Synthesis and anti-inflammatory activities of two new N-acetyl glucosamine derivatives.

N-acetyl glucosamine (NAG) is a natural amino sugar found in various human tissues with previously described anti-inflammatory effects. Various chemical modifications of NAG have been made to promote its biomedical applications. In this study, we synthesized two bi-deoxygenated NAG, BNAG1 and BNAG2 and investigated their anti-inflammatory properties, using an in vivo and in vitro inflammation mouse model induced by lipopolysaccharide (LPS). Among the parent molecule NAG, BNAG1 and BNAG2, BNAG1 showed the highest inhibition against serum levels of IL-6 and TNF α and the leukocyte migration to lungs and peritoneal cavity in LPS challenged mice, as well as IL-6 and TNF α production in LPS-stimulated primary peritoneal macrophages. BNAG2 displayed an anti-inflammatory effect which was comparable to NAG. These findings implied potential application of these novel NAG derivatives, especially BNAG1, in treatment of certain inflammation-related diseases.

In Vitro Antiviral and Anti-Inflammatory Activities of N-Acetylglucosamine: Development of an Alternative and Safe Approach to Fight Viral Respiratory Infections.

Viral respiratory tract infections (RTIs) are responsible for significant morbidity and mortality worldwide. A prominent feature of severe respiratory infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is the cytokine release syndrome. Therefore, there is an urgent need to develop different approaches both against viral replication and against the consequent inflammation. N-acetylglucosamine (GlcNAc), a glucosamine (GlcN) derivative, has been developed as an immunomodulatory and anti-inflammatory inexpensive and non-toxic drug for non-communicable disease treatment and/or prevention. Recent studies have suggested that GlcN, due to its anti-inflammatory activity, could be potentially useful for the control of respiratory virus infections. Our present study aimed to evaluate in two different immortalized cell lines whether GlcNAc could inhibit or reduce both viral infectivity and the inflammatory response to viral infection. Two different viruses, frequent cause of upper and lower respiratory tract infections, were used: the H1N1 Influenza A virus (IAV) (as model of enveloped RNA virus) and the Human adenovirus type 2 (Adv) (as model of naked DNA virus). Two forms of GlcNAc have been considered, bulk GlcNAc and GlcNAc in nanoform to overcome the possible pharmacokinetic limitations of GlcNAc. Our study suggests that GlcNAc restricts IAV replication but not Adv infection, whereas nano-GlcNAc inhibits both viruses. Moreover, GlcNAc and mainly its nanoformulation were able to reduce the pro-inflammatory cytokine secretion stimulated by viral infection. The correlation between inflammatory and infection inhibition is discussed.

[O-linked N-acetylglucosamine modification induced by lipopolysaccharide is involved in inflammatory signaling pathway in endothelial cells].

OBJECTIVE: To explore whether the lipopolysaccharide (LPS)-induced modification of O-linked N-acetylglucosamine (O-GlcNAc) is involved in the inflammatory signaling pathway of endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured in vitro, and cells in logarithmic growth phase were used for experiments. Cells were divided into blank control group, LPS group (2 000 mg/L LPS), O-GlcNAc transferase (OGT) overexpression (OGT-OE)+LPS group (plasmid transfection OGT+2 000 mg/L LPS), protein kinase C (PKC) inhibitor+LPS group (10 µmol/L Go 6983+2 000 mg/L LPS), RhoA inhibitor+LPS group (40 µmol/L Rhoin hydrochloride+2 000 mg/L LPS), phosphatidylinositol-3-kinase (PI3K) inhibitor+LPS group (1 µmol/L SL-2052+2 000 mg/L LPS), serine/threonine kinase (Akt) inhibitor+LPS group (10 µmol/L PP2+2 000 mg/L LPS) and small interfering RNA (siRNA) treated Akt (si-AKT)+LPS group (si-Akt+2 000 mg/L LPS). After 24 hours of LPS treatment, real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the transcription levels of inflammatory cytokines [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. The protein expression or phosphorylation of OGT, O-GlcNAc, Akt, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), nuclear factor-κB p65 (NF-κB p65), and signal transducer and activator of transcription 3 (STAT3) were determined by Western blotting. RESULTS: Compared with the blank control group, the expression of OGT and the modification of O-GlcNAc in the LPS group were decreased, while the expressions of phosphorylated ERK, p38MAPK, and STAT3 were increased, and the transcript levels of inflammatory cytokines were also significantly increased [IL-6 mRNA (2-ΔΔCt): 4.71±0.60 vs. 1.03±0.29, TNF-α mRNA (2-ΔΔCt): 1.89±0.11 vs. 1.04±0.35, ICAM-1 mRNA (2-ΔΔCt): 2.06±0.18 vs. 1.02±0.21, VCAM-1 mRNA (2-ΔΔCt): 2.94±0.57 vs. 1.01±0.17, all P < 0.05], indicating that LPS could decrease O-GlcNAc modification, activate inflammatory signaling pathways and increase inflammatory cytokines expression. Compared with the LPS group, the expressions of phosphorylated ERK, p38MAPK, NF-κB p65, and STAT3 in the endothelial cells of the OGT-OE+LPS group were decreased, and the expression of inflammatory factors were significantly decreased [IL-6 mRNA (2-ΔΔCt): 0.12±0.01 vs. 0.90±0.17, TNF-α mRNA (2-ΔΔCt): 0.31±0.01 vs. 0.91±0.14, ICAM-1 mRNA (2-ΔΔCt): 0.64±0.02 vs. 1.13±0.16, VCAM-1 mRNA (2-ΔΔCt): 0.11±0.01 vs. 0.93±0.11, all P < 0.05], indicating that the increase of OGT level could inhibit the partial activation of the endothelial inflammatory signal pathway under the LPS stimulation. Compared with the blank control group, the phosphorylation level of Akt in the LPS group was increased. Compared with the LPS group, both OGT expression and O-GlcNAc modification were down-regulated after pretreatment of PKC inhibitor, RhoA inhibitor, PI3K inhibitor, or Akt inhibitor. Compared with the LPS group, the transcript levels of IL-6, TNF-α and ICAM-1 in the PP2+LPS group were significantly decreased [IL-6 mRNA (2-ΔΔCt): 1.46±0.16 vs. 3.55±0.87, TNF-α mRNA (2-ΔΔCt): 0.98±0.14 vs. 1.76±0.10, ICAM-1 mRNA (2-ΔΔCt): 1.39±0.24 vs. 2.04±0.13, all P < 0.05], but there was no significant change in VCAM-1. Compared with the LPS group, the expression of OGT and O-GlcNAc modification in the si-Akt+LPS group were decreased, while the transcript levels of inflammatory cytokines were also significantly decreased [IL-6 mRNA (2-ΔΔCt): 0.75±0.03 vs. 0.99±0.09, TNF-α mRNA (2-ΔΔCt): 0.69±0.01 vs. 1.10±0.08, ICAM-1 mRNA (2-ΔΔCt): 0.76±0.01 vs. 0.99±0.02, VCAM-1 mRNA (2-ΔΔCt): 0.93±0.08 vs. 1.20±0.21, all P < 0.05], indicating that Akt participated in the action process of LPS on OGT and affected the inflammatory factor expression. CONCLUSIONS: The decreased level of O-GlcNAc modification in endothelial cells stimulated with LPS promotes partial activation of inflammatory signaling pathways, mainly involving ERK, p38MAPK, and STAT3, and affects the expression of inflammatory factors. AKT may be involved in the effect of LPS on the inhibition of O-GlcNAc modification.

In-silico screening and microsecond molecular dynamics simulations to identify single point mutations that destabilize ß-hexosaminidase A causing Tay-Sachs disease.

ß-hexosaminidase A (HexA) protein is responsible for the degradation of GM2 gangliosides in the central and peripheral nervous systems. Tay-Sachs disease occurs when HexA within Hexosaminidase does not properly function and harmful GM2 gangliosides begin to build up within the neurons. In this study, in silico methods such as SIFT, PolyPhen-2, PhD-SNP, and MutPred were utilized to analyze the effects of nonsynonymous single nucleotide polymorphisms (nsSNPs) on HexA in order to identify possible pathogenetic and deleterious variants. Molecular dynamics (MD) simulations showed that two mutants, P25S and W485R, experienced an increase in structural flexibility compared to the native protein. Particularly, there was a decrease in the overall number and frequencies of hydrogen bonds for the mutants compared to the wildtype. MM/GBSA calculations were performed to help assess the change in binding affinity between the wildtype and mutant structures and a mechanism-based inhibitor, NGT, which is known to help increase the residual activity of HexA. Both of the mutants experienced a decrease in the binding affinity from -23.8 kcal/mol in wildtype to -20.9 and -18.7 kcal/mol for the P25S and W485R variants of HexA, respectively.

Cholestenone functions as an antibiotic against Helicobacter pylori by inhibiting biosynthesis of the cell wall component CGL.

Helicobacter pylori, a pathogen responsible for gastric cancer, contains a unique glycolipid, cholesteryl-α-D-glucopyranoside (CGL), in its cell wall. Moreover, O-glycans having α1,4-linked N-acetylglucosamine residues (αGlcNAc) are secreted from gland mucous cells of gastric mucosa. Previously, we demonstrated that CGL is critical for H. pylori survival and that αGlcNAc serves as antibiotic against H. pylori by inhibiting CGL biosynthesis. In this study, we tested whether a cholesterol analog, cholest-4-en 3-one (cholestenone), exhibits antibacterial activity against H. pylori in vitro and in vivo. When the H. pylori standard strain ATCC 43504 was cultured in the presence of cholestenone, microbial growth was significantly suppressed dose-dependently relative to microbes cultured with cholesterol, and cholestenone inhibitory effects were not altered by the presence of cholesterol. Morphologically, cholestenone-treated H. pylori exhibited coccoid forms. We obtained comparable results when we examined the clarithromycin-resistant H. pylori strain "2460." We also show that biosynthesis of CGL and its derivatives cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside and cholesteryl-6-O-phosphatidyl-α-D-glucopyranoside in H. pylori is remarkably inhibited in cultures containing cholestenone. Lastly, we asked whether orally administered cholestenone eradicated H. pylori strain SS1 in C57BL/6 mice. Strikingly, mice fed a cholestenone-containing diet showed significant eradication of H. pylori from the gastric mucosa compared with mice fed a control diet. These results overall strongly suggest that cholestenone could serve as an oral medicine to treat patients infected with H. pylori, including antimicrobial-resistant strains.

Interplay between NOD1 and TLR4 Receptors in Macrophages: Nonsynergistic Activation of Signaling Pathways Results in Synergistic Induction of Proinflammatory Gene Expression.

Interactions between pattern-recognition receptors shape innate immune responses to pathogens. NOD1 and TLR4 are synergistically interacting receptors playing a pivotal role in the recognition of Gram-negative bacteria. However, mechanisms of their cooperation are poorly understood. It is unclear whether synergy is produced at the level of signaling pathways downstream of NOD1 and TLR4 or at more distal levels such as gene transcription. We analyzed sequential stages of human macrophage activation by a combination of NOD1 and TLR4 agonists (N-acetyl-d-muramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid [M-triDAP] and LPS, respectively). We show that events preceding or not requiring activation of transcription, such as activation of signaling kinases, rapid boost of glycolysis, and most importantly, nuclear translocation of NF-κB, are regulated nonsynergistically. However, at the output of the nucleus, the combination of M-triDAP and LPS synergistically induces expression of a subset of M-triDAP- and LPS-inducible genes, particularly those encoding proinflammatory cytokines (TNF, IL1B, IL6, IL12B, and IL23A). This synergistic response develops between 1 and 4 h of agonist treatment and requires continuous signaling through NOD1. The synergistically regulated genes have a lower basal expression and higher inducibility at 4 h than those regulated nonsynergistically. Both gene subsets include NF-κB-inducible genes. Therefore, activation of the NF-κB pathway does not explain synergistic gene induction, implying involvement of other transcription factors. Inhibition of IKKß or p38 MAPK lowers agonist-induced TNF mRNA expression but does not abolish synergy. Thus, nonsynergistic activation of NOD1- and TLR4-dependent signaling pathways results in the synergistic induction of a proinflammatory transcriptional program.

Synthesis and cytotoxicity of the conjugates of diterpenoid isosteviol and N-acetyl-D-glucosamine.

A series of conjugates of diterpenoid isosteviol (16-oxo-ent-beyeran-19-oic acid) and N-acetyl-D-glucosamine was synthesised and their cytotoxicity against several human cancer cell lines (M-Hela, MCF-7, Hep G2, Panc-1, PC-3), as well as normal human cell lines (WI-38, Chang liver) was assayed. Most of the conjugates were found to be cytotoxic against the mentioned cancer cell lines in the range of IC50 values 13-89 µM. Two lead compounds 14a and 14b showed selective cytotoxicity against M-Hela (IC50 13 and 14 µM) that was two times as high as the cytotoxicity of the anti-cancer drug Tamoxifen in control (IC50 28 µM). It was found that cytotoxic activity of the lead compounds against M-Hela cells is due to induction of apoptosis.

Glucosamine regulates hepatic lipid accumulation by sensing glucose levels or feeding states of normal and excess.

Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.

Effects of glucuronic acid and N-acetyl-D-glucosamine supplementation on the perivitelline space during the IVM of pig oocytes.

The objective of this study was to minimise polyspermic penetration by increasing the perivitelline space (PVS) thickness through supplementation of the hyaluronic acid components glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Oocytes (n=4690) were supplemented during the first 24h and/or the remainder of maturation (final 16-18h) with 0.01mM glucuronic acid and 0.01mM GlcNAc and then evaluated for PVS thickness, hyaluronic acid, glutathione and glutathione peroxidase concentrations. Fertilised oocytes were evaluated for polyspermic penetration and embryo development. The PVS thickness and amount of hyaluronic acid was significantly (P<0.05) greater in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. In addition, polyspermic penetration was significantly (P<0.05) less in oocytes supplemented with 0.01mM glucuronic acid and 0.01mM GlcNAc during the second part or all of maturation compared with the other treatments. Supplementing 0.01mM glucuronic acid and GlcNAc during maturation significantly (P<0.05) increased the percentage of cleaved embryos by 48h after IVF and blastocysts formed by 144h after IVF compared those not supplemented. These results indicate that supplementing PVS components during maturation decreases polyspermic penetration by increasing PVS thickness.

Purification and Assays of Tachylectin-2.

Tachylectin-2, a 27-kDa protein consisting of a five-bladed ß-propeller structure, is purified by three steps of chromatography, including dextran sulfate-Sepharose CL-6B, CM-Sepharose CL-6B, and Mono S. Three isolectins of tachylectin-2 including tachylectin-2a, -2b, and -2c are purified. These isolectins exhibit hemagglutinating activity against human A-type erythrocytes in a Ca2+-independent manner with tachylectin-2b showing the highest activity. Tachylectin-2b specifically agglutinates Staphylococcus saprophyticus KD. The tachylectin-2b-mediated hemagglutination is inhibited in the presence of GlcNAc and GalNAc. The association constants for GlcNAc and GalNAc are Ka = 1.95 × 104 M-1 and Ka = 1.11 × 103 M-1, respectively. Ultracentrifugation analysis shows that tachylectin-2b is present in monomer form in solution.

O-GlcNAc Signaling Augmentation Protects Human Corneal Endothelial Cells from Oxidative Stress via AKT Pathway Activation.

Purpose: To investigate the effect of inhibitor of O-glycosylation on human corneal endothelial cells (HCECs) under oxidative stress.Methods: HCECs were cultured and treated with 10 mM tert-butyl hydroperoxide (tBHP) with or without PUGNAc, a known inhibitor of OGA. Cell viability was assessed. Mitochondrial membrane potential (ΔΨm) was measured. Intracellular Ca2+ levels and mitochondrial Ca2+ levels were measured. Intracellular reactive oxygen species formation was measured. Levels of O-linked ß-N-acetylglucosamine (O-GlcNAc), AKT, and pAKT were evaluated by Western blotting.Results: O-GlcNAc augmentation by PUGNAc increased cell viability, attenuated the loss of ΔΨm, and intracellular ROS against tBHP-induced oxidative stress (p < .05). O-GlcNAc augmentation reduced tBHP-induced mitochondrial calcium overload (p < .05) while it did not have any effect on intracellular calcium overload with tBHP. Furthermore, AKT signaling was activated in the cells with O-GlcNAc augmentation.Conclusions: O-GlcNAc signaling augmentation protects HCECs from oxidative stress via activation of AKT pathways.

Carbamothioic S-acid derivative and kigamicins, the activated production of silent metabolites in Amycolatopsis alba DSM 44262Δabm9 elicited by N-acetyl-D-glucosamine.

One new carbamothioic S-acid derivative (1) and five known kigamicin derivatives (2-6) were isolated from the fermentation extract of Amycolatopsis alba DSM 44262Δabm9 elicited by N-acetyl-D-glucosamine. HPLC-DAD-UV analyses indicated that the DSM 44262Δabm9 strain did not produce these metabolites originally and the production of 1-6 was induced by adding 25 mM N-acetyl-D-glucosamine in the culture medium. The structures of 1-6 were identified on the basis of NMR spectroscopic data and high-resolution ESIMS. These results highlight that addition of N-acetyl-D-glucosamine in the microbial culture medium could activate cryptic gene expression, induce and increase the production of new or known secondary metabolites.

A Novel Nanofiber Hydrogel Loaded with Platelet-Rich Plasma Promotes Wound Healing Through Enhancing the Survival of Fibroblasts.

BACKGROUND Hydrogels are ideal biological carriers in vivo and have been widely used in the treatment of wound healing through loading with or without bioactive substances. Platelet-rich plasma (PRP) is purified from autologous plasma and has known curative efficacy for wound healing. The combined efficacy of shorten poly-N-acetyl glucosamine (sNAG) hydrogels and PRP in the treatment of wound healing has not been previously assessed. MATERIAL AND METHODS The cytotoxic and proliferative effects of PRP on fibroblasts were detected using Cell Counting Kit-8 assays and flow cytometry. The levels of cyclin D1 and cyclin D3 were assessed to evaluate cell proliferation. Protein expression was assessed by western blot analysis. Adenosine levels were assessed by enzyme-linked immunosorbent assay. Cell apoptosis was assessed by flow cytometry and western blot analysis. Rat wound models were performed, and the effects of PRP, single hydrogels, and sNAG hydrogels loaded with PRP were respectively detected through the assessment of wound closure. Hematoxylin eosin staining was used to measure the depth and width of regenerative scars. RESULTS Our results demonstrated that PRP promotes fibroblast proliferation and inhibits apoptosis. PRP contains abundant levels of adenosine, which has a positive role on fibroblast function, whilst the inhibition of adenosine A2A receptors impairs the efficacy of PRP. sNAG hydrogels loaded with PRP showed curative efficacy during wound healing in mice. Mice treated with hydrogels loaded with PRP showed high levels of regeneration with scarless healing. CONCLUSIONS Our results indicate that sNAG hydrogels loaded with PRP promote wound healing. The pro-proliferative, and anti-apoptotic effects of the fibroblasts are mediated by the activating A2A receptor in response to elevated adenosine levels.

The relationship between the structural characteristics of lactobacilli-EPS and its ability to induce apoptosis in colon cancer cells in vitro.

Colon cancer is one of the most common cancer around the world. Exopolysaccharides (EPSs) produced by lactobacilli as potential prebiotics have been found to have an anti-tumor effect. In this study, lyophilized EPSs of four Lactobacillus spp. for their impact on apoptosis in colon cancer cells (HT-29) was evaluated using flow cytometry. The relationship between capability of a lactobacilli-EPS to induce apoptosis and their monosaccharide composition, molecular weight (MW), and linkage type was investigated by HPLC, SEC, and NMR, respectively. Changes in apoptotic-markers were examined by qPCR and Western Blotting. EPSs were capable of inhibiting proliferation in a time-dependent manner and induced apoptosis via increasing the expression of Bax, Caspase 3 and 9 while decreasing Bcl-2 and Survivin. All EPSs contained mannose, glucose, and N-acetylglucosamine with different relative proportions. Some contained arabinose or fructose. MW ranged from 102-104Da with two or three fractions. EPS of L. delbrueckii ssp. bulgaricus B3 having the highest amount of mannose and the lowest amount of glucose, showed the highest apoptosis induction. In conclusion, lactobacilli-EPSs inhibit cell proliferation in HT-29 via apoptosis. Results suggest that a relationship exists between the ability of EPS to induce apoptosis and its mannose and glucose composition.

Ac4GlcNAcF3, an OGT-tolerated but OGA-resistant regulator for O-GlcNAcylation.

O-Linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslationalmonosaccaride-modification found on Ser or Thr residues of intracellular proteins in most eukaryotes. The dynamic nature of O-GlcNAc has enabled researchers to modulate the stoichiometry of O-GlcNAc on proteins in order to investigate its function. Cell permeable small moleculars have proven invaluable tools to increase O-GlcNAc levels. Herein, using in vitro substrate screening, we identified GlcNAcF3 as an OGT-accepted but OGA-resistant sugar mimic. Cellular experiments with cell-permeable peracetylated-GlcNAcF3 (Ac4GlcNAcF3) displayed that Ac4GlcNAcF3 was a potent tool to increase O-GlcNAc levels in several cell lines. Further, NIH3T3 cells interfered with OGT (siOGT) showed significant decreasing of O-GlcNAc levels with Ac4GlcNAcF3 treatment, indicating O-GlcNAcF3 was an OGT-dependent modification. In addition, cellular toxic assay confirmed O-GlcNAcF3 production has no significant effect on cell proliferation or viability. Thus, Ac4GlcNAcF3 represents a safe and dual regulator for both OGT and OGA, which will benefit the study of O-GlcNAc.

Serenoa repens and N-acetyl glucosamine/milk proteins complex differentially affect the paracrine communication between endothelial and follicle dermal papilla cells.

Current treatments for hair follicle (HF) disruption are based on 5-α reductase inhibitors and prostaglandin modulators. Botanicals and nutraceutical compounds interfere with hair loss or stimulate its partial regrowth. Here, we used in vitro cocultures to investigate the activity of Serenoa repens ( SR) and N-acetyl glucosamine + milk proteins (NAG/Lac) on the paracrine interactions between human microvascular endothelial cells (HMVEC) and HF dermal papilla cells (FDPC). Both SR and NAG/Lac-induced endothelial tubulogenesis were enhanced by FDPC. SR promoted proliferation of both the cell types, while NAG/Lac was effective on endothelium. Vascular endothelial growth factor production, enhanced by SR, was further augmented by FDPC. In FDPC 5-α reductase-II and ß-catenin expressions were modified by SR and less by NAG/Lac, with no additional effect by HMVEC. SR and NAG/Lac prevented lipid peroxidation, whereas NAG/Lac was effective on interleukin 1ß production. Finally, SR and NAG/Lac differentially affected HMVEC permeability and tight junction proteins content. These data provide a mechanistic background for the potential use of these compounds as promoters of HF vascularization.

Glucosamine prevents polarization of cytotoxic granules in NK-92 cells by disturbing FOXO1/ERK/paxillin phosphorylation.

Glucosamine (GlcN) is a naturally occurring derivative of glucose and an over-the-counter food additive. However, the mechanism underlying GlcN action on cells is unknown. In this study, we investigated the effect of GlcN on natural killer (NK) cells. We demonstrate that GlcN affects NK-92 cell cytotoxicity by altering the distribution of cathepsin C, a cysteine protease required for granzyme processing in cytotoxic granules. The relocation of cathepsin C due to GlcN was shown to be accompanied by a decrease in the intracellular enzyme activity and its extracellular secretion. Similarly, the relocation of endosomal aspartic cathepsin E was observed. Furthermore, we elucidated that repositioning of cathepsin C is a consequence of altered signaling pathways of cytotoxic granule movement. The inhibition of phosphorylation upstream and downstream of ERK by GlcN disturbed the polarized release of cytotoxic vesicles. Considerable changes in the ERK phosphorylation dynamics, but not in those of p38 kinase or JNK, were observed in the IL2-activated NK-92 cells. We found decreased phosphorylation of the transcription factor FOXO1 and simultaneous prolonged phosphorylation of ERK as well as its nuclear translocation. Additionally, a protein downstream of the ERK phosphorylation cascade, paxillin, was less phosphorylated, resulting in a diffuse distribution of cytotoxic granules. Taken together, our results suggest that dietary GlcN affects signaling pathway activation of NK-92 immune cells.

N-Acetyl-Glucosamine Sensitizes Non-Small Cell Lung Cancer Cells to TRAIL-Induced Apoptosis by Activating Death Receptor 5.

BACKGROUND/AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anti-cancer agent due to its selective toxicity. However, many human non-small cell lung cancer (NSCLC) cells are partially resistant to TRAIL, thereby limiting its clinical application. Therefore, there is a need for the development of novel adjuvant therapeutic agents to be used in combination with TRAIL. METHODS: In this study, the effect of N-acetyl-glucosamine (GlcNAc), a type of monosaccharide derived from chitosan, combined with TRAIL was evaluated in vitro and in vivo. Thirty NSCLC clinical samples were used to detect the expression of death receptor (DR) 4 and 5. After GlcNAc and TRAIL co-treatment, DR expression was determined by real-time PCR and western blotting. Cycloheximide was used to detect the protein half-life to further understand the correlation between GlcNAc and the metabolic rate of DR. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect receptor clustering, and the localization of DR was visualized by immunofluorescence under a confocal microscope. Furthermore, a co-immunoprecipitation assay was performed to analyze the formation of death-inducing signaling complex (DISC). O-linked glycan expression levels were evaluated following DR5 overexpression and RNA interference mediated knockdown. RESULTS: We found that the clinical samples expressed higher levels of DR5 than DR4, and GlcNAc co-treatment improved the effect of TRAIL-induced apoptosis by activating DR5 accumulation and clustering, which in turn recruited the apoptosis-initiating protease caspase-8 to form DISC, and initiated apoptosis. Furthermore, GlcNAc promoted DR5 clustering by improving its O-glycosylation. CONCLUSION: These results uncovered the molecular mechanism by which GlcNAc sensitizes cancer cells to TRAIL-induced apoptosis, thereby highlighting a novel effective agent for TRAIL-mediated NSCLC-targeted therapy.

Novel glycopolymer sensitizes Burkholderia cepacia complex isolates from cystic fibrosis patients to tobramycin and meropenem.

Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8-128 µg/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (≤4 µg/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.

The comparison of the effects of a novel hydrogel compound and traditional hyaluronate following micro-fracture procedure in a rat full-thickness chondral defect model.

PURPOSE: The aim of this experimental study was to investigate the impact of HA-CS-NAG compound (hyaluronate, sodium chondroitin sulfate, N-acetyl-d-glucosamine) on the quality of repair tissue after micro-fracture and to compare it with HA (hyaluronat), in a rat full-thickness chondral defect model. METHODS: Full-thickness chondral defects were created in a non-weight bearing area by using a handle 2.7-mm drill bit, in the right knees of 33 Sprague-Dawley rats. Each specimen then underwent micro-fracture using a needle. Two weeks after surgery, 3 groups were randomly formed among the rats (n = 33). In Group 1, 0.2 mL of sterile saline solution (0.9%) was injected. In Group 2, 0.2 mL HA with a mean molecular weight of 1.2 Mda was injected. In Group 3, 0.2 mL of HA-CS-NAG compound (hyaluronate, sodium chondroitin sulfate, N-acetyl-d-glucosamine) was injected. The injections were applied on the 14th, the 21st and the 28th postoperative days. All rats were sacrificed on the 42nd postoperative day. Histological analysis of the repair tissue was performed for each specimen by two blinded observers using Wakitani scoring system. RESULTS: There was significantly improved repair tissue in both Group 3 and Group 2 when compared with Group 1. Group 3 showed statistically significant improvement in terms of 'cell morphology' and 'integration of donor with host' when compared to Group 2 (p < 0.001). CONCLUSION: Intra-articular injection of HA-CS-NAG compound after micro-fracture results in significantly improved repair tissue in rats' chondral defects when compared to HA regarding the donor integration and cell morphology.