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Different Cell Lines (different + cell_line)
Selected AbstractsPhosphatidylinositol-3-OH kinase regulatory subunits are differentially expressed during development of the rat cerebellumDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2001José L. Trejo Abstract Recent evidence implicates a central role for PI3K signalling in mediating cell survival during the process of neuronal differentiation. Although PI3K activity is stimulated by a wide range of growth factors and cytokines in different cell lines and tissues, activation of this pathway by insulin-like growth factor I (IGF-I) most likely represents the main survival signal during neuronal differentiation. IGF-I is highly expressed during development of the central nervous system, and thus is a critical factor for the development and maturation of the cerebellum. Upon ligand binding, the IGF-I receptor phosphorylates tyrosine residues in SHC and insulin receptor substrates (IRSs) initiating two main signalling cascades, the MAP kinase and the phosphatidylinositol 3-kinase (PI3K) pathways. Activated PI3K is composed of a catalytic subunit (p110, or ,) associated with one of a large family of regulatory subunits (p85,, p85,, p55,, p55,, and p50,). To evaluate the contributions of these various regulatory subunits to neuronal differentiation, we have used antibodies specific for each of the PI3K subunits. Using these antisera, we now demonstrate that PI3K subunits are differentially regulated in cerebellar development, and that the expression level of the p55, regulatory subunit reaches a maximum during postnatal development, decreasing thereafter to low levels in the adult cerebellum. Furthermore, our studies reveal that the distribution of the various PI3K regulatory subunits varies during development of the cerebellum. Interestingly, p55, is expressed in both glial and neuronal cells; moreover, in Purkinje neurones, this subunit colocalises with the IGF-IR. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 39,50, 2001 [source] MAP-kinase-activated protein kinase 2 expression and activity is induced after neuronal depolarizationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2008Tobias Thomas Abstract Mitogen-activated protein kinase-activated protein kinase (MK)2 is one of several downstream targets of p38 mitogen-activated protein kinase and has a well documented role in inflammation. Here, we describe a possible new function of MK2. We show that triggering depolarization by potassium chloride or increasing the cellular cAMP by forskolin treatment led to elevated levels of expression and activity of mouse MK2. In both treatments, the kinase inhibitor H89 completely prevented the up-regulation of MK2 at the transcript level. By the use of different cell lines we demonstrated that the induction of MK2 expression is characteristic of neuronal cells and is absent in fibroblasts, macrophages and kidney cells. In vivo, induction of a status epilepticus by systemic administration of the chemoconvulsant kainic acid resulted in markedly reduced neurodegeneration in the pyramidal layer of the hippocampus, dentate gyrus and hilus of MK2-deficient mice compared with wild-type mice. Together, our data suggest a possible role of MK2 in the cellular response after neuronal depolarization, in particular in excitotoxicity. [source] Acute activation of Erk1/Erk2 and protein kinase B/akt proceed by independent pathways in multiple cell typesFEBS JOURNAL, Issue 17 2005Doris Chiu We used two inhibitors of the signaling enzyme phosphatidylinositol 3-kinase (PtdIns3K), wortmannin and LY294002, to evaluate the potential involvement of PtdIns3K in the activation of the MAP kinases (MAPK), Erk1 and Erk2. In dose,response studies carried out on six different cell lines and a primary cell culture, we analyzed the ability of the inhibitors to block phosphorylation of protein kinase B/akt (PKB/akt) at Ser473 as a measure of PtdIns3K activity, or the phosphorylation of Erk1/2 at activating Thr/Tyr sites as a measure of the extent of activation of MAPK/Erk kinase (MEK/Erk). In three different hemopoietic cell lines stimulated with cytokines, and in HEK293 cells, stimulated with serum, either wortmannin or LY294002, but never both, could partially block phosphorylation of Erks. The same observations were made in a B-cell line and in primary fibroblasts. In only one cell type, the A20 B cells, was there a closer correlation between the PtdIns3K inhibition by both inhibitors, and their corresponding effects on Erk phosphorylation. However, this stands out as an exception that gives clues to the mechanism by which cross-talk might occur. In all other cells, acute activation of the pathway leading to Erk phosphorylation could proceed independently of PtdIns3K activation. In a biological assay comparing these two pathways, the ability of LY294002 and the MEK inhibitor, U0126, to induce apoptosis were tested. Whereas LY294002 caused death of cytokine-dependent hemopoietic cells, U0126 had little effect, but both inhibitors together had a synergistic effect. The data show that these two pathways are regulating very different downstream events involved in cell survival. [source] The A-subunit of surface-bound Shiga toxin stimulates clathrin-dependent uptake of the toxinFEBS JOURNAL, Issue 16 2005Maria L. Torgersen Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin,receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed. [source] A comparative proteome analysis of human metaphase chromosomes isolated from two different cell lines reveals a set of conserved chromosome-associated proteinsGENES TO CELLS, Issue 3 2007Hideaki Takata A comparative proteome analysis of human metaphase chromosomes between a typical epithelial-like cell, HeLa S3, and a lymphoma-type cell, BALL-1, was performed. One-dimensional (1-D) SDS-PAGE and radical-free and highly reducing two-dimensional electrophoresis (RFHR 2-DE) detected more than 200 proteins from chromosomes isolated from HeLa S3 cells, among which 189 proteins were identified by mass spectrometry (MS). Consistent with our recent four-layer structural model of a metaphase chromosome, all the identified proteins were grouped into four distinct levels of abundance. Both HeLa S3 and BALL-1 chromosomes contained specific sets of abundant chromosome structural and peripheral proteins in addition to less abundant chromosome coating proteins (CCPs). Furthermore, titin array analysis and a proteome analysis of the ultra-high molecular mass region indicated an absence of titin with their molecular weight (MW) more than 1000 kDa. Consequently, the present proteome analyses together with previous information on chromosome proteins provide the comprehensive list of proteins essential for the metaphase chromosome architecture. [source] DNA repair pathways involved in anaphase bridge formationGENES, CHROMOSOMES AND CANCER, Issue 6 2007Ceyda Acilan Cancer cells frequently exhibit gross chromosomal alterations such as translocations, deletions, or gene amplifications an important source of chromosomal instability in malignant cells. One of the better-documented examples is the formation of anaphase bridges,chromosomes pulled in opposite directions by the spindle apparatus. Anaphase bridges are associated with DNA double strand breaks (DSBs). While the majority of DSBs are repaired correctly, to restore the original chromosome structure, incorrect fusion events also occur leading to bridging. To identify the cellular repair pathways used to form these aberrant structures, we tested a requirement for either of the two major DSB repair pathways in mammalian cells: homologous recombination (HR) and nonhomologous end joining (NHEJ). Our observations show that neither pathway is essential, but NHEJ helps prevent bridges. When NHEJ is compromised, the cell appears to use HR to repair the break, resulting in increased anaphase bridge formation. Moreover, intrinsic NHEJ activity of different cell lines appears to have a positive trend with induction of bridges from DNA damage. © 2007 Wiley-Liss, Inc. [source] Antisense therapeutics for neurofibromatosis type 1 caused by deep intronic mutations,HUMAN MUTATION, Issue 3 2009Eva Pros Abstract Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting 1:3,500 individuals. Disease expression is highly variable and complications are diverse. However, currently there is no specific treatment for the disease. NF1 is caused by mutations in the NF1 gene, approximately 2.1% of constitutional mutations identified in our population are deep intronic mutations producing the insertion of a cryptic exon into the mature mRNA. We used antisense morpholino oligomers (AMOs) to restore normal splicing in primary fibroblast and lymphocyte cell lines derived from six NF1 patients bearing three deep intronic mutations in the NF1 gene (c.288+2025T>G, c.5749+332A>G, and c.7908-321C>G). AMOs were designed to target the newly created 5, splice sites to prevent the incorporation of cryptic exons. Our results demonstrate that AMO treatment effectively restored normal NF1 splicing at the mRNA level for the three mutations studied in the different cell lines analyzed. We also found that AMOs had a rapid effect that lasted for several days, acting in a sequence-specific manner and interfering with the splicing mechanism. Finally, to test whether the correction of aberrant NF1 splicing also restored neurofibromin function to wild-type levels, we measured the amount of Ras-GTP after AMO treatment in primary fibroblasts. The results clearly show an AMO-dependent decrease in Ras-GTP levels, which is consistent with the restoration of neurofibromin function. To our knowledge this is the first time that an antisense technique has been usedsuccessfully to correct NF1 mutations opening the possibility of a therapeutic strategy for this type of mutation not only for NF1 but for other genetic disorders. Hum Mutat 30, 454,462, 2009. © 2009 Wiley-Liss, Inc. [source] In vitro evaluation of the chemoprotective action mechanisms of leontopodic acid against aflatoxin B1 and deoxynivalenol-induced cell damageJOURNAL OF APPLIED TOXICOLOGY, Issue 1 2009Stefano Costa Abstract Several in vitro studies showed that free radical scavengers possess chemopreventive properties against mycotoxin-induced cell damage which are at least partially associated with the induction of phase II detoxifying enzymes and antioxidant enzymes like glutathione S -transferase (GST) and glutathione peroxidase (GPx). The aim of this project was to study the chemopreventive effects of leontopodic acid (LA), a potent natural occurring free radical scavenger isolated from the aerial parts of Leontopodium alpinum. Different mycotoxins were evaluated in two different cell lines on the basis of their specific toxicity: aflatoxin B1 (AFB1) on HepG2 cells and deoxynivalenol (DON) on U937 cells. Cell viability and reactive oxygen species concentration were determined, and the effects of pre-treatment with LA on these parameters were investigated together with the GST and GPx activity as well as the concentration of reduced glutathione. The results show that LA protects U937 cells from DON-induced cell damage but not HepG2 cells from AFB1. Moreover LA is able to enhance GPx activity in U937, but not GST activity in HepG2. We hypothesize that the increase in detoxifying enzymes is probably the main mechanism of antioxidant mediated chemoprevention. Copyright © 2008 John Wiley & Sons, Ltd. [source] Candidate cis -elements for human renin gene expression in the promoter regionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Tadashi Konoshita Abstract The regulation of renin gene expression, the rate-limiting enzyme of the system, is thought to be fundamental to the total system. Previously, we mapped six putative cis -elements in the promoter region of the human renin gene with nuclear proteins from human chorionic cells and human renal cortex by DNase I protection assay (footprint A,F). Each footprint contains Ets motif like site (A), HOXñPBX recognition sequence (B), unknown sequence as DNA binding consensus (C), CRE (D), COUP-TFII (ARP-1) motif like site (E), and AGE3 like site (F). Footprint D has been characterized by means of functional studies as the genuine human renin gene CRE interacting with CREB in cooperation with the site of footprint B. To obtain further clues to the specific expression in the promoter region, these putative cis -elements were conducted to a consensus-specific binding assay to compare renin-producing and non-renin-producing cells by EMSA and electromobility super-shift assay. Different sequence-specific DNA/protein binding was obtained among the different cell lines with footprint B site, with COUP-TFII (ARP-1) motif like site and possibly with footprint F site. The results implicate these putative cis -elements and each corresponding trans -factor in the specific expression of the human renin gene in the promoter region. Further functional characterization of these elements would provide important data for a better understanding of human renin gene expression. © 2004 Wiley-Liss, Inc. [source] Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2003Kwok Keung Chan Abstract SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder. © 2003 Wiley-Liss, Inc. [source] Growth of malignant oral epithelial stem cells after seeding into organotypical cultures of normal mucosaJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 2 2004Ian C. Mackenzie Background:, Oral squamous cell carcinoma (OSCC) is associated both with the local expansion of clones of malignant cells and with their further migration to regional and distant sites. The interactions that occur between normal and malignant cells during these events are not well modelled by standard culture conditions, but organotypical cultures, in which epithelial cells are grown on a matrix containing fibroblasts, provide a suitable environment for such investigations. Methods:, Cells from five cell lines, each derived from OSCC and marked by retroviral transduction with alkaline phosphatase, were incorporated as small subpopulations (0.1,5%) in uniformly differentiating organotypical cultures constructed from normal oral mucosal cells. The patterns of growth of the malignant cells within the normal epithelium were examined for 3 weeks. Results:, There was variation between the different cell lines in their rates and patterns of growth, but all cell lines produced clusters of malignant cells that had expanded within 3 weeks to replace the normal epithelium. The appearance and spacing of these clusters suggested that each was derived from a single progenitor cell. The number of malignant cells initially present within a given area of organotypical epithelium was much greater than the number of expanding cell clusters subsequently formed. Cluster-forming cells thus represented only a subpopulation of the tumour cells. Conclusions:, The organotypical model allows examination of interactions occurring between cells derived from OSCC and normal epithelia. The three-dimensional nature of organotypical cultures, together with their more normal patterns of differentiation, provides an environment that more closely mimics the in vivo environment in which tumours develop. The finding that only a subpopulation of tumour cells forms expanding tumour colonies suggests a range of growth potentials within a tumour population and may provide preliminary evidence for some form of stem and amplifying cell pattern. [source] Difference in susceptibilities of different cell lines to bilirubin damageJOURNAL OF PAEDIATRICS AND CHILD HEALTH, Issue 1 2000K-C Ngai Objective: To investigate if there are differences in susceptibilities to bilirubin toxicity of different cell lines. Methodology: A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method was adopted to study the cytotoxic effect of bilirubin on several commercially available cell lines including human glioblastoma (ATCC CRL 1690, T98G), human neuroblastoma (ATCC HTB-10, SK-N-MC), human liver (ATCC CCL 13, Chang Liver, HeLa markers) and a mouse fibroblast (ATCC CCL-1, NCTC Colon 929). Results: Cytotoxicity was observed when certain bilirubin:albumin molar ratios were exceeded in the medium of a cell line in culture. Different cells exhibited different susceptibilities to the cytotoxic effects of bilirubin; neuroblastoma and glioblastoma were most susceptible, fibroblasts were the least vulnerable. Conclusions: Our findings have confirmed the clinical impression that different cells sustain different degrees of cytotoxicities caused by bilirubin. [source] Solubilizing efficiency and in vitro cytotoxicity of Peptoad GJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2010Radhakrishna K. Maroju Abstract A novel non-ionic surfactant, Peptoad G, is evaluated for its solubilizing capacity and cytotoxicity in order to explore its possible use in aqueous formulation of hydrophobic drugs. Solubility studies were carried out using ten model hydrophobic drugs, and cytotoxicity of the surfactant was evaluated in three different cell lines using the MTT assay. It was shown that peptoad G enhances the solubility of the ten model drugs to different extents, ranging from 20- to 1100-fold, which correlated with the number of hydrogen-bonding sites on the drug molecules. The in vitro cytotoxicity studies revealed comparable cytotoxicity of peptoad G to that of cremophor EL. The results suggest peptoad G possesses potential as an alternative to conventional solubilizers in hydrophobic drug formulations. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2196,2198, 2010 [source] From CT Scan to Ceramic Bone GraftJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 7 2003Jens Darsell An indirect fused deposition process was used to fabricate controlled-porosity alumina bone grafts using a computer-aided-design file created from computed tomography (CT) scans of a horse's short pastern bone. Structures with both uniform and gradient porosity were fabricated to show the effectiveness of this process for the fabrication of custom orthopedic implants. Cytotoxicity and cell proliferation studies were conducted with different cell lines to show that these bone grafts are biocompatible. Uniaxial compression tests were also conducted to understand the influence of porosity on the mechanical properties of these structures. [source] Proteomic analysis of pancreatic endocrine tumor cell lines treated with the histone deacetylase inhibitor trichostatin APROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2007Daniela Cecconi Abstract Effects of the histone-deacetylases inhibitor trichostatin A (TSA) on the growth of three different human pancreatic endocrine carcinoma cell lines (CM, BON, and QGP-1) have been assessed via dosage-dependent growth inhibition curves. TSA determined strong inhibition of cell growth with similar IC50 values for the different cell lines: 80.5,nM (CM), 61.6,nM (BON), and 86,nM (QGP-1), by arresting the cell cycle in G2/M phase and inducing apoptosis. 2DE and nano-RP-HPLC-ESI-MS/MS analysis revealed 34, 33, and 38 unique proteins differentially expressed after TSA treatment in the CM, BON, and QGP-1 cell lines, respectively. The most important groups of modulated proteins belong to cell proliferation, cell cycle, and apoptosis classes (such as peroxiredoxins 1 and 2, the diablo protein, and HSP27). Other proteins pertain to processes such as regulation of gene expression (nucleophosmin, oncoprotein dek), signal transduction (calcium-calmodulin), chromatin, and cytoskeleton organization (calgizzarin, dynein, and lamin), RNA splicing (nucleolin, HNRPC), and protein folding (HSP70). The present data are in agreement with previous proteomic analyses performed on pancreatic ductal carcinoma cell lines (Cecconi, D. et al.., Electrophoresis 2003; Cecconi, D. et al., J. Proteome Res. 2005) and place histone-deacetylases inhibitors among the potentially most powerful drugs for the treatment of pancreatic tumors. [source] Signal transduction responses to lysophosphatidic acid and sphingosine 1-phosphate in human prostate cancer cellsTHE PROSTATE, Issue 14 2009Terra C. Gibbs Abstract BACKGROUND Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lipid mediators that bind to G-protein-coupled receptors. In this study, signaling responses to 18:1 LPA and S1P were examined in parallel in three human prostate cancer cell lines: PC-3, Du145, and LNCaP. METHODS Receptor expression was assessed by RT-PCR, Northern blotting, and immunoblotting. Cellular responses to mediators were studied by proliferation assays, phosphoprotein immunoblotting, and phospholipid metabolism assays. RESULTS All cell lines express mRNA for both LPA and S1P receptors. PC-3 and Du145, but not LNCaP, proliferate in response to LPA and S1P. Epidermal growth factor (EGF), phorbol 12-myristate 13-acetate (PMA), LPA, and S1P induce activation of Erks in PC-3 and Du145; only EGF and PMA activate Erks in LNCaP. In Du145 and PC-3, Akt is activated by EGF, LPA, and S1P. Akt is constitutively active in LNCaP; EGF but not LPA or S1P stimulates further phosphorylation. FAK is phosphorylated in response to both LPA and S1P in PC-3 and Du145, but not in LNCaP. LPA and S1P stimulate phospholipase D (PLD) activity to varying extents in the different cell lines. Notably, both lipid mediators activate PLD in LNCaP. In Du145, LPA, but not S1P, activates PLD and enhances cellular production of LPA. CONCLUSIONS Although both LPA and S1P induce signal transduction in all prostate cancer cell lines studied, a proliferation response is observed only when the Erk, Akt, and FAK pathways are activated. Other responses to the lipid mediators, such as PLD activation, likely contribute to other cellular outcomes. Prostate 69: 1493,1506, 2009. © 2009 Wiley-Liss, Inc. [source] Effect of inhibitors of mitogen-activated protein kinase kinase on ,1B -adrenoceptor phosphorylationAUTONOMIC & AUTACOID PHARMACOLOGY, Issue 1-2 2009R. Alcántara-Hernández Summary 1,Mitogen-activated protein kinases mediate hormone/neurotransmitter action on proliferation and differentiation and participate in receptor regulation. The effect of inhibitors of mitogen-activated kinase kinase (MEK) on ,1B -adrenoceptor phosphorylation state and function was studied using different cell lines. It was observed that at nanomolar concentrations the MEK inhibitors, PD98059 (2,-amino-3,-methoxyflavone) and UO126 [1,4-(diamino-2,3-dicyano/1,4-bis-(2-aminophenylthio)-butadiene], increased ,1B -adrenoceptor phosphorylation and diminished the functional response of this receptor to noradrenaline. These agents did not alter the action of lysophosphatidic acid. 2,Staurosporine (IC50 , 0.8 nm) (a general protein kinase inhibitor) and bis-indolyl-maleimide I (IC50 , 200 nm) (a selective protein kinase C inhibitor) inhibited PD98059-induced ,1B -adrenoceptor phosphorylation. In contrast, neither wortmannin (phosphoinositide 3-kinase inhibitor) nor genistein (protein tyrosine kinase inhibitor) had any effect. The data suggest the possibility that MEK might exert control on the activity of the enzymes that regulate receptor phosphorylation, such as G-protein-coupled receptor kinases, protein kinase C or serine/threonine protein phosphatases. 3,Coimmunoprecipitation studies showed a constant association of total extracellular signal-regulated kinase 2 (ERK2) with ,1B -adrenoceptors. Association of phospho-ERK 1/2 to ,1B -adrenoceptors increased not only in response to agonist but also in response to agents that increase ,1B -adrenoceptor and ERK1/2 phosphorylation [such as endothelin-1, phorbol 12-myristate-13-acetate (PMA) and epidermal growth factor (EGF)]; not surprisingly, PD98059 decreased this effect. 4,Our data show that blockade of MEK activity results in increased ,1B -adrenoceptor phosphorylation, diminished adrenoceptor function and perturbation of receptor,ERK1/2 interaction. [source] Ultra scale-down approaches for clarification of mammalian cell culture broths in disc-stack centrifugesBIOTECHNOLOGY PROGRESS, Issue 6 2009Ferhana Zaman Abstract Ultra-scale down (USD) methodology developed by University College London for cell broth clarification with industrial centrifuges was applied to two common cell lines (NS0 and GS-CHO) expressing various therapeutic monoclonal antibodies. A number of centrifuges at various scales were used with shear devices operating either by high speed rotation or flow-through narrow channels. The USD methodology was found effective in accounting for both gravitational and shear effects on clarification performance with three continuous centrifuges at pilot and manufacturing scales. Different shear responses were observed with the two different cell lines and even with the same cell line expressing different products. Separate particle size analysis of the treated broths seems consistent with the shear results. Filterability of the centrifuged solutions was also evaluated to assess the utility of the USD approach for this part of the clarification operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] N-linked glycosylation is an important parameter for optimal selection of cell lines producing biopharmaceutical human IgGBIOTECHNOLOGY PROGRESS, Issue 1 2009Patrick H. C. van Berkel Abstract We studied the variations in N-linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one of the six different antibodies. Antibody expression was based on glutamine synthetase selection technology in suspension growing CHO-K1SV cells. The glycans detected on the Fc fragment were mainly of the core-fucosylated complex type containing zero or one galactose and little to no sialic acid. The glycosylation was highly consistent for the same cell line when grown multiple times, indicating the robustness of the production and glycan analysis procedure. However, a twofold to threefold difference was observed in the level of galactosylation and/or non-core-fucosylation between the 105 different cell lines, suggesting clone-to-clone variation. These differences may change the Fc-mediated effector functions by such antibodies. Large variation was also observed in the oligomannose-5 glycan content, which, when present, may lead to undesired rapid clearance of the antibody in vivo. Statistically significant differences were noticed between the various glycan parameters for the six different antibodies, indicating that the variable domains and/or light chain isotype influence Fc glycosylation. The glycosylation altered when batch production in shaker was changed to fed-batch production in bioreactor, but was consistent again when the process was scaled from 400 to 5,000 L. Taken together, the observed clone-to-clone glycosylation variation but batch-to-batch consistency provides a rationale for selection of optimal production cell lines for large-scale manufacturing of biopharmaceutical human IgG. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] High-Level Expression of Proteins in Mammalian Cells Using Transcription Regulatory Sequences from the Chinese Hamster EF-1, GeneBIOTECHNOLOGY PROGRESS, Issue 3 2004Jennifer Running Deer High-level expression of a recombinant protein in Chinese hamster ovary (CHO) cells typically requires the laborious and time-consuming procedure of stepwise gene amplification. We hypothesized that use of transcription control regions from a highly expressed gene in CHO cells to drive expression of a gene of interest might reduce the requirement for gene amplification. To this end, we cloned a 19 kb DNA fragment containing the Chinese hamster elongation factor-1, (EF-1,) gene, as well as 12 kb of 5, flanking sequence and 4 kb of 3, flanking sequence. Expression vectors containing 5, and 3, flanking sequences from the Chinese hamster EF-1, (CHEF1) gene were constructed and, after insertion of six different reporter genes, transfected into CHO cells. For comparison, CHO cells were also transfected with the same six reporter genes inserted into commercial vectors utilizing either the immediate early promoter from cytomegalovirus (CMV) or the human EF-1, promoter. The striking result from these studies was that average expression levels from pooled, stable transfectants of CHEF1 vectors were 6- to 35-fold higher than expression levels from commercial vectors that utilize the CMV or the human EF-1, promoters. We also used a CHEF1 vector to express a secreted and a membrane-bound protein in stably transfected non-CHO cell lines. CHEF1-driven expression of secreted alkaline phosphatase (SEAP) in three of four cell lines tested (HEK 293, K562, L1.2, and HCT 116) was 13- to 280-fold greater than that from a commercial vector employing the CMV promoter. After transfection of four different cell lines of hematopoietic origin (K562, L1.2, JY, and Jurkat), the CHEF1 vector was found to express the chemokine receptor CCR4 at >10-fold higher levels than that driven from a commercial vector utilizing the CMV promoter. Results from these experiments suggest that the CHEF1 vectors will be useful for high-level protein expression not only in CHO cells, but also in a variety of other mammalian cell lines. [source] Combined targeting of MAPK and AKT signalling pathways is a promising strategy for melanoma treatmentBRITISH JOURNAL OF DERMATOLOGY, Issue 6 2007F. Meier Summary Background, In melanoma, several signalling pathways are constitutively activated. Among them, the RAS/RAF/MEK/ERK (MAPK) and PI3K/AKT (AKT) signalling pathways are activated through multiple mechanisms and appear to play a major role in melanoma development and progression. Objectives, In this study, we examined whether targeting the MAPK and/or AKT signalling pathways would have therapeutic effects against melanoma. Methods, Using a panel of pharmacological inhibitors (BAY 43-9006, PD98059, U0126, wortmannin, LY294002) we inhibited the MAPK and AKT signalling pathways at different levels and evaluated the effects on growth, survival and invasion of melanoma cells in monolayer and organotypic skin culture. Results, Antiproliferative and proapoptotic effects of inhibitors alone in monolayer culture were disappointing and varied among the different cell lines. In contrast, combined targeting of the MAPK and AKT signalling pathways significantly inhibited growth and enhanced apoptosis in monolayer culture. To verify our data in a more physiological context we incorporated melanoma cells into regenerated human skin mimicking the microenvironment of human melanoma. Combinations of MAPK and AKT inhibitors completely suppressed invasive tumour growth of melanoma cells in regenerated human skin. Conclusions, Combined targeting of MAPK and AKT signalling pathways is a promising strategy for melanoma treatment and should encourage further in-depth investigations. [source] Antitumor activity of sequence-specific alkylating agents: Pyrolle-imidazole CBI conjugates with indole linkerCANCER SCIENCE, Issue 3 2006Ken-ichi Shinohara DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2,6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of , log GI50 (50% growth inhibition concentration) for compounds 1,6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the , log GI50 of compound 1 and its conjugates 2,6 showed extremely low values (R < 0). These results suggest that differences in the sequence specificity of DNA alkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis. (Cancer Sci 2006; 97: 219,225) [source] Disubstituted 4(3H) Quinazolones: A Novel Class of Antitumor AgentsCHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2009Vikas Srivastava A series of disubstituted 4(3H) quinazolines were designed for potential application in tumors. Firstly, N -benzoyl anthranilic acid is formed, which undergoes cyclization in the presence of pyridine. Subsequently, nucleophilic attack by semicarbazide on the carbonyl carbon gives 2-substituted 3-carbamido 4(3H) quinazolones, which gives final compound with appropriate substitution. The final as well as intermediate products were confirmed by NMR, FT-IR, and mass spectrometry. In vitro toxicity was performed with different cell lines and showed that the connection of hydrophilic styryl to quinazoline moiety increases its efficacy. [source] Immunostimulatory cellular responses of cured Leishmania -infected patients and hamsters against the integral membrane proteins and non-membranous soluble proteins of a recent clinical isolate of Leishmania donovaniCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2005R. Garg Summary Development of an effective immunoprophylactic agent for visceral leishmaniasis (VL) has become imperative due to the increasing number of cases of drug resistance and relapse. Live and killed whole parasites as well as fractionated and recombinant preparations have been evaluated for vaccine potential. However, a successful vaccine against the disease has been elusive. Because protective immunity in human and experimental leishmaniasis is predominantly of the Th1 type, immunogens with Th1 stimulatory potential would make good vaccine candidates. In the present study, the integral membrane proteins (IMPs) and non-membranous soluble proteins (NSPs), purified from promastigotes of a recent field isolate, Leishmania donovani stain 2001, were evaluated for their ability to induce cellular responses in cured patients (n = 9), endemic controls (n = 5) of visceral leishmaniasis (VL) and treated hamsters (n = 10). IMPs and NSPs induced significant proliferative responses (SI 6·3 ± 4·1 and 5·6 ± 2·3, respectively; P < 0·01) and IFN-, production (356·3 ± 213·4 and 294·29 ± 107·6 pg/ml, respectively) in lymphocytes isolated from cured VL patients. Significant lymphoproliferative responses against IMPs and NSPs were also noticed in cured Leishmania animals (SI 7·2 ± 4·7 & 6·4 ± 4·1, respectively; P < 0·01). In addition, significant NO production in response both IMPs and NSPs was also noticed in macrophages of hamsters and different cell lines (J774A-1 and THP1). These results suggest that protective, immunostimulatory molecules are present in the IMP and NSP fractions, which may be exploited for development of a subunit vaccine for VL. [source] Comparison of Rickettsia conorii growth within different cell lines by real-time quantitative PCRCLINICAL MICROBIOLOGY AND INFECTION, Issue 2009P. Balraj No abstract is available for this article. [source] | ||||||||||||||||