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Mammalian Cells (mammalian + cell)
Kinds of Mammalian Cells Terms modified by Mammalian Cells Selected AbstractsUptake and Release of Double-Walled Carbon Nanotubes by Mammalian CellsADVANCED FUNCTIONAL MATERIALS, Issue 19 2010Vera Neves Abstract Efforts to develop carbon nanotubes (CNTs) as nano-vehicles for precise and controlled drug and gene delivery, as well as markers for in vivo biomedical imaging, are currently hampered by uncertainties with regard to their cellular uptake, their fate in the body, and their safety. All of these processes are likely to be affected by the purity of CNT preparation, as well as the size and concentration of CNTs used, parameters that are often poorly controlled in biological experiments. It is demonstrated herein that under the experimental conditions of standard transfection methods, DWNTs are taken up by cultured cells but are then released after 24 h with no discernable stress response. The results support the potential therapeutic use of CNTs in many biomedical settings, such as cancer therapy. [source] Induction of Persistent Double Strand Breaks Following Multiphoton Irradiation of Cycling and G1 -arrested Mammalian Cells,Replication-induced Double Strand BreaksPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2008Jane V. Harper DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of ,-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for >4 h in addition to those induced at replication. [source] Fluorometric Analysis of DNA Unwinding (FADU) as a Method for Detecting Repair-induced DNA Strand Breaks in UV-irradiated Mammalian Cells,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2000Christa Baumstark-Khan ABSTRACT Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray,induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20°C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation). [source] Patterning Mammalian Cells for Modeling Three Types of Naturally Occurring Cell,Cell Interactions,ANGEWANDTE CHEMIE, Issue 44 2009Zhenling Chen Dr. Verschiedene Streifen: Mikrofluidiktechniken und oberflächenchemische Verfahren wurden angewendet, um verschiedenartige Zellmuster auf einem Substrat aufzubauen und dreierlei natürliche Zell-Zell-Wechselwirkungen nachzustellen. PDMS=Poly(dimethylsiloxan)-Stempel, FN=Fibronectin als Promotor der Zelladhäsion, SAM=selbstorganisierte Monoschicht eines Alkanthiols, das die Zelladhäsion verhindert. [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] Corrigendum: A Genetically Encoded ,- N -Methyl Lysine in Mammalian CellsCHEMBIOCHEM, Issue 8 2010Dan Groff Dr. No abstract is available for this article. [source] Cell culture monitoring via an auto-sampler and an integrated multi-functional off-line analyzerBIOTECHNOLOGY PROGRESS, Issue 1 2010Gayle E. Derfus Abstract Mammalian cell-based bioprocesses are used extensively for production of therapeutic proteins. Off-line monitoring of such cultivations via manual sampling is often labor-intensive and can introduce operator-dependent error into the process. An integrated multi-functional off-line analyzer, the BioProfile FLEX (NOVA Biomedical, Waltham MA) has been developed, which combines the functionality of three off-line analyzers (a cell counter, an osmometer, and a gas/electrolyte & nutrient/metabolite bio-profile analyzer) into one device. In addition, a novel automated sampling system has also been developed that allows the BioProfile FLEX to automatically analyze the culture conditions in as many as ten bioreactors. This is the first report on the development and function of this integrated analyzer and an auto-sampler prototype for monitoring of mammalian cell cultures. Evaluation of the BioProfile FLEX was conducted in two separate laboratories and involved two BioProfile FLEX analyzers and two sets of reference analyzers (Nova BioProfile 400, Beckman-Coulter Vi-Cell AS, and Advanced Instruments Osmometer 3900), 13 CHO cell lines and over 20 operators. In general, BioProfile FLEX measurements were equivalent to those obtained using reference analyzers, and the auto-sampler did not alter the samples it provided to the BioProfile FLEX. These results suggest that the system has the potential to dramatically reduce the manual labor involved in monitoring mammalian cell bioprocesses without altering the quality of the data obtained, and integration with a bioreactor control system will allow feedback control of parameters previously available only for off-line monitoring. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Biochemical insights into the mechanisms central to the response of mammalian cells to cold stress and subsequent rewarmingFEBS JOURNAL, Issue 1 2009Anne Roobol Mammalian cells cultured in vitro are able to recover from cold stress. However, the mechanisms activated during cold stress and recovery are still being determined. We here report the effects of hypothermia on cellular architecture, cell cycle progression, mRNA stability, protein synthesis and degradation in three mammalian cell lines. The cellular structures examined were, in general, well maintained during mild hypothermia (27,32 °C) but became increasingly disrupted at low temperatures (4,10 °C). The degradation rates of all mRNAs and proteins examined were much reduced at 27 °C, and overall protein synthesis rates were gradually reduced with temperature down to 20 °C. Proteins involved in a range of cellular activities were either upregulated or downregulated at 32 and 27 °C during cold stress and recovery. Many of these proteins were molecular chaperones, but they did not include the inducible heat shock protein Hsp72. Further detailed investigation of specific proteins revealed that the responses to cold stress and recovery are at least partially controlled by modulation of p53, Grp75 and eIF3i levels. Furthermore, under conditions of severe cold stress (4 °C), lipid-containing structures were observed that appeared to be in the process of being secreted from the cell that were not observed at less severe cold stress temperatures. Our findings shed light on the mechanisms involved and activated in mammalian cells upon cold stress and recovery. [source] Identification of a novel REV1-interacting motif necessary for DNA polymerase , functionGENES TO CELLS, Issue 2 2009Eiji Ohashi When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a "polymerase switch" recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Pol,, Pol,, Pol, and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Pol,, Pol,, and Pol, and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Pol,. We have identified novel REV1-interacting regions (RIRs) present in Pol,, Pol, and Pol,. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. The consensus sequence for REV1-binding is denoted by x-x-x-F-F-y-y-y-y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Pol, mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk -null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Pol, function in vivo. [source] Mechanisms of actin stress fibre assemblyJOURNAL OF MICROSCOPY, Issue 3 2008P. NAUMANEN Summary Stress fibres are contractile acto-myosin structures found from many types of non-muscle cells, where they are involved in adhesion, motility and morphogenesis. Stress fibres typically display a periodic ,-actinin,myosin II pattern and are thus suggested to resemble the sarcomeric actin filament structures of muscle cells. Mammalian cells contain three categories of stress fibres: ventral stress fibres that are attached to focal adhesions at both ends, dorsal stress fibres that are attached to focal adhesions typically at one end and transverse arcs that are curved acto-myosin bundles, which do not directly attach to focal adhesions. In this review, we discuss the definition of stress fibres, organization of actin filaments and other components within these contractile structures, and the mechanisms of stress fibre assembly. [source] Circulating cell wall components derived from gram-negative, not gram-positive, bacteria cause a profound induction of the gene-encoding Toll-like receptor 2 in the CNSJOURNAL OF NEUROCHEMISTRY, Issue 3 2001Nathalie Laflamme The recent characterization of human homologs of Toll may be the missing link for the transduction events leading to nuclear factor-,B (NF-,B) activity and proinflammatory gene transcription during innate immune response. Mammalian cells may express as many as 10 distinct Toll-like receptors (TLRs), although TLR2 is a key receptor for recognizing cell wall components of Gram-positive bacteria. The present study investigated the effects of circulating bacterial cell wall components on the expression of the gene-encoding TLR2 across the mouse brain. Surprisingly, while Gram-negative components caused a robust increase in TLR2 transcription within the cerebral tissue, peptidoglycan (PGN) and lipoteichoic acid (LTA), either alone or combined, failed to modulate the receptor transcript. Indeed, the mRNA levels for TLR2 in the choroid plexus and few other regions of the brain remained similar between vehicle-, LTA-, PGN-, and LTA/PGN-administered mice at all the times evaluated (i.e. 30 min to 24 h post-intraperitoneal injection). This contrasts with the profound de novo expression of TLR2 following a single systemic injection of the lipopolysaccharide (LPS). The signal was first detected in regions devoid of blood,brain barrier and few blood vessels and microcapillaries. A second wave of TLR2 expression was also detected from these structures to their surrounding parenchymal cells that stained for a microglial marker iba1. The rapid induction of I,B, (index of NF-,B activity) and up-regulation of the adaptor protein MyD88 suggest that LPS-induced TLR2 transcription may be dependent on the NF-,B pathway. These data provide the evidence that TLR2 is not only present in the brain, but its encoding gene is regulated by cell wall components derived from Gram-negative, not Gram-positive, bacteria. The robust wave of TLR2-expressing microglial cells may have a determinant impact on the innate immune response that occurs in the brain during systemic infection by Gram-negative, not Gram-positive, bacteria. [source] Induction of Persistent Double Strand Breaks Following Multiphoton Irradiation of Cycling and G1 -arrested Mammalian Cells,Replication-induced Double Strand BreaksPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2008Jane V. Harper DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of ,-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for >4 h in addition to those induced at replication. [source] Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases,BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Pei-Qi Liu Abstract Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require sequential rounds of homologous recombination and drug selection to isolate rare desired events,a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi-locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97,105. © 2009 Wiley Periodicals, Inc. [source] Infection with Toxoplasma gondii results in dysregulation of the host cell cycleCELLULAR MICROBIOLOGY, Issue 5 2008Robert E. Molestina Summary Mammalian cells infected with Toxoplasma gondii are characterized by a profound reprogramming of gene expression. We examined whether such transcriptional responses were linked to changes in the cell cycle of the host. Human foreskin fibroblasts (HFFs) in the G0/G1 phase of the cell cycle were infected with T. gondii and FACS analysis of DNA content was performed. Cell cycle profiles revealed a promotion into the S phase followed by an arrest towards the G2/M boundary with infection. This response was markedly different from that of growth factor stimulation which caused cell cycle entry and completion. Transcriptional profiles of T. gondii -infected HFF showed sustained increases in transcripts associated with a G1/S transition and DNA synthesis coupled to an abrogation of cell cycle regulators critical in G2/M transition relative to growth factor stimulation. These divergent responses correlated with a distinct temporal modulation of the critical cell cycle regulator kinase ERK by infection. While the kinetics of ERK phosphorylation by EGF showed rapid and sustained activation, infected cells displayed an oscillatory pattern of activation. Our results suggest that T. gondii infection induces and maintains a ,proliferation response' in the infected cell which may fulfill critical growth requirements of the parasite during intracellular residence. [source] Fluorescent Plasmodium berghei sporozoites and pre-erythrocytic stages: a new tool to study mosquito and mammalian host interactions with malaria parasitesCELLULAR MICROBIOLOGY, Issue 6 2001Ramya Natarajan To track malaria parasites for biological studies within the mosquito and mammalian hosts, we constructed a stably transformed clonal line of Plasmodium berghei, PbFluspo, in which sporogonic and pre-erythrocytic liver-stage parasites are autonomously fluorescent. A cassette containing the structural gene for the FACS-adapted green fluorescent protein mutant 2 (GFPmut2), expressed from the 5, and 3, flanking sequences of the circumsporozoite (CS) protein gene, was integrated and expressed at the endogenous CS locus. Recombinant parasites, which bear a wild-type copy of CS, generated highly fluorescent oocysts and sporozoites that invaded mosquito salivary glands and were transmitted normally to rodent hosts. The parasites infected cultured hepatocytes in vitro, where they developed into fluorescent pre-erythrocytic forms. Mammalian cells infected by these parasites can be separated from non-infected cells by fluorescence activated cell sorter (FACS) analysis. These fluorescent insect and mammalian stages of P. berghei should be useful for phenotypic studies in their respective hosts, as well as for identification of new genes expressed in these parasite stages. [source] Oxidative stress as a multiple effector in Fanconi anaemia clinical phenotypeEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2005Giovanni Pagano Abstract:, Fanconi anaemia (FA) is a genetic disease characterised by bone marrow failure with excess risk of myelogenous leukaemia and solid tumours. A widely accepted notion in FA research invokes a deficiency of response to DNA damage as the fundamental basis of the ,crosslinker sensitivity' observed in this disorder. However, such an isolated defect cannot readily account for the full cellular and clinical phenotype, which includes a number of other abnormalities, such as malformations, endocrinopathies, and typical skin spots. An extensive body of evidence pointing toward an involvement of oxidative stress in the FA phenotype includes the following: (i) In vitro and ex vivo abnormalities in a number of redox status endpoints; (ii) the functions of several FA proteins in protecting cells from oxidative stress; (iii) redox-related toxicity mechanisms of the xenobiotics evoking excess toxicity in FA cells. The clinical features in FA and the in vivo abnormalities of redox parameters are here reconsidered in view of the pleiotropic clinical phenotype and known biochemical and molecular links to an in vivo prooxidant state, which causes oxidative damage to biomolecules, resulting in an excessive number of acquired abnormalities that may overwhelm the cellular repair capacity rather than a primary deficiency in DNA repair. FA may thus represent a unique model disease in testing the integration between the acquisition of macromolecular damage as a result of oxidative stress and the ability of the mammalian cell to respond effectively to such damage. [source] Improved In vitro Model Systems for Gastrointestinal Infection by Choice of Cell Line, pH, Microaerobic Conditions, and Optimization of Culture ConditionsHELICOBACTER, Issue 4 2007Sara K. Lindén Abstract Background:, Commonly used in vitro infection cultures do not mimic the human gastrointestinal tract with regard to pH and microaerobic conditions. Furthermore, despite the importance of mucin,Helicobacter interactions, the cell lines used have not been selected for appropriate mucin expression. To make in vitro studies more applicable to human disease, we have developed coculture methods taking these factors into account. Materials and methods:, Nine human gastrointestinal epithelial cell lines (MKN1, MKN7, MKN28, MKN45, KATO3, HFE145, PCAA/C11 Caco-2, and LS513) were investigated. Expression and glycosylation of mucins (MUC1, 2, 3, 4, 5AC, 5B, 6, 12, 13, and 16) were determined by immunohistochemistry. We analyzed the effect of microaerobic conditions and acidic pH on cell proliferation, viability, and apoptosis. Results:, Microaerobic culture, which is more physiological for the bacteria, did not adversely affect mammalian cell viability, proliferation, or induce apoptosis The cell lines varied in mucin expression, with MKN7 and MKN45 being most similar to gastric mucosa and Caco-2 and LS513 to intestinal mucosa, although none exactly matched normal mucosa. However, changes in culture conditions did not cause major changes in the mucin expression within cell lines. Conclusions:, Culture conditions mimicking the natural environment and allowing the bacterial cells to thrive had no effect on cell viability or apoptosis, and very little influence on mucin expression of human gastrointestinal cells. Thus, it is feasible, using the simple methods we present here, to substantially improve bacterial,mammalian cell in vitro coculture studies to make them more reflective of human infection. [source] MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay,HUMAN MUTATION, Issue 2 2010Sara Molatore Abstract MUTYH -associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8-hydroxyguanine (8-oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh,/, mouse defective cells. Several parameters, including accumulation of 8-oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell-based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild-type protein. Our cell-based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1,8, 2009. © 2009 Wiley-Liss, Inc. [source] Targeted optical injection of gold nanoparticles into single mammalian cellsJOURNAL OF BIOPHOTONICS, Issue 12 2009Craig McDougall Abstract We present an all optical technique for the targeted delivery of single 100 nm diameter gold nanoparticles into a specified region of the interior of an individual mammalian cell through a combination of optical tweezing and optical injection. The internalisation of the nanoparticle is verified by confocal laser scanning microscopy and confocal laser scanning reflectance microscopy. This represents the first time that nano sized particles have been tweezed and optically injected into mammalian cells using only light, and provides a novel methodology for internalising nanosphere based biosensors within specific intracellular regions of a mammalian cell. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Characterization of tissue-specific LIM domain protein (FHL1C) which is an alternatively spliced isoform of a human LIM-only protein (FHL1)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001Enders Kai On Ng Abstract We have cloned and characterized another alternatively spliced isoform of the human four-and-a-half LIM domain protein 1 (FHL1), designated FHL1C. FHL1C contains a single zinc finger and two tandem repeats of LIM domains at the N-terminus followed by a putative RBP-J binding region at the C-terminus. FHL1C shares the same N-terminal two-and-a-half LIM domains with FHL1 but different C-terminal protein sequences. Due to the absence of the exon 4 in FHL1C, there is a frame-shift in the 3, coding region. Sequence analysis indicated that FHL1C is the human homolog of murine KyoT2. The Northern blot and RT-PCR results revealed that FHL1 is widely expressed in human tissues, including skeletal muscle and heart at a high level, albeit as a relatively low abundance transcript in brain, placenta, lung, liver, kidney, pancreas, and testis. In contrast, FHL1C is specifically expressed in testis, skeletal muscle, and heart at a relatively low level compared with FHL1. The expression of FHL1C transcripts was also seen in aorta, left atrium, left, and right ventricles of human heart at low level. Immunoblot analysis using affinity-purified anti-FHL1C antipeptide antibodies confirmed a 20 kDa protein of FHL1C in human skeletal muscle and heart. Unlike FHL1B, which is another FHL1 isoform recently reported by our group and localized predominantly in the nucleus [Lee et al., 1999], FHL1C is localized both in the nucleus and cytoplasm of mammalian cell. J. Cell. Biochem. 82: 1,10, 2001. © 2001 Wiley-Liss, Inc. [source] Increasing the sialylation of therapeutic glycoproteins: The potential of the sialic acid biosynthetic pathwayJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2009Kaya Bork Abstract The number of therapeutic proteins has increased dramatically over the past years and most of the therapeutic proteins in the market today are glycoproteins. Usually, recombinant glycoproteins are produced in mammalian cell lines, such as Chinese-hamster-ovary-cells to obtain mammalian-type of glycosylation. The terminal monosaccharide of N-linked complex glycans is typically occupied by sialic acid. Presence of this sialic acid affects absorption, serum half-life, and clearance from the serum, as well as the physical, chemical and immunogenic properties of the respective glycoprotein. From a manufacturing perspective, the degree of sialylation is crucial since sialylation varies the function of the product. In addition, insufficient or inconsistent sialylation is also a major problem for the process consistency. Sialylation of over-expressed glycoproteins in all mammalian cell lines commonly used in biotechnology for the production of therapeutic glycoproteins is incomplete and there is a need for strategies leading to homogenous, naturally sialylated glycoproteins. This review will shortly summarize the biosynthesis of sialic acids and describe some recent strategies to increase or modify sialylation of specific therapeutic glycoproteins. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3499,3508, 2009 [source] LplA1-dependent utilization of host lipoyl peptides enables Listeria cytosolic growth and virulenceMOLECULAR MICROBIOLOGY, Issue 3 2007Kristie M. Keeney Summary The bacterial pathogen Listeria monocytogenes replicates within the cytosol of mammalian cells. Mechanisms by which the bacterium exploits the host cytosolic environment for essential nutrients are poorly defined. L. monocytogenes is a lipoate auxotroph and must scavenge this critical cofactor, using lipoate ligases to facilitate attachment of the lipoyl moiety to metabolic enzyme complexes. Although the L. monocytogenes genome encodes two putative lipoate ligases, LplA1 and LplA2, intracellular replication and virulence require only LplA1. Here we show that LplA1 enables utilization of host-derived lipoyl peptides by L. monocytogenes. LplA1 is dispensable for growth in the presence of free lipoate, but necessary for growth on low concentrations of mammalian lipoyl peptides. Furthermore, we demonstrate that the intracellular growth defect of the ,lplA1 mutant is rescued by addition of exogenous lipoic acid to host cells, suggesting that L. monocytogenes dependence on LplA1 is dictated by limiting concentrations of available host lipoyl substrates. Thus, the ability of L. monocytogenes and other intracellular pathogens to efficiently use host lipoyl peptides as a source of lipoate may be a requisite adaptation for life within the mammalian cell. [source] Evaluation of biocompatibility of a pectin/polyvinyl alcohol composite hydrogel as a new nucleus materialORTHOPAEDIC SURGERY, Issue 3 2009Nv-zhao Yao MD Objective:, To evaluate the biocompatibility of a new kind of prosthetic nucleus: a pectin/polyvinyl alcohol composite (CoPP) hydrogel. Methods:, According to Chinese national standard GB/-T16886 documents, the toxicity of the CoPP prosthetic nucleus material was examined by cytotoxicity, sensitization, Ames, mice marrow micronucleus, chromosome aberration test of mammalian cell and implantation tests. Results:, Cell growth was similar in the CoPP culture and control groups. No significant difference was found between the CoPP culture and control groups at each time point (P > 0.05). The cell proliferation rate was greater than 100%. In accordance with the relationship between cytotoxicity to proliferation rate, it was confirmed that the cytotoxicity of CoPP was 0 grade. Mice had no allergic reaction when injected with an extract of CoPP. A reverse mutation test with Salmonella typhimurium showed that no significant effect on the number of histidine revertants of TA97, TA98, TA100 and TA102 strains after CoPP was added. The micronucleus rate in bone marrow cells was less than 5%; there was no significant difference compared with the negative control group (P > 0.05). The rate of chromosome aberration was less than 5%; no significant difference was found between the CoPP culture and the control groups. All experimental animal wounds achieved primary healing without exudation, infection or sinus formation. On macroscopic observation, no abscess or hematoma formed at the implantation site. Conclusion:, The CoPP prosthetic nucleus has good biocompatibility and can potentially be used as an implant material. [source] NF-,B DNA-binding activity after high peak power pulsed microwave (8.2 GHz) exposure of normal human monocytesBIOELECTROMAGNETICS, Issue 4 2002Mohan Natarajan Abstract The hypothesis investigated is that exposure of a mammalian cell to high peak power pulsed RF, at the frequency of 8.2 GHz, can result in the activation of an important eukaryotic transcriptional regulator, nuclear factor kappa B (NF-,B). This DNA-binding protein controls genes involved in long term cellular regulation. The selection of 8.2 GHz was based on the availability of a high peak power pulsed RF transmitter. In these studies, triplicate cultures of human monocytes (Mono Mac-6) were exposed to the pulsed wave radiation. The peak to average power ratio was 455:1 (2.2 ,s pulse width and pulse repetition rate of 1000 pulses/s). The average power density at the position of exposure was 50 W/m2, and the mean SAR at the bottom of the culture flask was 10.8,±,7.1 W/kg. The FDTD analysis indicated that 10% of the cells had an SAR of 22,29 W/kg. The cells were exposed continuously for 90 min at 37 °C, reincubated at this temperature, and harvested 4 h postexposure. The nuclear extracts were analyzed by electrophoretic mobility shift assay. The results showed a profound increase (3.6-fold) in the DNA binding activity of NF-,B in monocytes at 4 h after the pulsed RF exposure compared to sham irradiated controls. Competition experiments with cold NF-,B- specific oligonucleotides confirmed the specificity of the DNA binding activity. These results provide evidence that high peak power pulsed radiofrequency radiation can perturb the cell and initiate cell signaling pathways. However, at this point, we are not prepared to advocate that the cause is a nonthermal mechanism. Because of the broad distribution of SAR's in the flask, experiments need to be performed to determine if the changes observed are associated with cells exposed to high or low SARs. Bioelectromagnetics 23:271,277, 2002. © 2002 Wiley-Liss, Inc. [source] Flow characterization of a wavy-walled bioreactor for cartilage tissue engineeringBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2006Bahar Bilgen Abstract Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0,0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties. © 2006 Wiley Periodicals, Inc. [source] Activation of PLA2 isoforms by cell swelling and ischaemia/hypoxiaACTA PHYSIOLOGICA, Issue 1-2 2006I. H. Lambert Abstract Phospholipase A2 (PLA2) activity is increased in mammalian cells in response to numerous stimuli such as osmotic challenge, oxidative stress and exposure to allergens. The increased PLA2 activity is seen as an increased release of free, polyunsaturated fatty acids, e.g. arachidonic acid and membrane-bound lysophospholipids. Even though arachidonic acid acts as a second messenger in its own most mammalian cells seem to rely on oxidation of the fatty acid into highly potent second messengers via, e.g. cytochrome P450, the cyclo-oxygenase, or the lipoxygenase systems for downstream signalling. Here, we review data that illustrates that stress-induced PLA2 activity involves various PLA2 subtypes and that the PLA2 in question is determined by the cell type and the physiological stress condition. [source] Novel interactors and a role for supervillin in early cytokinesis,CYTOSKELETON, Issue 6 2010Tara C. Smith Abstract Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, FLNA, HAX1, and MIF4GD also mimic supervillin over-expression by inhibiting cell spreading. Most new interactors have known roles in supervillin-associated processes, e.g. cell motility, membrane trafficking, ERK signaling, and matrix invasion; three (KIF14, KIFC3, STARD9/KIF16A) have kinesin motor domains; and five (EPLIN, KIF14, BUB1, ODF2/cenexin, RHAMM) are important for cell division. GST fusions of the supervillin G2-G3 or G4-G6 repeats co-sediment KIF14 and EPLIN, respectively, consistent with a direct association. Supervillin depletion leads to increased numbers of bi- and multi-nucleated cells. Cytokinesis failure occurs predominately during early cytokinesis. Supervillin localizes with endogenous myosin II and EPLIN in the cleavage furrow, and overlaps with the oncogenic kinesin, KIF14, at the midbody. We conclude that supervillin, like its interactors, is important for efficient cytokinesis. Our results also suggest that supervillin and its interaction partners coordinate actin and microtubule motor functions throughout the cell cycle. © 2010 Wiley-Liss, Inc. [source] Rab6 family proteins interact with the dynein light chain protein DYNLRB1CYTOSKELETON, Issue 3 2008Bas Wanschers Abstract The small GTPase Rab6 is a key regulator in the retrograde transfer from endosomes via the Golgi to the ER. Three isoforms of Rab6 have been identified, the ubiquitously expressed Rab6A and Rab6A,, and the brain specific Rab6B. Recent studies have shown that Rab6A, is the major isoform regulating this retrograde transport. Cytoplasmic dynein is the main motor protein complex for this transport. Dynein consists of two heavy chains, two intermediate chains, four light intermediatechains and several light chains, called roadblock/LC7 proteins or DYNLRB proteins. In mammalian cells two light chain isoforms have been identified, DYNLRB1 and DYNLRB2. We here show with yeast-two-hybrid, co-immunoprecipitation and pull down studies that DYNLRB1 specifically interacts with all three Rab6 isoforms and co-localises at the Golgi. This is the first example of a direct interaction between Rab6 isoforms and the dynein complex. Pull down experiments showed further preferred association of DYNLRB1 with GTP-bound Rab6A and interestingly GDP-bound Rab6A, and Rab6B. In addition DYNLRB1 was found in the Golgi apparatus where it co-localises with EYFP-Rab6 isoforms. DYNLRB is a putative modulator of the intrinsic GTPase activity of GTP-binding proteins. In vitro we were not able to reproduce this effect on Rab6 GTPase activity. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Long-term culture of Xenopus presumptive ectoderm in a nutrient-supplemented culture mediumDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Yasuto Fukui Animal cap assay is a useful experimental model for investigating the activity of inducers in amphibian development. This assay has revealed that activin A is a potent mesoderm-inducing factor. However, it has been very difficult to induce highly differentiated tissues such as cartilage in a 3,4 day culture period. It was recently reported that jaw cartilage was induced in vitro in an animal cap that had been cultured for 14 days in Steinberg's solution using the sandwich culture method and activin A. Under these conditions, necrosis was occasionally observed in the explants. In this study, we have achieved long-term animal cap cultures in a nutrient-supplemented culture medium designated RDX. This medium was made by modifying the saline concentration of the RD medium previously developed as a basal medium for the serum-free culture of various kinds of mammalian cells. The explants cultured in RDX grew more vigorously compared with those in Steinberg's solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinberg's solution, and also increased the frequency of cartilage induction. Therefore, the supplemental nutrients may support and promote the differentiation of cartilage. This long-term culture method using RDX medium is useful for studying the differentiation of tissues or organs such as cartilage in vitro. [source] Pleiotropic function of FGF-4: Its role in development and stem cellsDEVELOPMENTAL DYNAMICS, Issue 2 2009Nobuyoshi Kosaka Abstract Fibroblast growth factors (FGFs) were initially recognized as fibroblast-specific growth factor, and it is now apparent that these growth factors regulate multiple biological functions. The diversity of FGFs function is paralleled by the emerging diversity of interactions between FGF ligands and their receptors. FGF-4 is a member of the FGF superfamily and is a mitogen exhibiting strong action on numerous different cell types. It plays a role in various stages of development and morphogenesis, as well as in a variety of biological processes. Recent studies reveal the molecular mechanisms of FGF-4 gene regulation in mammalian cells, which is involved in the developmental process. Furthermore, FGF-4 also acts on the regulation of proliferation and differentiation in embryonic stem cells and tissue stem cells. In this review, we focus on the diverse biological functions of FGF-4 in the developmental process and also discuss its putative roles in stem cell biology. Developmental Dynamics 238:265,276, 2009. © 2008 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||||||||||||||