructions after massive weight loss. Mean age was 29.1 years, mean preoperative body mass index was 29.2, and mean preoperative weight loss was 63.9 kg. The chest wall improvement was rated "very good" by eight patients. No major complications occurred in all nine patients. Male chest deformities after massive weight loss can be dealt by several approaches. The optimal scar positioning and the preservation of NAC may be the most challenging aspects of these procedures. Therefore, the preservation of the NAC on a central dermoglandular pedicle with a horizontal submammary scar course may optimize the esthetic outcome.

Mesolimbic leptin signaling negatively regulates cocaine-conditioned reward.

PubMed

Shen, M; Jiang, C; Liu, P; Wang, F; Ma, L

2016-12-06

The regulatory mechanisms underlying the response to addictive drugs are complex, and increasing evidence indicates that there is a role for appetite-regulating pathways in substance abuse. Leptin, an important adipose hormone that regulates energy balance and appetite, exerts its physiological functions via leptin receptors. However, the role of leptin signaling in regulating the response to cocaine remains unclear. Here we examined the potential role of leptin signaling in cocaine reward using a conditioned place preference (CPP) procedure. Our results showed that inhibition of leptin signaling by intracerebroventricular infusion of the leptin receptor (LepR) antagonist SMLA during cocaine conditioning increased the cocaine-CPP and upregulated the level of dopamine and its metabolites in the nucleus accumbens (NAc). We then selectively knocked down the LepR in the mesolimbic ventral tegmental area (VTA), NAc core and central amygdala (CeA) by injecting AAV-Cre into Lepr flox/flox mice. LepR deletion in the VTA increased the dopamine levels in the NAc and enhanced the cocaine-conditioned reward. LepR deletion in the NAc core enhanced the cocaine-conditioned reward and impaired the effect of the D2-dopamine receptor on cocaine-CPP, whereas LepR deletion in the CeA had no effect on cocaine-CPP but increased the anxiety level of mice. In addition, prior exposure to saccharin increased LepR mRNA and STAT3 phosphorylation in the NAc and VTA and impaired cocaine-CPP. These results indicate that leptin signaling is critically involved in cocaine-conditioned reward and the regulation of drug reward by a natural reward and that these effects are dependent on mesolimbic LepR.

The selective solubilization of different murine splenocyte membrane fractions with lubrol WX and triton X-100 distinguishes two forms of Ia antigens. A cell surface (alpha, beta) and an intracellular (alpha, Ii, beta).

PubMed

Moosic, J P; Sung, E; Nilson, A; Jones, P P; McKean, D J

1982-08-25

The selective solubilization of different murine lymphocyte membrane compartments with several nonionic detergents was used to study the subcellular distribution of two distinct forms of lymphocyte cell recognition structures (Ia antigens). Ia antigens were isolated with a monoclonal anti-Ia immunoadsorbent from murine splenocytes that had been solubilized with four different nonionic detergents. Analyses of the immunoprecipitates indicated that Lubrol WX was selectively solubilizing a subpopulation of Ia consisting of mature highly glycosylated alpha and beta polypeptides which were not associated with Ii polypeptide. A second Ia subpopulation consisting of less glycosylated cytoplasmic precursor alpha and beta polypeptides associated with Ii polypeptide was immunoprecipitated from the Lubrol WX-insoluble material after solubilizing this material with Triton X-100. Comparable results were obtained when HLA-DR antigens were similarly isolated from cultured human lymphoblastoid cells. This selective solubilization phenomenon was not unique to Ia antigens. Only mature highly glycosylated H-2K molecules were immunoprecipitated from the Lubrol WX-soluble material while the less glycosylated precursor H-2K molecules were immunoprecipitated from the Triton X-100-solubilized Lubrol-insoluble material. These data directly demonstrate that the Ii polypeptide is exclusively associated with the intracellular Ia antigen cytoplasmic precursor molecules. These data also indicate that, under the conditions used in these experiments, Lubrol WX does not completely solubilize integral membrane proteins that have previously been shown to be associated with the rough endoplasmic reticulum.

Probing the organic-mineral interface at the molecular level in model biominerals.

PubMed

Metzler, Rebecca A; Kim, Il Won; Delak, Katya; Evans, John Spencer; Zhou, Dong; Beniash, Elia; Wilt, Fred; Abrecht, Mike; Chiou, Jau-Wern; Guo, Jinghua; Coppersmith, Susan N; Gilbert, P U P A

2008-03-18

It is widely known that macromolecules, such as proteins, can control the nucleation and growth of inorganic solids in biomineralizing organisms. However, what is not known are the complementary molecular interactions, organization, and rearrangements that occur when proteins interact with inorganic solids during the formation of biominerals. The organic-mineral interface (OMI) is expected to be the site for these phenomena, and is therefore extraordinarily interesting to investigate. In this report, we employ X-ray absorption near edge (XANES) spectromicroscopy to investigate the electronic structure of both calcium carbonate mineral crystals and polypeptides, and detect changing bonds at the OMI during crystal growth in the presence of polypeptides. We acquired XANES spectra from calcium carbonate crystals grown in the presence of three mollusk nacre-associated polypeptides (AP7N, AP24N, n16N) and in the presence of a sea urchin spicule matrix protein, LSM34. All these model biominerals gave similar results, including the disruption of CO bonds in calcite and enhancement of the peaks associated with C-H bonds and C-O bonds in peptides, indicating ordering of the amino acid side chains in the mineral-associated polypeptides and carboxylate binding. This is the first evidence of the mutual effect of calcite on peptide chain and peptide chain on calcite during biomineralization. We also show that these changes do not occur when Asp and Glu are replaced in the n16N sequence with Asn and Gln, respectively, demonstrating that carboxyl groups in Asp and Glu do participate in polypeptide-mineral molecular associations.

The effects of GABAA and NMDA receptors in the shell-accumbens on spatial memory of METH-treated rats.

PubMed

Heysieattalab, Soomaayeh; Naghdi, Nasser; Zarrindast, Mohammad-Reza; Haghparast, Abbas; Mehr, Shahram Ejtemaei; Khoshbouei, Habibeh

2016-03-01

Methamphetamine (METH) is a highly addictive and neurotoxic psychostimulant. Its use in humans is often associated with neurocognitive impairment and deficits in hippocampal plasticity. Striatal dopamine system is one of the main targets of METH. The dopamine neurons in the striatum directly or indirectly regulate the GABA and glutamatergic signaling in this region and thus their outputs. This is consistent with previous reports showing modification of neuronal activity in the striatum modulates the expression of hippocampal LTP and hippocampal-dependent memory tasks such as Morris water maze (MWM). Therefore, reversing or preventing METH-induced synaptic modifications via pharmacological manipulations of the shell-nucleus accumbens (shell-NAc) may introduce a viable therapeutic target to attenuate the METH-induced memory deficits. This study is designed to investigate the role of intra-shell NAc manipulation of GABAA and NMDA receptors and their interaction with METH on memory performance in MWM task. Pharmacological manipulations were performed in rats received METH or saline. We found systemic saline plus intra-shell NAc infusions of muscimol dose-dependently impaired performance, while bicuculline had no effect. Surprisingly, the intra-NAc infusions of 0.005μg/rat muscimol that has no effect on memory performance (ineffective dose) prevented METH-induced memory impairment. In the contrary, the intra-NAc infusions of bicuculline (0.2μg/rat) increased METH-induced memory impairment. However, pre-training intra-NAc infusions of D-AP5 dose-dependently impaired performance, while NMDA had no effect in rats received systemic saline (control group). The intra-NAc infusions with an ineffective dose of NMDA (0.1μg/rat) increased METH-induced memory impairment. Furthermore, intra-NAc infusions of D-AP5 with an ineffective dose (0.1μg/rat) prevented METH-induced memory impairment. Our result is consistent with the interpretation that METH-mediated learning deficit might be due to modification of hippocampus-VTA loop and that augmentation of GABAA receptor function in the shell-NAc may provide a new therapeutic target for alleviating METH-induced memory deficits. Copyright © 2015. Published by Elsevier Inc.

Improvement of cognitive function in schizophrenia with N-acetylcysteine: A theoretical review.

PubMed

Yolland, Caitlin O B; Phillipou, Andrea; Castle, David J; Neill, Erica; Hughes, Matthew E; Galletly, Cherrie; Smith, Zoe M; Francis, Paul S; Dean, Olivia M; Sarris, Jerome; Siskind, Dan; Harris, Anthony W F; Rossell, Susan L

2018-05-30

Schizophrenia is a debilitating psychiatric illness associated with positive and negative symptoms as well as significant impairments in cognition. Current antipsychotic medications do not alleviate these cognitive deficits, and more effective therapeutic options are required. Increased oxidative stress and altered antioxidant levels, including glutathione (GSH) have been observed both in individuals with cognitive impairment and in people with schizophrenia. A GSH precursor, the antioxidant N-acetylcysteine (NAC) has been investigated as a novel treatment for the cognitive symptoms of schizophrenia, and recent research suggests that NAC may be a promising adjunctive treatment option. However, the current literature lacks integration as to why NAC may effectively improve cognition in schizophrenia. The present theoretical synthesis aimed to address this gap by examining the processes by which NAC may improve cognitive function in schizophrenia. The schizophrenia literature was reviewed in three key domains: cognitive impairment, the relationship between oxidative stress and cognition, and the efficacy of NAC as a novel treatment. This led to a theoretical analysis of the neurobiological processes by which NAC may improve cognition in schizophrenia. This theoretical review concluded that improved cognition may result from a combination of factors, including decreased oxidative stress, neuroprotection of cognitive networks and an increase in glutamatergic modulation of the N-methyl-d-aspartate receptor system. Whilst a number of mechanisms by which NAC may improve cognition and symptoms in schizophrenia have been proposed, there is still limited understanding of the specific metabolic pathways involved and how they interrelate and modify specific symptomology. Exploration of how NAC treatment may act to improve cognitive function could guide clinical trials by investigation of the specific neurotransmitter systems and processes involved, allowing for targeted neurological outcome measures. Future research would benefit from the investigation of both in vivo cortical GSH concentration and peripheral plasma GSH in a population of individuals with chronic schizophrenia.

Metabolomic Analysis of N-acetylcysteine Protection of Injury from Gadolinium-DTPA Contrast Agent in Rats with Chronic Renal Failure.

PubMed

Wan, Chuanling; Xue, Rong; Zhan, Youyang; Wu, Yijie; Li, Xiaojing; Pei, Fengkui

2017-09-01

Gadolinium-based contrast agents (GBCAs) are frequently used to enhance the diagnostic efficacy of magnetic resonance imaging. On the other hand, the association between GBCA administration in patients with advanced renal disease and nephrogenic systemic fibrosis (NSF) was also noted. NSF is a systemic disorder characterized by widespread tissue fibrosis that may lead to death. N-acetylcysteine (NAC) protects rats from injury induced by gadolinium-based contrast agents, but the underlying mechanisms remain unclear. In this study, a nuclear magnetic resonance-based metabolomic approach was used to systematically investigate the protective effects of NAC on Gd-DTPA-induced injury. Thirty-two male Sprague-Dawley rats were given adenine (200 mg·kg -1 body weight) by oral gavage once a day for 3 weeks to induce chronic renal failure (CRF). NAC (600 mg/L in drinking water for 9 days) pretreatment was initiated 2 days before Gd-DTPA injection (a single tail vein injection, 2 mmol/kg body weight). Serum and liver samples were collected on day 7 after Gd-DTPA injection. By study design, the serum and hepatic metabolic changes of rats were measured in four groups of eight each: CRF, CRF-Gd, CRF-Gd-NAC, and CRF-NAC. Gd-DTPA administration to rats with CRF resulted in disturbances of several metabolic pathways, including glucose, lipid, glutamate, choline, gut microbiota, one-carbon, and purine metabolism. NAC pretreatment reversed the abundance changes of high-density lipoprotein, low-density lipoprotein, very low-density lipoprotein, glutamate, glutamine, oxidized glutathione, choline, phosphocholine, glycerophosphocholine, trimethylamine, and trimethylamine-N-oxide induced by Gd-DTPA. It is noteworthy, however, that the ameliorating effects of NAC on the disturbance of glutamate, choline, and gut microbiota metabolism may be specific to Gd-DTPA. In all, these findings could be potentially useful to decipher the underlying mechanisms of NAC protective effects from the injury induced by gadolinium-based contrast agents.

Rapid risk household screening by neonatal arm circumference: results from a cohort study in rural Burkina Faso.

PubMed

Benzler, J; Sauerborn, R

1998-12-01

Neonatal arm circumference (NAC) and other attributes of the newborn and its household were analysed as potential predictors of child death in a cohort of 1367 newborn children representing the majority of births in a rural area of Burkina Faso from 1992 to 1994. During 3872 person years observed 264 children died, resulting in an average mortality rate of 6.8% per year. 90 mm was chosen as the best cut-off to differentiate low NAC associated with high mortality from normal NAC. The hazard ratio of children with low NAC (15.7%) compared to others was 1.7 (P < 0.001) in Cox regression. Kaplan-Meier curves of cumulative survival showed that this higher risk lasted throughout the first two years of life. Multivariate Cox regression comparing NAC with other variables known or suspected to influence child survival yielded a model including mother's death, twin birth, affiliation to a particular health centre, home delivery and birth during the rainy or harvest season as other significant risk factors beside NAC. Protective factors were mother's participation in antenatal care despite considerable distance to the health centre, medium household size (5-7 members) and household cash crop production. We propose a simple risk score for rapid household screening in rural Burkina Faso and comparable settings elsewhere for identifying households at risk of experiencing child death. As much of the other variables' contribution to the explanation of survival pattern is absorbed by NAC in more parsimonious models, even simpler screening strategies based on NAC make sense. In the study area risk households will be offered periodical home visits by the local nurse promoting immunization, treatment of illness and strengthening the mothers' competence to recognize and manage frequent health problems of their children as part of a 'Shared Care' concept.

Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

PubMed Central

Labokha, Aksana A; Gradmann, Sabine; Frey, Steffen; Hülsmann, Bastian B; Urlaub, Henning; Baldus, Marc; Görlich, Dirk

2013-01-01

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ⩾30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability. PMID:23202855

The effect of molecular subtype and body mass index on neo-adjuvant chemotherapy in breast cancer patients.

PubMed

Iwase, Toshiaki; Nakamura, Rikiya; Yamamoto, Naohito; Yoshi, Atushi; Itami, Makiko; Miyazaki, Masaru

2014-06-01

The aim of the present study was to analyze the effect of subtype and body mass index (BMI) on neo-adjuvant chemotherapy (NAC) and postoperative prognosis. Two-hundred and forty nine patients who underwent surgery after NAC were included. A multivariate analysis and survival analysis were used to clarify the relationship between BMI, subtype, and NAC. In the logistic regression model, the pCR rate had a significant relationship with the subtype and tumor stage. In the non-pCR group, more overweight patients had significantly a worse disease-free survival (DFS) compared to normal range patients (Log lank test, p < 0.05). In the Cox proportional hazards model, subtype and tumor stage were significantly associated with decreased DFS. In conclusion, patients with the ER (+), HER (-) type and a high BMI had a high risk for recurrence when they achieved non-pCR after NAC. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nail-biting stuff? The effect of N-acetyl cysteine on nail-biting.

PubMed

Berk, Michael; Jeavons, Sue; Dean, Olivia M; Dodd, Seetal; Moss, Kirsteen; Gama, Clarissa S; Malhi, Gin S

2009-07-01

N-acetyl cysteine (NAC) is a widely available nutraceutical with a variety of actions. As a precursor of cysteine and glutathione, it has antioxidant properties that may impact on mood and contribute to an effect on impulsivity and obsessive behaviour. Via its additional effect on glutamate via the cystine-glutamate exchange system, NAC has been shown to mediate impulsivity in preclinical models of addiction, reduce craving, and cue extinction. Further, by boosting glutathione, NAC acts as a potent antioxidant and has been shown in two positive, large-scale randomized placebo-controlled trials to affect negative symptoms in schizophrenia and depression in bipolar disorder. We describe three cases in which its actions specifically on nail-biting and associated anxiety may offer a potential treatment. The spontaneous findings are reported as part of an ongoing treatment trial examining the utility of NAC in bipolar disorder. Its actions, if robustly replicated, also point to potential treatment targets in glutathione or glutamate pathways in the brain.

Creating Future Stem Leaders: The National Astronomy Consortium:

NASA Astrophysics Data System (ADS)

Sheth, Kartik; Mills, Elisabeth A. C.; Boyd, Patricia T.; Strolger, Louis-Gregory; Benjamin, Robert A.; Brisbin, Drew; Giles, Faye; National Astronomy Consortium

2016-01-01

The National Astronomy Consortium (NAC) is a program led by the National Radio Astronomy Observatory (NRAO) and Associated Universities Inc., (AUI) in partnership with the National Society of Black Physicists (NSBP), and a number of minority and majority universities to increase the numbers of students from underrepresented groups and those otherwise overlooked by the traditional academic pipeline into STEM or STEM-related careers. The seed for the NAC was a partnership between NRAO and Howard University which began with an exchange of a few summer students five years ago. Since then the NAC has grown tremendously. Today the NAC aims to host between 4 to 5 cohorts nationally in an innovative model in which the students are mentored throughout the year with multiple mentors and peer mentoring, continued engagement in research and professional development / career training throughout the academic year and throughout their careers. We will summarize the results from this innovative and highly succesful program and provide lessons learned.

Effects of dexpanthenol and N-acetylcysteine pretreatment in rats before renal ischemia/reperfusion injury.

PubMed

Sen, Huseyin; Deniz, Suleyman; Yedekci, A Erturk; Inangil, Gokhan; Muftuoglu, Tuba; Haholu, Aptullah; Ozkan, Sezai

2014-11-01

We investigated the anti-inflammatory and protective effects of concomitant use of dexpanthenol (DXP) and N-acetylcysteine (NAC) induced ischemia/reperfusion (I/R) injury of kidney. Forty rats were randomly divided into 5 groups. In all groups except for Group 1(Sham), renal arteries bilaterally occluded with vascular clamp for IR injury. Group 1(Sham), received a single dose of 10 mL/kg isotonic saline daily by intraperitoneal (IP) injection for three days. Group 2(IR), received a single dose of 10 mL/kg isotonic saline daily by IP injection for three days. Group 3(IR + NAC), received 300 mg/kg NAC daily by IP injection for three days. Group 4(IR + DXP), received 500 mg/kg DXP daily by IP injection for three days. Group 5(IR + NAC + DXP), received 500 mg/kg DXP and 300 mg/kg NAC daily by IP injection for three days. Serum urea (BUN), creatinine (Cr) and neutrophil gelatinase-associated lipocalin (NGAL, lipocalin 2, siderocalin) levels were measured as kidney function tests. TNF-α levels were measured as inflammatory marker. Tissue sections were evaluated histopathologically under light microscopy. IR + NAC + DXP group received both NAC and DXP before induction of renal I/R and as the biochemical and histopathological data revealed the results of the IR + NAC + DXP group and sham group were similar. Biochemically and histopathologically, combined use of NAC and DXP has better results when each of them used alone. We concluded that concomitant use of DXP and NAC plays a major role against I/R injury and may be useful in acute treatment of I/R induced renal failure.

N-acetylcysteine ameliorates contrast‑induced kidney injury in rats with unilateral hydronephrosis.

PubMed

Xia, Qiang; Liu, Chunxiao; Zheng, Xia

2018-02-01

The aim of the present study was to investigate the protective effects of N‑acetylcysteine (NAC) on contrast‑induced acute kidney injury in rats with unilateral hyronephrosis. Eighty‑two male Sprague Dawley rats were randomized to undergo sham operation (n=14) or unilateral ureteral obstruction (UUO) (n=68). After 3 weeks, the UUO animals were randomized to three groups: NAC gastric perfusion, UUO+iohexol+NAC (n=24); normal saline perfusion, UUO+iohexol (n=24); and controls, UUO (n=20). After 3 days, UUO+iohexol+NAC and UUO+iohexol rats were injected with iohexol. One day after contrast, half of the rats were sacrificed to assess the pathological changes to the kidneys, serum creatinine, serum neutrophil gelatinase‑associated lipocalin (NGAL), renal cell apoptosis rate and expression of apoptosis regulators Bcl‑2/Bax. The remaining rats underwent obstruction relief and were analyzed 3 weeks later. Compared with the controls, serum NGAL levels were high in UUO+iohexol rats 1 day following injection and 3 weeks after obstruction relief, but UUO+iohexol+NAC rats exhibited lower serum NGAL levels compared with UUO+iohexol rats (all P<0.05). Following modeling, UUO+iohexol rats exhibited a significantly higher apoptosis rate of renal tubular cells, higher expression of Bax mRNA, and lower ratio of Bcl‑2/Bax (all P<0.05). Three weeks after obstruction relief, UUO+iohexol+NAC rats exhibited a lower apoptosis rate, lower Bax mRNA expression, higher expression of Bcl‑2 mRNA and higher ratio of Bcl‑2/Bax (all P<0.05) compared with day 1 following drug administration. The prophylactic use of NAC reduced the apoptotic rate of renal tubular cells following contrast exposition, which was accompanied by changes in the expression of Bcl‑2/Bax mRNA.

N-Acetylcysteine for Nonsuicidal Self-Injurious Behavior in Adolescents: An Open-Label Pilot Study.

PubMed

Cullen, Kathryn R; Klimes-Dougan, Bonnie; Westlund Schreiner, Melinda; Carstedt, Patricia; Marka, Nicholas; Nelson, Katharine; Miller, Michael J; Reigstad, Kristina; Westervelt, Ana; Gunlicks-Stoessel, Meredith; Eberly, Lynn E

2018-03-01

Nonsuicidal self-injury (NSSI) is common in adolescents and young adults, and few evidence-based treatments are available for this significant problem. N-acetylcysteine (NAC) is a widely available nutritional supplement that has been studied in some psychiatric disorders relevant to NSSI including mood and addictive disorders. This pilot study tested the use of NAC as a potential treatment for NSSI in youth. Thirty-five female adolescents and young adults with NSSI aged 13-21 years were enrolled in this study that had an open-label, single-arm study design. All participants were given oral NAC as follows: 600 mg twice daily (weeks 1-2), 1200 mg twice daily (weeks 3-4), and 1800 mg twice daily (weeks 5-8). Patients were seen every 2 weeks throughout the trial, at which time youth reported the frequency of NSSI episodes. Levels of depression, impulsivity, and global psychopathology were measured at baseline and at the end of the trial using the Beck Depression Inventory-II (BDI-II), Barratt Impulsivity Scale, and Symptoms Checklist-90 (SCL-90). About two-thirds of the enrolled female youth completed the trial (24/35). NAC was generally well tolerated in this sample. NAC treatment was associated with a significant decrease in NSSI frequency at visit 6 and visit 8 compared to baseline. We also found that depression scores and global psychopathology scores (but not impulsivity scores) decreased after NAC treatment. Decrease in NSSI was not correlated with decrease in BDI-II or SCL-90 scores, suggesting these might be independent effects. We provide preliminary evidence that NAC may have promise as a potential treatment option for adolescents with NSSI. The current results require follow-up with a randomized, placebo-controlled trial to confirm efficacy.

Locomotor sensitization to intermittent ketamine administration is associated with nucleus accumbens plasticity in male and female rats.

PubMed

Strong, C E; Schoepfer, K J; Dossat, A M; Saland, S K; Wright, K N; Kabbaj, M

2017-07-15

Clinical evidence suggests superior antidepressant response over time with a repeated, intermittent ketamine treatment regimen as compared to a single infusion. However, the club drug ketamine is commonly abused. Therefore, the abuse potential of repeated ketamine injections at low doses needs to be investigated. In this study, we investigated the abuse potential of repeated exposure to either 0, 2.5, or 5 mg/kg ketamine administered once weekly for seven weeks. Locomotor activity and conditioned place preference (CPP) were assayed to evaluate behavioral sensitization to the locomotor activating effects of ketamine and its rewarding properties, respectively. Our results show that while neither males nor females developed CPP, males treated with 5 mg/kg and females treated with either 2.5 or 5 mg/kg ketamine behaviorally sensitized. Furthermore, dendritic spine density was increased in the NAc of both males and females administered 5 mg/kg ketamine, an effect specific to the NAc shell (NAcSh) in males but to both the NAc core (NAcC) and NAcSh in females. Additionally, males administered 5 mg/kg ketamine displayed increased protein expression of ΔfosB, calcium calmodulin kinase II alpha (CaMKIIα), and brain-derived neurotrophic factor (BDNF), an effect not observed in females administered either dose of ketamine. However, males and females administered 5 mg/kg ketamine displayed increased protein expression of AMPA receptors (GluA1). Taken together, low-dose ketamine, when administered intermittently, induces behavioral sensitization at a lower dose in females than males, accompanied by an increase in spine density in the NAc and protein expression changes in pathways commonly implicated in addiction. Copyright © 2017. Published by Elsevier Ltd.

N-acetylcysteine reverses diastolic dysfunction and hypertrophy in familial hypertrophic cardiomyopathy

PubMed Central

Wilder, Tanganyika; Ryba, David M.; Wieczorek, David F.; Wolska, Beata M.

2015-01-01

S-glutathionylation of cardiac myosin-binding protein C (cMyBP-C) induces Ca2+ sensitization and a slowing of cross-bridge kinetics as a result of increased oxidative signaling. Although there is evidence for a role of oxidative stress in disorders associated with hypertrophic cardiomyopathy (HCM), this mechanism is not well understood. We investigated whether oxidative myofilament modifications may be in part responsible for diastolic dysfunction in HCM. We administered N-acetylcysteine (NAC) for 30 days to 1-mo-old wild-type mice and to transgenic mice expressing a mutant tropomyosin (Tm-E180G) and nontransgenic littermates. Tm-E180G hearts demonstrate a phenotype similar to human HCM. After NAC administration, the morphology and diastolic function of Tm-E180G mice was not significantly different from controls, indicating that NAC had reversed baseline diastolic dysfunction and hypertrophy in our model. NAC administration also increased sarco(endo)plasmic reticulum Ca2+ ATPase protein expression, reduced extracellular signal-related kinase 1/2 phosphorylation, and normalized phosphorylation of phospholamban, as assessed by Western blot. Detergent-extracted fiber bundles from NAC-administered Tm-E180G mice showed nearly nontransgenic (NTG) myofilament Ca2+ sensitivity. Additionally, we found that NAC increased tension cost and rate of cross-bridge reattachment. Tm-E180G myofilaments were found to have a significant increase in S-glutathionylation of cMyBP-C, which was returned to NTG levels upon NAC administration. Taken together, our results indicate that oxidative myofilament modifications are an important mediator in diastolic function, and by relieving this modification we were able to reverse established diastolic dysfunction and hypertrophy in HCM. PMID:26432840

N-acetylcysteine protects against star fruit-induced acute kidney injury.

PubMed

Shimizu, Maria Heloisa Massola; Gois, Pedro Henrique França; Volpini, Rildo Aparecido; Canale, Daniele; Luchi, Weverton Machado; Froeder, Leila; Heilberg, Ita Pfeferman; Seguro, Antonio Carlos

2017-11-01

Star fruit (SF) is a popular fruit, commonly cultivated in many tropical countries, that contains large amount of oxalate. Acute oxalate nephropathy and direct renal tubular damage through release of free radicals are the main mechanisms involved in SF-induced acute kidney injury (AKI). The aim of this study was to evaluate the protective effect of N-acetylcysteine (NAC) on SF-induced nephrotoxicity due to its potent antioxidant effect. Male Wistar rats received SF juice (4 mL/100 g body weight) by gavage after a 12 h fasting and water deprivation. Fasting and water deprivation continued for 6 h thereafter to warrant juice absorption. Thereafter, animals were allocated to three experimental groups: SF (n = 6): received tap water; SF + NAC (n = 6): received NAC (4.8 g/L) in drinking water for 48 h after gavage; and Sham (n = 6): no interventions. After 48 h, inulin clearance studies were performed to determine glomerular filtration rate. In a second series of experiment, rats were housed in metabolic cages for additional assessments. SF rats showed markedly reduced inulin clearance associated with hyperoxaluria, renal tubular damage, increased oxidative stress and inflammation. NAC treatment ameliorated all these alterations. Under polarized light microscopy, SF rats exhibited intense calcium oxalate birefringence crystals deposition, dilation of renal tubules and tubular epithelial degeneration, which were attenuate by NAC therapy. Our data show that therapeutic NAC attenuates renal dysfunction in a model of acute oxalate nephropathy following SF ingestion by reducing oxidative stress, oxaluria, and inflammation. This might represent a novel indication of NAC for the treatment of SF-induced AKI.

N-acetyl-cysteine increases cellular dysfunction in progressive chronic kidney damage after acute kidney injury by dampening endogenous antioxidant responses.

PubMed

Small, David M; Sanchez, Washington Y; Roy, Sandrine F; Morais, Christudas; Brooks, Heddwen L; Coombes, Jeff S; Johnson, David W; Gobe, Glenda C

2018-05-01

Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI), but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI, but their benefits in CKD are controversial since clinical and preclinical investigations often conflict. Here we examined the influence of the antioxidant N-acetyl-cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-min bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice. NAC (5% in diet) was given to mice 7 days prior and up to 21 days post-IR (21d-IR). NAC treatment resulted in the following: prevented proximal tubular epithelial cell apoptosis at early IR (40-min postischemia), yet enhanced interstitial cell proliferation at 21d-IR; increased transforming growth factor-β1 expression independent of IR time; and significantly dampened nuclear factor-like 2-initiated cytoprotective signaling at early IR. In the long term, NAC enhanced cellular metabolic impairment demonstrated by increased peroxisome proliferator activator-γ serine-112 phosphorylation at 21d-IR. Intravital multiphoton microscopy revealed increased endogenous fluorescence of nicotinamide adenine dinucleotide (NADH) in cortical tubular epithelial cells during ischemia, and at 21d-IR that was not attenuated with NAC. Fluorescence lifetime imaging microscopy demonstrated persistent metabolic impairment by increased free/bound NADH in the cortex at 21d-IR that was enhanced by NAC. Increased mitochondrial dysfunction in remnant tubular cells was demonstrated at 21d-IR by tetramethylrhodamine methyl ester fluorimetry. In summary, NAC enhanced progression to CKD following AKI not only by dampening endogenous cellular antioxidant responses at time of injury but also by enhancing persistent kidney mitochondrial and metabolic dysfunction.

Normobaric Hyperoxia Extends Neuro- and Vaso-Protection of N-Acetylcysteine in Transient Focal Ischemia.

PubMed

Liu, Yushan; Liu, Wen-Cao; Sun, Yanyun; Shen, Xianzhi; Wang, Xiaona; Shu, Hui; Pan, Rong; Liu, Chun-Feng; Liu, Wenlan; Liu, Ke Jian; Jin, Xinchun

2017-07-01

N-acetylcysteine (NAC), a precursor of glutathione that reduces reperfusion-induced injury, has been shown protection when it was administered pre-ischemia. However, less is known about the effect when it was given post-ischemia and there is no positive result associated with anti-oxidant in clinical trials. This study investigated the neuro- and vaso-protection of post-ischemia NAC administration as well as combining NAC with normobaric hyperoxia (NBO). Male Sprague-Dawley rats were exposed to NBO or normoxia during 2-h occlusion of the middle cerebral artery, followed by 48-h reperfusion. NAC or vehicle was intraperitoneally administered to rats immediately before reperfusion onset. NAC and NBO treatments produced 1.2 and 30 % reduction of infarction volume, respectively, and combination treatment showed greater reduction (59.8 %) as well as more decrease of hemispheric swelling volume. Of note, combination therapy showed improved neurological assessment and motor function which were sustained for 7 days after reperfusion. We also determined that the combination therapy showed greater inhibitory effects on tight junction protein degradation accompanied by Evan's blue extravasation, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) induction, and poly ADP-ribose polymerase (PARP)-1 activation in ischemic brain tissue. Our results showed that although post-ischemia NAC administration had limited protection, combination treatment of NAC plus NBO effectively prevented blood-brain barrier (BBB) damage and significantly improved the outcome of brain injury, providing new evidence to support the concept that "cocktail" treatment targeting different stages provides better neuro- and vaso-protection than current individual treatment that has all failed in their clinical trials.

Reduced Slc6a15 in Nucleus Accumbens D2-Neurons Underlies Stress Susceptibility.

PubMed

Chandra, Ramesh; Francis, T Chase; Nam, Hyungwoo; Riggs, Lace M; Engeln, Michel; Rudzinskas, Sarah; Konkalmatt, Prasad; Russo, Scott J; Turecki, Gustavo; Iniguez, Sergio D; Lobo, Mary Kay

2017-07-05

Previous research demonstrates that Slc6a15, a neutral amino acid transporter, is associated with depression susceptibility. However, no study examined Slc6a15 in the ventral striatum [nucleus accumbens (NAc)] in depression. Given our previous characterization of Slc6a15 as a striatal dopamine receptor 2 (D2)-neuron-enriched gene, we examined the role of Slc6a15 in NAc D2-neurons in mediating susceptibility to stress in male mice. First, we showed that Slc6a15 mRNA was reduced in NAc of mice susceptible to chronic social defeat stress (CSDS), a paradigm that produces behavioral and molecular adaptations that resemble clinical depression. Consistent with our preclinical data, we observed Slc6a15 mRNA reduction in NAc of individuals with major depressive disorder (MDD). The Slc6a15 reduction in NAc occurred selectively in D2-neurons. Next, we used Cre-inducible viruses combined with D2-Cre mice to reduce or overexpress Slc6a15 in NAc D2-neurons. Slc6a15 reduction in D2-neurons caused enhanced susceptibility to a subthreshold social defeat stress (SSDS) as observed by reduced social interaction, while a reduction in social interaction following CSDS was not observed when Slc6a15 expression in D2-neurons was restored. Finally, since both D2-medium spiny neurons (MSNs) and D2-expressing choline acetyltransferase (ChAT) interneurons express Slc6a15, we examined Slc6a15 protein in these interneurons after CSDS. Slc6a15 protein was unaltered in ChAT interneurons. Consistent with this, reducing Slc5a15 selectively in NAc D2-MSNs, using A2A-Cre mice that express Cre selectively in D2-MSNs, caused enhanced susceptibility to SSDS. Collectively, our data demonstrate that reduced Slc6a15 in NAc occurs in MDD individuals and that Slc6a15 reduction in NAc D2-neurons underlies stress susceptibility. SIGNIFICANCE STATEMENT Our study demonstrates a role for reduced Slc6a15, a neutral amino acid transporter, in nucleus accumbens (NAc) in depression and stress susceptibility. The reduction of Slc6a15 occurs selectively in the NAc D2-neurons. Genetic reduction of Slc6a15 induces susceptibility to a subthreshold stress, while genetic overexpression in D2-neurons prevents social avoidance after chronic social defeat stress. Copyright © 2017 the authors 0270-6474/17/376527-12$15.00/0.

Reduced Slc6a15 in Nucleus Accumbens D2-Neurons Underlies Stress Susceptibility

PubMed Central

Nam, Hyungwoo; Engeln, Michel; Konkalmatt, Prasad; Iniguez, Sergio D.

2017-01-01

Previous research demonstrates that Slc6a15, a neutral amino acid transporter, is associated with depression susceptibility. However, no study examined Slc6a15 in the ventral striatum [nucleus accumbens (NAc)] in depression. Given our previous characterization of Slc6a15 as a striatal dopamine receptor 2 (D2)-neuron-enriched gene, we examined the role of Slc6a15 in NAc D2-neurons in mediating susceptibility to stress in male mice. First, we showed that Slc6a15 mRNA was reduced in NAc of mice susceptible to chronic social defeat stress (CSDS), a paradigm that produces behavioral and molecular adaptations that resemble clinical depression. Consistent with our preclinical data, we observed Slc6a15 mRNA reduction in NAc of individuals with major depressive disorder (MDD). The Slc6a15 reduction in NAc occurred selectively in D2-neurons. Next, we used Cre-inducible viruses combined with D2-Cre mice to reduce or overexpress Slc6a15 in NAc D2-neurons. Slc6a15 reduction in D2-neurons caused enhanced susceptibility to a subthreshold social defeat stress (SSDS) as observed by reduced social interaction, while a reduction in social interaction following CSDS was not observed when Slc6a15 expression in D2-neurons was restored. Finally, since both D2-medium spiny neurons (MSNs) and D2-expressing choline acetyltransferase (ChAT) interneurons express Slc6a15, we examined Slc6a15 protein in these interneurons after CSDS. Slc6a15 protein was unaltered in ChAT interneurons. Consistent with this, reducing Slc5a15 selectively in NAc D2-MSNs, using A2A-Cre mice that express Cre selectively in D2-MSNs, caused enhanced susceptibility to SSDS. Collectively, our data demonstrate that reduced Slc6a15 in NAc occurs in MDD individuals and that Slc6a15 reduction in NAc D2-neurons underlies stress susceptibility. SIGNIFICANCE STATEMENT Our study demonstrates a role for reduced Slc6a15, a neutral amino acid transporter, in nucleus accumbens (NAc) in depression and stress susceptibility. The reduction of Slc6a15 occurs selectively in the NAc D2-neurons. Genetic reduction of Slc6a15 induces susceptibility to a subthreshold stress, while genetic overexpression in D2-neurons prevents social avoidance after chronic social defeat stress. PMID:28576941

Molecular description of the LCST behavior of an elastin-like polypeptide.

PubMed

Li, Nan K; García Quiroz, Felipe; Hall, Carol K; Chilkoti, Ashutosh; Yingling, Yaroslava G

2014-10-13

Elastin-like polypeptides (ELPs) with the repeat sequence of VPGVG are widely used as a model system for investigation of lower critical solution temperature (LCST) transition behavior. In this paper, the effect of temperature on the structure, dynamics and association of (VPGVG)18 in aqueous solution is investigated using atomistic molecular dynamics simulations. Our simulations show that as the temperature increases the ELP backbones undergo gradual conformational changes, which are attributed to the formation of more ordered secondary structures such as β-strands. In addition, increasing temperature changes the hydrophobicity of the ELP by exposure of hydrophobic valine-side chains to the solvent and hiding of proline residues. Based on our simulations, we conclude that the transition behavior of (VPGVG)18 can be attributed to a combination of thermal disruption of the water network that surrounds the polypeptide, reduction of solvent accessible surface area of the polypeptide, and increase in its hydrophobicity. Simulations of the association of two (VPGVG)18 molecules demonstrated that the observed gradual changes in the structural properties of the single polypeptide chain are enough to cause the aggregation of polypeptides above the LCST. These results lead us to propose that the LCST phase behavior of poly(VPGVG) is a collective phenomenon that originates from the correlated gradual changes in single polypeptide structure and the abrupt change in properties of hydration water around the peptide and is a result of a competition between peptide-peptide and peptide-water interactions. This is a computational study of an important intrinsically disordered peptide system that provides an atomic-level description of structural features and interactions that are relevant in the LCST phase behavior.

Cross regulation between mTOR signaling and O-GlcNAcylation.

PubMed

Very, Ninon; Steenackers, Agata; Dubuquoy, Caroline; Vermuse, Jeanne; Dubuquoy, Laurent; Lefebvre, Tony; El Yazidi-Belkoura, Ikram

2018-06-01

The hexosamine biosynthetic pathway (HBP) integrates glucose, amino acids, fatty acids and nucleotides metabolisms for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is the nucleotide sugar donor for O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) processes. O-GlcNAc transferase (OGT) is the enzyme which transfers the N-acetylglucosamine (O-GlcNAc) residue onto target proteins. Several studies previously showed that glucose metabolism dysregulations associated with obesity, diabetes or cancer correlated with an increase of OGT expression and global O-GlcNAcylation levels. Moreover, these diseases present an increased activation of the nutrient sensing mammalian target of rapamycin (mTOR) pathway. Other works demonstrate that mTOR regulates protein O-GlcNAcylation in cancer cells through stabilization of OGT. In this context, we studied the cross-talk between these two metabolic sensors in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells. We report that levels of OGT and O-GlcNAcylation are increased in obese mice colon tissues and colon cancer cells and are associated with a higher activation of mTOR signaling. In parallel, treatments with mTOR regulators modulate OGT and O-GlcNAcylation levels in both normal and colon cancer cells. However, deregulation of O-GlcNAcylation affects mTOR signaling activation only in cancer cells. Thus, a crosstalk exists between O-GlcNAcylation and mTOR signaling in contexts of metabolism dysregulation associated to obesity or cancer.

In vitro synthesis of intermediates involved in the assembly of enterobacterial common antigen (ECA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barr, K.; Wolski, S.; Kroto, J.

1986-05-01

ECA is a cell surface antigen found in all bacteria belonging to the family Enterobacteriaceae. The serological specificity of ECA is determined by a linear heteropolysaccharide comprised of trisaccharide repeat units; the component sugars are N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcUA), and 4-acetamido-D-fucose (Fuc4NAc). In vivo studies have suggested that GlcNAc-pyrophosphorylundecaprenol (GlcNAc-PP-lipid) is an intermediate in ECA synthesis. More recently, they have demonstrated UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase activity in cell envelope preparations of E. coli. Radioactivity from UDP-(/sup 3/H)Glc-NAc was incorporated into endogenous lipid acceptor, and the labeled product was characterized as GlcNAc-PP-lipid (lipid I). Transferase activity was inhibited by tunicamycin andmore » UMP, but it was unaffected by UDP. The reaction was reversible, and the synthesis of UDP-(/sup 3/H)GlcNAc from UMP and (/sup 3/H)GlcNAc-PP-lipid was also sensitive to tunicamycin. The simultaneous addition of UDP-(/sup 14/C)ManNAcUA and UDP-(/sup 3/H)GlcNAc to cell envelope preparations resulted in the synthesis of a more polar lipid (lipid II) that contained both labeled sugars in equimolar amounts. Synthesis of lipid II was dependent on prior synthesis of lipid I. Accordingly, (/sup 3/H)GlcNAc-PP-lipid that had been synthesized in vivo served as an acceptor in vitro of ManNAcUA residues from UDP-ManNAcUA. Lipid II has been tentatively identified as ManNAcUA-GlcNAc-pyrophosphorylundecaprenol.« less

Dopamine in the nucleus accumbens core, but not shell, increases during signaled food reward and decreases during delayed extinction.

PubMed

Biesdorf, C; Wang, A-L; Topic, B; Petri, D; Milani, H; Huston, J P; de Souza Silva, M A

2015-09-01

Microdialysis studies in rat have generally shown that appetitive stimuli release dopamine (DA) in the nucleus accumbens (NAc) shell and core. Here we examined the release of DA in the NAc during delivery of reward (food) and during extinction of food reward in the freely moving animal by use of in vivo microdialysis and HPLC. Fifty-two male Wistar rats were trained to receive food reward associated with appearance of cue-lights in a Skinner-box during in vivo microdialysis. Different behavioral protocols were used to assess the effects of extinction on DA and its metabolites. Results Exp. 1: (a) During a 20-min period of cued reward delivery, DA increased significantly in the NAc core, but not shell subregion; (b) for the next 60min period half of the rats underwent immediate extinction (with the CS light presented during non-reward) and the other half did not undergo extinction to the cue lights (CS was not presented during non-reward). DA remained significantly increased in both groups, providing no evidence for a decrease in DA during extinction in either NAc core or shell regions. (c) In half of the animals of the group that was not subjected to extinction, the cue lights were turned on for 30min, thus, initiating extinction to cue CS at a 1h delay from the period of reward. In this group DA in the NAc core, but not shell, significantly decreased. Behavioral analysis showed that while grooming is an indicator of extinction-induced behavior, glances toward the cue-lights (sign tracking) are an index of resistance to extinction. Results Exp. 2: (a) As in Exp. 1, during a 30-min period of cued reward delivery, DA levels again increased significantly in the NAc core but not in the NAc shell. (b) When extinction (the absence of reward with the cue lights presented) was administered 24h after the last reward session, DA again significantly decreased in the NAc core, but not in the NAc shell. (a) These results confirm the importance of DA release in the NAc for reward-related states, with DA increasing in the core, but not shell subregion. (b) They provide first evidence that during the withholding of expected reward, DA decreases in the NAc core, but not shell region. (c) This decrease in DA appears only after a delay between delivery of reward and extinction likely due to it being masked by persisting DA release. We hypothesize the decrease in extinction-induced release of DA in the NAc core to be a marker for the despair/depression that is known to accompany the failure to obtain expected rewards/reinforcers. Copyright © 2015 Elsevier Inc. All rights reserved.

Serine and alanine racemase activities of VanT: a protein necessary for vancomycin resistance in Enterococcus gallinarum BM4174.

PubMed

Arias, C A; Weisner, J; Blackburn, J M; Reynolds, P E

2000-07-01

Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 degrees C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 degrees C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.

Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Acα2-6Galβ1-4GlcNAc human-type influenza receptor

PubMed Central

Kadirvelraj, Renuka; Grant, Oliver C; Goldstein, Irwin J; Winter, Harry C; Tateno, Hiroaki; Fadda, Elisa; Woods, Robert J

2011-01-01

Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7 Å) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding. PMID:21436237

Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting.

PubMed Central

Clarkson, G H; Neagle, J; Lindsay, J G

1991-01-01

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1996968

N-Acetylcysteine Reverses Anxiety and Oxidative Damage Induced by Unpredictable Chronic Stress in Zebrafish.

PubMed

Mocelin, Ricieri; Marcon, Matheus; D'ambros, Simone; Mattos, Juliane; Sachett, Adrieli; Siebel, Anna M; Herrmann, Ana P; Piato, Angelo

2018-06-06

There is accumulating evidence on the use of N-acetylcysteine (NAC) in the treatment of patients with neuropsychiatric disorders. As a multi-target drug and a glutathione precursor, NAC is a promising molecule in the management of stress-related disorders, for which there is an expanding field of research investigating novel therapies targeting oxidative pathways. The deleterious effects of chronic stress in the central nervous system are a result of glutamatergic hyperactivation, glutathione (GSH) depletion, oxidative stress, and increased inflammatory response, among others. The aim of this study was to investigate the effects of NAC in zebrafish submitted to unpredictable chronic stress (UCS). Animals were initially stressed or not for 7 days, followed by treatment with NAC (1 mg/L, 10 min) or vehicle for 7 days. UCS decreased the number of entries and time spent in the top area in the novel tank test, which indicate increased anxiety levels. It also increased reactive oxygen species (ROS) levels and lipid peroxidation (TBARS) while decreased non-protein thiols (NPSH) and superoxide dismutase (SOD) activity. NAC reversed the anxiety-like behavior and oxidative damage observed in stressed animals. Additional studies are needed to investigate the effects of this agent on glutamatergic modulation and inflammatory markers related to stress. Nevertheless, our study adds to the existing body of evidence supporting the clinical evaluation of NAC in mood disorders, anxiety, post-traumatic stress disorder, and other conditions associated with stress.

Excimer-based peptide beacons: a convenient experimental approach for monitoring polypeptide-protein and polypeptide-oligonucleotide interactions.

PubMed

Oh, Kenneth J; Cash, Kevin J; Plaxco, Kevin W

2006-11-01

While protein-polypeptide and nucleic acid-polypeptide interactions are of significant experimental interest, quantitative methods for the characterization of such interactions are often cumbersome. Here we described a relatively simple means of optically monitoring such interactions using excimer-based peptide beacons (PBs). The design of PBs is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed with macromolecular targets. Using this binding-induced folding to segregate two pyrene moieties and therefore inhibit excimer formation, we have produced PBs directed against both anti-HIV antibodies and the retroviral transactive response (TAR) RNA hairpin. For both polypeptides, target recognition is accompanied by a roughly 2-fold decrease in excimer emission, thus allowing the detection of their respective targets at concentrations of a few nanomolar. Because excimer emission requires the formation of a tight, precisely oriented pyrene dimer, even relatively trivial binding-induced segregation reduces fluorescence significantly. This suggests that the PB approach will be suitable for monitoring a wide range of peptide-macromolecule recognition events. Moreover, the synthesis of excimer-based PBs utilizes commercially available modified pyrenes in a simple and well-established protocol, making the approach well suited for routine laboratory applications.

Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thoden, James B.; Holden, Hazel M.

2010-09-08

The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 {angstrom} resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimericmore » quaternary structure and belongs to the L{beta}H superfamily of N-acyltransferases. Each subunit contains 27 {beta}-strands, 23 of which form the canonical left-handed {beta}-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O{sup {delta}1} of Asn 84 and the sugar C-3{prime} amino group and the second between the backbone amide group of Arg 94 and the sugar C-5{prime} carboxylate. The sugar C-3{prime} amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.« less

High performance liquid chromatography analysis of aliphatic thiols in alimentary supplements and pharmaceuticals using menadione as a new useful derivatization reagent.

PubMed

Gatti, Rita; Vitellaro, Valeria

2013-12-04

The use of menadione (MD) as a pre-column reagent for high performance liquid chromatography (HPLC) analysis of aliphatic thiols is proposed. The reaction was carried out for 5 min at room temperature and pH 8.5. The developed method was applied to the N-acetylcysteine (NAC) analysis of alimentary supplements and pharmaceutical formulations. The effect of the complex matrix was evaluated by the study of the thiol derivatization reaction both in standard and in placebo solutions. The yield of NAC-MD adduct was found to be quantitative at a reagent to thiol molar ratio of about 4 in comparison with an authentic specimen of synthesized NAC adduct, which was characterized by (1)H NMR, IR and UV. The routine chromatographic separations were performed on a Synergi MAX-RP column using a mobile phase consisting of methanol/triethylammonium (TEA) phosphate buffer (pH 3; 0.05 mol L(-1)) 70:30 (v/v) at a flow-rate of 0.4 mL min(-1). UV-diode array detection was used setting the wavelength at λ=260 nm. The validation parameters such as linearity, sensitivity, accuracy, precision, selectivity and ruggedness were found to be highly satisfactory. Similar linear responses were observed by standard and placebo solutions (determination coefficient: 0.9996). Limit of detection was about 0.019 μg g(-1). Intra-day precision (relative standard deviation, R.S.D.) was ≤0.81% for NAC to internal standard (IS) peak area ratio, ≤0.28% and ≤0.32%, respectively, for NAC and IS retention times (tR), without significant differences between intra- and inter-day data. NAC recovery studies gave good results (100.12%) with R.S.D.=1.05%. Copyright © 2013 Elsevier B.V. All rights reserved.

Transcript Profiling Identifies NAC-Domain Genes Involved in Regulating Wall Ingrowth Deposition in Phloem Parenchyma Transfer Cells of Arabidopsis thaliana

PubMed Central

Wu, Yuzhou; Hou, Jiexi; Yu, Fen; Nguyen, Suong T. T.; McCurdy, David W.

2018-01-01

Transfer cells (TCs) play important roles in facilitating enhanced rates of nutrient transport at key apoplasmic/symplasmic junctions along the nutrient acquisition and transport pathways in plants. TCs achieve this capacity by developing elaborate wall ingrowth networks which serve to increase plasma membrane surface area thus increasing the cell's surface area-to-volume ratio to achieve increased flux of nutrients across the plasma membrane. Phloem parenchyma (PP) cells of Arabidopsis leaf veins trans-differentiate to become PP TCs which likely function in a two-step phloem loading mechanism by facilitating unloading of photoassimilates into the apoplasm for subsequent energy-dependent uptake into the sieve element/companion cell (SE/CC) complex. We are using PP TCs in Arabidopsis as a genetic model to identify transcription factors involved in coordinating deposition of the wall ingrowth network. Confocal imaging of pseudo-Schiff propidium iodide-stained tissue revealed different profiles of temporal development of wall ingrowth deposition across maturing cotyledons and juvenile leaves, and a basipetal gradient of deposition across mature adult leaves. RNA-Seq analysis was undertaken to identify differentially expressed genes common to these three different profiles of wall ingrowth deposition. This analysis identified 68 transcription factors up-regulated two-fold or more in at least two of the three experimental comparisons, with six of these transcription factors belonging to Clade III of the NAC-domain family. Phenotypic analysis of these NAC genes using insertional mutants revealed significant reductions in levels of wall ingrowth deposition, particularly in a double mutant of NAC056 and NAC018, as well as compromised sucrose-dependent root growth, indicating impaired capacity for phloem loading. Collectively, these results support the proposition that Clade III members of the NAC-domain family in Arabidopsis play important roles in regulating wall ingrowth deposition in PP TCs. PMID:29599795

Transcript Profiling Identifies NAC-Domain Genes Involved in Regulating Wall Ingrowth Deposition in Phloem Parenchyma Transfer Cells of Arabidopsis thaliana.

PubMed

Wu, Yuzhou; Hou, Jiexi; Yu, Fen; Nguyen, Suong T T; McCurdy, David W

2018-01-01

Transfer cells (TCs) play important roles in facilitating enhanced rates of nutrient transport at key apoplasmic/symplasmic junctions along the nutrient acquisition and transport pathways in plants. TCs achieve this capacity by developing elaborate wall ingrowth networks which serve to increase plasma membrane surface area thus increasing the cell's surface area-to-volume ratio to achieve increased flux of nutrients across the plasma membrane. Phloem parenchyma (PP) cells of Arabidopsis leaf veins trans -differentiate to become PP TCs which likely function in a two-step phloem loading mechanism by facilitating unloading of photoassimilates into the apoplasm for subsequent energy-dependent uptake into the sieve element/companion cell (SE/CC) complex. We are using PP TCs in Arabidopsis as a genetic model to identify transcription factors involved in coordinating deposition of the wall ingrowth network. Confocal imaging of pseudo-Schiff propidium iodide-stained tissue revealed different profiles of temporal development of wall ingrowth deposition across maturing cotyledons and juvenile leaves, and a basipetal gradient of deposition across mature adult leaves. RNA-Seq analysis was undertaken to identify differentially expressed genes common to these three different profiles of wall ingrowth deposition. This analysis identified 68 transcription factors up-regulated two-fold or more in at least two of the three experimental comparisons, with six of these transcription factors belonging to Clade III of the NAC-domain family. Phenotypic analysis of these NAC genes using insertional mutants revealed significant reductions in levels of wall ingrowth deposition, particularly in a double mutant of NAC056 and NAC018 , as well as compromised sucrose-dependent root growth, indicating impaired capacity for phloem loading. Collectively, these results support the proposition that Clade III members of the NAC-domain family in Arabidopsis play important roles in regulating wall ingrowth deposition in PP TCs.

Chemistry Notes

ERIC Educational Resources Information Center

School Science Review, 1977

1977-01-01

Includes methods for determining melting and boiling points, illustrating the Finkelstein reaction, choosing acid-base indicators, growing perfect NaC1 cubes and "whiskers," bromination of alkenes, using vanadium in the laboratory, preparing phenylamine-copper (II) sulfate (VI) complex, simulating first-order chemical kinetics on a programmable…

Isolation and expression profiling of GhNAC transcription factor genes in cotton (Gossypium hirsutum L.) during leaf senescence and in response to stresses.

PubMed

Shah, Syed Tariq; Pang, Chaoyou; Fan, Shuli; Song, Meizhen; Arain, Saima; Yu, Shuxun

2013-12-01

NAC (NAM, ATAF, and CUC) is a plant-specific transcription factor family with diverse roles in plant development and stress regulation. In this report, stress-responsive NAC genes (GhNAC8-GhNAC17) isolated from cotton (Gossypium hirsutum L.) were characterised in the context of leaf senescence and stress tolerance. The characterisation of NAC genes during leaf senescence has not yet been reported for cotton. Based on the sequence characterisation, these GhNACs could be classified into three groups belonging to three known NAC sub-families. Their predicted amino acid sequences exhibited similarities to NAC genes from other plant species. Senescent leaves were the sites of maximum expression for all GhNAC genes except GhNAC10 and GhNAC13, which showed maximum expression in fibres, collected from 25 days post anthesis (DPA) plants. The ten GhNAC genes displayed differential expression patterns and levels during natural and induced leaf senescence. Quantitative RT-PCR and promoter analyses suggest that these genes are induced by ABA, ethylene, drought, salinity, cold, heat, and other hormonal treatments. These results support a role for cotton GhNAC genes in transcriptional regulation of leaf senescence, stress tolerance and other developmental stages of cotton. © 2013.

Association of time-dependent changes in mu opioid receptor mRNA, but not BDNF, TrkB, or MeCP2 mRNA and protein expression in the rat nucleus accumbens with incubation of heroin craving.

PubMed

Theberge, Florence R M; Pickens, Charles L; Goldart, Evan; Fanous, Sanya; Hope, Bruce T; Liu, Qing-Rong; Shaham, Yavin

2012-12-01

Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial prefrontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence. We trained rats to self-administer heroin or saline for 9-10 days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA (Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone's (0-1.0 mg/kg) effect on extinction responding after 1 and 15 days. Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day 11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day 1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1 day. Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving.

Association of time-dependent changes in mu opioid receptor mRNA, but not BDNF, TrkB, or MeCP2 mRNA and protein expression in the rat nucleus accumbens with incubation of heroin craving

PubMed Central

Theberge, Florence R. M.; Pickens, Charles L.; Goldart, Evan; Fanous, Sanya; Hope, Bruce T.; Liu, Qing-Rong

2013-01-01

Rationale and objectives Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial pre-frontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence. Methods We trained rats to self-administer heroin or saline for 9–10 days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA (Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone’s (0–1.0 mg/kg) effect on extinction responding after 1 and 15 days. Results Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day 11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day 1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1 day. Conclusions Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving. PMID:22790874

AMPA Receptor Plasticity in Accumbens Core Contributes to Incubation of Methamphetamine Craving.

PubMed

Scheyer, Andrew F; Loweth, Jessica A; Christian, Daniel T; Uejima, Jamie; Rabei, Rana; Le, Tuan; Dolubizno, Hubert; Stefanik, Michael T; Murray, Conor H; Sakas, Courtney; Wolf, Marina E

2016-11-01

The incubation of cue-induced drug craving in rodents provides a model of persistent vulnerability to craving and relapse in human addicts. After prolonged withdrawal, incubated cocaine craving depends on strengthening of nucleus accumbens (NAc) core synapses through incorporation of Ca 2+ -permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (CP-AMPARs). Through metabotropic glutamate receptor 1 (mGluR1)-mediated synaptic depression, mGluR1 positive allosteric modulators remove CP-AMPARs from these synapses and thereby reduce cocaine craving. This study aimed to determine if similar plasticity accompanies incubation of methamphetamine craving. Rats self-administered saline or methamphetamine under extended-access conditions. Cue-induced seeking tests demonstrated incubation of methamphetamine craving. After withdrawal periods ranging from 1 to >40 days, rats underwent one of the following procedures: 1) whole-cell patch clamp recordings to characterize AMPAR transmission, 2) intra-NAc core injection of the CP-AMPAR antagonist 1-naphthyl acetyl spermine followed by a seeking test, or 3) systemic administration of a mGluR1 positive allosteric modulator followed by a seeking test. Incubation of methamphetamine craving was associated with CP-AMPAR accumulation in NAc core, and both effects were maximal after ~1 week of withdrawal. Expression of incubated craving was decreased by intra-NAc core 1-naphthyl acetyl spermine injection or systemic mGluR1 positive allosteric modulator administration. These results are the first to demonstrate a role for the NAc in the incubation of methamphetamine craving and describe adaptations in synaptic transmission associated with this model. They establish that incubation of craving and associated CP-AMPAR plasticity occur much more rapidly during withdrawal from methamphetamine compared with cocaine. However, a common mGluR1-based therapeutic strategy may be helpful for recovering cocaine and methamphetamine addicts. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Rab-GDI complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes.

PubMed

Malagnac, Fabienne; Fabret, Céline; Prigent, Magali; Rousset, Jean-Pierre; Namy, Olivier; Silar, Philippe

2013-01-01

In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild.

Bidirectional synaptic structural plasticity after chronic cocaine administration occurs through Rap1 small GTPase signaling

PubMed Central

Cahill, Michael E.; Bagot, Rosemary C.; Gancarz, Amy M.; Walker, Deena M.; Sun, HaoSheng; Wang, Zi-Jun; Heller, Elizabeth A.; Feng, Jian; Kennedy, Pamela J.; Koo, Ja Wook; Cates, Hannah M.; Neve, Rachael L.; Shen, Li; Dietz, David M.

2016-01-01

Summary Dendritic spines are the sites of most excitatory synapses in the CNS, and opposing alterations in the synaptic structure of medium spiny neurons (MSNs) of the nucleus accumbens, a primary brain reward region, are seen at early vs. late time points after cocaine administration. Here we investigate the time-dependent molecular and biochemical processes that regulate this bidirectional synaptic structural plasticity of NAc MSNs and associated changes in cocaine reward in response to chronic cocaine exposure. Our findings reveal key roles for the bidirectional synaptic expression of the Rap1b small GTPase and an associated local-synaptic protein translation network in this process. The transcriptional mechanisms and pathway-specific inputs to NAc that regulate Rap1b expression are also characterized. Collectively, these findings provide a precise mechanism by which nuclear to synaptic interactions induce “metaplasticity” in NAc MSNs, and we reveal the specific effects of this plasticity on reward behavior in a brain circuit-specific manner. PMID:26844834

Identification of glycopeptides as post-translationally modified neoantigens in leukemia

PubMed Central

Malaker, Stacy A.; Penny, Sarah A.; Steadman, Lora G.; Myers, Paisley T.; Loke, Justin C; Raghavan, Manoj; Bai, Dina L.; Shabanowitz, Jeffrey; Hunt, Donald F.; Cobbold, Mark

2017-01-01

Leukemias are highly immunogenic but have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these post-translationally modified neoantigens provide logical targets for cancer immunotherapy. PMID:28314751

Peptidergic innervation of the human male genital tract.

PubMed

Gu, J; Polak, J M; Probert, L; Islam, K N; Marangos, P J; Mina, S; Adrian, T E; McGregor, G P; O'Shaughnessy, D J; Bloom, S R

1983-08-01

Four peptides--vasoactive intestinal polypeptide, substance P, somatostatin and a peptide-like avian pancreatic polypeptide--have been found in nerves of the human male genitalia using highly sensitive and specific methods of immunocytochemistry and radioimmunoassay. Five other peptides (met-enkephalin, leu-enkephalin, neurotensin, bombesin and cholecystokinin-8) were absent. Vasoactive intestinal polypeptide was the most abundant peptide, its highest concentration being in the proximal corpus cavernosum. Immunoelectron microscopy localized this peptide to large (97 +/- 20 nm), round, electron-dense granules of p-type nerve terminals. Vasoactive intestinal polypeptide-immunoreactive neuronal cell bodies were found in the prostate gland and the root of the corpus cavernosum. Substance P immunoreactive material was present in smaller concentration and was mainly localized in nerves around the corpuscular receptors of the glans penis. Somatostatin immunoreactive nerves were associated mainly with the smooth muscle of the seminal vesicle and the vas deferens. When antiserum to avian pancreatic polypeptide was applied, certain nerves were stained, particularly in the vas deferens, the prostate gland and the seminal vesicle. However, chromatography detected no pure avian pancreatic polypeptide suggesting the presence of a structurally related substance, possibly neuropeptide Y, which cross-reacts with the avian pancreatic polypeptide antiserum. Similar distributions between vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves and between avian pancreatic polypeptide-immunoreactive and adrenergic nerves were observed. A general neuronal marker, neuron-specific enolase, was used to investigate the general pattern of the organ's innervation. The abundance and distribution patterns of these peptide-immunoreactive nerves indicate that they may play important roles in the male sexual physiology.

Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N- and O-Glycans without Direct Incorporation*

PubMed Central

Barthel, Steven R.; Antonopoulos, Aristotelis; Cedeno-Laurent, Filiberto; Schaffer, Lana; Hernandez, Gilberto; Patil, Shilpa A.; North, Simon J.; Dell, Anne; Matta, Khushi L.; Neelamegham, Sriram; Haslam, Stuart M.; Dimitroff, Charles J.

2011-01-01

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLeX), and related lectin ligands on effector leukocytes. Based on anti-sLeX antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLeX formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLeX (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLeX structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLeX on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis. PMID:21493714

Expression of a plant-derived peptide harboring water-cleaning and antimicrobial activities.

PubMed

Suarez, M; Entenza, J M; Doerries, C; Meyer, E; Bourquin, L; Sutherland, J; Marison, I; Moreillon, P; Mermod, N

2003-01-05

Drinking water is currently a scarce world resource, the preparation of which requires complex treatments that include clarification of suspended particles and disinfection. Seed extracts of Moringa oleifera Lam., a tropical tree, have been proposed as an environment-friendly alternative, due to their traditional use for the clarification of drinking water. However, the precise nature of the active components of the extract and whether they may be produced in recombinant form are unknown. Here we show that recombinant or synthetic forms of a cationic seed polypeptide mediate efficient sedimentation of suspended mineral particles and bacteria. Unexpectedly, the polypeptide was also found to possesses a bactericidal activity capable of disinfecting heavily contaminated water. Furthermore, the polypeptide has been shown to efficiently kill several pathogenic bacteria, including antibiotic-resistant isolates of Staphylococcus, Streptococcus, and Legionella species. Thus, this polypeptide displays the unprecedented feature of combining water purification and disinfectant properties. Identification of an active principle derived from the seed extracts points to a range of potential for drinking water treatment or skin and mucosal disinfection in clinical settings. Copyright 2002 Wiley Periodicals, Inc.

The Efficacy of Inositol and N-Acetyl Cysteine Administration (Ovaric HP) in Improving the Ovarian Function in Infertile Women with PCOS with or without Insulin Resistance

PubMed Central

Lico, Daniela; Di Cello, Annalisa; Rania, Erika; Cirillo, Roberto

2014-01-01

Objective. Substances such as inositol and N-acetylcysteine (NAC) have been recently shown to be effective in treatment of PCOS patients. The aim of this prospective trial is to evaluate the efficacy of NAC + Inositol + folic acid on ovulation rate and menstrual regularity in PCOS patients with and without insulin resistance. Methods. Among the 91 PCOS patients treated with NAC + Inositol + folic, insulin resistance was present in 44 subjects (A) and absent in 47 (B). The primary endpoint was the ovulation rate/year, determined by menstrual diary, serum progesterone performed between 21° and 24° days, ultrasound findings of growth follicular or luteal cysts, and luteal ratio. HOMA-index assessment after 6 and 12 months of treatment was evaluated as secondary endpoint. Results. In both groups there was a significant increase in ovulation rate and no significant differences were found in the primary outcome between two groups. In group A, a significant reduction of HOMA-index was observed. Conclusions. The association NAC + Inositol + folic, regardless of insulin-resistance state, seems to improve ovarian function in PCOS patients. Therefore, inositol and NAC may have additional noninsulin-related mechanisms of action that allow achieving benefits also in those patients with negative HOMA-index. PMID:24876842

The Efficacy of Inositol and N-Acetyl Cysteine Administration (Ovaric HP) in Improving the Ovarian Function in Infertile Women with PCOS with or without Insulin Resistance.

PubMed

Sacchinelli, Angela; Venturella, Roberta; Lico, Daniela; Di Cello, Annalisa; Lucia, Antonella; Rania, Erika; Cirillo, Roberto; Zullo, Fulvio

2014-01-01

Objective. Substances such as inositol and N-acetylcysteine (NAC) have been recently shown to be effective in treatment of PCOS patients. The aim of this prospective trial is to evaluate the efficacy of NAC + Inositol + folic acid on ovulation rate and menstrual regularity in PCOS patients with and without insulin resistance. Methods. Among the 91 PCOS patients treated with NAC + Inositol + folic, insulin resistance was present in 44 subjects (A) and absent in 47 (B). The primary endpoint was the ovulation rate/year, determined by menstrual diary, serum progesterone performed between 21° and 24° days, ultrasound findings of growth follicular or luteal cysts, and luteal ratio. HOMA-index assessment after 6 and 12 months of treatment was evaluated as secondary endpoint. Results. In both groups there was a significant increase in ovulation rate and no significant differences were found in the primary outcome between two groups. In group A, a significant reduction of HOMA-index was observed. Conclusions. The association NAC + Inositol + folic, regardless of insulin-resistance state, seems to improve ovarian function in PCOS patients. Therefore, inositol and NAC may have additional noninsulin-related mechanisms of action that allow achieving benefits also in those patients with negative HOMA-index.

Protein O-GlcNAcylation: A critical regulator of the cellular response to stress.

PubMed

Chatham, John C; Marchase, Richard B

2010-01-01

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of chronic disease processes. However, there is increasing evidence that O-GlcNAc levels are increased in response to stress and that acute augmentation of this response is cytoprotective, at least in the short term. Conversely, a reduction in O-GlcNAc levels appears to be associated with decreased cell survival in response to an acute stress. Here we summarize our current understanding of protein O-GlcNAcylation on the cellular response to stress and in mediating cellular protective mechanisms focusing primarily on the cardiovascular system as an example. We consider the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis and explore the parallels between O-GlcNAc signaling and redox signaling. We also discuss the apparent paradox between the reported adverse effects of increased O-GlcNAcylation with its recently reported role in mediating cell survival mechanisms.

O-GlcNAcylation enhances anaplastic thyroid carcinoma malignancy.

PubMed

Cheng, Y U; Li, Honglun; Li, Jianlin; Li, Jisheng; Gao, Yan; Liu, Baodong

2016-07-01

O-linked N -acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation), a dynamic post-translational modification of nuclear and cytoplasmic proteins, may have a critical role in the regulation of biological cell processes and human cancer. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Accumulating evidence suggests that O-GlcNAcylation is involved in a variety of types of human cancer. However, the exact role of O-GlcNAcylation in tumor pathogenesis or progression remains to be established. Computed tomography scans of patients with anaplastic thyroid carcinoma (ATC) reveal a rapid growth rate and invasion. The present study demonstrated that O-GlcNAcylation accelerates the progression of ATC. The global O-GlcNAc level of intracellular proteins was increased by overexpression of OGT or downregulation of OGA activity with the specific inhibitor Thiamet-G. By contrast, the global O-GlcNAc level was decreased by silencing of OGT. MTT assay indicated that O-GlcNAcylation significantly promotes cell proliferation. Furthermore, O-GlcNAcylation enhanced cellular biological functions, such as colony formation ability, migration and invasion, of ATC cells in vitro . The findings of the present study suggest that O-GlcNAcylation is associated with malignant properties of thyroid cancer, and may be a potential target for the diagnosis and treatment of thyroid cancer.

Peptides having reduced toxicity that stimulate cholesterol efflux

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bielicki, John K.; Johansson, Jan; Danho, Waleed

The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABCA1 that parallels that of full-length apolipoproteins. Further, the peptides of the invention have little or no toxicity when administered at therapeutic and higher doses. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

Wall-associated kinase-like polypeptide mediates nutritional status perception and response

DOEpatents

Yang, Zhenbiao; Karr, Stephen

2014-02-11

The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

The glatiramoid class of immunomodulator drugs.

PubMed

Varkony, Haim; Weinstein, Vera; Klinger, Ety; Sterling, Jeffrey; Cooperman, Helena; Komlosh, Turi; Ladkani, David; Schwartz, Rivka

2009-03-01

Glatiramer acetate (GA) is a complex heterogenous mixture of polypeptides with immunomodulatory activity approved for treatment of relapsing-remitting multiple sclerosis. GA is the first, and was until recently, the only member of the glatiramoids, a family of synthetic copolymer mixtures comprising the four amino acids, L-glutamic acid, L-alanine, L-lysine and L-tyrosine, in a defined molar ratio. Another glatiramoid, protiramer, was recently evaluated in preclinical studies and in two small Phase II clinical trials with relapsing-remitting multiple sclerosis patients. Due to the complexity and heterogeneity of GA and other glatiramoids, the clinically active epitopes within the mixture cannot be identified and the consistency of polypeptide sequences within the mixture is dependent on a tightly controlled manufacturing process. Although no two glatiramoids can be proved identical, it is possible to differentiate among members of the glatiramoid class using analytical methods and immunological and biological markers. Even slight differences in the distribution of molecular masses or in the composition of antigenic polypeptide sequences among glatiramoids can significantly influence their efficacy, toxicity and immunogenicity profiles. Experience with GA may be instructive regarding important safety and efficacy considerations for new glatiramoid mixtures now in development.

Natural polypeptide scaffolds: beta-sheets, beta-turns, and beta-hairpins.

PubMed

Rotondi, Kenneth S; Gierasch, Lila M

2006-01-01

This paper provides an introduction to fundamental conformational states of polypeptides in the beta-region of phi,psi space, in which the backbone is extended near to its maximal length, and to more complex architectures in which extended segments are linked by turns and loops. There are several variants on these conformations, and they comprise versatile scaffolds for presentation of side chains and backbone amides for molecular recognition and designed catalysts. In addition, the geometry of these fundamental folds can be readily mimicked in peptidomimetics. Copyright 2005 Wiley Periodicals, Inc.

Photooxidation of Methionine

ERIC Educational Resources Information Center

Lewis, Catherine; Scouten, William H.

1976-01-01

Describes an experiment in which the photooxidation of methionine using free methylene blue as the sensitizer is applied to the isolated amino acid or to the methionyl residues of a complex polypeptide. (MLH)

Association of nonribosomal nucleolar proteins in ribonucleoprotein complexes during interphase and mitosis.

PubMed

Piñol-Roma, S

1999-01-01

rRNA precursors are bound throughout their length by specific proteins, as the pre-rRNAs emerge from the transcription machinery. The association of pre-rRNA with proteins as ribonucleoprotein (RNP) complexes persists during maturation of 18S, 5.8S, and 28S rRNA, and through assembly of ribosomal subunits in the nucleolus. Preribosomal RNP complexes contain, in addition to ribosomal proteins, an unknown number of nonribosomal nucleolar proteins, as well as small nucleolar RNA-ribonucleoproteins (sno-RNPs). This report describes the use of a specific, rapid, and mild immunopurification approach to isolate and analyze human RNP complexes that contain nonribosomal nucleolar proteins, as well as ribosomal proteins and rRNA. Complexes immunopurified with antibodies to nucleolin-a major nucleolar RNA-binding protein-contain several distinct specific polypeptides that include, in addition to nucleolin, the previously identified nucleolar proteins B23 and fibrillarin, proteins with electrophoretic mobilities characteristic of ribosomal proteins including ribosomal protein S6, and a number of additional unidentified proteins. The physical association of these proteins with one another is mediated largely by RNA, in that the complexes dissociate upon digestion with RNase. Complexes isolated from M-phase cells are similar in protein composition to those isolated from interphase cell nuclear extracts. Therefore, the predominant proteins that associate with nucleolin in interphase remain in RNP complexes during mitosis, despite the cessation of rRNA synthesis and processing in M-phase. In addition, precursor rRNA, as well as processed 18S and 28S rRNA and candidate rRNA processing intermediates, is found associated with the immunopurified complexes. The characteristics of the rRNP complexes described here, therefore, indicate that they represent bona fide precursors of mature cytoplasmic ribosomal subunits.

Intratumor partitioning and texture analysis of dynamic contrast-enhanced (DCE)-MRI identifies relevant tumor subregions to predict pathological response of breast cancer to neoadjuvant chemotherapy.

PubMed

Wu, Jia; Gong, Guanghua; Cui, Yi; Li, Ruijiang

2016-11-01

To predict pathological response of breast cancer to neoadjuvant chemotherapy (NAC) based on quantitative, multiregion analysis of dynamic contrast enhancement magnetic resonance imaging (DCE-MRI). In this Institutional Review Board-approved study, 35 patients diagnosed with stage II/III breast cancer were retrospectively investigated using 3T DCE-MR images acquired before and after the first cycle of NAC. First, principal component analysis (PCA) was used to reduce the dimensionality of the DCE-MRI data with high temporal resolution. We then partitioned the whole tumor into multiple subregions using k-means clustering based on the PCA-defined eigenmaps. Within each tumor subregion, we extracted four quantitative Haralick texture features based on the gray-level co-occurrence matrix (GLCM). The change in texture features in each tumor subregion between pre- and during-NAC was used to predict pathological complete response after NAC. Three tumor subregions were identified through clustering, each with distinct enhancement characteristics. In univariate analysis, all imaging predictors except one extracted from the tumor subregion associated with fast washout were statistically significant (P < 0.05) after correcting for multiple testing, with area under the receiver operating characteristic (ROC) curve (AUC) or AUCs between 0.75 and 0.80. In multivariate analysis, the proposed imaging predictors achieved an AUC of 0.79 (P = 0.002) in leave-one-out cross-validation. This improved upon conventional imaging predictors such as tumor volume (AUC = 0.53) and texture features based on whole-tumor analysis (AUC = 0.65). The heterogeneity of the tumor subregion associated with fast washout on DCE-MRI predicted pathological response to NAC in breast cancer. J. Magn. Reson. Imaging 2016;44:1107-1115. © 2016 International Society for Magnetic Resonance in Medicine.

Evidence that biosynthesis of the second and third sugars of the archaellin Tetrasaccharide in the archaeon Methanococcus maripaludis occurs by the same pathway used by Pseudomonas aeruginosa to make a di-N-acetylated sugar.

PubMed

Siu, Sarah; Robotham, Anna; Logan, Susan M; Kelly, John F; Uchida, Kaoru; Aizawa, Shin-Ichi; Jarrell, Ken F

2015-05-01

Methanococcus maripaludis has two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354 and mmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions in mmp0350, mmp0351, mmp0352, or mmp0353 were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352 strain confirmed a glycan with only GalNAc, suggesting mmp0350 to mmp0353 were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357 mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr). M. maripaludis mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are proposed to be functionally equivalent to Pseudomonas aeruginosa wbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of the P. aeruginosa pathway with mmp0357 supports this hypothesis. This work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins of Methanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

PubMed Central

Siu, Sarah; Robotham, Anna; Logan, Susan M.; Kelly, John F.; Uchida, Kaoru; Aizawa, Shin-Ichi

2015-01-01

ABSTRACT Methanococcus maripaludis has two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354 and mmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions in mmp0350, mmp0351, mmp0352, or mmp0353 were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352 strain confirmed a glycan with only GalNAc, suggesting mmp0350 to mmp0353 were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357 mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr). M. maripaludis mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are proposed to be functionally equivalent to Pseudomonas aeruginosa wbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of the P. aeruginosa pathway with mmp0357 supports this hypothesis. IMPORTANCE This work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins of Methanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway. PMID:25733616

Cell Wall and Membrane-Associated Exo-β-d-Glucanases from Developing Maize Seedlings1

PubMed Central

Kim, Jong-Bum; Olek, Anna T.; Carpita, Nicholas C.

2000-01-01

A β-d-glucan exohydrolase was purified from the cell walls of developing maize (Zea mays L.) shoots. The cell wall enzyme preferentially hydrolyzes the non-reducing terminal glucosyl residue from (1→3)-β-d-glucans, but also hydrolyzes (1→2)-, (1→6)-, and (1→4)-β-d-glucosyl units in decreasing order of activity. Polyclonal antisera raised against the purified exo-β-d-glucanase (ExGase) were used to select partial-length cDNA clones, and the complete sequence of 622 amino acid residues was deduced from the nucleotide sequences of the cDNA and a full-length genomic clone. Northern gel-blot analysis revealed what appeared to be a single transcript, but three distinct polypeptides were detected in immunogel-blot analyses of the ExGases extracted from growing coleoptiles. Two polypeptides appear in the cell wall, where one polypeptide is constitutive, and the second appears at the time of the maximum rate of elongation and reaches peak activity after elongation has ceased. The appearance of the second polypeptide coincides with the disappearance of the mixed-linkage (1→3),(1→4)-β-d-glucan, whose accumulation is associated with cell elongation in grasses. The third polypeptide of the ExGase is an extrinsic protein associated with the exterior surface of the plasma membrane. Although the activity of the membrane-associated ExGase is highest against (1→3)-β-d-glucans, the activity against (1→4)-β-d-glucan linkages is severely attenuated and, therefore, the enzyme is unlikely to be involved with turnover of the (1→3),(1→4)-β-d-glucan. We propose three potential functions for this novel ExGase at the membrane-wall interface. PMID:10859178

TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis.

PubMed

Huang, Quanjun; Wang, Yan; Li, Bin; Chang, Junli; Chen, Mingjie; Li, Kexiu; Yang, Guangxiao; He, Guangyuan

2015-11-04

NAC (NAM, ATAF, and CUC) transcription factors play important roles in plant biological processes, including phytohormone homeostasis, plant development, and in responses to various environmental stresses. TaNAC29 was introduced into Arabidopsis using the Agrobacterium tumefaciens-mediated floral dipping method. TaNAC29-overexpression plants were subjected to salt and drought stresses for examining gene functions. To investigate tolerant mechanisms involved in the salt and drought responses, expression of related marker genes analyses were conducted, and related physiological indices were also measured. Expressions of genes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). A novel NAC transcription factor gene, designated TaNAC29, was isolated from bread wheat (Triticum aestivum). Sequence alignment suggested that TaNAC29 might be located on chromosome 2BS. TaNAC29 was localized to the nucleus in wheat protoplasts, and proved to have transcriptional activation activities in yeast. TaNAC29 was expressed at a higher level in the leaves, and expression levels were much higher in senescent leaves, indicating that TaNAC29 might be involved in the senescence process. TaNAC29 transcripts were increased following treatments with salt, PEG6000, H2O2, and abscisic acid (ABA). To examine TaNAC29 function, transgenic Arabidopsis plants overexpressing TaNAC29 were generated. Germination and root length assays of transgenic plants demonstrated that TaNAC29 overexpression plants had enhanced tolerances to high salinity and dehydration, and exhibited an ABA-hypersensitive response. When grown in the greenhouse, TaNAC29-overexpression plants showed the same tolerance response to salt and drought stresses at both the vegetative and reproductive period, and had delayed bolting and flowering in the reproductive period. Moreover, TaNAC29 overexpression plants accumulated lesser malondialdehyde (MDA), H2O2, while had higher superoxide dismutase (SOD) and catalase (CAT) activities under high salinity and/or dehydration stress. Our results demonstrate that TaNAC29 plays important roles in the senescence process and response to salt and drought stresses. ABA signal pathway and antioxidant enzyme systems are involved in TaNAC29-mediated stress tolerance mechanisms.

Identification of GIG1, a GlcNAc-Induced Gene in Candida albicans Needed for Normal Sensitivity to the Chitin Synthase Inhibitor Nikkomycin Z▿§

PubMed Central

Gunasekera, Angelo; Alvarez, Francisco J.; Douglas, Lois M.; Wang, Hong X.; Rosebrock, Adam P.; Konopka, James B.

2010-01-01

The amino sugar N-acetylglucosamine (GlcNAc) is known to be an important structural component of cells from bacteria to humans, but its roles in cell signaling are less well understood. GlcNAc induces two pathways in the human fungal pathogen Candida albicans. One activates cyclic AMP (cAMP) signaling, which stimulates the formation of hyphal cells and the expression of virulence genes, and the other pathway induces genes needed to catabolize GlcNAc. Microarray analysis of gene expression was carried out under four different conditions in order to characterize the transcriptional changes induced by GlcNAc. The most highly induced genes include those that encode a GlcNAc transporter (NGT1) and the GlcNAc catabolic enzymes (HXK1, DAC1, and NAG1). GlcNAc also activated most of the genes whose expression is increased when cells are triggered with other stimuli to form hyphae. Surprisingly, GlcNAc also induced a subset of genes that are regulated by galactose (GAL1, GAL7, and GAL10), which may be due to cross talk between signaling pathways. A novel GlcNAc-induced gene, GIG1, which is not essential for GlcNAc catabolism or the induction of hyphae, was identified. However, a Gig1-green fluorescent protein (GFP) fusion protein was specifically induced by GlcNAc, and not by other sugars. Gig1-GFP localized to the cytoplasm, where GlcNAc metabolism occurs. Significantly, a gig1Δ mutant displayed increased resistance to nikkomycin Z, which inhibits chitin synthase from converting UDP-GlcNAc into cell wall chitin. Gig1 is highly conserved in fungi, especially those that contain GlcNAc catabolic genes. These results implicate Gig1 in GlcNAc metabolism. PMID:20675577

Isolation and Expression of NAC Genes during Persimmon Fruit Postharvest Astringency Removal

PubMed Central

Min, Ting; Wang, Miao-Miao; Wang, Hongxun; Liu, Xiaofen; Fang, Fang; Grierson, Donald; Yin, Xue-Ren; Chen, Kun-Song

2015-01-01

NAC genes have been characterized in numerous plants, where they are involved in responses to biotic and abiotic stress, including low oxygen stress. High concentration of CO2 is one of the most effective treatments to remove astringency of persimmon fruit owing to the action of the accumulated anoxia metabolite acetaldehyde. In model plants, NAC genes have been identified as being responsive to low oxygen. However, the possible relationship between NAC transcription factors and persimmon astringency removal remains unexplored. In the present research, treatment with a high concentration of CO2 (95%) effectively removed astringency of “Mopan” persimmon fruit by causing decreases in soluble tannin. Acetaldehyde content increased in response to CO2 treatment concomitantly with astringency removal. Using RNA-seq and Rapid amplification of cDNA ends (RACE), six DkNAC genes were isolated and studied. Transcriptional analysis indicated DkNAC genes responded differentially to CO2 treatment; DkNAC1, DkNAC3, DkNAC5 and DkNAC6 were transiently up-regulated, DkNAC2 was abundantly expressed 3 days after treatment, while the DkNAC4 was suppressed during astringency removal. It is proposed that DkNAC1/3/5/6 could be important candidates as regulators of persimmon astringency removal and the roles of other member are also discussed. PMID:25599529

Identification and characterization of plant-specific NAC gene family in canola (Brassica napus L.) reveal novel members involved in cell death.

PubMed

Wang, Boya; Guo, Xiaohua; Wang, Chen; Ma, Jieyu; Niu, Fangfang; Zhang, Hanfeng; Yang, Bo; Liang, Wanwan; Han, Feng; Jiang, Yuan-Qing

2015-03-01

NAC transcription factors are plant-specific and play important roles in plant development processes, response to biotic and abiotic cues and hormone signaling. However, to date, little is known about the NAC genes in canola (or oilseed rape, Brassica napus L.). In this study, a total of 60 NAC genes were identified from canola through a systematical analysis and mining of expressed sequence tags. Among these, the cDNA sequences of 41 NAC genes were successfully cloned. The translated protein sequences of canola NAC genes with the NAC genes from representative species were phylogenetically clustered into three major groups and multiple subgroups. The transcriptional activities of these BnaNAC proteins were assayed in yeast. In addition, by quantitative real-time RT-PCR, we further observed that some of these BnaNACs were regulated by different hormone stimuli or abiotic stresses. Interestingly, we successfully identified two novel BnaNACs, BnaNAC19 and BnaNAC82, which could elicit hypersensitive response-like cell death when expressed in Nicotiana benthamiana leaves, which was mediated by accumulation of reactive oxygen species. Overall, our work has laid a solid foundation for further characterization of this important NAC gene family in canola.

Intra-accumbens Raclopride Administration Prevents Behavioral Changes Induced by Intermittent Access to Sucrose Solution.

PubMed

Suárez-Ortiz, Josué O; Cortés-Salazar, Felipe; Malagón-Carrillo, Ariadna L; López-Alonso, Verónica E; Mancilla-Díaz, Juan M; Tejas-Juárez, Juan G; Escartín-Pérez, Rodrigo E

2018-01-01

Overeating is one of the most relevant clinical features in Binge Eating Disorder and in some obesity patients. According to several studies, alterations in the mesolimbic dopaminergic transmission produced by non-homeostatic feeding behavior may be associated with changes in the reward system similar to those produced by drugs of abuse. Although it is known that binge-eating is related with changes in dopaminergic transmission mediated by D2 receptors in the nucleus accumbens shell (NAcS), it has not been determined whether these receptors may be a potential target for the treatment of eating pathology with binge-eating. Accordingly, the aim of the present study was to evaluate whether sugar binging induced by intermittent access to a sucrose solution produced changes in the structure of feeding behavior and whether blocking D2 receptors prevented these changes. We used the intermittent access model to a 10% sucrose solution (2 h/day for 4 weeks) to induce sugar binging in Sprague Dawley female rats. Experimental subjects consumed in a 2-h period more than 50% of the caloric intake consumed by the subjects with ad-lib access to the sweetened solution without any increase in body weight or fat accumulation. Furthermore, we evaluated whether sugar binging was associated to the estrous cycle and we did not find differences in caloric intake (estrous vs. diestrus). Subsequently, we characterized the structure of feeding behavior (microstructural analysis) and the motivation for palatable food (breakpoints) of the subjects with sugar binging and found that feeding episodes had short latencies, high frequencies, as well as short durations and inter-episode intervals. The intermittent access model did not increase breakpoints, as occurred in subjects with ad-lib access to the sucrose. Finally, we evaluated the effects of D2 receptor blockade in the NAcS, and found that raclopride (18 nM) prevented the observed changes in the frequency and duration of episodes induced by intermittent access to the sucrose solution. Our results suggest that alterations in behavioral patterns associated with binge-eating behavior depend in part on the dopaminergic transmission in the NAcS and that the antagonism of D2 receptors may be a therapeutic tool for feeding pathology with binge-eating.

Intra-accumbens Raclopride Administration Prevents Behavioral Changes Induced by Intermittent Access to Sucrose Solution

PubMed Central

Suárez-Ortiz, Josué O.; Cortés-Salazar, Felipe; Malagón-Carrillo, Ariadna L.; López-Alonso, Verónica E.; Mancilla-Díaz, Juan M.; Tejas-Juárez, Juan G.; Escartín-Pérez, Rodrigo E.

2018-01-01

Overeating is one of the most relevant clinical features in Binge Eating Disorder and in some obesity patients. According to several studies, alterations in the mesolimbic dopaminergic transmission produced by non-homeostatic feeding behavior may be associated with changes in the reward system similar to those produced by drugs of abuse. Although it is known that binge-eating is related with changes in dopaminergic transmission mediated by D2 receptors in the nucleus accumbens shell (NAcS), it has not been determined whether these receptors may be a potential target for the treatment of eating pathology with binge-eating. Accordingly, the aim of the present study was to evaluate whether sugar binging induced by intermittent access to a sucrose solution produced changes in the structure of feeding behavior and whether blocking D2 receptors prevented these changes. We used the intermittent access model to a 10% sucrose solution (2 h/day for 4 weeks) to induce sugar binging in Sprague Dawley female rats. Experimental subjects consumed in a 2-h period more than 50% of the caloric intake consumed by the subjects with ad-lib access to the sweetened solution without any increase in body weight or fat accumulation. Furthermore, we evaluated whether sugar binging was associated to the estrous cycle and we did not find differences in caloric intake (estrous vs. diestrus). Subsequently, we characterized the structure of feeding behavior (microstructural analysis) and the motivation for palatable food (breakpoints) of the subjects with sugar binging and found that feeding episodes had short latencies, high frequencies, as well as short durations and inter-episode intervals. The intermittent access model did not increase breakpoints, as occurred in subjects with ad-lib access to the sucrose. Finally, we evaluated the effects of D2 receptor blockade in the NAcS, and found that raclopride (18 nM) prevented the observed changes in the frequency and duration of episodes induced by intermittent access to the sucrose solution. Our results suggest that alterations in behavioral patterns associated with binge-eating behavior depend in part on the dopaminergic transmission in the NAcS and that the antagonism of D2 receptors may be a therapeutic tool for feeding pathology with binge-eating. PMID:29515353

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

PubMed Central

Siu, Fai Y.; Spanggord, Richard J.; Doudna, Jennifer A.

2007-01-01

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP–receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP–receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP–receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo. PMID:17164479

O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry.

PubMed

Borodkin, Vladimir S; Rafie, Karim; Selvan, Nithya; Aristotelous, Tonia; Navratilova, Iva; Ferenbach, Andrew T; van Aalten, Daan M F

2018-05-18

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.

O-GlcNAc in cancer: An Oncometabolism-fueled vicious cycle.

PubMed

Hanover, John A; Chen, Weiping; Bond, Michelle R

2018-06-01

Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueled by oncometabolism and elevated uptake of glucose and glutamine, the hexosamine biosynthetic pathway (HBP) sustains glycosylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of key targets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymes of intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription, signaling, proteostasis, and bioenergetics, key 'hallmarks of cancer'. In this review, we summarize accumulating evidence that many cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrient and oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis, Kreb's cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a 'vicious cycle'. Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. The elevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selective advantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment.

Isolation and characterization of spinach photosystem II membrane-associated catalase and polyphenol oxidase.

PubMed

Sheptovitsky, Y G; Brudvig, G W

1996-12-17

Photosystem II (PSII) membranes exhibit catalase and polyphenol oxidase (PPO) activities. Mild heat treatment of PSII membranes for 90 min at 30 degrees C releases most of these enzyme activities into the supernatant, accompanied by a 7-fold activation of PPO. In contrast, mild heat treatment of thylakoid membranes does not release significant amounts of either activity, indicating that both enzymes are bound to the luminal surface of the thylakoid membrane. The heat-released PSII membrane-associated catalase and PPO have been purified and characterized. Catalase activity was correlated with a 63 kDa polypeptide which was purified by batch adsorption to anion-exchange beads followed by gel filtration. The PSII membrane-associated catalase is unstable in solution, probably due to irreversible aggregation. The enzyme was characterized in terms of molecular and subunit size, amino-acid composition, UV-visible absorption, heme content, pH optimum, inhibitor sensitivity, and K(m) value for H2O2. Its properties indicate that the PSII membrane-associated catalase is a luminal thylakoid membrane-bound heme enzyme that has not been identified previously. The residual catalase activity of PSII membranes after mild heat treatment is irreversibly inhibited with 3-amino-1,2,4-triazole, a specific inhibitor of heme catalases, without inhibition of O2-evolution activity. This result indicates that little, if any, of the catalase activity from PSII membranes in the dark is catalyzed by the O2-evolving center of PSII. PPO activity was correlated with a 48 kDa polypeptide. However, the 48 kDa polypeptide and another heat-released polypeptide of 72 kDa have the same N-terminal sequence, which is also identical to that of a known 64 kDa protein [Hind, G., Marshak, D. R., & Coughlan, S. J. (1995) Biochemistry 34, 8157-8164]. During heat treatment of PSII membranes and further manipulations it was found that the 72 kDa polypeptide was largely converted into the 48 kDa polypeptide. Thus, the 72 kDa polypeptide appears to be a latent precursor of the active 48 kDa PPO. The PSII membrane-associated PPO was purified by anion-exchange chromatography and was characterized in terms of substrate specificity, pH optimum, inhibitor sensitivity and native molecular weight. The heat-released PPO appears to be identical to the enzyme previously isolated from spinach thylakoid membranes [Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977-984].

O-GlcNAcylation and oxidation of proteins: is signalling in the cardiovascular system becoming sweeter?

PubMed Central

Lima, Victor V.; Spitler, Kathryn; Choi, Hyehun; Webb, R. Clinton; Tostes, Rita C.

2012-01-01

O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury. PMID:22757958