Enhancement of O-GlcNAcylation on Mitochondrial Proteins with 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-d-pyranoside, Contributes to the Mitochondrial Network, Cellular Bioenergetics and Stress Response in Neuronal Cells under Ischemic-like Conditions.

O-GlcNAcylation is a nutrient-driven post-translational modification known as a metabolic sensor that links metabolism to cellular function. Recent evidences indicate that the activation of O-GlcNAc pathway is a potential pro-survival pathway and that acute enhancement of this response is conducive to the survival of cells and tissues. 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-ß-d-pyranoside (SalA-4g), is a salidroside analogue synthesized in our laboratory by chemical structure-modification, with a phenyl ring containing a para-methoxy group and a sugar ring consisting of N-acetylglucosamine. We have previously shown that SalA-4g elevates levels of protein O-GlcNAc and improves neuronal tolerance to ischemia. However, the specific target of SalA-4g regulating O-GlcNAcylation remains unknown. To address these questions, in this study, we have focused on mitochondrial network homeostasis mediated by O-GlcNAcylation in SalA-4g's neuroprotection in primary cortical neurons under ischemic-like conditions. O-GlcNAc-modified mitochondria induced by SalA-4g demonstrated stronger neuroprotection under oxygen glucose deprivation and reoxygenation stress, including the improvement of mitochondrial homeostasis and bioenergy, and inhibition of mitochondrial apoptosis pathway. Blocking mitochondrial protein O-GlcNAcylation with OSMI-1 disrupted mitochondrial network homeostasis and antagonized the protective effects of SalA-4g. Collectively, these data demonstrate that mitochondrial homeostasis mediated by mitochondrial protein O-GlcNAcylation is critically involved in SalA-4g neuroprotection.

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.

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.

Dietary Natural N-Acetyl-d-Glucosamine Prevents Bone Loss in Ovariectomized Rat Model of Postmenopausal Osteoporosis.

Postmenopausal osteoporosis has seriously affected the life quality of elderly women. A natural polymer, chitin, obtained from shells of crab and shrimp, has been widely used in the biomedical field owing to its nontoxicity, biocompatibility, and biodegradability. In this study, natural N-acetyl-d-glucosamine (NAG) was prepared from liquefied chitin. The protective activities of NAG in postmenopausal osteoporosis were evaluated on Sprague Dawley rats and osteoblast-based models. Results showed that oral administration of NAG boosted trabecular bone volume and trabecular numbers. Additionally, the calcium content in the femur and tibia increased, and femoral biomechanical properties improved. Furthermore, NAG supplementation significantly lowered alkaline phosphatase levels and increased calcium content in the serum of ovariectomized rats. In vitro studies showed that NAG markedly promoted cell proliferation and stimulated osteoblast differentiation of mouse calvaria origin MC3T3-E1 cells with increased alkaline phosphatase activity in a concentration-dependent manner. Moreover, NAG effectively protected osteoblasts from oxidative damage induced by hydrogen peroxide. In conclusion, our data provide an additional foundation for dietary supplementation of NAG, which could protect and reverse osteopenia in postmenopausal women.

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.

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.

An Insight into the Changes in Skin Texture and Properties following Dietary Intervention with a Nutricosmeceutical Containing a Blend of Collagen Bioactive Peptides and Antioxidants.

BACKGROUND: Skin aging is a multifactorial phenomenon which causes alterations in skin physiological functions and, most visibly, phenotypic changes. In particular, during the aging process, hyaluronic acid, collagen, and elastin fibers undergo structural and functional changes. AIMS: This study aimed to give an insight into the photo-protective benefits and efficacy of an oral liquid nutricosmeceutical containing collagen bioactive peptides and antioxidants to counteract the signs of aging. METHODS: A double-blind, randomized, placebo-controlled clinical trial was conducted by an independent esthetic clinic on 120 healthy volunteer subjects for 90 days. Subjects were divided into 2 groups: 60 subjects consumed 1 bottle (50 mL) of the nutricosmeceutical daily and the other 60 consumed 1 bottle (50 mL) of the placebo. Outcome measures were related to skin elasticity (expressed as Young's elasticity modulus) and skin architecture (histological analysis). In addition, the subjects recruited in this study underwent observational assessments through self-assessment questionnaires. RESULTS AND CONCLUSIONS: Overall, we demonstrated a significant increase in skin elasticity (+7.5%), p ≤ 0.001 and an improvement in skin texture after daily oral consumption of the nutricosmeceutical. We also obtained a positive patient feedback through the self-assessment questionnaires. Taken together these results show that this nutricosmeceutical supplement may have photo-protective effects and help improve skin health.

Augmented O-GlcNAc signaling via glucosamine attenuates oxidative stress and apoptosis following contrast-induced acute kidney injury in rats.

Contrast-induced acute kidney injury (CI-AKI) is an iatrogenic renal injury and associated with substantial morbidity and mortality in susceptible individuals. Despite extensive study of a variety of agents for renal protection, limited strategies have been shown to be effective in the reduction of CI-AKI. O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational regulatory modification of intracellular proteins and governs the function of numerous proteins, both cytosolic and nuclear. Increasing evidence suggests that O-GlcNAc levels are increased in response to stress and that acute augmentation of this reaction is cytoprotective. However, the underlying mechanisms by which augmented OGlcNAc signaling provides renoprotection against contrast media insults is still unknown. Here, we investigated the effect of augmented O-GlcNAc signaling via glucosamine on CI-AKI and explored the underlying molecular mechanisms, particularly its relationship with PI3-kinase (PI3K)/Akt signaling. We used a novel and reliable CI-AKI model consisting of 5/6 nephrectomized (NE) rats, and a low-osmolar contrast media (iohexol, 10mL/kg, 3.5gI) injected via the tail vein after dehydration for 48h. The results showed that augmented O-GlcNAc signaling by glucosamine prevented the kidneys against iohexol-induced injury characterized by the attenuation of renal dysfunction, tubular damage, apoptosis and oxidative stress. Furthermore, this renoprotection was blocked by treatment with alloxan, an O-GlcNAc transferase inhibitor. Augmented O-GlcNAc signaling also increased the protein expression levels of phospho-Akt (Ser473, but not Thr308 and Thr450), phospho-GSK-3ß, Nrf2, and Bcl-2, and decreased the levels of Bax and cleaved caspase-3. Both alloxan and specific inhibitors of PI3K (Wortmannin and LY294002) blocked the protection of glucosamine via inhibiting Akt signaling pathway. We further identified O-GlcNAcylated Akt through immunoprecipitation and western blot. We confirmed that Akt was modified by O-GlcNAcylation, and glucosamine pretreatment increased the O-GlcNAcylation of Akt. Collectively, the results demonstrate that glucosamine induces renoprotection against CI-AKI through augmented O-GlcNAc and activation of PI3K/Akt signaling, making it a promising strategy for preventing CI-AKI.

The repair of full-thickness articular cartilage defect using intra-articular administration of N-acetyl-D-glucosamine in the rabbit knee: randomized controlled trial.

BACKGROUND: Although various alterative models of therapy are used for cartilage repair, no definite conclusion has been reached. Glucosamine (GlcN) is widely used as a nutritional supplement. However, the clinical- evidence-based outcome of GlcN administration remains controversial. N-acetyl-D-glucosamine (GlcNAc), a derivative of GlcN, shows chondroprotective activity and mediates the activation of articular chondrocytes. Therefore, we investigated the effect of intra-articular administration of GlcNAc in rabbits' knee joints with experimental full-thickness articular cartilage (FTAC) defects. METHODS: Twelve male adult New Zealand white rabbits, providing 24 knees, were used in this study. FTAC defects were created in the high-weight-bearing area of the medial femoral condyles of bilateral knees. All rabbits were randomly allocated to analysis at postsurgical week 4 or postsurgical week 12. In the week 4 group, rabbits' knees (six per group) were intra-articularly injected with normal saline or with GlcNAc twice per week for 3 weeks, beginning 1 week postoperatively. In the week 12 group, the rabbits' knees (six in each group) were intra-articularly injected with normal saline or with GlcNAc twice per week for 4 weeks, beginning 1 week postoperatively. Rabbits were sacrificed at 4 or 12 weeks after surgery for macroscopic, histological and radiological examinations of the knee joints. RESULTS: All rabbits had no systemic or local adverse effects. The saline and GlcNAc groups showed visible differences in healing of the FTAC defect at the end of testing. At week 4, the GlcNAc group had a higher level of collagen type II (COL II) and showed up-regulated production of transforming growth factor (TGF)-ß2 and TGF-ß3, suggesting the involvement of endogenous growth factors. At week 12, the GlcNAc group displayed formation of hyaline-like cartilage regeneration with mature chondrocytes (SOX9+), robust glycosaminoglycan (GAG) content, and positive COL II content in both the adjacent cartilage and reparative sites. However, the saline group demonstrated mainly fibrocartilage scar tissue, indicating COL I expression. Furthermore, the GlcNAc group had significantly higher bone volume per tissue volume and higher trabecular thickness than the saline group. CONCLUSIONS: Intra-articular GlcNAc may promote the repair of experimental FTAC defects in the rabbit knee joint model.

Novel phosphoramidate prodrugs of N-acetyl-(D)-glucosamine with antidegenerative activity on bovine and human cartilage explants.

(D)-Glucosamine and other nutritional supplements have emerged as safe alternative therapies for osteoarthritis (OA), a chronic and degenerative articular joint disease. In our preceding paper, a series of novel O-6 phosphate N-acetyl (d)-glucosamine prodrugs aimed at improving the oral bioavailability of N-acetyl-(d)-glucosamine as its putative bioactive phosphate form were shown to have greater chondroprotective activity in vitro when compared to the parent agent. In order to extend the SAR studies, this work focuses on the O-3 and O-4 phosphate prodrugs of N-acetyl-(d)-glucosamine bearing a 4-methoxy phenyl group and different amino acid esters on the phosphate moiety. Among the compounds, the (l)-proline amino acid-containing prodrugs proved to be the most active of the series, more effective than the prior O-6 compounds, and well processed in chondrocytes in vitro. Data on human cartilage support the notion that these novel O-3 and O-4 regioisomers may represent novel promising leads for drug discovery for osteoarthritis.

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.

Cellular content of UDP-N-acetylhexosamines controls hyaluronan synthase 2 expression and correlates with O-linked N-acetylglucosamine modification of transcription factors YY1 and SP1.

Hyaluronan, a high molecular mass polysaccharide on the vertebrate cell surface and extracellular matrix, is produced at the plasma membrane by hyaluronan synthases using UDP-GlcNAc and UDP-GlcUA as substrates. The availability of these UDP-sugar substrates can limit the synthesis rate of hyaluronan. In this study, we show that the cellular level of UDP-HexNAc also controls hyaluronan synthesis by modulating the expression of HAS2 (hyaluronan synthase 2). Increasing UDP-HexNAc in HaCaT keratinocytes by adding glucosamine down-regulated HAS2 gene expression, whereas a decrease in UDP-HexNAc, realized by mannose treatment or siRNA for GFAT1 (glutamine:fructose-6-phosphate amidotransferase 1), enhanced expression of the gene. Tracing the UDP-HexNAc-initiated signal to the HAS2 promoter revealed no change in the binding of STAT3, NF-κB, and cAMP response element-binding protein, shown previously to mediate growth factor and cytokine signals on HAS2 expression. Instead, altered binding of SP1 and YY1 to the promoter correlated with cellular UDP-HexNAc content and inhibition of HAS2 expression. siRNA silencing of YY1 and SP1 confirmed their inhibitory effects on HAS2 expression. Reduced and increased levels of O-GlcNAc-modified SP1 and YY1 proteins were associated with stimulation or inhibition of HAS2 expression, respectively. Our data are consistent with the hypothesis that, by regulating the level of protein O-GlcNAc modifications, cellular UDP-HexNAc content controls HAS2 transcription and decreases the effects on hyaluronan synthesis that would result from cellular fluctuations of this substrate.

N-Acetylglucosamine suppress collagenases activation in ultraviolet B-irradiated human dermal fibroblasts: Involvement of calcium ions and mitogen-activated protein kinases.

BACKGROUND: N-Acetylglucosamine (GlcNAc) and its derivates have been utilized in dietary supplements and for therapeutic development due to their unique characteristics. GlcNAc is recognized primarily for its function as a precursor to hyaluronic acid, which plays a significant role in the structure and hydration of the extracellular matrix in skin, in both the epidermis and the dermis. OBJECTIVE: We investigated the protective effects of GlcNAc on immortalized human skin fibroblasts (HS68) against UVB damage. We then explored the inhibitory effects of GlcNAc on UVB-induced collagenases and investigated the molecular mechanism underlying those effects. METHODS: Those effects were assessed by semi-quantitative PCR, Western blotting and enzymatic activity assays. RESULTS: GlcNAc increased the viability of, and inhibited ROS production in, HS68 cells exposed to UVB irradiation. Pre-treatment of HS68 cells with GlcNAc inhibited UVB-induced production of the collagenases MMP-1 and MMP-13. Western blot analysis further revealed that GlcNAc markedly suppressed the enhancement of collagen degradation in UVB-exposed HS68 cells. GlcNAc also suppressed UVB-induced activation of c-Jun, c-Fos and NF-κB and the phosphorylation of MAPKs and PI3K/Akt, upstream modulators of AP-1 and NF-κB. Moreover, GlcNAc decreased the UVB-induced influx of Ca(2+) into HS68 cells and the phosphorylation of Ca(2+)/calmodulin-dependent kinases (CaMKs). CONCLUSION: The results indicate that GlcNAc inhibited UVB-induced collagenolytic MMP production by interfering with Ca(2+)-dependent Akt and MAPKs/AP-1 and NF-κB signaling. They may thus be potentially useful in the prevention and treatment of skin photoaging.

A 4-deoxy analogue of N-acetyl-D-glucosamine inhibits heparan sulphate expression and growth factor binding in vitro.

Heparan sulphate (HS) is a long, linear polysaccharide, which has a basic backbone of -beta1-4GlcA-alpha1-4GlcNAc- units. The involvement of HS in many steps of tumourigenesis, including growth and angiogenesis, makes it an appealing target for cancer therapy. To target the biosynthesis of HS by interfering with its chain elongation, a 4-deoxy analogue of N-acetyl-D-glucosamine (4-deoxy-GlcNAc) was synthesized. Using immunocytochemistry and agarose gel electrophoresis it was shown that incubation with the 4-deoxysugar resulted in a dose dependent reduction of HS expression of MV3 melanoma cells, 1 mM resulting in an almost nullified HS expression. The parent sugar GlcNAc had no effect. 4-deoxysugar treated cells were viable and proliferated at the same rate as control cells. Other glycan structures appeared to be only mildly affected, as staining by various lectins was generally not or only modestly inhibited. At 1 mM of the 4-deoxysugar, the capacity of cells to bind the HS-dependent pro-angiogenic growth factors FGF-2 and VEGF was greatly compromised. Using an in vitro angiogenesis assay, 4-deoxysugar treated endothelial cells showed a sharp reduction of FGF-2-induced sprout formation. Combined, these data indicate that an inexpensive, easily synthesized, water-soluble monosaccharide analogue can interfere with HS expression and pro-angiogenic growth factor binding.

Allium sativum L. extract prevents methyl mercury-induced cytotoxicity in peripheral blood leukocytes (LS).

Adenosine deaminase (ADA) is involved in purine metabolism and plays a significant role in the immune system. The focus of this investigation was to examine the effects of low concentrations of organic mercury on ADA activity in human leukocytes and to investigate the relationship between these effects and cell death. We have examined the protective potential effects of Allium sativum extract (GaE) against Methylmercury (MeHg)-induced cytotoxic effects on human leucocytes under in vitro conditions. MeHg (0.05-10 microM) significantly decreased leukocyte viability (58.97% for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and 51.67% for Alamar Blue (AB) and this decrease was positively correlated to the MeHg-induced inhibition of ADA activity. N-acetylcysteine (NAC) and GaE prevented both the MeHg-induced cytotoxic effects on leukocytes according to MTT and AB assays and the effects on the ADA activity. The present results suggest that the protective effects of GaE against MeHg-induced leukocyte damage is related to the removal of oxidant species generated in the presence of MeHg due to the antioxidant efficacy of garlic constituents. It is important to point out that the intense presence of ADA in Leukocyte suspension (LS) highlights the relevant effects in the immune system and in vitro cytotoxicity of MeHg exposure.

Prebiotic effect of lacto-N-biose I on bifidobacterial growth.

We demonstrated the prebiotic effect of lacto-N-biose I (Galbeta1-3GlcNAc) on bifidobacteria in vitro. Lacto-N-biose I, a building unit of the type-I milk oligosaccharides, enhanced the growth of many bifidobacteria, especially Bifidobacterium bifidum, B. breve, and B. longum, which are predominant in the intestines of breast-fed infants. It might be a substantial, natural prebiotic in human colostrums.

[Callose accumulation during treatment of tomato (Lycopersicon esculentum L.) cells with biotic elicitors].

Time-course of induced accumulation of callose in tomato cells has been studied. Localization of callose in L. esculenthum cells was investigated by fluorescent microscopy technique, and the optimal time for its determination was found. Callose accumulation in tomato cells treated with different biotic elicitors was determined. Nonlinear dependence between callose accumulation and concentration of chitin oligomers (with 3-5 N-acetylglucosamine fragments) was established. Increasing of callose accumulation in tomato cells was proportional to the increase of concentration ofchitin dimer and chitosan in the culture medium.

Isolation of an N-acetyl-D-glucosamine specific lectin from the rhizomes of Arundo donax with antiproliferative activity.

A lectin with antiproliferative activity towards human cancer cell lines and mitogenic towards human peripheral blood mononuclear cells was purified from the rhizomes of Arundo donax (Linn.) by affinity chromatography on N-acetyl-d-glucosamine linked to epoxy-activated sepharose-6B. The pure preparation apparently yielded a single band of approximately 15 kDa on SDS-PAGE, pH 8.3, under both reducing and non-reducing conditions. The molecular mass of native lectin was 32 kDa as determined by gel filtration chromatography. This showed the lectin to be a dimer, with subunits not held together by disulphide linkages. The A. donax lectin (ADL) agglutinated rabbit erythrocytes and the agglutination was inhibited by N-acetyl-d-glucosamine and its di- and trimer. The lectin was thermostable upto 55 degrees C and showed optimum activity in the range of pH 7.0-9.0 and comprised of 2.1% carbohydrate content.

Novel glycosaminoglycan precursors as antiamyloid agents: Part IV.

In vivo amyloids consist of two classes of constituents. The first is the disease-defining protein, beta-amyloid (Abeta), in Alzheimer's disease. The second is a set of common structural components that usually are the building blocks of basement membrane (BM), a tissue structure that serves as a scaffold onto which cells normally adhere. In vitro binding interactions between one of these BM components and amyloidogenic proteins rapidly change the conformation of the amyloidogenic protein into amyloid fibrils. The offending BM component is a heparan sulfate (HS) proteoglycan, part of which is protein and the remainder a specific linear polysaccharide, which is the portion responsible for binding and imparting the typical amyloid structure to the amyloid precursor protein/peptide. Our past work has demonstrated that agents that inhibit the binding between HS and the amyloid precursor are effective antiamyloid compounds both in vitro and in vivo. Similarly, 4-deoxy analogs of glucosamine (a precursor of HS biosynthesis) are effective antiamyloid compounds both in culture and in vivo. Our continuing work concerns (1) the testing of our 4-deoxy compounds in a mouse transgenic model of Alzheimer's disease, and (2) the continuing design and synthesis of modified sugar precursors of HS, which when incorporated into the polysaccharide will alter its structure so that it affects its amyloid-inducing properties. Since our previous report, 22 additional compounds have been designed and synthesized based on the known steps involved in HS biosynthesis. Of these, 12 soluble compounds have been assessed for their effect on HS biosynthesis in hepatocyte tissue cultures. In addition, one anomer of a 4-deoxy-d-glucosamine analog, which possesses AA-amyloid inhibitory properties in vivo is in the process of being assessed for its anti-Abeta activity using a murine transgenic model of brain Abeta amyloidogenesis. The majority of the novel sugars prepared to date are analogs of N-acetylglucosamine. They have been modified at the 2-N, C-3, C-4, C-3 and C-4, or C-6 positions. One compound modified at the 2-N position (QS231), which inhibits HS synthesis in hepatocyte cultures, has shown marked enhancing properties vis-à-vis AA amyloid deposition in vivo. Very instructive results with regard to HS structure and its relation to AA amyloid deposition should be forthcoming from analyses of the AA-associated HS generated with this compound.

Poly-N-acetyl glucosamine-mediated red blood cell interactions.

BACKGROUND: Investigations were performed to assess the effect of poly-N-acetyl glucosamine (p-GlcNAc) fiber slurry-mediated hemostasis by interactions with red blood cells. METHODS: Red blood cell aggregation studies were performed using test material-coated microscope slides and multiphoton microscopic measurements. Enzymatic removal of red blood cell surface proteins was achieved using trypsin and neuraminidase treatment. Zeta-potential measurements (surface charge) were performed. RESULTS: Red blood cells interact directly with poly-N-acetyl glucosamine polymers through ionic interactions and cell-surface proteins. The effective concentration of poly-N-acetyl glucosamine fiber material for 50% red blood cell aggregation was 0.28 mg/mL. The p-GlcNAc beta-configuration fibers and an alpha-configuration structural modification of the fibers both produced maximal responses because of their zeta-potentials, whereas other chemically modified p-GlcNAcs and chitosans were ineffective. CONCLUSION: Poly-N-acetyl glucosamine-induced red blood cell aggregation is mediated by interactions with red blood cell surface charges.