Blue Assay Research Articles

A pH-Sensitive, Biobased Calcium Carbonate Aragonite Nanocrystal as a Novel Anticancer Delivery System

The synthesised biobased calcium carbonate nanocrystals had demonstrated to be an effective carrier for delivery of anticancer drug doxorubicin (DOX). The use of these nanocrystals displayed high levels of selectivity and specificity in achieving effective cancer cell death without nonspecific toxicity. These results confirmed that DOX was intercalated into calcium carbonate nanocrystals at high loading and encapsulation efficiency (4.8 and 96%, resp.). The CaCO3/DOX nanocrystals are relatively stable at neutral pH (7.4), resulting in slow release, but the nanocrystals progressively dissociated in acidic pH (4.8) regimes, triggering faster release of DOX. The CaCO3/DOX nanocrystals exhibited high uptake by MDA MB231 breast cancer cells and a promising potential delivery of DOX to target cells. In vitro chemosensitivity using MTT, modified neutral red/trypan blue assay, and LDH on MDA MB231 breast cancer cells revealed that CaCO3/DOX nanocrystals are more sensitive and gave a greater reduction in cell growth than free DOX. Our findings suggest that CaCO3 nanocrystals hold tremendous promise in the areas of controlled drug delivery and targeted cancer therapy.

Aqueous Extract from a Chaga Medicinal Mushroom, Inonotus obliquus (Higher Basidiomyetes), Prevents Herpes Simplex Virus Entry Through Inhibition of Viral-Induced Membrane Fusion

Chaga medicinal mushroom, Inonotus obliquus, a popular prescription in traditional medicine in Europe and Asia, was used to reduce inflammation in the nasopharynx and to facilitate breathing. The aqueous extract from I. obliquus (AEIO) exhibited marked decrease in herpes simplex virus (HSV) infection (the 50% inhibitory concentration was 3.82 μg/mL in the plaque reduction assay and 12.29 μg/mL in the HSV-1/blue assay) as well as safety in Vero cells (the 50% cellular cytotoxicity was > 1 mg/mL, and selection index was > 80). Using a time course assay, effective stage analysis, and fusion inhibition assay, the mechanism of anti-HSV activity was found against the early stage of viral infection through inhibition of viral-induced membrane fusion. Therefore, AEIO could effectively prevent HSV-1 entry by acting on viral glycoproteins, leading to the prevention of membrane fusion, which is different from nucleoside analog antiherpetics.

Antioxidant and Antiradical Properties of Methanolic Extracts from Algerian Wild Edible Desert Truffles (Terfezia and Tirmania, Ascomycetes)

Wild edible truffles (namely, Terfezia leonis, Tirmania pinoyi, and T. nivea) were commercially obtained from Southern Algeria and methanolic extracts were prepared from these truffles. Their antioxidant and antiradical properties were studied by using five analytical methods: scavenging capacity on 1,1-diphenyl-2-picrylhydrazyl (DPPH·), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS·+), superoxide anion (O2·-) radicals, ferric reducing antioxidant power (FRAP), and ferricyanide/Prussian blue assay. Chemical constituents contributing to these activities were also investigated. T. leonis had the highest total phenolics, total carotenoids, and anthocyanin contents. At 2.6 mg/mL, scavenging effects on the DPPH· radical were 92.47%, 53.06%, and 41.34% for T. leonis, T. pinoyi, and T. nivea, respectively. T. leonis showed the most potent radical scavenging activities on DPPH·, ABTS·+, and O2·- radicals, with EC50 values of 1.08, 1.35, and 7.27 mg/mL, respectively. On the other hand, T. leonis exhibited the highest reductive capabilities. On the basis of the EC50 values, T. leonis had good antioxidant and antiradical properties. These results showed that methanolic extracts from these three truffles species had effective antioxidant and antiradical properties. Therefore, wild edible desert truffles could serve as an easily accessible item of food rich in natural antioxidants, as a possible food supplement, or even as a pharmaceutical agent.

Effects of low power laser radiation of blue, green and red ranges on free radical processes in rat blood in endotoxic shock

This study was performed to investigate the effects of low power laser radiation in blue (441.2 nm), green (532.5 nm) and red (632.8 nm) wavelength ranges on free radical processes in experimental endotoxic shock in rats. The experimental model was produced by intraperitoneal injection of lipopolysacharide B (25 mg/kg) (LPS). The following parameters were assayed in the study: the chemiluminescent assay (to evaluate the free radical production by blood leukocytes), nitro blue tetrazolium assay (to monitor the superoxide dismutase activity of plasma) and cis-parinaric acid fluorescence (to estimate the intensity of lipid peroxidation in erythrocyte membranes). It was found that the low power laser radiation significantly influenced all investigated processes, in animals both treated and untreated without LPS injection. The most pronounced effects were observed in all groups of animals subjected to the low power laser radiation: at the dose of 0.75 J/cm2 green laser was most effective and at the dose of 1.5 J/cm2 green and red lasers provided maximal effects. The mechanisms of the observed phenomena are discussed.

Urinary Screening of Mucopolysaccharidoses (MPS) in Cases Suspected of Inborn Errors of Metabolism Children in HUSM

Mucopolysaccharidoses (MPS) is a disease of inborn errors of metabolism (IEM). It constitutes a large and hetero- genous subgroup among the lysosomal storage diseases. For the detection of this disease, urinary glycosaminoglycan (GAG) is measured by dimethlmethylene blue (DMB) assay and high resolution electrophoresis (HRE) is done to characterize the different types of MPS. To screen for MPS in all urine samples suspected of inborn errors of metabolism sent to the metabolic laboratory from 1998 to 2008 and to look into the association between the positivity of DMB assay and HRE in the same sample, All urine samples sent to the metabolic laboratory that fulfilled the inclusion criteria were analyzed for GAG and were further subjected to HRE. Measurement of observed agreement using Cohen's kappa coefficient (k) was used for the association between both DMB and HRE method. A total of 134 urine samples were obtained however only 90 samples were analyzed. 28 (31.1%) of the samples had normal GAG levels, 59 (65.6%) had GAG levels between 1-2 folds above normal limits and 3 (3.3%) samples had more than 2 folds increment above the normal limits for age. Three samples showed abnormal bands when subjected to HRE. Poor association between both methods (k = 0.027) was observed. This study shows that all samples with high index of suspicion of MPS and elevated values of GAG of two folds or more should be subjected to HRE. Characterization of MPS through HRE gives the clinician some presumptive diagnosis and prognosis in managing these patients.

Iris Nertschinskia Ethanol Extract Differentially Induces Cytotoxicity in Human Breast Cancer Cells Depending on AKT1/2 Activity

Recently, we reported that an ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line. However, the detailed mechanisms were not fully explored. Here, we demonstrate another aspect of the activity of I. nertschinskia in breast cancer cells. We compared the response to an ethanol extract of I. nertschinskia in two different human breast cancer cell lines, Hs578Tand MDA-MB231, respectively with relatively low and high AKT1/2 activity by trypan blue exclusion assay and FACS analysis. Knockdown of endogenous AKT1 or AKT2 in breast cancer cells by RNA interference determined the sensitivity to I. nertschinskia ethanol extract compared to control cells. The I. nertschinskia ethanol extract induced cell death in a manner that depended on the level of phosphorylated AKT1/2 protein and was associated with a significant increase in the sub-G1 cell population, indicative of apoptosis. Our results indicate that an ethanol extract of I. nertschinskia differentially induces cell death in breast cancer cells depending on their level of phosphorylated AKT1/2.

Hyaluronic acid stimulates the formation of calcium phosphate on CoCrMo alloy in simulated physiological solution

The behaviour of CoCrMo alloy has been studied in two simulated physiological solutions-NaCl and Hanks' solutions-each containing the sodium salt of hyaluronic acid. Hyaluronic acid is a component of synovial joint fluid, so the behaviour of orthopaedic alloys in its presence needs to be assessed. Electrochemical methods, X-ray photoelectron spectroscopy and scanning electron microscopy have been used to analyse the composition, thickness and morphology of any layers formed on the alloy. The addition of hyaluronic acid shifts the corrosion potential and increases the value of polarization resistance. The presence of hyaluronic acid in simulated Hanks' physiological solution stimulates the formation of a calcium phosphate layer, opening up the possibility for tailoring the surface properties of CoCrMo alloy. The viability of human osteoblast-like was determined using the Alamar(®) Blue Assay, while the osteogenic activity was evaluated by alkaline phosphatase activity. The presence of hyaluronic acid affects the alkaline phosphatase activity.

The role of salvia extraction fluid in the benign biliary stricture

Objective To explore the role of salvia extraction fluid in the benign biliary stricture.Methods A model of trauma of bile duct was established in dogs and then repaired.The myofibroblast (MFB) were extracted by enzymatic digestion and in intro different velocity adherence methods.Through the technique of cell culture in vitro,to establish the model MFB fibrosis induced by Aid,then to add disposal factors. The MFB were divided into 3 groups:control group, experimental group and blank group.After 24 hours of action,proliferation was measured by thiazolyl blue (MTT) assay; the mRNA expression of TGF- β 1 and α -SMA was determined by RT-PCR. Results MTT results showed that cell proliferation rate and apoptosis enhanced in experimental group； gene expression of TGF- β 1 and α -SMA significantly decreased in MFB cells.Conclusions The high expression of TGF- β 1 and α -SMA is closely related to active proliferation of broblasts,extracellular matrix over deposition and scar contracture of bile duct.Salvia extraction fluid could effectively inhibit the proliferation of MBF in a concentration dependent manner,which may play an important role in the regression of cells apoptosis and preventing benign biliary stricture. Key words: Myofibroblast (MFB); Benign biliary stricture; Salvia extraction fluid; Transforming growth factor β 1(TGF- β 1); α -SMA

Low-intensity ultrasound enhances the anticancer activity of cetuximab in human head and neck cancer cells

The potential clinical use of ultrasound in inducing cell apoptosis and enhancing the effects of anticancer drugs in the treatment of cancers has previously been investigated. In this study, the combined effects of low-intensity ultrasound (LIU) and cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody, on cell killing and induction of apoptosis in HSC-3 and HSC-4 head and neck cancer cells, and its mechanisms were investigated. Experiments were divided into 4 groups: non-treated (CNTRL), cetuximab-treated (CETU), ultrasound-treated (UST) and the combination of cetuximab and US-treated (COMB). Cell viability was assessed by trypan blue staining assay and induction of apoptosis was detected by fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI) staining assay at 24 h after cetuximab and/or US treatment. To elucidate the effect of cetuximab and US on EGFR signaling and apoptosis in head and neck cancer cells after the treatments, the expression of EGFR, phospho-EGFR, and the activation of caspase-3 were evaluated with western blotting. More cell killing features were evident in the COMB group in HSC-3 and HSC-4 cells compared with the other groups. No differences in EGFR expression among the CETU, UST and COMB groups was observed, while the expression of phospho-EGFR in the CETU group was downregulated compared with that in the CNTRL group. Phospho-EGFR expression was much more downregulated in the COMB group compared with that in the other groups. In addition, the activation of caspase-3 in the UST group was upregulated compared with that in the CNTRL group. Caspase-3 activation was much more upregulated in the COMB group than that in the other groups. These data indicated that LIU was able to enhance the anticancer effect of cetuximab in HSC-3 and HSC-4 head and neck cancer cells.

Concentration-dependent effects of tiludronate on equine articular cartilage explants incubated with and without interleukin-1β

To determine concentration-dependent effects of tiludronate on cartilage explants incubated with or without recombinant equine interleukin-1β (rEq IL-1). Articular cartilage explants from the femorotibial joints of 3 young adult horses. Cartilage explants were incubated with 1 of 6 concentrations (0, 0.19, 1.9, 19, 190, or 1,900 mg/L) of tiludronate and with or without rEq IL-1 (0.01 ng/mL) for 96 hours. Prostaglandin E(2) (PGE(2)) concentrations in culture medium and explant digests were analyzed via PGE(2) enzyme immunoassay. Sulfated glycosaminoglycan (sGAG) concentrations in culture medium were quantified via 1,9-dimethylmethylene blue assay. Chondrocyte apoptosis in paraffin embedded explant sections was measured via terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Relative gene expression of matrix metalloproteinases (MMPs), interleukin (IL)-6, and IL-8 was determined via the comparative cycle threshold method. rEq IL-1 increased PGE(2) concentration, sGAG release from explants, chondrocyte apoptosis, and MMP gene expression. Lower tiludronate concentrations reduced rEq IL-1-induced sGAG release and chondrocyte apoptosis, whereas the higher tiludronate concentrations increased sGAG release and chondrocyte apoptosis. At the highest tiludronate concentration evaluated, IL-8 gene expression was increased independent of whether rEq IL-1 was present. Tiludronate had biphasic concentration-dependent effects on cartilage explants that were independent of PGE(2) secretion or MMP gene expression. Low tiludronate concentrations had some chondroprotective effects, whereas high tiludronate concentrations were detrimental to equine articular cartilage. Administration of tiludronate intra-articularly to horses may be detrimental, dependent on the dose used. In vivo studies are needed before intra-articular tiludronate administration to horses can be recommended.

Effects of icotinib hydrochloride on the proliferation and apoptosis of human lung cancer cell lines

To explore the effects of icotinib on the proliferation and apoptosis of various lung cancer cell lines. Human lung cancer cell lines HCC827, H1650, H1975, A549 and human epidermal cancer cell line A431 were treated in vitro with icotinib or gefitinib at a concentration gradient of 0 - 40 µmol/L. Their proliferation effects were analyzed by the thiazolyl blue (MTT) assay and the apoptotic effects detected by flow cytometer. The downstream signaling proteins were detected by Western blot. The median inhibitory concentrations (IC(50)) of icotinib for A431 and HCC827 cell lines were (0.04 ± 0.02) and (0.15 ± 0.06) µmol/L respectively. No significant differences existed between the inhibitions of gefitinib and icotinib on A431, HCC827, H1650, H1975 and A549 cell lines (all P > 0.05). Compared with H1650, H1975 and A549 cell lines, icotinib significantly inhibited A431 (P = 0.009, 0.005 and 0.000) and HCC827 (P = 0.001, 0.001 and 0.000) cell lines. And it lowered the expressions of p-AKT, p-ERK and survivin protein expression through the inhibited activity of p-EGFR protein. Icotinib can arrest the proliferation of lung adenocarcinoma cells with EGFR mutation or over-expression by inhibiting the signal pathways of AKT-ERK and survivin.

Synadenium umbellatum Pax. promotes cell cycle arrest and induces apoptosis in K-562 leukemia cells

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease that shows apoptosis resistance. The introduction of imatinib mesylate has revolutionized the treatment of CML, but imatinib resistance may develop at any time and inevitably leads to disease progression. Synadenium umbellatum Pax. belongs to the Euphorbiaceae family and is popularly used in Brazil for the treatment of cancer. The cytotoxicity of Euphorbiaceae is associated with the ability of these plants and their bioactive compounds to induce apoptotic tumor cell death. Therefore, we aimed to investigate the cytotoxicity and the mechanisms of death induced by S. umbellatum extract in leukemic cells. S. umbellatum cytotoxicity was evaluated by trypan blue exclusion assay and flow cytometric analysis of the cell cycle; the mechanisms involved in K-562 cell death were investigated by light microscopy and flow cytometry. The results demonstrate that S. umbellatum is cytotoxic to leukemic cells in a concentration-dependent manner. Morphological analysis revealed that S. umbellatum treatment induced K-562 cell death by an apoptotic pathway. Furthermore, data indicate ROS overproduction, alterations in mitochondrial membrane potential, phosphatidylserine externalization and activation of caspase 9. Taken together, the results demonstrate that S. umbellatum extract arrested the cell cycle and triggered apoptosis at several levels in K-562 cells.

Abstract 307: Endothelial mPGES-1 Mediates Thiazolidinedione-Induced Adipose-Specific Capillary Hyperpermeability and Fluid Retention

Thiazolidinedione-induced fluid retention results from two prominent mechanisms: increased distal tubular fluid reabsorption and adipose-specific increases in capillary permeability; the later mechanism requires activation of endothelial but not smooth muscle PPARγ as demonstrated by using conditional knockout mice (Yang et al. 63rd High Blood Pressure Research Conference. #063, 2009). The present study examined the role of microsomal prostaglandin E synthase-1 (mPGES-1) in TZD-induced capillary hyperperbeability in the adipose tissues. Following rosiglitazone (RGZ) treatment, remarkable COX-2 and mPGES-1 inductions were detected at day 1 and peaked at day 2 (COX-2:104-fold for mRNA and 98-fold for protein; mPGES-1: 2.5-fold for mRNA and 30-fold for protein) in parallel with a 64-fold increase in PGE2 production in apidydimal fat depot (AFD) but not in other tissues tested, and declined at day 14. Body weight gain (BWG) in response to RGZ in WT mice were 1.62 g on day 14. In contrast, the BWG in mPGES-1 KO mice was completely blocked on day 7 and was reduced by 60% on day 14. RGZ-treated WT mice exhibited a 60% increase in water content in the AFD, which was significantly attenuated in the KO mice. In parallel, the capillary perbeability in the adipose tissue, as assessed by Evans blue leakage assay, exhibited a 60% increase in WT mice after RGZ treatment. As compared with WT controls, the adipose capillary permeability in the KO mice was 60% less at baseline and 100% less after RGZ treatment. RGZ-induced fluid retention was further tested in mice lacking mPGES-1 in endothelium or smooth muscle generated respectively by crossing mPGES-1 floxed mice with Tie-2-Cre or SM22-Cre mice (termed EC KO or SM KO). In EC KO mice, TZD-induced capillary hyperpermeability in adipose tissue was completely abolished, associated with a 50% reduction of the BWG and complete blockade of PGE2 stimulation in the ADF, a phenotype analogous to that of endothelial PPARγ KO mice. In contrast, neither water content nor capillary permeability in the skeletal muscle was affected by RGZ treatment. Taken together, this study has defined endothelial mPGES-1 as a key mediator of TZD-induced adipose-specific capillary hyperperbeability and fluid retention.

Overall Sulfation of Heparan Sulfate from Pancreatic Islet β-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.

Comparative effects of gamma and electron beam irradiation on the antioxidant potential of Portuguese chestnuts (Castanea sativa Mill.)

Chestnuts (Castanea sativa Mill.) are widely consumed all over the world, and have been recently studied for their antioxidant potential. The present study reports the effect of e-beam and gamma radiation (doses of 0, 0.5, 1 and 3kGy) on the antioxidant potential of Portuguese chestnuts. Irradiation might be an alternative preservation method, since Methyl Bromide, a widely used fumigant, was banished by the European Union in 2010 due to its toxicity. The antioxidant activity was evaluated through 2,2-diphenyl-1-pycrylhydrazyl (DPPH) free radical scavenging activity assay, reducing power by the Ferricyanide/Prussian blue assay, and lipid peroxidation inhibition by β-carotene/linoleate and thiobarbituric acid reactive substances (TBARS) assays. The analysis of total phenolics and flavonoids was performed by spectrophotometric assays. Irradiated samples preserved total phenolics content (but not flavonoids) and revealed higher antioxidant activity (lower EC50 values) than the control samples. The most indicated doses to maintain antioxidants content, and to increase antioxidant activity were 1 and 3kGy for electron beam and gamma radiation, respectively.

The effect of low power laser radiation of blue, green, and red ranges on free radical processes in blood of rats with experimental endotoxic shock

This study was performed to investigate the effects of low power laser radiation in blue (441.2 nm), green (532.5 nm) and red (632.8 nm) wavelength ranges on free radical processes in experimental endotoxic shock in rats. The experimental model was induced by intraperitoneal injection of lipopolysaccharide B (25 mg/kg) (LPS). Functional activity of blood leukocytes was evaluated by the method of luminol-dependent chemiluminescence, plasma superoxide dismutase activity was determined by the nitro blue tetrazolium assay, intensity of lipid peroxidation in erythrocyte membranes was estimated by cis-parinaric acid fluorescence. It was found that the low power laser radiation significantly influenced all investigated processes, in LPS-treated and control animals. The most pronounced effects were observed in all groups of LPS-treated animals, in which the laser radiation increased all investigated parameters. At the radiation dose 0.75 J/cm2 green laser was the most effective, while at the dose of 1.5 J/cm2 both green and red lasers produced potent effects. Possible mechanisms of the observed phenomena are discussed.

Cell Surface Glycosylation and Glycosaminoglycan Composition Profiles in Immature and Mature Intervertebral Disks

Introduction Back pain is a predominant cause of disability, and the major cause of this disability is intervertebral disk (IVD) degeneration.1 Proteoglycan (PG) content of the IVD is decreased during degeneration, and glycosaminoglycan (GAG) composition is altered.2 In this study, the cell surface glycosylation and GAG composition profiles of immature and mature ovine IVD tissue were profiled in an effort to understand potential signals involved in cell-extracellular matrix cross-talk during maturation and degeneration. Materials and Methods IVD tissue from L3-L4 and L4-L5 lumbar segments were harvested from 3- and 11-month-old animals. One portion of the tissue was fixed for histochemical profiling with a panel of fluorescently-labeled lectins ( Maackia amurensis agglutinin, Sambucus nigra agglutinin I, Ulex europaeus agglutinin I, Concanavalin A (jack bean lectin), Wisteria floribunda agglutinin). The remaining portion was digested with proteinase K and the sulfated GAG (s-GAG) content was quantified by dimethylmethylene blue (DMMB) assay. The digested tissue was treated with chondroitinase ABC (ChABC) and analyzed by HPLC. Results The glycosylation expression level differed between annulus fibrosus (AF) and nucleus pulposus (NP) tissue as indicated by quantitative analysis using lectin histochemistry. All lectins stained cells and extracellular matrix (ECM) of the IVD independently of the tissue type. The cells demonstrated greater lectin binding compared to the ECM and the intensity of staining differed according to the tissue-type. With maturity, the expression profiles of cell surface glycosylation changed, notably for the n a-(2?6)-linked sialic acid (SNA-I) and terminal GalNAc (WFA staining) expression with a 10-fold and 2-fold increase of expression at 11 months old, respectively. DMMB assay showed that NP tissue contained threefold more s-GAGs than AF and cartilage in 3-month-old samples. In the 11-month-old IVD samples, no difference was observed between AF and NP, but overall content of s-GAG was higher than cartilage. HPLC analysis of the ChABC-digested IVD tissue revealed that the quantity and ratio of s-GAG components differed from NP to AF tissue. Moreover, a change in sulfation pattern was observed with the maturity of the disk ( Fig. 1 ). Indeed, an inversion of the occurrence of Ddi-4S and Ddi-6S disaccharides was observed. A significant increase of Ddi-4S disaccharides with a significant decrease of Ddi-6S disaccharides was noted in the 11-month-old IVD AF tissue compared to 3 month old. A similar trend, albeit not significant, was observed for the NP tissue. Cartilage tissue did not present a significant difference in percentage of sulfated disaccharides with maturity. [Figure: see text] Conclusion GAG sulfation pattern was shown to change with maturation. Sulfation is critical for maintaining water content and specific patterns of sulfation are influential in cellular signaling and differentiation events. Furthermore, high changes in glycosylation profiles at a cellular and extracellular level were observed. A better understanding of these phenomena (sulfation and glycosylation profiles changes) will allow us to design therapeutic strategies for IVD regeneration. Acknowledgements AO Foundation (S-09-7P), Science Foundation Ireland, Research Frontiers Programme (07/RFP/ENMF482), Science Foundation Ireland, 07/SRC/B1163. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Fritz JM, et al., Physical therapy for ccute low back pain: Associations with subsequent healthcare costs. Spine 2008;33(16):1800–1805 O'Halloran D, et al. Tissue-engineering approach to regenerating the intervertebral disk. Tissue Engineering 2007;13(8):1927–1954

Study of <i>In Vitro</i> Reaction between Bone Cell and Poly(vinyl alcohol)-Matrix Composite Biomaterials

Aim of the study is to investigate the in vitro reaction between rat bone cell and poly (vinyl alcohol) (PVA)-matrix composites. Sample of Hydroxyapatite (HA) /PVA gel composites were prepared by the in situ growth method and Experiments of Alamar Blue Activity and Alkaline Phosphatase Assay were carefully made in our lab. SEM results displays that PVA-matrix composites containing HA fillers and pure PVA as a control group have an excellent biocompatible bone reaction in vitro with rat bone cells and HA filler particles in composites promote reaction between bone cell and composites. In vitro biology experiment resulted proves that adhesion, growth and differentiation of bone cells on all PVA-matrix composites are promoted by HA filler particles.

Effect of ADM on Proliferation and Apoptosis of Human Gastric Cancer Cell SGC-7901

To observe the effect of ADM on cells apoptosis of SGC-7901 cells. The SGC-7901 cells were treated by ADM. And the inhibitory ratio of cells was measured by trypan blue stain assay, the IC50 value was calculated. Cells apoptosis were detected by DNA agarose gel electrophoresis.The cell cycles were analyzed by flow cytometry system after treatment with ADM. Morphologic changes were observed using phase-contrast microscopy . The SGC-7901 cells proliferation were remarkably inhibited by ADM. The IC50 values were 5.7 μg / mL. The typical DNA ladder on agarose gel electrophoresis for analysis of cellular apoptosis were significantly appeared. ADM could restrain the SGC-7901 cells proliferation, and to cause the morphologic changes of apoptosis. Apoptosis peaks appeared with flow cytometry analysis.

Evaluation of MLN4924, an investigational NEDD8-activating enzyme (NAE) inhibitor, as a novel targeted agent with single-agent antitumor activity in mantle cell lymphoma (MCL) and Hodgkin lymphoma preclinical models.

e18539 Background: MLN4924 is a novel, potent, and selective inhibitor of NAE, an essential component of the ubiquitin-proteosome system (UPS). We have previously demonstrated that the UPS plays a pivotal role in the development of rituximab and chemotherapy resistance in B-cell lymphomas. There is a scientific need to target the UPS more efficiently in an attempt to reverse acquired resistance to biological and chemotherapeutic agents in relapsed/refractory aggressive lymphoma. To this end, we studied the anti-tumor activity of MLN4924 in rituximab-chemotherapy sensitive and resistant pre-clinical models. Methods: A panel of Burkitt (BL), diffuse large B-cell (DLBCL), MCL and HL lymphoma cell lines and primary tumor cells isolated from patients with non-Hodgkin lymphoma (NHL) (N=10) were exposed to escalating doses of MLN4924 alone or in combination with a panel of chemotherapeutic agents for up to 72 hrs. Cell viability was determined by alamar Blue reduction or CellTiter-glo assay. Apoptosis was determined by Western blotting. Cell cycle analysis was performed by flow cytometry. Results: MLN4924 demonstrated activity in all cell lines in a time-and dose-dependent manner, including the rituximab/chemotherapy-resistant cell lines. The most potent activity was noted in MCL and HL cell lines (IC50 doses were tenfold lower as compared to DLBCL or BL cell lines). A variable degree of anti-tumor activity was observed in primary tumor cells isolated from NHL patients. Induction of apoptosis was observed in rituximab-resistant cell lines. MLN4924 exhibited synergistic anti-tumor activity when combined with bortezomib, bendamustine, and cytarabine in MCL cell lines. Conclusions: MLN4924 exhibits potent in vitro cytotoxic activity against a variety of human B-cell lymphoma cell lines and primary tumor cells isolated from NHL patients. Significant activity was observed in MCL cell lines. Experiments investigating the in vivo activity of MLN4924 are ongoing. MLN4924 is a highly promising agent for the treatment of relapsed/refractory MCL or HL.