MicroSPECT Imaging-Guided Treatment of Idiopathic Pulmonary Fibrosis in Mice with a Vimentin-Targeting 99mTc-Labeled N-Acetylglucosamine-Polyethyleneimine.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease with poor prognosis. Evidence has shown that vimentin is a key regulator of lung fibrogenesis. 99mTc-labeled N-acetylglucosamine-polyethyleneimine (NAG-PEI), a vimentin-targeting radiotracer, was used for the early diagnosis of IPF, and NAG-PEI was also used as a therapeutic small interfering RNA (siRNA) delivery vector for the treatment of IPF in this study. Single-photon emission-computed tomography (SPECT) imaging of bleomycin (BM)- and silica-induced IPF mice with 99mTc-labeled NAG-PEI was performed to visualize pulmonary fibrosis and monitor the treatment efficiency of siRNA-loaded NAG-PEI, lipopolysaccharide (LPS, a tolerogenic adjuvant), or zymosan (ZYM, an immunostimulant). The lung uptakes of 99mTc-NAG-PEI in the BM- and silica-induced IPF mice were clearly and directly correlated with IPF progression. The lung uptake of 99mTc-NAG-PEI in the NAG-PEI/TGF-ß1-siRNA treatment group or LPS treatment group was evidently lower than that in the control group, while the lung uptake of 99mTc-NAG-PEI was significantly higher in the ZYM treatment group compared to that in the control group. These results demonstrate that NAG-PEI is a potent MicroSPECT imaging-guided theranostic platform for IPF diagnosis and therapy.

GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) Is a GlcNAc-Containing Glycosylinositol Phosphorylceramide Glycosyltransferase.

Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structures, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis (Arabidopsis thaliana) and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain GlcNAc and GlcN [collectively GlcN(Ac)]. In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, "rice-like" GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.

Glucosamine and N-acetyl glucosamine as new CEST MRI agents for molecular imaging of tumors.

The efficacy of glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) as agents for chemical exchange saturation transfer (CEST) magnetic resonance molecular imaging of tumors is demonstrated. Both agents reflect the metabolic activity and malignancy of the tumors. The method was tested in two types of tumors implanted orthotopically in mice: 4T1 (mouse mammary cancer cells) and MCF7 (human mammary cancer cells). 4T1 is a more aggressive type of tumor than MCF7 and exhibited a larger CEST effect. Two methods of administration of the agents, intravenous (IV) and oral (PO), gave similar results. The CEST MRI observation of lung metastasis was confirmed by histology. The potential of the clinical application of CEST MRI with these agents for cancer diagnosis is strengthened by their lack of toxicity as can be indicated from their wide use as food supplements.

Novel biologically active series of N-acetylglucosamine derivatives for the suppressive activities on GAG release.

(d)-Glucosamine and other nutritional supplements have emerged as safe alternative therapies for osteoarthritis, a chronic and degenerative articular joint disease. N-acetyl-(d)-glucosamine, a compound that can be modified at the N position, is considered to improve the oral bioavailability of (d)-glucosamine and has been proven to possess greater in vitro chondroprotective activity compared with the parent agent. In this study, to further utilize these properties, we focus on the modification of the N position with a benzenesulfonyl and different isoxazole formyl groups. Among these compounds, the 3-(2-chlorobenzene)-5-methyl-isoxazole formyl chloride and p-methoxybenzenesulfonyl chloride modifying structures proved to be the most active of the series and efficiently processed the chondrocytes in vitro. These novel N-position substitution compounds may represent promising leads for osteoarthritis drug development.

Triggering effect of N-acetylglucosamine on retarded drug release from a lectin-anchored chitosan nanoparticles-in-microparticles system.

The aim of this study was to investigate the use of N-acetylglucosamine (NAG) to accelerate drug release from a lectin-modified carrier. A wheat germ agglutinin (WGA)-anchored salmeterol xinafoate (SalX)-loaded nanoparticles-in-microparticles system (NiMS) was prepared with an ionotropic gelation technique combined with a spray drying method. The formulated microparticles were spherical, with diameters ranging mainly from 2 to 8 µm; the drug entrapment efficiency was >70% (w/w), and the loading capacity was approximately 8% (w/w). Drug release from WGA-SalX-NiMS, within the first 4h, was approximately 30% less than that from SalX-NiMS, indicating an effect of lectin-modification to retard drug release from the NiMS. Due to "sugar-lectin" interactions, drug release from WGA-SalX-NiMS was substantially increased after the addition of NAG to the release medium. However, no significant influence of NAG was observed on the drug release profile of SalX-NiMS without WGA anchorage. The characteristics of NAG-WGA interaction may provide valuable insights into the "triggering-effects" of specific sugars on drug release from lectin-anchored carriers. These results suggest that it is possible to control drug release from a lectin-anchored drug delivery system using a specific sugar, and that the designed novel WGA-SalX-NiMS may be a suitable formulation for chronotherapy of asthma.

N-acetylglucosamine biofunctionalized CdSeTe quantum dots as fluorescence probe for specific protein recognition.

Hsp70 proteins are implicated in resistance to chemotherapy in cancers, the detection of which is important for cancer treatment and prognosis. In this work, we report the study on the detection of specific intracellular target protein in fixed cells using GlcNAc-conjugated CdSeTe QDs. The QDs were coupled with Con A via a carbodiimide reaction and then were further assembled with GlcNAc by lectin-carbohydrate interaction between Con A and GlcNAc. The obtained QDs-Con A-GlcNAc conjugates have an emission wavelength at 650 nm that is close to the near-infrared (NIR) regions and a specific recognition for Hsp70. These results show that the QDs-Con A-GlcNAc probe can be a promising tool for direct localization of the Hsp70 protein.

De novo biosynthesis of starch chains without a primer and the mechanism for its biosynthesis by potato starch-synthase.

The starch-synthase enzymes used in this study were the second acetone precipitate and Fractions 21 and 23, Table 1, [Mukerjea, Ru.; Falconer, D. J.; Yoon, S.-H.; Robyt, J. F. Carbohydr. Res. 2010, 345, 1555-1563]. Fractions 21 and 23 had high specific activities of 544 and 944 International Units/mg, respectively. When the enzymes and buffer and substrate were treated with immobilized α-amylase and glucoamylase for 30 min, they all had the same activity, before and after treatment, indicating that the enzymes were free of putative primers and synthesized amylose chains de novo, without the addition of primers. Starch-synthase was immobilized and reacted with ADP-[(14)C]Glc; the immobilized enzyme was removed, washed and treated at pH 2 and 50 °C for 30 min, giving the release of (14)C-D-glucopyranose and (14)C-amylose, showing that during catalysis they were covalently attached to the enzyme active-site. Pulse and chase reactions of starch-synthase with ADP-[(14)C]Glc and ADPGlc, respectively, followed by reduction and acid hydrolysis of the starch-chain product, gave (14)C-D-glucitol from the pulse reaction and a significant decrease of (14)C-D-glucitol from the chase reaction, showing that the addition of D-glucose from ADPGlc was to the reducing-ends of the growing amylose chains. Reactions of four different concentrations of starch-synthase, with constant ADPGlc concentration and temperature, gave four amylose chains, each with different number average molecular weights that were inversely proportional to the concentration of the enzyme, indicating that the synthesis was processive. From the results, a two catalytic-site, insertion mechanism is proposed for the biosynthesis of starch chains.

Glycomics for drug discovery: metabolic perturbation in androgen-independent prostate cancer cells induced by unnatural hexosamine mimics.

Inhibited: N-acetylglucosamine (GlcNAc) derivatives with a fluorine atom at the C4 position (2-4) were synthesized, and their ability to inhibit cancer-cell growth was investigated. The administration of these 4F-GlcNAc derivatives to cells led to the unnatural sugar nucleotide 1. Furthermore, N-glycan profiles of cells were determined by using a glycoblotting-based enrichment analysis, which is suitable for high-throughput screenings for drug discovery.

Investigations into an alternate approach to target mannose receptors on macrophages using 4-sulfated N-acetyl galactosamine more efficiently in comparison with mannose-decorated liposomes: an application in drug delivery.

In this study the potential of 2 different ligands, i.e., palmitoyl mannose (Man-Lip) and 4-SO(4)GalNAc (Sulf-Lip) to target resident macrophages was investigated after surface decoration of Amphotericin B (AmB) loaded liposomes. In the case of Sulf-Lip, the 4-SO(4)GalNAc was adsorbed through electrostatic interaction on cationic liposomes, which was confirmed by change in zeta potential from +48.2 ± 3.7 mV for Lip to +12.2 ± 1.3 mV for Sulf-Lip. The mean particle size of Sulf-Lip and Man-Lip was found to be 139.4 ± 7.4 nm and 147.4 ± 8.6 nm, respectively. Flow cytometric data reveal enhanced uptake of Sulf-Lip in both J774 and RAW cell lines in comparison with the uptake of Man-Lip. Intracellular localization studies indicate that the fluorescence intensity of Sulf-Lip was much higher in comparison with that of Man-Lip and Lip formulations. Sulf-Lip and Man-Lip showed significantly higher localization of AmB at all time points in comparison with Lip (P < 0.05) after intravenous (IV) administration. The studies provide evidence that 4-SO(4)GalNAc possesses a promising feature for targeting resident macrophages and its application in the conditions of leishmaniasis is in the offing. FROM THE CLINICAL EDITOR: This in vivo study compares two different ligands to deliver Amphotericin B l(AmB) loaded liposomes to resident macrophages. Targeted approaches showed significantly higher localization of AmB at all time points in comparison to non-targeted liposomes, and future applications in leishmaniasis are already under preparation.

Entadosides A-D, triterpene saponins and a glucoside of the sulphur-containing amide from the kernel nuts of Entada phaseoloides (L.) Merrill.

From a MeOH extract of kernel nuts of Entada phaseoloides (L.) Merrill, one new and one known sulphur-containing glucoside were isolated. From the 1-BuOH-soluble fraction of a H(2)O extract, four new triterpene saponins containing N-acetylglucosamine in their sugar chains were isolated. The antiproliferative activities of the triterpene saponins were assayed.

[Bio-based pharmaceutical polymers, possibility of their chemical modification and the applicability of modified polymers]. / Természetes alapú gyógyszerészeti polimerek, kémiai módosításuk lehetoségei és a módosított segédanyagok alkalmazhatósága.

Different types of polymers are widely used in biomedical, pharmaceutical and cosmetic purposes. Their applications are curbed, if the polymers can not break down by the body or if the polymer itself is harmful or decompose to harmful material. Authors provide an overview of different types of pharmaceutical polymers of various sources, of the structural characterization and possibilities of their chemical modification and of the classical and instrumental analytical examination methods. The paper deals with the limitations of the use of biopolymers, as well.

Synthesis and macrophage activation of lentinan-mimic branched amino polysaccharides: curdlans having N-Acetyl-d-glucosamine branches.

N-Acetyl-d-glucosamine branches were incorporated at the C-6 position of curdlan, a linear ß-1,3-d-glucan, and the resulting nonnatural branched polysaccharides were evaluated in terms of the immunomodulation activities in comparison with lentinan, a ß-1,3-d-glucan having d-glucose branches at C-6. To incorporate the amino sugar branches, we conducted a series of regioselective protection-deprotections of curdlan involving triphenylmethylation at C-6, phenylcarbamoylation at C-2 and C-4, and detriphenylmethylation. Subsequent glycosylation with a d-glucosamine-derived oxazoline, followed by deprotection gave rise to the branched curdlans with various substitution degrees. The products exhibited remarkable solubility in both organic solvents and water. Their immunomodulation activities were determined using mouse macrophagelike cells, and the secretions of both the tumor necrosis factor and nitric oxide proved to be significantly higher than those with lentinan. These results conclude that the amino sugar/curdlan hybrid materials are promising as a new type of polysaccharide immunoadjuvants useful for cancer chemotherapy.

Medicinal plants of Thailand. I structures of rheedeiosides A-D and cis-entadamide A ß-D-glucopyranoside from the seed kernels of Entada rheedei.

Four new oleanane-type triterpene oligoglycosides, named rheedeiosides A, B, C and D, and one new thioamide glycoside, cis-entadamide A ß-D-glucopyranoside, were isolated from the seed kernels of a Thai medicinal plant, Entada rheedei SPRENGEL. The rheedeiosides were found to contain N-acetylglucosamine as a sugar component. Their structures were elucidated based on spectral and chemical evidence.

Screening from the world's largest TCM database against H1N1 virus.

The swine influenza virus (H1N1) 2009 pandemic highlights the importance of having effective anti-viral strategies. Recently, oseltamivir (Tamiflu) resistant influenza viruses are identified; which further emphasizes the urgency in developing new antiviral agents. In influenza virus replication cycle, viral surface glycoprotein, hemagglutinin, is responsible for viral entry into host cells. Hence, a potentially effective antiviral strategy is to inhibit viral entry mechanism. To develop novel antiviral agent that inhibits viral entry, we analyzed 20,000 traditional Chinese medicine (TCM) ingredients in hemagglutinin subtype H1 sialic acid binding site found on H1N1 virus. We then performed molecular dynamics simulations to investigate receptor-ligand interaction of the candidates obtained from docking. Here, we report three TCM derivatives that have high binding affinities to H1 sialic acid binding site residues based on structure-based calculations. The top three derivatives, xylopine_2, rosmaricine_14 and rosmaricine_15, all have an amine group that interact with Glu83 and a pyridinium group that interact with Asp103. Molecular dynamics simulations show that these derivatives form strong hydrogen bonding with Glu83 but interact transiently with Asp103. We therefore suggest that an enhanced hemagglutinin inhibitor, based on our scaffold, should be designed to bind both Glu83 and Asp103 with high affinity.

Antitumor activity of N-acetyl-D-glucosamine-substituted glycoconjugates and combined therapy with keyhole limpet hemocyanin in B16F10 mouse melanoma model.

N-Acetyl-D-glucosamine-substituted glycoconjugates (GCJs) with the polyamidoamine (GN8P) or calix[4]arene (GN4C) scaffold represent ligands for NKR-P1 molecule and induce NK cell-mediated cytotoxicity in vitro. The in vivo effect of these GCJs on mouse melanoma model was determined when administered either alone or in combination with non-specific immunostimulator keyhole limpet hemocyanin (KLH). All types of treatment significantly reduced the tumor growth on day 23, while GN4C as well as KLH were effective continuously (from day 14). The GN4C also induced the longest mean survival time (46.3 ± 11.1 d), followed by KLH+GN4C (36.4 ± 12.1), KLH (35.6 ± 6.5), KLH+GN8P (35.6 ± 6.7), and GN8P (32.4 ± 7.0), compared to controls (29.8 ± 3.6). The B16F10 specific cytotoxicity of peripheral blood cells was significantly elevated by both KLH and GN8P, whereas not by GN4C. KLH increased the effect of the GN4C, but did not influence that of GN8P. GN4C was proved to exert anticancer activity in mouse melanoma model. The combination of KLH with GCJs did not generate synergism.

Quantification of O-glycosylation stoichiometry and dynamics using resolvable mass tags.

Mechanistic studies of O-GlcNAc glycosylation have been limited by an inability to monitor the glycosylation stoichiometries of proteins obtained from cells. Here we describe a powerful method to visualize the O-GlcNAc-modified protein subpopulation using resolvable polyethylene glycol mass tags. This approach enables rapid quantification of in vivo glycosylation levels on endogenous proteins without the need for protein purification, advanced instrumentation or expensive radiolabels. In addition, it establishes the glycosylation state (for example, mono-, di-, tri-) of proteins, providing information regarding overall O-GlcNAc site occupancy that cannot be obtained using mass spectrometry. Finally, we apply this strategy to rapidly assess the complex interplay between glycosylation and phosphorylation and discover an unexpected reverse 'yin-yang' relationship on the transcriptional repressor MeCP2 that was undetectable by traditional methods. We anticipate that this mass-tagging strategy will advance our understanding of O-GlcNAc glycosylation, as well as other post-translational modifications and poorly understood glycosylation motifs.

Modulation of transcription factor function by O-GlcNAc modification.

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of nuclear and cytoplasmic proteins is important for many cellular processes, and the number of proteins that contain this modification is steadily increasing. This modification is dynamic and reversible, and in some cases competes for phosphorylation of the same residues. O-GlcNAc modification of proteins is regulated by cell cycle, nutrient metabolism, and other extracellular signals. Compared to protein phosphorylation, which is mediated by a large number of kinases, O-GlcNAc modification is catalyzed only by one enzyme called O-linked N-acetylglucosaminyl transferase or OGT. Removal of O-GlcNAc from proteins is catalyzed by the enzyme beta-N-acetylglucosaminidase (O-GlcNAcase or OGA). Altered O-linked GlcNAc modification levels contribute to the establishment of many diseases, such as cancer, diabetes, cardiovascular disease, and neurodegeneration. Many transcription factors have been shown to be modified by O-linked GlcNAc modification, which can influence their transcriptional activity, DNA binding, localization, stability, and interaction with other co-factors. This review focuses on modulation of transcription factor function by O-linked GlcNAc modification.

Hyaluronidase binds differently DPPC, DPPS or GlcNAc-bearing glycolipid biomimetic monolayers.

Bovine testis hyaluronidase (btHyal) had been shown to have direct effects on cancer cells and to be a useful adjuvant in several medicines. Furthermore this enzyme had been found to be membrane-associated. Thus, in this work, the interactions between btHyal and membranes were analyzed by using lipid monolayers at the air-water interface as a biomimetic membrane system. This allowed us to define the btHyal interactions with two residues of hyaluronic acid (a btHyal substrate), GlcNAc and carboxylic group, which are present in cholesteryl-triethoxy-N-acetylglucosamine (Chol-E3-GlcNAc) and in DPPS, respectively. btHyal bound preferentially Chol-E3-GlcNAc monolayers and showed a decreasing affinity for Chol-E3-GlcNAc-DPPC monolayers containing decreasing amount of glycolipid, suggesting a crucial role of the glycolipid GlcNAc. Furthermore the significant btHyal binding to DPPS was not affected by the presence of free GlcNAc in the subphase. These results and the absence of significant binding of btHyal to pure DPPC monolayer suggest that the protein interacts with the lipid monolayer by mimicking the enzyme-substrate interactions or by electrostatic interactions.

Effect on the Ras/Raf signaling pathway of post-translational modifications of neurofibromin: in silico study of protein modification responsible for regulatory pathways.

Mapping and chemical characterization of post-translational modifications (PTMs) in proteins are critical to understand the regulatory mechanisms involving modified proteins and their role in disease. Neurofibromatosis type 1 (NF-1) is an autosomal dominantly inherited disorder, where NF1 mutations usually result in a reduced level of the tumor suppressor protein, neurofibromin (NF). NF is a multifunctional cytoplasmic protein that regulates microtubule dynamics and participates in several signaling pathways, particularly the RAS signaling pathway. NF is a Ras GTPase-activating protein (GAP) that prevents oncogenesis by converting GTP-Ras to GDP-Ras. This function of NF is regulated by phosphorylation. Interplay of phosphorylation with O-GlcNAc modification on the same or vicinal Ser/Thr residues, the Yin Yang sites, is well known in cytoplasmic and nuclear proteins. The dynamic aspects of PTMs and their interplay being difficult to follow in vivo, we undertook this in silico work to predict and define the possible role of Yin Yang sites in NF-1. Interplay of phosphorylation and O-GlcNAc modification is proposed as a mechanism controlling the Ras signaling pathway.

In silico modulation of HMGN-1 binding to histones and gene expression by interplay of phosphorylation and O-GlcNAc modification.

Utilizing different computational methods; phosphorylation, O-GlcNAc modification and Yin Yang sites are predicted in HMGN-1. Prediction results suggest that interplay of phosphorylation and O-GlcNAc modification regulates binding and removal of HMGN-1 with the nucleosome and its translocation from nucleus to cytoplasm and back to nucleus, consequently modulating gene expression.