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Selected AbstractsEffects of a new 1,3,4-thiadiazolium mesoionic compound, MI-D, on the acute inflammatory responseDRUG DEVELOPMENT RESEARCH, Issue 4 2004Júlio C. Cardoso Abstract A new mesoionic compound, 4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine (MI-D), is described along with some of its biological properties. Its effects on hepatic metabolism, on O and nitric oxide (NO) production, and in in vivo models for potential antinociceptive, antipyretic, and antiinflammatory activities were determined. In perfused rat liver, MI-D (25 µM) stimulated glycogenolysis (95%), and inhibited oxygen uptake (37%) with affecting glycolysis. In phorbol 12-myristate 13-acetate-stimulated macrophages, O generation was reduced (95%) by MI-D (15 µM), whereas the production of NO was unaffected. MI-D (2 mg/kg) inhibited (55%) the number of abdominal writhings induced by acetic acid. At 1 mg/kg, MI-D inhibited the febrile response (5 h) induced by lipopolysaccharide (LPS) and was also effective against a preexisting febrile response. Treatment with MI-D (1 mg/kg) reduced by 67% prostaglandin (PGE2) levels in the cerebrospinal fluid of LPS-exposed mice, and at a higher dose (8 mg/kg) MI-D inhibited paw edema formation (2 h) induced by carrageenan. MI-D has a spectrum of activities similar to other nonsteroidal antiinflammatory drugs, qualifying it as a potential anti-inflammatory drug. Drug Dev. Res. 61:207,217, 2004. © 2004 Wiley-Liss, Inc. [source] Skin sensitization potency of isoeugenol and its dimers evaluated by a non-radioisotopic modification of the local lymph node assay and guinea pig maximization testJOURNAL OF APPLIED TOXICOLOGY, Issue 4 2008Masahiro Takeyoshi Abstract Allergic contact dermatitis is the serious unwanted effect arising from the use of consumer products such as cosmetics. Isoeugenol is a fragrance chemical with spicy, carnation-like scent, is used in many kinds of cosmetics and is a well-known moderate human sensitizer. It was previously reported that the dimerization of eugenol yielded two types of dimer possessing different sensitization potencies. This study reports the differences in skin sensitization potencies for isoeugenol and two types of dimer, , -O-4-dilignol and dehydrodiisoeugenol (DIEG), as evaluated by the non-radioisotopic local lymph node assay (non-RI LLNA) and guinea pig maximization test. In the guinea pig maximization test, isoeugenol, , -O-4-dilignol and DIEG were classified as extreme, weak and moderate sensitizers, respectively. As for the results of non-RI LLNA, the EC3 for isoeugenol, , -O-4-dilignol and DIEG were calculated as 12.7%, >30% and 9.4%, respectively. The two types of isoeugenol dimer showed different sensitizing activities similar to the case for eugenol dimers. A reduction of sensitization potency achieved by dimerization may lead to developing safer cosmetic ingredients. Isoeugenol dimers are not currently used for fragrance chemicals. However, the dimerization of isoeugenol may yield a promising candidate as a cosmetic ingredient with low sensitization risk. The data may also provide useful information for the structure-activity relationship (SAR) in skin sensitization. Copyright © 2007 John Wiley & Sons, Ltd. [source] FREE RADICAL-SCAVENGING ACTIVITIES OF LOW MOLECULAR WEIGHT CHITIN OLIGOSACCHARIDES LEAD TO ANTIOXIDANT EFFECT IN LIVE CELLSJOURNAL OF FOOD BIOCHEMISTRY, Issue 2010DAI-NGHIEP NGO ABSTRACT Chitin oligosaccharides (NA-COS) with low molecular weight distribution of 229.21,593.12 Da were produced from crab chitin by acid hydrolysis. They showed reducing power and scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Sigma Chemical Co., St. Louis, MO), hydroxyl and alkyl radicals. It was observed that the radical-scavenging activity of NA-COS increased in a dose-dependent manner. Their IC50 values for DPPH, hydroxyl and alkyl radicals were 0.8, 1.75 and 1.14 mg/mL, respectively. Furthermore, NA-COS exhibited the inhibitory effect on the oxidative damage of DNA from human lymphoma U937 (American Type Culture Collection, Manassas, VA) and the direct radical-scavenging effect in human fibrosarcoma cells (HT1080) (American Type Culture Collection) in 2,,7,-dichlorofluorescin diacetate (DCFH-DA) assay (Molecular Probes Inc., Eugene, OR). The results suggest that NA-COS can exert antioxidant effect in live cells and have the potential to be applied to food supplements or nutraceuticals. PRACTICAL APPLICATIONS Chitin oligosaccharides (NA-COS) are the hydrolyzed products of chitin (KEUMHO chemical products Co. Ltd., Gyeongbuk, Korea) of which derivatives have shown antioxidant, antimicrobial, anticancer, anti-inflammatory and immunostimulant effects. According to previous studies, NA-COS have beneficial biological activities similar to those of chitin. Furthermore, they are easily soluble in water because of their shorter chain length. Therefore, NA-COS are potentially applicable to improve food quality and human health. [source] EGCG inhibits protein synthesis, lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cellsMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 9 2009Chi-Hung Huang Abstract In the previous studies, (,)-epigallocatechin-3-gallate (EGCG) has been shown to have anticarcinogenic effects via modulation in protein expression of p53. Using p53 positive Hep G2 and p53 negative Hep 3B cells, we found that treatment of EGCG resulted in dose-dependent inhibition of cellular proliferation, which suggests that the interaction of EGCG with p53 may not fully explain its inhibitory effect on proliferation. Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents. EGCG has multiple beneficial activities similar to those associated with CR. One key enzyme thought to be activated during CR is AMP-activated kinase (AMPK), a sensor of cellular energy levels. Here, we showed that EGCG activated AMPK in both p53 positive and negative human hepatoma cells. The activation of AMPK suppressed downstream substrates, such as mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and a general decrease in mRNA translation. Moreover, EGCG activated AMPK decreases the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). Interestingly, the decision between apoptosis and growth arrest following AMPK activation is greatly influenced by p53 status. In p53 positive Hep G2 cells, EGCG blocked the progression of cell cycle at G1 phase by inducing p53 expression and further up-regulating p21 expression. However, EGCG inducted apoptosis in p53 negative Hep 3B cells. Based on these results, we have demonstrated that EGCG has a potential to be a chemoprevention and anti-lipogenesis agent for human hepatoma cells. [source] Synthesis and Biological Activities of 2,4-Diaminopteridine DerivativesARCHIV DER PHARMAZIE, Issue 5 2009Fei Ma Abstract Substituted 2,4-diaminopteridine derivatives 10a,10l were prepared in moderate to good yield. Their structures were confirmed by 1H-NMR and MS spectroscopy, as well as by elemental analysis. Their inhibitory properties against inducible nitric oxide synthase (iNOS) were evaluated in vitro. Biological tests indicated that compound 10a, 10d, 10e, 10h, 10i, and 10l showed potent inhibitory activities similar to that of methotrexate (MTX), while the activities of compound 10b, 10c, 10f, 10g, 10j, and 10k are stronger than MTX. Two compounds, i. e., 10b (IC50 = 18.85 ,M) and 10i (IC50 = 24.08 ,M) were further studied for their effect on septic shock in rats and immunologically liver injured mice (in vivo). The results demonstrated that 10b and 10i had the capacity to increase the blood pressure in septic shock and showed notable protective activities on immunological hepatic injury. [source] Retinoid ameliorates experimental autoimmune myositis, with modulation of Th cell differentiation and antibody production in vivoARTHRITIS & RHEUMATISM, Issue 10 2009Naho Ohyanagi Objective Polymyositis and dermatomyositis are chronic inflammatory muscle diseases. Retinoids are compounds that bind to the retinoic acid binding site of retinoic acid receptors and have biologic activities similar to those of vitamin A. Recent studies indicate that retinoids promote Th2 differentiation and suppress Th1 and Th17 differentiation in vitro. The present study was undertaken to examine the effects of a synthetic retinoid, Am80, on experimental autoimmune myositis as well as on Th phenotype development and antibody production. Methods Experimental autoimmune myositis was induced in SJL/J mice by immunization with rabbit myosin. Am80 was administered orally once daily. Its effects were evaluated by measurement of the numbers of infiltrating inflammatory cells, production of inflammatory cytokines in muscle, production of Th-specific cytokines by myosin-stimulated splenic T cells, and production of antimyosin antibodies in serum. Results In mice with experimental autoimmune myositis, orally administered Am80 significantly reduced the number of infiltrating inflammatory cells and the expression of tumor necrosis factor , and interleukin-1, (IL-1,) in muscle. Moreover, Am80 increased production of interferon-,, IL-4, and IL-10, but not IL-17, by myosin-stimulated splenic T cells of mice with experimental autoimmune myositis, suggesting that it could enhance differentiation into Th1 and Th2, but not Th17, in vivo. Am80 also decreased serum levels of IgG2a and IgG2b antimyosin antibodies, but did not affect levels of IgG1 antimyosin antibodies. In addition, it suppressed chemokine expression and activator protein 1 activity in myoblasts in vitro. Conclusion The synthetic retinoid Am80 has an inhibitory effect on experimental autoimmune myositis. It might regulate the development of Th phenotype and antibody production in vivo, in addition to its effects on cytokine and chemokine production. [source] Controlling International Joint Ventures: An Investigation of Australian Parent PartnersAUSTRALIAN ACCOUNTING REVIEW, Issue 2 2009Francesco Giacobbe International joint ventures (IJVs) are an important modern organisational form, with their complexity presenting significant management and control issues. Our research uses a cross-sectional survey of Australian parent partners of IJVs to provide insights into the characteristics of Australian companies and their IJVs, performance outcomes and choices of parent partners management control systems (MCS) design in terms of operational areas they seek to control (focus), the extent to which they exercise control (extent) and the mechanisms of control used (formal and informal). Australian parent partners of IJVs generally are, partnering in countries close to Australia in the Asia-Pacific, in activities similar to those they undertake in Australia, contributing a significant amount of resources, but overall have only limited IJV experience. Australian parent partners focus on a limited set of controls, often with tighter formalised control structures. Finally, overall they are satisfied with the performance of their IJVs, with higher satisfaction for organisational learning and product/customer dimensions than financial performance. [source] Type III IFNs: New layers of complexity in innate antiviral immunityBIOFACTORS, Issue 1 2009Nina Ank Abstract Cytokines are small secreted molecules, which mediate cross-talk between cells involved in the immune response. Interferons (IFN)s, constitute a class of cytokines with antiviral activities, and the type I IFNs have been ascribed particularly important roles in the innate antiviral response. Type III IFNs (also known as IFN-, or interleukin 28/29) represent a class of novel cytokines with biological activities similar to the type I IFNs, but seem to have a more specialized role in antiviral defense by exerting host-protection primarily at epithelial surfaces. In this review, we describe the current knowledge on the role of type III IFNs in antiviral defense. © 2009 International Union of Biochemistry and Molecular Biology, Inc. [source] Synthesis of Benzamides Related to Anacardic Acid and Their Histone Acetyltransferase (HAT) Inhibitory ActivitiesCHEMMEDCHEM, Issue 9 2008Abstract A group of benzamides related to anacardic acid amide CTPB with alkyl chains of defined length were prepared by a five-step sequence starting from 2,6-dihydroxybenzoic acid, and their activities were compared with those reported for the HAT inhibitor anacardic acid (AA). The subset of 4-cyano-3-trifluoromethylphenylbenzamides with shorter chains exhibited activities similar to that of AA, as they behaved as human p300 inhibitors, induced a decrease in histone acetylation levels in immortalized HEK cells, and counteracted the action of the HDAC inhibitor SAHA in MCF7 breast cancer cells. Moreover, an analogue with the shortest alkyl chain induced significant apoptosis at 50,,M in U937 leukemia cells. [source] Role of interleukin-15 in the development of mouse olfactory nerveCONGENITAL ANOMALIES, Issue 4 2009Tsuyoshi Umehara ABSTRACT Interleukin (IL)-15 interacts with components of the IL-2 receptor (R) and exhibits T cell-stimulating activity similar to that of IL-2. In addition, IL-15 is widely expressed in many cell types and tissues, including the central nervous system. We provide evidence of a novel role of IL-15 in olfactory neurogenesis. Both IL-15 and IL-15R, were expressed in neuronal precursor cells of the developing olfactory epithelium in mice. Adult IL-15R, knockout mice had fewer mature olfactory neurons and proliferating cells than wild-type. Our results suggest that IL-15 plays an important role in regulating cell proliferation in olfactory neurogenesis. [source] Characteristics of mutations generated through digestion with restriction enzyme and ligation in plasmid DNAENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2001Shingo Nakamura Abstract Recently, the use of restriction enzymes has been extended to studies in which rare events such as mutation and mistakes in DNA repair are examined. In these studies, the specificity of restriction enzymes becomes critical. To clarify the nature of the rare unexpected events occurring in the process of cutting of DNA with restriction enzymes then ligating it, we studied the molecular characteristics of unexpected plasmid DNAs that were retrieved as mutants of the plasmid after transfection to E. coli. The plasmid used was pUR288, containing lacZ as a marker of mutation. It was digested with restriction enzymes under the conditions recommended by the supplier of the enzymes and under the presence of DMSO, which is known to induce star activity of the enzymes. Comparisons of mutant frequencies and of nucleotide sequences of the mutants found in the different conditions indicated that nonspecific endonucleolytic activity similar to that found under star activity was present under the recommended conditions and, further, was responsible for the creation of deletion-type mutations. The frequency of these events ranged from 10,5 to 10,3, depending on the kind of restriction enzymes analyzed. Although the levels of the nonspecificity were not high, they should be considered in assays such as mutation and mistakes in DNA repair, where rare events are examined. Environ. Mol. Mutagen. 38:46,54, 2001 © 2001 Wiley-Liss, Inc. [source] Salt-inducible kinase-1 represses cAMP response element-binding protein activity both in the nucleus and in the cytoplasmFEBS JOURNAL, Issue 21 2004Yoshiko Katoh Salt-inducible kinase-1 (SIK1) is phosphorylated at Ser577 by protein kinase A in adrenocorticotropic hormone-stimulated Y1 cells, and the phospho-SIK1 translocates from the nucleus to the cytoplasm. The phospho-SIK1 is dephosphorylated in the cytoplasm and re-enters the nucleus several hours later. By using green-fluorescent protein-tagged SIK1 fragments, we found that a peptide region (586,612) was responsible for the nuclear localization of SIK1. The region was named the ,RK-rich region' because of its Arg- and Lys-rich nature. SIK1s mutated in the RK-rich region were localized mainly in the cytoplasm. Because SIK1 represses cAMP-response element (CRE)-mediated transcription of steroidogenic genes, the mutants were examined for their effect on transcription. To our surprise, the cytoplasmic mutants strongly repressed the CRE-binding protein (CREB) activity, the extent of repression being similar to that of SIK1(S577A), a mutant localized exclusively in the nucleus. Several chimeras were constructed from SIK1 and from its isoform SIK2, which was localized mainly in the cytoplasm, and they were examined for intracellular localization as well as CREB-repression activity. A SIK1-derived chimera, where the RK-rich region had been replaced with the corresponding region of SIK2, was found in the cytoplasm, its CREB-modulating activity being similar to that of wild-type SIK1. On the other hand, a SIK2-derived chimera with the RK-rich region of SIK1 was localized in both the nucleus and the cytoplasm, and had a CREB-repressing activity similar to that of the wild-type SIK2. Green fluorescent protein-fused transducer of regulated CREB activity 2 (TORC2), a CREB-specific co-activator, was localized in the cytoplasm and nucleus of Y1 cells, and, after treatment with adrenocorticotropic hormone, cytoplasmic TORC2 entered the nucleus, activating CREB. The SIK1 mutants, having a strong CRE-repressing activity, completely inhibited the adrenocorticotropic hormone-induced nuclear entry of green fluorescent protein-fused TORC2. This suggests that SIK1 may regulate the intracellular movement of TORC2, and as a result modulates the CREB-dependent transcription activity. Together, these results indicate that the RK-rich region of SIK1 is important for determining the nuclear localization and attenuating CREB-repressing activity, but the degree of the nuclear localization of SIK1 itself does not necessarily reflect the degree of SIK1-mediated CREB repression. [source] The catalytic role of the distal site asparagine-histidine couple in catalase-peroxidasesFEBS JOURNAL, Issue 5 2003Christa Jakopitsch Catalase-peroxidases (KatGs) are unique in exhibiting an overwhelming catalase activity and a peroxidase activity of broad specificity. Similar to other peroxidases the distal histidine in KatGs forms a hydrogen bond with an adjacent conserved asparagine. To investigate the catalytic role(s) of this potential hydrogen bond in the bifunctional activity of KatGs, Asn153 in Synechocystis KatG was replaced with either Ala (Asn153,Ala) or Asp (Asn153,Asp). Both variants exhibit an overall peroxidase activity similar with wild-type KatG. Cyanide binding is monophasic, however, the second-order binding rates are reduced to 5.4% (Asn153,Ala) and 9.5% (Asn153,Asp) of the value of native KatG [(4.8 ± 0.4) × 105 m,1·s,1 at pH 7 and 15 °C]. The turnover number of catalase activity of Asn153,Ala is 6% and that of Asn153,Asp is 16.5% of wild-type activity. Stopped-flow analysis of the reaction of the ferric forms with H2O2 suggest that exchange of Asn did not shift significantly the ratio of rates of H2O2 -mediated compound I formation and reduction. Both rates seem to be reduced most probably because (a) the lower basicity of His123 hampers its function as acid-base catalyst and (b) Asn153 is part of an extended KatG-typical H-bond network, the integrity of which seems to be essential to provide optimal conditions for binding and oxidation of the second H2O2 molecule necessary in the catalase reaction. [source] Mammalian CLASPs are required for mitotic spindle organization and kinetochore alignmentGENES TO CELLS, Issue 8 2006Yuko Mimori-Kiyosue CLASP1 and CLASP2 are homologous mammalian proteins, which associate with the ends of growing microtubules, as well as the cell cortex and the kinetochores of mitotic chromosomes. Previous studies have shown that in interphase cells CLASPs can attach microtubule plus ends to the cortex and stabilize them by repeatedly rescuing them from depolymerization. Here we show that CLASP1 and 2 play similar and redundant roles in organizing the mitotic apparatus in HeLa cells. Simultaneous depletion of both CLASPs causes mitotic spindle defects and a significant metaphase delay, which often results in abnormal exit from mitosis. Metaphase delay is associated with decreased kinetochore tension, increased kinetochore oscillations and more rapid microtubule growth. We show that the association of CLASP2 with the kinetochores relies on its C-terminal domain, but is independent of microtubules or association with CLIP-170. We propose that CLASPs exhibit at the kinetochores an activity similar to that at the cortex, providing apparent stabilization of microtubules by locally reducing the amplitude of growth/shortening episodes at the microtubule ends. This local stabilization of microtubules is essential for the formation of normal metaphase spindle, completion of anaphase and cytokinesis. [source] Cloning and Functional Analysis of a Family of Nuclear Matrix Transcription Factors (NP/NMP4) that Regulate Type I Collagen Expression in OsteoblastsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2001Pasutha Thunyakitpisal Abstract Collagen expression is coupled to cell structure in connective tissue. We propose that nuclear matrix architectural transcription factors link cell shape with collagen promoter geometry and activity. We previously indicated that nuclear matrix proteins (NP/NMP4) interact with the rat type I collagen ,1(I) polypeptide chain (COL1A1) promoter at two poly(dT) sequences (sites A and B) and bend the DNA. Here, our objective was to determine whether NP/NMP4- COL1A1 binding influences promoter activity and to clone NP/NMP4. Promoter-reporter constructs containing 3.5 kilobases (kb) of COL1A1 5, flanking sequence were fused to a reporter gene. Mutation of site A or site B increased promoter activity in rat UMR-106 osteoblast-like cells. Several full-length complementary DNAs (cDNAs) were isolated from an expression library using site B as a probe. These clones expressed proteins with molecular weights and COL1A1 binding activity similar to NP/NMP4. Antibodies to these proteins disrupted native NP/NMP4- COL1A1 binding activity. Overexpression of specific clones in UMR-106 cells repressed COL1A1 promoter activity. The isolated cDNAs encode isoforms of Cys2His2 zinc finger proteins that contain an AT-hook, a motif found in architectural transcription factors. Some of these isoforms recently have been identified as Cas-interacting zinc finger proteins (CIZ) that localize to fibroblast focal adhesions and enhance metalloproteinase gene expression. We observed NP/NMP4/CIZ expression in osteocytes, osteoblasts, and chondrocytes in rat bone. We conclude that NP/NMP4/CIZ is a novel family of nuclear matrix transcription factors that may be part of a general mechanical pathway that couples cell structure and function during extracellular matrix remodeling. [source] Recombinant Human Lactoferrin is Effective in the Treatment of Helicobacter felis -infected MiceJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 12 2000E. J. DIAL Recombinant human lactoferrin possesses in-vitro antibiotic and anti-inflammatory activity similar to the native form. It was tested for in-vivo activity in mice infected with the gastritis-inducing bacterium Helicobacter felis. A two-week course of treatment with lactoferrin was sufficient to partially reverse both infection-induced gastritis and the infection rate, and fully reverse gastric surface hydro-phobicity changes. A comparison of lactoferrin with amoxicillin and standard triple therapy revealed no differences in infection rate. These results show that recombinant human lactoferrin is effective in a mouse model of Helicobacter infection, and support further testing of this promising agent for this application. [source] Structural characterization of human general transcription factor TFIIF in solutionPROTEIN SCIENCE, Issue 3 2008Satoko Akashi Abstract Human general transcription factor IIF (TFIIF), a component of the transcription pre-initiation complex (PIC) associated with RNA polymerase II (Pol II), was characterized by size-exclusion chromatography (SEC), electrospray ionization mass spectrometry (ESI-MS), and chemical cross-linking. Recombinant TFIIF, composed of an equimolar ratio of , and , subunits, was bacterially expressed, purified to homogeneity, and found to have a transcription activity similar to a natural one in the human in vitro transcription system. SEC of purified TFIIF, as previously reported, suggested that this protein has a size >200 kDa. In contrast, ESI-MS of the purified sample gave a molecular size of 87 kDa, indicating that TFIIF is an ,, heterodimer, which was confirmed by matrix-assisted laser desorption/ionization (MALDI) MS of the cross-linked TFIIF components. Recent electron microscopy (EM) and photo-cross-linking studies showed that the yeast TFIIF homolog containing Tfg1 and Tfg2, corresponding to the human , and , subunits, exists as a heterodimer in the PIC, so the human TFIIF is also likely to exist as a heterodimer even in the PIC. In the yeast PIC, EM and photo-cross-linking studies showed different results for the mutual location of TFIIE and TFIIF along DNA. We have examined the direct interaction between human TFIIF and TFIIE by ESI-MS, SEC, and chemical cross-linking; however, no direct interaction was observed, at least in solution. This is consistent with the previous photo-cross-linking observation that TFIIF and TFIIE flank DNA separately on both sides of the Pol II central cleft in the yeast PIC. [source] Effect of oleocanthal and its derivatives on inflammatory response induced by lipopolysaccharide in a murine chondrocyte cell lineARTHRITIS & RHEUMATISM, Issue 6 2010Anna Iacono Objective In joint diseases, cartilage homeostasis is disrupted by mechanisms that are driven by combinations of biologic factors that vary according to the disease process. In osteoarthritis (OA), biomechanical stimuli predominate, with up-regulation of both catabolic and anabolic factors. Likewise, OA progression is characterized by increased nitric oxide (NO) production, which has been associated with cartilage degradation. Given the relevance of cartilage degenerative diseases in our society, the development of a novel pharmacologic intervention is a critically important public health goal. Recently, oleocanthal isolated from extra virgin olive oil was found to display nonsteroidal antiinflammatory drug activity similar to that of ibuprofen, a drug widely used in the therapeutic management of joint inflammatory diseases. We undertook this study to evaluate the effect of oleocanthal and its derivatives on the modulation of NO production in chondrocytes. Methods Cultured ATDC-5 chondrocytes were tested with different doses of oleocanthal and its derivatives. Cell viability was evaluated using the MTT assay. Nitrite accumulation was determined in culture supernatant using the Griess reaction. Inducible NO synthase (NOS2) protein expression was examined using Western blotting analysis. Results Oleocanthal and its derivatives decreased lipopolysaccharide-induced NOS2 synthesis in chondrocytes without significantly affecting cell viability at lower concentrations. Among the derivatives we examined, derivative 231 was the most interesting, since its inhibitory effect on NOS2 was devoid of cytotoxicity even at higher concentrations. Conclusion This class of molecules shows potential as a therapeutic weapon for the treatment of inflammatory degenerative joint diseases. [source] Development and validation of a sensitive ELISA for quantitation of Grastim® (rhG-CSF) in rat plasma: application to a pharmacokinetic study,BIOMEDICAL CHROMATOGRAPHY, Issue 9 2006Chandrasekar Nirmala Abstract Grastim® is bacterially produced recombinant counterpart of human granulocyte colony stimulating factor (G-CSF). It has biological activity similar to that of endogenous G-CSF. In the present work a sensitive, accurate, precise and enzyme-linked immunosorbent assay (ELISA) for the quantitation of G-CSF in rat plasma was developed and validated. The ELISA method employed a technique in which anti-human-G-CSF was adsorbed onto 96-well maxisorp plates and used to capture the G-CSF in rat plasma samples. The captured G-CSF was then detected using streptavidin-HRP amplification system. Absolute recovery was >90% from rat plasma. The validation includes assessments of method accuracy and precision, range of reliable response, lower limit of quantitation (LLOQ), storage stability (30 days) in rat plasma and assay specificity. The standard curve for G-CSF was linear (R2 > 0.996) in the concentration range 4.88,625 pg/mL. The LLOQ was established at 4.88 pg/mL. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 15, 250 and 500 pg/mL, were in the range 3.00,8.66% relative standard deviation (RSD) and 1.03,4.69% RSD, respectively. Accuracy in the measurement of QC samples was in the range 87.28,110.79% of the nominal values. The assay shows dilutional linearity and specificity. Stability of G-CSF was established for 30 days at ,80°C and through three freeze,thaw cycles. The validated assay was successfully employed for the assessment of pharmacokinetic disposition of G-CSF in rats. Copyright © 2006 John Wiley & Sons, Ltd. [source] Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Nicholas E. Timmins Abstract Dose-intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34+ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum-free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800-fold expansion in cell number was achieved for UCB, and up to 4,000-fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10-fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave-type bioreactor at a volume of 10,L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients. Biotechnol. Bioeng. 2009; 104: 832,840 © 2009 Wiley Periodicals, Inc. [source] Structure-Aided Optimization of Kinase Inhibitors Derived from AlsterpaulloneCHEMBIOCHEM, Issue 3 2005Conrad Kunick Prof. Abstract In order to perform computer-aided design of novel alsterpaullone derivatives, the vicinity of the entrance to the ATP-binding site was scanned for areas that could be useful as anchoring points for additional protein,ligand interactions. Based on the alignment of alsterpaullone in a CDK1/cyclin B homology model, substituents were attached to the 2-position of the parent scaffold to enable contacts within the identified areas. Synthesis of the designed structures revealed three derivatives (3,5) with kinase-inhibitory activity similar to alsterpaullone. The novel 2-cyanoethylalsterpaullone (7) proved to be the most potent paullone described so far, exhibiting inhibitory concentrations for CDK1/ cyclin B and GSK-3, in the picomolar range. [source] Structure,Activity Relationship Studies on the Immune Stimulatory Effects of Base-Modified CpG Toll-Like Receptor 9 AgonistsCHEMMEDCHEM, Issue 9 2006Marion Jurk Dr. Abstract Synthetic oligodeoxynucleotides containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are able to stimulate potent immune responses through a signaling pathway involving Toll-like receptor 9 (TLR9). We have investigated the structure,activity relationship (SAR) of base-modified CpG oligonucleotides with TLR9 by measuring TLR9 activation by 20-mer oligonucleotides having just a single human recognition motif (5,-GTCGTT-3,) in functional cell-based TLR9 assays. Substitution of guanine by hypoxanthine and 6-thioguanine resulted in activity similar to the unmodified parent molecule, whereas purine, 2-aminopurine, 2,6-diaminopurine, and 8-oxo-7,8-dihydroguanine substitution resulted in approximately 40,60,% reduction in activity, and 7-deazaguanine substitution led to the strongest (80,%) reduction in TLR9 stimulation. Furthermore, none of the investigated modifications at C5 and N4 of cytosine were well tolerated with respect to human TLR9 stimulation. Our results are compatible with a SAR model in which guanine is recognized by the Hoogsteen site, and C5 is most critical for recognition of cytosine. In addition, we found significant species-specific differences between human and murine TLR9 recognition, which demonstrates the importance of choosing appropriate assay systems for SAR studies. [source] What is the role of the hevein-like domain of fruit class I chitinases in their allergenic capacity?CLINICAL & EXPERIMENTAL ALLERGY, Issue 3 2002A. Děaz-Perales Background Class I chitinases are the major panallergens in fruits associated with the latex,fruit syndrome. These enzymes contain an N-terminal hevein-like domain homologous to latex hevein, and a larger catalytic domain. The role of these domains in their allergenic capacity is still controversial. Objective We sought to evaluate the role of both domains of class I chitinases in their IgE-binding properties, using Cas s 5, the major allergen from chestnut, as a model. Methods Recombinant Cas s 5 and its deleted form, lacking the hevein-like domain, designated rCat, were expressed in Pichia pastoris using the pPIC 9 vector. Both recombinant products were purified from the supernatants of transformed yeast cultures by gel-filtration and cation-exchange chromatography. The isolated proteins were characterized by N-terminal sequencing, enzymatic activity and N-glycosylation tests, anti-chitinase and specific IgE immunodetection. Immunoblot, RAST and CAP inhibition assays were also performed. Results Both purified rCas s 5 and rCat showed the expected N-terminal amino acid sequences and an enzymatic activity similar to that of their natural counterparts isolated from chestnut seeds, and were strongly recognized by anti-chitinase antibodies. In contrast, only rCas s 5, but not rCat, bound specific IgE from sera of patients suffering from the latex,fruit syndrome, and fully inhibited IgE-binding to natural Cas s 5 in immunoblot inhibition assays. Latex hevein also exerted a strong immunoblot inhibition of IgE-binding to chestnut Cas s 5. RAST and CAP inhibition using whole chestnut extract on the solid phase, rendered inhibition levels around 70,90% for rCas s 5 and 60% for rCat, in contrast to the immunoblotting results. Conclusions Recombinant Cas s 5 behaves like natural Cas s 5 in IgE-binding assays in vitro. The hevein-like domain of allergenic class I chitinases seems to include all their main IgE-binding epitopes when tested by immunodetection and immunoblot inhibition experiments. RAST and CAP inhibition assays, on the contrary, suggest that relevant epitopes are also harboured in the catalytic domain of these allergens. [source] RESEARCH: Zopiclone (Cyclopyrrolone): A Novel Hypnosedative; Hypnosedation Caused by Zopiclone Does Not Impair Memory-Learning in Albino MiceCNS: NEUROSCIENCE AND THERAPEUTICS, Issue 5 2010Uma Kadam SUMMARY Objectives: To evaluate hypnosedative action of Zopiclone by using animal models for hypnosis and sedation (anxiolysis); and to further evaluate whether this hypnosedation impairs memory-learning in albino mice like conventional hypnosedatives. Methods: For evaluation of hypnosedation, following experiments were performed in albino mice: (1) righting reflex test, (2) pentobarbitone sleeping time potentiation, (3) open field apparatus behavior, and (4) elevated plus maze performance. For evaluation of effects on impairment of memory-learning, elevated plus maze retention test was performed in albino mice. Results: Zopiclone (7.5 mg/kg p.o.) did not inhibit the righting reflex. Significant (P < 0.001) potentiation of pentobarbitone sleeping time and increase in exploration in open field apparatus was observed. Elevated plus maze performance also showed significant (P < 0.01) increase in number of entries to open arm at the same time significant (P < 0.02) increase in time spent in open arm was observed. Elevated plus maze retention test showed significant (P < 0.01) increase in transfer latency on second day of experiment. Conclusions: Zopiclone (7.5 mg/kg p.o.) has selective hypnosedative activity but not CNS-depressant activity similar to BZDs. Hypnosedative action of Zopiclone does not impair memory-learning in albino mice like conventional hypnosedatives. [source] | ||||||||||||||||