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Sample records for cells express enzymatic

  1. ENZYMATIC CATALYSIS BY PERMEABILIZED CELLS

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    Wilberg K. Q.

    1997-01-01

    Full Text Available This paper presents an enzymatic process for sorbitol and gluconic acid production using cells of Zymomonas mobilis permeabilized with CTAB. Equimolar solutions of glucose and fructose (from 96.0 to 422.2 g/L were used. In a batch reactor, conversions of 97% were attained after 15 to 20 hours of reaction. The effect of the initial concentration of the substrates was evaluated in experiments using 20% more and 20% less glucose than fructose. It was observed that the reaction performed with more fructose reached completion faster and with a higher value of conversion

  2. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  3. Analytical techniques for characterizing enzymatic biofuel cells.

    Science.gov (United States)

    Moehlenbrock, Michael J; Arechederra, Robert L; Sjöholm, Kyle H; Minteer, Shelley D

    2009-12-01

    Enzymatic biofuel cells, which replace expensive metal catalysts with enzymes, are still in an early stage of development. This article details the analytical techniques that are often employed for evaluating and characterizing enzymatic biofuel cells and their corresponding bioanodes and biocathodes. (To listen to a podcast about this feature, please go to the Analytical Chemistry multimedia page at pubs.acs.org/page/ancham/audio/index.html.).

  4. Recent development of miniatured enzymatic biofuel cell

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    Song, Yin; Penmatsa, Varun; Wang, Chunlei

    2011-06-01

    Enzymatic biofuel cells (EBFCs) that oxidize biological fuels using enzyme-modified electrodes are considered a promising candidate for implantable power sources. However, there are still challenges to overcome before biofuel cells become competitive in any practical applications. Currently, the short lifespan of the catalytic enzymes and poor power density are the most critical issues in developing EBFCs. In this paper, we will review the recent development of biofuel cells and highlight the progress in Carbon-microelectromechanical system (C-MEMS) based micro biofuel cells by both computational modeling and experimental work. Also, our effort on utilizing a covalent immobilization technique for the attachment of enzymes onto the substrate which is expected to increase the enzyme loading efficiency and the power density of devices is discussed in this paper.

  5. Cloning and expression of the enzymatic region of Streptococcal hyaluronidase

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    Nafiseh Al-Sadat Mirjamali

    2014-09-01

    Full Text Available Objective(s: Streptococcus pyogenes produces extracellular hyaluronidase enzyme. This enzyme is directly associated with the spread of the organism during infection. The objective of the present study was to clone and express the nucleotide sequence of the enzyme which is involved in hyaluronidase enzymatic activity. Materials and Methods: The enzymatic region of hyaluronidase gene was detected by bioinformatics method. The PCR method was used to amplify enzymatic region of hyaluronidase gene from chromosomal DNA of Streptococcus pyogenes. The eluted product was cloned into the prokaryotic expression vector pET32a which was digested by BamHI and HindIII restriction endonuclease enzymes. The target protein was expressed in the Escherichia coli. The bacteria including pET32a-hylA (hylA is abbreviation of Streptococcus pyogenes hyaluronidase gene and hylA is abbreviation of Streptococcus pyogenes hyaluronidase protein plasmids were induced by IPTG and analyzed by SDS-PAGE. The enzymatic evaluation and antigenicity was finally studied. Results: Enzymes digestion analysis, sequencing results showed that the target gene (1296 base pair was inserted correctly into the recombinant vector. The expressed protein (65 KDa was purified successfully via affinity chromatography. Data also indicated that enzymatic region of hyaluronidase protein from Streptococcus pyogenes was recognized in all 5 patient’s sera. Conclusion: In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in E. coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.

  6. Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

    DEFF Research Database (Denmark)

    Norén, K; Hansen, Gert Helge; Clausen, H;

    1997-01-01

    In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...

  7. Genome-Wide Identification, 3D Modeling, Expression and Enzymatic Activity Analysis of Cell Wall Invertase Gene Family from Cassava (Manihot esculenta Crantz

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    Yuan Yao

    2014-04-01

    Full Text Available The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6 were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(VD, in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6, and Val residue from the WECVD (MeCWINV3 and 4 are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.

  8. Genome-wide identification, 3D modeling, expression and enzymatic activity analysis of cell wall invertase gene family from cassava (Manihot esculenta Crantz).

    Science.gov (United States)

    Yao, Yuan; Geng, Meng-Ting; Wu, Xiao-Hui; Liu, Jiao; Li, Rui-Mei; Hu, Xin-Wen; Guo, Jian-Chun

    2014-04-28

    The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.

  9. Enzymatic Modification of Plant Cell Wall Polysaccharides

    DEFF Research Database (Denmark)

    Øbro, Jens; Hayashi, Takahisa; Mikkelsen, Jørn Dalgaard

    2011-01-01

    for sustainable processes that replace chemical treatments with white biotechnology. Plants can contribute significantly to this sustainable process by producing plant or microbialenzymes in planta that are necessary for plant cell wall modification or total degradation. This will give rise to superior food......Plant cell walls are intricate structures with remarkable properties, widely used in almost every aspect of our life. Cell walls consist largely of complex polysaccharides and there is often a need for chemical and biochemical processing before industrial use. There is an increasing demand...... fibres, hydrocolloids, paper,textile, animal feeds or biofuels. Classical microbial-based fermentation systems could in the future face serious competition from plant-based expression systems for enzyme production. Plant expressed enzymes can either be targeted to specific cellular compartments...

  10. Enzymatic Modification of Plant Cell Wall Polysaccharides

    DEFF Research Database (Denmark)

    Øbro, Jens; Hayashi, Takahisa; Mikkelsen, Jørn Dalgaard

    2011-01-01

    fibres, hydrocolloids, paper,textile, animal feeds or biofuels. Classical microbial-based fermentation systems could in the future face serious competition from plant-based expression systems for enzyme production. Plant expressed enzymes can either be targeted to specific cellular compartments......Plant cell walls are intricate structures with remarkable properties, widely used in almost every aspect of our life. Cell walls consist largely of complex polysaccharides and there is often a need for chemical and biochemical processing before industrial use. There is an increasing demand...... for sustainable processes that replace chemical treatments with white biotechnology. Plants can contribute significantly to this sustainable process by producing plant or microbialenzymes in planta that are necessary for plant cell wall modification or total degradation. This will give rise to superior food...

  11. Recent Advances in Enzymatic Fuel Cells: Experiments and Modeling

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    Ivan Ivanov

    2010-04-01

    Full Text Available Enzymatic fuel cells convert the chemical energy of biofuels into electrical energy. Unlike traditional fuel cell types, which are mainly based on metal catalysts, the enzymatic fuel cells employ enzymes as catalysts. This fuel cell type can be used as an implantable power source for a variety of medical devices used in modern medicine to administer drugs, treat ailments and monitor bodily functions. Some advantages in comparison to conventional fuel cells include a simple fuel cell design and lower cost of the main fuel cell components, however they suffer from severe kinetic limitations mainly due to inefficiency in electron transfer between the enzyme and the electrode surface. In this review article, the major research activities concerned with the enzymatic fuel cells (anode and cathode development, system design, modeling by highlighting the current problems (low cell voltage, low current density, stability will be presented.

  12. Enzymatic cell disruption of microalgae biomass in biorefinery processes.

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    Demuez, Marie; Mahdy, Ahmed; Tomás-Pejó, Elia; González-Fernández, Cristina; Ballesteros, Mercedes

    2015-10-01

    When employing biotechnological processes for the procurement of biofuels and bio-products from microalgae, one of the most critical steps affecting economy and yields is the "cell disruption" stage. Currently, enzymatic cell disruption has delivered effective and cost competitive results when compared to mechanical and chemical cell disruption methods. However, the introduction of enzymes implies additional associated cost within the overall process. In order to reduce this cost, autolysis of microalgae is proposed as alternative enzymatic cell disruption method. This review aims to provide the state of the art of enzymatic cell disruption treatments employed in biorefinery processes and highlights the use of endopeptidases. During the enzymatic processes of microalgae life cycle, some lytic enzymes involved in cell division and programmed cell death have been proven useful in performing cell lysis. In this context, the role of endopeptidases is emphasized. Mirroring these natural events, an alternative cell disruption approach is proposed and described with the potential to induce the autolysis process using intrinsic cell enzymes. Integrating induced autolysis within biofuel production processes offers a promising approach to reduce overall global costs and energetic input associated with those of current cell disruption methods. A number of options for further inquiry are also discussed. © 2015 Wiley Periodicals, Inc.

  13. Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway

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    Zhu, Zhiguang; Wang, Yiran; Minteer, Shelley D.; Percival Zhang, Y.-H.

    Enzymatic fuel cells (EFCs) use a variety of fuels to generate electricity through oxidoreductase enzymes, such as oxidases or dehydrogenases, as catalysts on electrodes. We have developed a novel synthetic enzymatic pathway containing two free enzymes (maltodextrin phosphorylase and phosphoglucomutase) and one immobilized glucose-6-phosphate dehydrogenase that can utilize an oligomeric substrate maltodextrin for producing electrons mediated via a diaphorase and vitamin K 3 electron shuttle system. Three different enzyme immobilization approaches were compared based on electrostatic force entrapment, chemical cross-linking, and cross-linking with the aid of carbon nanotubes. At 10 mM glucose-6-phosphate (G6P) as a substrate concentration, the maximum power density of 0.06 mW cm -2 and retaining 42% of power output after 11 days were obtained through the method of chemical cross-linking with carbon nanotubes, approximately 6-fold and 3.5-fold better than those of the electrostatic force-based method, respectively. When changed to maltodextrin (degree of polymerization = 19) as the substrate, the EFC achieved a maximum power density of 0.085 mW cm -2. With the advantages of stable, low cost, high energy density, non-inhibitor to enzymes, and environmental friendly, maltodextrin is suggested to be an ideal fuel to power enzymatic fuel cells.

  14. Multicompartment Artificial Organelles Conducting Enzymatic Cascade Reactions inside Cells

    DEFF Research Database (Denmark)

    Gallardo, Maria Godoy; Labay, Cédric Pierre; Trikalitis, Vasileios

    2017-01-01

    Cell organelles are subcellular structures entrapping a set of enzymes to achieve a specific functionality. The incorporation of artificial organelles into cells is a novel medical paradigm which might contribute to the treatment of various cell disorders by replacing malfunctioning organelles....... In particular, artificial organelles are expected to be a powerful solution in the context of enzyme replacement therapy since enzymatic malfunction is the primary cause of organelle dysfunction. Although several attempts have been made to encapsulate enzymes within a carrier vehicle, only few intracellularly...... active artificial organelles have been reported to date and they all consist of single-compartment carriers. However, it is noted that biological organelles consist of multicompartment architectures where enzymatic reactions are executed within distinct subcompartments. Compartmentalization allows...

  15. Cell multiplication following partial enzymatic removal of surface coat.

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    Wyroba, E

    1978-08-01

    Treatment of Paramecium aurelia with trypsin or pronase (1 mg per 10(5) cells, at 0 to 4 degrees C) partially removes the surface coat and modifies significantly multiplication of cells. The division rate after 24 hours of cultivation is diminished approximately twice in the case of pronase-treated cells and 1.5 for tyrpsin-digested ciliates as compared with the control. On the second day the division rate increases rapidly and number of cell divisions exceeds the values observed in the control. After 72 hours of cultivation the division rate in both untreated and enzyme-treated cells is almost the same. It is concluded that the observed inhibition of cell fission results from the enzymatic removal of the surface coat--the integrity of this surface coat seems to be necessary in the process of cell division. The influence of environmental factors on the rate of growth is presented.

  16. Recent advances in Carbon Nanotube based Enzymatic Fuel Cells

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    Serge eCosnier

    2014-10-01

    Full Text Available This review summarizes recent trends in the field of enzymatic fuel cells. Thanks to the high specificity of enzymes, biofuel cells can generate electrical energy by oxidation of a targeted fuel (sugars, alcohols or hydrogen at the anode and reduction of oxidants (O2, H2O2 at the cathode in complex media. The combination of carbon nanotubes, enzymes and redox mediators was widely exploited to develop biofuel cells since the electrons, involved in the bio-electrocatalytic processes, can be efficiently transferred from or to an external circuit. Original approaches to construct electron transfer based CNT-bioelectrodes and impressive biofuel cell performances are reported as well as biomedical applications.

  17. Molecular expression and enzymatic characterization of thioredoxin from the carcinogenic human liver fluke Opisthorchis viverrini.

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    Suttiprapa, Sutas; Matchimakul, Pitchaya; Loukas, Alex; Laha, Thewarach; Wongkham, Sopit; Kaewkes, Sasithorn; Brindley, Paul J; Sripa, Banchob

    2012-03-01

    The human liver fluke, Opisthorchis viverrini, induces inflammation of the hepatobiliary system. Despite being constantly exposed to inimical oxygen radicals released from inflammatory cells, the parasite survives for years. Defense against oxidative damage can be mediated through glutathione and/or thioredoxin utilizing systems. Here, we report the molecular expression and biochemical characterization of a thioredoxin (Trx) from O. viverrini. O. viverrini Trx cDNA encoded a polypeptide of 105 amino acid residues, of molecular mass 11.63 kDa. The predicted protein has similarity to previously characterized thioredoxins with 26-51% identity. Recombinant O. viverrini Trx (Ov-Trx-1) was expressed as soluble protein in E. coli. The recombinant protein showed insulin reduction activity and supported the enzymatic function of O. viverrini thioredoxin peroxidase. Expression of Ov-Trx-1 at mRNA and protein levels was observed in all obtainable developmental stages of the liver fluke. Ov-Trx-1 was also detected in excretory-secretory products released by adult O. viverrini. Immunohistochemistry, Ov-Trx-1 was expressed in nearly all parasite tissue excepted ovary and mature sperms. Interestingly, Ov-Trx-1 was observed in the infected biliary epithelium but not in normal bile ducts. These results suggest that Ov-Trx-1 is essential for the parasite throughout the life cycle. In the host-parasite interaction aspect, Ov-Trx-1 may support thioredoxin peroxidase in protecting the parasite against damage induced by reactive oxygen species from inflammation.

  18. An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

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    Ying Huang

    2013-11-01

    Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage. Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and

  19. Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris.

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    Irina Borodina

    Full Text Available Wasp venom allergy is the most common insect venom allergy in Europe. It is manifested by large local reaction or anaphylactic shock occurring after a wasp sting. The allergy can be treated by specific immunotherapy with whole venom extracts. Wasp venom is difficult and costly to obtain and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form in yeast Pichia pastoris, which can give high yields of correctly folded protein on defined minimal medium and secretes relatively few native proteins simplifying purification.Residual amounts of enzymatically active phospholipase A1 could be expressed, but the venom protein had a deleterious effect on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A. All the three mutated forms could be expressed in P. pastoris. The H229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom allergic patients and could inhibit the binding of wasp venom to IgE antibodies specific for phospholipase A1 as shown by Enzyme Allergo-Sorbent Test (EAST. Moreover, the recombinant protein was allergenic in a biological assay as demonstrated by its capability to induce histamine release of wasp venom-sensitive basophils.The recombinant phospholipase A1 presents a good candidate for wasp venom immunotherapy.

  20. Multichannel expression analysis of submicrogram total RNA samples without enzymatic amplification using a one-day protocol

    Science.gov (United States)

    Getts, Robert C.

    2001-06-01

    Typical gene expression array analysis requires relatively large quantities of total or poly A+RNA. Samples prepared by techniques such as laser capture microdissection (LCM) and single cell expression analysis yield relatively little RNA and traditionally require the purification of poly A+ message and subsequent enzymatic amplification before analysis on an array. This type of analysis may be biased to the detection of certain messages, is labor intensive, time consuming and requires considerable expertise for reproducible success. The 3DNATM SubmicroTM expression detection kit has been developed to detect low level expression from a microgram or less of total RNA in one day. The method does not require enzymatic amplification or the direct incorporation of a modified nucleotide during probe synthesis and is simple and easy to use. The 3DNATM detection system is based on patented DNA dendrimers that contain hundreds of fluorescent labels. Signal is generated by the dendrimer after it binds to the cDNA probe (sample) via hybridization of the dendrimer to a capture sequence that is part of the original reverse transcription primer. 50-200 fold improvement of specific signal over noise compared to direct incorporation methods has been demonstrated. The theory and use of the 3DNATM SubmicroTM technology will be discussed for 2,3 and 4 channel analysis.

  1. Regional effects of enzymatic digestion on knee meniscus cell yield and phenotype for tissue engineering.

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    Sanchez-Adams, Johannah; Athanasiou, Kyriacos A

    2012-03-01

    An abundant cell source is the cornerstone of most tissue engineering strategies, but extracting cells from the knee meniscus is hindered by its dense fibrocartilaginous matrix. Identifying a method to efficiently isolate meniscus cells is important, as it can reduce the cost and effort required to perform meniscus engineering research. In this study, six enzymatic digestion regimens used for cartilaginous cell isolation were used to isolate cells from the outer, middle, and inner regions of the bovine knee meniscus. Each regimen in each region was assessed in terms of cell yield, impact on cell phenotype, and cytotoxicity. All digestion regimens caused an overall upregulation of cartilage-specific genes Sox9, collagen type I (Col 1), collagen type II (Col 2), cartilage oligomeric matrix protein, and aggrecan (AGC) in cells from all meniscus regions, but was highest for cells isolated using 1075 U/mL of collagenase for 3 h (high collagenase). In response to isolation, outer meniscus cells showed highest upregulation of Sox9 and Col 1 genes, whereas greatest upregulation for middle meniscus cells was seen in Col 1 expression, and Col 2 expression for inner cells. Cell yield was highest in all regions when subjected to 45 min of 61 U/mL pronase followed by 3 h of 1075 U/mL collagenase (pronase/collagenase [P/C]) digestion regimen (outer: 6.57±0.37, middle: 12.77±1.41, inner: 22.17±1.47×10(6) cells/g tissue). The second highest cell yield was achieved using the low collagenase (LC) digestion regimen that applied 433 U/mL of collagenase for 18 h (outer: 1.95±0.54, middle: 3.3±4.4, inner: 6.06±2.44×10(6) cells/g tissue). Cytotoxicity analysis showed higher cell death in the LC group compared with the P/C group. Self-assembled constructs formed from LC-isolated cells were less dense than constructs formed from P/C-isolated cells, and P/C constructs showed higher glycosaminoglycan content and compressive moduli than LC constructs. All isolation methods tested

  2. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    DEFF Research Database (Denmark)

    Thygesen, Lisbeth Garbrecht; E. Thybring, Emil; Johansen, Katja Salomon

    2014-01-01

    Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understo...

  3. Enzymatic Cell Isolation and Explant Cultures of Rat Calvarial Osteoblast Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Osteoblast cells were isolated from the calvarial bones of newborn Wistar rats and cultured in vitro via both collagenase digestion method and explant technique, and a comparative study was carried out on the two culture methods. The biologic characteristics of tbs osteoblast cells were studied via cell number counting,morphology observation, alkaline phosphatase staining of the cells and alizarine- red staining of the calcified nodules. The results show that osteoblast cells can be cultured in vitro via collagenase digestion method and explant technique, and the obtained cells are of good biologic characteristics. In comparison with the explant techniqne,the operative procedure of the enzymatic digestion method is more complicated. The digestion time must be carefully controlled. However, with this method, one can obtain a lager number of cells in a short time. The operative procedure of the explant technique is simpler, but it usually takes longer time to obtain cells of desirable number.

  4. EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis.

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    Galeano, E; Barroso, A A M; Vasconcelos, T S; López-Rubio, A; Albrecht, A J P; Victoria Filho, R; Carrer, H

    2016-08-12

    Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance.

  5. Periodontal tissue regeneration using enzymatically solidified chitosan hydrogels with or without cell loading

    NARCIS (Netherlands)

    Yan, X.Z.; Beucken, J.J.J.P van den; Cai, X; Yu, N.; Jansen, J.A.; Yang, F.

    2015-01-01

    This study is aimed to evaluate the in vivo biocompatibility and periodontal regenerative potential of enzymatically solidified chitosan hydrogels with or without incorporated periodontal ligament cells (PDLCs). To this end, chitosan hydrogels, with (n=8; CHIT+CELL) or without (n=8; CHIT) fluorescen

  6. Periodontal tissue regeneration using enzymatically solidified chitosan hydrogels with or without cell loading

    NARCIS (Netherlands)

    Yan, X.Z.; Beucken, J.J.J.P van den; Cai, X; Yu, N.; Jansen, J.A.; Yang, F.

    2015-01-01

    This study is aimed to evaluate the in vivo biocompatibility and periodontal regenerative potential of enzymatically solidified chitosan hydrogels with or without incorporated periodontal ligament cells (PDLCs). To this end, chitosan hydrogels, with (n=8; CHIT+CELL) or without (n=8; CHIT)

  7. Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens.

    Directory of Open Access Journals (Sweden)

    Brigitte Mack

    Full Text Available The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm(3 cubes termed explants, which are cultured 1-3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.

  8. Extracting signal from noise: kinetic mechanisms from a Michaelis-Menten-like expression for enzymatic fluctuations.

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    Moffitt, Jeffrey R; Bustamante, Carlos

    2014-01-01

    Enzyme-catalyzed reactions are naturally stochastic, and precision measurements of these fluctuations, made possible by single-molecule methods, promise to provide fundamentally new constraints on the possible mechanisms underlying these reactions. We review some aspects of statistical kinetics: a new field with the goal of extracting mechanistic information from statistical measures of fluctuations in chemical reactions. We focus on a widespread and important statistical measure known as the randomness parameter. This parameter is remarkably simple in that it is the squared coefficient of variation of the cycle completion times, although it places significant limits on the minimal complexity of possible enzymatic mechanisms. Recently, a general expression has been introduced for the substrate dependence of the randomness parameter that is for rate fluctuations what the Michaelis-Menten expression is for the mean rate of product generation. We discuss the information provided by the new kinetic parameters introduced by this expression and demonstrate that this expression can simplify the vast majority of published models. © 2013 FEBS.

  9. Expression and characterization of recombinant Rhizopus oryzae lipase for enzymatic biodiesel production.

    Science.gov (United States)

    Li, Zhilin; Li, Xun; Wang, Ye; Wang, Youdong; Wang, Fei; Jiang, Jianchun

    2011-10-01

    The Rhizopus oryzae lipase containing prosequence was expressed in Pichia pastoris. Recombinant lipase subunit showed a molecular mass of 32 kDa. The maximum activity of recombinant lipase obtained from Mut(s) recombinant was 90 IU/ml. The enzyme was stable in broad ranges of temperatures and pH, with the optimal temperature at 35 °C and pH 7.0. The crude recombinant R. oryzae lipase can be directly used for the transesterification of plant oils at high-water content of 60-100% (w/w) based on oil weight. The addition of 80% water to the transesterification systems resulted in the yield of methyl ester of 95%, 94% and 92% after 72 h using soybean oil, Jatropha curcas seed raw oil and Pistacia chinensis seed raw oil as raw material, respectively. These results indicate that the recombinant lipase is an effective biocatalyst for enzymatic biodiesel production.

  10. Effect of Brahman genetic influence on collagen enzymatic crosslinking gene expression and meat tenderness.

    Science.gov (United States)

    Gonzalez, J M; Johnson, D D; Elzo, M A; White, M C; Stelzleni, A M; Johnson, S E

    2014-01-01

    The objective of the study was to examine the effect of Brahman genetics on collagen enzymatic crosslinking gene expression and meat tenderness. Steers were randomly selected to represent a high percentage Brahman genetics (n = 13), Half-Blood genetics (n = 13), Brangus genetics (n = 13), and a high percentage Angus genetics (n = 13). Muscle samples from the Longissimus lumborum muscle were collected at weaning and harvest and reverse transcription quantitative PCR (qPCR) analysis was conducted to measure the mRNA expression of lysyl oxidase (LOX), bone morphogenetic protein 1 (BMP1), and cystatin C (CYS). Steaks from subject animals were collected at harvest, aged for 14 d and subjected to collagen analysis, Warner-Bratzler Shear Force (WBS) and trained sensory panel analysis (tenderness, juiciness, and connective tissue). Data indicated that Half-Blood and Brahman steers had greater (PBrahman and Half-Blood steaks when compared to Angus and Brangus steaks (P 0.10). At harvest, Brangus and Angus steers had greater LOX mRNA expression than Brahman cattle (P Brahman genetic influence.

  11. Cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp.

    Directory of Open Access Journals (Sweden)

    Hamed Esmaeil Lashgarian

    2016-10-01

    Full Text Available Cholesterol oxidase (CHO is one of the valuable enzymes that play an important role in: measurement of serum cholesterol, food industry as a biocatalyst and agriculture as a biological larvicide. This enzyme was produced by several bacterial strains. Wild type enzyme produced by Rhodococcus sp. secret two forms of CHO enzyme: extra cellular and membrane bound type which its amount is low and unstable. The goal of the study was cloning, expression, and enzymatic activity evaluation of cholesterol oxidase gene isolated from a native Rhodococcus sp. CHO gene was isolated from native bacteria and cloned into pET23a. In the next step, the construct was expressed in E.coli BL21 and induced by different concentration of IPTG ranges from 0.1 - 0.9 mM. This gene contains 1642 bp and encodes a protein consists of 533 amino acids. It has about 96 % homology with CHO gene isolated from Rhodococcus equi. The high expression was obtained in 0.5 mM concentration of IPTG after 4 hour induction. This recombinant enzyme had a molecular weight of 55 kDa, that secretion of intra cellular type is much more than extracellular form. The optimum pH and temperature conditions for the recombinant enzyme were 7.5 and 45°C, respectively. CHO enzyme obtained from Rhodococcus sp. is a cheap enzyme with medical and industrial applications that can be produced easily and purified in large scale with simple methods.

  12. Sustained transgene expression using non-viral enzymatic systems for stable chromosomal integration.

    Science.gov (United States)

    Palazzoli, Fabien; Carnus, Elodie; Wells, Dominic J; Bigot, Yves

    2008-10-01

    Gene delivery technologies have been developed for various biotechnology applications. In gene therapy, they are promising for the treatment of several inherited and acquired human diseases. When therapies require the transfection of a transgene, the vector integration is one of the solutions that is used for maintaining and sustaining expression. On the basis of their origin, vectorisation technologies are currently divided in two fields, gathering on one hand viral vectors and, on the other hand, non-viral approaches. In the case of the non-viral therapies, three main sub-fields are in progress to integrate transgenes. The first uses oligonucleotides to stimulate targeted gene repair by homologous recombination. The second is based on site-specific endonucleases for which the cleavage activity is used to stimulate the host recombination mechanisms in the presence of a DNA vector. The third one is developed from phage and transposon enzymatic systems. The two lasts sub-fields use non-viral enzymes and are the scope of this review. Here, our objective was to overview the main non-viral enzymatic systems able to integrate DNA cassettes. Their molecular and functional characteristics are summarized, and their properties and limits in the current state of the art highlighted. An overview of the safety and quality issues is also presented and discussed, taking into account the solutions that might circumvent problems, intellectual property and economic status for each system. As a conclusion, we propose projections of the future technological developments in the context of the different interests for public and private bodies.

  13. RECOVERY OF POLYHYDROXYALKANOATES (PHAs FROM BACTERIAL CELLS USING ENZYMATIC PROCESS

    Directory of Open Access Journals (Sweden)

    S. Marsudi

    2012-02-01

    Full Text Available Polyhydroxyalkanoates (PHAs are intracellular material accumulated by several bacteria. Commercial production of PHAs faces the issue of high production cost especially substrate cost and recovery/separation cost. An alternative to reduce the production cost is to use enzyme and or chemical to recover PHAs from bacterial cells. Recovery of PHAs from bacterial cells was done using enzyme, chemical, and a mixture of enzyme and chemical. Enzyme (s and or chemical(s were added into culture broth to disrupt cells after adjusting pH and temperature of the culture broth. Treatment by adding enzyme or chemical only into culture broth showed a low level of PHAs recovered from bacterial cells. Treatment by adding a mixture of enzymes and chemicals showed the best result among 22 examined combinations, i.e. a mixture of EDTA, lisozyme, papain enzyme, and SDS. This combination gave a PHA recovery of 65 % w/w.

  14. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    DEFF Research Database (Denmark)

    Thygesen, Lisbeth Garbrecht; E. Thybring, Emil; Johansen, Katja Salomon;

    2014-01-01

    . Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry......, particularly when it comes to up-scaling of processes based on insoluble feed stocks....

  15. Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens.

    Science.gov (United States)

    Lee, Jung-Min; Lee, Jaejung; Nam, Gyeong-Hwa; Son, Byung-Sam; Jang, Myoung-Uoon; Lee, So-Won; Hurh, Byung-Serk; Kim, Tae-Jip

    2017-02-01

    γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of γ-glutamyl compounds and the transfer of γ-glutamyl moiety to water or to amino acid/peptide acceptors. GGT can be utilized for the generation of γ-glutamyl peptides or glutamic acid, which are used as food taste enhancers. In the present study, Bacillus amyloliquefaciens SMB469 with high GGT activity was isolated from Doenjang, a traditional fermented soy food of Korea. The gene encoding GGT from B. amyloliquefaciens SMB469 (BaGGT469) was cloned from the isolate, and heterologously expressed in E. coli and B. subtilis. For comparison, three additional GGT genes were cloned from B. subtilis 168, B. licheniformis DSM 13, and B. amyloliquefaciens FZB42. The BaGGT469 protein was composed of 591 amino acids. The final protein comprises two separate polypeptide chains of 45.7 and 19.7 kDa, generated via autocatalytic cleavage. The specific activity of BaGGT469 was determined to be 17.8 U/mg with γ-L-glutamyl-p-nitroanilide as the substrate and diglycine as the acceptor. GGTs from B. amyloliquefaciens showed 1.4- and 1.7-fold higher transpeptidase activities than those from B. subtilis and B. licheniformis, respectively. Especially, recombinant B. subtilis expressing BaGGT469 demonstrated 11- and 23-fold higher GGT activity than recombinant E. coli and the native B. amyloliquefaciens, respectively, did. These results suggest that BaGGT469 can be utilized for the enzymatic production of various γ-glutamyl compounds.

  16. Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39.

    Science.gov (United States)

    Zhong, Xiaotian; Buddha, Madhavan; Guidotti, Guido; Kriz, Ron; Somers, Will; Mosyak, Lidia

    2008-11-01

    CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction-quality crystals of sCD39 were produced by the vapor-diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P3(2), with unit-cell parameters a = b = 118.1, c = 81.6 A and with two sCD39 copies in the asymmetric unit. Several low- to medium-resolution diffraction data sets were collected using an in-house X-ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular-replacement search was performed against the complete 3.2 A data set using a maximum-likelihood molecular-replacement method as implemented in Phaser. The initial model of the two sCD39 monomers was placed into the P3(2) lattice and rigid-body refined and position-minimized with PHENIX.

  17. Regulation of T Cell Trafficking by Enzymatic Synthesis of O-Glycans

    Directory of Open Access Journals (Sweden)

    Samuel J. Hobbs

    2017-05-01

    Full Text Available Selectins constitute a family of oligosaccharide binding proteins that play critical roles in regulating the trafficking of leukocytes. In T cells, L-selectin (CD62L controls the capacity for naive and memory T cells to actively survey peripheral lymph nodes, whereas P- and E-selectin capture activated T cells on inflamed vascular endothelium to initiate extravasation into non-lymphoid tissues. The capacity for T cells to interact with all of these selectins is dependent on the enzymatic synthesis of complex O-glycans, and thus, this protein modification plays an indispensable role in regulating the distribution and homing of both naive and previously activated T cells in vivo. In contrast to neutrophils, O-glycan synthesis is highly dynamic in T cell populations and is largely controlled by extracellular stimuli such as antigen recognition or signaling though cytokine receptors. Herein, we review the basic principles of enzymatic synthesis of complex O-glycans, discuss tools and reagents for studying this type of protein modification and highlight our current understanding of how O-glycan synthesis is regulated and subsequently impacts the trafficking potential of diverse T cell populations.

  18. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    Science.gov (United States)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  19. Improved expression of His(6)-tagged strictosidine synthase cDNA for chemo-enzymatic alkaloid diversification.

    Science.gov (United States)

    Yang, Liuqing; Zou, Hongbin; Zhu, Huajian; Ruppert, Martin; Gong, Jingxu; Stöckigt, Joachim

    2010-04-01

    Strictosidine synthase (STR1) catalyzes the stereoselective formation of 3alpha(S)-strictosidine from tryptamine and secologanin. Strictosidine is the key intermediate in the biosynthesis of 2,000 plant monoterpenoid indole alkaloids, and it is a key precursor of enzyme-mediated synthesis of alkaloids. An improved expression system is described which leads to optimized His(6)-STR1 synthesis in Escherichia coli. Optimal production of STR1 was achieved by determining the impact of co-expression of chaperones pG-Tf2 and pG-LJE8. The amount and activity of STR1 was doubled in the presence of chaperone pG-Tf2 alone. His(6)-STR1 immobilized on Ni-NTA can be used for enzymatic synthesis of strictosidines on a preparative scale. With the newly co-expressed His(6)-STR1, novel 3alpha(S)-12-azastrictosidine was obtained by enzymatic catalysis of 7-azatryptamine and secologanin. The results obtained are of significant importance for application to chemo-enzymatic approaches leading to diversification of alkaloids with novel improved structures.

  20. Enzymatic biofuel cell based on anode and cathode powered by ethanol.

    Science.gov (United States)

    Ramanavicius, Arunas; Kausaite, Asta; Ramanaviciene, Almira

    2008-12-01

    Enzymatic biofuel cell based on enzyme modified anode and cathode electrodes are both powered by ethanol and operate at ambient temperature is described. The anode of the presented biofuel cell was based on immobilized quino-hemoprotein-alcohol dehydrogenase (QH-ADH), while the cathode on co-immobilized alcohol oxidase (AOx) and microperoxidase (MP-8). Two enzymes AOx and MP-8 acted in the consecutive mode and were applied in the design of the biofuel cell cathode. The ability of QH-ADH to transfer electrons directly towards the carbon-based electrode and the ability of MP-8 to accept electrons directly from the same type of electrodes was exploited in this biofuel cell design. Direct electron transfer (DET) to/from enzymes was the basis for generating an electric potential between the anode and cathode. Application of immobilized enzymes and the harvesting of the same type of fuel at both electrodes (cathode and anode) avoided the compartmentization of enzymatic biofuel cell. The maximal open circuit potential of the biofuel cell was 240mV.

  1. Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3

    DEFF Research Database (Denmark)

    Marzec, Michal; Kasprzycka, Monika; Ptasznik, Andrzej;

    2005-01-01

    Aberrant expression of the ALK tyrosine kinase as a chimeric protein with nucleophosmin (NPM) and other partners plays a key role in malignant cell transformation of T-lymphocytes and other cells. Here we report that two small-molecule, structurally related, quinazoline-type compounds, WHI-131...... and WHI-154, directly inhibit enzymatic activity of NPM/ALK as demonstrated by in vitro kinase assays using a synthetic tyrosine-rich oligopeptide and the kinase itself as the substrates. The inhibition of NPM/ALK activity resulted in malignant T cells in suppression of their growth, induction...... of apoptosis and inhibition of tyrosine phosphorylation of STAT3, the key effector of the NPM/ALK-induced oncogenesis. We also show that the STAT3 tyrosine phosphorylation is mediated in the malignant T cells by NPM/ALK independently of Jak3 kinase as evidenced by the presence of STAT3 phosphorylation...

  2. Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function.

    Directory of Open Access Journals (Sweden)

    Anna Zaidman-Rémy

    Full Text Available Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs and, in some cases, to efficiently degrade it (catalytic PGRPs. In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

  3. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

    Science.gov (United States)

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-11-30

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  4. PRODUCTION OF EMBBRYONIC STEM CELLS FROM INNER CELL MASS OF BLASTOCYST ISOLATED BY ENZYMATIC AND IMMUNOSURGERY METHODS

    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine

    2008-03-01

    Full Text Available The objective of the research is determining the ICM isolation method to produce ESC. Blastocyst stage of DDy mice embryos were used in this study. Zona pellucida of blastocysts were removed by 0.25% pronase, the ICM isolation were done by enzimatic or immunosurgery method, and then they were cultured in DMEM-high glucose supplemented with mercaptoethanol, gentamycin, fetal bovine serum, and cumulus cells as feeder layer. The result of the research indicated that immunosurgery method yielding attachment rate and number ESC colony 93.85% and 43.08%, respectively, higher (P<0.05 than enzimatic method that weree 79.63% and 18.52%, respectively, but the viability of ICM cells were equal (P >0.05 that are 93.59% in enzymatic method and 98.56% in immunosurgery method. This research concluded that immunosurgery more effective method for isolation of ICM and ESC production than enzymatic method.

  5. Enzymatic production of fructose 1,6-diphosphate using crude cell extract of Bacillus stearothermophilus.

    Science.gov (United States)

    Widjaja, A; Yasuda, M; Ogino, H; Nakajima, H; Ishikawa, H

    1999-01-01

    The enzymatic production of fructose 1,6-diphosphate (FDP) from glucose was performed in a batch reactor and a semibatch reactor using the crude cell extract of Bacillus stearothermophilus which contains all four enzymes required for the synthesis. The experimental results of the yield and the time courses of FDP production obtained using various enzyme concentrations were in good agreement with the theoretical predictions calculated based on the differential equations including the rate equations of the four enzymes, which were determined using the purified enzymes of B. stearothermophilus.

  6. FT-IR spectroscopic analysis for studying Clostridium cell response to conversion of enzymatically hydrolyzed hay

    Science.gov (United States)

    Grube, Mara; Gavare, Marita; Nescerecka, Alina; Tihomirova, Kristina; Mezule, Linda; Juhna, Talis

    2013-07-01

    Grass hay is one of assailable cellulose containing non-food agricultural wastes that can be used as a carbohydrate source by microorganisms producing biofuels. In this study three Clostridium strains Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium tetanomorphum, capable of producing acetone, butanol and ethanol (ABE) were adapted to convert enzymatically hydrolyzed hay used as a growth media additive. The results of growth curves, substrate degradation kinetics and FT-IR analyses of bacterial biomass macromolecular composition showed diverse strain-specific cell response to the growth medium composition.

  7. Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

    Directory of Open Access Journals (Sweden)

    Farnaz Nikzad Jamnani

    2013-11-01

    Full Text Available Introduction: Fossil fuel is an expensive and finite energy source. Therefore, the use of renewable energy and biofuels production has been taken into consideration. One of the most suitable raw materials for biofuels is cellulosic compounds. Only microorganisms that contain cellulose enzymes can decompose cellulose and fungus of Trichodermareesei is the most important producer of this enzyme. Methods: In this study the nucleotide sequence of endoglucanase II, which is the starter of attack to cellulose chains, synthesized from amino acid sequence of this enzyme in fungus T.reesei and based on codon usage in the host; yeast Pichiapastoris. To produce optimized enzyme and to decrease the production time and enzyme price, protein engineering will be used. There are some methods to improve the enzymatic properties like site-directed mutagenesis in which amino-acid replacement occur. In this study two mutations were induced in endoglucanase enzyme gene by PCR in which free syctein positions 169 and 393 were switched to valine and histidine respectively. Then this gene was inserted into the pPinka expression vector and cloned in Escherichia coli. The recombinant plasmids were transferred into P.pastoris competent cells with electroporation, recombinant yeasts were cultured in BMMY medium and induced with methanol. Results: The sequencing of gene proved the induction of the two mutations and the presence of recombinant enzyme was confirmed by dinitrosalicilic acid method and SDS-PAGE. Conclusion: Examination of biochemical properties revealed that the two mutations simultaneously decreased catalytic power, thermal stability and increased the affinity of enzyme and substrate.

  8. Enzymatic Synthesis of Agmatine by Immobilized Escherichia coli Cells with Arginine Decarboxylase Activity

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei-guo; ZHAO Gen-hai; LIU Jun-zhong; LIU Qian; JIAO Qing-cai

    2011-01-01

    A new method for the enzymatic synthesis of agmatine by immobilized Escherichia coli cells with arginine decarboxylase(ADC)activity was established and a series of optimal reaction conditions was set down.The arginine decarboxylase showed the maximum activity when the pyridoxal phosphate(PLP)concentration was 50 mmol/L,pH=7 and 45 ℃.The arginine decarboxylase exhibited the maximum production efficiency when the substrate concentration was 100 mmol/L and the reaction time was 15 h.It was also observed that the appropriate concentration of Mg2+,especially at 0.5 mmol/L promoted the arginine decarboxylase activity; Mn2+ had little effect on the arginine decarboxylase activity.The inhibition of Cu2+ and Zn2+ to the arginine decarboxylase activity was significant.The immobilized cells were continuously used 6 times and the average conversion rate during the six-time usage was 55.6%.The immobilized cells exhibited favourable operational stability.After optimization,the maximally cumulative amount of agmatine could be up to 20 g/L.In addition,this method can also catalyze D,L-arginine to agmatine,leaving the pure optically D-arginine simultaneously.The method has a very important guiding significance to the enzymatic preparation of agmatine.

  9. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    Energy Technology Data Exchange (ETDEWEB)

    Marcondes, Marcelo F. [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Torquato, Ricardo J.S. [Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Assis, Diego M.; Juliano, Maria A. [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Hayashi, Mirian A.F. [Department of Pharmacology, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Oliveira, Vitor, E-mail: vitor@biofis.epm.br [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil)

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  10. Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris

    DEFF Research Database (Denmark)

    Borodina, Irina; Jensen, Bettina M.; Wagner, Tim;

    and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form...... on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H......229A mutant did not have any detectable phospholipase A1 activity and was secreted up to the level of 4 mg/L in shake flask culture. It was purified by nickel‐affinity chromatography and its identity was confirmed by MALDI‐TOF mass spectrometry. The protein could bind IgE antibodies from wasp venom...

  11. Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia pastoris

    DEFF Research Database (Denmark)

    Borodina, Irina; Jensen, Bettina M; Wagner, Tim;

    2011-01-01

    and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form...... on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H......229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from...

  12. Expression of Enzymatically Inactive Wasp Venom Phospholipase A1 in Pichia pastoris

    DEFF Research Database (Denmark)

    Borodina, Irina; Jensen, Bettina M.; Wagner, Tim;

    2011-01-01

    and is a subject to composition variation, therefore it can be advantageous to substitute it with a cocktail of recombinant allergens. One of the major venom allergens is phospholipase A1, which so far has been expressed in Escherichia coli and in insect cells. Our aim was to produce the protein in secreted form...... on growth of the yeast cells. To overcome the problem we introduced three different point mutations at the critical points of the active site, where serine137, aspartate165 or histidine229 were replaced by alanine (S137A, D165A and H229A). All the three mutated forms could be expressed in P. pastoris. The H......229A mutant did not have any detectable phospholipase A1 activity and was secreted at the level of several mg/L in shake flask culture. The protein was purified by nickel-affinity chromatography and its identity was confirmed by MALDI-TOF mass spectrometry. The protein could bind IgE antibodies from...

  13. Membraneless glucose/oxygen enzymatic fuel cells using redox hydrogel films containing carbon nanotubes.

    Science.gov (United States)

    MacAodha, Domhnall; Ó Conghaile, Peter; Egan, Brenda; Kavanagh, Paul; Leech, Dónal

    2013-07-22

    Co-immobilisation of three separate multiple blue copper oxygenases, a Myceliophthora thermophila laccase, a Streptomyces coelicolor laccase and a Myrothecium verrucaria bilirubin oxidase, with an [Os(2,2'-bipyridine)2 (polyvinylimidazole)10Cl](+/2+) redox polymer in the presence of multi-walled carbon nanotubes (MWCNTs) on graphite electrodes results in enzyme electrodes that produce current densities above 0.5 mA cm(-2) for oxygen reduction at an applied potential of 0 V versus Ag/AgCl. Fully enzymatic membraneless fuel cells are assembled with the oxygen-reducing enzyme electrodes connected to glucose-oxidising anodes based on co-immobilisation of glucose oxidase or a flavin adenine dinucleotide-dependent glucose dehydrogenase with an [Os(4,4'-dimethyl-2,2'-bipyridine)2(polyvinylimidazole)10Cl](+/2+) redox polymer in the presence of MWCNTs on graphite electrodes. These fuel cells can produce power densities of up to 145 μW cm(-2) on operation in pH 7.4 phosphate buffer solution at 37 °C containing 150 mM NaCl, 5 mM glucose and 0.12 mM O2. The fuel cells based on Myceliophthora thermophila laccase enzyme electrodes produce the highest power density if combined with glucose oxidase-based anodes. Although the maximum power density of a fuel cell of glucose dehydrogenase and Myceliophthora thermophila laccase enzyme electrodes decreases from 110 μW cm(-2) in buffer to 60 μW cm(-2) on testing in artificial plasma, it provides the highest power output reported to date for a fully enzymatic glucose-oxidising, oxygen-reducing fuel cell in artificial plasma.

  14. Epigallocatechin gallate incorporation into lignin enhances the alkaline delignification and enzymatic saccharification of cell walls

    Directory of Open Access Journals (Sweden)

    Elumalai Sasikumar

    2012-08-01

    Full Text Available Abstract Background Lignin is an integral component of the plant cell wall matrix but impedes the conversion of biomass into biofuels. The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers such as flavonoids into cell wall lignins that are consequently less recalcitrant to biomass processing. In the present study, epigallocatechin gallate (EGCG was evaluated as a potential lignin bioengineering target for rendering biomass more amenable to processing for biofuel production. Results In vitro peroxidase-catalyzed polymerization experiments revealed that both gallate and pyrogallyl (B-ring moieties in EGCG underwent radical cross-coupling with monolignols mainly by β–O–4-type cross-coupling, producing benzodioxane units following rearomatization reactions. Biomimetic lignification of maize cell walls with a 3:1 molar ratio of monolignols and EGCG permitted extensive alkaline delignification of cell walls (72 to 92% that far exceeded that for lignified controls (44 to 62%. Alkali-insoluble residues from EGCG-lignified walls yielded up to 34% more glucose and total sugars following enzymatic saccharification than lignified controls. Conclusions It was found that EGCG readily copolymerized with monolignols to become integrally cross-coupled into cell wall lignins, where it greatly enhanced alkaline delignification and subsequent enzymatic saccharification. Improved delignification may be attributed to internal trapping of quinone-methide intermediates to prevent benzyl ether cross-linking of lignin to structural polysaccharides during lignification, and to the cleavage of ester intra-unit linkages within EGCG during pretreatment. Overall, our results suggest that apoplastic deposition of EGCG for incorporation into lignin would be a promising plant genetic engineering target for improving the delignification and saccharification of biomass crops.

  15. CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

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    Prakash K Gupta

    2015-10-01

    Full Text Available Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

  16. Ultrasound-induced stress responses of Panax ginseng cells: enzymatic browning and phenolics production.

    Science.gov (United States)

    Wu, Jianyong; Lin, Lidong

    2002-01-01

    The stress metabolic activities of Panax ginseng (P. ginseng) cells induced by low-energy ultrasound (US) were examined. P. ginseng cells in suspension cultures were exposed to 38.5 kHz US at two power levels (power density 13.7 and 61 mW/cm(3)) for 2 min. The US treatment caused rapid increase in the intracellular levels of polyphenol oxidase (PPO), peroxidase (PO), and phenylalanine ammonia lyase (PAL) and the production of polyphenols (PP) and phenolic compounds. The US-induced enzyme activities and phenolics production are part of plant stress responses to a mechanical stimulus. The much higher PPO activity and rate of PP production in the sonicated cultures are correlated to enzymatic browning, suggestive of physical damage and membrane permeabilization of the cells by US. The cells after sonication also showed decreased water content and cell volume, which may also be attributed to US-induced cell membrane permeabilization and water release. High-pressure shock and fluid shear stress arising from acoustic cavitation were regarded as the major causes of the responses. Nevertheless, the US exposure caused only temporary cell growth depression but no net loss of biomass yield of the culture.

  17. Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts.

    Science.gov (United States)

    Nagel, Marie-Danielle; Verhoef, René; Schols, Henk; Morra, Marco; Knox, J Paul; Ceccone, Giacomo; Della Volpe, Claudio; Vigneron, Pascale; Bussy, Cyrill; Gallet, Marlène; Velzenberger, Elodie; Vayssade, Muriel; Cascardo, Giovanna; Cassinelli, Clara; Haeger, Ash; Gilliland, Douglas; Liakos, Ioannis; Rodriguez-Valverde, Miguel; Siboni, Stefano

    2008-01-01

    Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and MHR-alpha), carrot (MHR-C) and potato (MHR-P) cells. Polystyrene (PS) Petri dishes, aminated by a plasma deposition process, were surface modified by the covalent linking of the MHRs. Results clearly demonstrate that MHR-B induces cell adhesion, proliferation and survival, in contrast to the other MHRs. Moreover, MHR-alpha causes cells to aggregate, decrease proliferation and enter into apoptosis. Cells cultured in standard conditions with 1% soluble MHR-B or MHR-alpha show the opposite behaviour to the one observed on MHR-B and -alpha-grafted PS. Fibronectin was similarly adsorbed onto MHR-B and tissue culture polystyrene (TCPS) control, but poorly on MHR-alpha. The Fn cell binding site (RGD sequence) was more accessible on MHR-B than on TCPS control, but poorly on MHR-alpha. The disintegrin echistatin inhibited fibroblast adhesion and spreading on MHR-B-grafted PS, which suggests that MHRs control fibroblast behaviour via serum-adhesive proteins. This study provides a basis for the design of intelligently-tailored biomaterial coatings able to induce specific cell functions.

  18. Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

    Institute of Scientific and Technical Information of China (English)

    Zeng Yinxin; Yu Yong; Chen Bo; Li Huirong

    2004-01-01

    The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.

  19. Utilization of hydrolysate from lignocellulosic biomass pretreatment to generate electricity by enzymatic fuel cell system.

    Science.gov (United States)

    Kim, Sung Bong; Kim, Dong Sup; Yang, Ji Hyun; Lee, Junyoung; Kim, Seung Wook

    2016-04-01

    The waste hydrolysate after dilute acid pretreatment (DAP) of lignocellulosic biomass was utilized to generate electricity using an enzymatic fuel cell (EFC) system. During DAP, the components of biomass containing hemicellulose and other compounds are hydrolyzed, and glucose is solubilized into the dilute acid solution, called as the hydrolysate liquid. Glucose oxidase (GOD) and laccase (Lac) were assembled on the electrode of the anode and cathode, respectively. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were measured, and the maximum power density was found to be 1.254×10(3) μW/cm(2). The results indicate that the hydrolysate from DAP is a reliable electrolyte containing the fuel of EFC. Moreover, the impurities in the hydrolysate such as phenols and furans slightly affected the charge transfer on the surface of the electrode, but did not affect the power generation of the EFC system in principal.

  20. Optimisation of the enzymatic hydrolysis of blood cells with a neutral protease.

    Science.gov (United States)

    Zheng, Yanbin; Chen, Qiushi; Shan, Anshan; Zhang, Hao

    2013-01-01

    For utilizing the blood cells (BCs) effectively, enzymatic hydrolysis was applied to produce the enzymatically hydrolyzed blood cells (EHBCs) by using a neutral protease as a catalyst. The results of the single-factor experiments showed optimal substrate concentration, enzyme to substrate ratio (E/S), pH, temperature, and incubation period were 1.00%, 0.10, 7.00, 50.00°C, and 12.00 h, respectively. The optimized hydrolysis conditions from response surface methodology (RSM) were pH 6.50, E/S 0.11, temperature 45.00°C, and incubation period 12.00 h. Under these conditions (substrate concentration 1.00%), the degree of hydrolysis (DH) was 35.06%. The free amino acids (FAAs) content of the EHBCs (35.24%) was 40.46 times higher than BCs while the total amino acids (TAAs) content was lower than BCs. The scores of lysine (human 0.87; pig 0.97), valine (human 1.42; pig 1.38), leucine (human 1.50; pig 1.90), tyrosine (human 0.84; pig 1.09), and histidine (human 2.17; pig 2.50) indicated that the EHBCs basically fulfilled the adult human and pig nutritional requirements. The calculated protein efficiency ratios (C-PERs) of the EHBCs were 3.94, 6.19, 21.73, and 2.04. In summary, the EHBCs were produced successfully with optimized conditions and could be a novel protein source for humans and pigs.

  1. Optimisation of the Enzymatic Hydrolysis of Blood Cells with a Neutral Protease

    Directory of Open Access Journals (Sweden)

    Yanbin Zheng

    2013-01-01

    Full Text Available For utilizing the blood cells (BCs effectively, enzymatic hydrolysis was applied to produce the enzymatically hydrolyzed blood cells (EHBCs by using a neutral protease as a catalyst. The results of the single-factor experiments showed optimal substrate concentration, enzyme to substrate ratio (E/S, pH, temperature, and incubation period were 1.00%, 0.10, 7.00, 50.00°C, and 12.00 h, respectively. The optimized hydrolysis conditions from response surface methodology (RSM were pH 6.50, E/S 0.11, temperature 45.00°C, and incubation period 12.00 h. Under these conditions (substrate concentration 1.00%, the degree of hydrolysis (DH was 35.06%. The free amino acids (FAAs content of the EHBCs (35.24% was 40.46 times higher than BCs while the total amino acids (TAAs content was lower than BCs. The scores of lysine (human 0.87; pig 0.97, valine (human 1.42; pig 1.38, leucine (human 1.50; pig 1.90, tyrosine (human 0.84; pig 1.09, and histidine (human 2.17; pig 2.50 indicated that the EHBCs basically fulfilled the adult human and pig nutritional requirements. The calculated protein efficiency ratios (C-PERs of the EHBCs were 3.94, 6.19, 21.73, and 2.04. In summary, the EHBCs were produced successfully with optimized conditions and could be a novel protein source for humans and pigs.

  2. The influence of Fe2+ on growth and development of cells enzymatically isolated from Porphyra yezoensis blades

    Institute of Scientific and Technical Information of China (English)

    SHEN Songdong; DAI Jixun

    2005-01-01

    Fe2+ acted as an accessorial factor for many cellular enzymatic reactions is very important for seaweed growth and development, but the Fe2+ requirement in nori had not been seen. Porphyra yezoensis cells were separated enzymatically and cultured in a series of sterilized seawater media containing various concentrations of Fe2+. The growth development and cell were investigated in this work. Through this experiment, two biologically-meant concentration scales were found, one is low concentrations, 12.1-102.1 μg/L, 10-100 times than that in seawater, favoring the development of isolated cells of Porphyra and the other was high concentrations, more than 10mg/L inhibiting the cell growth, leading to the deformity and shrinkage of the cells. At the concentration of 50 mg/L, the cells stopped growing and died eventually.

  3. Gelatinase expression and proteolytic activity in giant‐cell arteritis

    Science.gov (United States)

    Segarra, Marta; García‐Martínez, Ana; Sánchez, Montse; Hernández‐Rodríguez, José; Lozano, Ester; Grau, Josep M; Cid, Maria C

    2007-01-01

    Objectives Gelatinases (MMP2 and MMP9) are expressed in giant‐cell arteritis (GCA) and are thought to play a role in vessel disruption. However, their activation status and enzymatic activity have not been evaluated. Our aim was to investigate the distribution and proteolytic activity of gelatinases in GCA lesions at different stages. Methods Expression of MMP2, MMP9, MMP2‐activator MMP14 and their natural inhibitors TIMP1 and TIMP2 was determined by real‐time PCR and immunohistochemistry in temporal artery sections from 46 patients and 12 controls. MMP activation status and enzymatic activity were assessed by gelatin and film in situ zymography. Results Vascular smooth muscle cells from normal specimens constitutively expressed pro‐MMP2 and its inhibitor TIMP2 with no resulting proteolytic activity. In GCA MMP2, MMP9 and MMP14 were strongly expressed in their active form by infiltrating leucocytes. Inflamed arteries also expressed TIMP1 and TIMP2. However, the MMP9/TIMP1 and MMP2/TIMP2 ratios were higher in patients compared with controls, indicating an increased proteolytic balance in GCA which was confirmed by in situ zymography. Maximal gelatinase expression and activity occurred at the granulomatous areas surrounding the internal elastic lamina (IEL). Myointimal cells also expressed MMPs and exhibited proteolytic activity, suggesting a role for gelatinases in vascular remodelling and repair. Conclusions GCA lesions show intense expression of gelatinases. Activators and inhibitors are regulated to yield enhanced gelatinase activation and proteolytic activity. Distribution of expression and proteolytic activity suggests that gelatinases have a major role not only in the progression of inflammatory infiltrates and vessel destruction but also in vessel repair. PMID:17502363

  4. Cloning and expression of human colon mast cell carboxypeptidase

    Institute of Scientific and Technical Information of China (English)

    Zhang-Quan Chen; Shao-Heng He

    2004-01-01

    AIM: To clone and express the human colon mast cell METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR). The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to conrtruct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The recombinant fusion protein expressed in E.coli was induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoris (P.pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid RESULTS: The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E.coli was purified with amylose affinity chromatography and heparin agarose chromatogphy.SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E. coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine.carboxypeptidase can be successfully cloned and expressed in E.coli and P. pastoris, which will contribute greatly to the fonctional study on hMC-CP.

  5. Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin.

    Science.gov (United States)

    Otsuka, Yuichiro; Matsuda, Motoki; Sonoki, Tomonori; Sato-Izawa, Kanna; Goodell, Barry; Jelison, Jody; Navarro, Ronald R; Murata, Hitoshi; Nakamura, Masaya

    2016-12-01

    This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H2O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

  6. Overcoming bottlenecks of enzymatic biofuel cell cathodes: crude fungal culture supernatant can help to extend lifetime and reduce cost.

    Science.gov (United States)

    Sané, Sabine; Jolivalt, Claude; Mittler, Gerhard; Nielsen, Peter J; Rubenwolf, Stefanie; Zengerle, Roland; Kerzenmacher, Sven

    2013-07-01

    Enzymatic biofuel cells (BFCs) show great potential for the direct conversion of biochemically stored energy from renewable biomass resources into electricity. However, enzyme purification is time-consuming and expensive. Furthermore, the long-term use of enzymatic BFCs is hindered by enzyme degradation, which limits their lifetime to only a few weeks. We show, for the first time, that crude culture supernatant from enzyme-secreting microorganisms (Trametes versicolor) can be used without further treatment to supply the enzyme laccase to the cathode of a mediatorless BFC. Polarization curves show that there is no significant difference in the cathode performance when using crude supernatant that contains laccase compared to purified laccase in culture medium or buffer solution. Furthermore, we demonstrate that the oxygen reduction activity of this enzymatic cathode can be sustained over a period of at least 120 days by periodic resupply of crude culture supernatant. This is more than five times longer than control cathodes without the resupply of culture supernatant. During the operation period of 120 days, no progressive loss of potential is observed, which suggests that significantly longer lifetimes than shown in this work may be possible. Our results demonstrate the possibility to establish simple, cost efficient, and mediatorless enzymatic BFC cathodes that do not require expensive enzyme purification procedures. Furthermore, they show the feasibility of an enzymatic BFC with an extended lifetime, in which self-replicating microorganisms provide the electrode with catalytically active enzymes in a continuous or periodic manner.

  7. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

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    Sumarheni

    2016-01-01

    Full Text Available Penton-dodecahedron (Pt-Dd derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents.

  8. Oxidant and enzymatic antioxidant status (gene expression and activity) in the brain of chickens with cold-induced pulmonary hypertension

    Science.gov (United States)

    Hassanpour, Hossein; Khalaji-Pirbalouty, Valiallah; Nasiri, Leila; Mohebbi, Abdonnaser; Bahadoran, Shahab

    2015-11-01

    To evaluate oxidant and antioxidant status of the brain (hindbrain, midbrain, and forebrain) in chickens with cold-induced pulmonary hypertension, the measurements of lipid peroxidation, protein oxidation, antioxidant capacity, enzymatic activity, and gene expression (for catalase, glutathione peroxidase, and superoxide dismutases) were done. There were high lipid peroxidation/protein oxidation and low antioxidant capacity in the hindbrain of cold-induced pulmonary hypertensive chickens compared to control ( P hypertensive chickens, superoxide dismutase activity was decreased (forebrain, midbrain, and hindbrain), while catalase activity was increased (forebrain and midbrain) ( P hypertensive chickens. Probably, these situations in the oxidant and antioxidant status of the brain especially hindbrain may change its function at cardiovascular center and sympathetic nervous system to exacerbate pulmonary hypertension.

  9. Novel β-N-acetylglucosaminidases from Vibrio harveyi 650: Cloning, expression, enzymatic properties, and subsite identification

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    Mizuhara Mamiko

    2010-09-01

    Full Text Available Abstract Background Since chitin is a highly abundant natural biopolymer, many attempts have been made to convert this insoluble polysaccharide into commercially valuable products using chitinases and β-N-acetylglucosaminidases (GlcNAcases. We have previously reported the structure and function of chitinase A from Vibrio harveyi 650. This study t reports the identification of two GlcNAcases from the same organism and their detailed functional characterization. Results The genes encoding two new members of family-20 GlcNAcases were isolated from the genome of V. harveyi 650, cloned and expressed at a high level in E. coli. VhNag1 has a molecular mass of 89 kDa and an optimum pH of 7.5, whereas VhNag2 has a molecular mass of 73 kDa and an optimum pH of 7.0. The recombinant GlcNAcases were found to hydrolyze all the natural substrates, VhNag2 being ten-fold more active than VhNag1. Product analysis by TLC and quantitative HPLC suggested that VhNag2 degraded chitooligosaccharides in a sequential manner, its highest activity being with chitotetraose. Kinetic modeling of the enzymic reaction revealed that binding at subsites (-2 and (+4 had unfavorable (positive binding free energy changes and that the binding pocket of VhNag2 contains four GlcNAc binding subsites, designated (-1,(+1,(+2, and (+3. Conclusions Two novel GlcNAcases were identified as exolytic enzymes that degraded chitin oligosaccharides, releasing GlcNAc as the end product. In living cells, these intracellular enzymes may work after endolytic chitinases to complete chitin degradation. The availability of the two GlcNAcases, together with the previously-reported chitinase A from the same organism, suggests that a systematic development of the chitin-degrading enzymes may provide a valuable tool in commercial chitin bioconversion.

  10. Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP-5M.

    Science.gov (United States)

    Nallapan Maniyam, Maegala; Sjahrir, Fridelina; Latif Ibrahim, Abdul; Cass, Anthony E G

    2015-01-01

    The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

  11. Enzymatic quantification of sphingosine in the picomole range in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Van Veldhoven, P.P.; Bishop, W.R.; Bell, R.M. (Duke Univ. Medical Center, Durham, NC (USA))

    1989-11-15

    An enzymatic method to quantify the mass levels of free sphingosine in cellular lipid extracts was developed. The assay is based upon the observation that ceramide is phosphorylated by Escherichia coli diacylglycerol kinase. Although sphingosine is not recognized by the enzyme, it can be converted to a substrate by acylation with hexanoic anhydride. Using a mixed micellar assay, previously reported for the mass quantification of diacylglycerol, the short-chain ceramide (N-C6-sphingosine), generated by acylation, is quantitatively phosphorylated to N-C6-(32P)sphingosine phosphate. This assay allows quantification of sphingosine over a broad range from 25 to 5000 pmol. When this assay was applied to standard compounds, reverse-phase thin-layer chromatography of the reaction products was adequate to separate the phosphorylated derivatives of long-chain ceramide and N-C6-sphingosine. However, the presence of other lipids in extracts from biological samples (mainly monoalkylglycerols which are also a substrate for the diacylglycerol kinase) interfered and necessitated an additional purification step. The most efficient purification step devised was a combination of anion- and cation-exchange chromatography. The mass levels of free sphingoid bases in different cultured cells were quantified using this assay. Levels varied between 8 to 20 pmol/10(6) cells. When normalized to phospholipids, sphingosine levels varied between 0.01 and 0.04 mol%. The lowest levels were found in L929 cells, while Schwann cells derived from Twitcher mice contained the highest levels. These levels were significantly higher than those of Schwann cells derived from normal mice.

  12. Design of a mediated enzymatic fuel cell to generate power from renewable fuel sources.

    Science.gov (United States)

    Korkut, Seyda; Kilic, Muhammet Samet

    2016-01-01

    The present work reported a compartment-less enzymatic fuel cell (EFC) based on newly synthesized Poly(pyrrole-2-carboxylic acid-co-3-thiophene acetic acid) film containing glucose oxidase and laccase effectively wired by p-benzoquinone incorporated into the copolymer structure. The resulting system generated a power density of 18.8 µW/cm(2) with 30 mM of glucose addition at +0.94 V at room temperature. Improvements to maximize the power output were ensured with step-by-step optimization of electrode fabrication design and operational parameters for operating the system with renewable fuel sources. We demonstrated that the improved fuel cell could easily harvest glucose produced during photosynthesis to produce electrical energy in a simple, renewable and sustainable way by generating a power density of 10 nW/cm(2) in the plant leaf within 2 min. An EFC for the first time was successfully operated in municipal wastewater which contained glycolytic substances to generate electrical energy with a power output of 3.3 µW/cm(2).

  13. Modulating in vitro bone cell and macrophage behavior by immobilized enzymatically tailored pectins.

    Science.gov (United States)

    Bussy, Cyrill; Verhoef, René; Haeger, Ash; Morra, Marco; Duval, Jean-Luc; Vigneron, Pascale; Bensoussan, Anne; Velzenberger, Elodie; Cascardo, Giovanna; Cassinelli, Clara; Schols, Henk; Knox, J Paul; Nagel, Marie-Danielle

    2008-09-01

    Previous work has reported the results of a multidisciplinary effort producing a proof-of-concept on the use of pectic polysaccharides in the surface modification of medical devices. This study was designed to learn more about the capability of engineered rhamnogalacturonan-I (RG-I) fractions of apple pectin to control bone cell and macrophage behavior. Thermanox or polystyrene Petri dishes were surface modified with two different modified hairy regions (MHRs) obtained by different enzymatic liquefaction processes of apples differing in relative amounts and lengths of their neutral side chains: (long-haired) MHR-alpha and (short-haired) MHR-B. Bone explants from 14-day-old chick embryos were cultured for 14 days on both pectic substrata. MHR-B promoted cell migration and differentiation, MHR-alpha did not. On MHR-alpha, J774.2 macrophages grew well, their percentage in G1 phase was decreased and in S phase increased, and they did not secrete either proinflammatory-cytokines or nitrites. Contrasting results were gained from macrophages on MHR-B, except for nitrite secretion. Thus, we conclude that coatings from tailored pectins show different biological activities in vitro and are potential innovative candidates for improving the biocompatibility of medical devices in various applications.

  14. Potential Biomedical Application of Enzymatically Treated Alginate/Chitosan Hydrosols in Sponges—Biocompatible Scaffolds Inducing Chondrogenic Differentiation of Human Adipose Derived Multipotent Stromal Cells

    Directory of Open Access Journals (Sweden)

    Anna Zimoch-Korzycka

    2016-08-01

    Full Text Available Current regenerative strategies used for cartilage repair rely on biomaterial functionality as a scaffold for cells that may have potential in chondrogenic differentiation. The purpose of the research was to investigate the biocompatibility of enzymatically treated alginate/chitosan hydrosol sponges and their suitability to support chondrogenic differentiation of human adipose derived multipotent stromal cells (hASCs. The alginate/chitosan and enzyme/alginate/chitosan sponges were formed from hydrosols with various proportions and were used as a biomaterial in this study. Sponges were tested for porosity and wettability. The porosity of each sponge was higher than 80%. An equal dose of alginate and chitosan in the composition of sponges improved their swelling ability. It was found that equal concentrations of alginate and chitosan in hydrosols sponges assure high biocompatibility properties that may be further improved by enzymatic treatment. Importantly, the high biocompatibility of these biomaterials turned out to be crucial in the context of hydrosols’ pro-chondrogenic function. After exposure to the chondrogenic conditions, the hASCs in N/A/C and L/A/C sponges formed well developed nodules and revealed increased expression of collagen type II, aggrecan and decreased expression of collagen type I. Moreover, in these cultures, the reactive oxygen species level was lowered while superoxide dismutase activity increased. Based on the obtained results, we conclude that N/A/C and L/A/C sponges may have prospective application as hASCs carriers for cartilage repair.

  15. High-level expression and characterization of recombinant acid urease for enzymatic degradation of urea in rice wine.

    Science.gov (United States)

    Yang, Yuqing; Kang, Zhen; Zhou, Jianli; Chen, Jian; Du, Guocheng

    2015-01-01

    Ethylcarbamate, a carcinogenic compound, is formed from urea and ethanol in rice wine, and enzymatic elimination of urea is always attractive. In the present work, we amplified the acid urease gene cluster ureABCEFGD from Lactobacillus reuteri CICC6124 and constructed robust Lactococcus lactis cell factories for the production of acid urease. The titer of the recombinant acid urease was increased from 1,550 to 11,560 U/L by optimization of the cultivation process. Meanwhile, the enzyme showed satisfied properties toward urea elimination in the rice wine model system. By incubating the enzyme (50 U/L) at 20 °C for 60 h, about 95.8% of urea in rice wine was removed. Interestingly, this acid urease also exhibited activity toward ethylcarbamate. The results demonstrated that this recombinant acid urease has great potential in the elimination of urea in rice wine.

  16. Changes in plant cell-wall structure of corn stover due to hot compressed water pretreatment and enhanced enzymatic hydrolysis.

    Science.gov (United States)

    Zhou, Wei; Yang, Maohua; Wang, Caixia; Liu, Jianfei; Xing, Jianmin

    2014-08-01

    Corn stover is a potential feedstock for biofuel production. This work investigated physical and chemical changes in plant cell-wall structure of corn stover due to hot compressed water (HCW) pretreatment at 170-190 °C in a tube reactor. Chemical composition analysis showed the soluble hemicellulose content increased with pretreatment temperature, whereas the hemicellulose content decreased from 29 to 7 % in pretreated solids. Scanning electron microscopy revealed the parenchyma-type second cell-wall structure of the plant was almost completely removed at 185 °C, and the sclerenchyma-type second cell wall was greatly damaged upon addition of 5 mmol/L ammonium sulfate during HCW pretreatment. These changes favored accessibility for enzymatic action. Enzyme saccharification of solids by optimized pretreatment with HCW at 185 °C resulted in an enzymatic hydrolysis yield of 87 %, an enhancement of 77 % compared to the yield from untreated corn stover.

  17. An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity.

    Science.gov (United States)

    Gazzaniga, Francesca S; Blackburn, Elizabeth H

    2014-12-11

    Telomerase is a ribonucleoprotein complex that adds telomeric DNA to the ends of linear chromosomes. It contains two core canonical components: the essential RNA component, hTR, which provides the template for DNA synthesis, and the reverse transcriptase protein component, hTERT. Low telomerase activity in circulating peripheral blood mononuclear cells has been associated with a variety of diseases. It is unknown, however, whether telomerase, in addition to its long-term requirement for telomere maintenance, is also necessary for short-term immune cell proliferation and survival. We report that overexpression of enzymatically inactive hTR mutants protected against dexamethasone-induced apoptosis in stimulated CD4 T cells. Furthermore, hTR knockdown reproducibly induced apoptosis in the absence of any detectable telomere shortening or DNA damage response. In contrast, hTERT knockdown did not induce apoptosis. Strikingly, overexpression of hTERT protein caused apoptosis that was rescued by overexpression of enzymatically inactive hTR mutants. Hence, we propose that hTR can function as a noncoding RNA that protects from apoptosis independent of its function in telomerase enzymatic activity and long-term telomere maintenance in normal human immune cells. These results imply that genetic or environmental factors that alter hTR levels can directly affect immune cell function to influence health and disease.

  18. Antioxidant, antimicrobial, cell viability and enzymatic inhibitory of antioxidant polymers as biological macromolecules.

    Science.gov (United States)

    Hashemi Gahruie, Hadi; Niakousari, Mehrdad

    2017-11-01

    Polymeric antioxidants such as Catechinaldehyde Polycondensates, Catechin-acelaldehydepolycondensates, Flavonoid-grafted chitosan fibers, Ferulate hydrogel, Dextran ferulate hydrogel, Starch-quercetin conjugate, Gallic acid- and Caffeic acid-functionalized chitosan, Gallic acid - chitosan conjugate, Poly(rutin), Gallic acid grafted chitosan, Dextran-Catechin Conjugate belong to biological macromolecules. These kinds of compounds have stronger antioxidant potential and pharmacokinetic activities, as compared to similar low molecular weight preservatives. Most of these compounds sources are either antioxidants with low molecules polymerization, or polymers conjugation such as synthetic or natural preservatives. Additives are well known as being an important ingredient of food products due to their strong preservative potential. Many researchers and industries attempt to find synthesize materials with the same antioxidant potential and higher stability than the similar compounds with low molecular weight. Recently, macromolecular antioxidants have received wide attention as food additives and dietary supplements in functional foods. It seems that the main usage of these compounds is in the food packaging industry. Most of these compounds have strong antioxidant, antimicrobial, cell viability and enzymatic inhibitory properties. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Amide group anchored glucose oxidase based anodic catalysts for high performance enzymatic biofuel cell

    Science.gov (United States)

    Chung, Yongjin; Ahn, Yeonjoo; Kim, Do-Heyoung; Kwon, Yongchai

    2017-01-01

    A new enzyme catalyst is formed by fabricating gold nano particle (GNP)-glucose oxidase (GOx) clusters that are then attached to polyethyleneimine (PEI) and carbon nanotube (CNT) with cross-linkable terephthalaldehyde (TPA) (TPA/[CNT/PEI/GOx-GNP]). Especially, amide bonds belonging to TPA play an anchor role for incorporating rigid bonding among GNP, GOx and CNT/PEI, while middle size GNP is well bonded with thiol group of GOx to form strong GNP-GOx cluster. Those bonds are identified by chemical and electrochemical characterizations like XPS and cyclic voltammogram. The anchording effect of amide bonds induces fast electron transfer and strong chemical bonding, resulting in enhancements in (i) catalytic activity, (ii) amount of immobilized GOx and (ii) performance of enzymatic biofuel cell (EBC) including the catalyst. Regarding the catalytic activity, the TPA/[CNT/PEI/GOx-GNP] produces high electron transfer rate constant (6 s-1), high glucose sensitivity (68 μA mM-1 cm-2), high maximum current density (113 μA cm-2), low charge transfer resistance (17.0 Ω cm2) and long-lasting durability while its chemical structure is characterized by XPS confirming large portion of amide bond. In EBC measurement, it has high maximum power density (0.94 mW cm-2) compatible with catalytic acitivity measurements.

  20. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    Science.gov (United States)

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  1. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;

    2009-01-01

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed...

  2. Enzymatically crosslinked gelatin hydrogel promotes the proliferation of adipose tissue-derived stromal cells

    Science.gov (United States)

    Ren, Xiaomei; Long, Haiyan; Qian, Hong; Ma, Kunlong

    2016-01-01

    Gelatin hydrogel crosslinked by microbial transglutaminase (mTG) exhibits excellent performance in cell adhesion, proliferation, and differentiation. We examined the gelation time and gel strength of gelatin/mTG hydrogels in various proportions to investigate their physical properties and tested their degradation performances in vitro. Cell morphology and viability of adipose tissue-derived stromal cells (ADSCs) cultured on the 2D gel surface or in 3D hydrogel encapsulation were evaluated by immunofluorescence staining. Cell proliferation was tested via Alamar Blue assay. To investigate the hydrogel effect on cell differentiation, the cardiac-specific gene expression levelsof Nkx2.5, Myh6, Gja1, and Mef2c in encapsulated ADSCs with or without cardiac induction medium were detected by real-time RT-PCR. Cell release from the encapsulated status and cell migration in a 3D hydrogel model were assessed in vitro. Results show that the gelatin/mTG hydrogels are not cytotoxic and that their mechanical properties are adjustable. Hydrogel degradation is related to gel concentration and the resident cells. Cell growth morphology and proliferative capability in both 2D and 3D cultures were mainly affected by gel concentration. PCR result shows that hydrogel modulus together with induction medium affects the cardiac differentiation of ADSCs. The cell migration experiment and subcutaneous implantation show that the hydrogels are suitable for cell delivery. PMID:27703850

  3. Influence of Different Lignocellulose Sources on Endo-1,4-β-Glucanase Gene Expression and Enzymatic Activity of Bacillus amyloliquefaciens B31C

    Directory of Open Access Journals (Sweden)

    Rosangela Di Pasqua

    2014-01-01

    Full Text Available Conversion of cellulose into fermentable sugars for ethanol production is currently performed by enzymatic hydrolysis catalyzed by cellulases. The cellulases are produced by a wide variety of microorganisms, playing a major role in the recycling of biomass. The endo-1,4-β-glucanase (CelB31C from Bacillus amyloliquefaciens B31C, isolated from compost and previously selected on the basis of highest cellulase activity levels among Bacillus isolated, was characterized as being a potential candidate for a biocatalyst in lignocellulose conversion for second-generation bioethanol production. The aim of this work was to evaluate the changes in production of enzymatic activity of the endo-1,4-β-glucanase (CelB31C and the expression of its gene (bglC using a carboxymethylcellulase activity assay and qRT-PCR analysis, respectively, during growth of B. amyloliquefaciens B31C on different cellulose sources: carboxymethylcellulose (CMC, pure cellulose from Arundo donax, pretreated Arundo donax biomass (Chemtex, and microcrystalline cellulose (Avicel. The results showed that both the expression of bglC gene and the enzymatic activity production are related to the type of cellulose source. The strain showed a high enzymatic activity on lignocellulosic biomass and on microcrystalline cellulose. Furthermore, the highest gene expression occurred during the exponential phase of growth, except in the presence of Avicel.

  4. Enzymatic, expression and structural divergences among carboxyl O-methyltransferases after gene duplication and speciation in Nicotiana.

    Science.gov (United States)

    Hippauf, Frank; Michalsky, Elke; Huang, Ruiqi; Preissner, Robert; Barkman, Todd J; Piechulla, Birgit

    2010-02-01

    Methyl salicylate and methyl benzoate have important roles in a variety of processes including pollinator attraction and plant defence. These compounds are synthesized by salicylic acid, benzoic acid and benzoic acid/salicylic acid carboxyl methyltransferases (SAMT, BAMT and BSMT) which are members of the SABATH gene family. Both SAMT and BSMT were isolated from Nicotiana suaveolens, Nicotiana alata, and Nicotiana sylvestris allowing us to discern levels of enzyme divergence resulting from gene duplication in addition to species divergence. Phylogenetic analyses showed that Nicotiana SAMTs and BSMTs evolved in separate clades and the latter can be differentiated into the BSMT1 and the newly established BSMT2 branch. Although SAMT and BSMT orthologs showed minimal change coincident with species divergences, substantial evolutionary change of enzyme activity and expression patterns occurred following gene duplication. After duplication, the BSMT enzymes evolved higher preference for benzoic acid (BA) than salicylic acid (SA) whereas SAMTs maintained ancestral enzymatic preference for SA over BA. Expression patterns are largely complementary in that BSMT transcripts primarily accumulate in flowers, leaves and stems whereas SAMT is expressed mostly in roots. A novel enzyme, nicotinic acid carboxyl methyltransferase (NAMT), which displays a high degree of activity with nicotinic acid was discovered to have evolved in N. gossei from an ancestral BSMT. Furthermore a SAM-dependent synthesis of methyl anthranilate via BSMT2 is reported and contrasts with alternative biosynthetic routes previously proposed. While BSMT in flowers is clearly involved in methyl benzoate synthesis to attract pollinators, its function in other organs and tissues remains obscure.

  5. Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination.

    Science.gov (United States)

    Tsuji, Akihiko; Tsukamoto, Kana; Iwamoto, Keiko; Ito, Yuka; Yuasa, Keizo

    2013-01-01

    Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination.

  6. Expression of an accessory cell phenotype by hairy cells during lymphocyte colony formation in agar culture.

    Science.gov (United States)

    Farcet, J P; Gourdin, M F; Testa, U; Andre, C; Jouault, H; Reyes, F

    1983-01-01

    Human T lymphocytes require the cooperation of accessory cells to generate lymphocyte colonies in agar culture under PHA stimulation. Various hairy cell enriched fractions, as well as normal monocytes, have been found to be able to initiate colony formation by normal lymphocytes. Leukemic monocytes from CMML patients were also effective, but not the leukemic lymphocytes from CLL patients. The phenotype expressed by HC in agar colonies was further studied using cell surface and enzymatic markers. We have concluded that HC in agar culture in the presence of both normal T lymphocytes and PHA lose the B phenotype that they express in vivo and function like an accessory cell in contrast to normal or leukemic B lymphocytes.

  7. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor

    Directory of Open Access Journals (Sweden)

    Lizeth Núñez-López

    2015-03-01

    Full Text Available Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0, mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200–300% more glucose, adult vegetative plants yielded 40–90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1, juvenile transition (SPL3-5 and meristematic identity (FUL, SOC1 but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production.

  8. Effect of pretreatment and enzymatic hydrolysis of wheat straw on cell wall composition, hydrophobicity and cellulase adsorption.

    Science.gov (United States)

    Heiss-Blanquet, Senta; Zheng, Dan; Lopes Ferreira, Nicolas; Lapierre, Catherine; Baumberger, Stéphanie

    2011-05-01

    The present study aimed to determine the impact of cell wall composition and lignin content on enzyme adsorption and degradability. Thioacidolysis analysis of residual lignins in wheat straw after steam-explosion or organosolv pretreatment revealed an increase in lignin condensation degree of 27% and 33%, respectively. Surface hydrophobicity assessed through wettability tests decreased after the pretreatments (contact angle decrease of 20-50%), but increased with enzymatic conversion (30% maximum contact angle increase) and correlatively to lignin content. Adsorption of the three major cellulases Cel7A, Cel6A and Cel7B from Trichoderma reesei decreased with increasing hydrolysis time, down to 7%, 31% and 70% on the sample with the highest lignin content, respectively. The fraction of unspecifically bound enzymes was dependent both on the enzyme and the lignin content. Adsorption and specific activity were shown to be inversely proportional to lignin content and hydrophobicity, suggesting that lignin is one of the factors restricting enzymatic hydrolysis.

  9. Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

    Science.gov (United States)

    Ormeño, Fernando; Barrientos, Camila; Ramirez, Santiago; Ponce, Iván; Valenzuela, Lucía; Sepúlveda, Sofía; Bitar, Mainá; Kemmerling, Ulrike; Machado, Carlos Renato; Cabrera, Gonzalo; Galanti, Norbel

    2016-01-01

    Trypanosoma cruzi, the etiological agent of Chagas’ disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing. PMID:27284968

  10. Performance of glucose/O2 enzymatic fuel cell based on supporting electrodes over-coated by polymer-nanogold particle composite with entrapped enzymes

    Science.gov (United States)

    Huo, W. S.; Zeng, H.; Yang, Y.; Zhang, Y. H.

    2017-03-01

    Enzymatic electrodes over-coated by thin film of nano-composite made up of polymer and functionalized nano-gold particle was prepared. Glucose/O2 membrane-free enzymatic fuel cell based on nano-composite based electrodes with incorporated glucose oxidase and laccase was assembled. This enzymatic fuel cell exhibited high energy out-put density even when applied in human serum. Catalytic cycle involved in enzymatic fuel cell was limited by oxidation of glucose occurred on bioanode resulting from impact of sophisticated interaction between active site in glucose oxidase and nano-gold particle on configuration of redox center of enzyme molecule which crippled catalytic efficiency of redox protein.

  11. Enzymatic degradation of plant cell wall polysaccharides: the kinetic effect of competitive adsorption

    DEFF Research Database (Denmark)

    Bergsøe, Merete Norsker; Bloch, Line; Adler-Nissen, Jens

    1999-01-01

    Insoluble potato dietary fibre, isolated from potato pulp, can be enzymatically hydrolysed with the pectolytic enzyme preparation Pectinex Ultra SP from Novo Nordisk A/S, in order to produce soluble fibre. The soluble fibre has valuable functional properties for the food industry. Cloned monocomp...

  12. Enzymatically-tailored pectins differentially influence the morphology, adhesion, cell cycle progression and survival of fibroblasts

    NARCIS (Netherlands)

    Nagel, M.D.; Verhoef, R.P.; Schols, H.A.; Morra, M.; Knox, J.P.; Ceccone, G.; Volpe, C.D.; Vigneron, P.; Bussy, C.; Gallet, M.; Velzenberger, E.; Vayssade, M.; Cascardo, G.; Cassinelli, C.; Haeger, A.; Gilliland, D.; Liakos, I.; Rodrigues-Valverde, M.; Siboni, S.

    2008-01-01

    Improved biocompatibility and performance of biomedical devices can be achieved through the incorporation of bioactive molecules on device surfaces. Five structurally distinct pectic polysaccharides (modified hairy regions (MHRs)) were obtained by enzymatic liquefaction of apple (MHR-B, MHR-A and

  13. Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.

    Directory of Open Access Journals (Sweden)

    Xiao-Yang Wang

    Full Text Available Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP(+ cells. Moreover, CCSP(+ cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP(-expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs in terminal bronchioles (TBs. We conclude that CCSP(+ cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

  14. Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility.

    Science.gov (United States)

    Nie, Daotai; Che, Mingxin; Zacharek, Alex; Qiao, Yan; Li, Li; Li, Xinglin; Lamberti, Mario; Tang, Keqin; Cai, Yilong; Guo, Yande; Grignon, David; Honn, Kenneth V

    2004-02-01

    Arachidonic acid metabolism through cyclooxygenase, lipoxygenase, or P-450 epoxygenase pathways can generate a variety of eicosanoids. Thromboxane synthase (TxS) metabolizes the cyclooxygenase product, prostanglandin H(2), into thromboxane A(2) (TXA(2)), which can cause vessel constriction, platelet activation, and aggregation. Here we demonstrate that human prostate cancer (PCa) cells express enzymatically active TxS and that this enzyme is involved in cell motility. In human PCa cell lines, PC-3, PC-3M, and ML-2 cells expressed higher levels of TxS than normal prostate epithelial cells or other established PCa cell lines such as DU145, LNCaP, or PPC-1. We cloned and sequenced the full-length TxS cDNA from PC-3 cells and found two changes in the amino acid residues. Immunohistochemical analysis of tumor specimens revealed that expression of TxS is weak or absent in normal differentiated luminal, or secretory cells, significantly elevated in less differentiated or advanced prostate tumors, and markedly increased in tumors with perineural invasion. TxS expressed in PC-3 cells was enzymatically active and susceptible to carboxyheptal imidazole, an inhibitor of TxS. The biosynthesis of TXA(2) in PC-3 cells was dependent on COX-2, and to a lesser extent, COX-1. Treatment of PC-3 cells with a COX-1 selective inhibitor, piroxicam, reduced TXA(2) synthesis by approximately 40%, while the COX-2 specific inhibitor NS398 reduced TXA(2) production by approximately 80%. Inhibition of TxS activity or blockade of TXA(2) function reduced PC-3 cell migration on fibronectin, while having minimal effects on cell cycle progression or survival. Finally, increased expression of TxS in DU145 cells increased cell motility. Our data suggest that human PCa cells express TxS and that this enzyme may contribute to PCa progression through modulating cell motility.

  15. Signal peptide peptidase-mediated nuclear localization of heme oxygenase-1 promotes cancer cell proliferation and invasion independent of its enzymatic activity.

    Science.gov (United States)

    Hsu, F-F; Yeh, C-T; Sun, Y-J; Chiang, M-T; Lan, W-M; Li, F-A; Lee, W-H; Chau, L-Y

    2015-04-30

    Heme oxygenase-1 (HO-1) is a heme-degrading enzyme anchored in the endoplasmic reticulum by a carboxyl-terminal transmembrane segment (TMS). HO-1 is highly expressed in various cancers and its nuclear localization is associated with the progression of some cancers. Nevertheless, the mechanism underlying HO-1 nuclear translocation and its pathological significance remain elusive. Here we show that the signal peptide peptidase (SPP) catalyzes the intramembrane cleavage of HO-1. Coexpression of HO-1 with wild-type SPP, but not a dominant-negative SPP, promoted the nuclear localization of HO-1 in cells. Mass spectrometry analysis of cytosolic HO-1 isolated from HeLa cells overexpressing HO-1 and SPP revealed two adjacent intramembrane cleavage sites located after S275 and F276 within the TMS. Mutations of S275F276 to A275L276 significantly hindered SPP-mediated HO-1 cleavage and nuclear localization. Nuclear HO-1 was detected in A549 and DU145 cancer cell lines expressing high levels of endogenous HO-1 and SPP. SPP knockdown or inhibition significantly reduced nuclear HO-1 localization in A549 and DU145 cells. The positive nuclear HO-1 stain was also evident in lung cancer tissues expressing high levels of HO-1 and SPP. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion. The effect of t-HO-1 was not affected by a mutation in the catalytic site. However, blockade of t-HO-1 nuclear localization abolished t-HO-1-mediated effect. The tumorigenic effect of t-HO-1 was also demonstrated in the mouse model. These findings disclose that SPP-mediated intramembrane cleavage of HO-1 promotes HO-1 nuclear localization and cancer progression independent of HO-1 enzymatic activity.

  16. Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls

    DEFF Research Database (Denmark)

    Thygesen, Lisbeth Garbrecht; Hidayat, Budi Juliman; Johansen, Katja Salomon

    2011-01-01

    The study of biomass deconstruction by enzymatic hydrolysis has hitherto not focussed on the importance of supramolecular structures of cellulose. In lignocellulose fibres, regions with a different organisation of the microfibrils are present. These regions are called dislocations or slip planes...... and they are known to be more susceptible to various forms of degradation such as acid hydrolysis. Traditionally the cellulose within these regions has been assumed to be amorphous, but in this study it is shown by use of polarized light microscopy that dislocations are birefringent. This indicates that they have...... the initial part of enzymatic hydrolysis of cellulose. The implications of this phenomenon have not yet been recognized or explored within cellulosic biofuels....

  17. Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors

    Institute of Scientific and Technical Information of China (English)

    HEShao-Heng; CHENPu; CHENHan-Qiu

    2003-01-01

    AIM: To investigate the actions of protease inhibitors on the enzymatic activities of tryptase and chymase in similarexperimental systems. METHODS: Human lung tryptase and human skin chymase were purified by a similarprocedure involving high salt extraction of tryptase, heparin agarose affinity chromatography, and S-200 Sephacrylgel filtration chromatography. Actions of protease inhibitors on tryptase and chymase activities were examined byenzyme assays. RESULTS: The specific activities of tryptase and chymase were 2.1 kU/g protein and 4.9 kU/g protein, respectively. Both preparations showed a single diffuse band on SDS-PAGE. Among non-native proteaseinhibitors, N-(1-hydroxy-2-naphthoyl)-L- arginyl-L-prolinamide hydrochloride (HNAP), leupeptin, antipain,benzamidine, and protamine inhibited more than 90 % enzymatic activity of tryptase, whereas soy bean trypsininhibitor (SBTI), Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPM) and chymostatin inhibited more than 95 % enzymaticactivity of chymase. Native protease inhibitors α-antitrypsin and secretory leukocyte protease inhibitor (SLPI)inhibited more than 90 % enzymatic activity of chymase, but lactoferrin appeared to enhance chymase enzymaticactivity. All the 3 inhibitors had weak inhibitory actions on tryptase. CONCLUSION: The protease inhibitorstested had relatively good selectivity to either tryptase or chymase.

  18. Construction of a highly sensitive non-enzymatic sensor for superoxide anion radical detection from living cells.

    Science.gov (United States)

    Liu, Yuelin; Liu, Xiuhui; Liu, Yidan; Liu, Guoan; Ding, Lan; Lu, Xiaoquan

    2017-04-15

    A novel non-enzymatic superoxide anion (O2•(-)) sensor was fabricated based on Ag nanoparticles (NPs)/L-cysteine functioned carbon nanotubes (Cys-MWCNTs) nanocomposites and used to measure the release of O2•(-) from living cells. In this strategy, AgNPs could be uniformly electrodeposited on the MWCNTs surface with average diameter of about 20nm as exhibited by scanning electronmicroscopy (SEM). Electrochemical study demonstrated that the AgNPs/Cys-MWCNTs modified glassy carbon electrode exhibited excellent catalytic activity toward the reduction of O2•(-) with a super wide linear range from 7.00×10(-11) to 7.41×10(-5)M and a low detection limit (LOD) of 2.33×10(-11)M (S/N=3). Meanwhile, the mechanism for O2•(-) reduction was also proposed for the first time. Importantly, this novel non-enzymatic O2•(-) sensor can detect O2•(-) release from cancer cells under both the external stimulation and the normal condition, which has the great potential application in clinical diagnostics to assess oxidative stress of living cells.

  19. The operation of enzymatic fuel cell fabricated with rationally designed poly(caprolactone-g-ethylene glycol) copolymers.

    Science.gov (United States)

    Korkut, Seyda; Kilic, Muhammet Samet; Sanal, Timur; Hazer, Baki

    2017-07-01

    This study describes construction of an enzymatic fuel cell comprised of poly(caprolactone-g-ethylene glycol) coated novel glucose oxidase anode and laccase cathode. Rationally designed poly(caprolactone-g-ethylene glycol) containing various poly(ethylene glycol) percentages ranging between 2.67 and 15.04% were synthesized chemically and tested separately for operation of the fuel cell system to achieve the best energy generation. The maximum power density was found to be 80.55μWcm(-2) at 0.91V (vs. Ag/AgCl) in pH5, 100mM citrate buffer (20°C) by the addition of 30mM of glucose from the electrodes coated with 11.34% poly(ethylene glycol) containing polymer with a quantity of 600μg. High poly(ethylene glycol) percentages with more numbers of long poly(ethylene glycol) brushes lead to the creation of a complexity in the polymer morphology and steric hindrance effect for electron transport. The graft copolymer was easily used for the fuel cell system owing to its biocompatible and microporous film morphology. The grafted polymer was able to facilitate enzymatic glucose oxidation and oxygen reduction while simultaneously producing high catalytic electrical currents. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Directory of Open Access Journals (Sweden)

    Jacob L Lilly

    Full Text Available Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  1. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    Science.gov (United States)

    Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  2. Expressão e atividade enzimática de glutationa s-transferase em tecidos de fêmeas de Boophilus microplus Expression and enzymatic activity of glutathione s-transferase in tissues of Boophilus microplus females

    Directory of Open Access Journals (Sweden)

    Daniela R. J. de Freitas

    2008-06-01

    Full Text Available Enzimas de detoxificação e excreção celular são importantes para a manutenção da homeostase celular. Neste trabalho foi caracterizada a transcrição de mRNA, a expressão da proteína e a atividade enzimática de glutationa S-transfersases (GSTs, enzimas que atuam em rotas de excreção de substâncias endo e xenobióticas, protegendo as células contra toxicidade química e estresse, em diferentes tecidos (glândula salivar, ovário e singânglio de fêmeas adultas semi-ingurgitadas e ingurgitadas do carrapato do bovino Boophilus microplus. Os resultados mostraram que a atividade de GST é mais alta em fêmeas semi-ingurgitadas e diminui em fêmeas ingurgitadas de acordo com o final do período de pré-postura. A expressão de mRNA de GST foi detectada em glândulas salivares e singânglios de fêmeas adultas semi-ingurgitadas e ingurgitadas, mas não em ovários. Estes dados podem ajudar a compreender melhor o papel de enzimas antioxidantes durante a preparação das fêmeas do carrapato para a postura.Cellular detoxification and excretion enzymes are important to the maintenance of cellular homeostasis. In this work mRNA transcription, protein expression and enzymatic activity of Glutathione S-transferases (GSTs, enzymes involved in the excretion of endo and xenobiotic compounds were analyzed. These parameters are elements believed to protect cells against chemical toxicity and oxidative stress in different tissues (salivary gland, ovary and synganglion from partially engorged females and engorged females of Boophilus microplus. The results presented showed elevated GST activity in partially engorged females. The enzymatic activity decreased during the preoviposition period in engorged females. GST mRNA transcription was detected in salivary glands and synganglion from partially engorged and engorged females, but not in ovary. The results of this work help to elucidate the role of GST in tick development and assist in the

  3. In vitro assay for HCV serine proteinase expressed in insect cells

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hou; Gui-Xin Du; Rong-Bin Guan; Yi-Gang Tong; Hai-Tao Wang

    2003-01-01

    AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition.After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells.The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purifed by metalchelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected.RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS.Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5×107 cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients′ sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity.CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.

  4. Evaluation of alcohol dehydrogenase and aldehyde dehydrogenase enzymes as bi-enzymatic anodes in a membraneless ethanol microfluidic fuel cell

    Science.gov (United States)

    Galindo-de-la-Rosa, J.; Arjona, N.; Arriaga, L. G.; Ledesma-García, J.; Guerra-Balcázar, M.

    2015-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldH) enzymes were immobilized by covalent binding and used as the anode in a bi-enzymatic membraneless ethanol hybrid microfluidic fuel cell. The purpose of using both enzymes was to optimize the ethanol electro-oxidation reaction (EOR) by using ADH toward its direct oxidation and AldH for the oxidation of aldehydes as by-products of the EOR. For this reason, three enzymatic bioanode configurations were evaluated according with the location of enzymes: combined, vertical and horizontally separated. In the combined configuration, a current density of 16.3 mA cm-2, a voltage of 1.14 V and a power density of 7.02 mW cm-2 were obtained. When enzymes were separately placed in a horizontal and vertical position the ocp drops to 0.94 V and to 0.68 V, respectively. The current density also falls to values of 13.63 and 5.05 mA cm-2. The decrease of cell performance of bioanodes with separated enzymes compared with the combined bioanode was of 31.7% and 86.87% for the horizontal and the vertical array.

  5. Salmonella Typhimurium Enzymatically Landscapes the Host Intestinal Epithelial Cell (IEC) Surface Glycome to Increase Invasion.

    Science.gov (United States)

    Park, Dayoung; Arabyan, Narine; Williams, Cynthia C; Song, Ting; Mitra, Anupam; Weimer, Bart C; Maverakis, Emanual; Lebrilla, Carlito B

    2016-12-01

    Although gut host-pathogen interactions are glycan-mediated processes, few details are known about the participating structures. Here we employ high-resolution mass spectrometric profiling to comprehensively identify and quantitatively measure the exact modifications of native intestinal epithelial cell surface N-glycans induced by S. typhimurium infection. Sixty minutes postinfection, select sialylated structures showed decreases in terms of total number and abundances. To assess the effect of cell surface mannosylation, we selectively rerouted glycan expression on the host using the alpha-mannosidase inhibitor, kifunensine, toward overexpression of high mannose. Under these conditions, internalization of S. typhimurium significantly increased, demonstrating that bacteria show preference for particular structures. Finally, we developed a novel assay to measure membrane glycoprotein turnover rates, which revealed that glycan modifications occur by bacterial enzyme activity rather than by host-derived restructuring strategies. This study is the first to provide precise structural information on how host N-glycans are altered to support S. typhimurium invasion. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Role of supramolecular cellulose structures in enzymatic hydrolysis of plant cell walls.

    Science.gov (United States)

    Thygesen, Lisbeth Garbrecht; Hidayat, Budi Juliman; Johansen, Katja Salomon; Felby, Claus

    2011-08-01

    The study of biomass deconstruction by enzymatic hydrolysis has hitherto not focussed on the importance of supramolecular structures of cellulose. In lignocellulose fibres, regions with a different organisation of the microfibrils are present. These regions are called dislocations or slip planes and they are known to be more susceptible to various forms of degradation such as acid hydrolysis. Traditionally the cellulose within these regions has been assumed to be amorphous, but in this study it is shown by use of polarized light microscopy that dislocations are birefringent. This indicates that they have a crystalline organisation. Dislocations may be entry points for endoglucanases. Using a fluorescent labelled endoglucanase combined with confocal fluorescence microscopy, it is shown that the enzyme selectively binds to dislocations during the initial phase of the hydrolysis. Using a commercial cellulase mixture on hydrothermally treated wheat straw, it was found that the fibres were cut into segments corresponding to the sections between the dislocations initially present, as has previously been observed for acid hydrolysis of softwood pulps. The results indicate that dislocations are important during the initial part of enzymatic hydrolysis of cellulose. The implications of this phenomenon have not yet been recognized or explored within cellulosic biofuels.

  7. Extractability and digestibility of plant cell wall polysaccharides during hydrothermal and enzymatic degradation of wheat straw (Triticum aestivum L.)

    DEFF Research Database (Denmark)

    Hansen, Mads A.T.; Ahl, Louise I.; Pedersen, Henriette L.

    2014-01-01

    , regardless their extractability in water or only alkali. Based on the results, AX and MLG appear to be loosely bound in the cell wall matrix while the other polysaccharides are bound more tightly and shielded from enzymatic attack by AX and MLG until pretreatment. The gradual solubilisation and digestion...... and by comprehensive microarray polymer profiling (CoMPP). This way, the effects of each degradation step to the intermolecular organisation of specific polysaccharides in the cell walls were elucidated. After pretreatment, the degree of polymerisation (DP) of released xylo-oligosaccharides in both samples was up...... to about 20, but mostly around 3-8, and notably more acetylated in stems. Arabinoxylan (AX) and mixed-linkage glucan (MLG) became water-extractable while xylan, xyloglucan (XG), mannan and glucan remained only alkali-extractable. All polysaccharides became partly digestible after pretreatment however...

  8. Cathepsin G Induces Cell Aggregation of Human Breast Cancer MCF-7 Cells via a 2-Step Mechanism: Catalytic Site-Independent Binding to the Cell Surface and Enzymatic Activity-Dependent Induction of the Cell Aggregation

    Directory of Open Access Journals (Sweden)

    Riyo Morimoto-Kamata

    2012-01-01

    Full Text Available Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.

  9. Highly Dispersed NiO Nanoparticles Decorating graphene Nanosheets for Non-enzymatic Glucose Sensor and Biofuel Cell

    Science.gov (United States)

    Zeng, Guisheng; Li, Weiping; Ci, Suqin; Jia, Jingchun; Wen, Zhenhai

    2016-11-01

    Nickel oxide-decorated graphene nanosheet (NiO/GNS), as a novel non-enzymatic electrocatalyst for glucose oxidation reaction (GOR), was synthesized through a facile hydrothermal route followed by the heat treatment. The successful synthesis of NiO/GNS was characterized by a series of techniques including XRD, BET, SEM and TEM. Significantly, the NiO/GNS catalyst show excellent catalytic activity toward GOR, and was employed to develop a sensitive non-enzymatic glucose sensor. The developed glucose sensor could response to glucose in a wide range from 5 μM-4.2 mM with a low detection limit (LOD) of 5.0 μM (S/N = 3). Importantly, compared with bare NiO, the catalytic activity of NiO/GNS was much higher. The reason might be that the 2D structure of graphene could prevent the aggregation of NiO and facilitate the electron transfer at electrode interface. Moreover, the outstanding catalytic activity of NiO/GNS was further demonstrated by applying it to construct a biofuel cell using glucose as fuel, which exhibited high stability and current density.

  10. Simultaneous decolorization and bioelectricity generation in a dual chamber microbial fuel cell using electropolymerized-enzymatic cathode.

    Science.gov (United States)

    Savizi, Iman Shahidi Pour; Kariminia, Hamid-Reza; Bakhshian, Sahar

    2012-06-19

    Effect of cathodic enzymatic decolorization of reactive blue 221 (RB221) on the performance of a dual-chamber microbial fuel cell (MFC) was investigated. Immobilized laccase on the surface of a modified graphite electrode was used in the cathode compartment in order to decolorize the azo dye and enhance the oxygen reduction reaction. First, methylene blue which is an electroactive polymer was electropolymerized on the surface of a graphite bar to prepare the modified electrode. Utilization of the modified electrode with no enzyme in the MFC increased the power density up to 57% due to the reduction of internal resistance from 1000 to 750 Ω. Using the electropolymerized-enzymatic cathode resulted in 65% improvement of the power density and a decolorization efficiency of 74%. Laccase could act as a biocatalyst for oxygen reduction reaction along with catalyzing RB221 decolorization. Treatment of RB221 with immobilized laccase reduced its toxicity up to 5.2%. Degradation products of RB221 were identified using GC-MS, and the decomposition pathway was proposed. A discussion was also provided as to the mechanism of dye decolorization on the enhancement of the MFC performance.

  11. Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis

    Energy Technology Data Exchange (ETDEWEB)

    Bouetard, Anthony, E-mail: anthony.bouetard@rennes.inra.fr [INRA, UMR INRA-Agrocampus Ouest ESE 0985, Equipe Ecotoxicologie et Qualite des Milieux Aquatiques, 65 rue de Saint-Brieuc, 35042 Rennes cedex (France); Besnard, Anne-Laure; Vassaux, Daniele; Lagadic, Laurent; Coutellec, Marie-Agnes [INRA, UMR INRA-Agrocampus Ouest ESE 0985, Equipe Ecotoxicologie et Qualite des Milieux Aquatiques, 65 rue de Saint-Brieuc, 35042 Rennes cedex (France)

    2013-01-15

    The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 {mu}g l{sup -1}) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased

  12. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    .05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...

  13. Human Neuroepithelial Cells Express NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Cappell B

    2003-11-01

    Full Text Available Abstract L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1 cerebral endothelial barrier and 2 cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR expression via immunohistochemistry and murine neuroepithelial cell line (V1 were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease.

  14. Transitional cell carcinoma express vitamin D receptors

    DEFF Research Database (Denmark)

    Hermann, G G; Andersen, C B

    1997-01-01

    Recently, vitamin D analogues have shown antineoplastic effect in several diseases. Vitamin D analogues exert its effect by interacting with the vitamin D receptor (VDR). Studies of VDR in transitional cell carcinoma (TCC) have not been reported. The purpose of the present study was therefore...... to examine whether human bladder tumor cells express VDR. Tumor biopsies were obtained from 26 patients with TCC. Expression of VDR was examined by immunohistochemical experiments. All tumors expressed VDR. Biopsies from advanced disease contained more VDR positive cells than low stage disease (p ....05). Similarly, also tumor grade appeared to be related to the number of cells expressing the receptor. Normal urothlium also expressed VDR but only with low intensity. Our study shows that TCC cells possess the VDR receptor which may make them capable to respond to stimulation with vitamin D, but functional...

  15. Expression profile of germ stem cell-specific genes in human spermatogonial stem cells after co culture with sertoli cells

    Directory of Open Access Journals (Sweden)

    Maria Zahiri

    2014-05-01

    Full Text Available Background: Human spermatogonial stem cells (SSCs, are the foundation of spermatogenesis. Because of low number and lack of significant marker in human SSCs, studying their characteristics, could provide better understanding about the biology of male fertility. This study was designed to examine the effects of in vitro co-culture with sertoli cells on SSC colonization and germ cells specific gene expression of human spermatogonial stem cells. Material and Methods: Testicular cells were isolated from testis biopsies by using two step enzymatic digestion and differential plating. two culture system were designed: co-culture with patient Sertoli cells and culture of SSC without co-culture(as control group. The number and diameter of colonies were evaluated during 3 weeks of culture. The expression of alpha 6 integrin, beta1 integrin and PLZF, as germ stem cell specific markers, was assessed using quantitative RT-PCR. Statistical analysis was performed using one way ANOVA in SPSS vesion 16 software with 95% Confidence interval . Result: Our results were showed that the number and diameter of colonies increased significantly in co-culture with sertoli cells (P<0.05. The expression profile of genes in 2nd and 3rd weeks of culture revealed that there is significant higher expression of germ stem cell markers in our co-culture group versus control group. Conclusion: Based on the optimal effects of sertoli cells on spermatogonial stem cells, co culture of the human SSCs with the feeder layer sertoli may be used as a suitable method for the enrichment of human spermatogonial stem cells.

  16. Rho GTPase expression in human myeloid cells.

    Directory of Open Access Journals (Sweden)

    Suzanne F G van Helden

    Full Text Available Myeloid cells are critical for innate immunity and the initiation of adaptive immunity. Strict regulation of the adhesive and migratory behavior is essential for proper functioning of these cells. Rho GTPases are important regulators of adhesion and migration; however, it is unknown which Rho GTPases are expressed in different myeloid cells. Here, we use a qPCR-based approach to investigate Rho GTPase expression in myeloid cells.We found that the mRNAs encoding Cdc42, RhoQ, Rac1, Rac2, RhoA and RhoC are the most abundant. In addition, RhoG, RhoB, RhoF and RhoV are expressed at low levels or only in specific cell types. More differentiated cells along the monocyte-lineage display lower levels of Cdc42 and RhoV, while RhoC mRNA is more abundant. In addition, the Rho GTPase expression profile changes during dendritic cell maturation with Rac1 being upregulated and Rac2 downregulated. Finally, GM-CSF stimulation, during macrophage and osteoclast differentiation, leads to high expression of Rac2, while M-CSF induces high levels of RhoA, showing that these cytokines induce a distinct pattern. Our data uncover cell type specific modulation of the Rho GTPase expression profile in hematopoietic stem cells and in more differentiated cells of the myeloid lineage.

  17. Human plasma cells express granzyme B.

    Science.gov (United States)

    Xu, Wei; Narayanan, Priya; Kang, Ning; Clayton, Sandra; Ohne, Yoichiro; Shi, Peiqing; Herve, Marie-Cecile; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Gorvel, Jean-Pierre; Oh, Sangkon; Pascual, Virginia; Banchereau, Jacques

    2014-01-01

    While studying the plasma cell (PC) compartment in human tonsils, we identified that immunoglobulin kappa or lambda chain-expressing PCs are the main cells expressing granzyme B (GrzB). In vitro studies revealed that activated B cells differentiated into GrzB-expressing PCs when co-cultured with macrophages and follicular helper T cells. This effect could be reproduced on combined stimulation of IL-15 (produced by macrophages) and IL-21 (produced by T follicular helper cells) in a STAT3-dependent manner. Whereas IL-21 triggers the transcription of mRNA of GrzB, IL-15 synergizes the translation of GrzB proteins. The precise role of GrzB in PC biology remains to be understood and studies in mice will not help as their PCs do not express GrzB.

  18. Cell Surface Enzymatic Engineering-Based Approaches to Improve Cellular Therapies

    KAUST Repository

    AbuElela, Ayman

    2014-06-06

    The cell surface represents the interface between the cell and its environment. As such, the cell surface controls cell–cell interactions and functions such as adhesion and migration, and will transfer external cues to regulate processes such as survival, death, and differentiation. Redefining the cell surface by temporarily (or permanently) modifying the molecular landscape of the plasma membrane affects the way in which the cell interacts with its environment and influences the information that is relayed into the cell along downstream signaling pathways. This chapter outlines the role of key enzymes, the glycosyltransferases, in posttranslationally modifying proteins and lipids to fine-tune cells, ability to migrate. These enzymes are critical in controlling the formation of a platform structure, sialyl Lewis x (sLex), on circulating cells that plays a central role in the recognition and recruitment by selectin counter receptors on endothelial cells that line blood vessels of tissues throughout the body. By developing methods to manipulate the activity of these enzymes and hence the cell surface structures that result, treatments can be envisioned that direct the migration of therapeutic cells to specific locations throughout the body and also to inhibit metastasis of detrimental cells such as circulating tumor cells.

  19. Behavior of some enzymatic systems to the action of the cytostatic active EGlCP glucanic biopreparation upon HeLa neoplastic cells

    Directory of Open Access Journals (Sweden)

    Daniela Gherghel

    2010-02-01

    Full Text Available Interference of an autochthonous cytostatic active EGlCP glucanic biopreparation (in dose of 1.5 mg/mL with the activity of some key enzymes, involved in the development of active transmembranary transport, of the intermediary and energetic metabolism, as well as in cellular answer to the oxidative stress, of HeLa neoplastic cells has been investigated. The study revealed: the intensification of the membranary Na+-K+-ATP-ase, of the cellular Mg2+-ATP-ase, of the superoxide dismutase activities; the operating level attenuation of the of catalase, peroxidase, glutathion peroxidase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase; the diminution of the malondialdehyde content. This functional interference with some cell enzymatic biomolecules has also induced the perturbation of the diverse membrane and metabolic processes, which was incompatible with the survival of HeLa tumoral cells The modulations of the cellular enzymatic equipment activity can be the consequences of the glucanic components direct (with the molecules of the miscellaneous enzymes or indirect interactions ( with membrane or genetic apparatus with some cell, subcell and molecular structures, implicated in the control and regulation of the biosynthesis and activity of the enzymatic biomolecules. The central element, which induces this enzymatic imbalance, appears to be the excess generation of the free radicals in the tumoral cells’ metabolism aggressed by glucanic constituents.

  20. Expression of lactoperoxidase in differentiated mouse colon epithelial cells.

    Science.gov (United States)

    Kim, Byung-Wook; Esworthy, R Steven; Hahn, Maria A; Pfeifer, Gerd P; Chu, Fong-Fong

    2012-05-01

    Lactoperoxidase (LPO) is known to be present in secreted fluids, such as milk and saliva. Functionally, LPO teams up with dual oxidases (DUOXs) to generate bactericidal hypothiocyanite in the presence of thiocyanate. DUOX2 is expressed in intestinal epithelium, but there is little information on LPO expression in this tissue. To fill the gap of knowledge, we have analyzed Lpo gene expression and its regulation in mouse intestine. In wild-type (WT) C57BL/6 (B6) mouse intestine, an appreciable level of mouse Lpo gene expression was detected in the colon, but not the ileum. However, in B6 mice deficient in glutathione peroxidase (GPx)-1 and -2, GPx1/2-double-knockout (DKO), which had intestinal pathology, the colon Lpo mRNA levels increased 5- to 12-fold depending on mouse age. The Lpo mRNA levels in WT and DKO 129S1/SvlmJ (129) colon were even higher, 9- and 5-fold, than in B6 DKO colon. Higher levels of Lpo protein and enzymatic activity were also detected in the 129 mouse colon compared to B6 colon. Lpo protein was expressed in the differentiated colon epithelial cells, away from the crypt base, as shown by immunohistochemistry. Similar to human LPO mRNA, mouse Lpo mRNA had multiple spliced forms, although only the full-length variant 1 was translated. Higher methylation was found in the 129 than in the B6 strain, in DKO than in control colon, and in older than in juvenile mice. However, methylation of the Lpo intragenic CpG island was not directly induced by inflammation, because dextran sulfate sodium-induced colitis did not increase DNA methylation in B6 DKO colon. Also, Lpo DNA methylation is not correlated with gene expression.

  1. Differential expression and enzymatic properties of GalNAc-4-sulfotransferase-1 and GalNAc-4-sulfotransferase-2.

    Science.gov (United States)

    Boregowda, Rajeev K; Mi, YiLing; Bu, Hongyin; Baenziger, Jacques U

    2005-12-01

    We have cloned two GalNAc-4-sulfotransferases, GalNAc-4-ST1 and GalNAc-4-ST2, that transfer sulfate to terminal beta1,4-linked GalNAc. In conjunction with the action of protein-specific beta1,4GalNAc-transferases, GalNAc-4-ST1 and GalNAc-4-ST2 account for the presence of terminal beta1,4-linked GalNAc-4-SO(4) on glycoproteins such as lutropin, thyrotropin (TSH), proopiomelanocortin (POMC), carbonic anhydratase-VI (CA-VI), and tenascin-R. GalNAc-4-ST1 and GalNAc-4-ST2 can be distinguished by their differing specificity for oligosaccharide acceptors and temperature lability. The differences in properties have been used to show that the levels of GalNAc-4-ST1 and GalNAc-4-ST2 activity are proportionate to the levels of their respective transcripts. Furthermore, we have found that both transcript and activity levels of GalNAc-4-ST1 and GalNAc-4-ST2 vary widely among different tissues indicating that the regulation of their expression differs. Differences in specificity and the regulation of expression may account for existence of two GalNAc-4-sulfotransferases in vivo. The highest levels of both GalNAc-4-ST1 and GalNAc-4-ST2 transcripts are present in the pituitary of the mouse with multiple cell types that produce glycoproteins terminating with GalNAc-4-SO(4). Genetic ablation of both GalNAc-4-ST1 and GalNAc-4-ST2 may be necessary to alter the pattern and/or extent of sulfate addition to terminal beta1,4GalNAc in tissues such as pituitary.

  2. Microanalysis of gene expression in cultured cells

    NARCIS (Netherlands)

    E. van der Veer (Eveliene)

    1982-01-01

    textabstractIn this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing g

  3. Purification of replication factors using insect and mammalian cell expression systems.

    Science.gov (United States)

    Uno, Shuji; You, Zhiying; Masai, Hisao

    2012-06-01

    Purification of factors for DNA replication in an amount sufficient for detailed biochemical characterization is essential to elucidating its mechanisms. Insect cell expression systems are commonly used for purification of the factors proven to be difficult to deal with in bacteria. We describe first the detailed protocols for purification of mammalian Mcm complexes including the Mcm2/3/4/5/6/7 heterohexamer expressed in insect cells. We then describe a convenient and economical system in which large-sized proteins and multi-factor complexes can be transiently overexpressed in human 293T cells and be rapidly purified in a large quantity. We describe various expression vectors and detailed methods for transfection and purification of various replication factors which have been difficult to obtain in a sufficient amount in other systems. Availability of efficient methods to overproduce and purify the proteins that have been challenging would facilitate the enzymatic analyses of the processes of DNA replication.

  4. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    Thymidine kinase (TK) is a key enzyme in the salvage pathway of the nucleoside metabolism catalyzing the first phosphorylation step in TTP synthesis. Human cytosolic TK (TKl) is highly cell cycle regulated. TKl is regulated on many different levels of expression and isoforms with altered enzymatic...... properties are found in cancer cells. Investigation of these factors offers possibilities to understand the molecular background for TKl expression including to clarify general regulation patterns. It also gives valuable information for constructing new nucleoside analogs for the therapy of cancer and virus...... infections. In the first part of the present investigation a sensitive test for quantitating TKl mRNA (competitive PCR) is developed and the results show that PHA stimulated lymphocytes reveal the same pattern concerning expression of TKl mRNA and TKl enzyme activity as serum-stimulated cells. This pattern...

  5. Altered Cell Cycle Arrest by Multifunctional Drug-Loaded Enzymatically-Triggered Nanoparticles.

    Science.gov (United States)

    Huang, Can; Sun, Ying; Shen, Ming; Zhang, Xiangyu; Gao, Pei; Duan, Yourong

    2016-01-20

    cRGD-targeting matrix metalloproteinase (MMP)-sensitive nanoparticles [PLGA-PEG1K-cRGD/PLGA-peptide-PEG5K (NPs-cRGD)] were successfully developed. Au-Pt(IV) nanoparticles, PTX, and ADR were encapsulated into NPs-RGD separately. The effects of the drug-loaded nanoparticles on the cell cycle were investigated. Here, we showed that higher cytotoxicity of drug-loaded nanoparticles was related to the cell cycle arrest, compared to that of free drugs. The NPs-cRGD studied here did not disrupt cell cycle progression. The cell cycle of Au-Pt(IV)@NPs-cRGD showed a main S phase arrest in all phases of the cell cycle phase, especially in G0/G1 phase. PTX@NPs-cRGD and ADR@NPs-cRGD showed a higher ratio of G2/M and S phase arrest than the free drugs, respectively. Cells in G0/G1 and S phases of the cell cycle had a higher uptake ratio of NPs-cRGD. A nutrient deprivation or an increase in the requirement of nutrients in tumor cells could promote the uptake of nanoparticles from the microenvironments. In vivo, NPs-cRGD could efficiently accumulate at tumor sites. The inhibition of tumor growth coupled with cell cycle arrest is in line with that in vitro. On the basis of our results, we propose that future studies on nanoparticle action mechanism should consider the cell cycle, which could be different from free drugs. Understanding the actions of cell cycle arrest could affect the application of nanomedicine in the clinic.

  6. Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

    DEFF Research Database (Denmark)

    Neuvonen, M.; Manna, M.; Mokkila, S.

    2014-01-01

    of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human...

  7. Recent advances on enzymatic glucose/oxygen and hydrogen/oxygen biofuel cells: Achievements and limitations

    Science.gov (United States)

    Cosnier, Serge; J. Gross, Andrew; Le Goff, Alan; Holzinger, Michael

    2016-09-01

    The possibility of producing electrical power from chemical energy with biological catalysts has induced the development of biofuel cells as viable energy sources for powering portable and implanted electronic devices. These power sources employ biocatalysts, called enzymes, which are highly specific and catalytic towards the oxidation of a biofuel and the reduction of oxygen or hydrogen peroxide. Enzymes, on one hand, are promising candidates to replace expensive noble metal-based catalysts in fuel cell research. On the other hand, they offer the exciting prospect of a new generation of fuel cells which harvest energy from body fluids. Biofuel cells which use glucose as a fuel are particularly interesting for generating electricity to power electronic devices inside a living body. Hydrogen consuming biofuel cells represent an emerging alternative to platinum catalysts due to comparable efficiencies and the capability to operate at lower temperatures. Currently, these technologies are not competitive with existing commercialised fuel cell devices due to limitations including insufficient power outputs and lifetimes. The advantages and challenges facing glucose biofuel cells for implantation and hydrogen biofuel cells will be summarised along with recent promising advances and the future prospects of these exotic energy-harvesting devices.

  8. Evaluation of single and stack membraneless enzymatic fuel cells based on ethanol in simulated body fluids.

    Science.gov (United States)

    Galindo-de-la-Rosa, J; Arjona, N; Moreno-Zuria, A; Ortiz-Ortega, E; Guerra-Balcázar, M; Ledesma-García, J; Arriaga, L G

    2017-02-08

    The purpose of this work is to evaluate single and double-cell membraneless microfluidic fuel cells (MMFCs) that operate in the presence of simulated body fluids SBF, human serum and blood enriched with ethanol as fuels. The study was performed using the alcohol dehydrogenase enzyme immobilised by covalent binding through an array composed of carbon Toray paper as support and a layer of poly(methylene blue)/tetrabutylammonium bromide/Nafion and glutaraldehyde (3D bioanode electrode). The single MMFC was tested in a hybrid microfluidic fuel cell using Pt/C as the cathode. A cell voltage of 1.035V and power density of 3.154mWcm(-2) were observed, which is the highest performance reported to date. The stability and durability were tested through chronoamperometry and polarisation/performance curves obtained at different days, which demonstrated a slow decrease in the power density on day 10 (14%) and day 20 (26%). Additionally, the cell was tested for ethanol oxidation in simulated body fluid (SBF) with ionic composition similar to human blood plasma. Those tests resulted in 0.93V of cell voltage and a power density close to 1.237mWcm(-2). The double cell MMFC (Stack) was tested using serum and human blood enriched with ethanol. The stack operated with blood in a serial connection showed an excellent cell performance (0.716mWcm(-2)), demonstrating the feasibility of employing human blood as energy source.

  9. Engineering Cellular Microenvironments with Photo- and Enzymatically Responsive Hydrogels: Toward Biomimetic 3D Cell Culture Models.

    Science.gov (United States)

    Tam, Roger Y; Smith, Laura J; Shoichet, Molly S

    2017-04-18

    Conventional cell culture techniques using 2D polystyrene or glass have provided great insight into key biochemical mechanisms responsible for cellular events such as cell proliferation, differentiation, and cell-cell interactions. However, the physical and chemical properties of 2D culture in vitro are dramatically different than those found in the native cellular microenvironment in vivo. Cells grown on 2D substrates differ significantly from those grown in vivo, and this explains, in part, why many promising drug candidates discovered through in vitro drug screening assays fail when they are translated to in vivo animal or human models. To overcome this obstacle, 3D cell culture using biomimetic hydrogels has emerged as an alternative strategy to recapitulate native cell growth in vitro. Hydrogels, which are water-swollen polymers, can be synthetic or naturally derived. Many methods have been developed to control the physical and chemical properties of the hydrogels to match those found in specific tissues. Compared to 2D culture, cells cultured in 3D gels with the appropriate physicochemical cues can behave more like they naturally do in vivo. While conventional hydrogels involve modifications to the bulk material to mimic the static aspects of the cellular microenvironment, recent progress has focused on using more dynamic hydrogels, the chemical and physical properties of which can be altered with external stimuli to better mimic the dynamics of the native cellular microenvironment found in vivo. In this Account, we describe our progress in designing stimuli-responsive, optically transparent hydrogels that can be used as biomimetic extracellular matrices (ECMs) to study cell differentiation and migration in the context of modeling the nervous system and cancer. Specifically, we developed photosensitive agarose and hyaluronic acid hydrogels that are activated by single or two-photon irradiation for biomolecule immobilization at specific volumes within the 3D

  10. A Bi-enzymatic Whole-Cell Algal Biosensor for Monitoring Waste Water Pollutants

    OpenAIRE

    2004-01-01

    International audience; Two algal whole cells biosensors are developed to measure specific toxicity of freshwater pollutants. Both optical and conductometric biosensors are based on inhibition of algal alkaline phosphatase (AP) and esterase activities. Chlorella vulgaris cells are immobilised on a membrane placed in front of an optical fiber bundle for optical sensing or deposited on the surface of an electrode for conductometric sensing. Phosphatase activity of the biosensor is strongly inhi...

  11. Assembly and Stacking of Flow-through Enzymatic Bioelectrodes for High Power Glucose Fuel Cells.

    Science.gov (United States)

    Abreu, Caroline; Nedellec, Yannig; Gross, Andrew J; Ondel, Olivier; Buret, Francois; Goff, Alan Le; Holzinger, Michael; Cosnier, Serge

    2017-07-19

    Bioelectrocatalytic carbon nanotube based pellets comprising redox enzymes were directly integrated in a newly conceived flow-through fuel cell. Porous electrodes and a separating cellulose membrane were housed in a glucose/oxygen biofuel cell design with inlets and outlets allowing the flow of electrolyte through the entire fuel cell. Different flow setups were tested and the optimized single cell setup, exploiting only 5 mmol L(-1) glucose, showed an open circuit voltage (OCV) of 0.663 V and provided 1.03 ± 0.05 mW at 0.34 V. Furthermore, different charge/discharge cycles at 500 Ω and 3 kΩ were applied to optimize long-term stability leading to 3.6 J (1 mW h) of produced electrical energy after 48 h. Under continuous discharge at 6 kΩ, about 0.7 mW h could be produced after a 24 h period. The biofuel cell design further allows a convenient assembly of several glucose biofuel cells in reduced volumes and their connection in parallel or in series. The configuration of two biofuel cells connected in series showed an OCV of 1.35 V and provided 1.82 ± 0.09 mW at 0.675 V, and when connected in parallel, showed an OCV of 0.669 V and provided 1.75 ± 0.09 mW at 0.381 V. The presented design is conceived to stack an unlimited amount of biofuel cells to reach the necessary voltage and power for portable electronic devices without the need for step-up converters or energy managing systems.

  12. Cell Wall Composition and Candidate Biosynthesis Gene Expression During Rice Development

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Fan; Manisseri, Chithra; Fagerström, Alexandra; Peck, Matthew L.; Vega-Sánchez, Miguel E.; Williams, Brian; Chiniquy, Dawn M.; Saha, Prasenjit; Pattathil, Sivakumar; Conlin, Brian; Zhu, Lan; Hahn, Michael G.; Willats, William G. T.; Scheller, Henrik V.; Ronald, Pamela C.; Bartley, Laura E.

    2016-08-01

    Cell walls of grasses, including cereal crops and biofuel grasses, comprise the majority of plant biomass and intimately influence plant growth, development and physiology. However, the functions of many cell wall synthesis genes, and the relationships among and the functions of cell wall components remain obscure. To better understand the patterns of cell wall accumulation and identify genes that act in grass cell wall biosynthesis, we characterized 30 samples from aerial organs of rice (Oryza sativa cv. Kitaake) at 10 developmental time points, 3-100 d post-germination. Within these samples, we measured 15 cell wall chemical components, enzymatic digestibility and 18 cell wall polysaccharide epitopes/ligands. We also used quantitative reverse transcription-PCR to measure expression of 50 glycosyltransferases, 15 acyltransferases and eight phenylpropanoid genes, many of which had previously been identified as being highly expressed in rice. Most cell wall components vary significantly during development, and correlations among them support current understanding of cell walls. We identified 92 significant correlations between cell wall components and gene expression and establish nine strong hypotheses for genes that synthesize xylans, mixed linkage glucan and pectin components. This work provides an extensive analysis of cell wall composition throughout rice development, identifies genes likely to synthesize grass cell walls, and provides a framework for development of genetically improved grasses for use in lignocellulosic biofuel production and agriculture.

  13. Microbial-enzymatic-hybrid biological fuel cell with optimized growth conditions for Shewanella oneidensis DSP-10.

    Science.gov (United States)

    Roy, Jared N; Luckarift, Heather R; Sizemore, Susan R; Farrington, Karen E; Lau, Carolin; Johnson, Glenn R; Atanassov, Plamen

    2013-07-10

    In this work we present a biological fuel cell fabricated by combining a Shewanella oneidensis microbial anode and a laccase-modified air-breathing cathode. This concept is devised as an extension to traditional biochemical methods by incorporating diverse biological catalysts with the aim of powering small devices. In preparing the biological fuel cell anode, novel hierarchical-structured architectures and biofilm configurations were investigated. A method for creating an artificial biofilm based on encapsulating microorganisms in a porous, thin film of silica was compared with S. oneidensis biofilms that were allowed to colonize naturally. Results indicate comparable current and power densities for artificial and natural biofilm formations, based on growth characteristics. As a result, this work describes methods for creating controllable and reproducible bio-anodes and demonstrates the versatility of hybrid biological fuel cells.

  14. Power Harvesting from Human Serum in Buckypaper-Based Enzymatic Biofuel Cell

    Directory of Open Access Journals (Sweden)

    Güray eGüven

    2016-02-01

    Full Text Available The requirement for a miniature, high density, long life, rechargeable power source is common to a vast majority of microsystems, including the implantable devices for medical applications. A model biofuel cell system operating in human serum has been studied for future applications of biomedical and implantable medical devices. Anodic and cathodic electrodes were made of carbon nanotube –buckypaper modified with PQQ-dependent glucose dehydrogenase and laccase, respectively. Modified electrodes were characterized electrochemically and assembled in a biofuel cell set-up. Power density of 16.12 μW/cm2 was achieved in human serum for lower than physiological glucose concentrations. Increasing the glucose concentration and biofuel cell temperature caused an increase on power output leading up to 49.16 μW/cm2.

  15. Enzymatic production of atranorin: a component of the oak moss absolute by immobilized lichen cells.

    Science.gov (United States)

    Vicente, C; Fontaniella, B; Millanes, A M; Sebastián, B; Legaz, M E

    2003-04-01

    Cells of the lichen, Evernia prunastri, immobilized in calcium alginate were able to produce the depside atranorin from acetate. The synthesis of the depside was enhanced by molecular oxygen and NADH. This enhancement suggested the participation of an oxidase and an alcohol dehydrogenase to produce an aldehyde-substituted phenolic acid, hematommic acid, as the most probable precursor of atranorin. The participation of both enzymes was confirmed by loading immobilized cells with sodium azide, an inhibitor of several metallo-oxidases, and pyrazole, an inhibitor of alcohol dehydrogenase, which impeded atranorin production and accumulated beta-methyl orsellinate (after azide loading) or its alcohol derivative (after pirazole treatment).

  16. CNPase Expression in Olfactory Ensheathing Cells

    Directory of Open Access Journals (Sweden)

    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  17. Combined Enzymatic and High-Pressure Processing Affect Cell Wall Polysaccharides in Berries

    NARCIS (Netherlands)

    Hilz, H.; Lille, M.; Poutanen, K.; Schols, H.A.; Voragen, A.G.J.

    2006-01-01

    The effect of high-pressure processing (HPP) on cell wall polysaccharides in berries was investigated. HPP decreased the degree of methyl esterification (DM), probably by activation of pectin methyl esterase (PME), and improved the extractability of pectins. When commercial enzyme mixtures were adde

  18. Designer cells for stereocomplementary de novo enzymatic cascade reactions based on laboratory evolution.

    Science.gov (United States)

    Agudo, Rubén; Reetz, Manfred T

    2013-12-04

    Designer cells for a synthetic cascade reaction harnessing selective redox reactions were devised, featuring two successive regioselective P450-catalyzed CH-activating oxidations of 1-cyclohexene carboxylic acid methyl ester followed by stereoselective olefin-reduction catalysed by (R)- or (S)-selective mutants of an enoate reductase.

  19. Enzymatically-gellable galactosylated chitosan: Hydrogel characteristics and hepatic cell behavior.

    Science.gov (United States)

    Khoshfetrat, Ali Baradar; Khanmohammadi, Mehdi; Sakai, Shinji; Taya, Masahito

    2016-11-01

    The influence of contents of galactose and phenolic hydroxyl (Ph) groups incorporated into chitosan was investigated on characteristics of the chitosan derivatives and the resultant gels as well as HepG2 cell attachment and growth behaviors. Introduction of galactose groups increased the solubility of the chitosan derivatives. The gelation time decreased with increasing content of Ph groups in the chitosan derivatives. The increase of galactose groups incorporated at a fixed content of Ph groups improved mechanical properties of the resultant gels. In vitro degradation rate of the resultant gels decreased by increasing Ph groups and decreasing galactose groups incorporated into the chitosan derivatives. The HepG2 cells formed dense spheroid cell clusters when the galactose groups were absent or incorporated at high level into chitosan (13.8mol%). However, the cells exhibited spreading morphology with spheroid formation on the gels containing 1.1 and 5.2mol% galactose groups. The albumin secretion level on a cellular basis also increased considerably when the galactose groups increased to 13.8mol%. The results demonstrated the potential of the chitosan derivative hydrogels for liver tissue engineering applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Biocatalyst including porous enzyme cluster composite immobilized by two-step crosslinking and its utilization as enzymatic biofuel cell

    Science.gov (United States)

    Chung, Yongjin; Christwardana, Marcelinus; Tannia, Daniel Chris; Kim, Ki Jae; Kwon, Yongchai

    2017-08-01

    An enzyme cluster composite (TPA/GOx) formed from glucose oxidase (GOx) and terephthalaldehyde (TPA) that is coated onto polyethyleneimine (PEI) and carbon nanotubes (CNTs) is suggested as a new catalyst ([(TPA/GOx)/PEI]/CNT). In this catalyst, TPA promotes inter-GOx links by crosslinking to form a large and porous structure, and the TPA/GOx composite is again crosslinked with PEI/CNT to increase the amount of immobilized GOx. Such a two-step crosslinking (i) increases electron transfer because of electron delocalization by π conjugation and (ii) reduces GOx denaturation because of the formation of strong chemical bonds while its porosity facilitates mass transfer. With these features, an enzymatic biofuel cell (EBC) employing the new catalyst is fabricated and induces an excellent maximum power density (1.62 ± 0.08 mW cm-2), while the catalytic activity of the [(TPA/GOx)/PEI]/CNT catalyst is outstanding. This is clear evidence that the two-step crosslinking and porous structure caused by adoption of the TPA/GOx composite affect the performance enhancement of EBC.

  1. Membrane-less and mediator-free enzymatic biofuel cell using carbon nanotube/porous silicon electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shiunchin C.; Yang, Fan; Silva, Manuel; Zarow, Anna; Wang, Yubing; Iqbal, Zafar [Department of Chemistry and Environmental Science, New Jersey Institute of Technology, Newark, NJ 07102 (United States)

    2009-01-15

    Membrane-less and mediator-free direct electron transfer enzymatic biofuel cells (BFCs) with bioelectrodes comprised of single wall carbon nanotubes (SWNTs) deposited by two methods on porous silicon (pSi) substrates, are reported. In one method the SWNTs were grown by chemical vapor deposition (CVD) and then functionalized with carboxylic groups, and in the second method, pre-synthesized carboxylated SWNTs (c-SWNTs) were electrophoretically deposited on gold-coated pSi. Anodic glucose oxidase (GOx) and cathodic laccase (Lac) were immobilized on the pSi/SWNT substrates to form BFCs in pH 7 phosphate buffer solution. A peak power density of 1.38 {mu}W/cm{sup 2} (with a lifetime of 24 h) down to 0.3 {mu}W/cm{sup 2} was obtained for a BFC comprised of c-SWNT/enzyme electrodes in 4 mM glucose solution as fuel, corresponding to normal blood sugar concentration, and air as oxidant. BFCs of this relatively simple architecture have the potential for further optimization of power output and lifetime. (author)

  2. Membrane/mediator-free rechargeable enzymatic biofuel cell utilizing graphene/single-wall carbon nanotube cogel electrodes.

    Science.gov (United States)

    Campbell, Alan S; Jeong, Yeon Joo; Geier, Steven M; Koepsel, Richard R; Russell, Alan J; Islam, Mohammad F

    2015-02-25

    Enzymatic biofuel cells (EBFCs) utilize enzymes to convert chemical energy present in renewable biofuels into electrical energy and have shown much promise in the continuous powering of implantable devices. Currently, however, EBFCs are greatly limited in terms of power and operational stability with a majority of reported improvements requiring the inclusion of potentially toxic and unstable electron transfer mediators or multicompartment systems separated by a semipermeable membrane resulting in complicated setups. We report on the development of a simple, membrane/mediator-free EBFC utilizing novel electrodes of graphene and single-wall carbon nanotube cogel. These cogel electrodes had large surface area (∼ 800 m(2) g(-1)) that enabled high enzyme loading, large porosity for unhindered glucose transport and moderate electrical conductivity (∼ 0.2 S cm(-1)) for efficient charge collection. Glucose oxidase and bilirubin oxidase were physically adsorbed onto these electrodes to form anodes and cathodes, respectively, and the EBFC produced power densities up to 0.19 mW cm(-2) that correlated to 0.65 mW mL(-1) or 140 mW g(-1) of GOX with an open circuit voltage of 0.61 V. Further, the electrodes were rejuvenated by a simple wash and reloading procedure. We postulate these porous and ultrahigh surface area electrodes will be useful for biosensing applications, and will allow reuse of EBFCs.

  3. Employing FAD-dependent glucose dehydrogenase within a glucose/oxygen enzymatic fuel cell operating in human serum.

    Science.gov (United States)

    Milton, Ross D; Lim, Koun; Hickey, David P; Minteer, Shelley D

    2015-12-01

    Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) is emerging as an oxygen-insensitive alternative to glucose oxidase (GOx) as the biocatalyst for bioelectrodes and bioanodes in glucose sensing and glucose enzymatic fuel cells (EFCs). Glucose EFCs, which utilize oxygen as the oxidant and final electron acceptor, have the added benefit of being able to be implanted within living hosts. These can then produce electrical energy from physiological glucose concentrations and power internal or external devices. EFCs were prepared with FAD-GDH and bilirubin oxidase (BOx) to evaluate the suitability of FAD-GDH within an implantable setting. Maximum current and power densities of 186.6±7.1 μA cm(-2) and 39.5±1.3 μW cm(-2) were observed when operating in human serum at 21 °C, which increased to 285.7±31.3 μA cm(-2) and 57.5±5.4 μW cm(-2) at 37 °C. Although good stability was observed with continual near-optimal operation of the EFCs in human serum at 21 °C for 24 h, device failure was observed between 13-14 h when continually operated at 37 °C.

  4. Enzymatic electrodes nanostructured with functionalized carbon nanotubes for biofuel cell applications

    Energy Technology Data Exchange (ETDEWEB)

    Nazaruk, E.; Bilewicz, R. [University of Warsaw, Faculty of Chemistry, Warsaw (Poland); Sadowska, K.; Biernat, J.F. [Gdansk University of Technology, Chemical Faculty, Gdansk (Poland); Rogalski, J. [Maria Curie Sklodowska University, Department of Biochemistry, Lublin (Poland); Ginalska, G. [Medical University of Lublin, Department of Biochemistry, Lublin (Poland)

    2010-10-15

    Nanostructured bioelectrodes were designed and assembled into a biofuel cell with no separating membrane. The glassy carbon electrodes were modified with mediator-functionalized carbon nanotubes. Ferrocene (Fc) and 2,2{sup '}-azino-bis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) bound chemically to the carbon nanotubes were found useful as mediators of the enzyme catalyzed electrode processes. Glucose oxidase from Aspergillus niger AM-11 and laccase from Cerrena unicolor C-139 were incorporated in a liquid-crystalline matrix-monoolein cubic phase. The carbon nanotubes-nanostructured electrode surface was covered with the cubic phase film containing the enzyme and acted as the catalytic surface for the oxidation of glucose and reduction of oxygen. Thanks to the mediating role of derivatized nanotubes the catalysis was almost ten times more efficient than on the GCE electrodes: catalytic current of glucose oxidation was 1 mA cm{sup -2} and oxygen reduction current exceeded 0.6 mA cm{sup -2}. The open circuit voltage of the biofuel cell was 0.43 V. Application of carbon nanotubes increased the maximum power output of the constructed biofuel cell to 100 {mu}W cm{sup -2} without stirring of the solution which was ca. 100 times more efficient than using the same bioelectrodes without nanotubes on the electrode surface. (orig.)

  5. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    Energy Technology Data Exchange (ETDEWEB)

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. (Beckman Research Institute of the City of Hope, Duarte, CA (USA))

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  6. Enzymatic electrodes nanostructured with functionalized carbon nanotubes for biofuel cell applications.

    Science.gov (United States)

    Nazaruk, E; Sadowska, K; Biernat, J F; Rogalski, J; Ginalska, G; Bilewicz, R

    2010-10-01

    Nanostructured bioelectrodes were designed and assembled into a biofuel cell with no separating membrane. The glassy carbon electrodes were modified with mediator-functionalized carbon nanotubes. Ferrocene (Fc) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) bound chemically to the carbon nanotubes were found useful as mediators of the enzyme catalyzed electrode processes. Glucose oxidase from Aspergillus niger AM-11 and laccase from Cerrena unicolor C-139 were incorporated in a liquid-crystalline matrix-monoolein cubic phase. The carbon nanotubes-nanostructured electrode surface was covered with the cubic phase film containing the enzyme and acted as the catalytic surface for the oxidation of glucose and reduction of oxygen. Thanks to the mediating role of derivatized nanotubes the catalysis was almost ten times more efficient than on the GCE electrodes: catalytic current of glucose oxidation was 1 mA cm(-2) and oxygen reduction current exceeded 0.6 mA cm(-2). The open circuit voltage of the biofuel cell was 0.43 V. Application of carbon nanotubes increased the maximum power output of the constructed biofuel cell to 100 μW cm(-2) without stirring of the solution which was ca. 100 times more efficient than using the same bioelectrodes without nanotubes on the electrode surface.

  7. Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells.

    Science.gov (United States)

    Heldring, Nina; Joseph, Bertrand; Hermanson, Ola; Kioussi, Chrissa

    2012-11-01

    Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

  8. Expression in aneuploid Drosophila S2 cells.

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    2010-02-01

    Full Text Available Extensive departures from balanced gene dose in aneuploids are highly deleterious. However, we know very little about the relationship between gene copy number and expression in aneuploid cells. We determined copy number and transcript abundance (expression genome-wide in Drosophila S2 cells by DNA-Seq and RNA-Seq. We found that S2 cells are aneuploid for >43 Mb of the genome, primarily in the range of one to five copies, and show a male genotype ( approximately two X chromosomes and four sets of autosomes, or 2X;4A. Both X chromosomes and autosomes showed expression dosage compensation. X chromosome expression was elevated in a fixed-fold manner regardless of actual gene dose. In engineering terms, the system "anticipates" the perturbation caused by X dose, rather than responding to an error caused by the perturbation. This feed-forward regulation resulted in precise dosage compensation only when X dose was half of the autosome dose. Insufficient compensation occurred at lower X chromosome dose and excessive expression occurred at higher doses. RNAi knockdown of the Male Specific Lethal complex abolished feed-forward regulation. Both autosome and X chromosome genes show Male Specific Lethal-independent compensation that fits a first order dose-response curve. Our data indicate that expression dosage compensation dampens the effect of altered DNA copy number genome-wide. For the X chromosome, compensation includes fixed and dose-dependent components.

  9. Feeding strategies enhance high cell density cultivation and protein expression in milliliter scale bioreactors.

    Science.gov (United States)

    Faust, Georg; Janzen, Nils H; Bendig, Christoph; Römer, Lin; Kaufmann, Klaus; Weuster-Botz, Dirk

    2014-10-01

    Miniature bioreactors under parallel fed-batch operations are not only useful screening tools for bioprocess development but also provide a suitable basis for eventual scale-up. In this study, three feeding strategies were investigated: besides the established intermittent feeding by a liquid handler, an optimized microfluidic device and a new enzymatic release system were applied for parallel fed-batch cultivation of Escherichia coli HMS174(DE3) and BL21(DE3) strains in stirred-tank bioreactors on a 10 mL scale. Lower fluctuation in dissolved oxygen (DO) and higher optical densities were measured in fed-batch processes applying the microfluidic device or the enzymatic glucose/fructose release system (conversion of intermittently added sucrose by an invertase), but no difference in dry cell weights (DCW) were observed. With all three feeding strategies high cell densities were realized on a milliliter scale with final optical density measured at 600 nm (OD600 ) of 114-133 and final DCW concentrations of 69-70 g L(-1) . The effect of feeding strategies on the expression of two heterologous proteins was investigated. Whereas no impact was observed on the expression of the spider silk protein eADF4(C16), the fluorescence of enhanced green fluorescence protein (eGFP) was reproducibly lower, if an intermittent glucose feed was applied. Thus, the impact of feeding strategy on expression is strongly dependent on the E. coli strain and/or expressed protein. As a completely continuous feed supply is difficult to realize in miniature bioreactors, the enzymatic release approach from this study can be easily applied in all microfluidic system to reduce fluctuations of glucose supply and DO concentrations.

  10. Enzymatic Hydrolysis of Lignocelluloses

    DEFF Research Database (Denmark)

    Kolasa, Marta; Ahring, Birgitte Kiær; Lübeck, Peter Stephensen

    2010-01-01

    bonds. Cellulose can be degraded to simple sugar components by means of enzymatic hydrolysis. However, due to its complex, crystalline structure it is difficult to break it down and the cooperative action of a variety of cellulolytic enzymes is necessary. Fungi are known to have potential in production......Lignocellulosic materials form a huge part of the plant biomass from agricultural and forestry wastes. They consist of three major components: cellulose, hemicellulose and lignin. Cellulose, the main constituent of plant cell wall, is a polymer of D–glucopyranose units linked by β-1,4-glucosidic...... of a variety of cellulolytic enzymes. The aim of this work is to discover new thermostable and robust cellulolytic enzymes for improved enzymatic hydrolysis of biomass. For this purpose two screening methods are applied in different fungal strains with high cellulolytic activities: an expression–based method...

  11. Mediatorless sugar/oxygen enzymatic fuel cells based on gold nanoparticle-modified electrodes.

    Science.gov (United States)

    Wang, Xiaoju; Falk, Magnus; Ortiz, Roberto; Matsumura, Hirotoshi; Bobacka, Johan; Ludwig, Roland; Bergelin, Mikael; Gorton, Lo; Shleev, Sergey

    2012-01-15

    We report on the fabrication and characterisation of a gold-nanoparticle (AuNP)-based mediatorless sugar/oxygen biofuel cell (BFC) operating in neutral sugar-containing buffers and human physiological fluids, such as blood and plasma. First, Corynascus thermophilus cellobiose dehydrogenase (CtCDH) and Myrothecium verrucaria bilirubin oxidase (MvBOx), used as anodic and cathodic bioelements, respectively, were immobilised on gold electrodes modified with 20 nm AuNPs. Detailed characterisation and optimisation of a new CDH/AuNP-based bioanode were performed and the following fundamental parameters were obtained: (i) the redox potential of the haem-containing centre of the enzyme was measured to be 75 mV vs. NHE, (ii) the surface coverage of CtCDH was found to be 0.65 pmol cm(-2) corresponding to a sub-monolayer coverage of the thiol-modified AuNPs by the enzyme, (iii) a turnover number for CtCDH immobilised on thiol-modified AuNPs was calculated to be ca. 0.5 s(-1), and (iv) the maximal current densities as high as 40 μA cm(-2) were registered in sugar-containing neutral buffers. Second, both biomodified electrodes, namely the CtCDH/AuNP-based bioanode and the MvBOx/AuNP-based biocathode, were combined into a functional BFC and the designed biodevices were carefully investigated. The following characteristics of the mediator-, separator- and membrane-less, miniature BFC were obtained: in phosphate buffer; an open-circuit voltage of 0.68 V, a maximum power density of 15 μW cm(-2) at a cell voltage of 0.52 V and in human blood; an open-circuit voltage of 0.65 V, a maximum power density of 3 μW cm(-2) at a cell voltage of 0.45 V, respectively. The estimated half-lives of the biodevices were found to be >12, <8, and <2 h in a sugar-containing buffer, human plasma, and blood, respectively. The basic characteristics of mediatorless sugar/oxygen BFCs were significantly improved compared with previously designed biodevices, because of the usage of three-dimensional Au

  12. Enhanced performance of electrospun carbon fibers modified with carbon nanotubes: promising electrodes for enzymatic biofuel cells.

    Science.gov (United States)

    Engel, A Both; Cherifi, A; Tingry, S; Cornu, D; Peigney, A; Laurent, Ch

    2013-06-21

    New nanostructured electrodes, promising for the production of clean and renewable energy in biofuel cells, were developed with success. For this purpose, carbon nanofibers were produced by the electrospinning of polyacrylonitrile solution followed by convenient thermal treatments (stabilization followed by carbonization at 1000, 1200 and 1400° C), and carbon nanotubes were adsorbed on the surfaces of the fibers by a dipping method. The morphology of the developed electrodes was characterized by several techniques (SEM, Raman spectroscopy, electrical conductivity measurement). The electrochemical properties were evaluated through cyclic voltammetry, where the influence of the carbonization temperature of the fibers and the beneficial contribution of the carbon nanotubes were observed through the reversibility and size of the redox peaks of K3Fe(CN)6 versus Ag/AgCl. Subsequently, redox enzymes were immobilized on the electrodes and the electroreduction of oxygen to water was realized as a test of their efficiency as biocathodes. Due to the fibrous and porous structure of these new electrodes, and to the fact that carbon nanotubes may have the ability to promote electron transfer reactions of redox biomolecules, the new electrodes developed were capable of producing higher current densities than an electrode composed only of electrospun carbon fibers.

  13. Engineering of pyranose dehydrogenase for application to enzymatic anodes in biofuel cells.

    Science.gov (United States)

    Yakovleva, Maria E; Gonaus, Christoph; Schropp, Katharina; ÓConghaile, Peter; Leech, Dónal; Peterbauer, Clemens K; Gorton, Lo

    2015-04-14

    In the search for improved glucose oxidising enzymes for biofuel cells, a number of Agaricus meleagris (Am) pyranose dehydrogenase mutants (mPDHs) exhibiting different degrees of glycosylation were produced using site-directed mutagenesis and electrochemically characterised. The response of electrodes modified with different mPDHs is compared in a mediated electron transfer mode, where the electrodes are modified with each of the mutants covalently attached to redox polymers based on polyvinylimidazole-bound osmium complexes using a cross-linking agent. Coating of each of the enzymes onto the graphite electrode surface is also used to screen for their capacity for direct electron transfer. The double mutant PDH exhibits the highest response to glucose at physiological pH in both direct and mediated electron transfer modes, producing a Jmax of ≈800 μA cm(-2) at room temperature and when "wired" to the Os-polymer having the highest formal potential. From the results obtained the double mPDH is proposed as the most suitable candidate for application to bioanode fabrication.

  14. Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression

    Directory of Open Access Journals (Sweden)

    Ana Carolina Irioda

    2016-01-01

    Full Text Available Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d, colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d, cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

  15. Melanopsin expressing human retinal ganglion cells

    DEFF Research Database (Denmark)

    Hannibal, Jens; Christensen, Anders Tolstrup; Heegaard, Steffen

    2017-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex and sleep/wake cycles. The different functions seem...

  16. Multiple enzymatic defects in mitochondria in hematological cells of patients with primary sideroblastic anemia.

    Science.gov (United States)

    Aoki, Y

    1980-07-01

    Activities of mitochondrial enzymes in blood cells from 69 patients with primary sideroblastic anemia were determined to elucidate the pathogenesis of the disease. In erythroblasts of patients with primary acquired type the activities of both delta-aminolevulinic acid synthetase and mitochondrial serine protease were inevitably decreased. The susceptibility to the protease of apo-delta-aminolevulinic acid synthetase prepared from erythroblasts of patients with this type was within the normal range, in contrast to that of pyridoxine-responsive anemia. The activities of mitochondrial enzymes such as cytochrome oxidase, serine protease, and oligomycin-sensitive ATPase, except citrate synthetase, were usually decreased in mature granulocytes of the patients. Patients with hereditary sideroblastic anemia also had decreased delta-aminolevulinic acid synthetase activity in erythroblasts, and decreased serine protease activity in both erythroblasts and mature granulocytes. Mature granulocytes obtained from patients with pyridoxine-responsive anemia before therapy had decreased cytochrome oxidase activity, however, the activity increased to a normal level when the patients were in remission. The activities of other mitochondrial enzymes in mature granulocytes were within normal range in these patients before pyridoxine therapy. The activities of these mitochondrial enzymes in lymphocytes were within normal range in all groups of patients with primary sideroblastic anemia. We suggest that patients with primary acquired, and possibly also those with hereditary sideroblastic anemia have impaired mitochondrial function in both erythroblasts and granulocytes. That only anemia is observed in these patients is because a functional abnormality of mitochondria in erythroblasts is most important because of the role of mitochondria in the formation of heme in erythrocyte development. In contrast to these two types of sideroblastic anemia, only delta-aminolevulinic acid synthetase in

  17. Dental enamel cells express functional SOCE channels.

    Science.gov (United States)

    Nurbaeva, Meerim K; Eckstein, Miriam; Concepcion, Axel R; Smith, Charles E; Srikanth, Sonal; Paine, Michael L; Gwack, Yousang; Hubbard, Michael J; Feske, Stefan; Lacruz, Rodrigo S

    2015-10-30

    Dental enamel formation requires large quantities of Ca(2+) yet the mechanisms mediating Ca(2+) dynamics in enamel cells are unclear. Store-operated Ca(2+) entry (SOCE) channels are important Ca(2+) influx mechanisms in many cells. SOCE involves release of Ca(2+) from intracellular pools followed by Ca(2+) entry. The best-characterized SOCE channels are the Ca(2+) release-activated Ca(2+) (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca(2+) uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca(2+) release mechanism. Passive depletion of ER Ca(2+) stores with thapsigargin resulted in a significant raise in [Ca(2+)]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca(2+) entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca(2+) uptake in enamel formation.

  18. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  19. Ultrasensitive detection of superoxide anion released from living cells using a porous Pt-Pd decorated enzymatic sensor.

    Science.gov (United States)

    Zhu, Xiang; Liu, Tingting; Zhao, Hongli; Shi, Libo; Li, Xiaoqing; Lan, Minbo

    2016-05-15

    Considering the critical roles of superoxide anion (O2(∙-)) in pathological conditions, it is of great urgency to establish a reliable and durable approach for real-time determination of O2(∙-). In this study, we propose a porous Pt-Pd decorated superoxide dismutase (SOD) sensor for qualitative and quantitative detection O2(∙-). The developed biosensor exhibits a fast, selective and linear amperometric response upon O2(∙-) in the concentration scope of 16 to 1,536 μM (R(2)=0.9941), with a detection limit of 0.13 μM (S/N=3) and a low Michaelis-Menten constant of 1.37 μM which indicating a high enzymatic activity and affinity to O2(∙-). Inspiringly, the proposed sensor possesses an ultrahigh sensitivity of 1270 μA mM(-1)cm(-2). In addition, SOD/porous Pt-Pd sensor exhibits excellent anti-interference property, reproducibility and long-term storage stability. Beyond our expectation, the trace level of O2(∙-) released from living cells has also been successfully captured. These satisfactory results are mainly ascribed to (1) the porous interface with larger surface area and more active sites to provide a biocompatible environment for SOD (2) the specific biocatalysis of SOD towards O2(∙-) and (3) porous Pt-Pd nanomaterials fastening the electron transfer. The superior electrochemical performance makes SOD/porous Pt-Pd sensor a promising candidate for monitoring the dynamic changes of O2(∙-)in vivo.

  20. Polymer-based protein engineering grown ferrocene-containing redox polymers improve current generation in an enzymatic biofuel cell.

    Science.gov (United States)

    Campbell, Alan S; Murata, Hironobu; Carmali, Sheiliza; Matyjaszewski, Krzysztof; Islam, Mohammad F; Russell, Alan J

    2016-12-15

    Enzymatic biofuel cells (EBFCs) are capable of generating electricity from physiologically present fuels making them promising power sources for the future of implantable devices. The potential application of such systems is limited, however, by inefficient current generation. Polymer-based protein engineering (PBPE) offers a unique method to tailor enzyme function through tunable modification of the enzyme surface with functional polymers. In this study, we report on the modification of glucose oxidase (GOX) with ferrocene-containing redox polymers to increase current generation efficiency in an enzyme-modified anode. Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide) (pFcAc) was grown from covalently attached, water-soluble initiator molecules on the surface of GOX in a "grafting-from" approach using atom transfer radical polymerization (ATRP). The covalently-coupled ferrocene-containing polymers on the enzyme surface promoted the effective "wiring" of the GOX active site to an external electrode. The resulting GOX-pFcAc conjugates generated over an order of magnitude increase in current generation efficiency and a 4-fold increase in maximum EBFC power density (≈1.7µWcm(-2)) with similar open circuit voltage (0.27V) compared to native GOX when physically adsorbed onto paddle-shaped electrodes made up of electrospun polyacrylonitrile fibers coated with gold nanoparticles and multi-wall carbon nanotubes. The formation of electroactive enzyme-redox polymer conjugates using PBPE represents a powerful new tool for the improvement of mediated enzyme-based bioelectronics without the need for free redox mediators or anode/cathode compartmentalization. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Omission and Resupply of Nitrogen Affect Physiological and Enzymatic Activities and the Gene Expression of Eucalypt Clones

    Directory of Open Access Journals (Sweden)

    Loane Vaz Fernandes

    Full Text Available ABSTRACT: The mineral nutrient uptake of plants in the field occurs in pulses, due to variations in the substance concentrations at the root surface. The fluctuations in nutrient supply probably induce changes in the plant, which are to date unknown for Eucalyptus. This study evaluated these changes in plant growth, nutritional status, photosynthesis, and gene expression, which can serve as biomarkers of the nitrogen status, of four eucalypt clones exposed to N omission and resupply. A greenhouse experiment with four Eucalyptus clones was installed, and after initial growth exposed to N omission for 21 d, followed by N resupply in nutrient solution for 14 d. Nitrogen omission decreased the total N and photosynthetic pigments, net photosynthesis and photochemical dissipation, and increased enzyme activity especially in leaves and the gene expression in leaves and roots. Nitrogen resupply decreased these variations, indicating recovery. The total N concentration was highly and significantly correlated with net photosynthesis, enzyme activity, expression of genes GS2;1 and Gln1;3 in the leaves and AMT1;2 in the roots, contents of chlorophyll a and b, and photochemical energy dissipation. The enzymes GS and NR in the leaves and the genes AMT1;2, GS2;1 and Gln1;3 proved to be sensitive N indicators.

  2. Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

    Science.gov (United States)

    Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng

    2015-10-01

    Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 μmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 μmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

  3. Angiogenesis gene expression in murine endothelial cells during post-pneumonectomy lung growth

    Directory of Open Access Journals (Sweden)

    Konerding Moritz A

    2011-07-01

    Full Text Available Abstract Although blood vessel growth occurs readily in the systemic bronchial circulation, angiogenesis in the pulmonary circulation is rare. Compensatory lung growth after pneumonectomy is an experimental model with presumed alveolar capillary angiogenesis. To investigate the genes participating in murine neoalveolarization, we studied the expression of angiogenesis genes in lung endothelial cells. After left pneumonectomy, the remaining right lung was examined on days 3, 6, 14 and 21days after surgery and compared to both no surgery and sham thoracotomy controls. The lungs were enzymatically digested and CD31+ endothelial cells were isolated using flow cytometry cell sorting. The transcriptional profile of the CD31+ endothelial cells was assessed using quantitative real-time polymerase chain reaction (PCR arrays. Focusing on 84 angiogenesis-associated genes, we identified 22 genes with greater than 4-fold regulation and significantly enhanced transcription (p

  4. Toward stable gene expression in CHO cells

    Science.gov (United States)

    Mariati; Koh, Esther YC; Yeo, Jessna HM; Ho, Steven CL; Yang, Yuansheng

    2014-01-01

    Maintaining high gene expression level during long-term culture is critical when producing therapeutic recombinant proteins using mammalian cells. Transcriptional silencing of promoters, most likely due to epigenetic events such as DNA methylation and histone modifications, is one of the major mechanisms causing production instability. Previous studies demonstrated that the core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene is effective to prevent DNA methylation. We generated one set of modified human cytomegalovirus (hCMV) promoters by insertion of one or two copies of IE in either forward or reverse orientations into different locations of the hCMV promoter. The modified hCMV with one copy of IE inserted between the hCMV enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability in CHO cells without comprising expression level when compared with the wild type hCMV. We also found that insertion of IE into a chimeric murine CMV (mCMV) enhancer and human elongation factor-1α core (hEF) promoter in reverse orientation did not enhance expression stability, indicating that the effect of IE on expression stability is possibly promoter specific. PMID:25482237

  5. MEMBRANE LEc EXPRESSION IN BREAST CANCER CELLS

    Directory of Open Access Journals (Sweden)

    Ya. A. Udalova

    2009-01-01

    Full Text Available Affine chromatography was used to isolate Lec antibodies from the sera of a healthy female donor with the high titers of these anti- bodies, which were labeled with biotin. The study enrolled 51 patients with primary breast cancer (BC. Antigen expression was found by immunohistochemistry and flow cytometry. With these two techniques being used, the detection rate of Lec expression in BC cells was 65% (33/51; the antigen was most frequently found by flow cytometry as compared with immunohistochemistry: 72 and 58% of cases, respectively.

  6. Optogenetics for gene expression in mammalian cells.

    Science.gov (United States)

    Müller, Konrad; Naumann, Sebastian; Weber, Wilfried; Zurbriggen, Matias D

    2015-02-01

    Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

  7. Upregulated expression of indoleamine 2, 3-dioxygenase in CHO cells induces apoptosis of competent T cells and increases proportion of Treg cells.

    Science.gov (United States)

    Sun, Jingyan; Yu, Jinpu; Li, Hui; Yang, Lili; Wei, Feng; Yu, Wenwen; Liu, Juntian; Ren, Xiubao

    2011-09-14

    The inflammatory enzyme indoleamine 2, 3-dioxygenase (IDO) participates in immune tolerance and promotes immune escape of IDO+ tumors. A recent hypothesis suggested that IDO may contribute to the differentiation of new T regulatory cells (Tregs) from naive CD4+ T cells. In this study we investigated the role of IDO in induction of immunosuppression in breast cancer by increasing the apoptosis of T cells and the proportion of Tregs. An IDO expression plasmid was constructed and Chinese hamster ovary (CHO) cells were stably transfected with human IDO. Purified CD3+ T cells were isolated from the peripheral blood monouclear cells of breast cancer patients. After co-culturing IDO expressing or untransfected (control) CHO cells with T cells, T cells apoptosis were determined by flow cytometry analysis and annexin-V and PI staining. The proportion of the regulatory T cell (Tregs [CD4 + CD25 + CD127⁻]) subset was measured by flow cytometry analysis. T cells total RNA and cellular protein samples were isolated for detecting Foxp3 gene and protein expression. IDO transgenic CHO cells yielded high levels of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in 79.07 ± 8.13% of CD3+T cells after co-cultured with IDO+ CHO cells for 3 days and the proportion of CD4 + CD25 + CD127⁻ T cells increased from 3.43 ± 1.07% to 8.98 ± 1.88% (P Tregs in vitro. Increased expression of Foxp3, a key molecular marker of Tregs, was confirmed by RT-PCR, real-time RT-PCR and Western blot analysis at the same time. These results suggest that IDO helps to create a tolerogenic milieu in breast tumors by directly inducing T cell apoptosis and enhancing Treg-mediated immunosuppression.

  8. Heterologous expression, purification, and enzymatic characterization of the acyclic carotenoid 1,2-hydratase from Rubrivivax gelatinosus.

    Science.gov (United States)

    Steiger, Sabine; Mazet, Andreas; Sandmann, Gerhard

    2003-06-01

    The carotenoid 1,2-hydratase CrtC from Rubrivivax gelatinosus has been expressed in Escherichia coli in an active form and purified by affinity chromatography. The enzyme catalyzes the conversion of various acyclic carotenes including 1-hydroxy derivatives. This broad substrate specificity reflects the participation of CrtC in 1'-HO-spheroidene and in spirilloxanthin biosynthesis. Enzyme kinetic studies including the determination of substrate specificity constants indicate that among the alternative biosynthetic routes to 1'-HO-spheroidene the one via spheroidene is the dominating pathway. In contrast to CrtC from Rvi. gelatinosus, the equivalent enzyme from Rhodobacter capsulatus, a closely related bacterium which lacks the biosynthetic branch to spirilloxanthin and accumulates spheroidene instead of substantial amounts of 1'-HO-spheroidene, is extremely poor in converting 1-HO-carotenoids. The individual catalytic properties of both carotenoid 1,2-hydratases reflect the in situ carotenogenic pathways in both purple photosynthetic bacteria.

  9. Direct Cell Lysis for Single-Cell Gene Expression Profiling

    OpenAIRE

    David eSvec; Daniel eAndersson; Milos ePekny; Robert eSjöback; Mikael eKubista; Anders eStåhlberg

    2013-01-01

    The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously express...

  10. DNA methylation analysis in the intestinal epithelium-effect of cell separation on gene expression and methylation profile.

    Directory of Open Access Journals (Sweden)

    Andreas C Jenke

    Full Text Available BACKGROUND: Epigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore desirable for all epigenetic studies. However, to date little information is available on whether separation protocols might influence epigenetic and/or gene expression signatures and hence might be less beneficial. We investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (IECs from 6 healthy individuals. MATERIALS AND METHODS: Epithelial cells were isolated from small bowel (i.e. terminal ileum biopsies using EDTA/DTT and enzymatic release followed by magnetic bead sorting via EPCAM labeled microbeads. Effects on gene/mRNA expression were analyzed using a real time PCR based expression array. DNA methylation was assessed by pyrosequencing of bisulfite converted DNA and methylated DNA immunoprecipitation (MeDIP. RESULTS: While cell purity was >95% using both cell separation approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable and only revealed subtle differences. Comparison of DNA methylation of the epithelial cell marker EPCAM in unseparated whole biopsy samples with separated epithelium (i.e. EPCAM positive and negative fraction demonstrated significant differences in DNA methylation between all three tissue fractions indicating cell type specific methylation patterns can be masked in unseparated tissue samples. CONCLUSIONS: Taken together, our data highlight the importance of considering the potential effect of cell separation on gene expression as well as DNA methylation signatures. The decision to separate tissue samples will therefore depend on study design and specific separation protocols.

  11. Enzymatic characterization of a novel Xaa-Pro aminopeptidase XpmA from Aspergillus oryzae expressed in Escherichia coli.

    Science.gov (United States)

    Matsushita-Morita, Mayumi; Tada, Sawaki; Suzuki, Satoshi; Hattori, Ryota; Kusumoto, Ken-Ichi

    2017-07-08

    Xaa-Pro aminopeptidases are peptidases responsible for the cleavage of any amino acid N-terminally adjacent to a proline residue. We identified a gene encoding a putative Xaa-Pro aminopeptidase in the genome of the filamentous fungus Aspergillus oryzae (genome database number: AO090701000720) and named this gene xpmA. We produced its enzyme in a C-terminally His6-tag-fused form in an Escherichia coli expression system and purified it. The purified recombinant XpmA (rXpmA) showed hydrolysis activity toward Xaa-Pro-oligopeptides, especially the two dipeptides Ala-Pro and Phe-Pro. The molecular weight of rXpmA was estimated to be 69 kDa by SDS-PAGE and 126 kDa by gel filtration, suggesting that it is a homodimer. The enzyme was activated by various divalent metal ions such as Mn(2+), Co(2+), and Mg(2+); in particular, the enzyme activity was increased 27.6-times relative to the no-addition control by 1 mM Mn(2+). Additionally, 10 mM EDTA suppressed its activity to 0.26-times of the control level. Therefore, rXpmA was a metalloprotease. Optimal hydrolytic activity of rXpmA was observed at 50°C and pH 8.5-9.0. The enzyme was stable up to 50°C and from pH 4.0 to 11.0. rXpmA showed substrate inhibition by Leu-Pro, Ser-Pro and Arg-Pro at concentrations over 4 mM, 10 mM, and 3 mM, respectively. NaCl increased the enzyme activity in the concentration range 0.5-3.0 M, suggesting that the enzyme is halophilic. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  12. Upregulated expression of indoleamine 2, 3-dioxygenase in CHO cells induces apoptosis of competent T cells and increases proportion of Treg cells

    Directory of Open Access Journals (Sweden)

    Liu Juntian

    2011-09-01

    Full Text Available Abstract Introduction The inflammatory enzyme indoleamine 2, 3-dioxygenase (IDO participates in immune tolerance and promotes immune escape of IDO+ tumors. A recent hypothesis suggested that IDO may contribute to the differentiation of new T regulatory cells (Tregs from naive CD4+ T cells. In this study we investigated the role of IDO in induction of immunosuppression in breast cancer by increasing the apoptosis of T cells and the proportion of Tregs. Methods An IDO expression plasmid was constructed and Chinese hamster ovary (CHO cells were stably transfected with human IDO. Purified CD3+ T cells were isolated from the peripheral blood monouclear cells of breast cancer patients. After co-culturing IDO expressing or untransfected (control CHO cells with T cells, T cells apoptosis were determined by flow cytometry analysis and annexin-V and PI staining. The proportion of the regulatory T cell (Tregs [CD4 + CD25 + CD127-] subset was measured by flow cytometry analysis. T cells total RNA and cellular protein samples were isolated for detecting Foxp3 gene and protein expression. Results IDO transgenic CHO cells yielded high levels of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in 79.07 ± 8.13% of CD3+T cells after co-cultured with IDO+ CHO cells for 3 days and the proportion of CD4 + CD25 + CD127- T cells increased from 3.43 ± 1.07% to 8.98 ± 1.88% (P Conclusions These results suggest that IDO helps to create a tolerogenic milieu in breast tumors by directly inducing T cell apoptosis and enhancing Treg-mediated immunosuppression.

  13. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  14. Ammonia metabolism capacity of HepG2 cells with high expression of human glutamine synthetase

    Institute of Scientific and Technical Information of China (English)

    Nan-Hong Tang; Xiao-Qian Wang; Xiu-Jin Li; Yan-Ling Chen

    2008-01-01

    BACKGROUND:Currently, one of the tough problems for the application of bioartiifcial liver (BAL) is the shortage of suitable hepatocytes. There are reports on different types of BAL assistance developed with porcine hepatocytes and HepG2 C3A cells, but their defects are obvious. In recent years, some studies focus more on liver cells with features of human origin and improved detoxiifcation. In this study, a hepatocyte line with high expression of human glutamine synthetase (hGS) was raised and its capacity for ammonia metabolism was investigated. METHODS:hGS cDNA and alpha-fetoprotein transcription regulatory element (AFP-TRE) were cloned with the designed primers. The eukaryotic expression vectors, pLNChGS and pLNAFhGS, were constructed and transfected into PA317 cells. Recombinant retroviruses (Retro-hGS and Retro-AFhGS) were produced and then infected into HepG2 cells. G418-resistant cell clones, HepG2/pLNChGS and HepG2/pLNAFhGS, were selected and ampliifed. Then hGS mRNA was measured by semi-quantitative RT-PCR;hGS enzymatic activity and ammonia metabolism analysis in different concentration of NH4+were detected with a quantitative biochemistry kit. The cell proliferation was also detected by MTT chromatometry. RESULTS:The expression of hGS mRNA in HepG2/pLNChGS cells (8.306±0.336) and HepG2/pLNAFhGS cells (21.358±1.716) was much stronger than in control cells (P CONCLUSION:The constructed hepatocytes (HepG2 cells) with speciifc high-expression of hGS have a powerful ability to degrade ammonia in vitro, and provide necessary experimental data for the selection of biomaterials in BAL.

  15. Coupling of an enzymatic biofuel cell to an electrochemical cell for self-powered glucose sensing with optical readout.

    Science.gov (United States)

    Pinyou, Piyanut; Conzuelo, Felipe; Sliozberg, Kirill; Vivekananthan, Jeevanthi; Contin, Andrea; Pöller, Sascha; Plumeré, Nicolas; Schuhmann, Wolfgang

    2015-12-01

    A miniaturized biofuel cell (BFC) is powering an electrolyser invoking a glucose concentration dependent formation of a dye which can be determined spectrophotometrically. This strategy enables instrument free analyte detection using the analyte-dependent BFC current for triggering an optical read-out system. A screen-printed electrode (SPE) was used for the immobilization of the enzymes glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) for the biocatalytic oxidation of glucose and reduction of molecular oxygen, respectively. The miniaturized BFC was switched-on using small sample volumes (ca. 60 μL) leading to an open-circuit voltage of 567 mV and a maximal power density of (6.8±0.6) μW cm(-2). The BFC power was proportional to the glucose concentration in a range from 0.1 to 1.0 mM (R(2)=0.991). In order to verify the potential instrument-free analyte detection the BFC was directly connected to an electrochemical cell comprised of an optically-transparent SPE modified with methylene green (MG). The reduction of the electrochromic reporter compound invoked by the voltage and current flow applied by the BFC let to MG discoloration, thus allowing the detection of glucose.

  16. Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To investigate the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cell lines,RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a basis for the chemoprevention of ovarian cancer.

  17. EXPRESSION OF Fas LIGAND IN HUMAN COLON CANCER CELL LINES

    Institute of Scientific and Technical Information of China (English)

    张建军; 丁尔迅; 王强; 陈学云; 付志仁

    2001-01-01

    To investigate the expression of Fas ligand in human colon carcinoma cell lines. Methods: A total of six human colon cancer cell lines were examined for the expression of Fas ligand mRNA and cell surface protein by using RT-PCR and flow cytometry respectively. Results: The results showed that Fas ligand mRNA was expressed in all of the six cancer cell lines and Fas ligand cell surface protein was expressed in part of them. Conclusion: These data suggest that Fas ligand was expressed, at least in part, in human colon cancer cell lines and might facilitate to escape from immune surveillance of the host.

  18. Direct cell lysis for single-cell gene expression profiling

    Directory of Open Access Journals (Sweden)

    David eSvec

    2013-11-01

    Full Text Available The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA to be the best lysis agent, resulting in efficient cell lysis, high RNA stability and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single cells as well as samples composed of small numbers of cells.

  19. ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.

    Science.gov (United States)

    Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O

    2016-08-02

    ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.

  20. Gene expression profiles in irradiated cancer cells

    Science.gov (United States)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C.

    2013-07-01

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  1. Gene expression profiles in irradiated cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Minafra, L.; Bravatà, V.; Russo, G.; Ripamonti, M.; Gilardi, M. C. [IBFM CNR - LATO, Cefalù, Segrate (Italy)

    2013-07-26

    Knowledge of the molecular and genetic mechanisms underlying cellular response to radiation may provide new avenues to develop innovative predictive tests of radiosensitivity of tumours and normal tissues and to improve individual therapy. Nowadays very few studies describe molecular changes induced by hadrontherapy treatments, therefore this field has to be explored and clarified. High-throughput methodologies, such as DNA microarray, allow us to analyse mRNA expression of thousands of genes simultaneously in order to discover new genes and pathways as targets of response to hadrontherapy. Our aim is to elucidate the molecular networks involved in the sensitivity/resistance of cancer cell lines subjected to hadrontherapy treatments with a genomewide approach by using cDNA microarray technology to identify gene expression profiles and candidate genes responsible of differential cellular responses.

  2. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    Directory of Open Access Journals (Sweden)

    Kimitoshi Kohno

    2011-10-01

    Full Text Available We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143 regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1, aurora kinase B (AURKB and some minichromosome maintenance complex components (MCM. However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  3. Forced Expression of ZNF143 Restrains Cancer Cell Growth

    Energy Technology Data Exchange (ETDEWEB)

    Izumi, Hiroto, E-mail: h-izumi@med.uoeh-u.ac.jp; Yasuniwa, Yoshihiro; Akiyama, Masaki; Yamaguchi, Takahiro; Kuma, Akihiro; Kitamura, Noriaki; Kohno, Kimitoshi [Department of Molecular Biology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 (Japan)

    2011-10-19

    We previously reported that the transcription factor Zinc Finger Protein 143 (ZNF143) regulates the expression of genes associated with cell cycle and cell division, and that downregulation of ZNF143 induces cell cycle arrest at G2/M. To assess the function of ZNF143 expression in the cell cycle, we established two cells with forced expression of ZNF143 derived from PC3 prostate cancer cell lines. These cell lines overexpress genes associated with cell cycle and cell division, such as polo-like kinase 1 (PLK1), aurora kinase B (AURKB) and some minichromosome maintenance complex components (MCM). However, the doubling time of cells with forced expression of ZNF143 was approximately twice as long as its control counterpart cell line. Analysis following serum starvation and re-seeding showed that PC3 cells were synchronized at G1 in the cell cycle. Also, ZNF143 expression fluctuated, and was at its lowest level in G2/M. However, PC3 cells with forced expression of ZNF143 synchronized at G2/M, and showed lack of cell cycle-dependent fluctuation of nuclear expression of MCM proteins. Furthermore, G2/M population of both cisplatin-resistant PCDP6 cells over-expressing ZNF143 (derived from PC3 cells) and cells with forced expression of ZNF143 was significantly higher than that of each counterpart, and the doubling time of PCDP6 cells is about 2.5 times longer than that of PC3 cells. These data suggested that fluctuations in ZNF143 expression are required both for gene expression associated with cell cycle and for cell division.

  4. Gastrin gene expression and regulation in rat islet cell lines.

    Science.gov (United States)

    Brand, S J; Wang, T C

    1988-11-15

    Gastrin gene expression was observed in two permanent rat insulinoma (RIN) cell lines derived from a rat insulinoma. Gastrin expression was selective; highest expression was seen in a cell line which did not express other islet cell hormones. Gastrin mRNA transcription initiated from the same promoter as antral gastrin mRNA. DNA transfection studies with a gastrin chloramphenicol acetyltransferase chimeric gene showed higher expression in gastrin-expressing RIN cells than non-gastrin-expressing islet cells. This implies that gastrin-expressing RIN cells selectively express a trans-acting transcriptional activator which binds to cis-acting regulatory sequences within the 5'-flanking DNA sequence and first exon of the gastrin gene. The gastrin peptide precursor synthesized in these RIN cell lines is subject to the same repertoire of posttranslational modifications within the cell's secretory apparatus (endoproteolytic cleavage, tyrosine sulfation, and C-terminal amidation) as seen in antral G cells. Gastrin mRNA levels in these RIN cells were selectively increased by increasing the extracellular calcium concentration. Membrane depolarization also stimulated gastrin mRNA levels, probably through activation of voltage-sensitive calcium channels. Thus, these gastrin-expressing RIN cell lines provide permanent cell lines useful in analyzing the cellular regulation of gastrin gene expression.

  5. Expression of basal cell keratins in human prostate cancer metastases and cell lines.

    NARCIS (Netherlands)

    Leenders, G.J.L.H. van; Aalders, M.W.; Hulsbergen-van de Kaa, C.A.; Ruiter, D.J.; Schalken, J.A.

    2001-01-01

    Within normal human prostate epithelium, basal and luminal cells can be discriminated by their expression of keratins (K). While basal cells express K5/14, luminal cells show expression of K8/18 and an intermediate cell population can be identified by co-expression of K5/18. Prostate cancer is predo

  6. T cells stimulate catabolic gene expression by the stromal cells from giant cell tumor of bone

    Energy Technology Data Exchange (ETDEWEB)

    Cowan, Robert W. [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Ghert, Michelle [Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada); Department of Surgery, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Singh, Gurmit, E-mail: gurmit.singh@jcc.hhsc.ca [Department of Pathology and Molecular Medicine, McMaster University, 1280 Main St. W., Hamilton, ON, Canada L8S 4L8 (Canada); Juravinski Cancer Centre, 699 Concession St., Hamilton, ON, Canada L8V 5C2 (Canada)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Two T cell lines stimulate PTHrP, RANKL, MMP13 gene expression in GCT cell cultures. Black-Right-Pointing-Pointer CD40 expressed by stromal cells; CD40L detected in whole tumor but not cultures. Black-Right-Pointing-Pointer Effect of CD40L treatment on GCT cells increased PTHrP and MMP13 gene expression. Black-Right-Pointing-Pointer PTHrP treatment increased MMP13 expression, while inhibition decreased expression. Black-Right-Pointing-Pointer T cells may stimulate GCT stromal cells and promote the osteolysis of the tumor. -- Abstract: The factors that promote the localized bone resorption by giant cell tumor of bone (GCT) are not fully understood. We investigated whether T cells could contribute to bone resorption by stimulating expression of genes for parathyroid hormone-related protein (PTHrP), matrix metalloproteinase (MMP)-13, and the receptor activator of nuclear-factor {kappa}B ligand (RANKL). Two cell lines, Jurkat clone E6-1 and D1.1, were co-cultured with isolated GCT stromal cells. Real-time PCR analyses demonstrated a significant increase of all three genes following 48 h incubation, and PTHrP and MMP-13 gene expression was also increased at 24 h. Further, we examined the expression of CD40 ligand (CD40L), a protein expressed by activated T cells, and its receptor, CD40, in GCT. Immunohistochemistry results revealed expression of the CD40 receptor in both the stromal cells and giant cells of the tumor. RNA collected from whole GCT tissues showed expression of CD40LG, which was absent in cultured stromal cells, and suggests that CD40L is expressed within GCT. Stimulation of GCT stromal cells with CD40L significantly increased expression of the PTHrP and MMP-13 genes. Moreover, we show that inhibition of PTHrP with neutralizing antibodies significantly decreased MMP13 expression by the stromal cells compared to IgG-matched controls, whereas stimulation with PTHrP (1-34) increased MMP-13 gene expression. These

  7. The expression of different superoxide dismutase forms is cell-type dependent in olive (Olea europaea L.) leaves.

    Science.gov (United States)

    Corpas, Francisco J; Fernández-Ocaña, Ana; Carreras, Alfonso; Valderrama, Raquel; Luque, Francisco; Esteban, Francisco J; Rodríguez-Serrano, María; Chaki, Mounira; Pedrajas, José R; Sandalio, Luisa M; del Río, Luis A; Barroso, Juan B

    2006-07-01

    Superoxide dismutase (SOD) is a key antioxidant enzyme present in prokaryotic and eukaryotic cells as a first line of defense against the accumulation of superoxide radicals. In olive leaves, the SOD enzymatic system was characterized and was found to be comprised of three isozymes, an Mn-SOD, an Fe-SOD and a CuZn-SOD. Transcript expression analysis of whole leaves showed that the three isozymes represented 82, 17 and 0.8% of the total SOD expressed, respectively. Using the combination of laser capture microdissection (LCM) and real-time quantitative reverse transcription-PCR (RT-PCR), the expression of these SOD isozymes was studied in different cell types of olive leaves, including spongy mesophyll, palisade mesophyll, xylem and phloem. In spongy mesophyll cells, the isozyme proportion was similar to that in whole leaves, but in the other cells the proportion of expressed SOD isozymes was different. In palisade mesophyll cells, Fe-SOD was the most abundant, followed by Mn-SOD and CuZn-SOD, but in phloem cells Mn-SOD was the most prominent isozyme, and Fe-SOD was present in trace amounts. In xylem cells, only the Mn-SOD was detected. On the other hand, the highest accumulation of superoxide radicals was localized in vascular tissue which was the tissue with the lowest level of SOD transcripts. These data show that in olive leaves, each SOD isozyme has a different gene expression depending on the cell type of the leaf.

  8. Membraneless enzymatic ethanol/O2 fuel cell: Transitioning from an air-breathing Pt-based cathode to a bilirubin oxidase-based biocathode

    Science.gov (United States)

    Aquino Neto, Sidney; Milton, Ross D.; Hickey, David P.; De Andrade, Adalgisa R.; Minteer, Shelley D.

    2016-08-01

    The bioelectrooxidation of ethanol was investigated in a fully enzymatic membraneless ethanol/O2 biofuel cell assembly using hybrid bioanodes containing multi-walled carbon nanotube (MWCNT)-decorated gold metallic nanoparticles with either a pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) enzyme or a nicotinamide adenine dinucleotide (NAD+)-dependent ADH enzyme. The biofuel cell anode was prepared with the PQQ-dependent enzyme and designed using either a direct electron transfer (DET) architecture or via a mediated electron transfer (MET) configuration through a redox polymer, 1,1‧-dimethylferrocene-modified linear polyethyleneimine (FcMe2-C3-LPEI). In the case of the bioanode containing the NAD+-dependent enzyme, only the mediated electron transfer mechanism was employed using an electropolymerized methylene green film to regenerate the NAD+ cofactor. Regardless of the enzyme being employed at the anode, a bilirubin oxidase-based biocathode prepared within a DET architecture afforded efficient electrocatalytic oxygen reduction in an ethanol/O2 biofuel cell. The power curves showed that DET-based bioanodes via the PQQ-dependent ADH still lack high current densities, whereas the MET architecture furnished maximum power density values as high as 226 ± 21 μW cm-2. Considering the complete membraneless enzymatic biofuel cell with the NAD+-dependent ADH-based bioanode, power densities as high as 111 ± 14 μW cm-2 were obtained. This shows the advantage of PQQ-dependent ADH for membraneless ethanol/O2 biofuel cell applications.

  9. Gene expression profile of renal cell carcinoma clear cell type

    Directory of Open Access Journals (Sweden)

    Marcos F. Dall’Oglio

    2010-08-01

    Full Text Available PURPOSE: The determination of prognosis in patients with renal cell carcinoma (RCC is based, classically, on stage and histopathological aspects. The metastatic disease develops in one third of patients after surgery, even in localized tumors. There are few options for treating those patients, and even the new target designed drugs have shown low rates of success in controlling disease progression. Few studies used high throughput genomic analysis in renal cell carcinoma for determination of prognosis. This study is focused on the identification of gene expression signatures in tissues of low-risk, high-risk and metastatic RCC clear cell type (RCC-CCT. MATERIALS AND METHODS: We analyzed the expression of approximately 55,000 distinct transcripts using the Whole Genome microarray platform hybridized with RNA extracted from 19 patients submitted to surgery to treat RCC-CCT with different clinical outcomes. They were divided into three groups (1 low risk, characterized by pT1, Fuhrman grade 1 or 2, no microvascular invasion RCC; (2 high risk, pT2-3, Fuhrman grade 3 or 4 with, necrosis and microvascular invasion present and (3 metastatic RCC-CCT. Normal renal tissue was used as control. RESULTS: After comparison of differentially expressed genes among low-risk, high-risk and metastatic groups, we identified a group of common genes characterizing metastatic disease. Among them Interleukin-8 and Heat shock protein 70 were over-expressed in metastasis and validated by real-time polymerase chain reaction. CONCLUSION: These findings can be used as a starting point to generate molecular markers of RCC-CCT as well as a target for the development of innovative therapies.

  10. A bi-enzymatic whole cell conductometric biosensor for heavy metal ions and pesticides detection in water samples.

    Science.gov (United States)

    Chouteau, Celine; Dzyadevych, Sergei; Durrieu, Claude; Chovelon, Jean-Marc

    2005-08-15

    A conductometric biosensor using immobilised Chlorella vulgaris microalgae as bioreceptors was used as a bi-enzymatic biosensor. Algae were immobilised inside bovine serum albumin membranes reticulated with glutaraldehyde vapours deposited on interdigitated conductometric electrodes. Local conductivity variations caused by algae alkaline phosphatase and acetylcholinesterase activities could be detected. These two enzymes are known to be inhibited by distinct families of toxic compounds: heavy metals for alkaline phosphatase, carbamates and organophosphorous (OP) pesticides for acetylcholinesterase. The bi-enzymatic biosensors were tested to study the influence of heavy metal ions and pesticides on the corresponding enzyme. It has finally appeared that these biosensors are quite sensitive to Cd2+ and Zn2+ (limits of detection (LOD) = 10 ppb for a 30 min long exposure) while Pb2+ gives no significant inhibition as this ion seems to adsorb on albumin preferably. For pesticides, first experiments showed that paraoxon-methyl inhibits C. vulgaris AChE contrary to parathion-methyl and carbofuran. Biosensors were then exposed to different mixtures (Cd2+/Zn2+, Cd2+/paraoxon-methyl) but no synergetic or antagonist effect could be observed. A good repeatability could be achieve with biosensors since the relative standard deviation did not exceed 8% while response time was 5-7 min. A comparison between inhibition levels obtained with biosensors (after a 30 min long exposure) and bioassays (after a 240 min long exposure) has finally shown a similar LOD for both Cd and Zn (LOD = 10 ppb).

  11. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Carolyn A. de Graaf

    2016-09-01

    Full Text Available Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest.

  12. EXPRESSION OF IMMUNE-RELATED MOLECULES IN GLIOBLASTOMA MULTIFORM CELLS

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective: To investigate the expression of immune- related molecules in glioblastoma multiform(GBM) cells. Methods: The expression of major histocompatibility complex (MHC), β2-microglobulin, Fas, CD80 and CD86 molecules on the surface of GBM cells were evaluated by flow cytometry. The expression of TAP-1, TAP-2 and Tapasin in the GBM cells were evaluated by RT-PCR method. Results: MHC class Ⅰ, β2 microglobulin, TAP-1, TAP-2 and tapasin were expressed in most GBM cell lines. Except U87, there was no MHC class Ⅱ molecule expression on any of the other GBM cell lines. Fas was expressed on all the GBM cell lines examined. Conclusion: The mechanism by which GBM escapes immune surveillance may involve down regulation of expression of MHC class Ⅰ molecules and MHC class Ⅱ molecules. MHC class Ⅰpositive GBM may be the suitable target of immunotherapy.

  13. High expression of markers of apoptosis in Langerhans cell histiocytosis

    DEFF Research Database (Denmark)

    Petersen, Bodil Laub; Lundegaard, Pia Rengtved; Bank, M I;

    2003-01-01

    53 and the number of cells in apoptosis detected with TUNEL. Langerhans cell histiocytosis cells showed strong expression of p53 and in some cases co-expression of Fas and Fas-L. The expression of Fas-L was significantly higher in infiltrates from patients with single-system disease. The actual...... number of pathological Langerhans cells in apoptosis as estimated by TUNEL was low. CONCLUSIONS: The low number of TUNEL-reactive cells can be explained by the rapid turnover of apoptotic cells in the tissue, not leaving the apoptotic cells long enough in the tissue to be detected. The co...

  14. Collagen type I and decorin expression in tenocytes depend on the cell isolation method

    Directory of Open Access Journals (Sweden)

    Wagenhäuser Markus U

    2012-08-01

    Full Text Available Abstract Backround The treatment of rotator cuff tears is still challenging. Tendon tissue engineering (TTE might be an alternative in future. Tenocytes seem to be the most suitable cell type as they are easy to obtain and no differentiation in vitro is necessary. The aim of this study was to examine, if the long head of the biceps tendon (LHB can deliver viable tenocytes for TTE. In this context, different isolation methods, such as enzymatic digestion (ED and cell migration (CM, are investigated on differences in gene expression and cell morphology. Methods Samples of the LHB were obtained from patients, who underwent surgery for primary shoulder arthroplasty. Using ED as isolation method, 0.2% collagenase I solution was used. Using CM as isolation method, small pieces of minced tendon were put into petri-dishes. After cell cultivation, RT-PCR was performed for collagen type I, collagen type III, decorin, tenascin-C, fibronectin, Scleraxis, tenomodulin, osteopontin and agreccan. Results The total number of isolated cells, in relation to 1 g of native tissue, was 14 times higher using ED. The time interval for cell isolation was about 17 hours using ED and approximately 50 days using CM. Cell morphology in vitro was similar for both isolation techniques. Higher expression of collagen type I could be observed in tenocyte-like cell cultures (TLCC using ED as isolation method (p  Conclusion In summary tenocyte-like cells can be obtained with both isolation methods (ED and CM from the LHB. As no obvious disadvantage could be seen using ED, this method is more suitable for clinical use, as time for cell isolation is shorter and a remarkably higher number of cells can be obtained. However, both isolation methods can further be improved.

  15. Expression and characterization of an endo-1,4-β-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans

    DEFF Research Database (Denmark)

    Michalak, Malwina; Thomassen, Lise Vestergaard; Roytio, Henna

    2012-01-01

    was to use potato β-1,4-galactan and the SPPP as substrates for enzymatic production of potentially prebiotic compounds of lower and narrower molecular weight. A novel endo-1,4-β-galactanase from Emericella nidulans (anamorph Aspergillus nidulans), GH family 53, was produced in a recombinant Pichia pastoris...

  16. Effect of low frequency (LF) electric fields on gene expression of a bone human cell line.

    Science.gov (United States)

    Caputo, Mariella; Zirpoli, Hylde; De Rosa, Maria Caterina; Rescigno, Tania; Chiadini, Francesco; Scaglione, Antonio; Stellato, Claudia; Giurato, Giorgio; Weisz, Alessandro; Tecce, Mario Felice; Bisceglia, Bruno

    2014-12-01

    We evaluated the effects, on cultured human SaOS-2 cells, of exposures to the low frequency (LF) electric signal (60 kHz sinusoidal wave, 24.5 V peak-to-peak voltage, amplitude modulated by a 12.5 Hz square wave, 50% duty cycle) from an apparatus of current clinical use in bone diseases requiring regenerating processes. Cells in flasks were exposed to a capacitively coupled electric field giving electric current density in the sample of 4 µA/cm(2). The whole expressed cellular mRNAs were systematically analyzed by "DNA microchips" technology to identify all individual species quantitatively affected by field exposure. Comparisons were made between RNA samples from exposed and control sham-exposed cells. Results indicated that immediately and 4 h after exposure there were almost no differentially modulated mRNA species. However, samples obtained at 24 h after exposure showed a small number of limitedly differential signals (7 down-regulated and 3 up-regulated with a cut-off value of ±1.5; 38 and 11, respectively, with a cut-off value of ±1.3), which included mostly mRNA encoding transcription factors and DNA binding proteins. Nevertheless, in identical experimental conditions, we previously demonstrated enzymatic changes of alkaline phosphatase occurring immediately after exposure and declining in a few hours. Therefore, since enzymatic changes occur before those observed at gene regulation level, it is conceivable that only earlier effects are directly due the treatment and then these effects are later able to affect gene expression only indirectly.

  17. Apoptosis induction in human breast cancer (MCF-7) cells by a novel venom L-amino acid oxidase (Rusvinoxidase) is independent of its enzymatic activity and is accompanied by caspase-7 activation and reactive oxygen species production.

    Science.gov (United States)

    Mukherjee, Ashis K; Saviola, Anthony J; Burns, Patrick D; Mackessy, Stephen P

    2015-10-01

    We report the elucidation of a mechanism of apoptosis induction in breast cancer (MCF-7) cells by an L-amino acid oxidase (LAAO), Rusvinoxidase, purified from the venom of Daboia russelii russelii. Peptide mass fingerprinting analysis of Rusvinoxidase, an acidic monomeric glycoprotein with a mass of ~57 kDa, confirmed its identity as snake venom LAAO. The enzymatic activity of Rusvinoxidase was completely abolished after two cycles of freezing and thawing; however, its cytotoxicity toward MCF-7 cells remained unaffected. Dose- and time-dependent induction of apoptosis by Rusvinoxidase on MCF-7 cells was evident from changes in cell morphology, cell membrane integrity, shrinkage of cells and apoptotic body formation accompanied by DNA fragmentation. Rusvinoxidase induced apoptosis in MCF-7 cells by both the extrinsic (death-receptor) and intrinsic (mitochondrial) signaling pathways. The former pathway of apoptosis operated through activation of caspase-8 that subsequently activated caspase-7 but not caspase-3. Rusvinoxidase-induced intrinsic pathway of apoptosis was accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species, followed by a decrease in cellular glutathione content and catalase activity, and down-regulation of expression of anti-apoptotic proteins Bcl-XL and heat-shock proteins (HSP-90 and HSP-70). Rusvinoxidase treatment resulted in increase of the pro-apoptotic protein Bax, subsequently leading to the release of cytochrome c from mitochondria to the cytosol and activating caspase-9, which in turn stimulated effector caspase-7. Rusvinoxidase at a dose of 4 mg/kg was non-toxic in mice, indicating that it may be useful as a model for the development of peptide-based anticancer drugs.

  18. An in vitro evaluation of anti-aging effect of guluronic acid (G2013) based on enzymatic oxidative stress gene expression using healthy individuals PBMCs.

    Science.gov (United States)

    Taeb, Mahsa; Mortazavi-Jahromi, Shahabeddin; Jafarzadeh, Abdollah; Mirzaei, Mohammad Reza; Mirshafiey, Abbas

    2017-06-01

    Aging is usually associated with increased levels of oxidants, and may result in damages caused by oxidative stress. There is a direct relationship between aging and increased incidence of inflammatory diseases. The present research intended to study the anti-aging and anti-inflammatory effects of the drug G2013 (guluronic acid) at low and high doses on the genes expression of a number of enzymes involved in oxidative stress (including SOD2, GPX1, CAT, GST, iNOS, and MPO) in peripheral blood mononuclear cells (PBMCs) of healthy individuals under in vitro conditions. Venous blood samples were taken from 20 healthy individuals, the PBMCs were isolated and their RNAs extracted and their cDNAs were synthesized, and the genes expression levels were measured using the qRT-PCR technique. Our results indicated that this drug could, at both low and high doses, significantly reduce the expression of the genes for SOD2, GPX1, CAT, and GST compared to the LPS group (p<0.0001). Moreover, it was noticed that the drug is able to significantly reduce gene expression levels at the high dose and at both doses (low and high), for iNOS and MPO compared to the LPS group (p<0.0001), respectively. The present research showed that G2013, as a novel NSAID drug with immunomodulatory properties, could modulate the expression levels of the genes for SOD2, GPX1, CAT, GST, iNOS, and MPO, to the level of healthy gene expression, and possibly it might reduce the pathological process of aging and age-related inflammatory diseases. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  19. Effect of LED irradiation on the expression of MMP-3 and MMP-13 in SW1353 cells in vitro

    Science.gov (United States)

    Zeng, Chang-chun; Guo, Zhou-yi; Zhang, Feng-xue; Deng, Wen-di; Liu, Song-hao

    2007-05-01

    Matrix Metalloproteinase (MMP) plays an active role in remodeling cartilage in osteoarthritic cartilage. To find an effective method of prevention of osteoclasia, this in vitro study focuses on the expression of MMP-3 and MMP-13 in the SW1353 cells by LED irradiation. The human chondrosarcoma cell line SW1353 were stimulated with the proinflammatory cytokine IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and were received the irradiation of LED (632nm, 4mW/cm2). The cell count was assessed over a 96-hour period by using Trypan blue dye exclusion assay, and the cell activity was evaluated with a Cell Counting Kit-8 Assays. The subsequent expression of MMP-3 and MMP-13 was quantified. Results of this experiment showed that the cultural cell activity was decreased, and the expression of MMP-3 and MMP-13 was increased by being stimulated with IL-1beta or TNF-alpha. After received LED irradiation, the death rate of cultural cell was increased and the expression of MMP-3 and MMP-13 was decreased significantly. The present study concluded that particular LED irradiation stimulates SW1353 cell proliferation activity and inhibit the MMP-3 and MMP-13 enzymatic activity. These findings might be clinically relevant, indicating that the low power laser irradiation treatment is likely to achieve the repair of articular cartilage in clinic.

  20. Human thymic epithelial cells express functional HLA-DP molecules

    DEFF Research Database (Denmark)

    Jørgensen, A; Röpke, C; Nielsen, M

    1996-01-01

    T lymphocytes, we examined whether human thymic epithelial cells (TEC) expressed HLA-DP molecules. We present evidence that TEC obtained from short time culture express low but significant levels of HLA-DP molecules. The expression of HLA-DP molecules was comparable to or higher than the expression...... of HLA-DP allospecific primed lymphocyte typing (PLT) CD4 T cell lines. IFN-gamma treatment strongly upregulated the HLA-DP allospecific PLT responses whereas other PLT responses remained largely unchanged. In conclusion, these data indicate that human thymus epithelial cells express significant levels...

  1. Bone cell expression on titanium surfaces is altered by sterilization treatments.

    Science.gov (United States)

    Stanford, C M; Keller, J C; Solursh, M

    1994-05-01

    Phenotypic responses of rat calvarial osteoblast-like cells (RCOB) were evaluated on commercially pure titanium (cpTi) surfaces when cultured at high density (5100 cells/mm2). These surfaces were prepared to three different clinically relevant surface preparations (1-micron, 600-grit, and 50-microns-grit sand-blast), followed by sterilization with either ultraviolet light, ethylene oxide, argon plasma-cleaning, or routine clinical autoclaving. Osteocalcin and alkaline phosphatase, but not collagen expression, were significantly affected by surface roughness when these surfaces were altered by argon plasma-cleaning. In general, plasma-cleaned cpTi surfaces demonstrated an inverse relationship between surface roughness and phenotypic markers for a bone-like response. On a per-cell basis, levels of the bone-specific protein, osteocalcin, and the enzymatic activity of alkaline phosphatase were highest on the smooth 1-micron polished surface and lowest on the roughest surfaces for the plasma-cleaned cpTi. Detectable bone cell expression can be altered by clinically relevant surfaces prepared by standard dental implant preparation techniques.

  2. Nobiletin inhibits human osteosarcoma cells metastasis by blocking ERK and JNK-mediated MMPs expression

    Science.gov (United States)

    Cheng, Hsin-Lin; Hsieh, Ming-Ju; Yang, Jia-Sin; Lin, Chiao-Wen; Lue, Ko-Haung; Lu, Ko-Hsiu; Yang, Shun-Fa

    2016-01-01

    Nobiletin, a polymethoxyflavone, has a few pharmacological activities, including anti-inflammation and anti-cancer effects. However, its effect on human osteosarcoma progression remains uninvestigated. Therefore, we examined the effectiveness of nobiletin against cellular metastasis of human osteosarcoma and the underlying mechanisms. Nobiletin, up to 100 μM without cytotoxicity, significantly decreased motility, migration and invasion as well as enzymatic activities, protein levels and mRNA expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in U2OS and HOS cells. In addition to inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the inhibitory effect of nobiletin on the DNA-binding activity of the transcription factor nuclear factor-kappa B (NF-κB), cAMP response element-binding protein (CREB), and specificity protein 1 (SP-1) in U2OS and HOS cells. Co-treatment with ERK and JNK inhibitors and nobiletin further reduced U2OS cells migration and invasion. These results indicated that nobiletin inhibits human osteosarcoma U2OS and HOS cells motility, migration and invasion by down-regulating MMP-2 and MMP-9 expressions via ERK and JNK pathways and through the inactivation of downstream NF-κB, CREB, and SP-1. Nobiletin has the potential to serve as an anti-metastatic agent for treating osteosarcoma. PMID:27144433

  3. Enzymatic oxidation of cephalosporin C using whole cells of the yeast Triginopsis variabilis within a "cross-flow filter-reactor".

    Science.gov (United States)

    Vicenzi, J T; Hansen, G J

    1993-04-01

    An economical process for the enzymatic oxidation of cephalosporin C to glutaryl-7-ACA was developed at a pilot plant scale. The process utilized nonviable whole cells of the yeast Triginopsis variabilis containing high levels of D-amino acid oxidase. Prior to use, the whole cells were permeabilized with a 25% acetone/water solution which enhanced their apparent activity by 20- to 50-fold. After permeabilization, the whole cells were incubated at pH 11, which served to selectively deactivate catalase which was present in very large quantities. Deactivation of catalase was critical to achieving high reaction yields. The whole cells were utilized within a "cross-flow filter-reactor" which allowed easy and economical recycle of the cells for repeated use. The overall yield of glutaryl-7-ACA from cephalosporin C was 90-95%. The overall productivity of the yeast was 13 kg cephalosporin C oxidized per kilogram yeast (dry basis). The reaction was run at a concentration of 40 g cephalosporin CL-1 and the overall reactor productivity was 11 g glutaryl-7-ACA l-1 h-1. The process has been thoroughly demonstrated on a 35-l scale, and it should be directly scaleable to 10,000 l or more.

  4. Enzymatic Treatment of Whey Proteins in Cow's Milk Results in Differential Inhibition of IgE-Mediated Mast Cell Activation Compared to T-Cell Activation

    NARCIS (Netherlands)

    Knipping, Karen; van Esch, Betty C. A. M.; van Ieperen-van Dijk, Adrie G.; van Hoffen, Els; van Baalen, Ton; Knippels, Leon M. J.; van der Heide, Sicco; Dubois, Anthony E. J.; Garssen, Johan; Knol, Edward F.

    2012-01-01

    Background: Cow's milk (CM) hydrolysates are frequently used as milk substitutes for children with CM allergy. In hydrolysates, allergenic epitopes within CM proteins are diminished by enzymatic treatment. The aim of this study was to examine the allergenic and immunogenic properties of whey protein

  5. Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

    Science.gov (United States)

    Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

    2000-04-01

    Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

  6. Insights into enzymatic thiamin catalysis

    OpenAIRE

    Wikner, Christer

    1997-01-01

    Thiamin diphosphate, the biologically active form of vitamin B,, functions as a cofactor in various enzymes in the cell. The protein enhances the reactivity of the cofactor by binding it in a very specific manner. In this work, based upon information from the crystal structure, the mechanism of the thiamin dependent enzyme transketolase from yeast has been investigated by various methods. In enzymatic thiamin catalysis, the protein has three major tasks in the formation of a...

  7. Stable protein expression in mammalian cells using baculoviruses.

    Science.gov (United States)

    Lackner, Andreas; Kreidl, Emanuel; Peter-Vörösmarty, Barbara; Spiegl-Kreinecker, Sabine; Berger, Walter; Grusch, Michael

    2012-01-01

    The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) has been widely used in biotechnology for protein expression in insect cells. Baculoviruses use arthropods as their natural hosts and are unable to replicate in mammalian cells. However, AcMNPV is able to enter many mammalian cell types and can be used for transgene expression if engineered to contain suitable expression cassettes. In this chapter, we describe the construction and application of a recombinant baculovirus containing a bicistronic expression cassette that can be used for stable protein expression in mammalian cells. As an example, the generation of glioblastoma and hepatocellular carcinoma cell lines stably expressing green fluorescent protein after puromycin selection is shown.

  8. Expression of HOX C homeobox genes in lymphoid cells.

    Science.gov (United States)

    Lawrence, H J; Stage, K M; Mathews, C H; Detmer, K; Scibienski, R; MacKenzie, M; Migliaccio, E; Boncinelli, E; Largman, C

    1993-08-01

    The class I homeobox genes located in four clusters in mammalian genomes (HOX A, HOX B, HOX C, and HOX D) appear to play a major role in fetal development. Previous surveys of homeobox gene expression in human leukemic cell lines have shown that certain HOX A genes are expressed only in myeloid cell lines, whereas HOX B gene expression is largely restricted to cells with erythroid potential. We now report a survey of the expression patterns of 9 homeobox genes from the HOX C locus in a panel of 24 human and 7 murine leukemic cell lines. The most striking observation is the lymphoid-specific pattern of expression of HOX C4, located at the 3' end of the locus. A major transcript of 1.9 kilobases is observed in both T-cell and B-cell lines. HOX C4 expression is also detected in normal human marrow and peripheral blood lymphocytes, but not in mature granulocytes or monocytes. HOX C8 is also expressed in human lymphoid cells but is expressed in other blood cell types as well. However, the HOX C8 transcript pattern is lineage specific. These data, in conjunction with earlier findings, suggest that homeobox gene expression influences lineage determination during hematopoiesis.

  9. Freedom of expression: cell-type-specific gene profiling.

    Science.gov (United States)

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling.

  10. In vitro cytotoxicity against Marek's disease lymphoblastoid cell lines after enzymatic removal of Marek's disease tumor-associated surface antigen.

    OpenAIRE

    Schat, K A; Murthy, K K

    1980-01-01

    Marek's disease tumor-associated surface antigen (MATSA) has been claimed to be the target of cytotoxic lymphocytes in in vitro tests for Marek's disease immunity. Treatment with papain, but not with trypsin or mixed glycosidases, removed MATSA from certain Marek's disease lymphoblastoid cell lines. Tumor cells with and without MATSA were used as target cells for in vitro studies on cell-mediated immune responses with sensitized spleen cells in a chromium release assay. The removal of MATSA d...

  11. Robust Inference of Cell-to-Cell Expression Variations from Single- and K-Cell Profiling.

    Directory of Open Access Journals (Sweden)

    Manikandan Narayanan

    2016-07-01

    Full Text Available Quantifying heterogeneity in gene expression among single cells can reveal information inaccessible to cell-population averaged measurements. However, the expression level of many genes in single cells fall below the detection limit of even the most sensitive technologies currently available. One proposed approach to overcome this challenge is to measure random pools of k cells (e.g., 10 to increase sensitivity, followed by computational "deconvolution" of cellular heterogeneity parameters (CHPs, such as the biological variance of single-cell expression levels. Existing approaches infer CHPs using either single-cell or k-cell data alone, and typically within a single population of cells. However, integrating both single- and k-cell data may reap additional benefits, and quantifying differences in CHPs across cell populations or conditions could reveal novel biological information. Here we present a Bayesian approach that can utilize single-cell, k-cell, or both simultaneously to infer CHPs within a single condition or their differences across two conditions. Using simulated as well as experimentally generated single- and k-cell data, we found situations where each data type would offer advantages, but using both together can improve precision and better reconcile CHP information contained in single- and k-cell data. We illustrate the utility of our approach by applying it to jointly generated single- and k-cell data to reveal CHP differences in several key inflammatory genes between resting and inflammatory cytokine-activated human macrophages, delineating differences in the distribution of 'ON' versus 'OFF' cells and in continuous variation of expression level among cells. Our approach thus offers a practical and robust framework to assess and compare cellular heterogeneity within and across biological conditions using modern multiplexed technologies.

  12. Photoelectrochemical enzymatic biosensors.

    Science.gov (United States)

    Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2017-06-15

    Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist.

  13. Advantages and Applications of CAR-Expressing Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Wolfgang eGlienke

    2015-02-01

    Full Text Available In contrast to donor T cells, natural killer (NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD. In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/ on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

  14. Expression of Pol(t) in tissues and cell lines of transitional cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:To explore the expression of DNA polymerase iota in transitional cell carcinoma cells and tissues; Methods: RT-PCR was applie to detect the expression of polymerase iota in BIU87 and T24 cells, then the expression of polymerase iota was also detected in the same way in transitional cell carcinoma which was derived from clinical bladder carcinoma and renal pelvic carcinoma. Results: The expression of Polt was low in bladder normal membrana mucosa but significantly elevated in transitional cell carcinoma cells. Compared with the expression of polymerase iota in bladder normal mucous membranes, the expression of polymerase iota was significantly increased in transitional cell carcinoma tissue (P<0.01)and associated with the grade of transitional cell carcinoma. Conclusion: The significantly increased expression of polymerase iota may be associated with the generation and development of transitional cell carcinoma, even with its high heterogenicity.

  15. Prognostic value of the CD4+/CD8+ ratio of tumour infiltrating lymphocytes in colorectal cancer and HLA-DR expression on tumour cells

    DEFF Research Database (Denmark)

    Diederichsen, Axel Cosmus Pyndt; Hjelmborg, J v B; Christensen, Per B

    2003-01-01

    clinical course, with significantly higher 5-year survival, p=0.046, independent of the Dukes stage and age. Our results have implications for tumour immunology; colorectal cancer cells might be a target for cytotoxic T-lymphocytes, however the tumour cells are not able to initiate an immune response......The purpose of this study was to clarify whether HLA-DR expression of colorectal tumour cells or the CD4+/CD8+ ratio of the tumour infiltrating lymphocytes is significantly associated with the prognosis of colorectal cancer. Using flow cytometry, we studied the tumour cell expression of the HLA...... class II in 70 enzymatically dissociated colorectal cancers and the phenotype of tumour infiltrating lymphocytes (TILs) in 41 cases. There was no trend in 5-year survival between three levels (low, medium, high) of HLA-DR expression on the tumour cells. Patients with low CD4+/CD8+ ratios had a better...

  16. Enhanced arsenic accumulation by engineered yeast cells expressing Arabidopsis thaliana phytochelatin synthase.

    Science.gov (United States)

    Singh, Shailendra; Lee, Wonkyu; Dasilva, Nancy A; Mulchandani, Ashok; Chen, Wilfred

    2008-02-01

    Phytochelatins (PCs) are naturally occurring peptides with high-binding capabilities for a wide range of heavy metals including arsenic (As). PCs are enzymatically synthesized by phytochelatin synthases and contain a (gamma-Glu-Cys)(n) moiety terminated by a Gly residue that makes them relatively proteolysis resistant. In this study, PCs were introduced by expressing Arabidopsis thaliana Phytochelatin Synthase (AtPCS) in the yeast Saccharomyces cerevisiae for enhanced As accumulation and removal. PCs production in yeast resulted in six times higher As accumulation as compared to the control strain under a wide range of As concentrations. For the high-arsenic concentration, PCs production led to a substantial decrease in levels of PC precursors such as glutathione (GSH) and gamma-glutamyl cysteine (gamma-EC). The levels of As(III) accumulation were found to be similar between AtPCS-expressing wild type strain and AtPCS-expressing acr3Delta strain lacking the arsenic efflux system, suggesting that the arsenic uptake may become limiting. This is further supported by the roughly 1:3 stoichiometric ratio between arsenic and PC2 (n = 2) level (comparing with a theoretical value of 1:2), indicating an excess availability of PCs inside the cells. However, at lower As(III) concentration, PC production became limiting and an additive effect on arsenic accumulation was observed for strain lacking the efflux system. More importantly, even resting cells expressing AtPCS pre-cultured in Zn(2+) enriched media showed PCs production and two times higher arsenic removal than the control strain. These results open up the possibility of using cells expressing AtPCS as an inexpensive sorbent for the removal of toxic arsenic.

  17. Chemokine receptor expression by inflammatory T cells in EAE.

    Science.gov (United States)

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4(+) T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8(+) T cells. CD8(+) T cells expressed CXCR3, which was also expressed by CD4(+) T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6(+) and CXCR3(+) CD4(+) T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8(+) T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4(+) T cells expressed CCR6 within infiltrates. CD4-negative CCR6(+) cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4(+) and CD8(+) T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE.

  18. Gene expression of manganese superoxide dismutase in human glioma cells

    Directory of Open Access Journals (Sweden)

    Novi S. Hardiany

    2010-02-01

    Full Text Available Aim This study analyze the MnSOD gene expression as endogenous antioxidant in human glioma cells compared with leucocyte cells as control.Methods MnSOD gene expression of 20 glioma patients was analyzed by measuring the relative expression of mRNA and enzyme activity of MnSOD in brain and leucocyte cells. The relative expression of mRNA MnSOD was determined by using quantitative Real Time RT-PCR and the enzyme activity of MnSOD using biochemical kit assay (xantine oxidase inhibition. Statistic analysis for mRNA and enzyme activity of MnSOD was performed using Kruskal Wallis test.Results mRNA of MnSOD in glioma cells of 70% sample was 0.015–0.627 lower, 10% was 1.002-1.059 and 20% was 1.409-6.915 higher than in leucocyte cells. Also the specific activity of MnSOD enzyme in glioma cells of 80% sample showed 0,064-0,506 lower and 20% sample was 1.249-2.718 higher than in leucocyte cells.Conclusion MnSOD gene expression in human glioma cells are significantly lower than its expression in leucocytes cells. (Med J Indones 2010; 19:21-5Keywords : MnSOD, glioma, gene expression

  19. Determining cell division symmetry through the dissection of dividing cells using single-cell expression analysis.

    Science.gov (United States)

    Jasnos, Lukasz; Sawado, Tomoyuki

    2014-03-01

    Symmetric cell divisions give rise to two sister cells that are identical to each other, whereas asymmetric divisions produce two sister cells with distinctive phenotypes. Although cell division symmetry is usually determined on the basis of a few markers or biological functions, the overall similarity between sister cells has not been thoroughly examined at a molecular level. Here we provide a protocol to separate sister embryonic stem cells (ESCs) and to conduct multiplexed gene expression analyses at the single-cell level by using 48 ESC genes. The procedure includes the dissection of dividing, paired sister cells by micromanipulation, followed by cell lysis, reverse transcription, gene-specific cDNA amplification and multiplexed quantitative PCR analyses. This protocol can be completed in 10 d, and it can be readily adapted to other cell types that are able to grow in suspension culture.

  20. Differential expression and function of CD27 in chronic lymphocytic leukemia cells expressing ZAP-70.

    Science.gov (United States)

    Lafarge, Sandrine T; Hou, Sen; Pauls, Samantha D; Johnston, James B; Gibson, Spencer B; Marshall, Aaron J

    2015-07-01

    Chronic lymphocytic leukemia is a malignancy driven by abberant B cell signaling and survival. Leukemic B cells accumulate in the peripheral blood and the lymphoid organs where contact with stromal cells and T cells provide critical survival signals. Clinical severity of CLL is associated with several prognostic markers including expression of the kinase ZAP-70. ZAP-70 expression enhances signaling via the B cell antigen receptor and is associated with increased cell adhesion and migration capacity. Here we report that ZAP-70-positive CLL patients display significantly higher expression of the TNF superfamily receptor and memory marker CD27 than do ZAP-70 negative patients. CD27 expression by CLL was acutely elevated upon BCR cross-linking, or upon ectopic expression of ZAP-70. CD27 expression correlated with functional capacity to adhere to stromal cells and antibody blockade of CD27 impaired CLL binding to stroma. These results provide the first evidence for differential expression of CD27 among CLL prognostic groups, suggest a role for ZAP-70 dependent signaling in CD27 induction and implicate CD27 in cell-cell interactions with the lymphoid tissue microenvironment.

  1. Paper-based enzymatic microfluidic fuel cell: From a two-stream flow device to a single-stream lateral flow strip

    Science.gov (United States)

    González-Guerrero, Maria José; del Campo, F. Javier; Esquivel, Juan Pablo; Giroud, Fabien; Minteer, Shelley D.; Sabaté, Neus

    2016-09-01

    This work presents a first approach towards the development of a cost-effective enzymatic paper-based glucose/O2 microfluidic fuel cell in which fluid transport is based on capillary action. A first fuel cell configuration consists of a Y-shaped paper device with the fuel and the oxidant flowing in parallel over carbon paper electrodes modified with bioelectrocatalytic enzymes. The anode consists of a ferrocenium-based polyethyleneimine polymer linked to glucose oxidase (GOx/Fc-C6-LPEI), while the cathode contains a mixture of laccase, anthracene-modified multiwall carbon nanotubes, and tetrabutylammonium bromide-modified Nafion (MWCNTs/laccase/TBAB-Nafion). Subsequently, the Y-shaped configuration is improved to use a single solution containing both, the anolyte and the catholyte. Thus, the electrolytes pHs of the fuel and the oxidant solutions are adapted to an intermediate pH of 5.5. Finally, the fuel cell is run with this single solution obtaining a maximum open circuit of 0.55 ± 0.04 V and a maximum current and power density of 225 ± 17 μA cm-2 and 24 ± 5 μW cm-2, respectively. Hence, a power source closer to a commercial application (similar to conventional lateral flow test strips) is developed and successfully operated. This system can be used to supply the energy required to power microelectronics demanding low power consumption.

  2. Correlation of electrical and physical properties of photoanode with hydrogen evolution in enzymatic photo-electrochemical cell

    Science.gov (United States)

    Bae, Sanghyun; Kang, Junwon; Shim, Eunjung; Yoon, Jaekyung; Joo, Hyunku

    2008-05-01

    In this study, the electrical and physical properties, including the current density, open-circuit voltage, morphology and crystalline structure, of an anodized TiO2 electrode on a titanium foil are correlated with the hydrogen production rate in an enzymatic photo-electrochemical system. The effect of light intensity at ca. 74 and ca. 146 mW cm-2 on the properties is also examined. Anodizing (20 V; bath temperature 5 °C; anodizing time 45 min) and subsequent annealing (350-850 °C for 5 h) of the Ti foils in an O2 atmosphere led to the formation of a tube-shaped, or a compact layered, TiO2 film on the Ti substrate depending on the annealing temperature. The annealing temperature has a similar effect on the properties of the sample and the hydrogen evolution rate. The generated electrical value, the chronoamperometry (CA), is +13 to -229 and +13 to -247 μA for light intensities of ca. 74 and ca. 146 mW cm-2, while the corresponding open-circuit voltage (OCV) is in the range of -41 to -687 and -144 to 738 mV, respectively. In the absence of light (dark), the CA is 13-29 μA and the OCV is +258 to -126 mW cm-2. The trend in the electrical properties for the different samples is well matched with the rate of hydrogen evolution. The samples with higher activities (450, 550, and 650 °C) have similar X-ray diffraction (XRD) patterns, which clearly indicates that the samples showing the highest evolution rate are composed of both anatase and rutile, while those showing a lower evolution rate are made of either anatase or rutile. Increasing the intensity of the irradiated light causes a remarkable enhancement in the rate of hydrogen production from 71 to 153 μmol h-1 cm-2.

  3. Calreticulin: Roles in Cell-Surface Protein Expression

    Directory of Open Access Journals (Sweden)

    Yue Jiang

    2014-09-01

    Full Text Available In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins.

  4. Chemokine receptor expression by inflammatory T cells in EAE

    DEFF Research Database (Denmark)

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20...... receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed...... whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays...

  5. Integrating Enzymatic Self-Assembly and Mitochondria Targeting for Selectively Killing Cancer Cells without Acquired Drug Resistance.

    Science.gov (United States)

    Wang, Huaimin; Feng, Zhaoqianqi; Wang, Youzhi; Zhou, Rong; Yang, Zhimou; Xu, Bing

    2016-12-14

    Targeting organelles by modulating the redox potential of mitochondria is a promising approach to kill cancer cells that minimizes acquired drug resistance. However, it lacks selectivity because mitochondria perform essential functions for (almost) all cells. We show that enzyme-instructed self-assembly (EISA), a bioinspired molecular process, selectively generates the assemblies of redox modulators (e.g., triphenyl phosphinium (TPP)) in the pericellular space of cancer cells for uptake, which allows selectively targeting the mitochondria of cancer cells. The attachment of TPP to a pair of enantiomeric, phosphorylated tetrapeptides produces the precursors (L-1P or D-1P) that form oligomers. Upon dephosphorylation catalyzed by ectophosphatases (e.g., alkaline phosphatase (ALP)) overexpressed on cancer cells (e.g., Saos2), the oligomers self-assemble to form nanoscale assemblies only on the surface of the cancer cells. The cancer cells thus uptake these assemblies of TPP via endocytosis, mainly via a caveolae/raft-dependent pathway. Inside the cells, the assemblies of TPP-peptide conjugates escape from the lysosome, induce dysfunction of mitochondria to release cytochrome c, and result in cell death, while the controls (i.e., omitting TPP motif, inhibiting ALP, or removing phosphate trigger) hardly kill the Saos2 cells. Most importantly, the repeated stimulation of the cancers by the precursors, unexpectedly, sensitizes the cancer cells to the precursors. As the first example of the integration of subcellular targeting with cell targeting, this study validates the spatial control of the assemblies of nonspecific cytotoxic agents by EISA as a promising molecular process for selectively killing cancer cells without inducing acquired drug resistance.

  6. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

    OpenAIRE

    1989-01-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer co...

  7. Enzymatically Modified Low-Density Lipoprotein Promotes Foam Cell Formation in Smooth Muscle Cells via Macropinocytosis and Enhances Receptor-Mediated Uptake of Oxidized Low-Density Lipoprotein.

    Science.gov (United States)

    Chellan, Bijoy; Reardon, Catherine A; Getz, Godfrey S; Hofmann Bowman, Marion A

    2016-06-01

    Enzyme-modified nonoxidized low-density lipoprotein (ELDL) is present in human atherosclerotic lesions. Our objective is to understand the mechanisms of ELDL uptake and its effects on vascular smooth muscle cells (SMC). Transformation of murine aortic SMCs into foam cells in response to ELDL was analyzed. ELDL, but not acetylated or oxidized LDL, was potent in inducing SMC foam cell formation. Inhibitors of macropinocytosis (LY294002, wortmannin, amiloride) attenuated ELDL uptake. In contrast, inhibitors of receptor-mediated endocytosis (dynasore, sucrose) and inhibitor of caveolae-/lipid raft-mediated endocytosis (filipin) had no effect on ELDL uptake in SMC, suggesting that macropinocytosis is the main mechanism of ELDL uptake by SMC. Receptor for advanced glycation end products (RAGE) is not obligatory for ELDL-induced SMC foam cell formation, but primes SMC for the uptake of oxidized LDL in a RAGE-dependent manner. ELDL increased intracellular reactive oxygen species, cytosolic calcium, and expression of lectin-like oxidized LDL receptor-1 in wild-type SMC but not in RAGE(-/-) SMC. The macropinocytotic uptake of ELDL is regulated predominantly by intracellular calcium because ELDL uptake was completely inhibited by pretreatment with the calcium channel inhibitor lacidipine in wild-type and RAGE(-/-) SMC. This is in contrast to pretreatment with PI3 kinase inhibitors which completely prevented ELDL uptake in RAGE(-/-) SMC, but only partially in wild-type SMC. ELDL is highly potent in inducing foam cells in murine SMC. ELDL endocytosis is mediated by calcium-dependent macropinocytosis. Priming SMC with ELDL enhances the uptake of oxidized LDL. © 2016 American Heart Association, Inc.

  8. Intraclonal protein expression heterogeneity in recombinant CHO cells.

    Directory of Open Access Journals (Sweden)

    Warren Pilbrough

    Full Text Available Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean, approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations. Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50

  9. Intraclonal Protein Expression Heterogeneity in Recombinant CHO Cells

    Science.gov (United States)

    Pilbrough, Warren; Munro, Trent P.; Gray, Peter

    2009-01-01

    Therapeutic glycoproteins have played a major role in the commercial success of biotechnology in the post-genomic era. But isolating recombinant mammalian cell lines for large-scale production remains costly and time-consuming, due to substantial variation and unpredictable stability of expression amongst transfected cells, requiring extensive clone screening to identify suitable high producers. Streamlining this process is of considerable interest to industry yet the underlying phenomena are still not well understood. Here we examine an antibody-expressing Chinese hamster ovary (CHO) clone at single-cell resolution using flow cytometry and vectors, which couple light and heavy chain transcription to fluorescent markers. Expression variation has traditionally been attributed to genetic heterogeneity arising from random genomic integration of vector DNA. It follows that single cell cloning should yield a homogeneous cell population. We show, in fact, that expression in a clone can be surprisingly heterogeneous (standard deviation 50 to 70% of the mean), approaching the level of variation in mixed transfectant pools, and each antibody chain varies in tandem. Phenotypic variation is fully developed within just 18 days of cloning, yet is not entirely explained by measurement noise, cell size, or the cell cycle. By monitoring the dynamic response of subpopulations and subclones, we show that cells also undergo slow stochastic fluctuations in expression (half-life 2 to 11 generations). Non-genetic diversity may therefore play a greater role in clonal variation than previously thought. This also has unexpected implications for expression stability. Stochastic gene expression noise and selection bias lead to perturbations from steady state at the time of cloning. The resulting transient response as clones reestablish their expression distribution is not ordinarily accounted for but can contribute to declines in median expression over timescales of up to 50 days. Noise

  10. Stem Leydig cell differentiation: gene expression during development of the adult rat population of Leydig cells.

    Science.gov (United States)

    Stanley, Erin L; Johnston, Daniel S; Fan, Jinjiang; Papadopoulos, Vassilios; Chen, Haolin; Ge, Ren-Shan; Zirkin, Barry R; Jelinsky, Scott A

    2011-12-01

    Leydig cells are the testosterone-producing cells in the adult male. Adult Leydig cells (ALCs) develop from stem Leydig cells (SLCs) through at least two intermediate cells, progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). Microarray gene expression was used to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to that in a well-established stem cell, bone marrow stem cells (BSCs). We show that the pattern of gene expression by SLCs was more similar to the expression by BSCs, an established stem cell outside the male reproductive tract, than to any of the cells in the Leydig cell developmental lineage. These results indicated that the SLCs have many of the molecular characteristics of other stem cells. Pathway analysis indicated that development of Leydig cells from SLCs to PLCs was associated with decreased expression of genes related to adhesion and increased expression of genes related to steroidogenesis. Gene expression changes between PLCs and ILCs and between ILCs and ALCs were relatively minimal, suggesting that these cells are highly similar. In contrast, gene expression changes between SLCs and ALCs were quite distinct.

  11. A transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins through N-glycosylation.

    Science.gov (United States)

    Hu, Jia-Biao; Zhang, Peng; Wang, Mei-Xian; Zhou, Fang; Niu, Yan-Shan; Miao, Yun-Gen

    2012-08-01

    Glycoproteins have been implicated in a wide variety of important biochemical and biological functions, including protein stability, immune function, enzymatic function, cellular adhesion and others. Unfortunately, there is no therapeutic protein produced in insect system to date, due to the expressed glycoproteins are paucimannosidic N-glycans, rather than the complex, terminally sialylated N-glycans in mammalian cells. In this paper, we cloned the necessary genes in glycosylation of mammalian cells, such as N-acetylglucosaminyltransferase II (Gn-TII), galactosyltransferases (Gal-Ts), 2,6-Sial-T (ST6 GalII)and 2,3-Sial-T (ST3GalIII), and transformed them to silkworm genome of BmN cell line through transgenesis to establish a transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins. The study supplied a new insect cell line which is practically to produce "bisected" complex N-glycans like in mammalian cells.

  12. Enzymatic synthesis of L-tryptophan by Enterobacter aerogenes tryptophanase highly expressed in Escherichia coli, and some properties of the purified enzyme.

    Science.gov (United States)

    Kawasaki, K; Yokota, A; Tomita, F

    1995-10-01

    We constructed two plasmids that have a strong tac promoter and a structural gene for tryptophanase of Enterobacter aerogenes SM-18 (pKT901EA) or Escherichia coli K-12 (pKT951EC). The tryptophanase activity of E. coli JM109 transformed with pKT901EA (JM109/pKT901EA) was inducible with isopropyl-beta-D-thiogalactopyranoside, and 3.6 times higher than that of E. aerogenes SM-18. Cells of JM109/pKT901EA induced for tryptophanase synthesized L-tryptophan from indole, ammonia, and pyruvate more efficiently than E. aerogenes SM-18. Although JM109/pKT951EC expressed a similar level of tryptophanase activity to that of JM109/pKT901EA, the synthesis of L-tryptophan by the cells of JM109/pKT951EC did not proceed well compared with JM109/pKT901EA. Tryptophanases from E. aerogenes and E. coli K-12 were purified, and their properties were investigated. The purified E. aerogenes tryptophanase showed higher stability against heat inactivation than E. coli tryptophanase.

  13. TRPM5-expressing microvillous cells in the main olfactory epithelium

    Directory of Open Access Journals (Sweden)

    Liman Emily R

    2008-11-01

    Full Text Available Abstract Background The main olfactory epithelium (MOE in the nasal cavity detects a variety of air borne molecules that provide information regarding the presence of food, predators and other relevant social and environmental factors. Within the epithelium are ciliated sensory neurons, supporting cells, basal cells and microvillous cells, each of which is distinct in morphology and function. Arguably, the least understood, are the microvillous cells, a population of cells that are small in number and whose function is not known. We previously found that in a mouse strain in which the TRPM5 promoter drives expression of the green fluorescent protein (GFP, a population of ciliated olfactory sensory neurons (OSNs, as well as a population of cells displaying microvilli-like structures is labeled. Here we examined the morphology and immunocytochemical properties of these microvillous-like cells using immunocytochemical methods. Results We show that the GFP-positive microvillous cells were morphologically diversified and scattered throughout the entire MOE. These cells immunoreacted to an antibody against TRPM5, confirming the expression of this ion channel in these cells. In addition, they showed a Ca2+-activated non-selective cation current in electrophysiological recordings. They did not immunoreact to antibodies that label cell markers and elements of the transduction pathways from olfactory sensory neurons and solitary chemosensory cells of the nasal cavity. Further, the TRPM5-expressing cells did not display axon-like processes and were not labeled with a neuronal marker nor did trigeminal peptidergic nerve fibers innervate these cells. Conclusion We provide morphological and immunocytochemical characterization of the TRPM5-expressing microvillous cells in the main olfactory epithelium. Our data demonstrate that these cells are non-neuronal and in terms of chemosensory transduction do not resemble the TRPM5-expressing olfactory sensory neurons

  14. Lab-specific gene expression signatures in pluripotent stem cells.

    Science.gov (United States)

    Newman, Aaron M; Cooper, James B

    2010-08-06

    Pluripotent stem cells derived from both embryonic and reprogrammed somatic cells have significant potential for human regenerative medicine. Despite similarities in developmental potential, however, several groups have found fundamental differences between embryonic stem cell (ESC) and induced-pluripotent stem cell (iPSC) lines that may have important implications for iPSC-based medical therapies. Using an unsupervised clustering algorithm, we further studied the genetic homogeneity of iPSC and ESC lines by reanalyzing microarray gene expression data from seven different laboratories. Unexpectedly, this analysis revealed a strong correlation between gene expression signatures and specific laboratories in both ESC and iPSC lines. Nearly one-third of the genes with lab-specific expression signatures are also differentially expressed between ESCs and iPSCs. These data are consistent with the hypothesis that in vitro microenvironmental context differentially impacts the gene expression signatures of both iPSCs and ESCs.

  15. Metformin suppresses sonic hedgehog expression in pancreatic cancer cells.

    Science.gov (United States)

    Nakamura, Masafumi; Ogo, Ayako; Yamura, Masahiro; Yamaguchi, Yoshiyuki; Nakashima, Hiroshi

    2014-04-01

    Metformin use has previously been associated with decreased cancer risk. The Hedgehog signaling pathway is a well-characterized early and late mediator of pancreatic cancer oncogenesis. The aim of the present study was to clarify the effect of metformin on factors involved in Hedgehog signaling. BxPC3 human pancreatic cancer cells were treated with metformin, and Sonic hedgehog (Shh) mRNA and protein levels were examined by real time reverse transcription-polymerase chain reaction, immunohistochemistry and immunoblotting, respectively. The effect of metformin on Shh levels was also examined in three other cancer cell lines. Shh protein and mRNA expression was suppressed by metformin in BxPC3 cells. This phenomenon was further confirmed in three other cancer cell lines. Shh mRNA expression was inhibited by metformin in a concentration-dependent manner in two cancer cell lines. Metformin reduces the expression of Shh in several cancer cell lines including pancreatic cancer cell.

  16. Two Novel Class II Hydrophobins from Trichoderma spp. Stimulate Enzymatic Hydrolysis of Poly(Ethylene Terephthalate) when Expressed as Fusion Proteins

    Science.gov (United States)

    Espino-Rammer, Liliana; Ribitsch, Doris; Przylucka, Agnieszka; Marold, Annemarie; Greimel, Katrin J.; Herrero Acero, Enrique; Guebitz, Georg M.; Kubicek, Christian P.

    2013-01-01

    Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET. PMID:23645195

  17. Oct-4 expression in pluripotent cells of the rhesus monkey.

    Science.gov (United States)

    Mitalipov, Shoukhrat M; Kuo, Hung-Chih; Hennebold, Jon D; Wolf, Don P

    2003-12-01

    The POU (Pit-Oct-Unc)-domain transcription factor, Oct-4, has become a useful marker of pluripotency in the mouse. It is found exclusively in mouse preimplantation-stage embryos after embryonic genome activation and is a characteristic of mouse embryonic stem (ES) cells, and its absence in knockout mice precludes inner cell mass (ICM) formation in blastocysts. Expression of Oct-4 has also been associated with pluripotency in primate cells. Here, we undertook a systematic study of Oct-4 expression in rhesus macaque preimplantation embryos produced by intracytoplasmic sperm injection and in ES cells before and after exposure to differentiating conditions in vitro. We also evaluated Oct-4 expression as a means of monitoring the extent of reprogramming following somatic cell nuclear transfer. Oct-4 was detected by reverse transcription-polymerase chain reaction and immunocytochemistry with a monoclonal antibody. Monkey pronuclear-stage zygotes and cleaving embryos up to the 8-cell stage showed no detectable Oct-4. Nuclear staining for Oct-4 first became obvious at the 16-cell stage, and a strong signal was observed in morula and compact morula stages. Both ICM and trophectodermal cell nuclei of monkey early blastocysts were positive for Oct-4. However, the signal was diminished in trophectodermal cells of expanded blastocysts, whereas expression remained high in ICM nuclei. Similar to the mouse, hatched monkey blastocysts showed strong Oct-4 expression in the ICM, with no detectable signal in the trophectoderm. Undifferentiated monkey ES cells derived from the ICM of in vitro-produced blastocysts expressed Oct-4, consistent with their pluripotent nature, whereas ES cell differentiation was associated with signal loss. Therefore, Oct-4 expression in the monkey, as in the mouse, provides a useful marker for pluripotency after activation of the embryonic genome. Finally, the observed lack or abnormal expression of Oct-4 in monkey nuclear transfer embryos suggests

  18. Neurohypophysial Receptor Gene Expression by Thymic T Cell Subsets and Thymic T Cell Lymphoma Cell Lines

    Directory of Open Access Journals (Sweden)

    I. Hansenne

    2004-01-01

    transcribed in thymic epithelium, while immature T lymphocytes express functional neurohypophysial receptors. Neurohypophysial receptors belong to the G protein-linked seven-transmembrane receptor superfamily and are encoded by four distinct genes, OTR, V1R, V2R and V3R. The objective of this study was to identify the nature of neurohypophysial receptor in thymic T cell subsets purified by immunomagnetic selection, as well as in murine thymic lymphoma cell lines RL12-NP and BW5147. OTR is transcribed in all thymic T cell subsets and T cell lines, while V3R transcription is restricted to CD4+ CD8+ and CD8+ thymic cells. Neither V1R nor V2R transcripts are detected in any kind of T cells. The OTR protein was identified by immunocytochemistry on thymocytes freshly isolated from C57BL/6 mice. In murine fetal thymic organ cultures, a specific OTR antagonist does not modify the percentage of T cell subsets, but increases late T cell apoptosis further evidencing the involvement of OT/OTR signaling in the control of T cell proliferation and survival. According to these data, OTR and V3R are differentially expressed during T cell ontogeny. Moreover, the restriction of OTR transcription to T cell lines derived from thymic lymphomas may be important in the context of T cell leukemia pathogenesis and treatment.

  19. A lower content of de-methylesterified homogalacturonan improves enzymatic cell separation and isolation of mesophyll protoplasts in Arabidopsis.

    Science.gov (United States)

    Lionetti, Vincenzo; Cervone, Felice; De Lorenzo, Giulia

    2015-04-01

    Cell adhesion occurs primarily at the level of middle lamella which is mainly composed by pectin polysaccharides. These can be degraded by cell wall degrading enzymes (CWDEs) during developmental processes to allow a controlled separation of plant cells. Extensive cell wall degradation by CWDEs with consequent cell separation is performed when protoplasts are isolated from plant tissues by using mixtures of CWDEs. We have evaluated whether modification of pectin affects cell separation and protoplast isolation. Arabidopsis plants overexpressing the pectin methylesterase inhibitors AtPMEI-1 or AtPMEI-2, and Arabidopsis pme3 plants, mutated in the gene encoding pectin methylesterase 3, showed an increased efficiency of isolation of viable mesophyll protoplasts as compared with Wild Type Columbia-0 plants. The release of protoplasts was correlated with the reduced level of long stretches of de-methylesterified homogalacturonan (HGA) present in these plants. Response to elicitation, cell wall regeneration and efficiency of transfection in protoplasts from transgenic plants was comparable to those of wild type protoplasts.

  20. Enzymatic desulfurization of coal

    Energy Technology Data Exchange (ETDEWEB)

    Marquis, J.K. (Boston Univ., MA (United States). School of Medicine); Kitchell, J.P. (Holometrix, Inc., Cambridge, MA (United States))

    1988-12-15

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  1. Expression and function of FERMT genes in colon carcinoma cells.

    Science.gov (United States)

    Kiriyama, Kenji; Hirohashi, Yoshihiko; Torigoe, Toshihiko; Kubo, Terufumi; Tamura, Yasuaki; Kanaseki, Takayuki; Takahashi, Akari; Nakazawa, Emiri; Saka, Eri; Ragnarsson, Charlotte; Nakatsugawa, Munehide; Inoda, Satoko; Asanuma, Hiroko; Takasu, Hideo; Hasegawa, Tadashi; Yasoshima, Takahiro; Hirata, Koichi; Sato, Noriyuki

    2013-01-01

    Invasion into the matrix is one of hallmarks of malignant diseases and is the first step for tumor metastasis. Thus, analysis of the molecular mechanisms of invasion is essential to overcome tumor cell invasion. In the present study, we screened for colon carcinoma-specific genes using a cDNA microarray database of colon carcinoma tissues and normal colon tissues, and we found that fermitin family member-1 (FERMT1) is overexpressed in colon carcinoma cells. FRRMT1, FERMT2 and FERMT3 expression was investigated in colon carcinoma cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that only FERMT1 had cancer cell-specific expression. Protein expression of FERMT1 was confirmed by western blotting and immunohistochemical staining. To address the molecular functions of FERMT genes in colon carcinoma cells, we established FERMT1-, FERMT2- and FERMT3-overexpressing colon carcinoma cells. FERMT1-overexpressing cells exhibited greater invasive ability than did FERMT2- and FERMT3-overexpressing cells. On the other hand, FERMT1-, FERMT2- and FERMT3-overexpressing cells exhibited enhancement of cell growth. Taken together, the results of this study indicate that FERMT1 is expressed specifically in colon carcinoma cells, and has roles in matrix invasion and cell growth. These findings indicate that FERMT1 is a potential molecular target for cancer therapy.

  2. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  3. Significance of Parafibromin Expression in Laryngeal Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    Inju Cho

    2016-07-01

    Full Text Available Background: Parafibromin is a product of the tumor suppressor gene that has been studied as a potential indicator of tumor aggressiveness in the parathyroid, breast, colorectum, and stomach. However, the clinical significance and potential function of parafibromin expression in head and neck squamous cell carcinomas remain largely unknown. The aim of this study was to evaluate the expression of parafibromin in laryngeal squamous cell carcinoma (LSCC and to verify its potential as a biomarker of tumor behavior. Methods: Parafibromin expression was evaluated in 30 cases of LSCC using immunohistochemistry. The correlations between parafibromin expression and clinicopathologic parameters were investigated. Results: Parafibromin expression was positive in 15 cases (50% and negative in 15 cases (50%. Tumor size and T stage showed a statistically significant inverse relationship with parafibromin expression (p=.028 and p.05. Conclusions: Our results indicate that the downregulation or loss of parafibromin expression can be employed as a novel marker of tumor progression or aggressiveness in LSCC.

  4. Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells

    Science.gov (United States)

    Zhong, Guocai; Wang, Haimin; Bailey, Charles C; Gao, Guangping; Farzan, Michael

    2016-01-01

    Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient cis-acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an aptazymes’s activity by integrating three characteristics of communication-module bases: hydrogen bonding, base stacking, and distance to the enzymatic core. We validated the power and generality of this approach by designing aptazymes responsive to three distinct ligands, each with markedly wider dynamic ranges than any previously reported. These aptayzmes efficiently regulated adeno-associated virus (AAV)-vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches. Our approach enables efficient, protein-independent control of gene expression by a range of small molecules. DOI: http://dx.doi.org/10.7554/eLife.18858.001 PMID:27805569

  5. [VEGF gene expression in transfected human multipotent stromal cells].

    Science.gov (United States)

    Smirnikhina, S A; Lavrov, A V; Bochkov, N P

    2011-01-01

    Dynamics of VEGF gene expression in transfected multipotent stromal cells from adipose tissue was examined using electroporation and lipofection. Differences in the potency and dynamics of plasmid elimination (up to day 9) between cell cultures were observed. All cultures were divided into fast and slow plasmid-eliminating ones. Interculture differences in VEGF expression were detected. The possibility of a 5-6-fold increase of VEGF expression was shown. There were no differences in transfection potency, plasmid elimination dynamics, and VEGF expression after transfection by both nonviral methods.

  6. HCMV Infection Depress NGF Expression in Human Glioma Cells

    Institute of Scientific and Technical Information of China (English)

    Hai-tao WANG; Bin WANG; Zhi-jun LIU; Zhi-qiang BAI; Ling LI; Dong-meng QIAN; Zhi-yong YAN; Xu-xia SONG

    2009-01-01

    Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia cells by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.

  7. Effect Of Microgravity On Aromatase Expression In Sertoli Cells.

    Science.gov (United States)

    Cirelli, Elisa; De Domenico, Emanuela; Botti, Flavia; Massoud, Renato; Geremia, Raffaele; Grimaldi, Paola

    2017-06-14

    Cytochrome P450-aromatase catalyzes estrogen biosynthesis from C19 steroids. In the testis, Sertoli cells express P450-aromatase and represent the primary source of estrogen during prepuberal age. This study focused on the effect of simulated microgravity (SM) on aromatase expression in primary mouse Sertoli cells. When cultured in Rotary Cell Culture System (RCCS), Sertoli cells, formed multicellular three dimensional spheroids (3D). Biological properties were first analyzed in terms of viability, cell cycle, expression of cytoskeletal components and growth factors in comparison to Sertoli cells cultured in spheroids at unit gravity (G). SM did not affect cell viability and proliferation, nor expression of the main cytoskeleton proteins and of growth factors like Kit Ligand (KL) and glial derived neurotrophic factor (GDNF). On the other hand, SM caused a strong increase in P450 aromatase mRNA and protein expression. Interestingly, P450-aromatase was no more inducible by 8-Br-cAMP. The presence of a functional aromatase was confirmed by enrichment of 17β-estradiol released in the medium by androgen precursors. We concluded that SM causes a significant upregulation of aromatase gene expression in Sertoli cells, leading to a consequent increase in 17β-estradiol secretion. High level of 17β-estradiol in the testis could have potentially adverse effects on male fertility and testicular cancer.

  8. Regulated expression of erythropoietin by two human hepatoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  9. 改良组织块酶消化法培养人龋损牙髓干细胞的实验研究%Culture of human caries dental pulp stem cells with combined explants method and enzymatic separation method

    Institute of Scientific and Technical Information of China (English)

    麻丹丹; 高杰; 吴补领

    2011-01-01

    AIM; To compare the successfulness and the growth of human caries dental pulp stem cells(hCDPSCs) cultured with three different methods. METHODS: Twenty-five normal and caries human third molars were collected, the dental pulp tissues were cultured by the tissue explant method, tissue-explan collagenase digestion method and the combination of explant method and enzymatic separation method, respectively. The adherence of the explants, the morphology and quantity of cells were observed under a phase-contrast microscope. Culture duration was recorded. The clones were identified by expression of Stro-1 and CD90 and the growth curve of normal DPSCs and CDPSCs was drawn. RESULTS; Human normal DPSCs and CDPSCs could be cultured by all the three methods. A large number of human normal DPSCs and CDPSCs were cultured by the com hined explant method and enzymatic separation method in a shorter time, and these cells exhibited more vitality and more different morphologies. The growth rate of CDPSCs was higher than that of normal DPSCs . CONCLISION: The improved combination of explant method and enzymatic separation method is an ideal method for the primary culture of hCDPSCs in vitro, it may provide a methodological foundation for studying the mechanism of the formation of the tertiary dentine when the tooth was damaged.%目的:比较3种方法培养人龋损牙髓干细胞的成功率和细胞生长状态,以探求人龋损牙髓干细胞的最佳培养方法.方法:取18~22岁成人新鲜正常和龋损离体第三磨牙各25个,采用组织块法、酶消化法、改良组织块酶消化法培养牙髓干细胞.通过倒置显微镜观察组织块的贴壁以及细胞的形态和数量,并记录培养所需时间;有限稀释法纯化牙髓干细胞,流式细胞仪检测正常和龋损牙髓干细胞表面标记物Stro-1、CD 90的表达情况,绘制正常和龋损牙髓干细胞生长曲线.结果:组织块法、酶消化法和改良组织块酶消化法均可以培养

  10. Expression of rd29A::AtDREB1A/CBF3 in tomato alleviates drought-induced oxidative stress by regulating key enzymatic and non-enzymatic antioxidants.

    Science.gov (United States)

    Rai, Govind Kumar; Rai, Neha Prakash; Rathaur, Sushma; Kumar, Sanjeev; Singh, Major

    2013-08-01

    Transgenic tomato lines (cv. Kashi Vishesh) over-expressing AtDREB1A/CBF3 driven by stress-inducible rd29A promoter showed significantly higher activities of key antioxidant enzymes when exposed to water-deficit for 7, 14, and 21 days. Transgenic tomato plants exposed to water-deficit recorded lower levels of hydrogen peroxide and superoxide anion formation compared to the non-transgenic plants, suggesting alleviation of reactive oxygen species (ROS). A significant increase in activities of enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) was observed in response to the different durations of water-deficit conditions. In contrast, enzyme guaiacol peroxidase (POD) activity was lower in the transgenic lines and showed a negative correlation with ROS, ascorbic acid (AsA), and glutathione levels. The concentrations of AsA, glutathione and their reduced forms were higher in the transgenic plants and increased with ROS levels. These results indicate that AtDREB1A transgenic tomato lines are better adapted to water-deficit as they showed lower drought-induced oxidative stress due to activation of the antioxidant response.

  11. Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility

    Directory of Open Access Journals (Sweden)

    Aymerick Eudes

    2016-07-01

    Full Text Available Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet. In this study, we demonstrate in Arabidopsis stems that targeted expression of S-adenosylmethionine hydrolase (AdoMetase, E.C. 3.3.1.2 in secondary cell-wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H units and a reduction of dimethylated syringyl (S units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock.

  12. Gamma-irradiation enhances transgene expression in leukemic cells.

    Science.gov (United States)

    Vereecque, R; Saudemont, A; Wickham, T J; Gonzalez, R; Hetuin, D; Fenaux, P; Quesnel, B

    2003-02-01

    The majority of immunotherapy-based gene therapy protocols consist of ex vivo gene transfer in tumor cells. To prevent further in vivo growth, modified cells must be irradiated before reinjection into patients. The present study examines the effects of gamma-irradiation on transgene expression in transduced leukemic cells. Human and murine leukemic cells were transfected with retroviral vectors or plasmids carrying beta-galactosidase, GM-CSF or CD80 genes. Fresh leukemic cells from patients with acute myeloid leukemia (AML) were transfected with AdZ.F(pK7) adenoviral vector. gamma-irradiation at various lethal doses enhanced transgene expression in leukemic cell lines and fresh AML cells when the gene of interest was under CMV promoter but not when SV40 promoter was used. Oxidative stress also enhanced transgene expression and both irradiation and oxidative stress effects were inhibited by addition of N-acetyl-L-cysteine, a thiol anti-oxidant, indicating the involvement of reactive oxygen species. Transgene expression was also enhanced in vivo 48 and 120 h after subcutaneous injection of irradiated leukemic cells in syngeneic mice. These results show that a cell vaccine protocol using ex vivo gene transfer of transduced cells might be feasible in acute leukemia even if leukemic cells must be irradiated at lethal doses prior to reinjection to patients.

  13. MicroRNA expression profiles in avian haemopoietic cells

    Directory of Open Access Journals (Sweden)

    Yongxiu eYao

    2013-08-01

    Full Text Available MicroRNAs (miRNAs are small, abundant, non-coding RNAs that modulate gene expression by interfering with translation or stability of mRNA transcripts in a sequence-specific manner. A total of 734 precursor and 996 mature miRNAs have so far been identified in the chicken genome. A number of these miRNAs are expressed in a cell type-specific manner, and understanding their function requires detailed examination of their expression in different cell types. We carried out deep sequencing of small RNA populations isolated from stimulated or transformed avian haemopoietic cell lines to determine the changes in the expression profiles of these important regulatory molecules during these biological events. There were significant changes in the expression of a number of miRNAs, including miR-155, in chicken B cells stimulated with CD40 ligand. Similarly, avian leukosis virus (ALV-transformed DT40 cells also showed changes in miRNA expression in relation to the naïve cells. Embryonic stem cell line BP25 demonstrated a distinct cluster of upregulated miRNAs, many of which were shown previously to be involved in embryonic stem cell development. Finally, chicken macrophage cell line HD11 showed changes in miRNA profiles, some of which are thought to be related to the transformation by v-myc transduced by the virus. This work represents the first publication of a catalog of microRNA expression in a range of important avian cells and provides insights into the potential roles of miRNAs in the hematopoietic lineages of cells in a model non-mammalian species.

  14. Modulation of Vascular Cell Function by Bim Expression

    Directory of Open Access Journals (Sweden)

    Margaret E. Morrison

    2013-01-01

    Full Text Available Apoptosis of vascular cells, including pericytes and endothelial cells, contributes to disease pathogenesis in which vascular rarefaction plays a central role. Bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix (ECM protein expression. Endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation. Here we set out to determine the cell autonomous impact of Bim expression on retinal endothelial cell and pericyte function using cells prepared from Bim deficient (Bim−/− mice. Bim−/− endothelial cells displayed an increased production of ECM proteins, proliferation, migration, adhesion, and VEGF expression but, a decreased eNOS expression and nitric oxide production. In contrast, pericyte proliferation decreased in the absence of Bim while migration, adhesion, and VEGF expression were increased. In addition, we demonstrated that the coculturing of either wild-type or Bim−/− endothelial cells with Bim−/− pericytes diminished their capillary morphogenesis. Thus, our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function.

  15. Modulation of vascular cell function by bim expression.

    Science.gov (United States)

    Morrison, Margaret E; Palenski, Tammy L; Jamali, Nasim; Sheibani, Nader; Sorenson, Christine M

    2013-01-01

    Apoptosis of vascular cells, including pericytes and endothelial cells, contributes to disease pathogenesis in which vascular rarefaction plays a central role. Bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix (ECM) protein expression. Endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation. Here we set out to determine the cell autonomous impact of Bim expression on retinal endothelial cell and pericyte function using cells prepared from Bim deficient (Bim(-/-)) mice. Bim(-/-) endothelial cells displayed an increased production of ECM proteins, proliferation, migration, adhesion, and VEGF expression but, a decreased eNOS expression and nitric oxide production. In contrast, pericyte proliferation decreased in the absence of Bim while migration, adhesion, and VEGF expression were increased. In addition, we demonstrated that the coculturing of either wild-type or Bim(-/-) endothelial cells with Bim(-/-) pericytes diminished their capillary morphogenesis. Thus, our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function.

  16. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells.

    Science.gov (United States)

    de Graaf, Carolyn A; Choi, Jarny; Baldwin, Tracey M; Bolden, Jessica E; Fairfax, Kirsten A; Robinson, Aaron J; Biben, Christine; Morgan, Clare; Ramsay, Kerry; Ng, Ashley P; Kauppi, Maria; Kruse, Elizabeth A; Sargeant, Tobias J; Seidenman, Nick; D'Amico, Angela; D'Ombrain, Marthe C; Lucas, Erin C; Koernig, Sandra; Baz Morelli, Adriana; Wilson, Michael J; Dower, Steven K; Williams, Brenda; Heazlewood, Shen Y; Hu, Yifang; Nilsson, Susan K; Wu, Li; Smyth, Gordon K; Alexander, Warren S; Hilton, Douglas J

    2016-09-13

    Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Impact of Annexin A3 expression in gastric cancer cells.

    Science.gov (United States)

    Yu, S Y; Li, Y; Fan, L Q; Zhao, Q; Tan, B B; Liu, Y

    2014-01-01

    Annexin A3 participates in various biological processes, including tumorigenesis, drug resistance, and metastasis. The aim of this study was to investigate the expression of Annexin A3 in gastric cancer and its relationship with cell differentiation, migration, and invasion of gastric cancer cells. Annexin A3 expression in gastric cancer tissues was detected by quantitative real-time PCR and Western blotting. The proliferation of gastric cancer cells was measured by the MTT assay. Cell migration and invasion were determined via wound healing and transwell assays, respectively. Knock down of endogenous Annexin A3 in gastric cancer BGC823 cells was performed using siRNA technology. The expression of Annexin A3 was significantly upregulated in gastric cancer tissues, and negatively correlated with the differentiation degree. Silencing of endogenous Annexin A3 suppressed the proliferation, migration, and invasion of BGC823 cells. Additionally, the expression of p21, p27, TIMP-1, and TIMP-2 was upregulated, and the expression of PCNA, cyclin D1, MMP-1, and MMP-2 decreased in cells treated with Annexin A3-siRNA. Annexin A3 was upregulated in gastric cancer cells. Deletion of endogenous Annexin A3 significantly inhibited gastric cancer cell proliferation, migration, and invasion.

  18. Expression and Characterization of Catalytic Domain of T Cell Protein Tyrosine Phosphatase(ΔTC-PTP)——Immunohistochemical Study of ΔTC-PTP Expression in Non-small Cell Lung Carcinomas

    Institute of Scientific and Technical Information of China (English)

    ZHU Zhi-cheng; SUN Mei; ZHANG Xing-yi; LIU Ke-xiang; SHI Dong-lei; LI Jin-dong; SU Ji-quan; XU Yue-chi; FU Xue-qi

    2007-01-01

    This study objective was to express and characterize the catalytic domain of the human T cell protein tyrosine phosphatase(ΔTC-PTP) and to study immunohistochemically the expression of ΔTC-PTP in human non-small cell lung cancers. ΔTC-PTP gene was PCR amplified with the cDNA of human TC-PTP as template, and cloned into the pT7 expression vector. The recombinant pT7-ΔTC-PTP was expressed in E.coli Rosetta(DE3) host cells and purified. The enzymatic characteristics of ΔTC-PTP including enzyme activity and kinetics assay were measured. The antiserum was prepared by immunizing rabbit with the purified recombinant ΔTC-PTP. Rabbit polyclonal antibody against ΔTC-PTP was purified by PVDF immobilized antigen affinity chromatography. Immunohistochemical staining of lung cancer tissues was performed with antibody against ΔTC-PTP protein. ΔTC-PTP gene was correctly cloned, expressed, and purified. The recombinant ΔTC-PTP had a highly catalytic activity of PTPase. Squamous cell lung carcinoma showed a significantly higher expression rate of ΔTC-PTP(76.92%, 10/13) than adenocarcinoma(57.14%, 4/7) and normal lung tissue(20%, 1/5). This study represents the first demonstration that ΔTC-PTP is highly expressed in human squamous cell lung carcinomas. In addition, this study provides an important basis for further studying the biological function of TC-PTP and its relationship with lung carcinomas and other diseases.

  19. Cardiomyocyte expression and cell-specific processing of procholecystokinin

    DEFF Research Database (Denmark)

    Gøtze, Jens P.; Johnsen, Anders H.; Kistorp, Caroline

    2015-01-01

    has only been suggested using transcriptional measures or methods, with the post-translational phase of gene expression unaddressed. In this study, we examined the cardiac expression of the CCK gene in adult mammals and its expression at the protein level. Using quantitative PCR, a library of sequence......Heart muscle cells produce peptide hormones such as natriuretic peptides. Developing hearts also express the gene for the classic intestinal hormone cholecystokinin (CCK) in amounts similar to those in the intestine and brain. However, cardiac expression of peptides other than natriuretic peptides...

  20. Beryllium sulfate induces p21 CDKN1A expression and a senescence-like cell cycle arrest in susceptible cancer cell types.

    Science.gov (United States)

    Gorjala, Priyatham; Gary, Ronald K

    2010-12-01

    In fibroblasts, beryllium salt causes activation of the p53 transcription factor and induction of a senescence-like state. It is not known whether Be(2+) can affect the proliferation of cancer cells, which are generally unsusceptible to senescence. A172 glioblastoma and RKO colon carcinoma cell lines each have wildtype p53, so these cell types have the potential to be responsive to agents that activate p53. In A172 cells, BeSO(4) produced a G(0)/G(1)-phase cell cycle arrest and increased expression of senescence-associated β-galactosidase, an enzymatic marker of senescence. BeSO(4) caused phosphorylation of serine-15 of p53, accumulation of p53 protein, and expression of p21, the cyclin-dependent kinase inhibitor that is prominent during senescence. BeSO(4) inhibited A172 growth with an IC(50) = 4.7 μM in a 6-day proliferation assay. In contrast, BeSO(4) had no effect on RKO cells, even though Be(2+) uptake was similar for the two cell types. This differential responsiveness marks BeSO(4) as a reagent capable of activating a separable branch of the p53 signaling network. A172 and RKO cells are known to exhibit p53-dependent upregulation of p21 in response to DNA damage. The RKO cells produced high levels of p21 when exposed to DNA damaging agents, yet failed to express p21 when treated with BeSO(4). Conversely, BeSO(4) did not cause DNA damage in A172 cells, yet it was a potent inducer of p21 expression. These observations indicate that the growth control pathway affected by BeSO(4) is distinct from the DNA damage response pathway, even though both ultimately converge on p53 and p21.

  1. Expression and clinical significance of sulfiredoxin expression in cervical squamous cell carcinoma tissue

    Directory of Open Access Journals (Sweden)

    Xiao-yan CHEN

    2015-10-01

    Full Text Available Objective To inquire into the expression and its clinical significance of sulfiredoxin (Srx in cervical squamous cell carcinoma tissue. Methods SABC immunohistochemical method was used to detect the expression levels of Srx in specimens of 104 cervical squamous cell carcinoma and the corresponding adjacent tissues, 15 cervical intraepithelial neoplasm (CIN Ⅲ, and 20 normal cervical squamous cell epithelium tissue. The relationship between the expression of Srx protein and clinical pathological parameters of the cancer was also analyzed. Results The positive expression rates of Srx in CIN Ⅲ and cervical squamous cell carcinoma [73.3%(11/15 and 82.7%(86/104, respectively] were significantly higher than that in normal cervical tissue [35.0%(7/20, χ2=17.778, P=0.000]. Meanwhile, Srx expression in cervical cancer specimens was significantly higher than that in normal adjacent tissues (χ2=56.224, P=0.000. The positive expression of Srx in cervical squamous cell carcinoma was significantly correlated with lymph node metastasis, the depth of cancer invasion, and the infiltration of blood vessels (P0.05. Conclusion The higher expression of Srx protein might be a valuable marker for the early diagnosis and evaluation of prognosis in patients with cervical squamous cell carcinoma. DOI: 10.11855/j.issn.0577-7402.2015.08.11

  2. Native and enzymatically modified wheat (Triticum aestivum L.) endogenous lipids in bread making: a focus on gas cell stabilization mechanisms.

    Science.gov (United States)

    Gerits, Lien R; Pareyt, Bram; Masure, Hanne G; Delcour, Jan A

    2015-04-01

    Lipopan F and Lecitase Ultra lipases were used in straight dough bread making to study how wheat lipids affect bread loaf volume (LV) and crumb structure setting. Lipase effects on LV were dose and dough piece weight dependent. The bread quality improving mechanisms exerted by endogenous lipids were studied in terms of gluten network strengthening, which indirectly stabilizes gas cells, and in terms of direct interfacial gas cell stabilization. Unlike diacetyl tartaric esters of mono- and diacylglycerols (DATEM, used as control), lipase use did not impact dough extensibility. The effect on dough extensibility was therefore related to its lipid composition at the start of mixing. Both lipases and DATEM strongly increase the levels of polar lipids in dough liquor and their availability for and potential accumulation at gas cell interfaces. Lipases form lysolipids that emulsify other lipids. We speculate that DATEM competes with (endogenous) polar lipids for interacting with gluten proteins.

  3. Expression of heparanase in basal cell carcinoma and squamous cell carcinoma*

    Science.gov (United States)

    Pinhal, Maria Aparecida Silva; Almeida, Maria Carolina Leal; Costa, Alessandra Scorse; Theodoro, Thérèse Rachell; Serrano, Rodrigo Lorenzetti; Machado Filho, Carlos D'Apparecida Santos

    2016-01-01

    Background Heparanase is an enzyme that cleaves heparan sulfate chains. Oligosaccharides generated by heparanase induce tumor progression. Basal cell carcinoma and squamous cell carcinoma comprise types of nonmelanoma skin cancer. Objectives Evaluate the glycosaminoglycans profile and expression of heparanase in two human cell lines established in culture, immortalized skin keratinocyte (HaCaT) and squamous cell carcinoma (A431) and also investigate the expression of heparanase in basal cell carcinoma, squamous cell carcinoma and eyelid skin of individuals not affected by the disease (control). Methods Glycosaminoglycans were quantified by electrophoresis and indirect ELISA method. The heparanase expression was analyzed by quantitative RT-PCR (qRTPCR). Results The A431 strain showed significant increase in the sulfated glycosaminoglycans, increased heparanase expression and decreased hyaluronic acid, comparing to the HaCaT lineage. The mRNA expression of heparanase was significantly higher in Basal cell carcinoma and squamous cell carcinoma compared with control skin samples. It was also observed increased heparanase expression in squamous cell carcinoma compared to the Basal cell carcinoma. Conclusion The glycosaminoglycans profile, as well as heparanase expression are different between HaCaT and A431 cell lines. The increased expression of heparanase in Basal cell carcinoma and squamous cell carcinoma suggests that this enzyme could be a marker for the diagnosis of such types of non-melanoma cancers, and may be useful as a target molecule for future alternative treatment. PMID:27828631

  4. Ganoderic acid Me induces the apoptosis of competent T cells and increases the proportion of Treg cells through enhancing the expression and activation of indoleamine 2,3-dioxygenase in mouse lewis lung cancer cells.

    Science.gov (United States)

    Que, Zujun; Zou, Fangyuan; Zhang, Anle; Zheng, Yuanhong; Bi, Ling; Zhong, Jianjiang; Tian, Jianhui; Liu, Jianwen

    2014-11-01

    The indoleamine 2,3-dioxygenase-(IDO-) mediated microenvironment plays an important role in tumor immune escape. It is known that ganoderic acid Me can enhance IFN-γ expression and IDO is preferentially induced by IFN-γ. However, whether GA-Me can induce IDO expression has not been clarified yet. We established stable clones of IDO-overexpressing 2 LL cells (2LL-EGFP-IDO). After co-culturing with IDO expressing or control vector-transfected 2LL-EGFP cells, T cell apoptosis was determined and the proportion of the regulatory T cells (Tregs) and CD8+ T cell subset was measured. The total cellular protein samples of 2 LL-EGFP-IDO cells were isolated for detecting JAK-STAT1 signalling pathway. Co-culture supernatants were used to detect amino acids and cytokines. IDO transfected 2 LL cells yielded high level of IDO enzymatic activity, resulting in complete depletion of tryptophan from the culture medium. We found that apoptosis occurred in T cells after cocultured with IDO+2LL cells and the proportion of CD4+CD25+ cells and FoxP3+ cells increased while CD8+ cells decreased. The specific inhibitor of IDO, 1-D-MT and GA-Me efficiently enhanced T cell apoptosis, increased Tregs, and reduced CD8+ T cells in vitro. Increased expression of IDO, p-JAK1 and p-STAT1 were confirmed by Western blot analysis. The levels of IFN-γ, IL-10, LDH and kynurenine in co-culture supernatant correspondingly increased, while tryptophan reduced. These results suggest that GA-Me contributing to IDO helps to create a tolerogenic milieu in lung tumors by directly inducing T cell apoptosis, restraining CD8+ T cell activation, and enhancing Treg-mediated immunosuppression. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Salmonella induces PD-L1 expression in B cells.

    Science.gov (United States)

    Lopez-Medina, Marcela; Perez-Lopez, Araceli; Alpuche-Aranda, Celia; Ortiz-Navarrete, Vianney

    2015-10-01

    Salmonella persists for a long time in B cells; however, the mechanism(s) through which infected B cells avoid effector CD8 T cell responses has not been characterized. In this study, we show that Salmonella infects and survives within all B1 and B2 cell subpopulations. B cells are infected with a Salmonella typhimurium strain expressing an ovalbumin (OVA) peptide (SIINFEKL) to evaluate whether B cells process and present Salmonella antigens in the context of MHC-I molecules. Our data showed that OVA peptides are presented by MHC class I K(b)-restricted molecules and the presented antigen is generated through proteasomal degradation and vacuolar processing. In addition, Salmonella-infected B cells express co-stimulatory molecules such as CD40, CD80, and CD86 as well as inhibitory molecules such as PD-L1. Thus, the cross-presentation of Salmonella antigens and the expression of activation molecules suggest that infected B cells are able to prime and activate specific CD8(+) T cells. However, the Salmonella infection-stimulated expression of PD-L1 suggests that the PD-1/PD-L1 pathway may be involved in turning off the cytotoxic effector response during Salmonella persistent infection, thereby allowing B cells to become a reservoir for the bacteria.

  6. Expression of SOST/sclerostin in compressed periodontal ligament cells

    Directory of Open Access Journals (Sweden)

    Masae Ueda

    2016-09-01

    Conclusion: These results indicate that PDL cells subjected to light compressive force exhibit increased expression of SOST/sclerostin, which inhibits bone formation on the compressed side during orthodontic tooth movement.

  7. Interdependence of cell growth and gene expression: origins and consequences.

    Science.gov (United States)

    Scott, Matthew; Gunderson, Carl W; Mateescu, Eduard M; Zhang, Zhongge; Hwa, Terence

    2010-11-19

    In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

  8. Expression of EPO Receptor in Pancreatic Cells and Its Effect on Cell Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hongxia SHUAI; Ji ZHANG; Yikai YU; Muxun ZHANG

    2008-01-01

    In order to explore the expression of erythropoietin receptor (EPOR) in pancreatic cell ine NIT-1 and its effect on cell apoptosis after binding with erythropoietin (EPO), NIT-1 cells were cultured and expanded. The expression of EPOR was detected using electrophoresis. NIT-1 apoptosis was induced by cytokines and their effects on cell apoptosis and cell insulin secretion were assayed after binding of EPO to EPOR. The results showed that EPOR was expressed in NIT-1 cells. Recom- binant human EPO (rHuEPO) had no effect on cell apoptosis but significantly inhibited apoptosis in- duced by cytokines, rHuEPO had no effect on cell insulin secretion but significantly improved insulin secretion inhibited by cytokines. From these findings, it was concluded that EPOR was expressed in NIT-1 cells and EPO could protect N1T-1 cells from apoptosis induced by cytokines.

  9. Expression of aquaporin-1 in SMMC-7221 liver carcinoma cells promotes cell migration

    Institute of Scientific and Technical Information of China (English)

    LI Yongming; FENG Xuechao; YANG Hong; MA Tonghui

    2006-01-01

    Migration of tumor cells is a crucial step in tumor invasion and metastasis. Here we provide evidence that aquaporin expression is involved in tumor cell migration. RT-PCR, immunofluorescence and Western blot analysis demonstrated the AQP1 protein expression on the plasma membrane of SMMC-7221 human hepatoma cells. SMMC-7221 cell clones with high (SMMC-7221hPf) and low (SMMC-7221/Pf) water permeability were identified by functional assays with corresponding high and low AQP1 expression. Cell migration rate was remarkably higher in SMMC-7221hPf cells than SMMC-7221/Pf cells, assessed by Boyden chamber and wound healing assays, whereas cell growth and adhesion were not different. Adenovirus-mediated AQP1 expression in SMMC-7221/Pf cells increased their water permeability and migration rate. These results provide the first evidence that aquaporin-mediated membrane water permeability enhances tumor cell migration and may be associated with tumor invasion and metastasis.

  10. Obstructor A Organizes Matrix Assembly at the Apical Cell Surface to Promote Enzymatic Cuticle Maturation in Drosophila*

    Science.gov (United States)

    Pesch, Yanina-Yasmin; Riedel, Dietmar; Behr, Matthias

    2015-01-01

    Assembly and maturation of the apical extracellular matrix (aECM) is crucial for protecting organisms, but underlying molecular mechanisms remain poorly understood. Epidermal cells secrete proteins and enzymes that assemble at the apical cell surface to provide epithelial integrity and stability during developmental growth and upon tissue damage. We analyzed molecular mechanisms of aECM assembly and identified the conserved chitin-binding protein Obst-A (Obstructor A) as an essential regulator. We show in Drosophila that Obst-A is required to coordinate protein and chitin matrix packaging at the apical cell surface during development. Secreted by epidermal cells, the Obst-A protein is specifically enriched in the apical assembly zone where matrix components are packaged into their highly ordered architecture. In obst-A null mutant larvae, the assembly zone is strongly diminished, resulting in severe disturbance of matrix scaffold organization and impaired aECM integrity. Furthermore, enzymes that support aECM stability are mislocalized. As a biological consequence, cuticle architecture, integrity, and function are disturbed in obst-A mutants, finally resulting in immediate lethality upon wounding. Our studies identify a new core organizing center, the assembly zone that controls aECM assembly at the apical cell surface. We propose a genetically conserved molecular mechanism by which Obst-A forms a matrix scaffold to coordinate trafficking and localization of proteins and enzymes in the newly deposited aECM. This mechanism is essential for maturation and stabilization of the aECM in a growing and remodeling epithelial tissue as an outermost barrier. PMID:25737451

  11. Non-enzymatic electrochemical biosensor based on Pt NPs/RGO-CS-Fc nano-hybrids for the detection of hydrogen peroxide in living cells.

    Science.gov (United States)

    Bai, Zhihao; Li, Guiyin; Liang, Jingtao; Su, Jing; Zhang, Yue; Chen, Huaizhou; Huang, Yong; Sui, Weiguo; Zhao, Yongxiang

    2016-08-15

    A highly sensitive non-enzymatic electrochemical sensor based on platinum nanoparticles/reduced graphene oxide-chitosan-ferrocene carboxylic acid nano-hybrids (Pt NPs/RGO-CS-Fc biosensor) was developed for the measurement of hydrogen peroxide (H2O2). The RGO-CS-Fc nano-hybrids was prepared and characterized by UV-vis spectrum, Fourier transform infrared spectroscopy, transmission electron microscopy, Raman spectrometer and electrochemical impedance spectroscopy. Under optimal experimental conditions, the Pt NPs/RGO-CS-Fc biosensor showed outstanding catalytic activity toward H2O2 reduction. The current response of the biosensor presented a linear relationship with H2O2 concentration from 2.0×10(-8)M to 3.0×10(-6)M with a correlation coefficient of R(2)=0.9968 and with logarithm of H2O2 concentration from 6.0×10(-6)M to 1.0×10(-2)M with a correlation coefficient of R(2)=0.9887, the low detection limit of 20nM was obtained at the signal/noise (S/N) ratio of 3. Moreover, the Pt NPs/RGO-CS-Fc biosensor exhibited excellent anti-interference capability and reproducibility for the detection of H2O2. The biosensor was also successfully applied for the detection of H2O2 from living cells containing normal and cancer cells. All these results prove that the Pt NPs/RGO-CS-Fc biosensor has the potential application in clinical diagnostics to evaluate oxidative stress of different living cells.

  12. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies that comp......It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies...... in cell adhesion and the cytoskeleton. If the proteins involved in tethering cells to the extracellular matrix are important in conferring drug resistance, it may be possible to improve chemotherapy by designing drugs that target these proteins....

  13. Isolation of a sesquiterpene synthase expressing in specialized epithelial cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

    Science.gov (United States)

    Uji, Yuya; Ozawa, Rika; Shishido, Hodaka; Taniguchi, Shiduku; Takabayashi, Junji; Akimitsu, Kazuya; Gomi, Kenji

    2015-05-15

    Volatile terpenoids such as monoterpenes and sesquiterpenes play multiple roles in plant responses and are synthesized by terpene synthases (TPSs). We have previously isolated a partial TPS gene, RlemTPS4, that responds to microbial attack in rough lemon. In this study, we isolated a full length RlemTPS4 cDNA from rough lemon. RlemTPS4 localized in the cytosol. The recombinant RlemTPS4 protein was obtained using a prokaryotic expression system and GC-MS analysis of the terpenes produced by the RlemTPS4 enzymatic reaction determined that RlemTPS4 produces some sesquiterpenes such as δ-elemene. The RlemTPS4 gene was specifically expressed in specialized epithelial cells surrounding the oil secretory cavities in rough lemon leaf tissue.

  14. Ctr2 Regulates Mast Cell Maturation by Affecting the Storage and Expression of Tryptase and Proteoglycans.

    Science.gov (United States)

    Öhrvik, Helena; Logeman, Brandon; Noguchi, Glyn; Eriksson, Inger; Kjellén, Lena; Thiele, Dennis J; Pejler, Gunnar

    2015-10-15

    Copper (Cu) is essential for multiple cellular functions. Cellular uptake of Cu(+) is carried out by the Ctr1 high-affinity Cu transporter. The mobilization of endosomal Cu pools is regulated by a protein structurally similar to Ctr1, called Ctr2. It was recently shown that ablation of Ctr2 caused an increase in the concentration of Cu localized to endolysosomes. However, the biological significance of excess endolysosomal Cu accumulation has not been assessed. In this study, we addressed this issue by investigating the impact of Ctr2 deficiency on mast cells, a cell type unusually rich in endolysosomal organelles (secretory granules). We show that Ctr2(-/-) mast cells have increased intracellular Cu concentrations and that the absence of Ctr2 results in increased metachromatic staining, the latter indicating an impact of Ctr2 on the storage of proteoglycans in the secretory granules. In agreement with this, the absence of Ctr2 caused a skewed ratio between proteoglycans of heparin and chondroitin sulfate type, with increased amounts of heparin accompanied by a reduction of chondroitin sulfate. Moreover, transmission electron microscopy analysis revealed a higher number of electron-dense granules in Ctr2(-/-) mast cells than in wild-type cells. The increase in granular staining and heparin content is compatible with an impact of Ctr2 on mast cell maturation and, in support of this, the absence of Ctr2 resulted in markedly increased mRNA expression, storage, and enzymatic activity of tryptase. Taken together, the present study introduces Ctr2 and Cu as novel actors in the regulation of mast cell maturation and granule homeostasis.

  15. Androgens enhance the glycolytic metabolism and lactate export in prostate cancer cells by modulating the expression of GLUT1, GLUT3, PFK, LDH and MCT4 genes.

    Science.gov (United States)

    Vaz, Cátia V; Marques, Ricardo; Alves, Marco G; Oliveira, Pedro F; Cavaco, José E; Maia, Cláudio J; Socorro, Sílvia

    2016-01-01

    The present study aims to investigate the role of androgens in controlling the glycolytic metabolism and lactate efflux in prostate cancer (PCa) cells. Androgen-responsive LNCaP cells were treated with 5α-dihydrotestosterone (DHT, 10 nM) for 12-48 h, and their glycolytic metabolism, lactate production and viability were analyzed. Intracellular and extracellular levels of glucose and lactate were determined spectrophotometrically, and the expression of glucose transporters (GLUT1/GLUT3), phosphofructokinase 1, lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) was analyzed by real-time PCR and Western blot. The enzymatic activity of LDH was determined by means of a colorimetric assay. Experiments were reproduced in androgen-non-responsive DU145 and PC3 cells. Androgens stimulated glucose consumption in LNCaP cells by increasing the expression of GLUT3, GLUT1 and PFK, which was underpinned by increased cell viability. Accordingly, lactate production by LNCaP cells was enhanced upon DHT stimulation as evidenced by the increased levels of lactate found in cell culture medium. Although LDH enzymatic activity decreased in LNCaP cells treated with DHT, the expression of MCT4 was significantly increased with androgenic treatment, which sustains the increase on lactate export. Glucose consumption and the expression of GLUTs and PFK remained unchanged in DHT-treated DU145 and PC3 cells. The results obtained establish androgens as modulators of glycolytic metabolism in PCa cells by stimulating glucose consumption, as well as the production and export of lactate, which may represent a crucial issue-driven prostate tumor development. These findings also highlight the importance of PCa therapies targeting AR and metabolism-related proteins.

  16. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

    Science.gov (United States)

    Wu, D Y; Ugozzoli, L; Pal, B K; Wallace, R B

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3' nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  17. Inducible expression of endomorphins in murine dendritic cells.

    Science.gov (United States)

    Yang, Xiaohuai; Xia, Hui; Chen, Yong; Liu, Xiaofen; Zhou, Cheng; Gao, Qin; Li, Zhenghong

    2012-12-15

    Bone marrow precursor cells were extracted from C57BL/6J mice aged 7-8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [(3)H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of µ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of µ-opioid receptors.

  18. Inducible expression of endomorphins in murine dendritic cells

    Institute of Scientific and Technical Information of China (English)

    Xiaohuai Yang; Hui Xia; Yong Chen; Xiaofen Liu; Cheng Zhou; Qin Gao; Zhenghong Li

    2012-01-01

    Bone marrow precursor cells were extracted from C57BL/6J mice aged 7–8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [3H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of μ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of μ-opioid receptors.

  19. Recombinant cells that highly express chromosomally-integrated heterologous gene

    Science.gov (United States)

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  20. Down-regulation of CXCR4 expression by tamoxifen is associated with DNA methyltransferase 3B up-regulation in MCF-7 breast cancer cells.

    Science.gov (United States)

    Kubarek, Ł; Kozłowska, A; Przybylski, M; Lianeri, M; Jagodzinski, P P

    2009-09-01

    The CXCR4 chemokine receptor is a seven transmembrane G protein-coupled receptor present on the surface of various cells including cancer cells. The CXCR4 receptor contributes to the induction of several intracellular signalling pathways that enhance survival, proliferation, and migration of malignant cells. We observed that tamoxifen (Tam) reduced the CXCR4 transcript and protein levels in MCF-7 breast cancer cells. However, we did not see a Tam effect on CXCR4 transcript and protein levels in MCF-7(LVMT3B) cells with RNA interference-mediated knockdown of DNMT3B. We also observed that Tam significantly increased, for several hours, the expression of enzymatically active DNMT3B splice variants in MCF-7 cells. However, there was no Tam effect on these DNMT3B splice variants' expression in MCF-7(LVMT3B) cells. Bisulfite sequencing suggests that Tam may reduce CXCR4 expression via increased methylation of cytosine in the cytosine-guanosine (CpG) dinucleotide island of the CXCR4 promoter of MCF-7 breast cancer cells. Our findings suggest that Tam induces an increase in DNMT3B expression that is associated with the increase of CpG dinucleotide methylation in the CXCR4 promoter and significant reduction of CXCR4 gene expression in MCF-7 cells.

  1. Cyp26b1 regulates retinoic acid-dependent signals in T cells and its expression is inhibited by transforming growth factor-β.

    Directory of Open Access Journals (Sweden)

    Hajime Takeuchi

    Full Text Available BACKGROUND: The vitamin A metabolite, retinoic acid (RA, plays important roles in the regulation of lymphocyte properties. Dendritic cells in gut-related lymphoid organs can produce RA, thereby imprinting gut-homing specificity on T cells and enhancing transforming growth factor (TGF-β-dependent induction of Foxp3+ regulatory T cells upon antigen presentation. In general, RA concentrations in cells and tissues are regulated by its degradation as well. However, it remained unclear if T cells could actively catabolize RA. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the expression of known RA-catabolizing enzymes in T cells from mouse lymphoid tissues. Antigen-experienced CD44+ T cells in gut-related lymphoid organs selectively expressed Cyp26b1, a member of the cytochrome P450 family 26. However, T cells in the spleen or skin-draining lymph nodes did not significantly express Cyp26b1. Accordingly, physiological levels of RA (1-10 nM could induce Cyp26b1 expression in naïve T cells upon activation in vitro, but could not do so in the presence of TGF-β. Overexpression of Cyp26b1 significantly suppressed the RA effect to induce expression of the gut-homing receptor CCR9 on T cells. On the other hand, knocking down Cyp26b1 gene expression with small interfering RNA or inhibiting CYP26 enzymatic activity led to enhancement of the RA-induced CCR9 expression. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a role for CYP26B1 in regulating RA-dependent signals in activated T cells but not during TGF-β-dependent differentiation to Foxp3+ regulatory T cells. Aberrant expression of CYP26B1 may disturb T cell trafficking and differentiation in the gut and its related lymphoid organs.

  2. Gene expression analysis of in vivo fluorescent cells.

    Directory of Open Access Journals (Sweden)

    Konstantin Khodosevich

    Full Text Available BACKGROUND: The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD. Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters. METHODOLOGY/PRINCIPAL FINDINGS: We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of approximately 2ratio1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR. CONCLUSIONS/SIGNIFICANCE: Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.

  3. Expression and function of nicotinic acetylcholine receptors in stem cells

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  4. HIV-1 induces DCIR expression in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Alexandra A Lambert

    Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

  5. 酶预消化连续组织块法培养大鼠脂肪来源干细胞的研究%Culture of rat adipose-diverted stem cells by serial explant after enzymatic predigestion

    Institute of Scientific and Technical Information of China (English)

    李伯休; 程飚; 陈峥嵘

    2008-01-01

    Objective To isolate and culture adipose-diverted stem cells (ADSCs) in SD rats using serial explant after enzymatic predigestion, and compare, the results with that from Zuk's method. Methods Adipose tissue from groin area of one-month-old SD rats was divided into two groups. In group A, the adipose tissue was snipped into 8mm3 blocks after washed with D-Hank's solution. The tissue blocks were digested by 0.25% trypsin for 5 minutes and 0.1% collagenase type 1 for 20 minutes, and put on 1mm grid nylon net for 3 to 4 days of cultivation. The tissues were then removed to another culture flask. They were repeatedly cultured by serial explant method for three or four times. In group B, the adipose tissue was treated by Zuk's method. MTT was used to measure bioactivity of the culture and flow cytometry was used to detect surface markers of the stem cells. Results In both groups, the cells had typical morphological characteristics of stem cells and expressed adipose stem cell surface markers CD105 and CD44. Conclusion The adipose-diverted stem cells cultured by technique of enzymatic digestion and serial explant in vitro have the same characteristics of those cultured with Zuk's method. This technique can yield plenty of ADSCs samples during a short period of time and thus enables its practical use in ADSCs cultivation.%目的 采用胰蛋白酶+I型胶原酶预消化后连续组织块法进行SD大鼠脂肪来源干细胞的体外培养,并和Zuk的胶原酶消化法培养效果相比较,为脂肪来源干细胞的体外培养方法提供参考依据.方法 取1月龄SD大鼠腹股沟处脂肪组织,分A、B两组.A组使用D-Hank's液冲洗净后剪成8mm3左右组织块.0.25%胰蛋白酶消化5min后,0.1%I型胶原酶消化组织块20min,将组织块置于孔径1mm的尼龙网放至培养皿贴壁培养,3-4d后将组织块连同滤网放至下一培养皿中以连续组织块法培养3-4次.B组使用Zuk的胶原酶消化法培养.采用MTT检测方法对细胞增

  6. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle

    -derived antigenic peptides, a function which is currently explored in immunotherapeutic approaches against cancer. Additionally, membrane-bound Hsp70 can stimulate antigen presenting cells to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells...... frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 cell surface expression on cancer...... cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC-inhibitor mediated Hsp70 cell surface expression was confined...

  7. Expression of stromelysin 3 in basal cell carcinomas.

    Science.gov (United States)

    Cribier, B; Noacco, G; Peltre, B; Grosshans, E

    2001-01-01

    Stromelysin 3 is a member of the metalloproteinase family, which is expressed in various remodelling processes. The prognosis of breast cancers and squamous cell carcinomas is correlated to the level of expression of this protein. The purpose of the present work was to evaluate the expression of stromelysin 3 in the major types of basal cell carcinomas. We selected cases of primary tumours that were fully excised, without previous biopsy: 40 Pinkus tumors, 40 superficial, 40 nodular, 38 morpheiform basal cell carcinomas and 10 cases showing deep subcutaneous or muscular invasion. Immunohistochemistry was carried out using monoclonal anti-ST3 antibodies (MC Rio, IGBMC Strasbourg), and evaluated on a semi-quantitative scale from 0 to 3. Positively stained cells were restricted to the periphery of the epithelial cells, which, by contrast, never expressed stromelysin 3. The global rate of expression was 27% in Pinkus tumors, 65% in superficial, 72.5% in nodular, 87% in morpheiform and 100% in deeply invasive carcinomas. The rates of tumours showing the highest number of positively stained cells (class 2 or 3) were respectively 7.5%, 20%, 45%, 63% and 100%. This systematic study of stromelysin3 expression in basal cell carcinomas confirms that it is a marker of poor prognosis, because the rate of positive tumours was much higher in aggressive carcinomas. Moreover, the majority of tumours showing an intense expression (i.e. the highest number of positively stained cells in their stroma) were of the morpheiform and deeply invasive types, which are of poor prognosis. Altogether, the studies performed on cutaneous tumours are consistent with the theory of stromelysin 3 playing an active role in tumour progression.

  8. Microarray gene expression profiling and analysis in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sadhukhan Provash

    2004-06-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  9. Performance of non-compartmentalized enzymatic biofuel cell based on buckypaper cathode and ferrocene-containing redox polymer anode

    Science.gov (United States)

    Bunte, Christine; Hussein, Laith; Urban, Gerald A.

    2014-02-01

    Novel single compartment Glucose/O2 biofuel cells (BFCs) were developed using immobilized enzymes and the mediated electron transfer (MET) approach. The bioanode was prepared through a ferrocene-containing redox polymer crosslinked in the presence of a biocatalyst on a glassy carbon support. Here, the redox polymer can physically entrap the enzyme and prevent it from leaching. Additionally it provides a biocompatible microenvironment and thus could extend the life time of enzyme. On the other side, the mediated biocathode was prepared based on bilirubin oxidase and 2,2‧-azinobis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS2-) system which has been physically entrapped in Nafion matrix and then adsorbed directly on a highly porous, conductive and functionalized buckypaper (fBP). Both electrodes were characterized physically and electrochemically. Employing these electrodes, the resulting BFC generates an open circuit voltage (Voc) of approximately 0.550 V and a peak power density of 26 μW cm-2 at 0.2 V at 37 °C in quiescent O2-saturated physiological buffer containing 5 mM glucose. The cell sustains a load up to 225 μA cm-2. Moreover, a high short circuit current (Isc) of 300 μA cm-2 is approached. This BFC can operate in mild conditions without using any toxic materials which makes it attractive for implantable devices.

  10. External nitrogen input affects pre- and post-harvest cell wall composition but not the enzymatic saccharification of wheat straw

    DEFF Research Database (Denmark)

    Baldwin, Laetitia Andrée; Glazowska, Sylwia Emilia; Mravec, Jozef

    2017-01-01

    Wheat is one of the most important crops for food and feed and its straw is a potential feedstock for biorefinery purposes. Nitrogen (N) is an essential input factor in wheat agriculture but no information is available on how it affects straw composition during maturation and at harvest. To inves......Wheat is one of the most important crops for food and feed and its straw is a potential feedstock for biorefinery purposes. Nitrogen (N) is an essential input factor in wheat agriculture but no information is available on how it affects straw composition during maturation and at harvest....... To investigate this, we conducted a large scale field experiment in which wheat plants were cultivated at three levels of externally applied N. The plants were harvested at different stages of maturation, spanning green straw at heading (ear emergence) to fully yellow straw at final maturity. Defined parts...... of the straw were analyzed for cell wall characteristics relevant for further biomass processing. The straw N concentration corroborated with the level of N input, but the yield of straw biomass was not largely affected. High N treatment modified cell wall composition, namely increased abundance...

  11. Cell-specific expression of TLR9 isoforms in inflammation.

    Science.gov (United States)

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

  12. Tff3 is Expressed in Neurons and Microglial Cells

    Directory of Open Access Journals (Sweden)

    Ting Fu

    2014-11-01

    Full Text Available Background/Aims: The trefoil factor family (TFF peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. Methods: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. Results: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. Conclusion: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

  13. Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

    Institute of Scientific and Technical Information of China (English)

    Jeeranan Manokawinchoke; Thanaphum Osathanon; Prasit Pavasant

    2016-01-01

    Objective: To investigate the influence of Notch signaling on osteoprotegerin(OPG)expression in a human oral squamous cell carcinoma cell line.Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a g-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG m RNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software.Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 m RNA expression, confirming the intracellular activation of Notch signaling. OPG m RNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a g-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 m RNA levels, and a marked increase in OPG protein expression was observed.These results implied that Notch signaling regulated OPG expression in HSC-4 cells.However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line(HSC-5) or a human head and neck squamous cell carcinoma cell line(HN22).Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

  14. Gene expression markers for Caenorhabditis elegans vulval cells.

    Science.gov (United States)

    Inoue, Takao; Sherwood, David R; Aspöck, Gudrun; Butler, James A; Gupta, Bhagwati P; Kirouac, Martha; Wang, Minqin; Lee, Pei-Yun; Kramer, James M; Hope, Ian; Bürglin, Thomas R; Sternberg, Paul W

    2002-12-01

    The analysis of cell fate patterning during the vulval development of Caenorhabditis elegans has relied mostly on the direct observation of cell divisions and cell movements (cell lineage analysis). However, reconstruction of the developing vulva from EM serial sections has suggested seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), many of which cannot be distinguished based on such observations. Here we report the vulval expression of seven genes, egl-17, cdh-3, ceh-2, zmp-1, B0034.1, T04B2.6 and F47B8.6 based on gfp, cfp and yfp (green fluorescent protein and color variants) reporter fusions. Each gene expresses in a specific subset of vulval cells, and is therefore useful as a marker for vulval cell fates. Together, expressions of markers distinguish six cell types, and reveal a strict temporal control of gene expression in the developing vulva.

  15. Effect of radiation quality on mutagenic joining of enzymatically-induced DNA double-strand breaks in previously irradiated human cells.

    Science.gov (United States)

    Li, Zhentian; Wang, Huichen; Wang, Ya; Murnane, John P; Dynan, William S

    2014-11-01

    Previous work has shown that high charge and energy particle irradiation of human cells evokes a mutagenic repair phenotype, defined by increased mutagenic repair of new double-strand breaks that are introduced enzymatically, days or weeks after the initial irradiation. The effect was seen originally with 600 MeV/u (56)Fe particles, which have a linear energy transfer (LET) value of 174 keV/μm, but not with X rays or γ rays (LET ≤ 2 keV/μm). To better define the radiation quality dependence of the phenomenon, we tested two ions with intermediate LET values, 1,000 MeV/u (48)Ti (LET = 108 keV/μm) and 300 MeV/u (28)Si (LET = 69 keV/μm). These experiments used a previously validated assay, where a rare-cutting nuclease introduces double-strand breaks in two reporter transgene cassettes, which are located on different chromosomes. Deletions of a block of sequence in one of the cassettes, or translocations between cassettes, are measured independently using a multicolor fluorescence assay. The results showed that (48)Ti was a potent, but transient, inducer of mutagenic repair, based on increased frequency of nuclease-induced translocations. The (48)Ti ions did not affect the frequency of nuclease-induced deletions. The (28)Si ions had no measurable effect on either endpoint. There was a close correlation between the induction of the mutagenic repair phenomenon and the frequency of micronuclei in the targeted population (R(2) = 0.74), whereas there was no apparent correlation with radiation-induced cell inactivation. Together, these results better define the radiation quality dependence of the mutagenic repair phenomenon and establish its correlation, or lack of correlation, with other endpoints.

  16. Fatty Acid Esters of Phloridzin Induce Apoptosis of Human Liver Cancer Cells through Altered Gene Expression

    Science.gov (United States)

    Nair, Sandhya V. G.; Ziaullah; Rupasinghe, H. P. Vasantha

    2014-01-01

    Phloridzin (phlorizin or phloretin 2′-O-glucoside) is known for blocking intestinal glucose absorption. We have investigated the anticarcinogenic effect of phloridzin and its novel derivatives using human cancer cell lines. We have synthesised novel acylated derivatives of phloridzin with six different long chain fatty acids by regioselective enzymatic acylation using Candida Antarctica lipase B. The antiproliferative effects of the new compounds were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids and chemotherapeutic drugs (sorafenib, doxorubicin and daunorubicin) using human hepatocellular carcinoma HepG2 cells, human breast adenocarcinoma MDA-MB-231 cells and acute monocytic leukemia THP-1 cells along with normal human and rat hepatocytes. The fatty acid esters of phloridzin inhibited significantly the growth of the two carcinoma and leukemia cells while similar treatment doses were not toxic to normal human or rat hepatocytes. The antiproliferative potency of fatty esters of phloridzin was comparable to the potency of the chemotherapeutic drugs. The fatty acid esters of phloridzin inhibited DNA topoisomerases IIα activity that might induce G0/G1 phase arrest, induced apoptosis via activation of caspase-3, and decreased ATP level and mitochondrial membrane potential in HepG2 cells. Based on the high selectivity on cancer cells, decosahexaenoic acid (DHA) ester of phloridzin was selected for gene expression analysis using RT2PCR human cancer drug target array. Antiproliferative effect of DHA ester of phloridzin could be related to the down regulation of anti-apoptotic gene (BCL2), growth factor receptors (EBFR family, IGF1R/IGF2, PDGFR) and its downstream signalling partners (PI3k/AKT/mTOR, Ras/Raf/MAPK), cell cycle machinery (CDKs, TERT, TOP2A, TOP2B) as well as epigenetics regulators (HDACs). These results suggest that fatty esters of phloridzin have potential chemotherapeutic effects mediated

  17. Fatty acid esters of phloridzin induce apoptosis of human liver cancer cells through altered gene expression.

    Directory of Open Access Journals (Sweden)

    Sandhya V G Nair

    Full Text Available Phloridzin (phlorizin or phloretin 2'-O-glucoside is known for blocking intestinal glucose absorption. We have investigated the anticarcinogenic effect of phloridzin and its novel derivatives using human cancer cell lines. We have synthesised novel acylated derivatives of phloridzin with six different long chain fatty acids by regioselective enzymatic acylation using Candida Antarctica lipase B. The antiproliferative effects of the new compounds were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids and chemotherapeutic drugs (sorafenib, doxorubicin and daunorubicin using human hepatocellular carcinoma HepG2 cells, human breast adenocarcinoma MDA-MB-231 cells and acute monocytic leukemia THP-1 cells along with normal human and rat hepatocytes. The fatty acid esters of phloridzin inhibited significantly the growth of the two carcinoma and leukemia cells while similar treatment doses were not toxic to normal human or rat hepatocytes. The antiproliferative potency of fatty esters of phloridzin was comparable to the potency of the chemotherapeutic drugs. The fatty acid esters of phloridzin inhibited DNA topoisomerases IIα activity that might induce G0/G1 phase arrest, induced apoptosis via activation of caspase-3, and decreased ATP level and mitochondrial membrane potential in HepG2 cells. Based on the high selectivity on cancer cells, decosahexaenoic acid (DHA ester of phloridzin was selected for gene expression analysis using RT2PCR human cancer drug target array. Antiproliferative effect of DHA ester of phloridzin could be related to the down regulation of anti-apoptotic gene (BCL2, growth factor receptors (EBFR family, IGF1R/IGF2, PDGFR and its downstream signalling partners (PI3k/AKT/mTOR, Ras/Raf/MAPK, cell cycle machinery (CDKs, TERT, TOP2A, TOP2B as well as epigenetics regulators (HDACs. These results suggest that fatty esters of phloridzin have potential chemotherapeutic effects

  18. Equine Adipose-Derived Mesenchymal Stem Cells: Phenotype and Growth Characteristics, Gene Expression Profile and Differentiation Potentials

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    Faezeh Alipour

    2015-01-01

    Full Text Available Objective: Because of the therapeutic application of stem cells (SCs, isolation and characterization of different types of SCs, especially mesenchymal stem cells (MSCs, have gained considerable attention in recent studies. Adipose tissue is an abundant and accessible source of MSCs which can be used for tissue engineering and in particular for treatment of musculoskeletal disorders. This study was aimed to isolate and culture equine adipose-derived MSCs (AT-MSCs from little amounts of fat tissue samples and determine some of their biological characteristics. Materials and Methods: In this descriptive study, only 3-5 grams of fat tissue were collected from three crossbred mares. Immediately, cells were isolated by mechanical means and enzymatic digestion and were cultured in optimized conditions until passage 3 (P3. The cells at P3 were evaluated for proliferative capacities, expression of specific markers, and osteogenic, chondrogenic and adipogenic differentiation potentials. Results: Results showed that the isolated cells were plastic adherent with a fibroblast-like phenotype. AT-MSCs exhibited expression of mesenchymal cluster of differentiation (CD markers (CD29, CD44 and CD90 and not major histocompatibility complex II (MHC-II and CD34 (hematopoietic marker. Cellular differentiation assays demonstrated the chondrogenic, adipogenic and osteogenic potential of the isolated cells. Conclusion: Taken together, our findings reveal that equine MSCs can be obtained easily from little amounts of fat tissue which can be used in the future for regenerative purposes in veterinary medicine.

  19. EOTAXIN AND EOTAXIN-2 EXPRESSION IN HUMAN BRONCHIAL EPITHELIAL CELL

    Institute of Scientific and Technical Information of China (English)

    TANG Wei; DENG Wei-wu; Albert CHAN; Stanley CHIK; Adrain WU

    2005-01-01

    Objective To study the role of eotaxin and eotaxin-2 expression by Th2 cytokine and analyze their relationship in normal human bronchial epithelial cell line-BEAS-2B cell. Methods Levels of eotaxin mRNA and protein expression in the bronchial epithelial cell line BEAS-2B cell were determined with RT-PCR and ELISA. We also used RT-PCR to evaluate eotaxin-2 expression under the regulation of Th2 cytokine IL-4 and IL-13 as well as proinflammatory agent-TNFα. Results Eotaxin mRNA expression was the highest at the time point of 12h under the stimulation of TNF-α. While Th2 cytokine IL-4 and IL-13 had the amplification effect on the expression. Eotaxin protein was also elevated with the combination stimulation of proinflammatory agent TNF-α and IL-4 in dose and time dependent manner(P<0.01). These results were also seen when the cells were stimulated by TNF-α and IL-13. Eotaxin-2 mRNA expression was the highest at the time point of 8h. The expression evaluated by semi-quantitative RT-PCR also elevated under the co-stimulation of TNF-α and IL-4 or TNF-α and IL-13 and it should significantly correlate with Eotaxin(P<0.05). Conclusion This study demonstrated that Th2 cytokine like IL-4 and IL-13 enhances eotaxin and eotaxin-2 expression when co-stimulated with proinflammatory agent TNF-α. These results showed that Th2 cytokines existence is the strong evidence for bronchial epithelial cells taking part in the allergic inflammation especially in eosinophils recruitment.

  20. Expression and purification of splicing proteins from mammalian cells.

    Science.gov (United States)

    Allemand, Eric; Hastings, Michelle L

    2014-01-01

    Pre-mRNA splicing is a complex process that is carried out by a large ribonucleoprotein enzyme, termed the spliceosome, which comprises up to 200 proteins. Despite this complexity, the role of individual spliceosomal proteins in the splicing reaction has been successfully investigated using cell-free assays. In many cases, the splicing factor of interest must be expressed and purified in order to study its function in vitro. Posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination of splicing factors are important for activity. Thus, their purification from mammalian cells presents numerous advantages. Here, we describe a method for expression and purification of splicing proteins from mammalian cells.

  1. Expression changes of dopaminergic system-related genes in PC12 cells induced by manganese, silver, or copper nanoparticles.

    Science.gov (United States)

    Wang, Jianyong; Rahman, Mohammed F; Duhart, Helen M; Newport, Glenn D; Patterson, Tucker A; Murdock, Richard C; Hussain, Saber M; Schlager, John J; Ali, Syed F

    2009-11-01

    Nanoparticles have received a great deal of attention for producing new engineering applications due to their novel physicochemical characteristics. However, the broad application of nanomaterials has also produced concern for nanoparticle toxicity due to increased exposure from large-scale industry production. This study was conducted to investigate the potential neurotoxicity of manganese (Mn), silver (Ag), and copper (Cu) nanoparticles using the dopaminergic neuronal cell line, PC12. Selective genes associated with the dopaminergic system were investigated for expression changes and their correlation with dopamine depletion. PC12 cells were treated with 10 microg/ml Mn-40 nm, Ag-15 nm, or Cu-90 nm nanoparticles for 24 h. Cu-90 nanoparticles induced dopamine depletion in PC12 cells, which is similar to the effect induced by Mn-40 shown in a previous study. The expression of 11 genes associated with the dopaminergic system was examined using real-time RT-PCR. The expression of Txnrd1 was up-regulated after the Cu-90 treatment and the expression of Gpx1 was down-regulated after Ag-15 or Cu-90 treatment. These alterations are consistent with the oxidative stress induced by metal nanoparticles. Mn-40 induced a down-regulation of the expression of Th; Cu-90 induced an up-regulation of the expression of Maoa. This indicates that besides the oxidation mechanism, enzymatic alterations may also play important roles in the induced dopamine depletion. Mn-40 also induced a down-regulation of the expression of Park2; while the expression of Snca was up-regulated after Mn-40 or Cu-90 treatment. These data suggest that Mn and Cu nanoparticles-induced dopaminergic neurotoxicity may share some common mechanisms associated with neurodegeneration.

  2. The regulation of CD5 expression in murine T cells

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    Herzenberg Leonard A

    2001-05-01

    Full Text Available Abstract Background CD5 is a pan-T cell surface marker that is also present on a subset of B cells, B-1a cells.Functional and developmental subsets of T cells express characteristic CD5 levels that vary over roughly a 30-fold range. Previous investigators have cloned a 1.7 Kb fragment containing the CD5 promoter and showed that it can confer similar lymphocyte-specific expression pattern as observed for endogenous CD5 expression. Results We further characterize the CD5 promoter and identify minimal and regulatory regions on the CD5 promoter. Using a luciferase reporter system, we show that a 43 bp region on the CD5 promoter regulates CD5 expression in resting mouse thymoma EL4 T cells and that an Ets binding site within the 43 bp region mediates the CD5 expression. In addition, we show that Ets-1, a member of the Ets family of transcription factors, recognizes the Ets binding site in the electrophoretic mobility shift assay (EMSA. This Ets binding site is directly responsible for the increase in reporter activity when co-transfected with increasing amounts of Ets-1 expression plasmid. We also identify two additional evolutionarily-conserved regions in the CD5 promoter (CD5X and CD5Y and demonstrate the respective roles of the each region in the regulation of CD5 transcription. Conclusion Our studies define a minimal and regulatory promoter for CD5 and show that the CD5 expression level in T cells is at least partially dependent on the level of Ets-1 protein. Based on the findings in this report, we propose a model of CD5 transcriptional regulation in T cells.

  3. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    , membrane-bound Hsp70 can stimulate antigen presenting cells (APCs) to release proinflammatory cytokines and can provide a target structure for NK cell-mediated lysis. Human cancer cells frequently express Hsp70 on their cell surface, whereas the corresponding normal tissues do not. In addition, several...... clinically applied reagents, such as alkyl-lysophospholipides, chemotherapeutic agents, and anti-inflammatory reagents, have been found to enhance Hsp70 surface expression on cancer cells. We have found that inhibition of histone deacetylase (HDAC) activity leads to surface expression of Hsp70 on various...... hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC-inhibitor mediated Hsp70 surface expression was confined to the apoptotic Annexin V positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis...

  4. Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines

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    Edyta Biskup

    2010-05-01

    Full Text Available We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa, Sézary syndrome (SeAx, and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK. Mac1 and Mac2a had the highest growth rate (doubling time 18-28 h, >90% cycling cells whereas SeAx was proliferating slowly (doub-ling time 55 h, approximately 35% cycling cells. Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma.

  5. Expression of ICAM-1 in colon epithelial cells

    DEFF Research Database (Denmark)

    Vainer, Ben; Sørensen, Susanne; Seidelin, Jakob;

    2003-01-01

    on monolayers of cancer cells. Conflicting results exist on epithelial ICAM-1 expression, and the aim of this study was to compare the expression in various models of colonic epithelium. MATERIALS AND METHODS: Colonic biopsies from four UC patients and four controls were examined by cryoimmuno......-electron microscopy using ICAM-1-antibodies. In four other controls, the epithelium was isolated from colonic biopsies, embedded in collagen, and evaluated similarly. Isolated crypts and cultured cancer cells were stimulated with interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha). RESULTS: ICAM-1......, both colonocytes and HT29 cells were capable of expressing ICAM-1 on their apical membranes in response to supraphysiologic cytokine concentrations. These observations question the justification of extrapolating observations from colon cancer cell lines to in vivo inflammatory conditions....

  6. Adipose tissue-derived stromal cells express neuronal phenotypes

    Institute of Scientific and Technical Information of China (English)

    杨立业; 刘相名; 孙兵; 惠国桢; 费俭; 郭礼和

    2004-01-01

    Background Adipose tissue-derived stromal cells (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons.Methods Adipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE).Results Nestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies.Conclusions The data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation.

  7. Oxygen-glucose deprivation increases the enzymatic activity and the microvesicle-mediated release of ectonucleotidases in the cells composing the blood-brain barrier.

    Science.gov (United States)

    Ceruti, Stefania; Colombo, Laura; Magni, Giulia; Viganò, Francesca; Boccazzi, Marta; Deli, Mária A; Sperlágh, Beáta; Abbracchio, Maria P; Kittel, Agnes

    2011-08-01

    The blood-brain barrier (BBB), the dynamic interface between the nervous tissue and the blood, is composed by endothelial cells, pericytes and astrocytes. Extracellular nucleotides and nucleosides and their receptors (the purinergic system) constitute a widely diffused signaling system involved in many pathophysiological processes. However, the role of this system in controlling BBB functions is still largely unknown. By using cultures of these three cell types grown separately and a BBB in vitro model consisting of triple co-cultures, we studied for the first time the expression and distribution of the ecto-enzymes nucleoside triphosphate diphosphohydrolases (NTPDases, the enzymes which hydrolyze extracellular nucleotides) under control and ischemic (oxygen-glucose deprivation in vitro; OGD) conditions. NTPDase1 was detected in all three cell types, whereas NTPDase2 was expressed by astrocytes and pericytes and, to a lesser extent, by endothelial cells. Endothelial cells were extremely susceptible to cell death when OGD was applied to mimic in vitro the cytotoxicity induced by ischemia, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted markedly increased e-ATPase activity following exposure to OGD in all three cell types, either when grown separately or when co-cultured together to resemble the composition of the BBB. Moreover, electron microscopy analysis showed that both endothelial cells and astrocytes shed microvesicles containing NTPDases from their membrane, which may suggest a novel mechanism to increase the breakdown of ATP released to toxic levels by damaged BBB cells. We hypothesize that this phenomenon could have a protective and/or modulatory effect for brain parenchymal cells. This in vitro model is therefore useful to study the role of extracellular nucleotides in modulating BBB responses to ischemic events, and to develop new effective purinergic-based approaches for brain ischemia.

  8. Human respiratory epithelial cells from nasal turbinate expressed stem cell genes even after serial passaging.

    Science.gov (United States)

    Ruszymah, B H I; Izham, B A Azrul; Heikal, M Y Mohd; Khor, S F; Fauzi, M B; Aminuddin, B S

    2011-12-01

    Current development in the field of tissue engineering led to the idea of repairing and regenerating the respiratory airway through in vitro reconstruction using autologous respiratory epithelial (RE). To ensure the capability of proliferation, the stem cell property of RE cells from the nasal turbinate should be evaluated. Respiratory epithelial cells from six human nasal turbinates were harvested and cultured in vitro. The gene expression of FZD-9 and BST-1 were expressed in passage 2 (P2) and passage 4 (P4). The levels of expression were not significant between both passages. The RE cells exhibit the stem cell properties, which remains even after serial passaging.

  9. Evaluating the Expression of Mesenchymal Stem Cells Markers in Human Hair Follicle Stem Cells

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    Mohammadreza Behvarz

    2014-12-01

    Full Text Available Background & objectives: Adult stem cells are undifferentiated cells that replace dead or injured cells. There are adult stem cells in some regions of human tissues and hair follicle is one of the tissues that have adult stem cell source and these cells have an important role in hair life cycle. In this study, we investigated the isolation of hair follicle stem cells (HFSCs and expression of mesenchymal stem cell markers on the isolated cells.   Methods : Human hair follicles obtained from men scalp tissue by micro punch technique. Hair follicles isolated and cultured in culture flasks in DMEM-F12 + FBS. After outgrowth of stem cells from hair bulges, they analyzed by flow cytometry for detection of stem cell markers.  Results: 23 to 27 days after isolation and culture of HFSCs in uncoated cell culture flasks, cell surface markers expression studied by flow cytometry. Flow cytometric analysis showed 25.26% Stro-1, 50.85% CD90, 45.24% CD105, 61.20% CD44, 8.20% CD45, 11.86% CD146, 2.72% CD106, 7.21% CD166 and 26.74% CD19 expression in HFSCs.   Conclusion: In this study, isolated stem cells significantly expressed some of the mesenchymal stem cell markers higher than other markers. These markers give certain characteristics to HFSCs, and introduce the cells as an alternative option for cell therapy, tissue engineering and regenerative medicine.

  10. Gene expression during development of fetal and adult Leydig cells.

    Science.gov (United States)

    Dong, Lei; Jelinsky, Scott A; Finger, Joshua N; Johnston, Daniel S; Kopf, Gregory S; Sottas, Chantal M; Hardy, Matthew P; Ge, Ren-Shan

    2007-12-01

    In rats and mice, Leydig cells are formed as two morphologically and functionally different generations. The first generation develops in utero, from undifferentiated stem Leydig cells (SLCs) that differentiate into fetal Leydig cells (FLCs). After birth, SLCs that may differ from the fetal SLCs undergo lineage-specific commitment and give rise to adult Leydig cells (ALCs). The intermediates of ALCs first become apparent by day 11 postpartum. These first-appearing intermediates, progenitor Leydig cells (PLCs), are spindle shaped and identifiable as steroidogenic because they express luteinizing hormone receptor (LHR) and 3beta-hydroxysteroid dehydrogenase (3betaHSD). The next step in the transition of PLCs to ALCs is the appearance of the immature Leydig cells (ILCs), most commonly seen in the testis during days 28 to 56 postpartum. ILCs have a more abundant smooth endoplasm reticulum (SER), the network of membranes providing a scaffold for steroidogenic enzyme localization, compared to PLCs, but are considered immature because they secrete higher levels of 5alpha-reduced androgen than testosterone. ILCs undergo a final division before ALC steroidogenic function matures by postnatal day 56. ALCs mark the point of maximum differentiation, and at this stage, the Leydig cell secretes testosterone at the highest rate. In this review, trends of gene expression during development of the two Leydig-cell generations, and recent information from gene profiling by microarray, are evaluated. The expression profiles are distinct, indicating that FLCs and ALCs may originate from separate pools of stem cells.

  11. Expression and roles of CCN2 in dental epithelial cells.

    Science.gov (United States)

    Shimo, Tsuyoshi; Koyama, Eiki; Kurio, Naito; Matsumoto, Kenichi; Okui, Tatsuo; Ibaragi, Soichiro; Yoshioka, Norie; Sasaki, Akira

    2015-01-01

    Connective tissue growth factor (CCN2) regulates diverse cellular functions, including tooth development. In order to delineate the precise role of CCN2 in the epithelium during odontogenesis, we investigated how it is expressed and what roles it may have in primary cultures of epithelial cells derived from developing tooth germ of the bovine fetus. Ccn2 mRNA and protein were strongly expressed in the inner dental epithelium, which is consistent with the expression of transforming growth factor-β2 mRNA and proliferating cell nuclear antigen. Bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) were also expressed in the inner dental epithelium, indicating that CCN2 functionally interacts with these factors in the epithelium. The stimulatory effects of FGF2 on cell proliferation and BMP4 on cell differentiation were additively up-regulated by CCN2 in a newly-established dental epithelium cell culture. Taken together, our data provide clear evidence that CCN2 is synthesized by inner dental epithelial cells, and appears to act as an autocrine factor, which regulates dental epithelial cell proliferation and differentiation in concert with growth factors. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  12. Foxd3 suppresses interleukin-10 expression in B cells.

    Science.gov (United States)

    Zhang, Yu; Wang, Zhiding; Xiao, He; Liu, Xiaoling; Zhu, Gaizhi; Yu, Dandan; Han, Gencheng; Chen, Guojiang; Hou, Chunmei; Ma, Ning; Shen, Beifen; Li, Yan; Wang, Tianxiao; Wang, Renxi

    2017-04-01

    Interleukin-10-positive (IL-10(+) ) regulatory B (Breg) cells play an important role in restraining excessive inflammatory responses by secreting IL-10. However, it is still unclear what key transcription factors determine Breg cell differentiation. Hence, we explore what transcription factor plays a key role in the expression of IL-10, a pivotal cytokine in Breg cells. We used two types of web-based prediction software to predict transcription factors binding the IL-10 promoter and found that IL-10 promoter had many binding sites for Foxd3. Chromatin immunoprecipitation PCR assay demonstrated that Foxd3 directly binds the predicted binding sites around the start codon upstream by -1400 bp. Further, we found that Foxd3 suppressed the activation of IL-10 promoter by using an IL-10 promoter report system. Finally, knocking out Foxd3 effectively promotes Breg cell production by up-regulating IL-10 expression. Conversely, up-regulated Foxd3 expression was negatively associated with IL-10(+) Breg cells in lupus-prone MRL/lpr mice. Hence, our data suggest that Foxd3 suppresses the production of IL-10(+) Breg cells by directly binding the IL-10 promoter. This study demonstrates the mechanism for Breg cell production and its application to the treatment of autoimmune diseases by regulating Foxd3 expression. © 2016 John Wiley & Sons Ltd.

  13. Enzymatic cleavage of cell surface proteins of pig and cow erythrocytes and its effect on concanavalin-mediated agglutinability.

    Science.gov (United States)

    Sharma, Savita; Gokhale, Sadashiv M

    2014-10-01

    Study was carried out to understand and compare architecture of the proteins of erythrocyte cell surface of some mammals viz., Homo sapiens (human), Sus scorfa domestica (pig) and Bos taurus domestica (cow). In this study, we investigated the action of proteinases viz., trypsin and chymotrypsin and neuraminidase on the erythrocyte surface proteins and erythrocyte agglutination tendency with a lectin (concanavalin A). The electrophoretic pattern of membrane proteins and glycophorins (analyzed by SDS-PAGE and visualized by Coomassie brilliant blue and periodic acid-schiff stains, respectively) and concanavalin A (Con A) agglutinability revealed that: (i) There were variations in the number and molecular weights of glycophorins in human, pig and cow, (ii) trypsin action on pig and cow erythrocyte membrane proteins was similar, unlike human, (iii) glycophorins degradation by trypsin and chymotrypsin was not similar in pig, as compared to that of human and cow, (iv) erythrocytes agglutination with Con A was significantly different due to differences in membrane composition and alterations in the surface proteins after enzyme treatment, (v) a direct correlation was found between degradation of glycophorins and Con A agglutinability, and (vi) removal of erythrocyte surface sialic acids by neuraminidase specifically indicated an increase in Con A agglutinability of pig and cow erythrocytes, similar to human.

  14. High Glucose Promotes Tumor Invasion and Increases Metastasis-Associated Protein Expression in Human Lung Epithelial Cells by Upregulating Heme Oxygenase-1 via Reactive Oxygen Species or the TGF-β1/PI3K/Akt Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Xiaowen Kang

    2015-02-01

    Full Text Available Background: Growing evidence indicates that heme oxygenase-1 (HO-1 is up-regulated in malignancies and subsequently alters tumor aggressiveness and various cancer-related factors, such as high glucose (HG levels. HO-1 expression can be induced when glucose concentrations are above 25 mM; however, the role of HO-1 in lung cancer patients with diabetes remains unknown. Therefore, in this study we investigated the promotion of tumor cell invasion and the expression of metastasis-associated proteins by inducing the up-regulation of HO-1 expression by HG treatment in A549 human lung epithelial cells. Methods: The expression of HO-1and metastasis-associated protein expression was explored by western blot analysis. HO-1 enzymatic activity, reactive oxygen species (ROS production and TGF-β1 production were examined by ELISA. Invasiveness was analyzed using a Transwell chamber. Results: HG treatment of A549 cells induced an increase in HO-1 expression, which was mediated by the HG-induced generation of reactive oxygen species (ROS and transforming growth factor-β1 (TGF-β1 in a concentration- and time-dependent manner. Following the increase in HO-1 expression, the enzymatic activity of HO-1 also increased in HG-treated cells. Pretreatment with N-acetyl-L-cysteine (NAC or with phosphatidylinositol 3-kinase (PI3K/Akt inhibitors attenuated the HG-induced increase in HO-1 expression. HG treatment of A549 cells enhanced the invasion potential of these cells, as shown with a Transwell assay, and increased metastasis-associated protein expression. However, HO-1 siRNA transfection significantly decreased these capabilities. Conclusion: this study is the first to demonstrate that HG treatment of A549 human lung epithelial cells promotes tumor cell invasion and increases metastasis-associated protein expression by up-regulating HO-1 expression via ROS or the TGF-β1/PI3K/Akt signaling pathway.

  15. Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity

    DEFF Research Database (Denmark)

    Jensen, Helle; Andresen, Lars; Hansen, Karen Aagaard

    2009-01-01

    We show that inhibition of HDAC activity leads to surface expression of Hsp70 on various hematopoietic cancer cells, an occurance that was not observed on naïve or activated peripheral blood cells. HDAC inhibitor-mediated Hsp70 surface expression was confined to the apoptotic Annexin V...... activity selectively induces surface expression of Hsp70 on hematopoietic cancer cells and that this may increase immunorecognition of these cells.......-positive cells and blocked by inhibition of apoptosis. Other chemotherapeutic inducers of apoptosis such as etoposide and camptothecin also led to a robust induction of Hsp70 surface expression. Hsp70 expression was, however, not caused by induction of apoptosis per se, as activated CD4 T cells remained Hsp70...

  16. Enzymatic dysfunction of mitochondrial complex I of the Candida albicans goa1 mutant is associated with increased reactive oxidants and cell death.

    Science.gov (United States)

    Li, Dongmei; Chen, Hui; Florentino, Abigail; Alex, Deepu; Sikorski, Patricia; Fonzi, William A; Calderone, Richard

    2011-05-01

    We have previously shown that deletion of GOA1 (growth and oxidant adaptation) of Candida albicans results in a loss of mitochondrial membrane potential, ATP synthesis, increased sensitivity to oxidants and killing by human neutrophils, and avirulence in a systemic model of candidiasis. We established that translocation of Goa1p to mitochondria occurred during peroxide stress. In this report, we show that the goa1Δ (GOA31), compared to the wild type (WT) and a gene-reconstituted (GOA32) strain, exhibits sensitivity to inhibitors of the classical respiratory chain (CRC), including especially rotenone (complex I [CI]) and salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase pathway (AOX), while potassium cyanide (KCN; CIV) causes a partial inhibition of respiration. In the presence of SHAM, however, GOA31 has an enhanced respiration, which we attribute to the parallel respiratory (PAR) pathway and alternative NADH dehydrogenases. Interestingly, deletion of GOA1 also results in a decrease in transcription of the alternative oxidase gene AOX1 in untreated cells as well as negligible AOX1 and AOX2 transcription in peroxide-treated cells. To explain the rotenone sensitivity, we measured enzyme activities of complexes I to IV (CI to CIV) and observed a major loss of CI activity in GOA31 but not in control strains. Enzymatic data of CI were supported by blue native polyacrylamide gel electrophoresis (BN-PAGE) experiments which demonstrated less CI protein and reduced enzyme activity. The consequence of a defective CI in GOA31 is an increase in reactive oxidant species (ROS), loss of chronological aging, and programmed cell death ([PCD] apoptosis) in vitro compared to control strains. The increase in PCD was indicated by an increase in caspase activity and DNA fragmentation in GOA31. Thus, GOA1 is required for a functional CI and partially for the AOX pathway; loss of GOA1 compromises cell survival. Further, the loss of chronological aging is new to

  17. Expression of cell cycle regulating factor mRNA in small cell lung cancer xenografts

    DEFF Research Database (Denmark)

    Krarup, M; Poulsen, H S; Spang-Thomsen, M

    1998-01-01

    We have investigated the expression of cyclins, cyclin dependent kinases (CDK), and CDK inhibitors (CKI) at the mRNA level in a panel of small-cell lung cancer (SCLC) cell lines in vitro and in vivo as xenografts in nude mice. The results showed that the cell lines expressed varying amounts of most...... cyclin and CDK's but only a few of the cell lines expressed cyclin D1 and/or D2 and some lacked expression of CDK6. Most cell lines expressed mRNA for the CKI's but two cell lines lacked expression of P15INK4B and p16INK4A. The mRNA expression differed for a few of the cell lines regarding cyclin D2...... and CDK6 when in vitro and in vivo data were compared. Two of the cell lines that express the retinoblastoma (Rb) protein had no sign of a deregulated Rb pathway but further studies at the protein level are necessary to demonstrate whether these two cell lines should have a normal Rb pathway or whether...

  18. ENZYMATIC HYDROLYSIS OF STARCH

    OpenAIRE

    Reyna M., L.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú; Robles, R.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú; Reyes P., M.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú; Mendoza R., Y.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú; Romero D., J.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú

    2014-01-01

    We obtained rate constant for enzymatic hydrolysis of barley at temperature of 70 ºC. Se ha llevado a cabo la hidrólisis enzimática de la cebada a 70 ºC y se determinó la constante cinética de reacción.

  19. Enzymatic synthesis of vanillin

    NARCIS (Netherlands)

    van den Heuvel, RHH; Fraaije, MW; Laane, C; van Berkel, WJH; Heuvel, Robert H.H. van den; Berkel, Willem J.H. van

    2001-01-01

    Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated conv

  20. Regulation of stem cell factor expression in inflammation and asthma

    Directory of Open Access Journals (Sweden)

    Carla A Da Silva

    2005-03-01

    Full Text Available Stem cell factor (SCF is a major mast cell growth factor, which could be involved in the local increase of mast cell number in the asthmatic airways. In vivo, SCF expression increases in asthmatic patients and this is reversed after treatment with glucocorticoids. In vitro in human lung fibroblasts in culture, IL-1beta, a pro-inflammatory cytokine, confirms this increased SCF mRNA and protein expression implying the MAP kinases p38 and ERK1/2 very early post-treatment, and glucocorticoids confirm this decrease. Surprisingly, glucocorticoids potentiate the IL-1beta-enhanced SCF expression at short term treatment, implying increased SCF mRNA stability and SCF gene transcription rate. This potentiation involves p38 and ERK1/2. Transfection experiments with the SCF promoter including intron1 also confirm this increase and decrease of SCF expression by IL-1beta and glucocorticoids, and the potentiation by glucocorticoids of the IL-1beta-induced SCF expression. Deletion of the GRE or kappaB sites abolishes this potentiation, and the effect of IL-1beta or glucocorticoids alone. DNA binding of GR and NF-kappaB are also demonstrated for these effects. In conclusion, this review concerns new mechanisms of regulation of SCF expression in inflammation that could lead to potential therapeutic strategy allowing to control mast cell number in the asthmatic airways.

  1. NFATc1 regulation of TRAIL expression in human intestinal cells.

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    Qingding Wang

    Full Text Available TNF-related apoptosis-inducing ligand (TRAIL; Apo2 has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA plus the calcium ionophore A23187 (Io increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA, an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.

  2. Comprehensive Profiling of GPCR Expression in Ghrelin-Producing Cells.

    Science.gov (United States)

    Koyama, Hiroyuki; Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Hosoda, Hiroshi; Ariyasu, Hiroyuki; Kusakabe, Toru; Son, Choel; Hosoda, Kiminori; Akamizu, Takashi; Kangawa, Kenji; Nakao, Kazuwa

    2016-02-01

    To determine the comprehensive G protein-coupled receptor (GPCR) expression profile in ghrelin-producing cells and to elucidate the role of GPCR-mediated signaling in the regulation of ghrelin secretion, we determined GPCR expression profiles by RNA sequencing in the ghrelin-producing cell line MGN3-1 and analyzed the effects of ligands for highly expressed receptors on intracellular signaling and ghrelin secretion. Expression of selected GPCRs was confirmed in fluorescence-activated cell-sorted fluorescently tagged ghrelin-producing cells from ghrelin-promoter CreERT2/Rosa-CAG-LSL-ZsGreen1 mice. Expression levels of GPCRs previously suggested to regulate ghrelin secretion including adrenergic-β1 receptor, GPR81, oxytocin receptor, GPR120, and somatostatin receptor 2 were high in MGN3-1 cells. Consistent with previous reports, isoproterenol and oxytocin stimulated the Gs and Gq pathways, respectively, whereas lactate, palmitate, and somatostatin stimulated the Gi pathway, confirming the reliability of current assays. Among other highly expressed GPCRs, prostaglandin E receptor 4 agonist prostaglandin E2 significantly stimulated the Gs pathway and ghrelin secretion. Muscarine, the canonical agonist of cholinergic receptor muscarinic 4, stimulated both the Gq and Gi pathways. Although muscarine treatment alone did not affect ghrelin secretion, it did suppress forskolin-induced ghrelin secretion, suggesting that the cholinergic pathway may play a role in counterbalancing the stimulation of ghrelin by Gs (eg, by adrenaline). In addition, GPR142 ligand tryptophan stimulated ghrelin secretion. In conclusion, we determined the comprehensive expression profile of GPCRs in ghrelin-producing cells and identified two novel ghrelin regulators, prostaglandin E2 and tryptophan. These results will lead to a greater understanding of the physiology of ghrelin and facilitate the development of ghrelin-modulating drugs.

  3. Expression and Fuactional Role of HERG1, K+ Channels in Leukemic Cells and Leukemic Stem Cells

    Institute of Scientific and Technical Information of China (English)

    LI Huiyu; LIU Liqiong; GUO Tiannan; ZHANG Jiahua; LI Xiaoqing; DU Wen; LIU Wei; CHEN Xiangjun; HUANG Shi'ang

    2007-01-01

    In order to investigate the expression and functional role of HERG1 K+ channels in leukemic cells and leukemic stem cells (LSCs), RT-PCR was used to detect the HERG1 K+ channels expression in leukemic cells and LSCs. The functional role of HERG1 K+ channels in leukemic cell proliferation was measured by MTT assay, and cell cycle and apoptosis were analyzed by flow cytometry. The results showed that herg mRNA was expressed in CD34+/CD38-, CD123+ LSCs but not in circulating CD34+ cells. Herg mRNA was also up-regulated in leukemia cell lines K562 and HL60 as well as almost all the primary leukemic cells while not in normal peripheral blood mononuclear cells (PBMNCs) and the expression of herg mRNA was not associated with the clinical and cytogenetic features of leukemia. In addition, leukemic cell proliferation was dramatically inhibited by HERG K+ channel special inhibitor E-4031. Moreover, E-4031 suppressed the cell growth by inducing a specific block at the G1/S transition phase of the cell cycle but had no effect on apoptosis in leukemic cells. The results suggested that HERG1 K+ channels could regulate leukemic cells proliferation and were necessary for leukemic cells to proceed with the cell cycle. HERG1 K+ channels may also have oncogenic potential and may be a biomarker for diagnosis of leukemia and a novel potential pharmacological target for leukemia therapy.

  4. Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

    Directory of Open Access Journals (Sweden)

    Dilyana Todorova

    Full Text Available BACKGROUND: Circulating CD34(+ cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN could target progenitor cell injury by Natural Killer (NK cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+-derived Endothelial Colony Forming Cells (ECFC can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+ progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+ cells expressing FKN was identified as an independent variable inversely correlated to CD34(+ progenitor cell count. We further showed that treatment of CD34(+ circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+ progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

  5. Mitochondrial dysfunction links ceramide activated HRK expression and cell death.

    Directory of Open Access Journals (Sweden)

    Farhan Rizvi

    Full Text Available PURPOSE: Cell death is an essential process in normal development and homeostasis. In eyes, corneal epithelial injury leads to the death of cells in underlying stroma, an event believed to initiate corneal wound healing. The molecular basis of wound induced corneal stromal cell death is not understood in detail. Studies of others have indicated that ceramide may play significant role in stromal cell death following LASIK surgery. We have undertaken the present study to investigate the mechanism of death induced by C6 ceramide in cultures of human corneal stromal (HCSF fibroblasts. METHODS: Cultures of HCSF were established from freshly excised corneas. Cell death was induced in low passage (p<4 cultures of HCSF by treating the cells with C6 ceramide or C6 dihydroceramide as a control. Cell death was assessed by Live/Dead cell staining with calcein AM and ethidium homodimer-1 as well as Annexin V staining, caspase activation and TUNEL staining Mitochondrial dysfunction was assessed by Mito Sox Red, JC-1 and cytochrome C release Gene expression was examined by qPCR and western blotting. RESULTS: Our data demonstrate ceramide caused mitochondrial dysfunction as evident from reduced MTT staining, cyto c release from mitochondria, enhanced generation of ROS, and loss in mitochondrial membrane potential (ΔΨm. Cell death was evident from Live -Dead Cell staining and the inability to reestablish cultures from detached cells. Ceramide induced the expression of the harikari gene(HRK and up-regulated JNK phosphorylation. In ceramide treated cells HRK was translocated to mitochondria, where it was found to interact with mitochondrial protein p32. The data also demonstrated HRK, p32 and BAD interaction. Ceramide-induced expression of HRK, mitochondrial dysfunction and cell death were reduced by HRK knockdown with HRK siRNA. CONCLUSION: Our data document that ceramide is capable of inducing death of corneal stromal fibroblasts through the induction of HRK

  6. Robust reconstitution of active cell-cycle control complexes from co-expressed proteins in bacteria

    Directory of Open Access Journals (Sweden)

    Harashima Hirofumi

    2012-06-01

    Full Text Available Abstract Background Cell proliferation is an important determinant of plant growth and development. In addition, modulation of cell-division rate is an important mechanism of plant plasticity and is key in adapting of plants to environmental conditions. One of the greatest challenges in understanding the cell cycle of flowering plants is the large families of CDKs and cyclins that have the potential to form many different complexes. However, it is largely unclear which complexes are active. In addition, there are many CDK- and cyclin-related proteins whose biological role is still unclear, i.e. whether they have indeed enzymatic activity. Thus, a biochemical characterization of these proteins is of key importance for the understanding of their function. Results Here we present a straightforward system to systematically express and purify active CDK-cyclin complexes from E. coli extracts. Our method relies on the concomitant production of a CDK activating kinase, which catalyzes the T-loop phosphorylation necessary for kinase activity. Taking the examples of the G1-phase cyclin CYCLIN D3;1 (CYCD3;1, the mitotic cyclin CYCLIN B1;2 (CYCB1;2 and the atypical meiotic cyclin SOLO DANCERS (SDS in conjunction with A-, B1- and B2-type CDKs, we show that different CDKs can interact with various cyclins in vitro but only a few specific complexes have high levels of kinase activity. Conclusions Our work shows that both the cyclin as well as the CDK partner contribute to substrate specificity in plants. These findings refine the interaction networks in cell-cycle control and pinpoint to particular complexes for modulating cell proliferation activity in breeding.

  7. A functional profile of gene expression in ARPE-19 cells

    Directory of Open Access Journals (Sweden)

    Johnson Dianna A

    2005-11-01

    Full Text Available Abstract Background Retinal pigment epithelium cells play an important role in the pathogenesis of age related macular degeneration. Their morphological, molecular and functional phenotype changes in response to various stresses. Functional profiling of genes can provide useful information about the physiological state of cells and how this state changes in response to disease or treatment. In this study, we have constructed a functional profile of the genes expressed by the ARPE-19 cell line of retinal pigment epithelium. Methods Using Affymetrix MAS 5.0 microarray analysis, genes expressed by ARPE-19 cells were identified. Using GeneChip® annotations, these genes were classified according to their known functions to generate a functional gene expression profile. Results We have determined that of approximately 19,044 unique gene sequences represented on the HG-U133A GeneChip® , 6,438 were expressed in ARPE-19 cells irrespective of the substrate on which they were grown (plastic, fibronectin, collagen, or Matrigel. Rather than focus our subsequent analysis on the identity or level of expression of each individual gene in this large data set, we examined the number of genes expressed within 130 functional categories. These categories were selected from a library of HG-U133A GeneChip® annotations linked to the Affymetrix MAS 5.0 data sets. Using this functional classification scheme, we were able to categorize about 70% of the expressed genes and condense the original data set of over 6,000 data points into a format with 130 data points. The resulting ARPE-19 Functional Gene Expression Profile is displayed as a percentage of ARPE-19-expressed genes. Conclusion The Profile can readily be compared with equivalent microarray data from other appropriate samples in order to highlight cell-specific attributes or treatment-induced changes in gene expression. The usefulness of these analyses is based on the assumption that the numbers of genes

  8. Influence of Feeding Enzymatically Hydrolyzed Yeast Cell Wall on Growth Performance and Digestive Function of Feedlot Cattle during Periods of Elevated Ambient Temperature.

    Science.gov (United States)

    Salinas-Chavira, J; Arzola, C; González-Vizcarra, V; Manríquez-Núñez, O M; Montaño-Gómez, M F; Navarrete-Reyes, J D; Raymundo, C; Zinn, R A

    2015-09-01

    In experiment 1, eighty crossbred steers (239±15 kg) were used in a 229-d experiment to evaluate the effects of increasing levels of enzymatically hydrolyzed yeast (EHY) cell wall in diets on growth performance feedlot cattle during periods of elevated ambient temperature. Treatments consisted of steam-flaked corn-based diets supplemented to provide 0, 1, 2, or 3 g EHY/hd/d. There were no effects on growth performance during the initial 139-d period. However, from d 139 to harvest, when 24-h temperature humidity index averaged 80, EHY increased dry matter intake (DMI) (linear effect, p0.10) on carcass characteristics. In experiment 2, four Holstein steers (292±5 kg) with cannulas in the rumen and proximal duodenum were used in a 4×4 Latin Square design experiment to evaluate treatments effects on characteristics of ruminal and total tract digestion in steers. There were no treatment effects (p>0.10) on ruminal pH, total volatile fatty acid, molar proportions of acetate, butyrate, or estimated methane production. Supplemental EHY decreased ruminal molar proportion of acetate (p = 0.08), increased molar proportion of propionate (p = 0.09), and decreased acetate:propionate molar ratio (p = 0.07) and estimated ruminal methane production (p = 0.09). It is concluded that supplemental EHY may enhance DMI and ADG of feedlot steers during periods of high ambient temperature. Supplemental EHY may also enhance ruminal fiber digestion and decrease ruminal acetate:propionate molar ratios in feedlot steers fed steam-flaked corn-based finishing diets.

  9. Influence of Feeding Enzymatically Hydrolyzed Yeast Cell Wall on Growth Performance and Digestive Function of Feedlot Cattle during Periods of Elevated Ambient Temperature

    Directory of Open Access Journals (Sweden)

    J. Salinas-Chavira

    2015-09-01

    Full Text Available In experiment 1, eighty crossbred steers (239±15 kg were used in a 229-d experiment to evaluate the effects of increasing levels of enzymatically hydrolyzed yeast (EHY cell wall in diets on growth performance feedlot cattle during periods of elevated ambient temperature. Treatments consisted of steam-flaked corn-based diets supplemented to provide 0, 1, 2, or 3 g EHY/hd/d. There were no effects on growth performance during the initial 139-d period. However, from d 139 to harvest, when 24-h temperature humidity index averaged 80, EHY increased dry matter intake (DMI (linear effect, p0.10 on carcass characteristics. In experiment 2, four Holstein steers (292±5 kg with cannulas in the rumen and proximal duodenum were used in a 4×4 Latin Square design experiment to evaluate treatments effects on characteristics of ruminal and total tract digestion in steers. There were no treatment effects (p>0.10 on ruminal pH, total volatile fatty acid, molar proportions of acetate, butyrate, or estimated methane production. Supplemental EHY decreased ruminal molar proportion of acetate (p = 0.08, increased molar proportion of propionate (p = 0.09, and decreased acetate:propionate molar ratio (p = 0.07 and estimated ruminal methane production (p = 0.09. It is concluded that supplemental EHY may enhance DMI and ADG of feedlot steers during periods of high ambient temperature. Supplemental EHY may also enhance ruminal fiber digestion and decrease ruminal acetate:propionate molar ratios in feedlot steers fed steam-flaked corn-based finishing diets.

  10. Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas

    Science.gov (United States)

    Azuma, Sakiko; Yokoyama, Yukihiro; Yamamoto, Akiko; Kyokane, Kazuhiro; Niida, Shumpei; Ishiguro, Hiroshi; Ko, Minoru S. H.

    2013-01-01

    We have recently identified the zinc finger and SCAN domain containing 4 (Zscan4), which is transiently expressed and regulates telomere elongation and genome stability in mouse embryonic stem (ES) cells. The aim of this study was to examine the expression of ZSCAN4 in the adult pancreas and elucidate the role of ZSCAN4 in tissue inflammation and subsequent regeneration. The expression of ZSCAN4 and other progenitor or differentiated cell markers in the human pancreas was immunohistochemically examined. Pancreas sections of alcoholic or autoimmune pancreatitis patients before and under maintenance corticosteroid treatment were used in this study. In the adult human pancreas a small number of ZSCAN4-positive (ZSCAN4+) cells are present among cells located in the islets of Langerhans, acini, ducts, and oval-shaped cells. These cells not only express differentiated cell markers for each compartment of the pancreas but also express other tissue stem/progenitor cell markers. Furthermore, the number of ZSCAN4+ cells dramatically increased in patients with chronic pancreatitis, especially in the pancreatic tissues of autoimmune pancreatitis actively regenerating under corticosteroid treatment. Interestingly, a number of ZSCAN4+ cells in the pancreas of autoimmune pancreatitis returned to the basal level after 1 yr of maintenance corticosteroid treatment. In conclusion, coexpression of progenitor cell markers and differentiated cell markers with ZSCAN4 in each compartment of the pancreas may indicate the presence of facultative progenitors for both exocrine and endocrine cells in the adult pancreas. PMID:23599043

  11. Connexin expression and gap-junctional intercellular communication in ES cells and iPS cells.

    Science.gov (United States)

    Oyamada, Masahito; Takebe, Kumiko; Endo, Aya; Hara, Sachiko; Oyamada, Yumiko

    2013-01-01

    Pluripotent stem cells, i.e., embryonic stem (ES) and induced pluripotent stem (iPS) cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC) during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming.

  12. Connexin expression and gap-junctional intercellular communication in ES cells and iPS cells

    Directory of Open Access Journals (Sweden)

    Masahito eOyamada

    2013-07-01

    Full Text Available Pluripotent stem cells, i.e., embryonic stem (ES and induced pluripotent stem (iPS cells, can indefinitely proliferate without commitment and differentiate into all cell lineages. ES cells are derived from the inner cell mass of the preimplantation blastocyst, whereas iPS cells are generated from somatic cells by overexpression of a few transcription factors. Many studies have demonstrated that mouse and human iPS cells are highly similar but not identical to their respective ES cell counterparts. The potential to generate basically any differentiated cell types from these cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. ES cells and iPS cells also provide useful models to study connexin expression and gap-junctional intercellular communication (GJIC during cell differentiation and reprogramming. In 1996, we reported connexin expression and GJIC in mouse ES cells. Because a substantial number of papers on these subjects have been published since our report, this Mini Review summarizes currently available data on connexin expression and GJIC in ES cells and iPS cells during undifferentiated state, differentiation, and reprogramming.

  13. Dynamic distribution and stem cell characteristics of Sox1-expressing cells in the cerebellar cortex

    Institute of Scientific and Technical Information of China (English)

    Joelle Alcock; Virginie Sottile

    2009-01-01

    Bergmann glia cells are a discrete radial glia population surrounding Purkinje cells in the cerebellar cortex. Al-though Bergmann glia are essential for the development and correct arborization of Purkinje cells, little is known about the regulation of this cell population after the developmental phase. In an effort to characterize this population at the molecular level, we have analyzed marker expression and established that adult Bergmann glia express Soxl, Sox2 and Sox9, a feature otherwise associated with neural stem cells (NSCs). In the present study, we have further analyzed the developmental pattern of Soxl-expressing cells in the developing cerebellum. We report that before be-coming restricted to the Purkinje cell layer, Soxl-positive cells are present throughout the immature tissue, and that these cells show characteristics of Bergmann glia progenitors. Our study shows that these progenitors express Soxl, Sox2 and Sox9, a signature maintained throughout cerebellar maturation into adulthood. When isolated in culture, the Soxl-expressing cerebellar population exhibited neurosphere-forming ability, NSC-marker characteristics, and demonstrated multipotency at the clonal level. Our results show that the Bergmann glia population expresses Soxl during cerebellar development, and that these cells can be isolated and show stem cell characteristics in vitro, sug-gesting that they could hold a broader potential than previously thought.

  14. Patterns of expression of cell wall related genes in sugarcane

    Directory of Open Access Journals (Sweden)

    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  15. Connective Tissue Growth Factor Expression in Human Bronchial Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Amrita DOSANJH

    2006-01-01

    Connective tissue growth factor (CTGF) is a cysteine-rich protein that promotes extracellular matrix deposition. CTGF is selectively induced by transforming growth factor β and des-Arg kallidin in lung fibroblasts and increases steady-state mRNA levels of α type I collagen, 5α-integrin and fibronectin in fibroblasts. Bronchial epithelial cells have been proposed to functionally interact with lung fibroblasts. We therefore investigated if bronchial epithelial cells are able to synthesize CTGF. Human bronchial epithelial cells were grown to subconfluence in standard growth media. Proliferating cells grown in small airway growth media were harvested following starvation for up to 24 h. Expression of CTGF transcripts was measured by PCR. Immunocytochemistry was also completed using a commercially available antibody.The cells expressed readily detectable CTGF transcripts. Starvation of these cells resulted in a quantitative decline of CTGF transcripts. Direct sequencing of the PCR product identified human CTGF. Immunocytochemistry confirmed intracellular CTGF in the cells and none in negative control cells. We conclude that bronchial epithelial cells could be a novel source of CTGF. Bronchial epithelial cell-derived CTGF could thus directly influence the deposition of collagen in certain fibrotic lung diseases.

  16. Regulation of cell-to-cell variability in divergent gene expression

    Science.gov (United States)

    Yan, Chao; Wu, Shuyang; Pocetti, Christopher; Bai, Lu

    2016-03-01

    Cell-to-cell variability (noise) is an important feature of gene expression that impacts cell fitness and development. The regulatory mechanism of this variability is not fully understood. Here we investigate the effect on gene expression noise in divergent gene pairs (DGPs). We generated reporters driven by divergent promoters, rearranged their gene order, and probed their expressions using time-lapse fluorescence microscopy and single-molecule fluorescence in situ hybridization (smFISH). We show that two genes in a co-regulated DGP have higher expression covariance compared with the separate, tandem and convergent configurations, and this higher covariance is caused by more synchronized firing of the divergent transcriptions. For differentially regulated DGPs, the regulatory signal of one gene can stochastically `leak' to the other, causing increased gene expression noise. We propose that the DGPs' function in limiting or promoting gene expression noise may enhance or compromise cell fitness, providing an explanation for the conservation pattern of DGPs.

  17. Human Neural Cells Transiently Express Reelin during Olfactory Placode Development.

    Directory of Open Access Journals (Sweden)

    M Cristina Antal

    Full Text Available Reelin, an extracellular glycoprotein is essential for migration and correct positioning of neurons during development. Since the olfactory system is known as a source of various migrating neuronal cells, we studied Reelin expression in the two chemosensory olfactory systems, main and accessory, during early developmental stages of human foetuses/embryos from Carnegie Stage (CS 15 to gestational week (GW 14. From CS 15 to CS 18, but not at later stages, a transient expression of Reelin was detected first in the presumptive olfactory and then in the presumptive vomeronasal epithelium. During the same period, Reelin-positive cells detach from the olfactory/vomeronasal epithelium and migrate through the mesenchyme beneath the telencephalon. Dab 1, an adaptor protein of the Reelin pathway, was simultaneously expressed in the migratory mass from CS16 to CS17 and, at later stages, in the presumptive olfactory ensheathing cells. Possible involvements of Reelin and Dab 1 in the peripheral migrating stream are discussed.

  18. Expression of cyclooxygenase-2 in human esophageal squamous cell carcinomas

    Institute of Scientific and Technical Information of China (English)

    Jian-Gang Jiang; Dao-Wen Wang; Jiang-Bo Tang; Chun-Lian Chen; Bao-Xing Liu; Xiang-Ning Fu; Zhi-Hui Zhu; Wei Qu; Katherine Cianflone; Michael P. Waalkes

    2004-01-01

    AIM: To determine whether cyclooxygenase-2 (COX-2) was expressed in human esophageal squamous cell carcinoma.METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, immunohistochemistry and immunofluorescence were used to assess the expression level of COX-2 in esophageal tissue.RESULTS: COX-2 mRNA levels were increased by >80-fold in esophageal squamous cell carcinoma when compared to adjacent noncancerous tissue. COX-2 protein was present in 21 of 30 cases of esophageal squamous cell carcinoma tissues, but was undetectable in noncancerous tissue. Immunohistochemistry was performed to directly show expression of COX-2 in tumor tissue.CONCLUSION: These results suggest that COX-2 may be an important factor for esophageal cancer and inhibition of COX-2 may be helpful for prevention and possibly treatment of this cancer.

  19. Human dental pulp cells response to mineral trioxide aggregate (MTA) and MTA Plus: cytotoxicity and gene expression analysis.

    Science.gov (United States)

    Rodrigues, E M; Cornélio, A L G; Mestieri, L B; Fuentes, A S C; Salles, L P; Rossa-Junior, C; Faria, G; Guerreiro-Tanomaru, J M; Tanomaru-Filho, M

    2017-08-01

    To investigate the cytotoxicity, osteogenic bioactivity and mRNA expression of osteogenic markers of bone morphogenetic protein 2 (BMP-2), osteocalcin (OC) and alkaline phosphatase (ALP) induced by the extracts of set MTA Plus (MTA P) (Avalon Biomed Inc. Bradenton, FL, USA) in comparison with MTA (Angelus, Londrina, PR, Brazil) on human dental pulp cells (hDPCs). Cell viability was assessed by mitochondrial dehydrogenase enzymatic (MTT) assay, and the mechanism of cell death was evaluated by flow cytometry. Bioactivity was evaluated by alkaline phosphatase (ALP) assay and detection of calcium deposits with alizarin red staining (ARS). The gene expression of BMP-2, OC and ALP was quantified with real-time PCR. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05). MTA and MTA P were not cytotoxic and did not induce apoptosis. MTA P had significant higher ALP activity in relation to MTA and the control (P MTA had a significantly higher percentage of mineralized area than MTA P (P MTA than MTA P after 1 day (P MTA P compared with MTA (P MTA and MTA Plus were noncytotoxic, increased mineralization processes in vitro and induced the expression of osteogenic markers. © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  20. Modulation of GLO1 Expression Affects Malignant Properties of Cells

    Science.gov (United States)

    Hutschenreuther, Antje; Bigl, Marina; Hemdan, Nasr Y. A.; Debebe, Tewodros; Gaunitz, Frank; Birkenmeier, Gerd

    2016-01-01

    The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed. PMID:27999356

  1. Modulation of GLO1 Expression Affects Malignant Properties of Cells

    Directory of Open Access Journals (Sweden)

    Antje Hutschenreuther

    2016-12-01

    Full Text Available The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO. Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1 that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

  2. Expression pattern of embryonic stem cell markers in DFAT cells and ADSCs.

    Science.gov (United States)

    Gao, Qian; Zhao, Lili; Song, Ziyi; Yang, Gongshe

    2012-05-01

    Mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability under the condition of ceiling method, named dedifferentiated fat cells (DFAT cells). These cells exhibit multilineage potential as adipose tissue-derived stromal cells (ADSCs). However, the stem molecular signature of DFAT cells and the difference distinct from ADSCs are still not sure. To study the molecular signature of DFAT cells better, highly purified mature adipocytes were obtained from rats and the purity was more than 98%, and about 98.6% were monocytes. These mature adipocytes dedifferentiated into fibroblast-like cells spontaneously by the ceiling culture method, these cells proliferated rapidly in vitro, grew in the same direction and formed vertex, and expressed extensively embryonic stem cell markers such as Oct4, Sox2, c-Myc, and Nanog, surface antigen SSEA-1, CD105, and CD31, moreover, these cells possessed ALP and telomerase activity. The expression level was Oct4 1.3%, Sox2 1.3%, c-Myc 1.2%, Nanog 1.2%, CD105 0.6%, CD31 0.6% and SSEA-1 0.4%, respectively, which was lower than that in ADSCs, but the purity of DFAT cells was much higher than that of ADSCs. In conclusion, DFAT cells is a highly purified stem cell population, and expressed some embryonic stem cell markers like ADSCs, which seems to be a good candidate source of adult stem cells for the future cell replacement therapy.

  3. FRAT1 expression regulates proliferation in colon cancer cells.

    Science.gov (United States)

    Zhu, Kongxi; Guo, Jianqiang; Wang, Hongjuan; Yu, Weihua

    2016-12-01

    Colorectal cancer is one of the most common gastric malignancies worldwide. However, the underlying mechanism of colon cancer development and valuable indicators of the disease remain unclear. In this study, the expression of frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) in colon cancer was investigated and the association between FRAT1 expression and biological properties of tumors was analyzed. A total of 147 colon cancer tissue samples and adjacent normal tissues were collected between January 2013 and June 2014. The FRAT1 gene and protein expression levels were analyzed in tissues with different TNM and pathological stages. Small hairpin RNAs (shRNAs) containing the human FRAT1 gene were constructed and transfected into colon cancer HT-29 cells. The proliferation and migration of the cells was also analyzed in relation to a reduction in FRAT1 expression. In colon cancer tissues, the expression of FRAT1 was significantly higher when compared with adjacent tissues. In addition, FRAT1 expression was found to positively correlate with the degree of tumor malignancy, and this difference was determined to be statistically significant (Pcolon cancer, FRAT1 may present a novel tool for analyzing the tumor progression and may be a novel therapeutic target for the treatment of colon cancer.

  4. Loss of c-KIT expression in thyroid cancer cells.

    Science.gov (United States)

    Franceschi, Sara; Lessi, Francesca; Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

    2017-01-01

    Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

  5. Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Møller, C; Bock, E

    1992-01-01

    characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were...... embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression....

  6. Regulation of gene expression in ovarian cancer cells by luteinizing hormone receptor expression and activation

    Directory of Open Access Journals (Sweden)

    Dam Phuongan

    2011-06-01

    Full Text Available Abstract Background Since a substantial percentage of ovarian cancers express gonadotropin receptors and are responsive to the relatively high concentrations of pituitary gonadotropins during the postmenopausal years, it has been suggested that receptor activation may contribute to the etiology and/or progression of the neoplasm. The goal of the present study was to develop a cell model to determine the impact of luteinizing hormone (LH receptor (LHR expression and LH-mediated LHR activation on gene expression and thus obtain insights into the mechanism of gonadotropin action on ovarian surface epithelial (OSE carcinoma cells. Methods The human ovarian cancer cell line, SKOV-3, was stably transfected to express functional LHR and incubated with LH for various periods of time (0-20 hours. Transcriptomic profiling was performed on these cells to identify LHR expression/activation-dependent changes in gene expression levels and pathways by microarray and qRT-PCR analyses. Results Through comparative analysis on the LHR-transfected SKOV-3 cells exposed to LH, we observed the differential expression of 1,783 genes in response to LH treatment, among which five significant families were enriched, including those of growth factors, translation regulators, transporters, G-protein coupled receptors, and ligand-dependent nuclear receptors. The most highly induced early and intermediate responses were found to occupy a network impacting transcriptional regulation, cell growth, apoptosis, and multiple signaling transductions, giving indications of LH-induced apoptosis and cell growth inhibition through the significant changes in, for example, tumor necrosis factor, Jun and many others, supportive of the observed cell growth reduction in in vitro assays. However, other observations, e.g. the substantial up-regulation of the genes encoding the endothelin-1 subtype A receptor, stromal cell-derived factor 1, and insulin-like growth factor II, all of which are

  7. CSTEA: a webserver for the Cell State Transition Expression Atlas.

    Science.gov (United States)

    Zhu, Guanghui; Yang, Hui; Chen, Xiao; Wu, Jun; Zhang, Yong; Zhao, Xing-Ming

    2017-05-09

    Cell state transition is one of the fundamental events in the development of multicellular organisms, and the transition trajectory path has recently attracted much attention. With the accumulation of large amounts of "-omics" data, it is becoming possible to get insights into the molecule mechanisms underlying the transitions between cell states. Here, we present CSTEA (Cell State Transition Expression Atlas), a webserver that organizes, analyzes and visualizes the time-course gene expression data during cell differentiation, cellular reprogramming and trans-differentiation in human and mouse. In particular, CSTEA defines gene signatures for uncharacterized stages during cell state transitions, thereby enabling both experimental and computational biologists to better understand the mechanisms of cell fate determination in mammals. To our best knowledge, CSTEA is the first webserver dedicated to the analysis of time-series gene expression data during cell state transitions. CSTEA is freely available at http://comp-sysbio.org/cstea/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Cell Adhesion Regulates Expression of the Androgen Receptor and Coregulators in Different Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sheng Li

    2007-02-01

    Full Text Available Prostate cancer cells adhere to a tumor basement membrane, while secretoryepithelial cells reside in a suprabasal cell compartment. Since tumor cells are derived fromsuprabasal epithelial cells, they experience de-novo substratum adhesion in the context ofoncogenesis. We therefore analyzed whether cell-matrix adhesion could affect the proteinexpression and activity of the AR. In this study, AR protein expression declined uponsuspension of BPH-1-AR cells, but not in PC-3-AR cells shown by Western blot. In a timecourse study, BPH-1 cell lost AR expression within 6 hours, and the synthetic androgen,R1881 reduced the loss of AR expression. We further explored the mechanism of AR loss insuspended BPH-1 cells. BPH-1-AR cells underwent apoptosis (anoikis when suspended for2 - 5 hours. Suspension did not induce significant apoptosis or decreasing of AR expressionin PC-3 cells. Inhibition of apoptosis in suspended BPH-1-AR cells, either by expression ofBcl-2 or Bcl-xl or by treatment with Z-VAD, a caspase inhibitor, prevented loss of ARprotein. In contrast, the calpain protease inhibitor , ALLN, accelerated the loss of AR proteinexpression. Additionally, cell-matrix adhesion changed the expression of coregulators of ARin the mRNA level of prostate cancer cells. Our results demonstrate that AR proteinexpression was reduced through activation of cell death pathways, and thus indirectly through cell suspension in BPH-AR cells. The activity of AR can also be regulated by adhesion in PC-3-AR and LNCaP cells through affecting the coregulators level.

  9. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

    Science.gov (United States)

    Marinov, Georgi K; Williams, Brian A; McCue, Ken; Schroth, Gary P; Gertz, Jason; Myers, Richard M; Wold, Barbara J

    2014-03-01

    Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.

  10. Constitutive expression and enzymatic activity of Tan protein in brain and epidermis of Ceratitis capitata and of Drosophila melanogaster wild-type and tan mutants.

    Science.gov (United States)

    Pérez, M M; Sabio, G; Badaracco, A; Quesada-Allué, L A

    2011-09-01

    The present report shows a partial biochemical characterization and life cycle expression of N-β-alanyldopamine hydrolase (Tan protein) in Ceratitis capitata and Drosophila melanogaster. This enzyme catalyzes the hydrolysis of N-β-alanyldopamine (NBAD), the main tanning precursor of insect brown cuticles. It also plays an important role in the metabolism of brain neurotransmitters, recycling dopamine and histamine. In contrast to NBAD-synthase, Tan is expressed constitutively in epidermis and does not respond directly to microbial challenge. Immunodetection experiments showed the novel localization of NBAD-hydrolase in the embryo central neural system and in different regions of the adult brain, in addition to optic lobes. We sequenced and characterized Drosophila mutants tan¹ and tan³. The latter appears to be a mutant with normal expression in neural tissue but weak one in epidermis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Immunglobulin Expression and Its Biological Significance in Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Duosha Hu; Hui Zheng; Haidan Liu; Ming Li; Wei Ren; Wei Liao; Zhi Duan; Lili Li; Ya Cao

    2008-01-01

    It is generally believed that the expression of a gene iS restricted "within the right place and at the right time".This principle has long been considered applicable as well to the expression of immunoglobulin(Ig)lymphocytes of B cell lineage.However,increasing evidence has shown Ig "paradoxically" expressed in malignant tumors of epitheliaI origin.We reviewed the recent progress in the study of cancer-derived Ig,and also discussed its mechanisms and possible functions,trying to arouse interest and attention to those working in the field of immunology and oncology.

  12. Expression of CIDE proteins in clear cell renal cell carcinoma and their prognostic significance.

    Science.gov (United States)

    Yu, Ming; Wang, Hui; Zhao, Jun; Yuan, Yuan; Wang, Chao; Li, Jing; Zhang, Lijun; Zhang, Liying; Li, Qing; Ye, Jing

    2013-06-01

    Clear cell renal cell carcinoma (ccRCC) is the major and aggressive subtype of renal cell carcinoma. It is known to derive its histologic appearance from accumulation of abundant lipids and glycogens. The cell death-inducing DFF45-like effector (CIDE) family has been characterized as the lipid droplet proteins involved in the metabolism of lipid storage droplets. The purpose of this study was to evaluate the expression of CIDE proteins in ccRCC cells and to investigate their prognostic significance. We examined consecutive patients with sporadic ccRCC, who underwent nephrectomy, to measure their mRNA and protein expression of CIDE proteins. We found that Cidec and ADRP expression were significantly up-regulated in ccRCC, compared with normal kidney tissues. Cideb was down-regulated. We also found that Cideb was expressed more in low-grade ccRCC than in high-grade tumors. To further clarify the relationship between Cideb expression and patient prognosis, we evaluated 57 ccRCC patients followed up for 120 months. Reduced ccRCC Cideb expression was associated with a higher Fuhrman nuclear grade. Patients with high Cideb expression had better overall survival rate than those with low expression (p < 0.05). Cideb expression was an independent predictor of survival (p = 0.001). Although the biologic function of Cideb in ccRCC remains unknown, the expression level of Cideb might be a novel predictor of prognosis in ccRCC.

  13. Amylase expression in taste receptor cells of rat circumvallate papillae.

    Science.gov (United States)

    Merigo, Flavia; Benati, Donatella; Cecchini, Maria Paola; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

    2009-06-01

    The chemical composition of the luminal content is now accepted to have a profound influence on the performance of chemosensory receptors. Gustatory and intestinal chemoreceptors have in common their expression of molecules involved in taste sensing and signal transduction pathways. The recent finding that enterocytes of the duodenal epithelium are capable of expressing luminal pancreatic amylase suggests that taste cells of the gustatory epithelium might, in the same way, express salivary amylase in the oral cavity. Therefore, we investigated amylase expression in rat circumvallate papillae by using analyses involving immunohistochemistry, Western blot, and reverse transcription with the polymerase chain reaction. In addition, we used double-labeling confocal laser microscopy to compare amylase immunolabeling with that of the following markers: protein gene product 9.5 (PGP 9.5) and chromogranin A (CgA) for endocrine cells, alpha-gustducin and phospholipase C beta 2 (PLC beta 2) as taste-signaling molecules, and cystic fibrosis transmembrane regulator (CFTR) and Clara-cell-specific secretory protein of 10-kDa (CC10) as secretory markers. The results showed that amylase was present in some taste bud cells; its immunoreactivity was observed in subsets of cells that expressed CgA, alpha-gustducin, PLC beta 2, CFTR, or CC10. PGP 9.5 immunoreactivity was never colocalized with amylase. The data suggest that amylase-positive cells constitute an additional subset of taste receptor cells also associated with chemoreceptorial and/or secretory molecules, confirming the occurrence of various pathways in taste buds.

  14. Implication of expression of Nanog in prostate cancer cells and their stem cells.

    Science.gov (United States)

    Gong, Chen; Liao, Hui; Guo, Fengjin; Qin, Liang; Qi, Jun

    2012-04-01

    Recent studies suggested that the prostate cancer may arise from prostate cancer stem cells that share some same characteristics with normal stem cells. The purpose of this study was to detect the differences of Nanog expression between PC3 prostate cancer cell line and its tumor stem cells, and the relationship was preliminarily examined between Nanog and prostate cancer and its tumor stem cells. By using magnetic active cell sorting (MACS), we isolated a population of CD44(+)/CD133(+) prostate cancer cells that display stem cell characteristics from PC3 cell line. Immunohistochemistry revealed positive expressions of CD44, CD133 and α(2)β(1)-integin in the isolated cells. CCK-8 analysis showed that isolated cells had a strong proliferative ability. The formation of the cell spheres in serum-free medium and holoclones in serum-supplied medium showed that the cells were capable of self-renewing, indicating that the isolated cells were a population of cancer stem-like cells derived from PC3 cell line. Western blotting exhibited that the isolated cells had higher experession of Nanog, an embryonic stem marker, as compared with PC3 cells. Our study showed that Nanog might be helpful in sustaining the self-renewal and the undifferentiation of prostate cancer stem cells, and may serve as a marker for prostate cancer stem cells for isolation and identification.

  15. Iron increases HMOX1 and decreases hepatitis C viral expression in HCV-expressing cells

    Institute of Scientific and Technical Information of China (English)

    Wei-Hong Hou; Lisa Rossi; Ying Shan; Jian-Yu Zheng; Richard W Lambrecht; Herbert L Bonkovsky

    2009-01-01

    AIM: To investigate effects of iron on oxidative stress,heme oxygenase-1 (HMOX1) and hepatitis C viral (HCV) expression in human hepatoma cells stably expressing HCV proteins.METHODS: Effects of iron on oxidative stress, HMOX1,and HCV expression were assessed in CON1 cells.Measurements included mRNA by quantitative reverse transcription-polymerase chain reaction, and protein levels by Western blots.RESULTS: Iron, in the form of ferric nitrilotriacetate,increased oxidative stress and up-regulated HMOX1 gene expression. Iron did not affect mRNA or protein levels of Bach1, a repressor of HMOX1. Silencing the up-regulation of HMOX1 nuclear factor-erythroid 2-related factor 2 (Nrf2) by Nrf2-siRNA decreased FeNTA-mediated up-regulation of HMOX1 mRNA levels. These iron effects were completely blocked by deferoxamine (DFO). Iron also significantly decreased levels of HCV core mRNA and protein by 80%-90%,nonstructural 5A mRNA by 90% and protein by about 50% in the Con1 full length HCV replicon cells,whereas DFO increased them.CONCLUSION: Excess iron up-regulates HMOX1 and down-regulates HCV gene expression in hepatoma cells. This probably mitigates liver injury caused by combined iron overload and HCV infection.

  16. Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

    Science.gov (United States)

    Shahrabi-Farahani, Shokoufeh; Wang, Lili; Zwaans, Bernadette M M; Santana, Jeans M; Shimizu, Akio; Takashima, Seiji; Kreuter, Michael; Coultas, Leigh; D'Amore, Patricia A; Arbeit, Jeffrey M; Akslen, Lars A; Bielenberg, Diane R

    2014-07-01

    Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity.

  17. Analysis of expression profiles of MAGE-A antigens in oral squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Reichert Torsten E

    2009-04-01

    Full Text Available Abstract Background The immunological response to solid tumours is insufficient. Therefore, tumour specific antigens have been explored to facilitate the activation of the immune system. The cancer/testis antigen class of MAGE-A antigens is a possible target for vaccination. Their differential expression profiles also modulate the course of the cancer disease and its response to antineoplastic drugs. Methods The expression profiles of MAGE-A2, -A3, -A4, -A6 and -A10 in five own oral squamous cell carcinoma cell lines were characterised by rt-PCR, qrt-PCR and immunocytochemistry with a global MAGE-A antibody (57B and compared with those of an adult keratinocyte cell line (NHEK. Results All tumour cell lines expressed MAGE-A antigens. The antigens were expressed in groups with different preferences. The predominant antigens expressed were MAGE-A2, -A3 and -A6. MAGE-A10 was not expressed in the cell lines tested. The MAGE-A gene products detected in the adult keratinocyte cell line NHEK were used as a reference. Conclusion MAGE-A antigens are expressed in oral squamous cell carcinomas. The expression profiles measured facilitate distinct examinations in forthcoming studies on responses to antineoplastic drugs or radiation therapy. MAGE-A antigens are still an interesting aim for immunotherapy.

  18. Polyclonal T-cells express CD1a in Langerhans cell histiocytosis (LCH lesions.

    Directory of Open Access Journals (Sweden)

    Jennifer A West

    Full Text Available Langerhans cell histiocytosis (LCH is a complex and poorly understood disorder that has characteristics of both inflammatory and neoplastic disease. By using eight-colour flow cytometry, we have identified a previously unreported population of CD1a(+/CD3(+ T-cells in LCH lesions. The expression of CD1a is regarded as a hallmark of this disease; however, it has always been presumed that it was only expressed by pathogenic Langerhans cells (LCs. We have now detected CD1a expression by a range of T-cell subsets within all of the LCH lesions that were examined, establishing that CD1a expression in these lesions is no longer restricted to pathogenic LCs. The presence of CD1a(+ T-cells in all of the LCH lesions that we have studied to date warrants further investigation into their biological function to determine whether these cells are important in the pathogenesis of LCH.

  19. Polyclonal T-Cells Express CD1a in Langerhans Cell Histiocytosis (LCH) Lesions

    Science.gov (United States)

    West, Jennifer A.; Olsen, Sharon L.; Mitchell, Jenée M.; Priddle, Ross E.; Luke, Jennifer M.; Åkefeldt, Selma Olsson; Henter, Jan-Inge; Turville, Christopher; Kannourakis, George

    2014-01-01

    Langerhans cell histiocytosis (LCH) is a complex and poorly understood disorder that has characteristics of both inflammatory and neoplastic disease. By using eight-colour flow cytometry, we have identified a previously unreported population of CD1a+/CD3+ T-cells in LCH lesions. The expression of CD1a is regarded as a hallmark of this disease; however, it has always been presumed that it was only expressed by pathogenic Langerhans cells (LCs). We have now detected CD1a expression by a range of T-cell subsets within all of the LCH lesions that were examined, establishing that CD1a expression in these lesions is no longer restricted to pathogenic LCs. The presence of CD1a+ T-cells in all of the LCH lesions that we have studied to date warrants further investigation into their biological function to determine whether these cells are important in the pathogenesis of LCH. PMID:25343480

  20. Expression of Neural Markers by Undifferentiated Rat Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Dana Foudah

    2012-01-01

    Full Text Available The spontaneous expression of neural markers by mesenchymal stem cells (MSCs has been considered to be a demonstration of MSCs’ predisposition to differentiate towards neural lineages. In view of their application in cell therapy for neurodegenerative diseases, it is very important to deepen the knowledge about this distinctive biological property of MSCs. In this study, we evaluated the expression of neuronal and glial markers in undifferentiated rat MSCs (rMSCs at different culture passages (from early to late. rMSCs spontaneously expressed neural markers depending on culture passage, and they were coexpressed or not with the neural progenitor marker nestin. In contrast, the number of rMSCs expressing mesengenic differentiation markers was very low or even completely absent. Moreover, rMSCs at late culture passages were not senescent cells and maintained the MSC immunophenotype. However, their differentiation capabilities were altered. In conclusion, our results support the concept of MSCs as multidifferentiated cells and suggest the existence of immature and mature neurally fated rMSC subpopulations. A possible correlation between specific MSC subpopulations and specific neural lineages could optimize the use of MSCs in cell transplantation therapy for the treatment of neurological diseases.

  1. Gene expression profiling of chicken primordial germ cell ESTs

    Directory of Open Access Journals (Sweden)

    Lim Dajeong

    2006-08-01

    Full Text Available Abstract Background Germ cells are the only cell type that can penetrate from one generation to next generation. At the early embryonic developmental stages, germ cells originally stem from primordial germ cells, and finally differentiate into functional gametes, sperm in male or oocyte in female, after sexual maturity. This study was conducted to investigate a large-scale expressed sequence tag (EST analysis in chicken PGCs and compare the expression of the PGC ESTs with that of embryonic gonad. Results We constructed 10,851 ESTs from a chicken cDNA library of a collection of highly separated embryonic PGCs. After chimeric and problematic sequences were filtered out using the chicken genomic sequences, there were 5,093 resulting unique sequences consisting of 156 contigs and 4,937 singlets. Pearson chi-square tests of gene ontology terms in the 2nd level between PGC and embryonic gonad set showed no significance. However, digital gene expression profiling using the Audic's test showed that there were 2 genes expressed significantly with higher number of transcripts in PGCs compared with the embryonic gonads set. On the other hand, 17 genes in embryonic gonads were up-regulated higher than those in the PGC set. Conclusion Our results in this study contribute to knowledge of mining novel transcripts and genes involved in germline cell proliferation and differentiation at the early embryonic stages.

  2. Progesterone Upregulates Gene Expression in Normal Human Thyroid Follicular Cells

    Directory of Open Access Journals (Sweden)

    Ana Paula Santin Bertoni

    2015-01-01

    Full Text Available Thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. There are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium-iodide symporter NIS, thyroglobulin TG, thyroperoxidase TPO, and KI-67 genes expression, in normal thyroid follicular cells, derived from human tissue. NIS, TG, TPO, and KI-67 mRNA expression increased significantly after TSH 20 μUI/mL, respectively: 2.08 times, P<0.0001; 2.39 times, P=0.01; 1.58 times, P=0.0003; and 1.87 times, P<0.0001. In thyroid cells treated with 20 μUI/mL TSH plus 10 nM progesterone, RNA expression of NIS, TG, and KI-67 genes increased, respectively: 1.78 times, P<0.0001; 1.75 times, P=0.037; and 1.95 times, P<0.0001, and TPO mRNA expression also increased, though not significantly (1.77 times, P=0.069. These effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. This work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.

  3. Detection of gene expression in an individual cell type within a cell mixture using microarray analysis.

    Directory of Open Access Journals (Sweden)

    Penelope A Bryant

    Full Text Available BACKGROUND: A central issue in the design of microarray-based analysis of global gene expression is the choice between using cells of single type and a mixture of cells. This study quantified the proportion of lipopolysaccharide (LPS induced differentially expressed monocyte genes that could be measured in peripheral blood mononuclear cells (PBMC, and determined the extent to which gene expression in the non-monocyte cell fraction diluted or obscured fold changes that could be detected in the cell mixture. METHODOLOGY/PRINCIPAL FINDINGS: Human PBMC were stimulated with LPS, and monocytes were then isolated by positive (Mono+ or negative (Mono- selection. The non-monocyte cell fraction (MonoD remaining after positive selection of monocytes was used to determine the effect of non-monocyte cells on overall expression. RNA from LPS-stimulated PBMC, Mono+, Mono- and MonoD samples was co-hybridised with unstimulated RNA for each cell type on oligonucleotide microarrays. There was a positive correlation in gene expression between PBMC and both Mono+ (0.77 and Mono- (0.61-0.67 samples. Analysis of individual genes that were differentially expressed in Mono+ and Mono- samples showed that the ability to detect expression of some genes was similar when analysing PBMC, but for others, differential expression was either not detected or changed in the opposite direction. As a result of the dilutional or obscuring effect of gene expression in non-monocyte cells, overall about half of the statistically significant LPS-induced changes in gene expression in monocytes were not detected in PBMC. However, 97% of genes with a four fold or greater change in expression in monocytes after LPS stimulation, and almost all (96-100% of the top 100 most differentially expressed monocyte genes were detected in PBMC. CONCLUSIONS/SIGNIFICANCE: The effect of non-responding cells in a mixture dilutes or obscures the detection of subtle changes in gene expression in an individual

  4. PENICILLIN-BINDING PROTEIN 2X OF STREPTOCOCCUS-PNEUMONIAE - EXPRESSION IN ESCHERICHIA-COLI AND PURIFICATION OF A SOLUBLE ENZYMATICALLY ACTIVE DERIVATIVE

    NARCIS (Netherlands)

    LAIBLE, G; KECK, W; LURZ, R; MOTTL, H; FRERE, JM; JAMIN, M; HAKENBECK, R

    1992-01-01

    A 2.5-kb DNA fragment including the structural gene coding for the penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae has been cloned into the vector pJDC9 and expressed in Escherichia coli. Mapping of RNA polymerase binding sites by electron microscopy indicated that the pbpX promot

  5. Gene cloning, characterization and expression and enzymatic activities related to trehalose metabolism during diapause of the onion maggot Delia antiqua (Diptera: Anthomyiidae).

    Science.gov (United States)

    Guo, Qiang; Hao, You-Jin; Li, Yuan; Zhang, Yu-Juan; Ren, Shuang; Si, Feng-Ling; Chen, Bin

    2015-07-01

    Trehalose represents the main hemolymph sugar in many insects, and it functions in energy metabolism and protection in extreme environmental conditions. To gain an insight into trehalose functions in Delia antiqua diapausing pupae, genes encoding trehalose-6-phosphate synthase (TPS), trehalose-6-phosphatase (TPP) and trehalase (TRE) were identified and characterized. Analysis of the deduced amino acid sequences indicated that these genes were highly similar to each homolog from Diptera insects. Gene expressions and their enzyme activities were also investigated. The differential expressions of TPS and TPP shared very similar trends for summer and winter diapausing pupae. Their enzyme activities were consistent with the gene expressions. Trehalose concentrations in summer- and winter-diapausing pupae were lower at the initial phase (4.37-5.09μg/mg) but increased gradually and peaked in the maintenance phase (10.59-14.36μg/mg); the concentrations then declined in the quiescence phase. We speculated that a higher trehalose content during the maintenance stage may contribute to protein and/or biological membrane stabilization in winter or to desiccation resistance in the summertime. Diapause termination requires a decrease in the trehalose concentration to promote pupal-adult development. The glucose content also varied during the diapausing processes. Our results provide an overview of the differential expression levels of trehalose metabolic enzymes, confirming the important roles of trehalose in diapausing pupae of the onion maggot. Further work remains to explore its actual functions.

  6. IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells.

    Science.gov (United States)

    Long, Jun; Zhang, Xulong; Wen, Mingjie; Kong, Qingli; Lv, Zhe; An, Yunqing; Wei, Xiao-Qing

    2013-01-01

    Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

  7. Differentially expressed genes in giant cell tumor of bone.

    Science.gov (United States)

    Babeto, Erica; Conceição, André Luis Giacometti; Valsechi, Marina Curado; Peitl Junior, Paulo; de Campos Zuccari, Débora Aparecida Pires; de Lima, Luiz Guilherme Cernaglia Aureliano; Bonilha, Jane Lopes; de Freitas Calmon, Marília; Cordeiro, José Antônio; Rahal, Paula

    2011-04-01

    Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.

  8. Phenolic acids enzymatic lipophilization.

    Science.gov (United States)

    Figueroa-Espinoza, Maria-Cruz; Villeneuve, Pierre

    2005-04-20

    Lipophilization is the esterification of a lipophilic moiety (fatty acid or fatty alcohol) on different substrates (phenolic acid, sugar, protein, ...), resulting in new molecules with modified hydrophilic/lipophilic balance. This reaction can be obtained chemically or enzymatically using different enzymes. Phenolic acids possess interesting biological properties (antioxidant, chelator, free radical scavenger, UV filter, antimicrobial, ...), but because of their relatively low solubility in aprotic media, their application in oil-based products is limited. Therefore, the esterification of their carboxylic acid function with a fatty alcohol enhances their hydrophobicity and results in a multifunctional amphiphilic molecule. Enzymatic lipophilization of phenolic acids is nowadays studied for potential industrial applications. Different systems have been proposed to perform the reaction yield [free or immobilized enzymes (lipase, feruloyl esterase, tannase, etc.), free or added organic solvent, addition of surfactant, microemulsion system, etc.]. Some of the functional properties of these esters have been demonstrated. This review presents a panorama of the advances in this field.

  9. Enzymatic desulfurization of coal

    Energy Technology Data Exchange (ETDEWEB)

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.

    1991-05-16

    The overall objective of this program was to investigate the feasibility of an enzymatic desulfurization process specifically intended for organic sulfur removal from coal. Toward that end, a series of specific objectives were defined: (1) establish the feasibility of (bio)oxidative pretreatment followed by biochemical sulfate cleavage for representative sulfur-containing model compounds and coals using commercially-available enzymes; (2) investigate the potential for the isolation and selective use of enzyme preparations from coal-utilizing microbial systems for desulfurization of sulfur-containing model compounds and coals; and (3) develop a conceptual design and economic analysis of a process for enzymatic removal of organic sulfur from coal. Within the scope of this program, it was proposed to carry out a portion of each of these efforts concurrently. (VC)

  10. Enzymatic production of cyclodextrins.

    Science.gov (United States)

    Biwer, A; Antranikian, G; Heinzle, E

    2002-09-01

    Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented.

  11. Effects of Trichostatin A on HDAC8 Expression, Proliferation and Cell Cycle of Molt-4 Cells

    Institute of Scientific and Technical Information of China (English)

    HE Jing; LIU Hongli; CHEN Yan

    2006-01-01

    The effects of Trichostatin A (TSA) on histone deacetylase 8 (HDAC8) expression, proliferation and cell cycle arrest in T-lymphoblastic leukemia cell line Molt-4 cells in vitro were investigated. The effect of TSA on the growth of Molt-4 cells was studied by MTT assay. Flow cytometry was used to examine the cell cycle. The expression of HDAC8 was detected by using immunocytochemistry and Western blot. The results showed that proliferation of Molt-4 cells was inhibited in TSA-treated group in a time- and dose-dependent manner. The IC50 of TSA exposures for 24 h and 36 h were 254.3236 and 199.257 μg/L respectively. The cell cycle analysis revealed that Molt-4 was mostly in G0/G1 phase, and after treatment with TSA from 50 to 400 μg/L for 24 h, the percents of G0/G1 cells were decreased and cells were arrested in G2/M phase. Treatment of TSA for 24 h could significantly inhibit the expression of HDAC8 protein in Molt-4 cells (P<0.01). It was concluded that TSA could decrease the expression of HDAC8 in Molt-4 cells, which contributed to the inhibition of proliferation and induction of cell cycle arrest in Molt-4 cells.

  12. AIRE expressing marginal zone dendritic cells balances adaptive immunity and T-follicular helper cell recruitment.

    Science.gov (United States)

    Lindmark, Evelina; Chen, Yunying; Georgoudaki, Anna-Maria; Dudziak, Diana; Lindh, Emma; Adams, William C; Loré, Karin; Winqvist, Ola; Chambers, Benedict J; Karlsson, Mikael C I

    2013-05-01

    Autoimmune polyendocrine syndrome Type I (APS I) results in multiple endocrine organ destruction and is caused by mutations in the Autoimmune regulator gene (AIRE). In the thymic stroma, cells expressing the AIRE gene dictate T cell education and central tolerance. Although this function is the most studied, AIRE is also expressed in the periphery in DCs and stromal cells. Still, how AIRE regulated transcription modifies cell behaviour in the periphery is largely unknown. Here we show that AIRE is specifically expressed by 33D1(+) DCs and dictates the fate of antibody secreting cell movement within the spleen. We also found that AIRE expressing 33D1(+) DCs expresses self-antigens as exemplified by the hallmark gene insulin. Also, as evidence for a regulatory function, absence of Aire in 33D1(+) DCs led to reduced levels of the chemokine CXCL12 and increased co-stimulatory properties. This resulted in altered activation and recruitment of T-follicular helper cells and germinal centre B cells. The altered balance leads to a change of the early response to a T cell-dependent antigen in Aire(-/-) mice. These findings add to the understanding of how specific DC subtypes regulate the early responses during T cell-dependent antibody responses within the spleen and further define the role of AIRE in the periphery as regulator of self-antigen expression and lymphocyte migration. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Functional bitter taste receptors are expressed in brain cells.

    Science.gov (United States)

    Singh, Nisha; Vrontakis, Maria; Parkinson, Fiona; Chelikani, Prashen

    2011-03-04

    Humans are capable of sensing five basic tastes which are sweet, sour, salt, umami and bitter. Of these, bitter taste perception provides protection against ingestion of potentially toxic substances. Bitter taste is sensed by bitter taste receptors (T2Rs) that belong to the G-protein coupled receptors (GPCRs) superfamily. Humans have 25 T2Rs that are expressed in the oral cavity, gastrointestinal (GI) neuroendocrine cells and airway cells. Electrophysiological studies of the brain neurons show that the neurons are able to respond to different tastants. However, the presence of bitter taste receptors in brain cells has not been elucidated. In this report using RT-PCR, and immunohistochemistry analysis we show that T2Rs are expressed in multiple regions of the rat brain. RT-PCR analysis revealed the presence of T2R4, T2R107 and T2R38 transcripts in the brain stem, cerebellum, cortex and nucleus accumbens. The bitter receptor T2R4 was selected for further analysis at the transcript level by quantitative real time PCR and at the protein level by immunohistochemistry. To elucidate if the T2R4 expressed in these cells is functional, assays involving G-protein mediated calcium signaling were carried out. The functional assays showed an increase in intracellular calcium levels after the application of exogenous ligands for T2R4, denatonium benzoate and quinine to these cultured cells, suggesting that endogenous T2R4 expressed in these cells is functional. We discuss our results in terms of the physiological relevance of bitter receptor expression in the brain.

  14. Improved Expression Systems for Regulated Expression in Salmonella Infecting Eukaryotic Cells

    Science.gov (United States)

    Medina, Carlos; Camacho, Eva María; Flores, Amando; Mesa-Pereira, Beatriz; Santero, Eduardo

    2011-01-01

    In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the Pm promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/Psal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i) the nasF transcriptional attenuator, which reduces basal expression levels, (ii) a strong ribosome binding site, and (iii) the Type III Secretion System (TTSS) signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies. PMID:21829692

  15. Improved expression systems for regulated expression in Salmonella infecting eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Carlos Medina

    Full Text Available In this work we describe a series of improvements to the Salmonella-based salicylate-inducible cascade expression system comprised of a plasmid-borne expression module, where target gene expression is driven by the P(m promoter governed by the XylS2 regulator, and a genome-integrated regulatory module controlled by the nahR/P(sal system. We have constructed a set of high and low-copy number plasmids bearing modified versions of the expression module with a more versatile multiple cloning site and different combinations of the following elements: (i the nasF transcriptional attenuator, which reduces basal expression levels, (ii a strong ribosome binding site, and (iii the Type III Secretion System (TTSS signal peptide from the effector protein SspH2 to deliver proteins directly to the eukaryotic cytosol following bacterial infection of animal cells. We show that different expression module versions can be used to direct a broad range of protein production levels. Furthermore, we demonstrate that the efficient reduction of basal expression by the nasF attenuator allows the cloning of genes encoding highly cytotoxic proteins such as colicin E3 even in the absence of its immunity protein. Additionally, we show that the Salmonella TTSS is able to translocate most of the protein produced by this regulatory cascade to the cytoplasm of infected HeLa cells. Our results indicate that these vectors represent useful tools for the regulated overproduction of heterologous proteins in bacterial culture or in animal cells, for the cloning and expression of genes encoding toxic proteins and for pathogenesis studies.

  16. PRL-3 expression in nasal sinus squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    Zi-Hui Chen; Min-Ying Li

    2016-01-01

    Objective:To investigate the relationship between liver regeneration phosphatase-3 (PRL-3) with differentiation extent of nasal sinus squamous cell carcinoma, and molecular biological effects on the pathogenesis of nasal sinus squamous cell carcinoma to comprehend its relevance, so as to make early diagnosis of patients, and to give guidance to the prognosis. Methods:Immunohistochemistry was used to detect PRL-3 in 30 cases of different degrees of sinus nasal squamous cell carcinoma. 20 cases of normal nasal cavity of mucosa tissues were set as control. Results:The PRL-3 in all levels of sinonasal squamous cell carcinoma tissues, there was a significant difference compared with the normal nasal mucosa (P<0.05), squamous cell carcinoma and its expression increased with the grade with enhanced trend. Conclusions:PRL-3 expression increased significantly in sinonasal squamous cell carcinoma than in nasal polyp tissue, showed that it may be associated with squamous cell carcinoma of nasal sinus squamous cell carcinoma, may be the early event.

  17. Expression of cell cycle regulator cdk2ap1 suppresses tumor cell phenotype by non-cell autonomous mechanisms

    OpenAIRE

    Zolochevska, Olga; Figueiredo, Marxa L

    2009-01-01

    We evaluated the effect of expressing the cell cycle regulator cdk2ap1 in epithelial or stromal cell compartments to reduce SCC growth in vitro and in vivo. Cell autonomous and/or non-cell autonomous expression of cdk2ap1 reduced tumor growth and invasion and altered cell cycle, adhesion, invasion, angiogenesis, and apoptotic gene expression, as assessed by several in vitro phenotype assays, quantitative real time PCR, and in vivo molecular imaging using a novel three-way xenograft animal mod...

  18. Endothelial cells downregulate apolipoprotein D expression in mural cells through paracrine secretion and Notch signaling.

    Science.gov (United States)

    Pajaniappan, Mohanasundari; Glober, Nancy K; Kennard, Simone; Liu, Hua; Zhao, Ning; Lilly, Brenda

    2011-09-01

    Endothelial and mural cell interactions are vitally important for proper formation and function of blood vessels. These two cell types communicate to regulate multiple aspects of vessel function. In studying genes regulated by this interaction, we identified apolipoprotein D (APOD) as one gene that is downregulated in mural cells by coculture with endothelial cells. APOD is a secreted glycoprotein that has been implicated in governing stress response, lipid metabolism, and aging. Moreover, APOD is known to regulate smooth muscle cells and is found in abundance within atherosclerotic lesions. Our data show that the regulation of APOD in mural cells is bimodal. Paracrine secretion by endothelial cells causes partial downregulation of APOD expression. Additionally, cell contact-dependent Notch signaling plays a role. NOTCH3 on mural cells promotes the downregulation of APOD, possibly through interaction with the JAGGED-1 ligand on endothelial cells. Our results show that NOTCH3 contributes to the downregulation of APOD and by itself is sufficient to attenuate APOD transcript expression. In examining the consequence of decreased APOD expression in mural cells, we show that APOD negatively regulates cell adhesion. APOD attenuates adhesion by reducing focal contacts; however, it has no effect on stress fiber formation. These data reveal a novel mechanism in which endothelial cells control neighboring mural cells through the downregulation of APOD, which, in turn, influences mural cell function by modulating adhesion.

  19. Changes in cell adhesion molecule expression on T cells associated with systemic virus infection

    DEFF Research Database (Denmark)

    Andersson, E C; Christensen, Jan Pravsgaard; Marker, O

    1994-01-01

    Virus-induced changes in adhesion molecule expression on T cells were investigated to understand how antiviral effector cells migrate into infectious foci. FACS analysis revealed that after systemic infection with lymphocytic choriomeningitis virus a number of cell adhesion molecules, including VLA...... analyses showed that T cells with a changed adhesion molecule profile tended to present other cell surface markers indicating a state of cellular activation, e.g., IL-2R, and included all virus-specific CTL effectors. Regarding the physiologic significance of these changes in adhesion molecule expression...

  20. GLUT-1 Expression in Cutaneous Basal and Squamous Cell Carcinomas.

    Science.gov (United States)

    Abdou, Asmaa Gaber; Eldien, Marwa Mohammad Serag; Elsakka, Daliah

    2015-09-01

    Glucose uptake is a key regulating step in glucose metabolism and is mediated by facilitative glucose transporters (GLUTs), and GLUT-1 is the predominant glucose transporter in many types of human cells. Cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) represent the most common skin cancer in Egypt. The present study aimed at evaluation of the pattern and distribution of GLUT-1 in cutaneous BCC (16 cases) and SCC (16 cases) by means of immunohistochemistry. GLUT-1 was expressed in all SCC (100%) and in 62.5% of BCC. Membranous pattern of GLUT-1 was seen in 62.5% of SCC and 31.25% of BCC. Positivity (P = .02) and percentage (P = .000) of GLUT-1 expression were in favor of SCC in comparison to BCC. The high percentage of GLUT-1 expression was associated with high grade in SCC (P = .03). The immunoreactivity for GLUT-1 was more in the periphery of malignant nests of SCC while it was more in the center of BCC nests. GLUT-1 is overexpressed in cutaneous non-melanoma skin cancer. Its expression in SCC is related to differentiation status, and its expression in BCC is intimately associated with squamous metaplastic areas.

  1. Ebola virus infection induces irregular dendritic cell gene expression.

    Science.gov (United States)

    Melanson, Vanessa R; Kalina, Warren V; Williams, Priscilla

    2015-02-01

    Filoviruses subvert the human immune system in part by infecting and replicating in dendritic cells (DCs). Using gene arrays, a phenotypic profile of filovirus infection in human monocyte-derived DCs was assessed. Monocytes from human donors were cultured in GM-CSF and IL-4 and were infected with Ebola virus Kikwit variant for up to 48 h. Extracted DC RNA was analyzed on SuperArray's Dendritic and Antigen Presenting Cell Oligo GEArray and compared to uninfected controls. Infected DCs exhibited increased expression of cytokine, chemokine, antiviral, and anti-apoptotic genes not seen in uninfected controls. Significant increases of intracellular antiviral and MHC I and II genes were also noted in EBOV-infected DCs. However, infected DCs failed to show any significant difference in co-stimulatory T-cell gene expression from uninfected DCs. Moreover, several chemokine genes were activated, but there was sparse expression of chemokine receptors that enabled activated DCs to home to lymph nodes. Overall, statistically significant expression of several intracellular antiviral genes was noted, which may limit viral load but fails to stop replication. EBOV gene expression profiling is of vital importance in understanding pathogenesis and devising novel therapeutic treatments such as small-molecule inhibitors.

  2. Human glucocerebrosidase: heterologous expression of active site mutants in murine null cells.

    Science.gov (United States)

    Fabrega, S; Durand, P; Codogno, P; Bauvy, C; Delomenie, C; Henrissat, B; Martin, B M; McKinney, C; Ginns, E I; Mornon, J P; Lehn, P

    2000-11-01

    Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.

  3. Enhancement of endothelial cell migration by constitutively active LPA{sub 1}-expressing tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Kitayoshi, Misaho; Kato, Kohei; Tanabe, Eriko; Yoshikawa, Kyohei; Fukui, Rie [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Mutated LPA{sub 1} stimulates cell migration of endothelial cells. Black-Right-Pointing-Pointer VEGF expressions are increased by mutated LPA{sub 1}. Black-Right-Pointing-Pointer LPA signaling via mutated LPA{sub 1} is involved in angiogenesis. Black-Right-Pointing-Pointer Mutated LPA{sub 1} promotes cancer cell progression. -- Abstract: Lysophosphatidic acid (LPA) receptors belong to G protein-coupled transmembrane receptors (LPA receptors; LPA{sub 1} to LPA{sub 6}). They indicate a variety of cellular response by the interaction with LPA, including cell proliferation, migration and differentiation. Recently, we have reported that constitutive active mutated LPA{sub 1} induced the strong biological effects of rat neuroblastoma B103 cells. In the present study, we examined the effects of mutated LPA{sub 1} on the interaction between B103 cells and endothelial F-2 cells. Each LPA receptor expressing B103 cells were maintained in serum-free DMEM and cell motility assay was performed with a Cell Culture Insert. When F-2 cells were cultured with conditioned medium from Lpar1 and Lpar3-expressing cells, the cell motility of F-2 cells was significantly higher than control cells. Interestingly, the motile activity of F-2 cells was strongly induced by mutated LPA{sub 1} than other cells, correlating with the expression levels of vascular endothelial growth factor (Vegf)-A and Vegf-C. Pretreatment of LPA signaling inhibitors inhibited F-2 cell motility stimulated by mutated LPA{sub 1}. These results suggest that activation of LPA signaling via mutated LPA{sub 1} may play an important role in the promotion of angiogenesis in rat neuroblastoma cells.

  4. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  5. Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

    DEFF Research Database (Denmark)

    Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad;

    2010-01-01

    HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

  6. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  7. Metastasis regulation by PPARD expression in cancer cells

    Science.gov (United States)

    Zuo, Xiangsheng; Xu, Weiguo; Xu, Min; Tian, Rui; Moussalli, Micheline J.; Mao, Fei; Zheng, Xiaofeng; Wang, Jing; Morris, Jeffrey S.; Eng, Cathy; Maru, Dipen M.; Rashid, Asif; Broaddus, Russell; Wei, Daoyan; Hung, Mien-Chie; Sood, Anil K.

    2017-01-01

    Peroxisome proliferator–activated receptor–δ (PPARD) is upregulated in many major human cancers, but the role that its expression in cancer cells has in metastasis remains poorly understood. Here, we show that specific PPARD downregulation or genetic deletion of PPARD in cancer cells significantly repressed metastasis in various cancer models in vivo. Mechanistically, PPARD promoted angiogenesis via interleukin 8 in vivo and in vitro. Analysis of transcriptome profiling of HCT116 colon cancer cells with or without genetic deletion of PPARD and gene expression patterns in The Cancer Genome Atlas colorectal adenocarcinoma database identified novel pro-metastatic genes (GJA1, VIM, SPARC, STC1, SNCG) as PPARD targets. PPARD expression in cancer cells drastically affected epithelial-mesenchymal transition, migration, and invasion, further underscoring its necessity for metastasis. Clinically, high PPARD expression in various major human cancers (e.g., colorectal, lung, breast) was associated with significantly reduced metastasis-free survival. Our results demonstrate that PPARD, a druggable protein, is an important molecular target in metastatic cancer. PMID:28097239

  8. Reduced expression of Slit2 in renal cell carcinoma.

    Science.gov (United States)

    Ma, Wei-Jie; Zhou, Yu; Lu, Dan; Dong, Dong; Tian, Xiao-Jun; Wen, Jie-Xi; Zhang, Jun

    2014-01-01

    Slit2, initially identified as an important axon guidance molecule in the nervous system, was suggested to be involved in multiple cellular processes. Recently, Slit2 was reported to function as a potential tumor suppressor in diverse tumors. In this study, we systematically analyzed the expression level of Slit2 in renal cell carcinoma. Compared to paired adjacent non-malignant tissues, both Slit2 mRNA and protein expression were significantly down-regulated in renal cell carcinoma (RCC). Methylation-specific PCR showed that Slit2 promoter was methylated in two renal carcinoma cell lines. Pharmacologic demethylation dramatically induced Slit2 expression in cancer cell lines with weak expression of Slit2. Besides, bisulfite genomic sequencing confirmed that dense methylation existed in Slit2 promoter. Furthermore, in paired RCC samples, Slit2 methylation was observed in 8 out of 38 patients (21.1 %), which was well correlated with the down-regulation of Slit2 in RCC. Therefore, Slit2 may also be a potential tumor suppressor in RCC, which is down-regulated in RCC partially due to promoter methylation.

  9. Gene expression analysis of dendritic/Langerhans cells and Langerhans cell histiocytosis

    NARCIS (Netherlands)

    Rust, Renata; Kluiver, J.; Visser, Lydia; Harms, G.; Blokzijl, T.; Kamps, W.A.; Poppema, Sibrand; van den Berg, Anke

    2006-01-01

    Langerhans cell histiocytosis (LCH) is a neoplastic disorder that results in clonal proliferation of cells with a Langerhans cell (LQ phenotype. The pathogenesis of LCH is still poorly understood. In the present study, serial analysis of gene expression (SAGE) was applied to LCs generated from umbil

  10. Enzymatic activity, gene expression and posttranslational modifications of photosynthetic and non-photosynthetic phosphoenolpyruvate carboxylase in ammonium-stressed sorghum plants.

    Science.gov (United States)

    Arias-Baldrich, Cirenia; de la Osa, Clara; Bosch, Nadja; Ruiz-Ballesta, Isabel; Monreal, José A; García-Mauriño, Sofía

    2017-07-01

    Sorghum plants grown with 5mM (NH4)2SO4 showed symptoms of stress, such as reduced growth and photosynthesis, leaf chlorosis, and reddish roots. Phosphoenolpyruvate carboxylase (PEPC) activity, by supplying carbon skeletons for ammonium assimilation, plays a pivotal role in tolerance to ammonium stress. This work investigated the effect of ammonium nutrition on PPC and PPCK gene expression, on PEPC activity, and on post-translational modifications (PTMs) of PEPC in leaves and roots of sorghum plants. Ammonium increased PEPC kinase (PEPCk) activity and the phosphorylation state of PEPC in leaves, both in light and in the dark, due to increased PPCK1 expression in leaves. This result resembled the effect of salinity on sorghum leaf PEPC and PEPCk, which is thought to allow a better functioning of PEPC in conditions that limit the income of reduced C. In roots, ammonium increased PEPC activity and the amount of monoubiquitinated PEPC. The first effect was related to increased PPC3 expression in roots. These results highlight the relevance of this specific isoenzyme (PPC3) in sorghum responses to ammonium stress. Although the role of monoubiquitination is not fully understood, it also increased in germinating seeds along with massive mobilization of reserves, a process in which the anaplerotic function of PEPC is of major importance. Copyright © 2017 Elsevier GmbH. All rights reserved.

  11. Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells

    DEFF Research Database (Denmark)

    Jørgensen, Claus; Sherman, Andrew; Chen, Ginny I

    2009-01-01

    Cells have self-organizing properties that control their behavior in complex tissues. Contact between cells expressing either B-type Eph receptors or their transmembrane ephrin ligands initiates bidirectional signals that regulate cell positioning. However, simultaneously investigating how...... information is processed in two interacting cell types remains a challenge. We implemented a proteomic strategy to systematically determine cell-specific signaling networks underlying EphB2- and ephrin-B1-controlled cell sorting. Quantitative mass spectrometric analysis of mixed populations of EphB2......- and ephrin-B1-expressing cells that were labeled with different isotopes revealed cell-specific tyrosine phosphorylation events. Functional associations between these phosphotyrosine signaling networks and cell sorting were established with small interfering RNA screening. Data-driven network modeling...

  12. Immune Killing Activity of Lymphocytes on Hela Cells Expressing Interleukin-12 In Vitro

    Institute of Scientific and Technical Information of China (English)

    Huiyan WANG; Suhua CHEN

    2008-01-01

    The killing effects of lymphocytes on Hela cells expressing intedeukin-12 (IL-12) in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL-12 between 24h and 72h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express IL-12 and were more easily killed by lymphocytes.

  13. Stem cell antigen 2 expression in adult and developing mice.

    Science.gov (United States)

    Antica, M; Wu, L; Scollay, R

    1997-01-01

    Stem cell antigen 2 (Sca-2) expression can distinguish the most immature T-lymphocyte precursors in the thymus from the hemopoietic stem cells. Sequence analysis of the Sca-2 protein showed that Sca-2 is a glycosylphosphatidylinositol (GPI) anchored molecule that shares some characteristics with the members of the Ly-6 multigene family, and that it is the same as the thymic shared antigen-1 (TSA-1). Here we extend these studies and critically reassess the expression of the Sca-2/TSA-1 antigen in hematopoietic tissues of adult and developing mice. With more sensitive methods we show that the distribution of Sca-2/TSA-1 differs from existing reports. We find especially high expression of Sca-2/TSA1 at day 14 of fetal development.

  14. The expression and regulation of glucose transporters in tumor cells

    Directory of Open Access Journals (Sweden)

    Pengfei Zhao

    2016-12-01

    Full Text Available Glucose transporter proteins are involved in many physiological and biochemical processes. In particular, the high expressions of sodium-glucose cotransporter and glucose transporter proteins in tumor cells show that these two transporters play a key role in tumor cell metabolism. Studying the crystal structure and conformation of human glucose transporter proteins has enabled the development of drugs based on specific binding sites, opening up a new path towards more effective cancer treatments. This mini review serves to summarize our existing understanding of the metabolic pathways of tumor cells, focusing on the roles of glucose transporter proteins.

  15. Unusual patterns of immunoglobulin gene rearrangement and expression during human B cell ontogeny: human B cells can simultaneously express cell surface kappa and lambda light chains

    OpenAIRE

    1993-01-01

    Immunoglobulin gene rearrangement during mammalian B cell development generally follows an ordered progression, beginning with heavy (H) chain genes and proceeding through kappa and lambda light (L) chain genes. To determine whether the predicted kappa-->lambda hierarchy was occurring in vitro, we generated Epstein-Barr virus-transformed cell lines from cultures undergoing human pre-B cell differentiation. A total of 143 cell lines were established. 24 expressed cell surface mu/lambda by flow...

  16. B cell lymphomas express CX3CR1 a non-B cell lineage adhesion molecule

    DEFF Research Database (Denmark)

    Andreasson, U.; Ek, S.; Merz, H.

    2008-01-01

    To study the differential expression of cell membrane-bound receptors and their potential role in growth and/or survival of the tumor cells, highly purified follicular lymphoma cells were analyzed, using gene expression analysis, and compared to non-malignant B cell populations. Filtering...... the genome for overexpressed genes coding for cell membrane-bound proteins/receptors resulted in a hit list of 27 identified genes. Among these, we have focused on the aberrant over expression of CX3CR1, in different types of B cell lymphoma, as compared to non-malignant B cells. We show that CX3CR1, which...... normally is not expressed on B cells, is expressed both at the mRNA and protein level in several subtypes of lymphoma. CX3CR1 has also shown to be involved in the homing to specific tissues that express the ligand, CX3CL1, in breast and prostate cancer and may thus be involved in dissemination of lymphoma...

  17. COX-2 expression positively correlates with PD-L1 expression in human melanoma cells.

    Science.gov (United States)

    Botti, Gerardo; Fratangelo, Federica; Cerrone, Margherita; Liguori, Giuseppina; Cantile, Monica; Anniciello, Anna Maria; Scala, Stefania; D'Alterio, Crescenzo; Trimarco, Chiara; Ianaro, Angela; Cirino, Giuseppe; Caracò, Corrado; Colombino, Maria; Palmieri, Giuseppe; Pepe, Stefano; Ascierto, Paolo Antonio; Sabbatino, Francesco; Scognamiglio, Giosuè

    2017-02-23

    The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma. Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAF(V600E) A375 and NRAS(Q61R) SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro. BRAF(V600E/V600K) and NRAS(Q61R/Q61L) were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAF(V600E) A375 and NRAS(Q61R) SK-MEL-2 melanoma cell lines. COX-2 expression

  18. Expression of Stem Cell Markers in Primo Vessel of Rat

    Directory of Open Access Journals (Sweden)

    Eun Seok Park

    2013-01-01

    Full Text Available Accumulating line of evidence support that adult tissues contain a rare population of pluripotent stem cells (PSCs, which differentiate into all types of cells in our body. Bonghan microcell (primo microcells (PMCs discovered in 1960s was reported to have a pluripotency like a stem cell in vivo as well as in vitro condition. Here, we describe the detailed morphology and molecular features of PMCs. PMCs reside in Bonghan duct (primo vessel (PV reported as a corresponding structure of acupuncture points and meridian system. We found that PMCs were frequently observed in the liver surface of the rat between 300 g and 400 g from April to June, suggesting that the their detection frequency depends on the weight, the season, and the organ of rat. As reported, PMCs freshly isolated from PVs were spherical ~1-2 μm microsized cells. In contrast, a unique bithread or budding-shaped PMCs emerged during tissue culture around 8 days. RT-PCR analysis demonstrated that PVs-derived cells express the Oct4, the most important PSCs gene, in addition to several PSCs markers (Sox2, Stella, Rex1, and Klf4. Thus, we for the first time provide the evidence about Oct4-expressing stem-like characteristics for cells resident in PVs, a possible novel stem cell enriched niche.

  19. Profiling helper T cell subset gene expression in deer mice

    Directory of Open Access Journals (Sweden)

    Hjelle Brian

    2006-08-01

    Full Text Available Abstract Background Deer mice (Peromyscus maniculatus are the most common mammals in North America and are reservoirs for several zoonotic agents, including Sin Nombre virus (SNV, the principal etiologic agent of hantavirus cardiopulmonary syndrome (HCPS in North America. Unlike human HCPS patients, SNV-infected deer mice show no overt pathological symptoms, despite the presence of virus in the lungs. A neutralizing IgG antibody response occurs, but the virus establishes a persistent infection. Limitations of detailed analysis of deer mouse immune responses to SNV are the lack of reagents and methods for evaluating such responses. Results We developed real-time PCR-based detection assays for several immune-related transcription factor and cytokine genes from deer mice that permit the profiling of CD4+ helper T cells, including markers of Th1 cells (T-bet, STAT4, IFNγ, TNF, LT, Th2 cells (GATA-3, STAT6, IL-4, IL-5 and regulatory T cells (Fox-p3, IL-10, TGFβ1. These assays compare the expression of in vitro antigen-stimulated and unstimulated T cells from individual deer mice. Conclusion We developed molecular methods for profiling immune gene expression in deer mice, including a multiplexed real-time PCR assay for assessing expression of several cytokine and transcription factor genes. These assays should be useful for characterizing the immune responses of experimentally- and naturally-infected deer mice.

  20. Expression of Connexin43 in Rat Epithelial Cells and Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy . And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n = 10) and electrical acupuncture group (EAgroup, n=10). EA ( 0.5-1.5 V, 4-16 Hz , 30 min) was applied to"Zusanli"acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected.The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91 % and 29.53 % respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

  1. Sequential Notch signalling at the boundary of fringe expressing and non-expressing cells.

    Directory of Open Access Journals (Sweden)

    Tobias Troost

    Full Text Available Wing development in Drosophila requires the activation of Wingless (Wg in a small stripe along the boundary of Fringe (Fng expressing and non-expressing cells (FB, which coincides with the dorso-ventral (D/V boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser and Delta (Dl respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, we analysed the function of Fng, Ser and Dl during wing development at an ectopic FB and at the D/V boundary. We find that Notch signalling is initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, our analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB.

  2. Tumor endothelial cells express high pentraxin 3 levels.

    Science.gov (United States)

    Hida, Kyoko; Maishi, Nako; Kawamoto, Taisuke; Akiyama, Kosuke; Ohga, Noritaka; Hida, Yasuhiro; Yamada, Kenji; Hojo, Takayuki; Kikuchi, Hiroshi; Sato, Masumi; Torii, Chisaho; Shinohara, Nobuo; Shindoh, Masanobu

    2016-12-01

    It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy.

  3. Effect of xanthohumol and 8-prenylnaringenin on MCF-7 breast cancer cells oxidative stress and mitochondrial complexes expression.

    Science.gov (United States)

    Blanquer-Rosselló, M Mar; Oliver, Jordi; Valle, Adamo; Roca, Pilar

    2013-12-01

    Xanthohumol (XN) and 8-prenylnaringenin (8PN) are hop (Humulus lupulus L.) polyphenols studied for their chemopreventive effects on certain cancer types. The breast cancer line MCF-7 was treated with doses ranging from 0.001 to 20 µM of XN or 8PN in order to assess the effects on cell viability and oxidative stress. Hoechst 33342 was used to measure cell viability and reactive oxygen species (ROS) production was determined by 2',7'-dichlorofluorescein diacetate. Catalase, superoxide dismutase, and glutathione reductase enzymatic activities were determined and protein expression of sirtuin1, sirtuin3, and oxidative phosphorylation system (OXPHOS) were done by Western blot. Treatments XN 0.01, 8PN 0.01, and 8PN 1 µM led to a decrease in ROS production along with an increase of OXPHOS and sirtuin expression; in contrast, XN 5 µM gave rise to an increase of ROS production accompanied by a decrease in OXPHOS and sirtuin expression. These results suggest that XN in low dose (0.01 µM) and 8PN at all assayed doses (0.001-20 µM) presumably improve mitochondrial function, whereas a high dose of XN (5 µM) worsens the functionality of this organelle.

  4. Hypertonic saline impedes tumor cell-endothelial cell interaction by reducing adhesion molecule and laminin expression.

    LENUS (Irish Health Repository)

    Shields, Conor J

    2012-02-03

    BACKGROUND: Hypertonic saline infusion dampens inflammatory responses and suppresses neutrophil-endothelial interaction by reducing adhesion molecule expression. This study tested the hypothesis that hypertonic saline attenuates tumor cell adhesion to the endothelium through a similar mechanism. METHODS: Human colon cancer cells (LS174T) were transfected with green fluorescent protein and exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 under hypertonic and isotonic conditions for 1 and 4 hours. Confluent human umbilical vein endothelial cells were similarly exposed. Cellular apoptosis and expression of adhesion molecules and laminin were measured by flow cytometry. Tumor cell adhesion to endothelium and laminin was assessed with fluorescence microscopy. Data are represented as mean +\\/- standard error of mean, and an ANOVA test was performed to gauge statistical significance, with P <.05 considered significant. RESULTS: Hypertonic exposure significantly reduced tumor cell adhesion despite the presence of the perioperative cell stressors (42 +\\/- 2.9 vs 172.5 +\\/- 12.4, P <.05), attenuated tumor cell beta-1 integrin (14.43 vs 23.84, P <.05), and endothelial cell laminin expression (22.78 +\\/- 2.2 vs 33.74 +\\/- 2.4, P <.05), but did not significantly alter cell viability. CONCLUSION: Hypertonic saline significantly attenuates tumor cell adhesion to endothelium by inhibiting adhesion molecule and laminin expression. This may halt the metastatic behavior of tumor cells shed at surgery.

  5. [The relationship between the effects of MTA on mRNA expression of four iconic proteins in cells of fetal rat skull and cell culture environment].

    Science.gov (United States)

    Zheng, Jun-Yuan; He, Li; Hu, Tu-qiang

    2016-02-01

    To discuss the influence of MTA on mRNA expression of Cbfa1, ALP, Col-Ⅰand BGP which are 4 kinds of iconic protein in cells of fetal rats skull, and explore its influence on cell culture environment and association of changes of calcium, phosphorus. Cells were obtained by 2 kinds of mixed enzymatic digestion for 3 steps from gestation fetal rat calvarial bone. The expression of Cbfa1mRNA, ALPmRNA, Col-1mRNA, BGP mRNA and extracellular calcium were detected. Phosphorus (P) and calcium concentration of fetal rat skull cells co-cultured with MTA for 3 weeks at different stages of cell differentiation was assessed atomic absorption spectrophotometry. The data was statistically analyzed using SPSS10.0 software package. At the 4th day, P3- content decreased significantly (PMTA group increased most greatly and was 40 times of the control group. At the 14th to 18th day, the Ca2+ and P3- content reduced significantly (PMTA group rised most greatly which was about 7.71 times of the control group. Then Cbfalpha l mRNA in MTA group increased most strongly later which was about 7.38 times of the control group. Col Ⅰ mRNA increased minimally in all time points. The change of P3- content may be the initiating factor when MTA promoted differentiation of fetal rats skull cells in vitro, and Ca2+ could greatly accelerate the process of mineralization when accumulated to a certain extent. At the same time, the expression of ALPmRNA, BGPmRNA, Col Ⅰ mRNA and Cbfalpha lmRNA were regulated accordingly, which is the key to explain osteogenetic mechanism of MTA.

  6. Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

    DEFF Research Database (Denmark)

    Rygaard, K; Vindeløv, L L; Spang-Thomsen, M

    1993-01-01

    A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse....... WE examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc-gene-family...... expression correlated with proliferative parameters. All tumours expressed at least one myc family member at the mRNA level. Exclusive c-myc mRNA expression was demonstrated in 8 tumours, L-myc in 7 and N-myc in I. Five tumours expressed both c-myc and L-myc, and 2 tumours expressed both c-myc and N...

  7. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer.

    Science.gov (United States)

    Zhang, Dingxiao; Park, Daechan; Zhong, Yi; Lu, Yue; Rycaj, Kiera; Gong, Shuai; Chen, Xin; Liu, Xin; Chao, Hsueh-Ping; Whitney, Pamela; Calhoun-Davis, Tammy; Takata, Yoko; Shen, Jianjun; Iyer, Vishwanath R; Tang, Dean G

    2016-02-29

    The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

  8. Expression and enzymatic activity of phenylalanine ammonia-lyase and p-coumarate 3-hydroxylase in mango (Mangifera indica 'Ataulfo') during ripening.

    Science.gov (United States)

    Palafox-Carlos, H; Contreras-Vergara, C A; Muhlia-Almazán, A; Islas-Osuna, M A; González-Aguilar, G A

    2014-05-16

    Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.

  9. Tangeretin induces cell cycle arrest and apoptosis through upregulation of PTEN expression in glioma cells.

    Science.gov (United States)

    Ma, Li-Li; Wang, Da-Wei; Yu, Xu-Dong; Zhou, Yan-Ling

    2016-07-01

    Tangeretin (TANG), present in peel of citrus fruits, has been shown to various medicinal properties such as chemopreventive and neuroprotective. However, the chemopreventive effect of TANG on glioblastoma cells has not been examined. The present study was designed to explore the anticancer potential of TANG in glioblastoma cells and to investigate the related mechanism. Human glioblastoma U-87MG and LN-18 cells were treated with 45μM concentration of TANG and cell growth was measured by MTT assay. The cell cycle distribution and cell death were measured by flow cytometry. The expression of cell cycle and apoptosis related genes were analyzed by quantitative RT-PCR and western blot. The cells treated with TANG were significantly increased cell growth suppression and cell death effects than vehicle treated cells. Further, TANG treatment increases G2/M arrest and apoptosis by modulating PTEN and cell-cycle regulated genes such as cyclin-D and cdc-2 mRNA and protein expressions. Moreover, the ability of TANG to decrease cell growth and to induce cell death was compromised when PTEN was knockdown by siRNA. Taken together, the chemopreventive effect of TANG is associated with regulation of cell-cycle and apoptosis in glioblastoma, thereby attenuating glioblastoma cell growth. Hence, the present findings suggest that TANG may be a therapeutic agent for glioblastoma treatment.

  10. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng, E-mail: oxyccc@163.com

    2015-12-04

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  11. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells

    Science.gov (United States)

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.

    2009-01-01

    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  12. Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.

    Science.gov (United States)

    Amer, Luke D; Holtzinger, Audrey; Keller, Gordon; Mahoney, Melissa J; Bryant, Stephanie J

    2015-08-01

    This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15)μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced under both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (∼ 2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1(+)/Nkx6.1(+) cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates.

  13. Differential expression of the klf6 tumor suppressor gene upon cell damaging treatments in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Gehrau, Ricardo C.; D' Astolfo, Diego S.; Andreoli, Veronica [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Bocco, Jose L., E-mail: jbocco@fcq.unc.edu.ar [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina); Koritschoner, Nicolas P. [Centro de Investigaciones en Bioquimica Clinica e Inmunologia (CIBICI-CONICET), Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Cordoba (Argentina)

    2011-02-10

    The mammalian Krueppel-like factor 6 (KLF6) is involved in critical roles such as growth-related signal transduction, cell proliferation and differentiation, development, apoptosis and angiogenesis. Also, KLF6 appears to be an emerging key factor during cancer development and progression. Its expression is thoroughly regulated by several cell-damaging stimuli. DNA damaging agents at lethal concentrations induce a p53-independent down-regulation of the klf6 gene. To investigate the impact of external stimuli on human klf6 gene expression, its mRNA level was analyzed using a cancer cell line profiling array system, consisting in an assortment of immobilized cDNAs from multiple cell lines treated with several cell-damaging agents at growth inhibitory concentrations (IC{sub 50}). Cell-damaging agents affected the klf6 expression in 62% of the cDNA samples, though the expression pattern was not dependent on the cell origin type. Interestingly, significant differences (p < 0.0001) in KLF6 mRNA levels were observed depending on the cellular p53 status upon cell damage. KLF6 expression was significantly increased in 63% of p53-deficient cells (122/195). Conversely, KLF6 mRNA level decreased nearly 4 fold in more than 70% of p53+/+ cells. In addition, klf6 gene promoter activity was down-regulated by DNA damaging agents in cells expressing the functional p53 protein whereas it was moderately increased in the absence of functional p53. Consistent results were obtained for the endogenous KLF6 protein level. Results indicate that human klf6 gene expression is responsive to external cell damage mediated by IC{sub 50} concentrations of physical and chemical stimuli in a p53-dependent manner. Most of these agents are frequently used in cancer therapy. Induction of klf6 expression in the absence of functional p53 directly correlates with cell death triggered by these compounds, whereas it is down-regulated in p53+/+ cells. Hence, klf6 expression level could represent a valuable

  14. Differential gene expression in stromal cells of human giant cell tumor of bone.

    Science.gov (United States)

    Wuelling, M; Delling, G; Kaiser, E

    2004-12-01

    Giant cell tumor (GCT) offers a unique model for the hematopoietic-stromal cell interaction in human bone marrow. Evidence has been presented that GCT stromal cells (GCTSCs) promote accumulation, size and activity of the giant cells. Although GCTSCs are considered the neoplastic component of GCT, little is known about their genetic basis and, to date, a tumor-specific gene expression pattern has not been characterized. Mesenchymal stem cells (MSCs) have been identified as the origin of the GCT neoplastic stromal cell. Using state of the art array technology, expression profiling was applied to enriched stromal cell populations from five different GCTs and two primary MSCs as controls. Of the 29 differentially expressed genes found, 25 showed an increased expression. Differential mRNA expression was verified by real-time polymerase chain reaction analysis of 10 selected genes, supporting the validity of cDNA arrays as a tool to identify tumor-related genes in GCTSCs. Increased expression of two oncogenes, JUN and NME2, was substantiated at the protein level, utilizing immunohistochemical evaluation of GCT sections and Western-blot analysis. Increased phosphorylation of JUN Ser-63 was also found.

  15. MHC-unrestricted lysis of MUC1-expressing cells by human peripheral blood mononuclear cells.

    Science.gov (United States)

    Wright, Stephen E; Rewers-Felkins, Kathleen A; Quinlin, Imelda S; Fogler, William E; Phillips, Catherine A; Townsend, Mary; Robinson, William; Philip, Ramila

    2008-01-01

    Many human adenocarcinomas can be killed in vitro by targeted cytotoxic T-lymphocytes (CTL); however, major histocompatibility complex (MHC)-restrictions are typically required. The MUC1 antigen is common in many human adenocarcinomas, and is associated with a variable number of tandem repeats. It has been proposed that antigens with such repeated epitopes may be vulnerable to cytotoxic T-lymphocyte killing without MHC-restriction. Therefore, it is possible that MUC1-expressing malignant cells may be killed by targeted cytotoxic T-lymphocyte in the absence of MHC-restriction. In this study, a human MUC1-expressing murine mammary carcinoma cell line was used to determine if cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells requires MHC-restriction. Specifically, MUC1-stimulated human mononuclear cells (M1SMC) were observed to kill human MUC1-transfected, MUC1-expressing murine mammary carcinoma cells, but not the mock-transfected, non-MUC1-expressing murine mammary carcinoma cells. Furthermore, the killing was blocked by antibody to MUC1, indicating MUC1-specific killing. In conclusion, cytotoxic T-lymphocyte killing of MUC1-expressing adenocarcinoma cells can be MHC-unrestricted.

  16. Characterization of cell subpopulations expressing progenitor cell markers in porcine cardiac valves.

    Directory of Open Access Journals (Sweden)

    Huan Wang

    Full Text Available Valvular interstitial cells (VICs are the main population of cells found in cardiac valves. These resident fibroblastic cells play important roles in maintaining proper valve function, and their dysregulation has been linked to disease progression in humans. Despite the critical functions of VICs, their cellular composition is still not well defined for humans and other mammals. Given the limited availability of healthy human valves and the similarity in valve structure and function between humans and pigs, we characterized porcine VICs (pVICs based on expression of cell surface proteins and sorted a specific subpopulation of pVICs to study its functions. We found that small percentages of pVICs express the progenitor cell markers ABCG2 (~5%, NG2 (~5% or SSEA-4 (~7%, whereas another subpopulation (~5% expresses OB-CDH, a type of cadherin expressed by myofibroblasts or osteo-progenitors. pVICs isolated from either aortic or pulmonary valves express most of these protein markers at similar levels. Interestingly, OB-CDH, NG2 and SSEA-4 all label distinct valvular subpopulations relative to each other; however, NG2 and ABCG2 are co-expressed in the same cells. ABCG2(+ cells were further characterized and found to deposit more calcified matrix than ABCG2(- cells upon osteogenic induction, suggesting that they may be involved in the development of osteogenic VICs during valve pathology. Cell profiling based on flow cytometry and functional studies with sorted primary cells provide not only new and quantitative information about the cellular composition of porcine cardiac valves, but also contribute to our understanding of how a subpopulation of valvular cells (ABCG2(+ cells may participate in tissue repair and disease progression.

  17. 15-Lipoxygenase-1 Activates Tumor Suppressor p53 Independent of Enzymatic Activity

    Science.gov (United States)

    Zhu, Hong; Glasgow, Wayne; George, Margaret D.; Chrysovergis, Kali; Olden, Kenneth; Roberts, John D.; Eling, Thomas

    2008-01-01

    15-LOX-1 and its metabolites are involved in colorectal cancer. Recently, we reported that 15-LOX-1 overexpression in HCT-116 human colorectal cancer cells inhibited cell growth by induction of p53 phosphorylation (4). To determine whether the 15-LOX-1 protein or its metabolites are responsible for phosphorylation of p53 in HCT-116 cells, we used HCT-116 cells that expressed a mutant 15-LOX-1. The mutant 15-LOX-1 enzyme, with a substitution of Leu at residue His361, was devoid of enzymatic activity. HCT-116 cells transiently transfected with either native or mutant 15-LOX-1 showed an increase in p53 phosphorylation and an increase in the expression of downstream genes. Thus 15-LOX-1 induces p53 phosphorylation independent of enzymatic activity. Treatment of A549 human lung carcinoma cells with IL-4 increased the expression of 15-LOX-1 and also increased the expression of downstream targets of p53. This confirmed that the activation of p53 was also observed in wild type cells expressing physiological 15-LOX-1. Immunoprecipitation experiments revealed that 15-LOX-1 interacts with, and binds to, DNA-dependent protein kinase (DNA-PK). The binding of 15-LOX-1 to DNA-PK caused an approximate 3.0 fold enhancement in kinase activity, resulting in increased p53 phosphorylation at Ser15. Knockdown of DNA-PK by small interfering RNA (siRNA) significantly reduced p53 phosphorylation. Furthermore, confocal microscopy demonstrated a co-localization of 15-LOX and DNA-PK in the cells. We propose that the 15-LOX-1 protein binds to DNA-PK, increasing its kinase activity, and results in downstream activation of the tumor suppressor p53, thus revealing a new mechanism by which lipoxygenases may influence the phenotype of tumor cells. PMID:18785202

  18. Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyatake, Kazumasa [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Tsuji, Kunikazu, E-mail: ktsuji.gcoe@tmd.ac.jp [International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan); Yamaga, Mika; Yamada, Jun; Matsukura, Yu; Abula, Kahaer [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); Sekiya, Ichiro [Section of Cartilage Regeneration, Tokyo Medical and Dental University, Tokyo (Japan); Muneta, Takeshi [Department of Joint Surgery and Sports Medicine, Tokyo Medical and Dental University, Tokyo (Japan); International Research Center for Molecular Science in Tooth and Bone Diseases (Global Center of Excellence Program), Tokyo Medical and Dental University, Tokyo (Japan)

    2013-02-01

    Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding the molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.

  19. Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: application to breast cancer cells.

    Science.gov (United States)

    Recchi, M A; Harduin-Lepers, A; Boilly-Marer, Y; Verbert, A; Delannoy, P

    1998-01-01

    In many cases of human cancer, the appearance of hypersialylated glycan structures is related to a precise stage of the disease; this may depend on altered regulation of one or more sialyltransferases genes. Since several distinct sialyltransferase enzymes arising from different unique genes transfer sialic acid residues in the same linkage onto the same acceptor, it is impossible to precisely determine which enzyme is involved in the observed phenotype based on enzymatic assays. We have developed a very sensitive and highly reproducible multiplex reverse transcriptase-polymerase chain reaction technique in order to monitor the expression of four human sialyltransferases genes ST6Gal I, ST3Gal I, ST3Gal III and ST3Gal IV in small cell samples. Multiplex PCR amplification using specific primers for each sialyltransferase and detection of amplification products by polyacrylamide gel electrophoresis is a method that is fast and easy to handle and has proven to be useful for establishing sialyltransferase patterns of expression in breast immortalized cell line HBL100 as well as in breast cancer cell lines MCF-7/6, MCF-7/AZ and MDA.

  20. ADAM17 deletion in thymic epithelial cells alters aire expression without affecting T cell developmental progression.

    Directory of Open Access Journals (Sweden)

    David M Gravano

    Full Text Available BACKGROUND: Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach. METHODOLOGY/PRINCIPAL FINDINGS: We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted. CONCLUSIONS/SIGNIFICANCE: In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and

  1. Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells.

    Science.gov (United States)

    Schroeder, Insa S; Sulzbacher, Sabine; Nolden, Tobias; Fuchs, Joerg; Czarnota, Judith; Meisterfeld, Ronny; Himmelbauer, Heinz; Wobus, Anna M

    2012-01-01

    Embryonic stem (ES) cells offer a valuable source for generating insulin-producing cells. However, current differentiation protocols often result in heterogeneous cell populations of various developmental stages. Here we show the activin A-induced differentiation of mouse ES cells carrying a homologous dsRed-IRES-puromycin knock-in within the Sox17 locus into the endoderm lineage. Sox17-expressing cells were selected by fluorescence-assisted cell sorting (FACS) and characterized at the transcript and protein level. Treatment of ES cells with high concentrations of activin A for 10 days resulted in up to 19% Sox17-positive cells selected by FACS. Isolated Sox17-positive cells were characterized by defini- tive endoderm-specific Sox17/Cxcr4/Foxa2 transcripts, but lacked pluripotency-associated Oct4 mRNA and protein. The Sox17-expressing cells showed downregulation of extraembryonic endoderm (Sox7, Afp, Sdf1)-, mesoderm (Foxf1, Meox1)- and ectoderm (Pax6, NeuroD6)-specific transcripts. The presence of Hnf4α, Hes1 and Pdx1 mRNA demonstrated the expression of primitive gut/foregut cell-specific markers. Ngn3, Nkx6.1 and Nkx2.2 transcripts in Sox17-positive cells were determined as properties of pancreatic endocrine progenitors. Immunocytochemistry of activin A-induced Sox17-positive embryoid bodies revealed coexpression of Cxcr4 and Foxa2. Moreover, the histochemical demonstration of E-cadherin-, Cxcr4-, Sox9-, Hnf1β- and Ngn3-positive epithelial-like structures underlined the potential of Sox17-positive cells to further differentiate into the pancreatic lineage. By reducing the heterogeneity of the ES cell progeny, Sox17-expressing cells are a suitable model to evaluate the effects of growth and differentiation factors and of culture conditions to delineate the differentiation process for the generation of pancreatic cells in vitro.

  2. Discrimination of meniscal cell phenotypes using gene expression profiles

    Directory of Open Access Journals (Sweden)

    M Son

    2012-03-01

    Full Text Available The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus. The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

  3. Performance analysis of Pluto/Express, multitube AMTEC cells

    Energy Technology Data Exchange (ETDEWEB)

    Tournier, J.-M.; El-Genk, M.S. [University of New Mexico, Albuquerque, NM (United States). Institute for Space and Nuclear Power Studies

    1999-10-01

    An AMTEC performance and evaluation analysis model (APEAM) was developed to predict the performance of next-generation Pluto/Express, multitube, vapor-anode alkali-metal thermal-to-electric conversion (AMTEC) cells. The model results compared well with the experimental data of PX-4C, PX-5A and PX-3A cells, tested at the Air Force Research Laboratory. The effects of various design changes on the performance of the cells were investigated. These changes were: (1) using a Creare condenser; (b) changing the number of BASE tubes and the electrode length; (c) using other electrode materials; (d) using molybdenum (Mo) structure on the hot side of the cell: and (e) using reflective rhodium coatings in the low vapor pressure cavity of the cell. The results showed that a stainless steel (SS) structure cell with improved electrodes (50% lower contact resistance than TiN, and an exchange current coefficient = 200 A.K{sup 1/2}/Pa.m{sup 2}) could deliver 7.1 We at 3.5 V and an efficiency of 19.5%. When Mo was substituted for the SS on the hot side of the cell, the electrical power output increased to 8.8 We, and the cell efficiency increased to 20.2%. Using rhodium coatings in the Mo/SS cell increased the electrical power output to 9.7 We and the conversion efficiency to 22.5%. (author)

  4. Expression of cubilin in mouse testes and Leydig cells.

    Science.gov (United States)

    Oh, Y S; Seo, J T; Ahn, H S; Gye, M C

    2016-04-01

    Cubilin (cubn) is a receptor for vitamins and various protein ligands. Cubn lacks a transmembrane domain but anchors to apical membranes by forming complexes with Amnionless or Megalin. In an effort to better understand the uptake of nutrients in testis, we analysed cubn expression in the developing mice testes. In testes, cubn mRNA increased from birth to adulthood. In the inter-stitium and isolated seminiferous tubules, neonatal increase in cubn mRNA until 14 days post-partum (pp) was followed by a marked increase at puberty (28 days pp). Cubn was found in the gonocytes, spermatogonia, spermatocytes and spermatids in the developing testes. In adult testes, strong Cubn immunoreactivity was found in the elongating spermatids, suggesting the role of Cubn in endocytosis during early spermiogenesis. In Sertoli cells and peritubular cells, Cubn immunoreactivity was weak throughout the testis development. In the inter-stitium, Cubn immunoreactivity was found in foetal Leydig cells, was weak to negligible in the stem cells and progenitor Leydig cells and was strong in immature and adult Leydig cells, demonstrating a positive association between Cubn and steroidogenic activity of Leydig cells. Collectively, these results suggest that Cubn may participate in the endocytotic uptake of nutrients in germ cells and somatic cells, supporting the spermatogenesis and steroidogenesis in mouse testes.

  5. Cervical cancer cells with positive Sox2 expression exhibit the properties of cancer stem cells.

    Directory of Open Access Journals (Sweden)

    Xiao-Fang Liu

    Full Text Available BACKGROUND: Although Sox2 expression has been found in several types of cancer, it has not yet been used to identify or isolate CSCs in somatic carcinoma. METHODS: SiHa and C33A cells stably transfected with a plasmid containing human Sox2 transcriptional elements driving the enhanced green fluorescent protein (EGFP reporter were sorted into the Sox2-positive and the Sox2-negative populations by FACS, and Sox2 expression was detected by western blot and immunohistochemistry. The differentiation, self-renewal and tumor formation abilities, as well as the expression of the stemness and the EMT related genes of the Sox2-positive and the Sox2-negative cervical cancer cells were characterized in vitro and in vivo. RESULTS: A pSox2/EGFP system was used to separate the Sox2-positive and the Sox2-negative cells from cervical cancer cell lines, SiHa and C33A cells. Compared with the Sox2-negative cells, the Sox2-positive SiHa and C33A cells exhibited greater capacities for self-renewal, differentiation and tumor formation. Furthermore, Sox2-positive SiHa and C33A cells expressed higher levels of stemness-related genes, such as Sox2/Bmi-1/Oct4/ALDH1, and EMT-related genes, such as vimentin/snail/β-catenin. Taken together, all these results indicated that cells expressing endogenous Sox2 are CSCs in cervical carcinomas. CONCLUSION: This study is the first to establish a functional link between endogenous Sox2 expression and CSCs in cervical carcinomas. Additionally, this study demonstrated that it is feasible to develop a tool to isolate CSCs from somatic tumors based on the expression of the endogenous nuclear protein Sox2 instead of cell surface markers.

  6. Chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells.

    Science.gov (United States)

    Funakoshi, Tadanao; Spector, Myron

    2010-06-01

    Reparative strategies for the treatment of injuries to tendons, including those of the rotator cuff of the shoulder, need to address the formation of the cartilage which serves as the attachment apparatus to bone and which forms at regions undergoing compressive loading. Moreover, recent work indicates that cells employed for rotator cuff repair may need to synthesize a lubricating glycoprotein, lubricin, which has recently been found to play a role in tendon tribology. The objective of the present study was to investigate the chondrogenic differentiation and lubricin expression of caprine infraspinatus tendon cells in monolayer and three-dimensional culture, and to compare the behavior with bone marrow-derived mesenchymal stem cells (MSCs). The results demonstrated that while tendon cells in various media, including chondrogenic medium, expressed lubricin, virtually none of the MSCs synthesized this important lubricating molecule. Also of interest was that the cartilage formation capacity of the tendon cells grown in pellet culture in chondrogenic medium was comparable with MSCs. These data inform the use of tendon cells for rotator cuff repair, including for fibrocartilaginous zones.

  7. Expression of activated Ras during Dictyostelium development alters cell localization and changes cell fate.

    Science.gov (United States)

    Jaffer, Z M; Khosla, M; Spiegelman, G B; Weeks, G

    2001-03-01

    There is now a body of evidence to indicate that Ras proteins play important roles in development. Dictyostelium expresses several ras genes and each appears to perform a distinct function. Previous data had indicated that the overexpression of an activated form of the major developmentally regulated gene, rasD, caused a major aberration in morphogenesis and cell type determination. We now show that the developmental expression of an activated rasG gene under the control of the rasD promoter causes a similar defect. Our results indicate that the expression of activated rasG in prespore cells results in their transdifferentiation into prestalk cells, whereas activated rasG expression in prestalk causes gross mislocalization of the prestalk cell populations.

  8. Paired Expression Analysis of Tumor Cell Surface Antigens

    Directory of Open Access Journals (Sweden)

    Rimas J. Orentas

    2017-08-01

    Full Text Available Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19 or antibody-based therapy (anti-CD20 in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling’s T-squared test (Hotelling–Lawley multivariate analysis of variance for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues. We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK

  9. Expression of basement membrane antigens in spindle cell melanoma.

    Science.gov (United States)

    Prieto, V G; Woodruff, J M

    1998-07-01

    Spindle cell melanoma (SCM) is an uncommon form of melanoma that may be confused histologically with other tumors, including malignant peripheral nerve sheath tumors (MPNST). Tumors with neural differentiation and melanocytic nevi may both show basement membrane immunohistochemically and at the ultrastructural level. However, most ultrastructural studies of melanoma have failed to demonstrate well formed basement membrane around tumor cells. The presence of basement membrane has been used by some authors as evidence favoring MPNST, as opposed to SCM. To evaluate this distinction immunohistochemically, 22 primary and metastatic cutaneous melanomas having a spindle cell component (SCM) were studied using monoclonal antibodies against laminin and Type IV collagen. S100 protein and HMB45 antigen expression were also studied. All but one of the SCM were reactive for S100 protein in at least 25% of the cells. Thirteen of 20 tumors (65%) were focally reactive with HMB45. Laminin was expressed in 42% of the tumors (only membranous pattern in 3; cytoplasmic and membranous in 5). Seventeen tumors (77%) expressed type IV collagen (only membranous pattern in 7; cytoplasmic and membranous pattern in 10). Laminin and type IV collagen, known components of basement membrane, are often found in SCM. Therefore, their detection cannot be used to distinguish SCM from MPNST.

  10. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    Science.gov (United States)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  11. Arsenic Trioxide Inhibits Proliferation in K562 Cells by Changing Cell Cycle and Survivin Expression

    Institute of Scientific and Technical Information of China (English)

    伍晓菲; 陈智超; 刘仲萍; 周浩; 游泳; 黎纬明; 邹萍

    2004-01-01

    To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As2O3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As2O3 induced cell apoptosis, K562 cells were cultured with As2O3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry.Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As2O3 (2-10 μmol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G2/M phase increased in proportion to As2O3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As2 O3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As2O3-induced apoptosis.

  12. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    Science.gov (United States)

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

  13. Enzymatic Modification of Sphingomyelin

    DEFF Research Database (Denmark)

    Zhang, Long; Hellgren, Lars; Xu, Xuebing

    Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. Currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost......-efficient, high yield production methods are of great interest. In the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin have been studied. Sphingomyelin (SM) is a ubiquitous membrane-lipid and dairy products or by-products is a rich source of sphingomyelin....... In present study, we have optimized the production of ceramide from sphingomyelin using Phospholipase C from Clostridium perfringens....

  14. Enzymatic cascade bioreactor

    Science.gov (United States)

    Simmons, Blake A.; Volponi, Joanne V.; Ingersoll, David; Walker, Andrew

    2007-09-04

    Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

  15. Mast cells express novel functional IL-15 receptor alpha isoforms.

    Science.gov (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Krause, Hans; Paus, Ralf; Bulfone-Paus, Silvia

    2003-05-15

    Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

  16. Binary gene induction and protein expression in individual cells

    Directory of Open Access Journals (Sweden)

    Conolly Rory B

    2006-04-01

    Full Text Available Abstract Background Eukaryotic gene transcription is believed to occur in either a binary or a graded fashion. With binary induction, a transcription activator (TA regulates the probability with which a gene template is switched from the inactive to the active state without affecting the rate at which RNA molecules are produced from the template. With graded, also called rheostat-like, induction the gene template has continuously varying levels of transcriptional activity, and the TA regulates the rate of RNA production. Support for each of these two mechanisms arises primarily from experimental studies measuring reporter proteins in individual cells, rather than from direct measurement of induction events at the gene template. Methods and results In this paper, using a computational model of stochastic gene expression, we have studied the biological and experimental conditions under which a binary induction mode operating at the gene template can give rise to differentially expressed "phenotypes" (i.e., binary, hybrid or graded at the protein level. We have also investigated whether the choice of reporter genes plays a significant role in determining the observed protein expression patterns in individual cells, given the diverse properties of commonly-used reporter genes. Our simulation confirmed early findings that the lifetimes of active/inactive promoters and half-lives of downstream mRNA/protein products are important determinants of various protein expression patterns, but showed that the induction time and the sensitivity with which the expressed genes are detected are also important experimental variables. Using parameter conditions representative of reporter genes including green fluorescence protein (GFP and β-galactosidase, we also demonstrated that graded gene expression is more likely to be observed with GFP, a longer-lived protein with low detection sensitivity. Conclusion The choice of reporter genes may determine whether protein

  17. Transcriptional expression of genes involved in cell invasion and migration by normal and tumoral trophoblast cells.

    Science.gov (United States)

    Janneau, Jean-Louis; Maldonado-Estrada, Juan; Tachdjian, Gérard; Miran, Isabelle; Motté, Nelly; Saulnier, Patrick; Sabourin, Jean-Christophe; Coté, Jean-François; Simon, Bénédicte; Frydman, René; Chaouat, Gérard; Bellet, Dominique

    2002-11-01

    Once initiated, invasion of trophoblast cells must be tightly regulated, particularly in early pregnancy. The mechanisms necessary for the invasion and migration of trophoblast cells are thought to be related to those involved in the invasive and metastatic properties of cancer cells. Quantitative PCR was used to measure, in trophoblast cells, the transcriptional expression profiles of four genes, INSL4, BRMS1, KiSS-1 and KiSS-1R, reported to be implicated in tumor invasion and metastasis. Laser capture microdissection and purification of trophoblast cells demonstrate that, as already known for INSL4, BRMS1, KiSS-1 and KiSS-1R are expressed by the trophoblast subset of placental tissues. Expression profiles of these genes studied in early placentas (7-9 weeks, n=55) and term placentas (n=11) showed that expression levels of BRMS1 are higher in term than in early placentas, while expression levels of KiSS-1R are higher in early than in term placentas. Low levels of expression of BRMS1 were observed in normal pregnancies, in molar pregnancies and in choriocarcinoma cell lines BeWo, JAR and JEG3 while, in striking contrast, the expression levels of INSL4, KiSS-1 and Kiss-1R were increased in both early placentas and molar pregnancies and were reduced in choriocarcinoma cells. These transcriptional expression profiles are in favor of a predominant role of INSL4, KiSS-1 and KiSS-1R in the control of the invasive and migratory properties of trophoblast cells.

  18. Characterization of protocadherin-1 expression in primary bronchial epithelial cells : association with epithelial cell differentiation

    NARCIS (Netherlands)

    Koning, Henk; Sayers, Ian; Stewart, Ceri E.; de Jong, Debora; ten Hacken, Nick H. T.; Postma, Dirkje S.; van Oosterhout, Antoon J. M.; Nawijn, Martijn C.; Koppelman, Gerard H.

    2012-01-01

    Protocadherin-1 (PCDH1) is a novel susceptibility gene for asthma that is expressed in airway epithelium. We aimed to characterize PCDH1 mRNA transcripts and protein expression in primary bronchial epithelial cells and to determine regulation of PCDH1 during mucociliary differentiation. Total RNA an

  19. Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma.

    Science.gov (United States)

    Kremer, Veronika; Ligtenberg, Maarten; Zendehdel, Rosa; Seitz, Christina; Duivenvoorden, Annet; Wennerberg, Erik; Colón, Eugenia; Scherman-Plogell, Ann-Helén; Lundqvist, Andreas

    2017-09-19

    Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer. Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays. We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells. To increase the success of NK

  20. Phytoestrogens regulate the proliferation and expression of stem cell factors in cell lines of malignant testicular germ cell tumors.

    Science.gov (United States)

    Hasibeder, Astrid; Venkataramani, Vivek; Thelen, Paul; Radzun, Heinz-Joachim; Schweyer, Stefan

    2013-11-01

    Phytoestrogens have been shown to exert anti-proliferative effects on different cancer cells. In addition it could be demonstrated that inhibition of proliferation is associated with downregulation of the known stem cell factors NANOG, POU5F1 and SOX2 in tumor cells. We demonstrate the potential of Belamcanda chinensis extract (BCE) and tectorigenin as anticancer drugs in cell lines of malignant testicular germ cell tumor cells (TGCT) by inhibition of proliferation and regulating the expression of stem cell factors. The TGCT cell lines TCam-2 and NTera-2 were treated with BCE or tectorigenin and MTT assay was used to measure the proliferation of tumor cells. In addition, the expression of stem cell factors was analyzed by quantitative PCR and western blot analysis. Furthermore, global expression analysis was performed by microarray technique. BCE and tectorigenin inhibited proliferation and downregulated the stem cell factors NANOG and POU5F1 in TGCT cells. In addition, gene expression profiling revealed induction of genes important for the differentiation and inhibition of oncogenes. Utilizing connectivity map in an attempt to elucidate mechanism underlying BCE treatments we found highly positive association to histone deacetylase inhibitors (HDACi) amongst others. Causing no histone deacetylase inhibition, the effects of BCE on proliferation and stem cell factors may be based on histone-independent mechanisms such as direct hyperacetylation of transcription factors. Based on these findings, phytoestrogens may be useful as new agents in the treatment of TGCT.

  1. Osteopontin Upregulates the Expression of Glucose Transporters in Osteosarcoma Cells

    Science.gov (United States)

    Hsieh, I-Shan; Yang, Rong-Sen; Fu, Wen-Mei

    2014-01-01

    Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients. PMID:25310823

  2. Inducible T-cell receptor expression in precursor T-cells for leukemia control

    Science.gov (United States)

    Hoseini, Shahabuddin S; Hapke, Martin; Herbst, Jessica; Wedekind, Dirk; Baumann, Rolf; Heinz, Niels; Schiedlmeier, Bernhard; Vignali, Dario AA; van den Brink, Marcel R.M.; Schambach, Axel; Blazar, Bruce R.; Sauer, Martin G.

    2015-01-01

    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. Since expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T cell development, was required to obtain a mature T cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity. PMID:25652739

  3. A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products.

    Science.gov (United States)

    Cyr, T; Menzies, A J; Calver, J; Whitehouse, L W

    2001-06-01

    The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that

  4. A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

    Science.gov (United States)

    Adachi, Daisuke; Koh, FookHee; Hama, Shinji; Ogino, Chiaki; Kondo, Akihiko

    2013-05-10

    To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.

  5. Gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells

    Institute of Scientific and Technical Information of China (English)

    胡庆柳; 朴英杰; 邹飞

    2003-01-01

    Objective To study the gene expression profiles of human bone marrow derived mesenchymal stem cells and tendon cells.Methods Total RNA extracted from human bone marrow derived mesenchymal stem cells and tendon cells underwent reverse transcription, and the products were labeled with α-32P dCTP. The cDNA probes of total RNA were hybridized to cDNA microarray with 1176 genes, and then the signals were analyzed by AtlasImage analysis software Version 1.01a.Results Fifteen genes associated with cell proliferation and signal transduction were up-regulated, and one gene that takes part in cell-to-cell adhesion was down-regulated in tendon cells.Conclusion The 15 up-regulated and one down-regulated genes may be beneficial to the orientational differentiation of mesenchymal stem cells into tendon cells.

  6. Migrating glioma cells express stem cell markers and give rise to new tumors upon xenografting

    DEFF Research Database (Denmark)

    Munthe, Sune; Sørensen, Mia D; Thomassen, Mads

    2016-01-01

    -related genes and the HOX-gene list in migrating cells compared to spheroids. Determination of GBM molecular subtypes revealed that subtypes of spheroids and migrating cells were identical. In conclusion, migrating tumor cells preserve expression of stem cell markers and functional CSC characteristics. Since......Glioblastoma (GBM) is the most frequent and malignant brain tumor with an overall survival of only 14.6 months. Although these tumors are treated with surgery, radiation and chemotherapy, recurrence is inevitable. A critical population of tumor cells in terms of therapy, the so-called cancer stem...... cells (CSCs), has been identified in gliomas and many other cancers. These tumor cells have a stem cell-like phenotype and are suggested to be responsible for tumor growth, chemo- and radio-resistance as well as recurrence. However, functional evidence for migrating glioma cells having a stem cell...

  7. Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

    Institute of Scientific and Technical Information of China (English)

    Claudia Lange; Philipp Bassler; Michael V. Lioznov; Helge Bruns; Dietrich Kluth; Axel R. Zander; Henning C. Fiegel

    2005-01-01

    AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore,we assessed the influence of cocultured liver cells on induction of liver-specific gene expression.METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF,EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19,and AFP was performed in the different cell populations.RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18,CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression.CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.

  8. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cells.

    Science.gov (United States)

    Yang, Chao; Ji, Lei; Yue, Wen; Shi, Shuang-Shuang; Wang, Ruo-Yong; Li, Yan-Hua; Xie, Xiao-Yan; Xi, Jia-Fei; He, Li-Juan; Nan, Xue; Pei, Xue-Tao

    2012-02-01

    Blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important methods for cell therapy. They are widely used in the treatment of incurable hematological disorder, infectious diseases, genetic diseases, and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. Recently, human embryonic stem cells (hESCs) have been shown to be an alternative resource for the generation of hematopoietic cells. In the current study, we describe a novel method for the efficient production of hematopoietic cells from hESCs. The stable human fetal liver stromal cell lines (hFLSCs) expressing erythropoietin (EPO) were established using the lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce the hESCs differentiation into hematopoietic cells, especially erythroid cells. They not only expressed fetal and embryonic globins but also expressed the adult-globin chain on further maturation. In addition, these hESCs-derived erythroid cells possess oxygen-transporting capacity, which indicated hESCs could generate terminally mature progenies. This should be useful for ultimately developing an animal-free culture system to generate large numbers of erythroid cells from hESCs and provide an experimental model to study early human erythropoiesis.

  9. Induction of T Cell Development In Vitro by Delta-Like (Dll)-Expressing Stromal Cells.

    Science.gov (United States)

    Mohtashami, Mahmood; Zarin, Payam; Zúñiga-Pflücker, Juan Carlos

    2016-01-01

    Recreating the thymic microenvironment in vitro poses a great challenge to immunologists. Until recently, the only approach was to utilize the thymic tissue in its three-dimensional form and to transfer the hematopoietic progenitors into this tissue to generate de novo T cells. With the advent of OP9-DL cells (bone marrow-derived cells that are transduced to express Notch ligand, Delta-like), hematopoietic stem cells (HSC) could be induced to differentiate into T cells in culture for the first time outside of the thymic tissue on a monolayer. We, as well as others, asked whether the ability to support T cell development in vitro in a monolayer is unique to BM-derived OP9 cells, and showed that provision of Delta-like expression to thymic epithelial cells and fibroblasts also allowed for T cell development. This provides the opportunity to design an autologous coculture system where the supportive stromal and the hematopoietic components are both derived from the same individual, which has obvious clinical implications. In this chapter, we describe methods for establishing a primary murine dermal fibroblast cell population that is transduced to express Delta-like 4, and describe the conditions for its coculture with HSCs to support T cell lineage initiation and expansion, while comparing it to the now classic OP9-DL coculture.

  10. Constitutive expression of IL-18 and IL-18R in differentiated IEC-6 cells: effect of TNF-alpha and IFN-gamma treatment.

    Science.gov (United States)

    Kolinska, Jirina; Lisa, Vera; Clark, Jessica A; Kozakova, Hana; Zakostelecka, Marie; Khailova, Ludmila; Sinkora, Marek; Kitanovicova, Andrea; Dvorak, Bohuslav

    2008-05-01

    The multifunctional cytokine interleukin-18 (IL-18) is an important mediator in intestinal inflammatory processes. The aim of this study was to evaluate the constitutive expression of IL-18 and its receptors (IL-18Ralpha and IL-18Rbeta) in intestinal epithelial cells (IEC) stimulated by tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In addition, cellular proliferation and evaluation of brush border enzymes as differentiation markers were studied. Nontransformed rat intestinal epithelial IEC-6 cells were grown on an extracellular matrix (ECM) in medium with or without TNF-alpha, IFN-gamma, or a combination of both. Gene expression of IL-18, its receptors and apoptotic markers was evaluated using real-time PCR. Expression of IL-18Ralpha protein was demonstrated by flow cytometry and Western blot. Enzymatic activities of brush border enzymes and caspase-1 were determined. The constitutive expression of IL-18, IL-18Ralpha and IL-18Rbeta mRNAs and proteins were detected in IEC-6 cells. The biologically active form of IL-18 was released in response to TNF-alpha and IFN-gamma treatment. Exogenous IL-18 had no effect on cellular proliferation, bru