Engineering Novel and Improved Biocatalysts by Cell Surface Display

PubMed Central

Smith, Mason R.; Khera, Eshita; Wen, Fei

2017-01-01

Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts. PMID:29056821

Biological Fuel Cells and Membranes.

PubMed

Ghassemi, Zahra; Slaughter, Gymama

2017-01-17

Biofuel cells have been widely used to generate bioelectricity. Early biofuel cells employ a semi-permeable membrane to separate the anodic and cathodic compartments. The impact of different membrane materials and compositions has also been explored. Some membrane materials are employed strictly as membrane separators, while some have gained significant attention in the immobilization of enzymes or microorganisms within or behind the membrane at the electrode surface. The membrane material affects the transfer rate of the chemical species (e.g., fuel, oxygen molecules, and products) involved in the chemical reaction, which in turn has an impact on the performance of the biofuel cell. For enzymatic biofuel cells, Nafion, modified Nafion, and chitosan membranes have been used widely and continue to hold great promise in the long-term stability of enzymes and microorganisms encapsulated within them. This article provides a review of the most widely used membrane materials in the development of enzymatic and microbial biofuel cells.

Biological Fuel Cells and Membranes

PubMed Central

Ghassemi, Zahra; Slaughter, Gymama

2017-01-01

Biofuel cells have been widely used to generate bioelectricity. Early biofuel cells employ a semi-permeable membrane to separate the anodic and cathodic compartments. The impact of different membrane materials and compositions has also been explored. Some membrane materials are employed strictly as membrane separators, while some have gained significant attention in the immobilization of enzymes or microorganisms within or behind the membrane at the electrode surface. The membrane material affects the transfer rate of the chemical species (e.g., fuel, oxygen molecules, and products) involved in the chemical reaction, which in turn has an impact on the performance of the biofuel cell. For enzymatic biofuel cells, Nafion, modified Nafion, and chitosan membranes have been used widely and continue to hold great promise in the long-term stability of enzymes and microorganisms encapsulated within them. This article provides a review of the most widely used membrane materials in the development of enzymatic and microbial biofuel cells. PMID:28106711

Monolignol 4-O-methyltransferases and uses thereof

DOEpatents

Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

2014-11-18

Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

Heparan sulfate proteoglycans undergo differential expression alterations in right sided colorectal cancer, depending on their metastatic character.

PubMed

Fernández-Vega, Iván; García-Suárez, Olivia; García, Beatriz; Crespo, Ainara; Astudillo, Aurora; Quirós, Luis M

2015-10-20

Heparan sulfate proteoglycans (HSPGs) are complex molecules involved in the growth, invasion and metastatic properties of cancerous cells. This study analyses the alterations in the expression patterns of these molecules in right sided colorectal cancer (CRC), both metastatic and non-metastatic. Twenty right sided CRCs were studied. A transcriptomic approach was used, employing qPCR to analyze both the expression of the enzymes involved in heparan sulfate (HS) chains biosynthesis, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate (CS) chains, we include the study of the genes involved in the biosynthesis of these glycosaminoglycans. Immunohistochemical techniques were also used to analyze tissue expression of particular genes showing significant expression differences, of potential interest. Changes in proteoglycan core proteins differ depending on their location; those located intracellularly or in the extracellular matrix show very similar alteration patterns, while those located on the cell surface vary greatly depending on the nature of the tumor: glypicans 1, 3, 6 and betaglycan are affected in the non-metastatic tumors, whereas in the metastatic, only glypican-1 and syndecan-1 are modified, the latter showing opposing alterations in levels of RNA and of protein, suggesting post-transcriptional regulation in these tumors. Furthermore, in non-metastatic tumors, polymerization of glycosaminoglycan chains is modified, particularly affecting the synthesis of the tetrasaccharide linker and the initiation and elongation of CS chains, HS chains being less affected. Regarding the enzymes responsible for the modificaton of the HS chains, alterations were only found in non-metastatic tumors, affecting N-sulfation and the isoforms HS6ST1, HS3ST3B and HS3ST5. In contrast, synthesis of the CS chains suggests changes in epimerization and sulfation of the C4 and C2 in both types of tumor. Right sided CRCs show alterations in the expression of HSPGs, including the expression of the cell surface core proteins, many glycosiltransferases and some enzymes that modify the HS chains depending on the metastatic nature of the tumor, resulting more affected in non-metastatic ones. However, matrix proteoglycans and enzymes involved in CS fine structure synthesis are extensively modified independetly of the presence of lymph node metastasis.

Enhanced cell adhesion on bioinert ceramics mediated by the osteogenic cell membrane enzyme alkaline phosphatase.

PubMed

Aminian, Alieh; Shirzadi, Bahareh; Azizi, Zahra; Maedler, Kathrin; Volkmann, Eike; Hildebrand, Nils; Maas, Michael; Treccani, Laura; Rezwan, Kurosch

2016-12-01

Functional bone and dental implant materials are required to guide cell response, offering cues that provide specific instructions to cells at the implant/tissue interface while maintaining full biocompatibility as well as the desired structural requirements and functions. In this work we investigate the influence of covalently immobilized alkaline phosphatase (ALP), an enzyme involved in bone mineralization, on the first contact and initial cell adhesion. To this end, ALP is covalently immobilized by carbodiimide-mediated chemoligation on two highly bioinert ceramics, alpha-alumina (Al2O3) and yttria-stabilized zirconia (Y-TZP) that are well-established for load-bearing applications. The physicochemical surface properties are evaluated by profilometry, zeta potential and water contact angle measurements. The initial cell adhesion of human osteoblasts (HOBs), human osteoblast-like cells (MG-63) and mesenchymal stromal cells (hMSCs) was investigated. Cell adhesion was assessed at serum free condition via quantification of percentage of adherent cells, adhesion area and staining of the focal adhesion protein vinculin. Our findings show that after ALP immobilization, the Al2O3 and Y-TZP surfaces gained a negative charge and their hydrophilicity was increased. In the presence of surface-immobilized ALP, a higher cell adhesion, more pronounced cell spreading and a higher number of focal contact points were found. Thereby, this work gives evidence that surface functionalization with ALP can be utilized to modify inert materials for biological conversion and faster bone regeneration on inert and potentially load-bearing implant materials. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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.

Preparation of immobilized glucose oxidase wafer enzyme on calcium-bentonite modified by surfactant

NASA Astrophysics Data System (ADS)

Widi, R. K.; Trisulo, D. C.; Budhyantoro, A.; Chrisnasari, R.

2017-07-01

Wafer glucose oxidase (GOx) enzymes was produced by addition of PAH (Poly-Allyamine Hydrochloride) polymer into immobilized GOx enzyme on modified-Tetramethylammonium Hydroxide (TMAH) 5%-calsium-bentonite. The use of surfactant molecul (TMAH) is to modify the surface properties and pore size distribution of the Ca-bentonite. These properties are very important to ensure GOx molecules can be bound on the Ca-bentonit surface to be immobilized. The addition of the polymer (PAH) is expected to lead the substrates to be adsorbed onto the enzyme. In this study, wafer enzymes were made in various concentration ratio (Ca-bentonite : PAH) which are 1:0, 1:1, 1:2 and 1:3. The effect of PAH (Poly-Allyamine Hydrochloride) polymer added with various ratios of concentrations can be shown from the capacitance value on LCR meter and enzyme activity using DNS method. The addition of the polymer (PAH) showed effect on the activity of GOx, it can be shown from the decreasing of capacitance value by increasing of PAH concentration.

Ferrocene-Modified Linear Poly(ethylenimine) for Enzymatic Immobilization and Electron Mediation.

PubMed

Hickey, David P

2017-01-01

Enzymatic glucose biosensors and biofuel cells make use of the electrochemical transduction between an oxidoreductase enzyme, such as glucose oxidase (GOx), and an electrode to either quantify the amount of glucose in a solution or generate electrical energy. However, many enzymes including GOx are not able to electrochemically interact with an electrode surface directly, but require an external electrochemical relay to shuttle electrons to the electrode. Ferrocene-modified linear poly(ethylenimine) (Fc-LPEI) redox polymers have been designed to simultaneously immobilize glucose oxidase (GOx) at an electrode and mediate electron transfer from their flavin adenine dinucleotide (FAD) active site to the electrode surface. Cross-linked films of Fc-LPEI create hydrogel networks that allow for rapid transport of glucose, while the covalently bound ferrocene moieties are able to facilitate rapid electron transfer due to the ability of ferrocene to exchange electrons between adjacent ferrocene residues. For these reasons, Fc-LPEI films have been widely used in the development of high current density bioanode materials. This chapter describes the synthesis of a commonly used dimethylferrocene-modified linear poly(ethylenimine), as well as the subsequent preparation and electrochemical characterization of a GOx bioanode film utilizing the synthesized polymer.

Redesigning of Microbial Cell Surface and Its Application to Whole-Cell Biocatalysis and Biosensors.

PubMed

Han, Lei; Zhao, Yukun; Cui, Shan; Liang, Bo

2018-06-01

Microbial cell surface display technology can redesign cell surfaces with functional proteins and peptides to endow cells some unique features. Foreign peptides or proteins are transported out of cells and immobilized on cell surface by fusing with anchoring proteins, which is an effective solution to avoid substance transfer limitation, enzyme purification, and enzyme instability. As the most frequently used prokaryotic and eukaryotic protein surface display system, bacterial and yeast surface display systems have been widely applied in vaccine, biocatalysis, biosensor, bioadsorption, and polypeptide library screening. In this review of bacterial and yeast surface display systems, different cell surface display mechanisms and their applications in biocatalysis as well as biosensors are described with their strengths and shortcomings. In addition to single enzyme display systems, multi-enzyme co-display systems are presented here. Finally, future developments based on our and other previous reports are discussed.

Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

PubMed

Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

2015-01-01

We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

SIALIC ACIDS AND AUTOIMMUNE DISEASE

PubMed Central

Mahajan, Vinay S.; Pillai, Shiv

2016-01-01

summary An important underlying mechanism that contributes to autoimmunity is the loss of inhibitory signaling in the immune system. Sialic acid-recognizing Ig superfamily lectins or Siglecs are a family of cell surface proteins largely expressed in hematopoietic cells. The majority of Siglecs are inhibitory receptors expressed in immune cells that bind to sialic acid containing ligands and recruit SH2-domain containing tyrosine phosphatases to their cytoplasmic tails. They deliver inhibitory signals that can contribute to the constraining of immune cells and thus protect the host from autoimmunity. The inhibitory functions of CD22/Siglec-2 and Siglec-G and their contributions to tolerance and autoimmunity, primarily in the B lymphocyte context, are considered in some detail in this review. The relevance to autoimmunity and unregulated inflammation of modified sialic acids, enzymes that modify sialic acid, and other sialic acid binding proteins are also reviewed. PMID:26683151

Gold-Coated M13 Bacteriophage as a Template for Glucose Oxidase Biofuel Cells with Direct Electron Transfer.

PubMed

Blaik, Rita A; Lan, Esther; Huang, Yu; Dunn, Bruce

2016-01-26

Glucose oxidase-based biofuel cells are a promising source of alternative energy for small device applications, but still face the challenge of achieving robust electrical contact between the redox enzymes and the current collector. This paper reports on the design of an electrode consisting of glucose oxidase covalently attached to gold nanoparticles that are assembled onto a genetically engineered M13 bacteriophage using EDC-NHS chemistry. The engineered phage is modified at the pIII protein to attach onto a gold substrate and serves as a high-surface-area template. The resulting "nanomesh" architecture exhibits direct electron transfer (DET) and achieves a higher peak current per unit area of 1.2 mA/cm(2) compared to most other DET attachment schemes. The final enzyme surface coverage on the electrode was calculated to be approximately 4.74 × 10(-8) mol/cm(2), which is a significant improvement over most current glucose oxidase (GOx) DET attachment methods.

Evidence of short-range electron transfer of a redox enzyme on graphene oxide electrodes.

PubMed

Martins, Marccus V A; Pereira, Andressa R; Luz, Roberto A S; Iost, Rodrigo M; Crespilho, Frank N

2014-09-07

Direct electron transfer (DET) between redox enzymes and electrode surfaces is of growing interest and an important strategy in the development of biofuel cells and biosensors. Among the nanomaterials utilized at electrode/enzyme interfaces to enhance the electronic communication, graphene oxide (GO) has been identified as a highly promising candidate. It is postulated that GO layers decrease the distance between the flavin cofactor (FAD/FADH2) of the glucose oxidase enzyme (GOx) and the electrode surface, though experimental evidence concerning the distance dependence of the rate constant for heterogeneous electron-transfer (k(het)) has not yet been observed. In this work, we report the experimentally observed DET of the GOx enzyme adsorbed on flexible carbon fiber (FCF) electrodes modified with GO (FCF-GO), where the k(het) between GO and electroactive GOx has been measured at a structurally well-defined interface. The curves obtained from the Marcus theory were used to obtain k(het), by using the model proposed by Chidsey. In agreement with experimental data, this model proved to be useful to systematically probe the dependence of electron transfer rates on distance, in order to provide an empirical basis to understand the origin of interfacial DET between GO and GOx. We also demonstrate that the presence of GO at the enzyme/electrode interface diminishes the activation energy by decreasing the distance between the electrode surface and FAD/FADH2.

Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics.

PubMed

Güleç, Hacı Ali

2013-04-01

The aim of this study was to investigate the effects of surface characteristics of plain and plasma modified cellulose acetate (CA) membranes on the immobilization yield of β-galactosidases from Kluyveromyces lactis (KLG) and its galacto-oligosaccharide (GOS) yield, respectively. Low pressure plasma treatments involving oxygen plasma activation, plasma polymerization (PlsP) of ethylenediamine (EDA) and PlsP of 2-mercaptoethanol were used to modify plain CA membrane surfaces. KLG enzyme was immobilized onto plain and oxygen plasma treated membrane surfaces by simple adsorption. Oxygen plasma activation increased the hydrophylicity of CA membrane surfaces and it improved the immobilization yield of the enzyme by 42%. KLG enzyme was also immobilized onto CA membrane surfaces through amino groups created by PlsP of EDA via covalent binding. Plasma action at 60W plasma power and 15 min. exposure time improved the amount of membrane bounded enzyme by 3.5-fold. The enrichment of the amount of amino groups via polyethyleneimine (PEI) addition enhanced this increase from 3.5-fold to 4.5-fold. Although high enzyme loading was achived (65-83%), both of the methods dramatically decreased the enzyme activity (11-12%) and GOS yield due to probably negative effects of active amino groups. KLG enzyme was more effectively immobilized onto thiolated CA membrane surface created by PlsP of 2-mercaptoethanol with high immobilization yield (70%) and especially high enzyme activity (46%). Immobilized enzymes on the CA membranes treated by PlsP were successively reutilized for 5-8 cycles at 25°C and enzymatic derivatives retained approximately 75-80% of their initial activites at the end of the reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Surface Plasmon Resonance based sensing of lysozyme in serum on Micrococcus lysodeikticus-modified graphene oxide surfaces.

PubMed

Vasilescu, Alina; Gáspár, Szilveszter; Gheorghiu, Mihaela; David, Sorin; Dinca, Valentina; Peteu, Serban; Wang, Qian; Li, Musen; Boukherroub, Rabah; Szunerits, Sabine

2017-03-15

Lysozyme is an enzyme found in biological fluids, which is upregulated in leukemia, renal diseases as well as in a number of inflammatory gastrointestinal diseases. We present here the development of a novel lysozyme sensing concept based on the use of Micrococcus lysodeikticus whole cells adsorbed on graphene oxide (GO)-coated Surface Plasmon Resonance (SPR) interfaces. M. lysodeikticus is a typical enzymatic substrate for lysozyme. Unlike previously reported sensors which are based on the detection of lysozyme through bioaffinity interactions, the bioactivity of lysozyme will be used here for sensing purposes. Upon exposure to lysozyme containing serum, the integrity of the bacterial cell wall is affected and the cells detach from the GO based interfaces, causing a characteristic decrease in the SPR signal. This allows sensing the presence of clinically relevant concentrations of lysozyme in undiluted serum samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Covalent immobilization of invertase on PAMAM-dendrimer modified superparamagnetic iron oxide nanoparticles

NASA Astrophysics Data System (ADS)

Uzun, K.; Çevik, E.; Şenel, M.; Sözeri, H.; Baykal, A.; Abasıyanık, M. F.; Toprak, M. S.

2010-10-01

In this study, polyamidoamine (PAMAM) dendrimer was synthesized on the surface of superparamagnetite nanoparticles to enhance invertase immobilization. The amount of immobilized enzyme on the surface-hyperbranched magnetite nanoparticle was up to 2.5 times (i.e., 250%) as much as that of magnetite nanoparticle modified with only amino silane. Maximum reaction rate ( V max) and Michaelis-Menten constant ( K m) were determined for the free and immobilized enzymes. Various characteristics of immobilized invertase such as; the temperature activity, thermal stability, operational stability, and storage stability were evaluated and results revealed that stability of the enzyme is improved upon immobilization.

Coupled reactions on bioparticles: Stereoselective reduction with cofactor regeneration on PhaC inclusion bodies.

PubMed

Spieler, Valerie; Valldorf, Bernhard; Maaß, Franziska; Kleinschek, Alexander; Hüttenhain, Stefan H; Kolmar, Harald

2016-07-01

Chiral alcohols are important building blocks for specialty chemicals and pharmaceuticals. The production of chiral alcohols from ketones can be carried out stereo selectively with alcohol dehydrogenases (ADHs). To establish a process for cost-effective enzyme immobilization on solid phase for application in ketone reduction, we used an established enzyme pair consisting of ADH from Rhodococcus erythropolis and formate dehydrogenase (FDH) from Candida boidinii for NADH cofactor regeneration and co-immobilized them on modified poly-p-hydroxybutyrate synthase (PhaC)-inclusion bodies that were recombinantly produced in Escherichia coli cells. After separate production of genetically engineered and recombinantly produced enzymes and particles, cell lysates were combined and enzymes endowed with a Kcoil were captured on the surface of the Ecoil presenting particles due to coiled-coil interaction. Enzyme-loaded particles could be easily purified by centrifugation. Total conversion of 4'-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol could be accomplished using enzyme-loaded particles, catalytic amounts of NAD(+) and formate as substrates for FDH. Chiral GC-MS analysis revealed that immobilized ADH retained enantioselectivity with 99 % enantiomeric excess. In conclusion, this strategy may become a cost-effective alternative to coupled reactions using purified enzymes. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antibacterial and antibiofilm surfaces through polydopamine-assisted immobilization of lysostaphin as an antibacterial enzyme.

PubMed

Yeroslavsky, Gil; Girshevitz, Olga; Foster-Frey, Juli; Donovan, David M; Rahimipour, Shai

2015-01-27

Antibiotic resistance and the colonization of bacteria on surfaces, often as biofilms, prolong hospitalization periods, increase mortality, and are thus major concerns for health care providers. There is an urgent need for antimicrobial and antibiofilm surface treatments that are permanent, can eradicate both biofilms and planktonic pathogens over long periods of time, and do not select for resistant strains. In this study, we have demonstrated a simple, robust, and biocompatible method that utilizes the adhesive property of polydopamine (PDA) to covalently attach the antimicrobial enzyme lysostaphin (Lst) to a variety of surfaces to generate antibacterial and antibiofilm interfaces. The immobilization of the recombinant Lst onto PDA-coated surfaces was carried out under physiological conditions, most probably through the C-terminal His6-tag fragment of the enzyme, minimizing the losses of bioagent activity. The modified surfaces were extensively characterized by X-ray photoelectron spectroscopy and peak force quantitative nanomechanical mapping (PeakForce QNM) AFM-based method, and the presence of Lst on the surfaces was further confirmed immunochemically using anti-Lst antibody. We also found that, in contrast to the physically adsorbed Lst, the covalently attached Lst does not leach from the surfaces and maintains its endopeptidase activity to degrade the staphylococcal cell wall, avoiding most intracellular bacterial resistance mechanisms. Moreover, the Lst-coated surfaces kill hospital strains of Staphylococcus aureus in less than 15 min and prevent biofilm formation. This immobilization method should be applicable also to other proteins and enzymes that are recombinantly expressed to include the His6-tag fragment.

Recent advances in material science for developing enzyme electrodes.

PubMed

Sarma, Anil Kumar; Vatsyayan, Preety; Goswami, Pranab; Minteer, Shelley D

2009-04-15

The enzyme-modified electrode is the fundamental component of amperometric biosensors and biofuel cells. The selection of appropriate combinations of materials, such as: enzyme, electron transport mediator, binding and encapsulation materials, conductive support matrix and solid support, for construction of enzyme-modified electrodes governs the efficiency of the electrodes in terms of electron transfer kinetics, mass transport, stability, and reproducibility. This review investigates the varieties of materials that can be used for these purposes. Recent innovation in conductive electro-active polymers, functionalized polymers, biocompatible composite materials, composites of transition metal-based complexes and organometallic compounds, sol-gel and hydro-gel materials, nanomaterials, other nano-metal composites, and nano-metal oxides are reviewed and discussed here. In addition, the critical issues related to the construction of enzyme electrodes and their application for biosensor and biofuel cell applications are also highlighted in this article. Effort has been made to cover the recent literature on the advancement of materials sciences to develop enzyme electrodes and their potential applications for the construction of biosensors and biofuel cells.

Pectin-modifying enzymes and pectin-derived materials: applications and impacts.

PubMed

Bonnin, Estelle; Garnier, Catherine; Ralet, Marie-Christine

2014-01-01

Pectins are complex branched polysaccharides present in primary cell walls. As a distinctive feature, they contain high amount of partly methyl-esterified galacturonic acid and low amount of rhamnose and carry arabinose and galactose as major neutral sugars. Due to their structural complexity, they are modifiable by many different enzymes, including hydrolases, lyases, and esterases. Their peculiar structure is the origin of their physicochemical properties. Among others, their remarkable gelling properties make them a key additive for food industries. Pectin-degrading enzymes and -modifying enzymes may be used in a wide variety of applications to modulate pectin properties or produce pectin derivatives and oligosaccharides with functional as well as nutritional interests. This paper reviews the scientific information available on pectin structure, pectin-modifying enzymes, and the use of enzymes to produce pectin with controlled structure or pectin-derived oligosaccharides, with functional or nutritional interesting properties.

Self-assembly of glucose oxidase on reduced graphene oxide-magnetic nanoparticles nanocomposite-based direct electrochemistry for reagentless glucose biosensor.

PubMed

Pakapongpan, Saithip; Poo-Arporn, Rungtiva P

2017-07-01

A novel approach of the immobilization of a highly selective and stable glucose biosensor based on direct electrochemistry was fabricated by a self-assembly of glucose oxidase (GOD) on reduced graphene oxide (RGO) covalently conjugated to magnetic nanoparticles (Fe 3 O 4 NPs) modified on a magnetic screen-printed electrode (MSPE). The RGO-Fe 3 O 4 nanocomposite has remarkable enhancement in large surface areas, is favorable environment for enzyme immobilization, facilitates electron transfer between enzymes and electrode surfaces and possesses superparamagnetism property. The morphology and electrochemical properties of RGO-Fe 3 O 4 /GOD were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, cyclic voltammetry (CV) and amperometry. The modified electrode was a fast, direct electron transfer with an apparent electron transfer rate constant (k s ) of 13.78s -1 . The proposed biosensor showed fast amperometric response (3s) to glucose with a wide linear range from 0.05 to 1mM, a low detection limit of 0.1μM at a signal to noise ratio of 3 (S/N=3) and good sensitivity (5.9μA/mM). The resulting biosensor has high stability, good reproducibility, excellent selectivity and successfully applied detection potential at -0.45V. This mediatorless glucose sensing used the advantages of covalent bonding and self-assembly as a new approach for immobilizing enzymes without any binder. It would be worth noting that it opens a new avenue for fabricating excellent electrochemical biosensors. This is a new approach that reporting the immobilization of glucose oxidase on reduced graphene oxide (RGO) covalently conjugated to magnetic nanoparticles (Fe 3 O 4 NPs) by electrostatic interaction and modified screen printed electrode. We propose the reagentless with fabrication method without binder and adhesive agents for immobilized enzyme. Fe 3 O 4 NPs increasing surface area to enhance the immobilization and prevent the leaching of enzymes at electrode surfaces by magnetic stickers which is improve the stability of the biosensor. Based on this synthesis technique, it is a good new strategy and simple used to fabrication of third-generation glucose biosensor and this nanocomposite could be used as a platform for disposable biosensor and biofuel cell applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Galactosyltransferase and Concanavalin A Agglutination of Cells

PubMed Central

Podolsky, Daniel K.; Weiser, Milton M.; Mont, J. Thomas La; Isselbacher, Kurt J.

1974-01-01

A correlation has been observed between concanavalin A agglutination of various cell types and the presence of surface membrane galactosyltransferase (1-O-α-D-Galactosyl-myo-inositol:raffinose galactosyltransferase, EC 2.4.1.67) activity. Moreover, a reduction to less than 50% of cell surface galactosyltransferase activity occurred after treatment with concanavalin A; other cell surface glycosyltransferase enzyme activities examined were unaffected by concanavalin A treatment. To confirm the participation of cell surface galactosyltransferase in concanavalin A-induced cell agglutination, the enzyme from rabbit erythrocytes was solubilized by sonication and purified by preparative polyacrylamide gel electrophoresis. It was possible to achieve a purified preparation of rabbit erythrocyte galactosyltransferase by separation on concanavalin A-Sepharose. The purified enzyme showed visible immunoprecipitation (Ouchterlony) with concanavalin A. Furthermore, human erythrocytes, which are not normally agglutinated by concanavalin A, became agglutinable by the lectin when the erythrocytes were preincubated with purified galactosyltransferase. These experiments suggest a direct and possible specific role of cell surface galactosyltransferase enzyme in the mechanism of concanavalin A agglutination of cells. Images PMID:4522801

Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application.

PubMed

Schüürmann, Jan; Quehl, Paul; Festel, Gunter; Jose, Joachim

2014-10-01

Despite the first report on the bacterial display of a recombinant peptide appeared almost 30 years ago, industrial application of cells with surface-displayed enzymes is still limited. To display an enzyme on the surface of a living cell bears several advantages. First of all, neither the substrate nor the product of the enzymatic reaction needs to cross a membrane barrier. Second, the enzyme being linked to the cell can be separated from the reaction mixture and hence the product by simple centrifugation. Transfer to a new substrate preparation results in multiple cycles of enzymatic conversion. Finally, the anchoring in a matrix, in this case, the cell envelope stabilizes the enzyme and makes it less accessible to proteolytic degradation and material adsorption resulting in continuous higher activities. These advantages in common need to balance some disadvantages before this application can be taken into account for industrial processes, e.g., the exclusion of the enzyme from the cellular metabolome and hence from redox factors or other co-factors that need to be supplied. Therefore, this digest describes the different systems in Gram-positive and Gram-negative bacteria that have been used for the surface display of enzymes so far and focuses on examples among these which are suitable for industrial purposes or for the production of valuable resources, not least in order to encourage a broader application of whole-cell biocatalysts with surface-displayed enzymes.

Surface modifications of magnesium alloys for biomedical applications.

PubMed

Yang, Jingxin; Cui, Fuzhai; Lee, In Seop

2011-07-01

In recent years, research on magnesium (Mg) alloys had increased significantly for hard tissue replacement and stent application due to their outstanding advantages. Firstly, Mg alloys have mechanical properties similar to bone which avoid stress shielding. Secondly, they are biocompatible essential to the human metabolism as a factor for many enzymes. In addition, main degradation product Mg is an essential trace element for human enzymes. The most important reason is they are perfectly biodegradable in the body fluid. However, extremely high degradation rate, resulting in too rapid loss of mechanical strength in chloride containing environments limits their applications. Engineered artificial biomaterials with appropriate mechanical properties, surface chemistry, and surface topography are in a great demand. As the interaction between the cells and tissues with biomaterials at the tissue--implant interface is a surface phenomenon; surface properties play a major role in determining both the biological response to implants and the material response to the physiological condition. Therefore, the ability to modify the surface properties while preserve the bulk properties is important, and surface modification to form a hard, biocompatible and corrosion resistant modified layer have always been an interesting topic in biomaterials field. In this article, attempts are made to give an overview of the current research and development status of surface modification technologies of Mg alloys for biomedical materials research. Further, the advantages/disadvantages of the different methods and with regard to the most promising method for Mg alloys are discussed. Finally, the scientific challenges are proposed based on own research and the work of other scientists.

Electrochemical Glucose Biosensor Based on Glucose Oxidase Displayed on Yeast Surface.

PubMed

Wang, Hongwei; Lang, Qiaolin; Liang, Bo; Liu, Aihua

2015-01-01

The conventional enzyme-based biosensor requires chemical or physical immobilization of purified enzymes on electrode surface, which often results in loss of enzyme activity and/or fractions immobilized over time. It is also costly. A major advantage of yeast surface display is that it enables the direct utilization of whole cell catalysts with eukaryote-produced proteins being displayed on the cell surface, providing an economic alternative to traditional production of purified enzymes. Herein, we describe the details of the display of glucose oxidase (GOx) on yeast cell surface and its application in the development of electrochemical glucose sensor. In order to achieve a direct electrochemistry of GOx, the entire cell catalyst (yeast-GOx) was immobilized together with multiwalled carbon nanotubes on the electrode, which allowed sensitive and selective glucose detection.

The Electrosome: A Surface-Displayed Enzymatic Cascade in a Biofuel Cell’s Anode and a High-Density Surface-Displayed Biocathodic Enzyme

PubMed Central

Szczupak, Alon; Aizik, Dror; Moraïs, Sarah; Vazana, Yael; Barak, Yoav; Bayer, Edward A.; Alfonta, Lital

2017-01-01

The limitation of surface-display systems in biofuel cells to a single redox enzyme is a major drawback of hybrid biofuel cells, resulting in a low copy-number of enzymes per yeast cell and a limitation in displaying enzymatic cascades. Here we present the electrosome, a novel surface-display system based on the specific interaction between the cellulosomal scaffoldin protein and a cascade of redox enzymes that allows multiple electron-release by fuel oxidation. The electrosome is composed of two compartments: (i) a hybrid anode, which consists of dockerin-containing enzymes attached specifically to cohesin sites in the scaffoldin to assemble an ethanol oxidation cascade, and (ii) a hybrid cathode, which consists of a dockerin-containing oxygen-reducing enzyme attached in multiple copies to the cohesin-bearing scaffoldin. Each of the two compartments was designed, displayed, and tested separately. The new hybrid cell compartments displayed enhanced performance over traditional biofuel cells; in the anode, the cascade of ethanol oxidation demonstrated higher performance than a cell with just a single enzyme. In the cathode, a higher copy number per yeast cell of the oxygen-reducing enzyme copper oxidase has reduced the effect of competitive inhibition resulting from yeast oxygen consumption. This work paves the way for the assembly of more complex cascades using different enzymes and larger scaffoldins to further improve the performance of hybrid cells. PMID:28644390

Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis.

PubMed

Partridge, Emily A; Le Roy, Christine; Di Guglielmo, Gianni M; Pawling, Judy; Cheung, Pam; Granovsky, Maria; Nabi, Ivan R; Wrana, Jeffrey L; Dennis, James W

2004-10-01

The Golgi enzyme beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor and transforming growth factor-beta receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5 expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition, cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which balances their surface retention against loss via endocytosis.

Catalytic activity of enzymes immobilized on AlGaN /GaN solution gate field-effect transistors

NASA Astrophysics Data System (ADS)

Baur, B.; Howgate, J.; von Ribbeck, H.-G.; Gawlina, Y.; Bandalo, V.; Steinhoff, G.; Stutzmann, M.; Eickhoff, M.

2006-10-01

Enzyme-modified field-effect transistors (EnFETs) were prepared by immobilization of penicillinase on AlGaN /GaN solution gate field-effect transistors. The influence of the immobilization process on enzyme functionality was analyzed by comparing covalent immobilization and physisorption. Covalent immobilization by Schiff base formation on GaN surfaces modified with an aminopropyltriethoxysilane monolayer exhibits high reproducibility with respect to the enzyme/substrate affinity. Reductive amination of the Schiff base bonds to secondary amines significantly increases the stability of the enzyme layer. Electronic characterization of the EnFET response to penicillin G indicates that covalent immobilization leads to the formation of an enzyme (sub)monolayer.

Novel soy protein scaffolds for tissue regeneration: Material characterization and interaction with human mesenchymal stem cells.

PubMed

Chien, Karen B; Shah, Ramille N

2012-02-01

Soy protein modified with heat treatment and enzyme crosslinking using transglutaminase in maltodextrin was used to fabricate novel, porous three-dimensional scaffolds through lyophilization. Physical properties of scaffolds were characterized using scanning electron microscopy, mercury intrusion porosimetry, moisture content analysis and mechanical testing. Human mesenchymal stem cells (hMSC) were seeded and cultured in vitro on the scaffolds for up to 2 weeks, and changes in stem cell growth and morphology were examined. The resulting scaffolds had rough surfaces, irregular pores with size distributions between 10 and 125 μm, <5% moisture content and compressive moduli ranging between 50 and 100 Pa. Enzyme treatment significantly lowered the moisture content. Increasing amounts of applied enzyme units lowered the median pore size. Although enzyme treatment did not affect the mechanical properties of the scaffolds, it did increase the degradation time by at least 1 week. These changes in scaffold degradation altered the growth and morphology of seeded hMSC. Cell proliferation was observed in scaffolds containing 3% soy protein isolate treated with 1 U of transglutaminase. These results demonstrate that controlling scaffold degradation rates is crucial for optimizing hMSC growth on soy protein scaffolds and that soy protein scaffolds have the potential to be used in tissue engineering applications. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Effect of enzymatic orientation through the use of syringaldazine molecules on multiple multi-copper oxidase enzymes.

PubMed

Ulyanova, Yevgenia; Babanova, Sofia; Pinchon, Erica; Matanovic, Ivana; Singhal, Sameer; Atanassov, Plamen

2014-07-14

The effect of proper enzyme orientation at the electrode surface was explored for two multi-copper oxygen reducing enzymes: Bilirubin Oxidase (BOx) and Laccase (Lac). Simultaneous utilization of "tethering" agent (1-pyrenebutanoic acid, succinimidyl ester; PBSE), for stable enzyme immobilization, and syringaldazine (Syr), for enzyme orientation, of both Lac and BOx led to a notable enhancement of the electrode performance. For Lac cathodes tested in solution it was established that PBSE-Lac and PBSE-Syr-Lac modified cathodes demonstrated approximately 6 and 9 times increase in current density, respectively, compared to physically adsorbed and randomly oriented Lac cathodes. Further testing in solution utilizing BOx showed an even higher increase in achievable current densities, thus BOx was chosen for additional testing in air-breathing mode. In subsequent air-breathing experiments the incorporation of PBSE and Syr with BOx resulted in current densities of 0.65 ± 0.1 mA cm(-2); 2.5 times higher when compared to an unmodified BOx cathode. A fully tethered/oriented BOx cathode was combined with a NAD-dependent Glucose Dehydrogenase anode for the fabrication of a complete enzymatic membraneless fuel cell. A maximum power of 1.03 ± 0.06 mW cm(-2) was recorded for the complete fuel cell. The observed significant enhancement in the performance of "oriented" cathodes was a result of proper enzyme orientation, leading to facilitated enzyme/electrode interface interactions.

Heparan sulfate proteoglycans mediate renal carcinoma metastasis.

PubMed

Qazi, Henry; Shi, Zhong-Dong; Song, Jonathan W; Cancel, Limary M; Huang, Peigen; Zeng, Ye; Roberge, Sylvie; Munn, Lance L; Tarbell, John M

2016-12-15

The surface proteoglycan/glycoprotein layer (glycocalyx) on tumor cells has been associated with cellular functions that can potentially enable invasion and metastasis. In addition, aggressive tumor cells with high metastatic potential have enhanced invasion rates in response to interstitial flow stimuli in vitro. Our previous studies suggest that heparan sulfate (HS) in the glycocalyx plays an important role in this flow mediated mechanostransduction and upregulation of invasive and metastatic potential. In this study, highly metastatic renal cell carcinoma cells were genetically modified to suppress HS production by knocking down its synthetic enzyme NDST1. Using modified Boyden chamber and microfluidic assays, we show that flow-enhanced invasion is suppressed in HS deficient cells. To assess the ability of these cells to metastasize in vivo, parental or knockdown cells expressing fluorescence reporters were injected into kidney capsules in SCID mice. Histological analysis confirmed that there was a large reduction (95%) in metastasis to distant organs by tumors formed from the NDST1 knockdown cells compared to control cells with intact HS. The ability of these cells to invade surrounding tissue was also impaired. The substantial inhibition of metastasis and invasion upon reduction of HS suggests an active role for the tumor cell glycocalyx in tumor progression. © 2016 UICC.

Modified Electrodes Used for Electrochemical Detection of Metal Ions in Environmental Analysis

PubMed Central

March, Gregory; Nguyen, Tuan Dung; Piro, Benoit

2015-01-01

Heavy metal pollution is one of the most serious environmental problems, and regulations are becoming stricter. Many efforts have been made to develop sensors for monitoring heavy metals in the environment. This review aims at presenting the different label-free strategies used to develop electrochemical sensors for the detection of heavy metals such as lead, cadmium, mercury, arsenic etc. The first part of this review will be dedicated to stripping voltammetry techniques, on unmodified electrodes (mercury, bismuth or noble metals in the bulk form), or electrodes modified at their surface by nanoparticles, nanostructures (CNT, graphene) or other innovative materials such as boron-doped diamond. The second part will be dedicated to chemically modified electrodes especially those with conducting polymers. The last part of this review will focus on bio-modified electrodes. Special attention will be paid to strategies using biomolecules (DNA, peptide or proteins), enzymes or whole cells. PMID:25938789

Acrolein Microspheres Are Bonded To Large-Area Substrates

NASA Technical Reports Server (NTRS)

Rembaum, Alan; Yen, Richard C. K.

1988-01-01

Reactive cross-linked microspheres produced under influence of ionizing radiation in aqueous solutions of unsaturated aldehydes, such as acrolein, with sodium dodecyl sulfate. Diameters of spheres depend on concentrations of ingredients. If polystyrene, polymethylmethacrylate, or polypropylene object immersed in solution during irradiation, microspheres become attached to surface. Resulting modified surface has grainy coating with reactivity similar to free microspheres. Aldehyde-substituted-functional microspheres react under mild conditions with number of organic reagents and with most proteins. Microsphere-coated macrospheres or films used to immobilize high concentrations of proteins, enzymes, hormones, viruses, cells, and large number of organic compounds. Applications include separation techniques, clinical diagnostic tests, catalytic processes, and battery separators.

From Agrobacterium to viral vectors: genome modification of plant cells by rare cutting restriction enzymes.

PubMed

Marton, Ira; Honig, Arik; Omid, Ayelet; De Costa, Noam; Marhevka, Elena; Cohen, Barry; Zuker, Amir; Vainstein, Alexander

2013-01-01

Researchers and biotechnologists require methods to accurately modify the genome of higher eukaryotic cells. Such modifications include, but are not limited to, site-specific mutagenesis, site-specific insertion of foreign DNA, and replacement and deletion of native sequences. Accurate genome modifications in plant species have been rather limited, with only a handful of plant species and genes being modified through the use of early genome-editing techniques. The development of rare-cutting restriction enzymes as a tool for the induction of site-specific genomic double-strand breaks and their introduction as a reliable tool for genome modification in animals, animal cells and human cell lines have paved the way for the adaptation of rare-cutting restriction enzymes to genome editing in plant cells. Indeed, the number of plant species and genes which have been successfully edited using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and engineered homing endonucleases is on the rise. In our review, we discuss the basics of rare-cutting restriction enzyme-mediated genome-editing technology with an emphasis on its application in plant species.

Modifying the red cell surface: towards an ABO-universal blood supply.

PubMed

Olsson, Martin L; Clausen, Henrik

2008-01-01

Eliminating the risk for ABO-incompatible transfusion errors and simplifying logistics by creating a universal blood inventory is a challenging idea. Goldstein and co-workers pioneered the field of enzymatic conversion of blood group A and B red blood cells (RBCs) to O (ECO). Using alpha-galactosidase from coffee beans to produce B-ECO RBCs, proof of principle for this revolutionary concept was achieved in clinical trials. However, because this enzyme has poor kinetic properties and low pH optimum the process was not economically viable. Conversion of group A RBCs was only achieved with the weak A2 subgroup with related enzymes having acidic pH optima. More recently, the identification of entirely new families of bacterial exoglycosidases with remarkably improved kinetic properties for cleaving A and B antigens has reinvigorated the field. Enzymatic conversion of groups A, B and AB RBCs with these novel enzymes resulting in ECO RBCs typing as O can now be achieved with low enzyme protein consumption, short incubation times and at neutral pH. Presently, clinical trials evaluating safety and efficacy of ECO RBCs are ongoing. Here, we review the status of the ECO technology, its impact and potential for introduction into clinical component preparation laboratories.

Bioelectrochemistry of heme peptide at seamless three-dimensional carbon nanotubes/graphene hybrid films for highly sensitive electrochemical biosensing.

PubMed

Komori, Kikuo; Terse-Thakoor, Trupti; Mulchandani, Ashok

2015-02-18

A seamless three-dimensional hybrid film consisting of carbon nanotubes grown at the graphene surface (CNTs/G) is a promising material for the application to highly sensitive enzyme-based electrochemical biosensors. The CNTs/G film was used as a conductive nanoscaffold for enzymes. The heme peptide (HP) was immobilized on the surface of the CNTs/G film for amperometric sensing of H2O2. Compared with flat graphene electrodes modified with HP, the catalytic current for H2O2 reduction at the HP-modified CNTs/G electrode increased due to the increase in the surface coverage of HP. In addition, microvoids in the CNTs/G film contributed to diffusion of H2O2 to modified HP, resulting in the enhancement of the catalytic cathodic currents. The kinetics of the direct electron transfer from the CNTs/G electrode to compound I and II of modified HP was also analyzed.

The heparan sulfate-modifying enzyme glucuronyl C5-epimerase HSE-5 controls Caenorhabditis elegans Q neuroblast polarization during migration.

PubMed

Wang, Xiangming; Liu, Jianhong; Zhu, Zhiwen; Ou, Guangshuo

2015-03-15

Directional cell migration is fundamental for neural development, and extracellular factors are pivotal for this process. Heparan sulfate proteoglycans (HSPGs) that carry long chains of differentially modified sugar residues contribute to extracellular matrix; however, the functions of HSPG in guiding cell migration remain elusive. Here, we used the Caenorhabditis elegans mutant pool from the Million Mutation Project and isolated a mutant allele of the heparan sulfate-modifying enzyme glucuronyl C5-epimerase HSE-5. Loss-of-function of this enzyme resulted in defective Q neuroblast migration. We showed that hse-5 controlled Q cell migration in a cell non-autonomous manner. By performing live cell imaging in hse-5 mutant animals, we found that hse-5 controlled initial polarization during Q neuroblast migration. Furthermore, our genetic epistasis analysis demonstrated that lon-2 might act downstream of hse-5. Finally, rescue of the hse-5 mutant phenotype by expression of human and mouse hse-5 homologs suggested a conserved function for this gene in neural development. Taken together, our results indicated that proper HSPG modification in the extracellular matrix by HSE-5 is essential for neuroblast polarity during migration. Copyright © 2015 Elsevier Inc. All rights reserved.

Effective Electrochemistry of Human Sulfite Oxidase Immobilized on Quantum-Dots-Modified Indium Tin Oxide Electrode.

PubMed

Zeng, Ting; Leimkühler, Silke; Koetz, Joachim; Wollenberger, Ulla

2015-09-30

The bioelectrocatalytic sulfite oxidation by human sulfite oxidase (hSO) on indium tin oxide (ITO) is reported, which is facilitated by functionalizing of the electrode surface with polyethylenimine (PEI)-entrapped CdS nanoparticles and enzyme. hSO was assembled onto the electrode with a high surface loading of electroactive enzyme. In the presence of sulfite but without additional mediators, a high bioelectrocatalytic current was generated. Reference experiments with only PEI showed direct electron transfer and catalytic activity of hSO, but these were less pronounced. The application of the polyelectrolyte-entrapped quantum dots (QDs) on ITO electrodes provides a compatible surface for enzyme binding with promotion of electron transfer. Variations of the buffer solution conditions, e.g., ionic strength, pH, viscosity, and the effect of oxygen, were studied in order to understand intramolecular and heterogeneous electron transfer from hSO to the electrode. The results are consistent with a model derived for the enzyme by using flash photolysis in solution and spectroelectrochemistry and molecular dynamic simulations of hSO on monolayer-modified gold electrodes. Moreover, for the first time a photoelectrochemical electrode involving immobilized hSO is demonstrated where photoexcitation of the CdS/hSO-modified electrode lead to an enhanced generation of bioelectrocatalytic currents upon sulfite addition. Oxidation starts already at the redox potential of the electron transfer domain of hSO and is greatly increased by application of a small overpotential to the CdS/hSO-modified ITO.

Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production.

PubMed

Gerken, Henri G; Donohoe, Bryon; Knoshaug, Eric P

2013-01-01

Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae.

Enzyme-modified electrolyte-gated organic field-effect transistors

NASA Astrophysics Data System (ADS)

Buth, Felix; Donner, Andreas; Stutzmann, Martin; Garrido, Jose A.

2012-10-01

Organic solution-gated field-effect transistors (SGFETs) can be operated at low voltages in aqueous environments, paving the way to the use of organic semiconductors in bio-sensing applications. However, it has been shown that these devices exhibit only a rather weak sensitivity to standard electrolyte parameters such as pH and ionic strength. In order to increase the sensitivity and to add specificity towards a given analyte, the covalent attachment of functional groups and enzymes to the device surface would be desirable. In this contribution we demonstrate that enzyme modified organic SGFETs can be used for the in-situ detection of penicillin in the low μM regime. In a first step, silane molecules with amine terminal groups are grafted to α-sexithiophene-based thin film transistors. Surface characterization techniques like X-ray photoemission confirm the modification of the surface with these functional groups, which are stable in standard aqueous electrolytes. We show that the presence of surface-bound amphoteric groups (e.g. amino or carboxylic moieties) increases the pH-sensitivity of the organic SGFETs. In addition, these groups serve as anchoring sites for the attachment of the enzyme penicillinase. The resulting enzyme-FETs are used for the detection of penicillin, enabling the study of the influence of the buffer strength and the pH of the electrolyte on the enzyme kinetics. The functionalization of the organic FETs shown here can be extended to a large variety of enzymes, allowing the specific detection of different chemical and biochemical analytes.

Multilayer Choline Phosphate Molecule Modified Surface with Enhanced Cell Adhesion but Resistance to Protein Adsorption.

PubMed

Chen, Xingyu; Yang, Ming; Liu, Botao; Li, Zhiqiang; Tan, Hong; Li, Jianshu

2017-08-22

Choline phosphate (CP), which is a new zwitterionic molecule, and has the reverse order of phosphate choline (PC) and could bind to the cell membrane though the unique CP-PC interaction. Here we modified a glass surface with multilayer CP molecules using surface-initiated atom-transfer radical polymerization (SI-ATRP) and the ring-opening method. Polymeric brushes of (dimethylamino)ethyl methacrylate (DMAEMA) were synthesized by SI-ATRP from the glass surface. Then the grafted PDMAEMA brushes were used to introduce CP groups to fabricate the multilayer CP molecule modified surface. The protein adsorption experiment and cell culture test were used to evaluate the biocompatibility of the modified surfaces by using human umbilical veinendothelial cells (HUVECs). The protein adsorption results demonstrated that the multilayer CP molecule decorated surface could prevent the adsorption of fibrinogen and serum protein. The adhesion and proliferation of cells were improved significantly on the multilayer CP molecule modified surface. Therefore, the biocompatibility of the material surface could be improved by the modified multilayer CP molecule, which exhibits great potential for biomedical applications, e.g., scaffolds in tissue engineering.

Controlling enzymatic activity by immobilization on graphene oxide

NASA Astrophysics Data System (ADS)

Bolibok, Paulina; Wiśniewski, Marek; Roszek, Katarzyna; Terzyk, Artur P.

2017-04-01

In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells' viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

Cellobiose Dehydrogenase Aryl Diazonium Modified Single Walled Carbon Nanotubes: Enhanced Direct Electron Transfer through a Positively Charged Surface

PubMed Central

2011-01-01

One of the challenges in the field of biosensors and biofuel cells is to establish a highly efficient electron transfer rate between the active site of redox enzymes and electrodes to fully access the catalytic potential of the biocatalyst and achieve high current densities. We report on very efficient direct electron transfer (DET) between cellobiose dehydrogenase (CDH) from Phanerochaete sordida (PsCDH) and surface modified single walled carbon nanotubes (SWCNT). Sonicated SWCNTs were adsorbed on the top of glassy carbon electrodes and modified with aryl diazonium salts generated in situ from p-aminobenzoic acid and p-phenylenediamine, thus featuring at acidic pH (3.5 and 4.5) negative or positive surface charges. After adsorption of PsCDH, both electrode types showed excellent long-term stability and very efficient DET. The modified electrode presenting p-aminophenyl groups produced a DET current density of 500 μA cm−2 at 200 mV vs normal hydrogen reference electrode (NHE) in a 5 mM lactose solution buffered at pH 3.5. This is the highest reported DET value so far using a CDH modified electrode and comes close to electrodes using mediated electron transfer. Moreover, the onset of the electrocatalytic current for lactose oxidation started at 70 mV vs NHE, a potential which is 50 mV lower compared to when unmodified SWCNTs were used. This effect potentially reduces the interference by oxidizable matrix components in biosensors and increases the open circuit potential in biofuel cells. The stability of the electrode was greatly increased compared with unmodified but cross-linked SWCNTs electrodes and lost only 15% of the initial current after 50 h of constant potential scanning. PMID:21417322

Cellobiose dehydrogenase aryl diazonium modified single walled carbon nanotubes: enhanced direct electron transfer through a positively charged surface.

PubMed

Tasca, Federico; Harreither, Wolfgang; Ludwig, Roland; Gooding, John Justin; Gorton, Lo

2011-04-15

One of the challenges in the field of biosensors and biofuel cells is to establish a highly efficient electron transfer rate between the active site of redox enzymes and electrodes to fully access the catalytic potential of the biocatalyst and achieve high current densities. We report on very efficient direct electron transfer (DET) between cellobiose dehydrogenase (CDH) from Phanerochaete sordida (PsCDH) and surface modified single walled carbon nanotubes (SWCNT). Sonicated SWCNTs were adsorbed on the top of glassy carbon electrodes and modified with aryl diazonium salts generated in situ from p-aminobenzoic acid and p-phenylenediamine, thus featuring at acidic pH (3.5 and 4.5) negative or positive surface charges. After adsorption of PsCDH, both electrode types showed excellent long-term stability and very efficient DET. The modified electrode presenting p-aminophenyl groups produced a DET current density of 500 μA cm(-2) at 200 mV vs normal hydrogen reference electrode (NHE) in a 5 mM lactose solution buffered at pH 3.5. This is the highest reported DET value so far using a CDH modified electrode and comes close to electrodes using mediated electron transfer. Moreover, the onset of the electrocatalytic current for lactose oxidation started at 70 mV vs NHE, a potential which is 50 mV lower compared to when unmodified SWCNTs were used. This effect potentially reduces the interference by oxidizable matrix components in biosensors and increases the open circuit potential in biofuel cells. The stability of the electrode was greatly increased compared with unmodified but cross-linked SWCNTs electrodes and lost only 15% of the initial current after 50 h of constant potential scanning. © 2011 American Chemical Society

Combining an optical resonance biosensor with enzyme activity kinetics to understand protein adsorption and denaturation.

PubMed

Wilson, Kerry A; Finch, Craig A; Anderson, Phillip; Vollmer, Frank; Hickman, James J

2015-01-01

Understanding protein adsorption and resultant conformation changes on modified and unmodified silicon dioxide surfaces is a subject of keen interest in biosensors, microfluidic systems and for medical diagnostics. However, it has been proven difficult to investigate the kinetics of the adsorption process on these surfaces as well as understand the topic of the denaturation of proteins and its effect on enzyme activity. A highly sensitive optical whispering gallery mode (WGM) resonator was used to study a catalytic enzyme's adsorption processes on different silane modified glass substrates (plain glass control, DETA, 13 F, and SiPEG). The WGM sensor was able to obtain high resolution kinetic data of glucose oxidase (GO) adsorption with sensitivity of adsorption better than that possible with SPR. The kinetic data, in combination with a functional assay of the enzyme activity, was used to test hypotheses on adsorption mechanisms. By fitting numerical models to the WGM sensograms for protein adsorption, and by confirming numerical predictions of enzyme activity in a separate assay, we were able to identify mechanisms for GO adsorption on different alkylsilanes and infer information about the adsorption of protein on nanostructured surfaces. Copyright © 2014 Elsevier Ltd. All rights reserved.

Tissue engineering of bladder using vascular endothelial growth factor gene-modified endothelial progenitor cells.

PubMed

Chen, Bai-Song; Xie, Hua; Zhang, Sheng-Li; Geng, Hong-Quan; Zhou, Jun-Mei; Pan, Jun; Chen, Fang

2011-12-01

This study assessed the use of vascular endothelial growth factor (VEGF) gene-modified endothelial progenitor cells (EPCs) seeded onto bladder acellular matrix grafts (BAMGs), to enhance the blood supply in tissue-engineered bladders in a porcine model. Autologous porcine peripheral EPCs were isolated, cultured, expanded, characterized, and modified with the VEGF gene using an adenovirus vector. The expression of VEGF was examined using reverse transcriptase polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay (ELISA). VEGF gene modified EPCs were seeded onto BAMG and cultured for 3 days before implantation into pigs for bladder tissue engineering. A partial bladder cystectomy was performed in 12 pigs. The experimental group (6 pigs) received VEGF gene-modified EPC-seeded BAMG. The control group (6 pigs) received BAMG without seeded EPCs. The resulting tissue-engineered bladders were subject to a general and histological analysis. Microvessel density (MVD) was assessed using immunohistochemistry. The ex vivo transfection efficiency of EPCs was greater than 60%-70% when concentrated adenovirus was used. The genetically modified cells expressed both VEGF and green fluorescent protein (GFP). Scanning electron microscopy (SEM) and Masson's trichrome staining of cross sections of the cultured cells seeded to BAMG showed cell attachment and proliferation on the surface of the BAMG. Histological examination revealed bladder regeneration in a time-dependent fashion. Significant increases in MVD were observed in the experimental group, in comparison with the control group. VEGF-modified EPCs significantly enhanced neovascularization, compared with BAMG alone. These results indicate that EPCs, combined with VEGF gene therapy, may be a suitable approach for increasing blood supply in the tissue engineering of bladders. Thus, a useful strategy to achieve a tissue-engineered bladder is indicated.

Exosomes Secreted by HeLa Cells Shuttle on Their Surface the Plasma Membrane-Associated Sialidase NEU3.

PubMed

Paolini, Lucia; Orizio, Flavia; Busatto, Sara; Radeghieri, Annalisa; Bresciani, Roberto; Bergese, Paolo; Monti, Eugenio

2017-12-05

Sialidases are glycohydrolases that remove terminal sialic acid residues from oligosaccharides, glycolipids, and glycoproteins. The plasma membrane-associated sialidase NEU3 is involved in the fine-tuning of sialic acid-containing glycans directly on the cell surface and plays relevant roles in important biological phenomena such as cell differentiation, molecular recognition, and cancer transformation. Extracellular vesicles are membranous structures with a diameter of 0.03-1 μm released by cells and can be detected in blood, urine, and culture media. Among extracellular vesicles, exosomes play roles in intercellular communication and maintenance of several physiological and pathological conditions, including cancer, and could represent a useful diagnostic tool for personalized nanomedicine approaches. Using inducible expression of the murine form of NEU3 in HeLa cells, a study of the association of the enzyme with exosomes released in the culture media has been performed. Briefly, NEU3 is associated with highly purified exosomes and localizes on the external leaflet of these nanovesicles, as demonstrated by enzyme activity measurements, Western blot analysis, and dot blot analysis using specific protein markers. On the basis of these results, it is plausible that NEU3 activity on exosome glycans enhances the dynamic biological behavior of these small extracellular vesicles by modifying the negative charge and steric hindrance of their glycocalyx. The presence of NEU3 on the exosomal surface could represent a useful marker for the detection of these nanovesicles and a tool for improving our understanding of the biology of these important extracellular carriers in physiological and pathological conditions.

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay.

PubMed

Liu, Daojun; Perdue, Robbyn K; Sun, Li; Crooks, Richard M

2004-07-06

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.

Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase

PubMed Central

Nelson, Jennifer; Francom, Lyndee L.; Anderson, Lynn; Damm, Kelly; Baker, Ryan; Chen, Joseph; Franklin, Sarah; Hamaker, Amy; Izidoro, Izadora; Moss, Eric; Orton, Mikayla; Stevens, Evan; Yeung, Celestine; Judd, Allan M.; Bell, John D.

2012-01-01

Summary Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt’s lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. PMID:22266334

Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins.

PubMed

Glenting, Jacob; Beck, Hans Christian; Vrang, Astrid; Riemann, Holger; Ravn, Peter; Hansen, Anne Maria; Antonsson, Martin; Ahrné, Siv; Israelsen, Hans; Madsen, Søren

2013-06-12

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated. Copyright © 2013 Elsevier GmbH. All rights reserved.

Reticulated vitreous carbon as a scaffold for enzymatic fuel cell designing.

PubMed

Kizling, Michal; Dzwonek, Maciej; Olszewski, Bartłomiej; Bącal, Paweł; Tymecki, Łukasz; Więckowska, Agnieszka; Stolarczyk, Krzysztof; Bilewicz, Renata

2017-09-15

Three - dimensional (3D) electrodes are successfully used to overcome the limitations of the low space - time yield and low normalized space velocity obtained in electrochemical processes with two - dimensional electrodes. In this study, we developed a three - dimensional reticulated vitreous carbon - gold (RVC-Au) sponge as a scaffold for enzymatic fuel cells (EFC). The structure of gold and the real electrode surface area can be controlled by the parameters of metal electrodeposition. In particular, a 3D RVC-Au sponge provides a large accessible surface area for immobilization of enzyme and electron mediators, moreover, effective mass diffusion can also take place through the uniform macro - porous scaffold. To efficiently bind the enzyme to the electrode and enhance electron transfer parameters the gold surface was modified with ultrasmall gold nanoparticles stabilized with glutathione. These quantum sized nanoparticles exhibit specific electronic properties and also expand the working surface of the electrode. Significantly, at the steady state of power generation, the EFC device with RVC-Au electrodes provided high volumetric power density of 1.18±0.14mWcm -3 (41.3±3.8µWcm -2 ) calculated based on the volume of electrode material with OCV 0.741±0.021V. These new 3D RVC-Au electrodes showed great promise for improving the power generation of EFC devices. Copyright © 2017 Elsevier B.V. All rights reserved.

Chemical modification of poly(ethylene terephthalate) and immobilization of the selected enzymes on the modified film

NASA Astrophysics Data System (ADS)

Irena, Gancarz; Jolanta, Bryjak; Karolina, Zynek

2009-07-01

Poly(ethylene terephthalate) (PET) film was modified by reaction with hydrazine (HD), ethylenediamine (EDA), 1,2-diaminopropane (1,2-DAP) and 1,3-diaminopropane (1,3-DAP). The maximal amount of amine functionalities introduced in the chosen conditions on the surface was found as 0.07, 3.35, 0.76 and 1.99 nmol cm -2 for HD, EDA, 1,2-DAP and 1,3-DAP respectively. During the modification process etching of the sample and an increase of stiffness takes place. FTIR-ATR spectra prove that the surface chemistry after modification in amine solution is very complex. The lack of clear correlation between the surface tension and surface concentration of amine functionalities seems to confirm that. For immobilization purpose invertase, laccase and tyrosinase were used. The amount of covalently attached proteins at first increases with the increase of surface concentration of amine groups but after reaching a certain level of amine groups, decrease of the immobilization level was observed. All enzymes tested showed highest activity for a moderate level of aminolysis and this activity had the highest values for EDA-modified PET.

Impacts of papain and neuraminidase enzyme treatment on electrohydrodynamics and IgG-mediated agglutination of type A red blood cells.

PubMed

Hyono, Atsushi; Gaboriaud, Fabien; Mazda, Toshio; Takata, Youichi; Ohshima, Hiroyuki; Duval, Jérôme F L

2009-09-15

The stability of native and enzyme-treated human red blood cells of type A (Rh D positive) against agglutination is investigated under conditions where it is mediated by immunoglobuline G (IgG) anti-D antibody binding. The propensity of cells to agglutinate is related to their interphasic (electrokinetic) properties. These properties significantly depend on the concentration of proteolytic papain enzyme and protease-free neuraminidase enzyme that the cells are exposed to. The analysis is based on the interpretation of electrophoretic data of cells by means of the numerical theory for the electrokinetics of soft (bio)particles. A significant reduction of the hydrodynamic permeability of the external soft glycoprotein layer of the cells is reported under the action of papain. This reflects a significant decrease in soft surface layer thickness and a loss in cell surface integrity/rigidity, as confirmed by nanomechanical AFM analysis. Neuraminidase action leads to an important decrease in the interphase charge density by removing sialic acids from the cell soft surface layer. This is accompanied by hydrodynamic softness modulations less significant than those observed for papain-treated cells. On the basis of these electrohydrodynamic characteristics, the overall interaction potential profiles between two native cells and two enzyme-treated cells are derived as a function of the soft surface layer thickness in the Debye-Hückel limit that is valid for cell suspensions under physiological conditions (approximately 0.16 M). The thermodynamic computation of cell suspension stability against IgG-mediated agglutination then reveals that a decrease in the cell surface layer thickness is more favorable than a decrease in interphase charge density for inducing agglutination. This is experimentally confirmed by agglutination data collected for papain- and neuraminidase-treated cells.

Novel materials to enhance corneal epithelial cell migration on keratoprosthesis.

PubMed

Karkhaneh, Akbar; Mirzadeh, Hamid; Ghaffariyeh, Alireza; Ebrahimi, Abdolali; Honarpisheh, Nazafarin; Hosseinzadeh, Masud; Heidari, Mohammad Hossein

2011-03-01

To introduce a new modification for silicone optical core Keratoprosthesis. Using mixtures of 2-hydroxyethyl methacrylate and acrylic acid polydimethylsiloxane (PDMS) films were modified with two-step oxygen plasma treatment, and then type I collagen was immobilised onto this modified surfaces. Both the biocompatibility of the modified films and cell behaviour on the surface of these films were investigated by in vitro tests, and formation of epithelial cell layer was evaluated by implantation of the modified films in the corneas of 10 rabbits. In vitro studies indicated that the number of attached and proliferated cells onto modified PDMS in comparison with the unmodified PDMS significantly increased. Histological studies showed that corneal epithelial cells migrated on the anterior surface of the modified films after 1week. The corneal epithelial cell formed an incomplete monolayer cellular sheet after 10days. A complete epithelialisation on the modified surface was formed after 21days. The epithelial layer persisted on the anterior surface of implant after 1-month and 3-month follow-up. This method may have potential use in silicone optical core Keratoprosthesis.

Molecular dynamics investigation of the ionic liquid/enzyme interface: application to engineering enzyme surface charge.

PubMed

Burney, Patrick R; Nordwald, Erik M; Hickman, Katie; Kaar, Joel L; Pfaendtner, Jim

2015-04-01

Molecular simulations of the enzymes Candida rugosa lipase and Bos taurus α-chymotrypsin in aqueous ionic liquids 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate were used to study the change in enzyme-solvent interactions induced by modification of the enzyme surface charge. The enzymes were altered by randomly mutating lysine surface residues to glutamate, effectively decreasing the net surface charge by two for each mutation. These mutations resemble succinylation of the enzyme by chemical modification, which has been shown to enhance the stability of both enzymes in ILs. After establishing that the enzymes were stable on the simulated time scales, we focused the analysis on the organization of the ionic liquid substituents about the enzyme surface. Calculated solvent charge densities show that for both enzymes and in both solvents that changing positively charged residues to negative charge does indeed increase the charge density of the solvent near the enzyme surface. The radial distribution of IL constituents with respect to the enzyme reveals decreased interactions with the anion are prevalent in the modified systems when compared to the wild type, which is largely accompanied by an increase in cation contact. Additionally, the radial dependence of the charge density and ion distribution indicates that the effect of altering enzyme charge is confined to short range (≤1 nm) ordering of the IL. Ultimately, these results, which are consistent with that from prior experiments, provide molecular insight into the effect of enzyme surface charge on enzyme stability in ILs. © 2015 Wiley Periodicals, Inc.

Depletion of regulatory T cells by anti-ICOS antibody enhances anti-tumor immunity of tumor cell vaccine in prostate cancer.

PubMed

Mo, Lijun; Chen, Qianmei; Zhang, Xinji; Shi, Xiaojun; Wei, Lili; Zheng, Dianpeng; Li, Hongwei; Gao, Jimin; Li, Jinlong; Hu, Zhiming

2017-10-13

ICOS + Treg cells exert important immunosuppressive effects in tumor immunity. We adopt a combination approach of ICOS + Treg cells depletion with tumor cell vaccine to evaluate anti-tumor immunity in mouse prostate cancer model. Streptavidin (SA)-mGM-CSF surface-modified RM-1 cells were prepared as the vaccine and the mouse subcutaneous prostate tumor model was used to evaluate the immunity. Tumor growth, flow cytometry, immunohistochemistry, immunofluorescence and enzyme linked immunosorbent assay (ELISA) were performed to evaluate the therapeutic effects. Our results demonstrated that SA-mGM-CSF vaccine was prepared successfully and tumor growth was inhibited. The tumor size in the combination group was much smaller than that in the vaccine with IgG mAb group. The portions of dendritic cells, CD8 + and CD4 + T cells in the mice blood and tumor tissues were increased after treatment with vaccine. There were more immune-suppressing Tregs infiltrated into tumor after treatment with tumor cell vaccine, and ICOS blocking could deplete the infiltrated Tregs, and T lymphocytes increased more dramatically in the combination therapy group. The concentrations of interferon-γ were increased in all vaccine group, the concentrations of Interleukin-10 and Interleukin-4 were much lower in the combination group. Our study demonstrated that ICOS blocking could deplete the tumor-infiltrated ICOS + Treg cells. Combining GM-CSF surface-modified RM-1 cell vaccine with Anti-ICOS antibody could induce better antitumor immunity than a vaccine alone. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

PubMed

Tanaka, Tsutomu; Kondo, Akihiko

2015-02-01

In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Novel amphiphilic poly(dimethylsiloxane) based polyurethane networks tethered with carboxybetaine and their combined antibacterial and anti-adhesive property

NASA Astrophysics Data System (ADS)

Jiang, Jingxian; Fu, Yuchen; Zhang, Qinghua; Zhan, Xiaoli; Chen, Fengqiu

2017-08-01

The traditional nonfouling materials are powerless against bacterial cells attachment, while the hydrophobic bactericidal surfaces always suffer from nonspecific protein adsorption and dead bacterial cells accumulation. Here, amphiphilic polyurethane (PU) networks modified with poly(dimethylsiloxane) (PDMS) and cationic carboxybetaine diol through simple crosslinking reaction were developed, which had an antibacterial efficiency of 97.7%. Thereafter, the hydrolysis of carboxybetaine ester into zwitterionic groups brought about anti-adhesive properties against bacteria and proteins. The surface chemical composition and wettability performance of the PU network surfaces were investigated by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and contact angle analysis. The surface distribution of PDMS and zwitterionic segments produced an obvious amphiphilic heterogeneous surface, which was demonstrated by atomic force microscopy (AFM). Enzyme-linked immunosorbent assays (ELISA) were used to test the nonspecific protein adsorption behaviors. With the advantages of the transition from excellent bactericidal performance to anti-adhesion and the combination of fouling resistance and fouling release property, the designed PDMS-based amphiphilic PU network shows great application potential in biomedical devices and marine facilities.

Expression of a cloned lipopolysaccharide antigen from Neisseria gonorrhoeae on the surface of Escherichia coli K-12.

PubMed Central

Palermo, D A; Evans, T M; Clark, V L

1987-01-01

A gonococcal gene bank maintained in Escherichia coli K-12 was screened by colony immunoblotting, and a transformant expressing a surface antigen reactive to anti-gonococcal outer membrane antiserum was isolated. The isolate carried a recombinant plasmid, pTME6, consisting of approximately 9 kilobases of Neisseria gonorrhoeae DNA inserted into the BamHI site of pBR322. Surface labeling of E. coli HB101(pTME6) confirmed that the antigen was expressed on the E. coli cell surface. The antigenic material was resistant to proteinase K digestion and sensitive to periodate oxidation, indicating that the material was carbohydrate. Purified lipopolysaccharide (LPS) from HB101(pTME6) produced a unique band on silver-stained polyacrylamide gels that contained immunoreactive material as seen on Western blots of LPS samples. Only two of three E. coli LPS mutant strains carrying pTME6 reacted with the antigonococcal antiserum, suggesting that a certain E. coli core structure is necessary for antigen expression. We conclude that pTME6 contains one or more gonococcal genes encoding an LPS core biosynthetic enzyme(s) which can modify E. coli core LPS to produce a gonococcuslike epitope(s). Images PMID:3117695

Getting to the Outer Leaflet: Physiology of Phosphatidylserine Exposure at the Plasma Membrane.

PubMed

Bevers, Edouard M; Williamson, Patrick L

2016-04-01

Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure. Copyright © 2016 the American Physiological Society.

Zwitterionic sulfobetaine polymer-immobilized surface by simple tyrosinase-mediated grafting for enhanced antifouling property.

PubMed

Kwon, Ho Joon; Lee, Yunki; Phuong, Le Thi; Seon, Gyeung Mi; Kim, Eunsuk; Park, Jong Chul; Yoon, Hyunjin; Park, Ki Dong

2017-10-01

Introducing antifouling property to biomaterial surfaces has been considered an effective method for preventing the failure of implanted devices. In order to achieve this, the immobilization of zwitterions on biomaterial surfaces has been proven to be an excellent way of improving anti-adhesive potency. In this study, poly(sulfobetaine-co-tyramine), a tyramine-conjugated sulfobetaine polymer, was synthesized and simply grafted onto the surface of polyurethane via a tyrosinase-mediated reaction. Surface characterization by water contact angle measurements, X-ray photoelectron spectroscopy and atomic force microscopy demonstrated that the zwitterionic polymer was successfully introduced onto the surface of polyurethane and remained stable for 7days. In vitro studies revealed that poly(sulfobetaine-co-tyramine)-coated surfaces dramatically reduced the adhesion of fibrinogen, platelets, fibroblasts, and S. aureus by over 90% in comparison with bare surfaces. These results proved that polyurethane surfaces grafted with poly(sulfobetaine-co-tyramine) via a tyrosinase-catalyzed reaction could be promising candidates for an implantable medical device with excellent bioinert abilities. Antifouling surface modification is one of the key strategy to prevent the thrombus formation or infection which occurs on the surface of biomaterial after transplantation. Although there are many methods to modify the surface have been reported, necessity of simple modification technique still exists to apply for practical applications. The purpose of this study is to modify the biomaterial's surface by simply immobilizing antifouling zwitterion polymer via enzyme tyrosinase-mediated reaction which could modify versatile substrates in mild aqueous condition within fast time period. After modification, pSBTA grafted surface becomes resistant to various biological factors including proteins, cells, and bacterias. This approach appears to be a promising method to impart antifouling property on biomaterial surfaces. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Novel optimization strategy for tannase production through a modified solid-state fermentation system.

PubMed

Wu, Changzheng; Zhang, Feng; Li, Lijun; Jiang, Zhedong; Ni, Hui; Xiao, Anfeng

2018-01-01

High amounts of insoluble substrates exist in the traditional solid-state fermentation (SSF) system. The presence of these substrates complicates the determination of microbial biomass. Thus, enzyme activity is used as the sole index for the optimization of the traditional SSF system, and the relationship between microbial growth and enzyme synthesis is always ignored. This study was conducted to address this deficiency. All soluble nutrients from tea stalk were extracted using water. The aqueous extract was then mixed with polyurethane sponge to establish a modified SSF system, which was then used to conduct tannase production. With this system, biomass, enzyme activity, and enzyme productivity could be measured rationally and accurately. Thus, the association between biomass and enzyme activity could be easily identified, and the shortcomings of traditional SSF could be addressed. Different carbon and nitrogen sources exerted different effects on microbial growth and enzyme production. Single-factor experiments showed that glucose and yeast extract greatly improved microbial biomass accumulation and that tannin and (NH 4 ) 2 SO 4 efficiently promoted enzyme productivity. Then, these four factors were optimized through response surface methodology. Tannase activity reached 19.22 U/gds when the added amounts of tannin, glucose, (NH 4 ) 2 SO 4 , and yeast extract were 7.49, 8.11, 9.26, and 2.25%, respectively. Tannase activity under the optimized process conditions was 6.36 times higher than that under the initial process conditions. The optimized parameters were directly applied to the traditional tea stalk SSF system. Tannase activity reached 245 U/gds, which is 2.9 times higher than our previously reported value. In this study, a modified SSF system was established to address the shortcomings of the traditional SSF system. Analysis revealed that enzymatic activity and microbial biomass are closely related, and different carbon and nitrogen sources have different effects on microbial growth and enzyme production. The maximal tannase activity was obtained under the optimal combination of nutrient sources that enhances cell growth and tannase accumulation. Moreover, tannase production through the traditional tea stalk SSF was markedly improved when the optimized parameters were applied. This work provides an innovative approach to bioproduction research through SSF.

Dithiothreitol-Regulated Coverage of Oligonucleotide-Modified Gold Nanoparticles To Achieve Optimized Biosensor Performance.

PubMed

Liang, Pingping; Canoura, Juan; Yu, Haixiang; Alkhamis, Obtin; Xiao, Yi

2018-01-31

DNA-modified gold nanoparticles (AuNPs) are useful signal-reporters for detecting diverse molecules through various hybridization- and enzyme-based assays. However, their performance is heavily dependent on the probe DNA surface coverage, which can influence both target binding and enzymatic processing of the bound probes. Current methods used to adjust the surface coverage of DNA-modified AuNPs require the production of multiple batches of AuNPs under different conditions, which is costly and laborious. We here develop a single-step assay utilizing dithiothreitol (DTT) to fine-tune the surface coverage of DNA-modified AuNPs. DTT is superior to the commonly used surface diluent, mercaptohexanol, as it is less volatile, allowing for the rapid and reproducible controlling of surface coverage on AuNPs with only micromolar concentrations of DTT. Upon adsorption, DTT forms a dense monolayer on gold surfaces, which provides antifouling capabilities. Furthermore, surface-bound DTT adopts a cyclic conformation, which reorients DNA probes into an upright position and provides ample space to promote DNA hybridization, aptamer assembly, and nuclease digestion. We demonstrate the effects of surface coverage on AuNP-based sensors using DTT-regulated DNA-modified AuNPs. We then use these AuNPs to visually detect DNA and cocaine in colorimetric assays based on enzyme-mediated AuNP aggregation. We determine that DTT-regulated AuNPs with lower surface coverage achieve shorter reaction times and lower detection limits relative to those for assays using untreated AuNPs or DTT-regulated AuNPs with high surface coverage. Additionally, we demonstrate that our DTT-regulated AuNPs can perform cocaine detection in 50% urine without any significant matrix effects. We believe that DTT regulation of surface coverage can be broadly employed for optimizing DNA-modified AuNP performance for use in biosensors as well as drug delivery and therapeutic applications.

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase.

PubMed

Nelson, Jennifer; Francom, Lyndee L; Anderson, Lynn; Damm, Kelly; Baker, Ryan; Chen, Joseph; Franklin, Sarah; Hamaker, Amy; Izidoro, Izadora; Moss, Eric; Orton, Mikayla; Stevens, Evan; Yeung, Celestine; Judd, Allan M; Bell, John D

2012-05-01

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. Copyright © 2012 Elsevier B.V. All rights reserved.

Restricting detergent protease action to surface of protein fibres by chemical modification.

PubMed

Schroeder, M; Lenting, H B M; Kandelbauer, A; Silva, C J S M; Cavaco-Paulo, A; Gübitz, G M

2006-10-01

Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1'-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (DeltaE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres.

Influence of surfaces modified with biomimetic extracellular matrices on adhesion and proliferation of mesenchymal stem cells and osteosarcoma cells.

PubMed

Cai, Rong; Kawazoe, Naoki; Chen, Guoping

2015-02-01

Preparation of surfaces modified with biomimetic extracellular matrices (ECMs) is important for investigation of the interaction between ECMs and cells. In the present study, surfaces modified with ECMs from normal somatic cells, stem cells and tumor cells were prepared by cell culture method. The ECMs derived from bone marrow-derived mesenchymal stem cells (MSCs), dermal fibroblasts (FBs), osteoblasts (OBs) and MG63 osteosarcoma cells were deposited on the surfaces of cell-culture polystyrene plates (TCPS). The ECMs from different cell types had different compositions. The effects of the ECM-deposited surfaces on the adhesion, spreading and proliferation of MSCs and MG63 human osteosarcoma cells were dependent on the type of both ECMs and cells. The surfaces deposited with ECMs from MSCs, FBs and OBs promoted cell adhesion more strongly than surfaces deposited with ECMs from MG63 cells and TCPS. Compared to TCPS, the ECM-deposited surfaces promoted proliferation of MSCs while they inhibited the proliferation of MG63 cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Host cells and methods for producing 3-methyl-2-buten-1-ol, 3-methyl-3-buten-1-ol, and 3-methyl-butan-1-ol

DOEpatents

Chou, Howard H [Berkeley, CA; Keasling, Jay D [Berkeley, CA

2011-07-26

The invention provides for a method for producing a 5-carbon alcohol in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses a first enzyme capable of catalyzing the dephosphorylation of an isopentenyl pyrophosphate (IPP) or dimethylallyl diphosphate (DMAPP), such as a Bacillus subtilis phosphatase (YhfR), under a suitable condition so that 5-carbon alcohol is 3-methyl-2-buten-1-ol and/or 3-methyl-3-buten-1-ol is produced. Optionally, the host cell may further comprise a second enzyme capable of reducing a 3-methyl-2-buten-1-ol to 3-methyl-butan-1-ol, such as a reductase.

Combining an Optical Resonance Biosensor with Enzyme Activity Kinetics to Understand Protein Adsorption and Denaturation

PubMed Central

Wilson, Kerry A.; Finch, Craig A.; Anderson, Phillip; Vollmer, Frank; Hickman, James J.

2014-01-01

Understanding protein adsorption and resultant conformation changes on modified and unmodified silicon dioxide surfaces is a subject of keen interest in biosensors, microfluidic systems and for medical diagnostics. However, it has been proven difficult to investigate the kinetics of the adsorption process on these surfaces as well as understand the topic of the denaturation of proteins and its effect on enzyme activity. A highly sensitive optical whispering gallery mode (WGM) resonator was used to study a catalytic enzyme’s adsorption processes on different silane modified glass substrates (plain glass control, DETA, 13F, and SiPEG). The WGM sensor was able to obtain high resolution kinetic data of glucose oxidase (GO) adsorption with sensitivity of adsorption better than that possible with SPR. The kinetic data, in combination with a functional assay of the enzyme activity, was used to test hypotheses on adsorption mechanisms. By fitting numerical models to the WGM sensograms for protein adsorption, and by confirming numerical predictions of enzyme activity in a separate assay, we were able to identify mechanisms for GO adsorption on different alkylsilanes and infer information about the adsorption of protein on nanostructured surfaces. PMID:25453976

Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord.

PubMed

Kadam, Sachin S; Tiwari, Shubha; Bhonde, Ramesh R

2009-01-01

The umbilical cord represents the link between mother and fetus during pregnancy. This cord is usually discarded as a biological waste after the child's birth; however, its importance as a "store house" of stem cells has been explored recently. We developed a method of simultaneous isolation of endothelial cells (ECs) from the vein and mesenchymal stem cells from umbilical cord Wharton's jelly of the same cord. The isolation protocol has been simplified, modified, and improvised with respect to choice of enzyme and enzyme mixture, digestion time, cell yield, cell growth, and culture medium. Isolated human umbilical vascular ECs (hUVECs) were positive for von-Willibrand factor, a classical endothelial marker, and could form capillary-like structures when seeded on Matrigel, thus proving their functionality. The isolated human umbilical cord mesenchymal stem cells (hUCMSCs) were found positive for CD44, CD90, CD 73, and CD117 and were found negative for CD33, CD34, CD45, and CD105 surface markers; they were also positive for cytoskeleton markers of smooth muscle actin and vimentin. The hUCMSCs showed multilineage differentiation potential and differentiated into adipogenic, chondrogenic, osteogenic, and neuronal lineages under influence of lineage specific differentiation medium. Thus, isolating endothelial cells as well as mesenchymal cells from the same umbilical cord could lead to complete utilization of the available tissue for the tissue engineering and cell therapy.

RAMP2 Influences Glucagon Receptor Pharmacology via Trafficking and Signaling.

PubMed

Cegla, Jaimini; Jones, Ben J; Gardiner, James V; Hodson, David J; Marjot, Thomas; McGlone, Emma R; Tan, Tricia M; Bloom, Stephen R

2017-08-01

Endogenous satiety hormones provide an attractive target for obesity drugs. Glucagon causes weight loss by reducing food intake and increasing energy expenditure. To further understand the cellular mechanisms by which glucagon and related ligands activate the glucagon receptor (GCGR), we investigated the interaction of the GCGR with receptor activity modifying protein (RAMP)2, a member of the family of receptor activity modifying proteins. We used a combination of competition binding experiments, cell surface enzyme-linked immunosorbent assay, functional assays assessing the Gαs and Gαq pathways and β-arrestin recruitment, and small interfering RNA knockdown to examine the effect of RAMP2 on the GCGR. Ligands tested were glucagon; glucagonlike peptide-1 (GLP-1); oxyntomodulin; and analog G(X), a GLP-1/glucagon coagonist developed in-house. Confocal microscopy was used to assess whether RAMP2 affects the subcellular distribution of GCGR. Here we demonstrate that coexpression of RAMP2 and the GCGR results in reduced cell surface expression of the GCGR. This was confirmed by confocal microscopy, which demonstrated that RAMP2 colocalizes with the GCGR and causes significant GCGR cellular redistribution. Furthermore, the presence of RAMP2 influences signaling through the Gαs and Gαq pathways, as well as recruitment of β-arrestin. This work suggests that RAMP2 may modify the agonist activity and trafficking of the GCGR, with potential relevance to production of new peptide analogs with selective agonist activities.

Response surface methodology as an approach to determine the optimal activities of xylose reductase and xylitol dehydrogenase enzymes from Candida Mogii.

PubMed

Mayerhoff, Zea D V L; Roberto, Inês C; Franco, Telma T

2006-05-01

A central composite experimental design leading to a set of 16 experiments with different combinations of pH and temperature was performed to attain the optimal activities of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes from Candida mogii cell extract. Under optimized conditions (pH 6.5 and 38 degrees C), the XR and XDH activities were found to be 0.48 U/ml and 0.22 U/ml, respectively, resulting in an XR to XDH ratio of 2.2. Stability, cofactor specificity and kinetic parameters of the enzyme XR were also evaluated. XR activity remained stable for 3 h under 4 and 38 degrees C and for 4 months of storage at -18 degrees C. Studies on cofactor specificity showed that only NADPH-dependent XR was obtained under the cultivation conditions employed. The XR present in C. mogii extracts showed a superior Km value for xylose when compared with other yeast strains. Besides, this parameter was not modified after enzyme extraction by aqueous two-phase system.

The heparanome--the enigma of encoding and decoding heparan sulfate sulfation.

PubMed

Lamanna, William C; Kalus, Ina; Padva, Michael; Baldwin, Rebecca J; Merry, Catherine L R; Dierks, Thomas

2007-04-30

Heparan sulfate (HS) is a cell surface carbohydrate polymer modified with sulfate moieties whose highly ordered composition is central to directing specific cell signaling events. The ability of the cell to generate these information rich glycans with such specificity has opened up a new field of "heparanomics" which seeks to understand the systems involved in generating these cell type and developmental stage specific HS sulfation patterns. Unlike other instances where biological information is encrypted as linear sequences in molecules such as DNA, HS sulfation patterns are generated through a non-template driven process. Thus, deciphering the sulfation code and the dynamic nature of its generation has posed a new challenge to system biologists. The recent discovery of two sulfatases, Sulf1 and Sulf2, with the unique ability to edit sulfation patterns at the cell surface, has opened up a new dimension as to how we understand the regulation of HS sulfation patterning and pattern-dependent cell signaling events. This review will focus on the functional relationship between HS sulfation patterning and biological processes. Special attention will be given to Sulf1 and Sulf2 and how these key editing enzymes might act in concert with the HS biosynthetic enzymes to generate and regulate specific HS sulfation patterns in vivo. We will further explore the use of knock out mice as biological models for understanding the dynamic systems involved in generating HS sulfation patterns and their biological relevance. A brief overview of new technologies and innovations summarizes advances in the systems biology field for understanding non-template molecular networks and their influence on the "heparanome".

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

PubMed Central

Gründel, Anne; Pfeiffer, Melanie; Jacobs, Enno

2015-01-01

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract. PMID:26667841

Therapeutic potential of intracerebroventricular replacement of modified human β-hexosaminidase B for GM2 gangliosidosis.

PubMed

Matsuoka, Kazuhiko; Tamura, Tomomi; Tsuji, Daisuke; Dohzono, Yukie; Kitakaze, Keisuke; Ohno, Kazuki; Saito, Seiji; Sakuraba, Hitoshi; Itoh, Kohji

2011-06-01

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

Therapeutic Potential of Intracerebroventricular Replacement of Modified Human β-Hexosaminidase B for GM2 Gangliosidosis

PubMed Central

Matsuoka, Kazuhiko; Tamura, Tomomi; Tsuji, Daisuke; Dohzono, Yukie; Kitakaze, Keisuke; Ohno, Kazuki; Saito, Seiji; Sakuraba, Hitoshi; Itoh, Kohji

2011-01-01

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB. PMID:21487393

Nickel-phendione complex covalently attached onto carbon nanotube/cross linked glucose dehydrogenase as bioanode for glucose/oxygen compartment-less biofuel cell

NASA Astrophysics Data System (ADS)

Korani, Aazam; Salimi, Abdollah; Hadadzadeh, Hasan

2015-05-01

Here, [Ni(phendion) (phen)]Cl2 complex, (phendion and phen are 1,10-phenanthroline-5,6-dione and 5-amino-1, 10-phenanthrolin) covalently attached onto carboxyl functionalized multi walls carbon nanotube modified glassy carbon electrode (GCE/MWCNTs-COOH) using solid phase interactions and combinatorial approaches.The attached [Ni(phendion) (phen)]Cl2 complex displays a surface controlled electrode process and it acts as an effective redox mediator for electrocatalytic oxidation of dihydronicotinamide adenine dinucleotide (NADH) at reduced overpotentials. With co-immobilization of glucose dehydrogenase enzyme (GDH) by crosslinking an effective biocatalyst for glucose oxidation designed. The onset potential and current density are -0.1 V versus Ag/AgCl electrode and 0.550 mA cm-2, which indicate the applicability of the proposed system as an efficient bioanode for biofuel cell (BFC) design. A GCE/MWCNTs modified with electrodeposited gold nanoparticles (AuNPs) as a platform for immobilization of bilirubin oxidase (BOD) and the prepared GCE/MWCNTs/AuNPs/BOD biocathode exhibits an onset potential of 0.56 V versus Ag/AgCl. The performance of the fabricated bioanode and biocathode in a membraneless enzyme based glucose/O2 biofuel cell is evaluated. The open circuit voltage of the cell and maximum current density are 520 mV and 0.233 mA cm-2, respectively, while maximum power density of 40 μWcm-2 achieves at voltage of 280 mV with stable output power after 24 h continues operation.

Surface functionalization of a polymeric lipid bilayer for coupling a model biological membrane with molecules, cells, and microstructures.

PubMed

Morigaki, Kenichi; Mizutani, Kazuyuki; Saito, Makoto; Okazaki, Takashi; Nakajima, Yoshihiro; Tatsu, Yoshiro; Imaishi, Hiromasa

2013-02-26

We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

PubMed

Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki

2014-02-01

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.

Engineered Joint Lubrication for OA Prevention and Treatment

DTIC Science & Technology

2015-09-01

Williams, C. G., Khan, M., Manson, P. & Elisseeff, J .H. In vivo chondrogenesis of mesenchymal stem cells in photopolymerized hydrogel. Plast...protecting cells from free-radical damage20–22. Coating surfaces with HA may also physically protect the surfaces from cytokines and degrading enzymes...modification provides a biomimetic mechanism to concentrate HA on the surface. Numerous endogenous enzymes and reactive oxygen species can degrade HA

Multijunction Solar Cell Technology for Mars Surface Applications

NASA Technical Reports Server (NTRS)

Stella, Paul M.; Mardesich, Nick; Ewell, Richard C.; Mueller, Robert L.; Endicter, Scott; Aiken, Daniel; Edmondson, Kenneth; Fetze, Chris

2006-01-01

Solar cells used for Mars surface applications have been commercial space qualified AM0 optimized devices. Due to the Martian atmosphere, these cells are not optimized for the Mars surface and as a result operate at a reduced efficiency. A multi-year program, MOST (Mars Optimized Solar Cell Technology), managed by JPL and funded by NASA Code S, was initiated in 2004, to develop tools to modify commercial AM0 cells for the Mars surface solar spectrum and to fabricate Mars optimized devices for verification. This effort required defining the surface incident spectrum, developing an appropriate laboratory solar simulator measurement capability, and to develop and test commercial cells modified for the Mars surface spectrum. This paper discusses the program, including results for the initial modified cells. Simulated Mars surface measurements of MER cells and Phoenix Lander cells (2007 launch) are provided to characterize the performance loss for those missions. In addition, the performance of the MER rover solar arrays is updated to reflect their more than two (2) year operation.

Genome-wide transcriptional profiling of Botrytis cinerea genes targeting plant cell walls during infections of different hosts

PubMed Central

Blanco-Ulate, Barbara; Morales-Cruz, Abraham; Amrine, Katherine C. H.; Labavitch, John M.; Powell, Ann L. T.; Cantu, Dario

2014-01-01

Cell walls are barriers that impair colonization of host tissues, but also are important reservoirs of energy-rich sugars. Growing hyphae of necrotrophic fungal pathogens, such as Botrytis cinerea (Botrytis, henceforth), secrete enzymes that disassemble cell wall polysaccharides. In this work we describe the annotation of 275 putative secreted Carbohydrate-Active enZymes (CAZymes) identified in the Botrytis B05.10 genome. Using RNAseq we determined which Botrytis CAZymes were expressed during infections of lettuce leaves, ripe tomato fruit, and grape berries. On the three hosts, Botrytis expressed a common group of 229 potentially secreted CAZymes, including 28 pectin backbone-modifying enzymes, 21 hemicellulose-modifying proteins, 18 enzymes that might target pectin and hemicellulose side-branches, and 16 enzymes predicted to degrade cellulose. The diversity of the Botrytis CAZymes may be partly responsible for its wide host range. Thirty-six candidate CAZymes with secretion signals were found exclusively when Botrytis interacted with ripe tomato fruit and grape berries. Pectin polysaccharides are notably abundant in grape and tomato cell walls, but lettuce leaf walls have less pectin and are richer in hemicelluloses and cellulose. The results of this study not only suggest that Botrytis targets similar wall polysaccharide networks on fruit and leaves, but also that it may selectively attack host wall polysaccharide substrates depending on the host tissue. PMID:25232357

Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

PubMed

Nguyen, Hoang-Minh; Mathiesen, Geir; Stelzer, Elena Maria; Pham, Mai Lan; Kuczkowska, Katarzyna; Mackenzie, Alasdair; Agger, Jane W; Eijsink, Vincent G H; Yamabhai, Montarop; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha

2016-10-04

Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry.

Host cells and methods for producing isoprenyl alkanoates

DOEpatents

Lee, Taek Soon; Fortman, Jeffrey L.; Keasling, Jay D.

2015-12-01

The invention provides for a method of producing an isoprenyl alkanoate in a genetically modified host cell. In one embodiment, the method comprises culturing a genetically modified host cell which expresses an enzyme capable of catalyzing the esterification of an isoprenol and a straight-chain fatty acid, such as an alcohol acetyltransferase (AAT), wax ester synthase/diacylglycerol acyltransferase (WS/DGAT) or lipase, under a suitable condition so that the isoprenyl alkanoate is produced.

In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth.

PubMed

Wang, Dong-an; Ji, Jian; Sun, Yong-hong; Shen, Jia-cong; Feng, Lin-xian; Elisseeff, Jennifer H

2002-01-01

A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.

Rational engineering of a mesohalophilic carbonic anhydrase to an extreme halotolerant biocatalyst

PubMed Central

Warden, Andrew C.; Williams, Michelle; Peat, Thomas S.; Seabrook, Shane A.; Newman, Janet; Dojchinov, Greg; Haritos, Victoria S.

2015-01-01

Enzymes expressed by highly salt-tolerant organisms show many modifications compared with salt-affected counterparts including biased amino acid and lower α-helix content, lower solvent accessibility and negative surface charge. Here, we show that halotolerance can be generated in an enzyme solely by modifying surface residues. Rational design of carbonic anhydrase II is undertaken in three stages replacing 18 residues in total, crystal structures confirm changes are confined to surface residues. Catalytic activities and thermal unfolding temperatures of the designed enzymes increase at high salt concentrations demonstrating their shift to halotolerance, whereas the opposite response is found in the wild-type enzyme. Molecular dynamics calculations reveal a key role for sodium ions in increasing halotolerant enzyme stability largely through interactions with the highly ordered first Na+ hydration shell. For the first time, an approach to generate extreme halotolerance, a trait with broad application in industrial biocatalysis, in a wild-type enzyme is demonstrated. PMID:26687908

Homogalacturonan-modifying enzymes: structure, expression, and roles in plants

PubMed Central

Sénéchal, Fabien; Wattier, Christopher; Rustérucci, Christine; Pelloux, Jérôme

2014-01-01

Understanding the changes affecting the plant cell wall is a key element in addressing its functional role in plant growth and in the response to stress. Pectins, which are the main constituents of the primary cell wall in dicot species, play a central role in the control of cellular adhesion and thereby of the rheological properties of the wall. This is likely to be a major determinant of plant growth. How the discrete changes in pectin structure are mediated is thus a key issue in our understanding of plant development and plant responses to changes in the environment. In particular, understanding the remodelling of homogalacturonan (HG), the most abundant pectic polymer, by specific enzymes is a current challenge in addressing its fundamental role. HG, a polymer that can be methylesterified or acetylated, can be modified by HGMEs (HG-modifying enzymes) which all belong to large multigenic families in all species sequenced to date. In particular, both the degrees of substitution (methylesterification and/or acetylation) and polymerization can be controlled by specific enzymes such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), polygalacturonases (PGs), or pectate lyases-like (PLLs). Major advances in the biochemical and functional characterization of these enzymes have been made over the last 10 years. This review aims to provide a comprehensive, up to date summary of the recent data concerning the structure, regulation, and function of these fascinating enzymes in plant development and in response to biotic stresses. PMID:25056773

Enhanced cell-surface display of a heterologous protein using SED1 anchoring system in SED1-disrupted Saccharomyces cerevisiae strain.

PubMed

Bamba, Takahiro; Inokuma, Kentaro; Hasunuma, Tomohisa; Kondo, Akihiko

2018-03-01

Yeast displaying enzymes on the cell surface are used for developing whole-cell biocatalysts. High enzyme activity on the cell surface is required in certain applications such as direct ethanol production from lignocellulosic materials. However, the cell surface enzyme activity is limited by several factors, one of which is the protein amount of the yeast cell wall. In this study, we attempted to improve the incorporation capacity of a displayed heterologous enzyme by disrupting a native cell-wall protein. β-Glucosidase (BGL1) from Aspergillus aculeatus was fused with Saccharomyces cerevisiae Sed1 and displayed on the cell surface of S. cerevisiae BY4741 strain and its SED1 disruptant. Sed1 is one of the most abundant stationary phase yeast cell wall protein. A time course analysis revealed that BGL1 activity of the control strain reached saturation after 48 h of cultivation. In contrast, the BGL1 activity of the SED1 disruptant increased until 72 h of cultivation and was 22% higher than that of the control strain. We also performed relative quantification of cell wall proteins of these strains by nanoscale ultra pressure liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nano-UPLC-MS E ). The amount of the cell wall-associated BGL1 per unit dry cell-weight of the SED1 disruptant was 19% higher than that of the control strain. These results suggested that the incorporation capacity of the cell wall for BGL1 was increased by disruption of SED1. Disruption of SED1 would be a promising approach for improving display efficiency of heterologous protein fused with Sed1. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Production of superparamagnetic nanobiocatalysts for green chemistry applications.

PubMed

Gasser, Christoph A; Ammann, Erik M; Schäffer, Andreas; Shahgaldian, Patrick; Corvini, Philippe F-X

2016-08-01

Immobilization of enzymes on solid supports is a convenient method for increasing enzymatic stability and enabling enzyme reuse. In the present work, a sorption-assisted surface conjugation method was developed and optimized to immobilize enzymes on the surface of superparamagnetic nanoparticles. An oxidative enzyme, i.e., laccase from Trametes versicolor was used as model enzyme. The immobilization method consists of the production of superparamagnetic nanoparticles by co-precipitation of FeCl2 and FeCl3. Subsequently, the particle surface is modified with an organosilane containing an amino group. Next, the enzymes are adsorbed on the particle surface before a cross-linking agent, i.e., glutaraldehyde is added which links the amino groups on the particle surface with the amino groups of the enzymes and leads to internal cross-linking of the enzymes as well. The method was optimized using response surface methodology regarding optimal enzyme and glutaraldehyde amounts, pH, and reaction times. Results allowed formulation of biocatalysts having high specific enzymatic activity and improved stability. The biocatalysts showed considerably higher stability compared with the dissolved enzymes over a pH range from 3 to 9 and in the presence of several chemical denaturants. To demonstrate the reusability of the immobilized enzymes, they were applied as catalysts for the production of a phenoxazinone dye. Virtually, 100 % of the precursor was transformed to the dye in each of the ten conducted reaction cycles while on average 84.5 % of the enzymatic activity present at the beginning of a reaction cycle was retained after each cycle highlighting the considerable potential of superparamagnetic biocatalysts for application in industrial processes.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

PubMed Central

Itoh, Takafumi; Hibi, Takao; Suzuki, Fumiko; Sugimoto, Ikumi; Fujiwara, Akihiro; Inaka, Koji; Tanaka, Hiroaki; Ohta, Kazunori; Fujii, Yutaka; Taketo, Akira; Kimoto, Hisashi

2016-01-01

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation. PMID:27907169

Dynamic moisture sorption characteristics of enzyme-resistant recrystallized cassava starch.

PubMed

Mutungi, Christopher; Schuldt, Stefan; Onyango, Calvin; Schneider, Yvonne; Jaros, Doris; Rohm, Harald

2011-03-14

The interaction of moisture with enzyme-resistant recrystallized starch, prepared by heat-moisture treatment of debranched acid-modified or debranched non-acid-modified cassava starch, was investigated in comparison with the native granules. Crystallinities of the powdered products were estimated by X-ray diffraction. Moisture sorption was determined using dynamic vapor sorption analyzer and data fitted to various models. Percent crystallinities of native starch (NS), non-acid-modified recrystallized starch (NAMRS), and acid-modified recrystallized starch (AMRS) were 39.7, 51.9, and 56.1%, respectively. In a(w) below 0.8, sorption decreased in the order NS > NAMRS > AMRS in line with increasing sample crystallinities but did not follow this crystallinity dependence at higher a(w) because of condensation and polymer dissolution effects. Adsorbed moisture became internally absorbed in NS but not in NAMRS and AMRS, which might explain the high resistance of the recrystallized starches to digestion because enzyme and starch cannot approach each other over fairly sufficient surface at the molecular level.

Nanopillar based electrochemical biosensor for monitoring microfluidic based cell culture

NASA Astrophysics Data System (ADS)

Gangadharan, Rajan

In-vitro assays using cultured cells have been widely performed for studying many aspects of cell biology and cell physiology. These assays also form the basis of cell based sensing. Presently, analysis procedures on cell cultures are done using techniques that are not integrated with the cell culture system. This approach makes continuous and real-time in-vitro measurements difficult. It is well known that the availability of continuous online measurements for extended periods of time will help provide a better understanding and will give better insight into cell physiological events. With this motivation we developed a highly sensitive, selective and stable microfluidic electrochemical glucose biosensor to make continuous glucose measurements in cell culture media. The performance of the microfluidic biosensor was enhanced by adding 3D nanopillars to the electrode surfaces. The microfluidic glucose biosensor consisted of three electrodes---Enzyme electrode, Working electrode, and Counter electrode. All these electrodes were enhanced with nanopillars and were optimized in their respective own ways to obtain an effective and stable biosensing device in cell culture media. For example, the 'Enzyme electrode' was optimized for enzyme immobilization via either a polypyrrole-based or a self-assembled-monolayer-based immobilization method, and the 'Working electrode' was modified with Prussian Blue or electropolymerized Neutral Red to reduce the working potential and also the interference from other interacting electro-active species. The complete microfluidic biosensor was tested for its ability to monitor glucose concentration changes in cell culture media. The significance of this work is multifold. First, the developed device may find applications in continuous and real-time measurements of glucose concentrations in in-vitro cell cultures. Second, the development of a microfluidic biosensor will bring technical know-how toward constructing continuous glucose monitoring devices. Third, the methods used to develop 3D electrodes incorporated with nanopillars can be used for other applications such as neural probes, fuel cells, solar cells etc., and finally, the knowledge obtained from the immobilization of enzymes onto nanostructures sheds some new insight into nanomaterial/biomolecule interactions.

Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope

PubMed Central

Navarre, William Wiley; Schneewind, Olaf

1999-01-01

The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. PMID:10066836

A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?

PubMed Central

Thygesen, Uffe H.; Riemann, Lasse; Stedmon, Colin A.

2015-01-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. PMID:26253668

Selective fibronectin adsorption against albumin and enhanced stem cell attachment on helium atmospheric pressure glow discharge treated titanium

NASA Astrophysics Data System (ADS)

Han, Inho; Vagaska, Barbora; Joo Park, Bong; Lee, Mi Hee; Jin Lee, Seung; Park, Jong-Chul

2011-06-01

Successful tissue integration of implanted medical devices depends on appropriate initial cellular response. In this study, the effect of helium atmospheric pressure glow discharge (He-APGD) treatment of titanium on selective protein adsorption and the initial attachment processes and focal adhesion formation of osteoprogenitor cells and stem cells were examined. Titanium disks were treated in a self-designed He-APGD system. Initial attachment of MC3T3-E1 mouse pre-osteoblasts and human mesenchymal stem cells (MSCs) was evaluated by MTT assay and plasma membrane staining followed by morphometric analysis. Fibronectin adsorption was investigated by Enzyme-Linked ImmunoSorbant Assay. MSCs cell attachment to treated and non-treated titanium disks coated with different proteins was verified also in serum-free culture. Organization of actin cytoskeleton and focal adhesions was evaluated microscopically. He-APGD treatment effectively modified the titanium surfaces by creating a super-hydrophilic surface, which promoted selectively higher adsorption of fibronectin, a protein of critical importance for cell/biomaterial interaction. In two different types of cells, the He-APGD treatment enhanced the number of attaching cells as well as their attachment area. Moreover, cells had higher organization of actin cytoskeleton and focal adhesions. Faster acceptance of the material by the progenitor cells in the early phases of tissue integration after the implantation may significantly reduce the overall healing time; therefore, titanium treatment with He-APGD seems to be an effective method of surface modification of titanium for improving its tissue inductive properties.

Fabrication of high performance bioanode based on fruitful association of dendrimer and carbon nanotube used for design O2/glucose membrane-less biofuel cell with improved bilirubine oxidase biocathode.

PubMed

Korani, Aazam; Salimi, Abdollah

2013-12-15

In this study, the preparation of an integrated modified electrode based on the covalent attachment of glucose dehydrogenase (GDH) enzyme and safranin O to amine-derivative multiwalled carbon nanotubes (MWCNTs-NH2) modified glassy carbon (GC) electrode using G2.5-carboxylated PAMAM dendrimer (Den) as linking agent is reported. The obtained results indicated that the proposed system has effective bioelectrocatalytic activity toward glucose oxidation at 100 mV with onset potential of -130 mV (vs. Ag/AgCl). The performance of the prepared hybrid system of GC/MWCNTs-NH2/Den/GDH/Safranin as anode in a membraneless enzyme-based glucose/O2 biofuel cell is further evaluated. The biocathode in this system was composed of bilirubin oxidase (BOX) enzyme immobilized onto a bilirubin modified carbon nanotube GC electrode. Immobilized BOX onto CNTs/bilirubin not only show direct electron transfer but also it has excellent electrocatalytic activity toward oxygen reduction at a positive potential of 610 mV. The open circuit voltage of the cell was 590 mV. The maximum current density was 0.5 mA cm(-2), while maximum power density of 108 μW cm(-2) was achieved at voltage of 330 mV. The immobilized enzymes in anode and cathode are very stable and output power of the BFC is approximately constant after 12 h continues operation. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparative investigation of two methods for Acetylcholinesterase enzyme immobilization on modified porous silicon

NASA Astrophysics Data System (ADS)

Khaldi, Khadidja; Sam, Sabrina; Lounas, Amel; Yaddaden, Chafiaa; Gabouze, Noure-Eddine

2017-11-01

In this work, Acetylcholinesterase enzyme (AChE) was immobilized on porous silicon (PSi) surface using two strategies. In the first method, acid chains were covalently grafted on the hydrogenated PSi by hydrosilylation reaction. The obtained acid-terminated surface was activated by a reaction with N-hydroxysuccinimide (NHS) in the presence of a peptide-coupling agent N-ethyl-N‧-(3-dimethylaminopropyl)-carbodiimide (EDC), and then reacted with the amino linker of the lysine residues AChE to anchor the enzyme by a covalent amide bond. In the second procedure, the PSi surface was first hydroxylated in piranha solution, followed by a silanization reaction with 3-aminopropyltriethoxysilane (APTES) to form amine-terminated surface. Finally, AChE was attached to the terminal amine groups by an aminolysis reaction with carboxylic acid groups of AChE in the presence of NHS/EDC mixture. Fourier transform infrared spectroscopy (FTIR) confirmed the efficiency of the surface modifications. The enzymatic activity of immobilized AChE was determined by means of a colorimetric test and was discussed according to the enzyme orientation on the surface which was revealed by contact angle measurements.

Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein.

PubMed

Urban, Johannes H; Moosmeier, Markus A; Aumüller, Tobias; Thein, Marcus; Bosma, Tjibbe; Rink, Rick; Groth, Katharina; Zulley, Moritz; Siegers, Katja; Tissot, Kathrin; Moll, Gert N; Prassler, Josef

2017-11-15

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.

The development, characterization, and application of biomimetic nanoscale enzyme immobilization

NASA Astrophysics Data System (ADS)

Haase, Nicholas R.

The utilization of enzymes is of interest for applications such as biosensors and biofuel cells. Immobilizing enzymes provides a means to develop these applications. Previous immobilization efforts have been accomplished by exposing surfaces on which silica-forming molecules are present to solutions containing an enzyme and a silica precursor. This approach leads to the enzyme being entrapped in a matrix three orders of magnitude larger than the enzyme itself, resulting in low retention of enzyme activity. The research herein introduces a method for the immobilization of enzymes during the layer-by-layer buildup of Si-O and Ti-O coatings which are nanoscale in thickness. This approach is an application of a peptide-induced mineral deposition method developed in the Sandhage and Kroger groups, and it involves the alternating exposure of a surface to solutions containing the peptide protamine and then an aqueous precursor solution of silicon- or titanium-oxide at near-neutral pH. A method has been developed that enables in situ immobilization of enzymes in the protamine/mineral oxide coatings. Depending on the layer and mineral (silica or titania) within which the enzyme is incorporated, the resulting multilayer biocatalytic hybrid materials retain 20 -- 100% of the enzyme activity. Analyses of kinetic properties of the immobilized enzyme, coupled with characterization of physical properties of the mineral-bearing layers (thickness, porosity, pore size distribution), indicates that the catalytic activities of the enzymes immobilized in the different layers are largely determined by substrate diffusion. The enzyme was also found to be substantially stabilized against heat-induced denaturation and largely protected from proteolytic attack. These functional coatings are then developed for use as antimicrobial materials. Glucose oxidase, which catalyzes production of the cytotoxic agent hydrogen peroxide, was immobilized with silver nanoparticles, can release antimicrobial silver ions. It is demonstrated that these two antimicrobial agents work in a synergistic manner for enhanced antimicrobial efficacy. Evidence of the proposed mechanism of synergy, namely enhanced release of silver ions by reaction of H2O2 with silver nanoparticles, is provided. Finally, the deployment of these materials in silk fibroins for development as wound dressings is also presented. Protamine cross-linking was then extended to the oxygen-reducing enzyme laccase to explore the use of this modified enzyme in an enzymatic biocathode. In this application laccase accepts electrons from the electrode and uses them to reduce oxygen to water molecules. The protamine-cross-linked enzyme exhibits a higher degree of immobilization, better retention of activity once immobilized, and superior electrochemical activity versus the native enzyme. Finally, preliminary research on the structure-function relationships of 16-mer peptides which adsorb to surfaces and deposit titanium oxide is presented. Specifically, the effect of content and distribution of arginine residues on the ability of peptides to adsorb to surfaces and subsequently deposit mineral oxides was investigated. The data demonstrate that surface adsorption of the peptides relies on both a critical number of arginine residues and their position within the peptide. Furthermore, the exchange of serine against arginine residues in surface-adsorbed peptides is detrimental to Ti-O deposition.

Structural and Functional Analysis of a Novel Interaction Motif within UFM1-activating Enzyme 5 (UBA5) Required for Binding to Ubiquitin-like Proteins and Ufmylation*

PubMed Central

Habisov, Sabrina; Huber, Jessica; Ichimura, Yoshinobu; Akutsu, Masato; Rogova, Natalia; Loehr, Frank; McEwan, David G.; Johansen, Terje; Dikic, Ivan; Doetsch, Volker; Komatsu, Masaaki; Rogov, Vladimir V.; Kirkin, Vladimir

2016-01-01

The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway. PMID:26929408

Miniaturized flow system based on enzyme modified PMMA microreactor for amperometric determination of glucose.

PubMed

Cerdeira Ferreira, Luís Marcos; da Costa, Eric Tavares; do Lago, Claudimir Lucio; Angnes, Lúcio

2013-09-15

This paper describes the development of a microfluidic system having as main component an enzymatic reactor constituted by a microchannel assembled in poly(methyl methacrylate) (PMMA) substrate connected to an amperometric detector. A CO2 laser engraving machine was used to make the channels, which in sequence were thermally sealed. The internal surfaces of the microchannels were chemically modified with polyethyleneimine (PEI), which showed good effectiveness for the immobilization of the glucose oxidase enzyme using glutaraldehyde as crosslinking agent, producing a very effective microreactor for the detection of glucose. The hydrogen peroxide generated by the enzymatic reaction was detected in an electrochemical flow cell localized outside of the reactor using a platinum disk as the working electrode. The proposed system was applied to the differential amperometric determination of glucose content in soft drinks showing good repeatability (DPR=1.72%, n=50), low detection limit (1.40×10(-6)molL(-1)), high sampling frequency (calculated as 345 samples h(-1)), and relatively good stability for long-term use. The results were in close agreement with those obtained by the classical spectrophotometric method utilized to quantify glucose in biological fluids. Copyright © 2013 Elsevier B.V. All rights reserved.

Modification of Glucose Oxidase biofuel cell by multi-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Lotfi, Ladan; Farahbakhsh, Afshin; Aghili, Sina

2018-01-01

Biofuel cells are a subset of fuel cells that employ biocatalysts. Enzyme-based biofuel cells (EBFCs) generate electrical energy from biofuels such as glucose and ethanol, which are renewable and sustainable energy sources. Glucose biofuel cells (GBFCs) are particularly interesting nowadays due to continuous harvesting of oxygen and glucose from bioavailable substrates, activity inside the human body, and environmental benign, which generate electricity through oxidation of glucose on the anode and reduction of oxygen on the cathode. Promoting the electron transfer of redox enzymes at modified electrode utilizing Nano size materials, such as carbon nanotubes (CNT), to achieve the direct electrochemistry of enzymes has been reported. The polypyrrole-MWCNTs-glucose oxidase (PY-CNT-GOx) electrode has been investigated in the present work. Cyclic voltammetry tests were performed in a three-electrode electrochemical set-up with modified electrode (Pt/PPy/MWCNTs/GOx) was used as working electrode. Platinum flat and Ag/AgCl (saturated KCl) were used as counter electrode and the reference electrode, respectively. The biofuel cells probe was prepared by immobilizing MWCNTs at the tip of a platinum (Pt) electrode (0.5 cm2) with PPy as the support matrix We have demonstrated a well-dispersed nanomaterial PPy/MWNT, which is able to immobilize GOx firmly under the condition of the absence of any other cross-linking agent.

Metabolic Glyco-Engineering in Eukaryotic Cells and Selected Applications.

PubMed

Piller, Friedrich; Mongis, Aline; Piller, Véronique

2015-01-01

By metabolic glyco-engineering cellular glycoconjugates are modified through the incorporation of synthetic monosaccharides which are usually analogues of naturally present sugars. In order to get incorporated, the monosaccharides need to enter the cytoplasm and to be substrates for the enzymes necessary for their transformation into activated sugars, most often nucleotide sugars. These have to be substrates for glycosyltransferases which finally catalyze their incorporation into glycans. Such pathways are difficult to reconstitute in vitro and therefore new monosaccharide analogues have to be tested in tissue culture for their suitability in metabolic glyco-engineering. For this, glycosylation mutants are the most appropriate since they are unable to synthesize specific glycans but through the introduction of the monosaccharide analogues they may express some glycans at the cell surface with the unnatural sugar incorporated. The presence of those glycans can be easily and quantitatively detected by lectin binding or by chemical methods identifying specific sugars. Monosaccharide analogues can also block the pathways leading to sugar incorporation, thus inhibiting the synthesis of glycan structures which is also easily detectable at the cell surface by lectin labeling. The most useful and most frequently employed application of metabolic glyco-engineering is the introduction of reactive groups which can undergo bio-orthogonal click reactions for the efficient labeling of glycans at the surface of live cells.

Arabidopsis TCH4, regulated by hormones and the environment, encodes a xyloglucan endotransglycosylase

NASA Technical Reports Server (NTRS)

Xu, W.; Purugganan, M. M.; Polisensky, D. H.; Antosiewicz, D. M.; Fry, S. C.; Braam, J.

1995-01-01

Adaptation of plants to environmental conditions requires that sensing of external stimuli be linked to mechanisms of morphogenesis. The Arabidopsis TCH (for touch) genes are rapidly upregulated in expression in response to environmental stimuli, but a connection between this molecular response and developmental alterations has not been established. We identified TCH4 as a xyloglucan endotransglycosylase by sequence similarity and enzyme activity. Xyloglucan endotransglycosylases most likely modify cell walls, a fundamental determinant of plant form. We determined that TCH4 expression is regulated by auxin and brassinosteroids, by environmental stimuli, and during development, by a 1-kb region. Expression was restricted to expanding tissues and organs that undergo cell wall modification. Regulation of genes encoding cell wall-modifying enzymes, such as TCH4, may underlie plant morphogenetic responses to the environment.

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared from refined beef fat; butterfat or...

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

21 CFR 184.1287 - Enzyme-modified fats.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Enzyme-modified fats. 184.1287 Section 184.1287... Listing of Specific Substances Affirmed as GRAS § 184.1287 Enzyme-modified fats. (a) Enzyme-modified refined beef fat, enzyme-modified butterfat, and enzyme-modified steam-rendered chicken fat are prepared...

Taxane recovery from cells of Taxus in micro- and hypergravity

NASA Technical Reports Server (NTRS)

Durzan, D. J.; Ventimiglia, F.; Havel, L.

1998-01-01

Cell suspension cultures of Taxus cuspidata produce taxanes that are released from the outer surface of cells into the culture medium as free and bound alkaloids. Paclitaxel (Taxol (TM)), is an anti-cancer drug in short supply. It has a taxane ring derived from baccatin III and a C-13 phenylisoserine side-chain. This drug is produced over a wide range of gravitational forces. Monoclonal and polyclonal antibodies to paclitaxel, baccatin III, and the C-13 phenylisoserine side chain were combined in multiple-labeling studies to localize taxanes and paclitaxel on cell surfaces or on particles released into the culture medium. Bioreactor vessel design altered the composition of taxanes recovered from cells in simulated microgravity. At 10(-2) and 2x10(-4)g, taxane recovery was reduced but biomass growth and percent paclitaxel was significantly increased. At 1 to 24g, growth was reduced with a significant recovery of total taxanes with low percent paclitaxel. Bound paclitaxel was also localized in endonuclease-rich fragmenting nuclei of individual apoptotic cells. A model is presented comprising TCH (touch) genes encoding enzymes that modify taxane-bearing xylan residues in cell walls, the calcium-sensing of gravitational forces by the cytoplasm, and the predisposition of nuclei to apoptosis. This integrates the adaptive physiological and biochemical responses of drug-producing genomes with gravitational forces.

Mechanistic insights into the regulation of metabolic enzymes by acetylation

PubMed Central

2012-01-01

The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

Rhamnogalacturonan I containing homogalacturonan inhibits colon cancer cell proliferation by decreasing ICAM1 expression

USDA-ARS?s Scientific Manuscript database

Pectin modified with pH, heat or enzymes, has previously been shown to exhibit anti-cancer activity. However, the structural requirements for bioactive modified pectins have rarely been addressed. In this study several pectin extracts representing different structural components of pectin were asses...

Viologen-modified electrodes for protection of hydrogenases from high potential inactivation while performing H2 oxidation at low overpotential.

PubMed

Oughli, Alaa A; Vélez, Marisela; Birrell, James A; Schuhmann, Wolfgang; Lubitz, Wolfgang; Plumeré, Nicolas; Rüdiger, Olaf

2018-06-08

In this work we present a viologen-modified electrode providing protection for hydrogenases against high potential inactivation. Hydrogenases, including O2-tolerant classes, suffer from reversible inactivation upon applying high potentials, which limits their use in biofuel cells to certain conditions. Our previously reported protection strategy based on the integration of hydrogenase into redox matrices enabled the use of these biocatalysts in biofuel cells even under anode limiting conditions. However, mediated catalysis required application of an overpotential to drive the reaction, and this translates into a power loss in a biofuel cell. In the present work, the enzyme is adsorbed on top of a covalently-attached viologen layer which leads to mixed, direct and mediated, electron transfer processes; at low overpotentials, the direct electron transfer process generates a catalytic current, while the mediated electron transfer through the viologens at higher potentials generates a redox buffer that prevents oxidative inactivation of the enzyme. Consequently, the enzyme starts the catalysis at no overpotential with viologen self-activated protection at high potentials.

Antibiotic Algae by Chemical Surface Engineering.

PubMed

Kerschgens, Isabel P; Gademann, Karl

2018-03-02

Chemical cell-surface engineering is a tool for modifying and altering cellular functions. Herein, we report the introduction of an antibiotic phenotype to the green alga Chlamydomonas reinhardtii by chemically modifying its cell surface. Flow cytometry and confocal microscopy studies demonstrated that a hybrid of the antibiotic vancomycin and a 4-hydroxyproline oligomer binds reversibly to the cell wall without affecting the viability or motility of the cells. The modified cells were used to inhibit bacterial growth of Gram-positive Bacillus subtilis cultures. Delivery of the antibiotic from the microalgae to the bacterial cells was verified by microscopy. Our studies provide compelling evidence that 1) chemical surface engineering constitutes a useful tool for the introduction of new, previously unknown functionality, and 2) living microalgae can serve as new platforms for drug delivery. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A model of extracellular enzymes in free-living microbes: which strategy pays off?

PubMed

Traving, Sachia J; Thygesen, Uffe H; Riemann, Lasse; Stedmon, Colin A

2015-11-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

PES Surface Modification Using Green Chemistry: New Generation of Antifouling Membranes.

PubMed

Nady, Norhan

2016-04-18

A major limitation in using membrane-based separation processes is the loss of performance due to membrane fouling. This drawback can be addressed thanks to surface modification treatments. A new and promising surface modification using green chemistry has been recently investigated. This modification is carried out at room temperature and in aqueous medium using green catalyst (enzyme) and nontoxic modifier, which can be safely labelled "green surface modification". This modification can be considered as a nucleus of new generation of antifouling membranes and surfaces. In the current research, ferulic acid modifier and laccase bio-catalyst were used to make poly(ethersulfone) (PES) membrane less vulnerable to protein adsorption. The blank and modified PES membranes are evaluated based on e.g., their flux and protein repellence. Both the blank and the modified PES membranes (or laminated PES on silicon dioxide surface) are characterized using many techniques e.g., SEM, EDX, XPS and SPM, etc. The pure water flux of the most modified membranes was reduced by 10% on average relative to the blank membrane, and around a 94% reduction in protein adsorption was determined. In the conclusions section, a comparison between three modifiers-ferulic acid, and two other previously used modifiers (4-hydroxybenzoic acid and gallic acid)-is presented.

An improved glucose/O2 membrane-less biofuel cell through glucose oxidase purification.

PubMed

Gao, Feng; Courjean, Olivier; Mano, Nicolas

2009-10-15

A key objective in any bioelectrochemical systems is to improve the current densities and mass transport limitation. Most of the work is focused on increasing the specific surface of the electrodes or improving the electron transfer between enzymes and electrodes. However, nothing is said about the comparison of purified and non-purified enzyme and their effects on the biosensor efficiency. To illustrate the effect of the enzyme purity, we studied the widely used commercial Glucose Oxidase (GOx) from Aspergillus niger that we are using in our miniature membrane-less biofuel cell. Our results indicate that even if additional compounds contained in the lyophilized enzyme powder do not interfere with its intrinsic catalytic properties, they could prevent a good electron transfer between the enzyme and the electrode surface. By introducing a purified glucose oxidase into a bioelectrocatalyst immobilized on an electrode surface, we show that we can increase the interaction between the enzyme and the redox polymer, forming a better homogenous, leather like gel. At 5mM glucose concentration and under oxygen atmosphere, the current is three-fold higher when using a purified enzyme than it is when using a non-purified enzyme. Built with this novel anode, we showed that a miniature implantable membrane-less glucose-O(2) biofuel cell could produce, under air, twice the power density that is usually obtained when using a non-purified GOx.

Engineering a biospecific communication pathway between cells and electrodes

NASA Astrophysics Data System (ADS)

Collier, Joel H.; Mrksich, Milan

2006-02-01

Methods for transducing the cellular activities of mammalian cells into measurable electronic signals are important in many biotechnical applications, including biosensors, cell arrays, and other cell-based devices. This manuscript describes an approach for functionally integrating cellular activities and electrical processes in an underlying substrate. The cells are engineered with a cell-surface chimeric receptor that presents the nonmammalian enzyme cutinase. Action of this cell-surface cutinase on enzyme substrate self-assembled monolayers switches a nonelectroactive hydroxyphenyl ester to an electroactive hydroquinone, providing an electrical activity that can be identified with cyclic voltammetry. In this way, cell-surface enzymatic activity is transduced into electronic signals. The development of strategies to directly interface the activities of cells with materials will be important to enabling a broad class of hybrid microsystems that combine living and nonliving components. biomaterial | extracellular matrix | signal transduction

The Role of Heparanase and Sulfatases in the Modification of Heparan Sulfate Proteoglycans within the Tumor Microenvironment and Opportunities for Novel Cancer Therapeutics

PubMed Central

Hammond, Edward; Khurana, Ashwani; Shridhar, Viji; Dredge, Keith

2014-01-01

Heparan sulfate proteoglycans (HSPGs) are an integral and dynamic part of normal tissue architecture at the cell surface and within the extracellular matrix. The modification of HSPGs in the tumor microenvironment is known to result not just in structural but also functional consequences, which significantly impact cancer progression. As substrates for the key enzymes sulfatases and heparanase, the modification of HSPGs is typically characterized by the degradation of heparan sulfate (HS) chains/sulfation patterns via the endo-6-O-sulfatases (Sulf1 and Sulf2) or by heparanase, an endo-glycosidase that cleaves the HS polymers releasing smaller fragments from HSPG complexes. Numerous studies have demonstrated how these enzymes actively influence cancer cell proliferation, signaling, invasion, and metastasis. The activity or expression of these enzymes has been reported to be modified in a variety of cancers. Such observations are consistent with the degradation of normal architecture and basement membranes, which are typically compromised in metastatic disease. Moreover, recent studies elucidating the requirements for these proteins in tumor initiation and progression exemplify their importance in the development and progression of cancer. Thus, as the influence of the tumor microenvironment in cancer progression becomes more apparent, the focus on targeting enzymes that degrade HSPGs highlights one approach to maintain normal tissue architecture, inhibit tumor progression, and block metastasis. This review discusses the role of these enzymes in the context of the tumor microenvironment and their promise as therapeutic targets for the treatment of cancer. PMID:25105093

Expression of E-selectin ligand-1 (CFR/ESL-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis.

PubMed

Antoine, Marianne; Tag, Carmen G; Gressner, Axel M; Hellerbrand, Claus; Kiefer, Paul

2009-02-01

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Ming-Hung; School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan; Haung, Chiung-Fang

In this study, neodymium-doped yttrium orthovanadate (Nd:YVO{sub 4}) as a laser source with different scanning speeds was used on biomedical Ti surface. The microstructural and biological properties of laser-modified samples were investigated by means of optical microscope, electron microscope, X-ray diffraction, surface roughness instrument, contact angle and cell cytotoxicity assay. After laser modification, the rough volcano-like recast layer with micro-/nanoporous structure and wave-like recast layer with nanoporous structure were generated on the surfaces of laser-modified samples, respectively. It was also found out that, an α → (α + rutile-TiO{sub 2}) phase transition occurred on the recast layers of laser-modified samples.more » The Ti surface becomes hydrophilic at a high speed laser scanning. Moreover, the cell cytotoxicity assay demonstrated that laser-modified samples did not influence the cell adhesion and proliferation behaviors of osteoblast (MG-63) cell. The laser with 50 mm/s scanning speed induced formation of rough volcano-like recast layer accompanied with micro-/nanoporous structure, which can promote cell adhesion and proliferation of MG-63 cell on Ti surface. The results indicated that the laser treatment was a potential technology to enhance the biocompatibility for titanium. - Highlights: • Laser induced the formation of recast layer with micro-/nanoporous structure on Ti. • An α → (α + rutile-TiO{sub 2}) phase transition was observed within the recast layer. • The Ti surface becomes hydrophilic at a high speed laser scanning. • Laser-modified samples exhibit good biocompatibility to osteoblast (MG-63) cell.« less

Clostridium perfringens Iota-Toxin: Mapping of Receptor Binding and Ia Docking Domains on Ib

PubMed Central

Marvaud, Jean-Christophe; Smith, Theresa; Hale, Martha L.; Popoff, Michel R.; Smith, Leonard A.; Stiles, Bradley G.

2001-01-01

Clostridium perfringens iota-toxin is a binary toxin consisting of iota a (Ia), an ADP-ribosyltransferase that modifies actin, and iota b (Ib), which binds to a cell surface protein and translocates Ia into a target cell. Fusion proteins of recombinant Ib and truncated variants were tested for binding to Vero cells and docking with Ia via fluorescence-activated cytometry and cytotoxicity experiments. C-terminal residues (656 to 665) of Ib were critical for cell surface binding, and truncated Ib variants containing ≥200 amino acids of the C terminus were effective Ib competitors and prevented iota cytotoxicity. The N-terminal domain (residues 1 to 106) of Ib was important for Ia docking, yet this region was not an effective competitor of iota cytotoxicity. Further studies showed that Ib lacking just the N-terminal 27 residues did not facilitate Ia entry into a target cell and subsequent cytotoxicity. Five monoclonal antibodies against Ib were also tested with each truncated Ib variant for epitope and structural mapping by surface plasmon resonance and an enzyme-linked immunosorbent assay. Each antibody bound to a linear epitope within the N terminus (residues 28 to 66) or the C terminus (residues 632 to 655). Antibodies that target the C terminus neutralized in vitro cytotoxicity and delayed the lethal effects of iota-toxin in mice. PMID:11254604

Rational design of engineered microbial cell surface multi-enzyme co-display system for sustainable NADH regeneration from low-cost biomass.

PubMed

Han, Lei; Liang, Bo; Song, Jianxia

2018-02-01

As an important cofactor, NADH is essential for most redox reactions and biofuel cells. However, supply of exogenous NADH is challenged, due to the low production efficiency and high cost of NADH regeneration system, as well as low stability of NADH. Here, we constructed a novel cell surface multi-enzyme co-display system with ratio- and space-controllable manner as exogenous NADH regeneration system for the sustainable NADH production from low-cost biomass. Dockerin-fused glucoamylase (GA) and glucose dehydrogenase (GDH) were expressed and assembled on the engineered bacterial surfaces, which displayed protein scaffolds with various combinations of different cohesins. When the ratio of GA and GDH was 3:1, the NADH production rate of the whole-cell biocatalyst reached the highest level using starch as substrate, which was three times higher than that of mixture of free enzymes, indicating that the highly ordered spatial organization of enzymes would promote reactions, due to the ratio of enzymes and proximity effect. To confirm performance of the established NADH regeneration system, the highly efficient synthesis of L-lactic acid (L-LA) was conducted by the system and the yield of L-LA (16 g/L) was twice higher than that of the mixture of free enzymes. The multi-enzyme co-display system showed good stability in the cyclic utilization. In conclusion, the novel sustainable NADH system would provide a cost-effective strategy to regenerate cofactor from low-cost biomass.

A design for the control of apoptosis in genetically modified Saccharomyces cerevisiae.

PubMed

Nishida, Nao; Noguchi, Misa; Kuroda, Kouichi; Ueda, Mitsuyoshi

2014-01-01

We have engineered a system that holds potential for use as a safety switch in genetically modified yeasts. Human apoptotic factor BAX (no homolog in yeast), under the control of the FBP1 (gluconeogenesis enzyme) promoter, was conditionally expressed to induce yeast cell apoptosis after glucose depletion. Such systems might prove useful for the safe use of genetically modified organisms.

Polymeric mercaptosilane-modified platinum electrodes for elimination of interferants in glucose biosensors.

PubMed

Jung, S K; Wilson, G S

1996-02-15

An oxidase-based glucose sensor has been developed that uses a mercaptosilane-modified platinum electrode to achieve selectivity of electrochemical interferants. A platinum-iridium (9:1) wire (0.178 mm o.d., sensing area of 1.12 mm2) is modified with (3-mercaptopropyl)trimethoxysilane. The modified sensors show excellent operational stability for more than 5 days. Glucose oxidase is immobilized on the modified surface (i) by using 3-maleimidopropionic acid as a linker or (ii) by cross-liking with bovine serum albumin using glutaraldehyde. Sensitivities in the range of 9.97 nA/mM glucose are observed when the enzyme is immobilized by method ii. Lower sensitivities (1.13 x 10(-1) nA/mM glucose) are observed when immobilization method i is employed. In terms of linear response range, the sensor enzyme-immobilized by method i is superior to that immobilized by method ii. The linearity is improved upon coating the enzyme layer with polyurethane. The sensor immobilized by method ii and coated with polyurethane exhibits a linear range to 15 mM glucose and excellent selectivity to glucose (0.47 nA/mM) against interferants such as ascorbic acid, uric acid, and acetaminophen.

Chemical and physical effects on the adhesion, maturation, and survival of monocytes, macrophages, and foreign body giant cells

NASA Astrophysics Data System (ADS)

Collier, Terry Odell, III

Injury caused by biomedical device implantation initiates inflammatory and wound healing responses. Cells migrate to the site of injury to degrade bacteria and toxins, create new vasculature, and form new and repair injured tissue. Blood-proteins rapidly adsorb onto the implanted material surface and express adhesive ligands which mediate cell adhesion on the material surface. Monocyte-derived macrophages and multi-nucleated foreign body giant cells adhere to the surface and degrade the surface of the material. Due to the role of macrophage and foreign body giant cell on material biocompatibility and biostability, the effects of surface chemistry, surface topography and specific proteins on the maturation and survival of monocytes, macrophages and foreign body giant cells has been investigated. Novel molecularly designed materials were used to elucidate the dynamic interactions which occur between inflammatory cells, proteins and surfaces. The effect of protein and protein adhesion was investigated using adhesive protein depleted serum conditions on RGD-modified and silane modified surfaces. The effects of surface chemistry were investigated using temperature responsive surfaces of poly (N-isopropylacrylamide) and micropatterned surfaces of N-(2 aminoethyl)-3-aminopropyltrimethoxysilane regions on an interpenetrating polymer network of polyacrylamide and poly(ethylene glycol). The physical effects were investigated using polyimide scaffold materials and polyurethane materials with surface modifying end groups. The depletion of immunoglobulin G caused decreased levels of macrophage adhesion, foreign body giant cell formation and increased levels of apoptosis. The temporal nature of macrophage adhesion was observed with changing effectiveness of adherent cell detachment with time, which correlated to increased expression of beta1 integrin receptors on detached macrophages with time. The limited ability of the micropatterned surface, polyimide scaffold and surface modified polyurethane materials to control macrophage adhesion indicates the complexity of macrophage adhesion and protein adsorption onto a surface. These studies have indicated components and adhesive mechanisms which can be utilized to create materials with enhanced resistance to macrophage adhesion and/or degradative abilities.

Skeletal stem cell and bone implant interactions are enhanced by LASER titanium modification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sisti, Karin E., E-mail: karinellensisti@gmail.com; Biomaterials Group, Institute of Chemistry, São Paulo State University; Federal University of Mato Grosso do Sul

Purpose: To evaluate the osteo-regenerative potential of Titanium (Ti) modified by Light Amplification by Stimulated Emission of Radiation (LASER) beam (Yb-YAG) upon culture with human Skeletal Stem Cells (hSSCs{sup 1}). Methods: Human skeletal cell populations were isolated from the bone marrow of haematologically normal patients undergoing primary total hip replacement following appropriate consent. STRO-1{sup +} hSSC{sup 1} function was examined for 10 days across four groups using Ti discs: i) machined Ti surface group in basal media (Mb{sup 2}), ii) machined Ti surface group in osteogenic media (Mo{sup 3}), iii) LASER-modified Ti group in basal media (Lb{sup 4}) and, iv)more » LASER-modified Ti group in osteogenic media (Lo{sup 5}). Molecular analysis and qRT-PCR as well as functional analysis including biochemistry (DNA, Alkaline Phosphatase (ALP{sup 6}) specific activity), live/dead immunostaining (Cell Tracker Green (CTG{sup 7})/Ethidium Homodimer-1 (EH-1{sup 8})), and fluorescence staining (for vinculin and phalloidin) were undertaken. Inverted, confocal and Scanning Electron Microscopy (SEM) approaches were used to characterise cell adherence, proliferation, and phenotype. Results: Enhanced cell spreading and morphological rearrangement, including focal adhesions were observed following culture of hSSCs{sup 1} on LASER surfaces in both basal and osteogenic conditions. Biochemical analysis demonstrated enhanced ALP{sup 6} specific activity on the hSSCs{sup 1}-seeded on LASER-modified surface in basal culture media. Molecular analysis demonstrated enhanced ALP{sup 6} and osteopontin expression on titanium LASER treated surfaces in basal conditions. SEM, inverted microscopy and confocal laser scanning microscopy confirmed extensive proliferation and migration of human bone marrow stromal cells on all surfaces evaluated. Conclusions: LASER-modified Ti surfaces modify the behaviour of hSSCs.{sup 1} In particular, SSC{sup 1} adhesion, osteogenic gene expression, cell morphology and cytoskeleton structure were affected. The current studies show Ti LASER modification can enhance the osseointegration between Ti and skeletal cells, with important implications for orthopaedic application. - Highlights: • Bone stem cells on LASER Ti surface display enhanced cell growth and viability. • Bone stem cells on LASER Ti surface exhibit marked biocompatibility. • Human bone stem cells on LASER Ti surface exhibit altered morphology. • LASER Ti enhance osteogenic differentiation of human bone skeletal stem cells. • LASER Ti provides a unique approach to enhance osseointegration with the material.« less

Effect of Surface Curvature and Chemistry on Protein Stability, Adsorption and Aggregation

NASA Astrophysics Data System (ADS)

Radhakrishna, Mithun

Enzyme immobilization has been of great industrial importance because of its use in various applications like bio-fuel cells, bio-sensors, drug delivery and bio-catalytic films. Although research on enzyme immobilization dates back to the 1970's, it has been only in the past decade that scientists have started to address the problems involved systematically. Most of the previous works on enzyme immobilization have been retrospective in nature i.e enzymes were immobilized on widely used substrates without a compatibility study between the enzyme and the substrate. Consequently, most of the enzymes lost their activity upon immobilization onto these substrates due to many governing factors like protein-surface and inter-protein interactions. These interactions also play a major role biologically in cell signaling, cell adhesion and inter-protein interactions specifically is believed to be the major cause for neurodegenerative diseases like Alzheimer's and Parkinson's disease. Therefore understanding the role of these forces on proteins is the need of the hour. In my current research, I have mainly focused on two factors a) Surface Curvature b) Surface Chemistry as both of these play a pivotal role in influencing the activity of the enzymes upon immobilization. I study the effect of these factors computationally using a stochastic method known as Monte Carlo simulations. My research work carried out in the frame work of a Hydrophobic-Polar (HP) lattice model for the protein shows that immobilizing enzymes inside moderately hydrophilic or hydrophobic pores results in an enhancement of the enzymatic activity compared to that in the bulk. Our results also indicate that there is an optimal value of surface curvature and hydrophobicity/hydrophilicity where this enhancement of enzymatic activity is highest. Further, our results also show that immobilization of enzymes inside hydrophobic pores of optimal sizes are most effective in mitigating protein-aggregation. These results provide us a rationale to understand the role of chaperonins in protein folding and disaggregation. Our results indicate that strong protein-surface interactions and confinement inducement stability inside pores makes it best suitable for enzyme immobilization.

Nanolayer formation on titanium by phosphonated gelatin for cell adhesion and growth enhancement

PubMed Central

Zhou, Xiaoyue; Park, Shin-Hye; Mao, Hongli; Isoshima, Takashi; Wang, Yi; Ito, Yoshihiro

2015-01-01

Phosphonated gelatin was prepared for surface modification of titanium to stimulate cell functions. The modified gelatin was synthesized by coupling with 3-aminopropylphosphonic acid using water-soluble carbodiimide and characterized by 31P nuclear magnetic resonance and gel permeation chromatography. Circular dichroism revealed no differences in the conformations of unmodified and phosphonated gelatin. However, the gelation temperature was changed by the modification. Even a high concentration of modified gelatin did not form a gel at room temperature. Time-of-flight secondary ion mass spectrometry showed direct bonding between the phosphonated gelatin and the titanium surface after binding. The binding behavior of phosphonated gelatin on the titanium surface was quantitatively analyzed by a quartz crystal microbalance. Ellipsometry showed the formation of a several nanometer layer of gelatin on the surface. Contact angle measurement indicated that the modified titanium surface was hydrophobic. Enhancement of the attachment and spreading of MC-3T3L1 osteoblastic cells was observed on the phosphonated gelatin-modified titanium. These effects on cell adhesion also led to growth enhancement. Phosphonation of gelatin was effective for preparation of a cell-stimulating titanium surface. PMID:26366080

Relating Nanoscale Accessibility within Plant Cell Walls to Improved Enzyme Hydrolysis Yields in Corn Stover Subjected to Diverse Pretreatments.

PubMed

Crowe, Jacob D; Zarger, Rachael A; Hodge, David B

2017-10-04

Simultaneous chemical modification and physical reorganization of plant cell walls via alkaline hydrogen peroxide or liquid hot water pretreatment can alter cell wall structural properties impacting nanoscale porosity. Nanoscale porosity was characterized using solute exclusion to assess accessible pore volumes, water retention value as a proxy for accessible water-cell walls surface area, and solute-induced cell wall swelling to measure cell wall rigidity. Key findings concluded that delignification by alkaline hydrogen peroxide pretreatment decreased cell wall rigidity and that the subsequent cell wall swelling resulted increased nanoscale porosity and improved enzyme binding and hydrolysis compared to limited swelling and increased accessible surface areas observed in liquid hot water pretreated biomass. The volume accessible to a 90 Å dextran probe within the cell wall was found to be correlated to both enzyme binding and glucose hydrolysis yields, indicating cell wall porosity is a key contributor to effective hydrolysis yields.

Cytocompatibility of Direct Laser Interference-patterned Titanium Surfaces for Implants.

PubMed

Hartjen, Philip; Nada, Ola; Silva, Thiago Gundelwein; Precht, Clarissa; Henningsen, Anders; Holthaus, Marzellus GROßE; Gulow, Nikolai; Friedrich, Reinhard E; Hanken, Henning; Heiland, Max; Zwahr, Christoph; Smeets, Ralf; Jung, Ole

2017-01-01

In an effort to generate titanium surfaces for implants with improved osseointegration, we used direct laser interference patterning (DLIP) to modify the surface of pure titanium grade 4 of four different structures. We assessed in vitro cytoxicity and cell attachment, as well as the viability and proliferation of cells cultured directly on the surfaces. Attachment of the cells to the modified surfaces was comparably good compared to that of cells on grit-blasted and acid-etched reference titanium surfaces. In concordance with this, viability and proliferation of the cells directly cultured on the specimens were similar on all the titanium surfaces, regardless of the laser modification, indicating good cytocompatibility. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Evolution of plant parasitism in the phylum Nematoda.

PubMed

Quist, Casper W; Smant, Geert; Helder, Johannes

2015-01-01

Within the species-rich and trophically diverse phylum Nematoda, at least four independent major lineages of plant parasites have evolved, and in at least one of these major lineages plant parasitism arose independently multiple times. Ribosomal DNA data, sequence information from nematode-produced, plant cell wall-modifying enzymes, and the morphology and origin of the style(t), a protrusible piercing device used to penetrate the plant cell wall, all suggest that facultative and obligate plant parasites originate from fungivorous ancestors. Data on the nature and diversification of plant cell wall-modifying enzymes point at multiple horizontal gene transfer events from soil bacteria to bacterivorous nematodes resulting in several distinct lineages of fungal or oomycete-feeding nematodes. Ribosomal DNA frameworks with sequence data from more than 2,700 nematode taxa combined with detailed morphological information allow for explicit hypotheses on the origin of agronomically important plant parasites, such as root-knot, cyst, and lesion nematodes.

Enzymatic Detachment of Staphylococcus epidermidis Biofilms

PubMed Central

Kaplan, Jeffrey B.; Ragunath, Chandran; Velliyagounder, Kabilan; Fine, Daniel H.; Ramasubbu, Narayanan

2004-01-01

The gram-positive bacterium Staphylococcus epidermidis is the most common cause of infections associated with catheters and other indwelling medical devices. S. epidermidis produces an extracellular slime that enables it to form adherent biofilms on plastic surfaces. We found that a biofilm-releasing enzyme produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans rapidly and efficiently removed S. epidermidis biofilms from plastic surfaces. The enzyme worked by releasing extracellular slime from S. epidermidis cells. Precoating surfaces with the enzyme prevented S. epidermidis biofilm formation. Our findings demonstrate that biofilm-releasing enzymes can exhibit broad-spectrum activity and that these enzymes may be useful as antibiofilm agents. PMID:15215120

Cell Wall and Secreted Proteins of Candida albicans: Identification, Function, and Expression

PubMed Central

Chaffin, W. Lajean; López-Ribot, José Luis; Casanova, Manuel; Gozalbo, Daniel; Martínez, José P.

1998-01-01

The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis. PMID:9529890

Synthesis and biological evaluation of sialyl-oligonucleotide conjugates targeting leukocyte B trans-membranal receptor CD22 as delivery agents for nucleic acid drugs.

PubMed

St-Pierre, Gabrielle; Pal, Sudip; Østergaard, Michael E; Zhou, Tianyuan; Yu, Jinghua; Tanowitz, Michael; Seth, Punit P; Hanessian, Stephen

2016-06-01

Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reversible logic gates based on enzyme-biocatalyzed reactions and realized in flow cells: a modular approach.

PubMed

Fratto, Brian E; Katz, Evgeny

2015-05-18

Reversible logic gates, such as the double Feynman gate, Toffoli gate and Peres gate, with 3-input/3-output channels are realized using reactions biocatalyzed with enzymes and performed in flow systems. The flow devices are constructed using a modular approach, where each flow cell is modified with one enzyme that biocatalyzes one chemical reaction. The multi-step processes mimicking the reversible logic gates are organized by combining the biocatalytic cells in different networks. This work emphasizes logical but not physical reversibility of the constructed systems. Their advantages and disadvantages are discussed and potential use in biosensing systems, rather than in computing devices, is suggested. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

From mannan to bioethanol: cell surface co-display of β-mannanase and β-mannosidase on yeast Saccharomyces cerevisiae.

PubMed

Ishii, Jun; Okazaki, Fumiyoshi; Djohan, Apridah Cameliawati; Hara, Kiyotaka Y; Asai-Nakashima, Nanami; Teramura, Hiroshi; Andriani, Ade; Tominaga, Masahiro; Wakai, Satoshi; Kahar, Prihardi; Yopi; Prasetya, Bambang; Ogino, Chiaki; Kondo, Akihiko

2016-01-01

Mannans represent the largest hemicellulosic fraction in softwoods and also serve as carbohydrate stores in various plants. However, the utilization of mannans as sustainable resources has been less advanced in sustainable biofuel development. Based on a yeast cell surface-display technology that enables the immobilization of multiple enzymes on the yeast cell walls, we constructed a recombinant Saccharomyces cerevisiae strain that co-displays β-mannanase and β-mannosidase; this strain is expected to facilitate ethanol fermentation using mannan as a biomass source. Parental yeast S. cerevisiae assimilated mannose and glucose as monomeric sugars, producing ethanol from mannose. We constructed yeast strains that express tethered β-mannanase and β-mannosidase; co-display of the two enzymes on the cell surface was confirmed by immunofluorescence staining and enzyme activity assays. The constructed yeast cells successfully hydrolyzed 1,4-β-d-mannan and produced ethanol by assimilating the resulting mannose without external addition of enzymes. Furthermore, the constructed strain produced ethanol from 1,4-β-d-mannan continually during the third batch of repeated fermentation. Additionally, the constructed strain produced ethanol from ivory nut mannan; ethanol yield was improved by NaOH pretreatment of the substrate. We successfully displayed β-mannanase and β-mannosidase on the yeast cell surface. Our results clearly demonstrate the utility of the strain co-displaying β-mannanase and β-mannosidase for ethanol fermentation from mannan biomass. Thus, co-tethering β-mannanase and β-mannosidase on the yeast cell surface provides a powerful platform technology for yeast fermentation toward the production of bioethanol and other biochemicals from lignocellulosic materials containing mannan components.

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A(1-17) as a non-substrate inhibitor.

PubMed

Gabrilovac, Jelka; Abramić, Marija; Uzarević, Branka; Andreis, Ana; Poljak, Ljiljana

2003-05-30

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V(max) = 39 micromol/min/10(6) cells, vs 3.7 micromol/min/10(6) cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A(1-17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A(1-17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A(1-17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A(1-17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.

Immobilization, stabilization and patterning techniques for enzyme based sensor systems.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flounders, A.W.; Carichner, S.C.; Singh, A.K.

1997-01-01

Sandia National Laboratories has recently opened the Chemical and Radiation Detection Laboratory (CRDL) in Livermore CA to address the detection needs of a variety of government agencies (e.g., Department of Energy, Environmental Protection Agency, Department of Agriculture) as well as provide a fertile environment for the cooperative development of new industrial technologies. This laboratory consolidates a variety of existing chemical and radiation detection efforts and enables Sandia to expand into the novel area of biochemically based sensors. One aspect of this biosensor effort is further development and optimization of enzyme modified field effect transistors (EnFETs). Recent work has focused uponmore » covalent attachment of enzymes to silicon dioxide and silicon nitride surfaces for EnFET fabrication. They are also investigating methods to pattern immobilized proteins; a critical component for development of array-based sensor systems. Novel enzyme stabilization procedures are key to patterning immobilized enzyme layers while maintaining enzyme activity. Results related to maximized enzyme loading, optimized enzyme activity and fluorescent imaging of patterned surfaces will be presented.« less

[Preparation of chicken red blood cells for calibration of flow cytometry].

PubMed

Yin, Jian; Zhao, Shutao; Wu, Xiaodong; Wang, Ce; Wu, Yunliang

2013-01-01

To prepare stable chicken red blood cells for the calibration of flow cytometry. The traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2+-free HBSS treatment and low-speed centrifugation. The effect of fluorescence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed cells. Chicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [(98.5∓3.5)% vs (93.5∓2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [(6.0∓0.3)% to 6.2∓0.4% vs (8.6∓0.5)% to (13.1∓1.4)%, P<0.01]. The chicken red blood cells isolated using the modified method can be applicable for calibration of flow cytometry.

PES Surface Modification Using Green Chemistry: New Generation of Antifouling Membranes

PubMed Central

Nady, Norhan

2016-01-01

A major limitation in using membrane-based separation processes is the loss of performance due to membrane fouling. This drawback can be addressed thanks to surface modification treatments. A new and promising surface modification using green chemistry has been recently investigated. This modification is carried out at room temperature and in aqueous medium using green catalyst (enzyme) and nontoxic modifier, which can be safely labelled “green surface modification”. This modification can be considered as a nucleus of new generation of antifouling membranes and surfaces. In the current research, ferulic acid modifier and laccase bio-catalyst were used to make poly(ethersulfone) (PES) membrane less vulnerable to protein adsorption. The blank and modified PES membranes are evaluated based on e.g., their flux and protein repellence. Both the blank and the modified PES membranes (or laminated PES on silicon dioxide surface) are characterized using many techniques e.g., SEM, EDX, XPS and SPM, etc. The pure water flux of the most modified membranes was reduced by 10% on average relative to the blank membrane, and around a 94% reduction in protein adsorption was determined. In the conclusions section, a comparison between three modifiers—ferulic acid, and two other previously used modifiers (4-hydroxybenzoic acid and gallic acid)—is presented. PMID:27096873

Surface-modified CMOS IC electrochemical sensor array targeting single chromaffin cells for highly parallel amperometry measurements.

PubMed

Huang, Meng; Delacruz, Joannalyn B; Ruelas, John C; Rathore, Shailendra S; Lindau, Manfred

2018-01-01

Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

Immobilization of cross linked Col-I-OPN bone matrix protein on aminolysed PCL surfaces enhances initial biocompatibility of human adipogenic mesenchymal stem cells (hADMSC)

NASA Astrophysics Data System (ADS)

Kim, Young-Hee; Jyoti, Md. Anirban; Song, Ho-Yeon

2014-06-01

In bone tissue engineering surface modification is considered as one of the important ways of fabricating successful biocompatible material. Addition of biologically active functionality on the surfaces has been tried for improving the overall biocompatibility of the system. In this study poly-ɛ-caprolactone film surfaces have been modified through aminolysis and immobilization process. Collagen type I (COL-I) and osteopontin (OPN), which play an important role in osteogenesis, was immobilized onto PCL films followed by aminolysis treatment using 1,6-hexanediamine. Characterization of animolysed and immobilized surfaces were done by a number techniques using scanning electron microscopy (SEM), FT-IR, XPS, ninhydrin staining, SDS-PAGE and confocal microscopy and compared between the modified and un-modified surfaces. Results of the successive experiments showed that aminolysis treatment was homogeneously achieved which helped to entrap or immobilize Col-I-OPN proteins on surfaces of PCL film. In vitro studies with human adipogenic mesenchymal stem cells (hADMSC) also confirmed the attachment and proliferation of cells was better in modified PCL surfaces than the unmodified surfaces. SEM, confocal microscopy and MTT assay showed a significant increase in cell spreading, attachment and proliferations on the biofunctionalized surfaces compared to the unmodified PCL surfaces at all-time points indicating the success of surface biofunctionalization.

Enhanced activity and stability of L-arabinose isomerase by immobilization on aminopropyl glass.

PubMed

Zhang, Ye-Wang; Jeya, Marimuthu; Lee, Jung-Kul

2011-03-01

Immobilization of Bacillus licheniformis L: -arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support⁻¹) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups on the surface of support. Reducing particle size increased the adsorption capacity (q(m)) and affinity (k(a)). The pH and temperature for immobilization were optimized to be pH 7.1 and 33 °C using response surface methodology (RSM). The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly, the catalytic efficiency (k(cat)/K(m)) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t₁/₂) increased from 2 to 275 h) at 50 °C following immobilization.

21 CFR 184.1063 - Enzyme-modified lecithin.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Enzyme-modified lecithin. 184.1063 Section 184... as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified lecithin is prepared by treating... percent of total phosphatides as determined by “Determination of Lysolecithin Content of Enzyme-Modified...

21 CFR 184.1063 - Enzyme-modified lecithin.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Enzyme-modified lecithin. 184.1063 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified... Lysolecithin Content of Enzyme-Modified Lecithin: Method I,” dated 1985, which is incorporated by reference in...

Synaptic structure and function are altered by the neddylation inhibitor MLN4924

PubMed Central

Scudder, Samantha L.; Patrick, Gentry N.

2015-01-01

The posttranslational modification of proteins by the ubiquitin-like small molecule NEDD8 has previously been shown to be vital in a number of cell signaling pathways. In particular, conjugation of NEDD8 (neddylation) serves to regulate protein ubiquitination through modifications to E3 ubiquitin ligases. Despite the prevalence of NEDD8 in neurons, very little work has been done to characterize the role of this modifier in these cells. Here, we use the recently developed NEDD8 Activating Enzyme (NAE) inhibitor MLN4924 and report evidence of a role for NEDD8 in regulating mammalian excitatory synapses. Application of this drug to dissociated rat hippocampal neurons caused reductions in synaptic strength, surface glutamate receptor levels, dendritic spine width, and spine density, suggesting that neddylation is involved in the maintenance of synapses. PMID:25701678

Integrated nanoparticle-biomolecule systems for biosensing and bioelectronics.

PubMed

Willner, Itamar; Baron, Ronan; Willner, Bilha

2007-04-15

The similar dimensions of biomolecules such as enzymes, antibodies or DNA, and metallic or semiconductor nanoparticles (NPs) enable the synthesis of biomolecule-NP hybrid systems where the unique electronic, photonic and catalytic properties of NPs are combined with the specific recognition and biocatalytic properties of biomolecules. The unique functions of biomolecule-NP hybrid systems are discussed with several examples: (i) the electrical contacting of redox enzymes with electrodes is the basis for the development of enzymatic electrodes for amperometric biosensors or biofuel cell elements. The reconstitution of the apo-glucose oxidase or apo-glucose dehydrogenase on flavin adenine dinucleotide (FAD)-functionalized Au NPs (1.4 nm) associated with electrodes, or on pyrroloquinoline quinone (PQQ)-functionalized Au NPs (1.4 nm) associated with electrodes, respectively, yields electrically contacted enzyme electrodes. The aligned, reconstituted enzymes on the electrode surfaces reveal effective electrical contacting, and the glucose oxidase and glucose dehydrogenase reveal turnover rates of 5000 and 11,800 s(-1), respectively. (ii) The photoexcitation of semiconductor nanoparticles yields fluorescence with a wavelength controlled by the size of the NPs. The fluorescence functions of semiconductor NPs are used to develop a fluorescence resonance energy transfer (FRET) assay for nucleic acids, and specifically, for analyzing telomerase activity in cancer cells. CdSe-ZnS NPs are functionalized by a primer recognized by telomerase, and this is elongated by telomerase extracted from HeLa cancer cells in the presence of dNTPs and Texas-red-functionalized dUTP. The dye integrated into the telomers allows the FRET process that is intensified as telomerization proceeds. Also, the photoexcited electron-hole pair generated in semiconductor NPs is used to generate photocurrents in a CdS-DNA hybrid system associated with an electrode. A redox-active intercalator, methylene blue, was incorporated into a CdS-duplex DNA monolayer associated with a Au electrode, and this facilitated the electron transfer between the electrode and the CdS NPs. The direction of the photocurrent was controlled by the oxidation state of the intercalator. (iii) Biocatalysts grow metallic NPs, and the absorbance of the NPs provides a means to assay the biocatalytic transformations. This is exemplified with the glucose oxidase-induced growth of Au NPs and with the tyrosinase-stimulated growth of Au NPs, in the presence of glucose or tyrosine, respectively. The biocatalytic growth of the metallic NPs is used to grow nanowires on surfaces. Glucose oxidase or alkaline phosphatase functionalized with Au NPs (1.4 nm) acted as 'biocatalytic inks' for the synthesis of metallic nanowires. The deposition of the Au NP-modified glucose oxidase, or the Au NP-modified alkaline phosphatase on Si surfaces by dip-pen nanolithography led to biocatalytic templates, that after interaction with glucose/AuCl4- or p-aminophenolphosphate/Ag+, allowed the synthesis of Au nanowires or Ag nanowires, respectively.

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

NASA Astrophysics Data System (ADS)

Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

2012-08-01

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

Immunolocalization of two hydrogenosomal enzymes of Trichomonas vaginalis.

PubMed

Brugerolle, G; Bricheux, G; Coffe, G

2000-01-01

Three monoclonal antibodies specific for malic enzyme and for the alpha- and beta-subunits, respectively, of the succinyl-coenzyme A (CoA) synthetase of Trichomonas vaginalis were used to immunolocalize these proteins in the cell. All antibodies labeled the hydrogenosome matrix as determined both by immunofluorescence and by immunogold staining. There was no labeling on the cell surface or in any other cell compartment. These results support the idea that these proteins are restricted to a hydrogenosomal function and do not play a role as adhesins at the plasma membrane surface.

Construction of conducting polymer/cytochrome C/thylakoid membrane based photo-bioelectrochemical fuel cells generating high photocurrent via photosynthesis.

PubMed

Cevik, Emre; Carbas, Buket Bezgin; Senel, Mehmet; Yildiz, Huseyin Bekir

2018-08-15

In this study, a photo-bioelectrochemical fuel cell was constructed for photocurrent generation by illuminating the electrodes within an aqueous solution. In this purpose, gold electrode was coated with poly 4-(4H-Dithieno [3,2-b:2',3'-d]pyrol-4-yl) aniline, P(DTP-Ph-NH 2 ) conductive polymer film by using electrochemical polymerization. Then, P(DTP-Ph-NH 2 ) conductive polymer film coated surface was electrochemically modified with cytochrome C which covalently linked onto the surface via bis-aniline functionality of the polymer film and formed crosslinked-structure. The thylakoid membrane was attached on the surface of this electrode by using bissulfosaxinimidyl suberate (BS 3 ) and used as photo-anode in photo-bioelectrochemical fuel cell. The photo-cathode of the photo-bioelectrochemical fuel cell fabrication was followed by the modification of conductive polymer poly[5-(4H-dithieno [3,2-b:2',3'-d]pyrol-4-yl) naphtalene-1-amine] film coating, glutaraldehyde activation, and bilirubin oxidase enzyme immobilization. During the photosynthesis occurring in thylakoid membrane under the light, water was oxidized and separated; while oxygen was released in anode side, the cathode side was reduced the oxygen gas into the water via a bio-electro-catalytic method. The cytochrome C was used for binding of thylakoid membrane to the electrode surface and play an important role for transferring of electrons released as a result of photosynthesis. Copyright © 2018 Elsevier B.V. All rights reserved.

21 CFR 184.1063 - Enzyme-modified lecithin.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Enzyme-modified lecithin. 184.1063 Section 184.1063... Listing of Specific Substances Affirmed as GRAS § 184.1063 Enzyme-modified lecithin. (a) Enzyme-modified... Lysolecithin Content of Enzyme-Modified Lecithin: Method I,” dated 1985, which is incorporated by reference in...

Degradation of native and modified forms of fructose-bisphosphate aldolase microinjected into HeLa cells.

PubMed Central

Hopgood, M F; Knowles, S E; Bond, J S; Ballard, F J

1988-01-01

The uptake and degradation of radiolabelled rabbit muscle fructose-bisphosphate aldolase (EC 4.1.2.13) was studied in HeLa cells microinjected by the erythrocyte ghost fusion system. Labelled aldolase was progressively modified by treatment with GSSG or N-ethylmaleimide (NEM) before microinjection to determine whether these agents, which inactivate and destabilize the enzyme in vitro, affect the half-life of the enzyme in vivo. Increasing exposure of aldolase to GSSG or NEM before microinjection increased the extent of aldolase transfer into the HeLa cells and decreased the proportion of the protein that could be extracted from the cells after water lysis. Some degradation of the GSSG- and NEM-inactivated aldolases was observed in the ghosts before microinjection; thus a family of radiolabelled proteins was microinjected in these experiments. In spite of the above differences, the 40 kDa subunit of each aldolase form was degraded with a half-life of 30 h in the HeLa cells. In contrast, the progressively modified forms of aldolase were increasingly susceptible to proteolytic action in vitro by chymotrypsin or by cathepsin B and in ghosts. These studies indicate that the rate of aldolase degradation in cells is not determined by attack by cellular proteinases that recognize vulnerable protein substrates; the results are most easily explained by a random autophagic process involving the lysosomal system. Images Fig. 3. Fig. 7. PMID:3223914

A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

PubMed

Huberman, Lori B; Murray, Andrew W

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

PubMed Central

Huberman, Lori B.; Murray, Andrew W.

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

Immobilization of cellulase on a silica gel substrate modified using a 3-APTES self-assembled monolayer

DOE PAGES

Zhang, Dezhi; Hegab, Hisham E.; Lvov, Yuri; ...

2016-01-20

Cellulase was immobilized onto silica gel surfaces pretreated with (3-aminopropyl) triethoxy-silane (3-APTES), and glutaraldehyde (GA) was used as a cross-linker. A carboxymethyl cellulose sodium salt (CMC) solution was used for activity experiments. Protein assay was performed to determine the mass immobilized and compare with free enzyme. Cellulase was successfully demonstrated to be immobilized on the modified silica gel surface, and no detectable amount of enzyme was stripped off during the hydrolysis of the CMC solution. The specific activity of the immobilized cellulase is 7 ± 2 % compared to the similar amount of free cellulase. Significant activity over multiple reusesmore » was observed. The seventh batch achieved 82 % activity of the initial batch, and the fifteenth batch retained 31 %. Lastly, it was observed that the immobilized cellulase retained 48 % of its initial activity after 4 days, and 22 % even after 14 days.« less

Surface-modified gold nanorods for specific cell targeting

NASA Astrophysics Data System (ADS)

Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

2012-05-01

Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines

PubMed Central

Gibson, Gary E.; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis; Zhang, Sheng

2015-01-01

Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins are unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced suc-cinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid (TCA) cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl CoA suggests that the catalysis due to the E2k suc-cinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995

Tailor-made resealable micro(bio)reactors providing easy integration of in situ sensors

NASA Astrophysics Data System (ADS)

Viefhues, Martina; Sun, Shiwen; Valikhani, Donya; Nidetzky, Bernd; Vrouwe, Elwin X.; Mayr, Torsten; Bolivar, Juan M.

2017-06-01

Flow microreactors utilizing immobilized enzymes are of great interest in biocatalysis development. Most of the common devices are permanently closed, single-use systems, which allow limited physical and chemical surface modifications and evaluation methods. In this paper we will present resealable flowcells that overcome these limitations and moreover allow a quick and easy integration of sensor systems, because of the use of modular building blocks. The devices were utilized to study the enzyme activity of glucose oxidase immobilized on chemically modified glass surfaces under flow conditions, employing integrated optical oxygen sensors for on-line monitoring.

Silica nanoparticle based techniques for extraction, detection, and degradation of pesticides.

PubMed

Bapat, Gandhali; Labade, Chaitali; Chaudhari, Amol; Zinjarde, Smita

2016-11-01

Silica nanoparticles (SiNPs) find applications in the fields of drug delivery, catalysis, immobilization and sensing. Their synthesis can be mediated in a facile manner and they display broad range compatibility and stability. Their existence in the form of spheres, wires and sheets renders them suitable for varied purposes. This review summarizes the use of silica nanostructures in developing techniques for extraction, detection and degradation of pesticides. Silica nanostructures on account of their sorbent properties, porous nature and increased surface area allow effective extraction of pesticides. They can be modified (with ionic liquids, silanes or amines), coated with molecularly imprinted polymers or magnetized to improve the extraction of pesticides. Moreover, they can be altered to increase their sensitivity and stability. In addition to the analysis of pesticides by sophisticated techniques such as High Performance Liquid Chromatography or Gas chromatography, silica nanoparticles related simple detection methods are also proving to be effective. Electrochemical and optical detection based on enzymes (acetylcholinesterase and organophosphate hydrolase) or antibodies have been developed. Pesticide sensors dependent on fluorescence, chemiluminescence or Surface Enhanced Raman Spectroscopic responses are also SiNP based. Moreover, degradative enzymes (organophosphate hydrolases, carboxyesterases and laccases) and bacterial cells that produce recombinant enzymes have been immobilized on SiNPs for mediating pesticide degradation. After immobilization, these systems show increased stability and improved degradation. SiNP are significant in developing systems for effective extraction, detection and degradation of pesticides. SiNPs on account of their chemically inert nature and amenability to surface modifications makes them popular tools for fabricating devices for 'on-site' applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

PubMed

Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

2017-06-16

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

Plasmid-mediated fosfomycin resistance is due to enzymatic modification of the antibiotic.

PubMed Central

Llaneza, J; Villar, C J; Salas, J A; Suarez, J E; Mendoza, M C; Hardisson, C

1985-01-01

The molecular mechanism of plasmid-mediated resistance to fosfomycin is described. The antibiotic was inactivated intracellularly and remained inside the cells. Modification was also obtained from cell extracts and was not energy dependent. The modifying enzyme seems to have sulfhydryl groups in its active center. PMID:3899003

Development of functional biointerfaces by surface modification of polydimethylsiloxane with bioactive chlorogenic acid.

PubMed

Wu, Ming; He, Jia; Ren, Xiao; Cai, Wen-Sheng; Fang, Yong-Chun; Feng, Xi-Zeng

2014-04-01

The effect of physicochemical surface properties and chemical structure on the attachment and viability of bacteria and mammalian cells has been extensively studied for the development of biologically relevant applications. In this study, we report a new approach that uses chlorogenic acid (CA) to modify the surface wettability, anti-bacterial activity and cell adhesion properties of polydimethylsiloxane (PDMS). The chemical structure of the surface was obtained by X-ray photoelectron spectroscopy (XPS), the roughness was measured by atomic force microscopy (AFM), and the water contact angle was evaluated for PDMS substrates both before and after CA modification. Molecular modelling showed that the modification was predominately driven by van der Waals and electrostatic interactions. The exposed quinic-acid moiety improved the hydrophilicity of CA-modified PDMS substrates. The adhesion and viability of E. coli and HeLa cells were investigated using fluorescence and phase contrast microscopy. Few viable bacterial cells were found on CA-coated PDMS surfaces compared with unmodified PDMS surfaces. Moreover, HeLa cells exhibited enhanced adhesion and increased spreading on the modified PDMS surface. Thus, CA-coated PDMS surfaces reduced the ratio of viable bacterial cells and increased the adhesion of HeLa cells. These results contribute to the purposeful design of anti-bacterial surfaces for medical device use. Copyright © 2013 Elsevier B.V. All rights reserved.

Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor

NASA Astrophysics Data System (ADS)

Jepsen, Morten Leth; Harmsen, Charlotte; Godbole, Adwait Anand; Nagaraja, Valakunja; Knudsen, Birgitta R.; Ho, Yi-Ping

2015-12-01

We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes.We present a quantum dot based DNA nanosensor specifically targeting the cleavage step in the reaction cycle of the essential DNA-modifying enzyme, mycobacterial topoisomerase I. The design takes advantages of the unique photophysical properties of quantum dots to generate visible fluorescence recovery upon specific cleavage by mycobacterial topoisomerase I. This report, for the first time, demonstrates the possibility to quantify the cleavage activity of the mycobacterial enzyme without the pre-processing sample purification or post-processing signal amplification. The cleavage induced signal response has also proven reliable in biological matrices, such as whole cell extracts prepared from Escherichia coli and human Caco-2 cells. It is expected that the assay may contribute to the clinical diagnostics of bacterial diseases, as well as the evaluation of treatment outcomes. Electronic supplementary information (ESI) available: Characterization of the QD-based DNA Nanosensor. See DOI: 10.1039/c5nr06326d

Microarray of neuroblastoma cells on the selectively functionalized nanocrystalline diamond thin film surface

NASA Astrophysics Data System (ADS)

Park, Young-Sang; Son, Hyeong-Guk; Kim, Dae-Hoon; Oh, Hong-Gi; Lee, Da-Som; Kim, Min-Hye; Lim, Ki-Moo; Song, Kwang-Soup

2016-01-01

Nanocrystalline diamond (NCD) film surfaces were modified with fluorine or oxygen by plasma treatment in an O2 or C3F8 gas environment in order to induce wettability. The oxygenated-NCD (O-NCD) film surface was hydrophilic and the fluorinated-NCD (F-NCD) surface was hydrophobic. The efficiency of early cell adhesion, which is dependent on the wettability of the cell culture plate and necessary for the growth and proliferation of cells, was 89.62 ± 3.92% on the O-NCD film and 7.78 ± 0.77% on the F-NCD film surface after 3 h of cell culture. The wettability of the NCD film surface was artificially modified using a metal mask and plasma treatment to fabricate a micro-pattern. Four types of micro-patterns were fabricated (line, circle, mesh, and word) on the NCD film surface. We precisely arrayed the neuroblastoma cells on the micro-patterned NCD film surfaces by controlling the surface wettability and cell seeding density. The neuroblastoma cells adhered and proliferated along the O-NCD film surface.

Lactobacillus plantarum 299v surface-bound GAPDH: a new insight into enzyme cell walls location.

PubMed

Saad, N; Urdaci, M; Vignoles, C; Chaignepain, S; Tallon, R; Schmitter, J M; Bressollier, P

2009-12-01

The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.

Improvement of the stability and activity of immobilized glucose oxidase on modified iron oxide magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Abbasi, Mahboube; Amiri, Razieh; Bordbar, Abdol-Kalegh; Ranjbakhsh, Elnaz; Khosropour, Ahmad-Reza

2016-02-01

Immobilized proteins and enzymes are widely investigated in the medical field as well as the food and environmental fields. In this study, glucose oxidase (GOX) was covalently immobilized on the surface of modified iron oxide magnetic nanoparticles (MIMNs) to produce a bioconjugate complex. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to the size, shape and structure characterization of the MIMNs. Binding of GOX to these MIMNs was confirmed by using FT-IR spectroscopy. The stability of the immobilized and free enzyme at different temperature and pH values was investigated by measuring the enzymatic activity. These studies reveal that the enzyme's stability is enhanced by immobilization. Further experiments showed that the storage stability of the enzyme is improved upon binding to the MIMNs. The results of kinetic measurements suggest that the effect of the immobilization process on substrate and product diffusion is small. Such bioconjugates can be considered as a catalytic nanodevice for accelerating the glucose oxidation reaction for biotechnological purposes.

Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cosgrove, Daniel J.

The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the ‘Young's modulus’ of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potentialmore » pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics.« less

Fabrication of hydrogels with elasticity changed by alkaline phosphatase for stem cell culture.

PubMed

Toda, Hiroyuki; Yamamoto, Masaya; Uyama, Hiroshi; Tabata, Yasuhiko

2016-01-01

The objective of this study is to design hydrogels whose elasticity can be changed by alkaline phosphatase (ALP) in cell culture and evaluate the effect of hydrogel elasticity on an osteogenic gene expression of cells. Hydrogels were prepared by the radical polymerization of acrylamide (AAm), N,N'-methylenebisacrylamide (BIS), and Phosmer™M containing phosphate groups (PE-PAAm hydrogels). The storage modulus of PE-PAAm hydrogels prepared was changed by the preparation conditions. When human mesenchymal stem cells (hMSC) were cultured on the ALP-responsive PE-PAAm hydrogels in the presence or absence of ALP, the morphology of hMSC was observed and one of the osteogenic differentiation markers, Runx2, was evaluated. By ALP addition into the culture medium, the morphology of hMSC was changed into an elongated shape without cell damage. ALP addition modified the level of Runx2 gene expression, which was influenced by the modulus of PE-PAAm hydrogels. It is concluded that the elasticity change of hydrogel substrates in cell culture had an influence on the Runx2 gene expression of hMSC. Stem cells sense the surface elasticity of culture substrates, and their differentiation fate is biologically modified by substrate properties. Most of experiments have been performed in static conditions during cell culture, while the in vivo microenvironment is dynamically changed. In this study, we established to design an enzyme-responsive hydrogel whose elasticity can be changed by alkaline phosphatase (ALP) in cell culture to mimic in vivo conditions. As a result, the cells were deformed and the gene expression level of an osteogenic maker, Runx2, was modified by ALP treatment. This is the novel report describing to demonstrate that the dynamic alteration of hydrogel substrate elasticity could modulate the osteoblastic gene expression of human MSC in vitro. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Rho-associated kinase (ROCK) inhibition reverses low cell activity on hydrophobic surfaces.

PubMed

Tian, Yu Shun; Kim, Hyun Jung; Kim, Hyun-Man

2009-08-28

Hydrophobic polymers do not offer an adequate scaffold surface for cells to attach, migrate, proliferate, and differentiate. Thus, hydrophobic scaffolds for tissue engineering have traditionally been physicochemically modified to enhance cellular activity. However, modifying the surface by chemical or physical treatment requires supplementary engineering procedures. In the present study, regulation of a cell signal transduction pathway reversed the low cellular activity on a hydrophobic surface without surface modification. Inhibition of Rho-associated kinase (ROCK) by Y-27632 markedly enhanced adhesion, migration, and proliferation of osteoblastic cells cultured on a hydrophobic polystyrene surface. ROCK inhibition regulated cell-cycle-related molecules on the hydrophobic surface. This inhibition also decreased expression of the inhibitors of cyclin-dependent kinases such as p21(cip1) and p27(kip1) and increased expression of cyclin A and D. These results indicate that defective cellular activity on the hydrophobic surface can be reversed by the control of a cell signal transduction pathway without physicochemical surface modification.

Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging.

PubMed

Jones, Krysten A; Porterfield, William B; Rathbun, Colin M; McCutcheon, David C; Paley, Miranda A; Prescher, Jennifer A

2017-02-15

Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate "hits" were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.

Biofuel cells based on direct enzyme-electrode contacts using PQQ-dependent glucose dehydrogenase/bilirubin oxidase and modified carbon nanotube materials.

PubMed

Scherbahn, V; Putze, M T; Dietzel, B; Heinlein, T; Schneider, J J; Lisdat, F

2014-11-15

Two types of carbon nanotube electrodes (1) buckypaper (BP) and (2) vertically aligned carbon nanotubes (vaCNT) have been used for elaboration of glucose/O2 enzymatic fuel cells exploiting direct electron transfer. For the anode pyrroloquinoline quinone dependent glucose dehydrogenase ((PQQ)GDH) has been immobilized on [poly(3-aminobenzoic acid-co-2-methoxyaniline-5-sulfonic acid), PABMSA]-modified electrodes. For the cathode bilirubin oxidase (BOD) has been immobilized on PQQ-modified electrodes. PABMSA and PQQ act as promoter for enzyme bioelectrocatalysis. The voltammetric characterization of each electrode shows current densities in the range of 0.7-1.3 mA/cm(2). The BP-based fuel cell exhibits maximal power density of about 107 µW/cm(2) (at 490 mV). The vaCNT-based fuel cell achieves a maximal power density of 122 µW/cm(2) (at 540 mV). Even after three days and several runs of load a power density over 110 µW/cm(2) is retained with the second system (10mM glucose). Due to a better power exhibition and an enhanced stability of the vaCNT-based fuel cells they have been studied in human serum samples and a maximal power density of 41 µW/cm(2) (390 mV) can be achieved. Copyright © 2014 Elsevier B.V. All rights reserved.

Secretion of acid phosphatase by axenic Entamoeba histolytica NIH-200 and properties of the extracellular enzyme.

PubMed

Agrawal, A; Pandey, V C; Kumar, S; Sagar, P

1989-01-01

Entamoeba histolytica (NIH-200) secreted large amounts of acid phosphatase in its external environment when grown axenically in modified TPS-II medium. Fractionation by DEAE-cellulose chromatography of the precipitate obtained from the cell-free medium at 60% ammonium sulfate saturation yielded 3 distinct peaks of enzyme activity. The enzyme in all the peaks showed resistance to tartrate but was inhibited by fluoride, cupric chloride, ethylene diamine-tetra acetic acid, ammonium molybdate and cysteine; however, enzyme associated with different peaks differed in its polyacrylamide gel electrophoretic profiles and behavior towards concanavalin A.

Toward the Reconstitution of a Two-Enzyme Cascade for Resveratrol Synthesis on Potyvirus Particles.

PubMed

Besong-Ndika, Jane; Wahlsten, Matti; Cardinale, Daniela; Pille, Jan; Walter, Jocelyne; Michon, Thierry; Mäkinen, Kristiina

2016-01-01

The highly ordered protein backbone of virus particles makes them attractive candidates for use as enzyme nano-carriers (ENCs). We have previously developed a non-covalent and versatile approach for adhesion of enzymes to virus particles. This approach makes use of z33, a peptide derived from the B-domain of Staphylococcus aureus protein A, which binds to the Fc domain of many immunoglobulins. We have demonstrated that with specific antibodies addressed against the viral capsid proteins (CPs) an 87% coverage of z33-tagged proteins can be achieved on potyvirus particles. 4-coumarate coenzyme A ligase (4CL2) and stilbene synthase (STS) catalyze consecutive steps in the resveratrol synthetic pathway. In this study, these enzymes were modified to carry an N-terminal z33 peptide and a C-terminal 6xHis tag to obtain (z)4CL2(His) and (z)STS(His), respectively. A protein chimera, (z)4CL2::STS(His), with the same modifications was also generated from the genetic fusion of both mono-enzyme encoding genes. All z33 enzymes were biologically active after expression in Escherichia coli as revealed by LC-MS analysis to identify resveratrol and assembled readily into macromolecular complexes with Potato virus A particles and α-PVA CP antibodies. To test simultaneous immobilization-purification, we applied the double antibody sandwich - ELISA protocol to capture active z33-containg mono-enzymes and protein chimera directly from clarified soluble cell lysates onto the virus particle surface. These immobilized enzymes were able to synthesize resveratrol. We present here a bottom up approach to immobilize active enzymes onto virus-based ENCs and discuss the potential to utilize this method in the purification and configuration of nano-devices.

[Effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterial on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells].

PubMed

Li, Jingfeng; Zheng, Qixin; Guo, Xiaodong; Chen, Liaobin

2014-10-01

In the present research, the effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterials on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells were investigated. The experiments were divided into three groups due to biomaterials used: Group A (composite materials of sintered bone modified with surface mineralization and P24, a peptide of bone morphogenetic protein-2); Group B (sintered bone modified with surface mineralization) and Group C (sintered bone only). The three groups were observed by scanning electron microscopy (SEM) before the experiments, respectively. Then MC3T3-E1 cells were cultured on the surfaces of the three kinds of material, respectively. The cell adhesion rate was assessed by precipitation method. The proliferative ability of MC3T3-E1 cells were measured with MTT assay. And the ALP staining and measurement of alkaline phosphatase (ALP) activity were performed to assess the differentiation of cells into osteoblasts. The SEM results showed that the materials in the three groups retained the natural pore structure and the pore sizes were in the range between 200-850 μm. The adhesive ratio measurements and MTT assay suggested that adhesion and proliferation of MC3T3-E1 cells in Group A were much higher than those in Group B and Group C (P < 0.05). The ALP staining and ALP activity of MC3T3-E1 cells in Group A were significantly higher than those in Group B and Group C (P < 0.05). The sintered bone modified with surface mineralization/P24 composite material was confirmed to improve the adhesion rate and proliferation and osteodifferentiation of MC3T3-E1 cells, and maintained their morphology.

Co-downregulation of the hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase and coumarate 3-hydroxylase significantly increases cellulose content in transgenic alfalfa (Medicago sativa L.).

PubMed

Tong, Zongyong; Li, Heng; Zhang, Rongxue; Ma, Lei; Dong, Jiangli; Wang, Tao

2015-10-01

Lignin is a component of the cell wall that is essential for growth, development, structure and pathogen resistance in plants, but high lignin is an obstacle to the conversion of cellulose to ethanol for biofuel. Genetically modifying lignin and cellulose contents can be a good approach to overcoming that obstacle. Alfalfa (Medicago sativa L.) is rich in lignocellulose biomass and used as a model plant for the genetic modification of lignin in this study. Two key enzymes in the lignin biosynthesis pathway-hydroxycinnamoyl -CoA:shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3H)-were co-downregulated. Compared to wild-type plants, the lignin content in the modified strain was reduced by 38%, cellulose was increased by 86.1%, enzyme saccharification efficiency was increased by 10.9%, and cell wall digestibility was increased by 13.0%. The modified alfalfa exhibited a dwarf phenotype, but normal above ground biomass. This approach provides a new strategy for reducing lignin and increasing cellulose contents and creates a new genetically modified crop with enhanced value for biofuel. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Efficient biological conversion of carbon monoxide (CO) to carbon dioxide (CO2) and for utilization in bioplastic production by Ralstonia eutropha through the display of an enzyme complex on the cell surface.

PubMed

Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

2015-06-25

An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of the CO2-utilizing Ralstonia eutropha and successfully resulted in a 3.3-fold increase in conversion efficiency. These results suggest that this complexed system may be a promising strategy for CO2 utilization as a biological tool for the production of bioplastics.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

PubMed

Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

2017-01-01

There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

Targeting Heparan Sulfate Proteoglycans and their Modifying Enzymes to Enhance Anticancer Chemotherapy Efficacy and Overcome Drug Resistance.

PubMed

Lanzi, Cinzia; Zaffaroni, Nadia; Cassinelli, Giuliana

2017-01-01

Targeting heparan sulfate proteoglycans (HSPGs) and enzymes involved in heparan sulfate (HS) chain editing is emerging as a new anticancer strategy. The involvement of HSPGs in tumor cell signaling, inflammation, angiogenesis and metastasis indicates that agents able to inhibit aberrant HSPG functions can potentially act as multitarget drugs affecting both tumor cell growth and the supportive boost provided by the microenvironment. Moreover, accumulating evidence supports that an altered expression or function of HSPGs, or of the complex enzyme system regulating their activities, can also depress the tumor response to anticancer treatments in several tumor types. Thereby, targeting HSPGs or HSPG modifying enzymes appears an appealing approach to enhance chemotherapy efficacy. A great deal of effort from academia and industry has led to the development of agents mimicking HS, and/or inhibiting HSPG modifying enzymes. Inhibitors of Sulf-2, an endosulfatase that edits the HS sulfation pattern, and inhibitors of heparanase, the endoglycosidase that produces functional HS fragments, appear particularly promising. In fact, a Sulf-2 inhibitor (OKN-007), and two heparanase inhibitors/HS mimics (roneparstat, PG545) are currently under early clinical investigation. In this review, we summarized preclinical studies in experimental tumor models of the main chemical classes of Sulf-2 and heparanase inhibitors. We described examples of different mechanisms through which heparanase and HSPGs, often in cooperation, may impact tumor sensitivity to various antitumor agents. Finally, we reported a few preclinical studies showing increased antitumor efficacy obtained with the use of candidate clinical HS mimics in combination regimens. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The Mevalonate Pathway and Innate Immune Hyper-Responsiveness in the Pathogenesis of COPD and Lung Cancer: Potential for Chemoprevention.

PubMed

Young, Robert P; Hopkins, Raewyn J

2017-01-01

Current evidence suggests that persisting and/or exaggerated inflammation in the lungs initiated by smoking, and up-regulated through genetic susceptibility, may result in lung remodelling and impaired repair. The mevalonate pathway, through its modifying effects on innate immune responsiveness, may be involved in these processes providing a plausible pathogenic link between the development of chronic obstructive pulmonary disease (COPD) and lung cancer. The mevalonate pathway, mediates these effects through important intra-cellular signalling molecules called guanine phosphate transferases (GTPases) such as Rho-A. Smoke exposure activates cell surface proteins which, through the mediating influence of GTPases, then modify the activation of NFkB and its downstream effects on genes underlying innate immunity, neutrophilic inflammation and carcinogenesis. The mevalonate pathway is readily and substantially modified by inhibition of the enzyme 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMGCo-A) reductase. This enzyme controls the rate limiting step of the mevalonate pathway and is subject to inhibition by statin drugs and small chain fatty acids derived from high dietary fibre intake. Thus inhibiting the mevelonate pathway, and dampening the innate immune response to smoking, may play a critical role in modifying pulmonary inflammation and lung remodelling. Such an action might slow the progression of COPD and reduce the tendency to the development of lung cancer. This review examines the pre-clinical and clinical data suggesting that HMGCoA-reductase inhibition and it's modification of the mevalonate pathway, may have a chemo-preventive effect on lung cancer, particularly in patients with COPD where pulmonary inflammation is increased and the risk of lung cancer is greatest. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Osteoblastlike cell adhesion on titanium surfaces modified by plasma nitriding.

PubMed

da Silva, Jose Sandro Pereira; Amico, Sandro Campos; Rodrigues, Almir Olegario Neves; Barboza, Carlos Augusto Galvao; Alves, Clodomiro; Croci, Alberto Tesconi

2011-01-01

The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nitriding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P < .05). Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion.

Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.

PubMed

Love, Kerry Routenberg; Pandya, Renuka K; Spooner, Eric; Ploegh, Hidde L

2009-04-17

Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.

Synaptic structure and function are altered by the neddylation inhibitor MLN4924.

PubMed

Scudder, Samantha L; Patrick, Gentry N

2015-03-01

The posttranslational modification of proteins by the ubiquitin-like small molecule NEDD8 has previously been shown to be vital in a number of cell signaling pathways. In particular, conjugation of NEDD8 (neddylation) serves to regulate protein ubiquitination through modifications to E3 ubiquitin ligases. Despite the prevalence of NEDD8 in neurons, very little work has been done to characterize the role of this modifier in these cells. Here, we use the recently developed NEDD8 Activating Enzyme (NAE) inhibitor MLN4924 and report evidence of a role for NEDD8 in regulating mammalian excitatory synapses. Application of this drug to dissociated rat hippocampal neurons caused reductions in synaptic strength, surface glutamate receptor levels, dendritic spine width, and spine density, suggesting that neddylation is involved in the maintenance of synapses. Copyright © 2015 Elsevier Inc. All rights reserved.

Cell penetrating peptide-modified poly(lactic-co-glycolic acid) nanoparticles with enhanced cell internalization.

PubMed

Steinbach, Jill M; Seo, Young-Eun; Saltzman, W Mark

2016-01-01

The surface modification of nanoparticles (NPs) can enhance the intracellular delivery of drugs, proteins, and genetic agents. Here we studied the effect of different surface ligands, including cell penetrating peptides (CPPs), on the cell binding and internalization of poly(lactic-co-glycolic) (PLGA) NPs. Relative to unmodified NPs, we observed that surface-modified NPs greatly enhanced cell internalization. Using one CPP, MPG (unabbreviated notation), that achieved the highest degree of internalization at both low and high surface modification densities, we evaluated the effect of two different NP surface chemistries on cell internalization. After 2h, avidin-MPG NPs enhanced cellular internalization by 5 to 26-fold relative to DSPE-MPG NP formulations. Yet, despite a 5-fold increase in MPG density on DSPE compared to Avidin NPs, both formulations resulted in similar internalization levels (48 and 64-fold, respectively) after 24h. Regardless of surface modification, all NPs were internalized through an energy-dependent, clathrin-mediated process, and became dispersed throughout the cell. Overall both Avidin- and DSPE-CPP modified NPs significantly increased internalization and offer promising delivery options for applications in which internalization presents challenges to efficacious delivery. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Evaluation of non-specific binding suppression schemes for neutravidin and alkaline phosphatase at the surface of reticulated vitreous carbon electrodes.

PubMed

Shedge, Hemangi Y; Creager, Stephen E

2010-01-11

Non-specific binding (NSB) of high-molecular-weight proteins onto electrode surfaces can complicate the application of electroanalytical techniques to clinical and environmental research, particularly in biosensor applications. We present herein various strategies to modify the surface of reticulated vitreous carbon (RVC) electrodes to suppress non-specific binding of biomolecules onto its surface. Non-specific binding and specific binding (SB) of two enzyme conjugates, neutravidin-alkaline phosphatase (NA-ALP) and biotinylated alkaline phosphatase (B-ALP), and also neutravidin itself, were studied using hydroquinone diphosphate (HQDP) as an enzyme substrate for ALP inside the pores of RVC electrodes that had been subjected to various modification schemes. The extent of NSB and SB of these biomolecules inside RVC pores was assessed by measuring the initial rate of generation of an electroactive product, hydroquinone (HQ), of the enzyme-catalyzed reaction, using linear scan voltammetry (LSV) for HQ detection. Electrodes functionalized with phenylacetic acid and poly(ethylene glycol) (PEG) showed low NSB and high SB (when biotin capture ligands were included in the modification scheme) in comparison with unmodified electrodes and RVC electrodes modified in other ways. A simple sandwich bioassay for neutravidin was performed on the RVC electrode with the lowest NSB. A concentration detection limit of 52+/-2 ng mL(-1) and an absolute detection limit of 5.2+/-0.2 ng were achieved for neutravidin when this assay was performed using a 100 microL sample size.

Direct Electron Transfer of Dehydrogenases for Development of 3rd Generation Biosensors and Enzymatic Fuel Cells.

PubMed

Bollella, Paolo; Gorton, Lo; Antiochia, Riccarda

2018-04-24

Dehydrogenase based bioelectrocatalysis has been increasingly exploited in recent years in order to develop new bioelectrochemical devices, such as biosensors and biofuel cells, with improved performances. In some cases, dehydrogeases are able to directly exchange electrons with an appropriately designed electrode surface, without the need for an added redox mediator, allowing bioelectrocatalysis based on a direct electron transfer process. In this review we briefly describe the electron transfer mechanism of dehydrogenase enzymes and some of the characteristics required for bioelectrocatalysis reactions via a direct electron transfer mechanism. Special attention is given to cellobiose dehydrogenase and fructose dehydrogenase, which showed efficient direct electron transfer reactions. An overview of the most recent biosensors and biofuel cells based on the two dehydrogenases will be presented. The various strategies to prepare modified electrodes in order to improve the electron transfer properties of the device will be carefully investigated and all analytical parameters will be presented, discussed and compared.

Application of Molecular Imprinted Magnetic Fe3O4@SiO2 Nanoparticles for Selective Immobilization of Cellulase.

PubMed

Tao, Qing-Lan; Li, Yue; Shi, Ying; Liu, Rui-Jiang; Zhang, Ye-Wang; Guo, Jianyong

2016-06-01

Magnetic Fe3O4@SiO2 nanoparticles were prepared with molecular imprinting method using cellulase as the template. And the surface of the nanoparticles was chemically modified with arginine. The prepared nanoparticles were used as support for specific immobilization of cellulase. SDS-PAGE results indicated that the adsorption of cellulase onto the modified imprinted nanoparticles was selective. The immobilization yield and efficiency were obtained more than 70% after the optimization. Characterization of the immobilized cellulase revealed that the immobilization didn't change the optimal pH and temperature. The half-life of the immobilized cellulase was 2-fold higher than that of the free enzyme at 50 degrees C. After 7 cycles reusing, the immobilized enzyme still retained 77% of the original activity. These results suggest that the prepared imprinted nanoparticles have the potential industrial applications for the purification or immobilization of enzymes.

Hybrid nanocatalysts containing enzymes and metallic nanoparticles for ethanol/O2 biofuel cell

NASA Astrophysics Data System (ADS)

Aquino Neto, S.; Almeida, T. S.; Palma, L. M.; Minteer, S. D.; de Andrade, A. R.

2014-08-01

We report the preparation of hybrid nanostructured bioanodes containing the enzyme alcohol dehydrogenase (ADH) with either Au, Pt, or Pt0.75Sn0.25 nanoparticles for use in ethanol/O2 hybrid biofuel cells. We describe two different methodologies for the preparation of the bioanodes: in a first case, multi walled carbon nanotubes (MWCNTs) were employed as a support for the metallic nanoparticles and TBAB-modified Nafion® aided enzyme immobilization. In the second case, we immobilized the enzymes using dendrimers-encapsulated nanoparticles as the agent for enzyme anchoring. The biofuel cell tests showed that the addition of metallic nanoparticles to the bioanode structure enhanced the overall biofuel cell performance. The bioelectrode containing Au nanoparticles displaying the best performance, with an open circuit potential of 0.61 ± 0.05 V and a maximum power density of 155 ± 11 μW cm-2. NADH cyclic voltammetric experiments indicated that Au nanoparticles behaved as a catalyst toward NADH oxidation. Comparing the two protocols we used to synthetized nanoparticles, the sample containing the Au nanoparticles supported on MWCNTs furnished fourfold higher values. Therefore, from the satisfactory results obtained, it can be inferred that the combination of small amounts of metallic nanoparticles with enzymes improve bioanode performance.

In vitro biocompatibility of the surface ion modified NiTi alloy

NASA Astrophysics Data System (ADS)

Gudimova, Ekaterina Yu.; Meisner, Ludmila L.; Lotkov, Aleksander I.; Matveeva, Vera A.; Meisner, Stanislav N.; Matveev, Andrey L.; Shabalina, Olga I.

2016-11-01

This paper presents the results of the chemical, topographic and structural properties of the NiTi alloy surface and their changes after surface treatments by ion implantation techniques with use of ions Ta+ and Si+. The influence of physicochemical properties of the surface ion modified NiTi alloy was studied on in vitro cultured mesenchymal stem cells of the rats' bone marrow. It is shown that the ion surface modification improves histocompatibility of the NiTi alloy and leads to increase of proliferative activity of mesenchymal stem cells on its surface. It was experimentally found that a major contribution to viability improvement mesenchymal stem cells of rat marrow has the chemical composition and the microstructure of the surface area.

Glucose oxidase immobilization on different modified surfaces of platinum nanowire for application in glucose detection

NASA Astrophysics Data System (ADS)

Thanh Tuyen Le, Thi; Duy Tran, Phu; Pham, Xuan Tung; Hien Tong, Duy; Chien Dang, Mau

2010-09-01

In this work, the surface of platinum (Pt) nanowires was modified by using several chemicals, including a compound of gelatin gel with SiO2, polyvinyl alcohol (PVA) with Prussian blue (PB) mediator and cysteamine self-assembled monolayers (SAM). Then, glucose oxidase (GOD) enzyme was immobilized on the modified surfaces of Pt nanowire electrodes by using techniques of electrochemical adsorption and chemical binding. The GOD immobilized Pt nanowires were used for application in glucose detection by performing a cyclic voltammetry measurement. The detection results showed that GOD was immobilized on all of the tested surfaces and the highest glucose detection sensitivity of 60 μM was obtained when the Pt nanowires were modified by PVA with PB mediator. Moreover, the sensors showed very high current response when the Pt nanowires were modified with the cysteamine SAM. The stability and catalyst activity of GOD are also reported here. For instance, the catalyst activity of GOD retained about 60% of its initial value after it was stored at 4 °C in a 100 mM PBS buffer solution with a pH of 7.2 for a period of 30 days.

Optimization of Culture Medium Enhances Viable Biomass Production and Biocontrol Efficacy of the Antagonistic Yeast, Candida diversa.

PubMed

Liu, Jia; Li, Guangkun; Sui, Yuan

2017-01-01

Viable biomass production is a key determinant of suitability of antagonistic yeasts as potential biocontrol agents. This study investigated the effects of three metal ions (magnesium, ferrous, and zinc) on biomass production and viability of the antagonistic yeast, Candida diversa . Using response surface methodology to optimize medium components, a maximum biomass was obtained, when the collective Mg 2+ , Fe 2+ , and Zn 2+ concentrations were adjusted in a minimal mineral (MM) medium. Compared with the unmodified MM, and three ion-deficient MM media, yeast cells cultured in the three ion-modified MM medium exhibited a lower level of cellular oxidative damage, and a higher level of antioxidant enzyme activity. A biocontrol assay indicated that C. diversa grown in the ion-modified MM exhibited the greatest level of control of gray mold on apple fruit. These results provide new information on culture medium optimization to grow yeast antagonists in order to improve biomass production and biocontrol efficacy.

Optimization of Culture Medium Enhances Viable Biomass Production and Biocontrol Efficacy of the Antagonistic Yeast, Candida diversa

PubMed Central

Liu, Jia; Li, Guangkun; Sui, Yuan

2017-01-01

Viable biomass production is a key determinant of suitability of antagonistic yeasts as potential biocontrol agents. This study investigated the effects of three metal ions (magnesium, ferrous, and zinc) on biomass production and viability of the antagonistic yeast, Candida diversa. Using response surface methodology to optimize medium components, a maximum biomass was obtained, when the collective Mg2+, Fe2+, and Zn2+ concentrations were adjusted in a minimal mineral (MM) medium. Compared with the unmodified MM, and three ion-deficient MM media, yeast cells cultured in the three ion-modified MM medium exhibited a lower level of cellular oxidative damage, and a higher level of antioxidant enzyme activity. A biocontrol assay indicated that C. diversa grown in the ion-modified MM exhibited the greatest level of control of gray mold on apple fruit. These results provide new information on culture medium optimization to grow yeast antagonists in order to improve biomass production and biocontrol efficacy. PMID:29089939

Retrovirally mediated correction of bone marrow-derived mesenchymal stem cells from patients with mucopolysaccharidosis type I.

PubMed

Baxter, Melissa A; Wynn, Robert F; Deakin, Jonathan A; Bellantuono, Ilaria; Edington, Kirsten G; Cooper, Alan; Besley, Guy T N; Church, Heather J; Wraith, J Ed; Carr, Trevor F; Fairbairn, Leslie J

2002-03-01

We have investigated the utility of bone marrow-derived mesenchymal stem cells (MSCs) as targets for gene therapy of the autosomal recessive disorder mucopolysaccharidosis type IH (MPS-IH, Hurler syndrome). Cultures of MSCs were initially exposed to a green fluorescent protein-expressing retrovirus. Green fluorescent protein-positive cells maintained their proliferative and differentiation capacity. Next we used a vector encoding alpha-L-iduronidase (IDUA), the enzyme that is defective in MPS-IH. Following transduction, MPS-IH MSCs expressed high levels of IDUA and secreted supernormal levels of this enzyme into the extracellular medium. Exogenous IDUA expression led to a normalization of glycosaminoglycan storage in MPS-IH cells, as evidenced by a dramatic decrease in the amount of (35)SO(4) sequestered within the heparan sulfate and dermatan sulfate compartments of these cells. Finally, gene-modified MSCs were able to cross-correct the enzyme defect in untransduced MPS-IH fibroblasts via protein transfer.

Angiotensin-converting enzyme in epithelial and neuroepithelial cells.

PubMed

Defendini, R; Zimmerman, E A; Weare, J A; Alhenc-Gelas, F; Erdös, E G

1983-07-01

Angiotensin-converting enzyme (CE) occurs in three types of cell: endothelial, epithelial, and neuroepithelial. In all three, it appears to be bound to plasma membrane. With antisera to the human enzyme, CE is demonstrated in paraffin sections on the apical surface of epithelial cells in the proximal tubule of the kidney, the mucosa of the small intestine, the syncytial trophoblast of the placenta, and the choroid plexus. Epithelial CE is characteristically found on microvillous surfaces in contact with an effluent, well placed to act on substrate in flux. In the brain, CE occurs in nerve fibers and terminals, mainly mesiobasally and in basal ganglia. Mesiobasal CE coincides with other components of the renin-angiotensin system (RAS) in the choroid/ventricular fluid, the subfornical organ, and the magnocellular neurosecretory system of the hypothalamus. Extrapyramidal CE, however, may not be related to the RAS. In the substantia nigra and the globus pallidus, the enzyme has the same cellular distribution as two putative neuromodulators, substance P and enkephalin, the latter a known substrate of CE.

Fibrinogen Motif Discriminates Platelet and Cell Capture in Peptide-Modified Gold Micropore Arrays.

PubMed

Adamson, Kellie; Spain, Elaine; Prendergast, Una; Moran, Niamh; Forster, Robert J; Keyes, Tia E

2018-01-16

Human blood platelets and SK-N-AS neuroblastoma cancer-cell capture at spontaneously adsorbed monolayers of fibrinogen-binding motifs, GRGDS (generic integrin adhesion), HHLGGAKQAGDV (exclusive to platelet integrin α IIb β 3 ), or octanethiol (adhesion inhibitor) at planar gold and ordered 1.6 μm diameter spherical cap gold cavity arrays were compared. In all cases, arginine/glycine/aspartic acid (RGD) promoted capture, whereas alkanethiol monolayers inhibited adhesion. Conversely only platelets adhered to alanine/glycine/aspartic acid (AGD)-modified surfaces, indicating that the AGD motif is recognized preferentially by the platelet-specific integrin, α IIb β 3 . Microstructuring of the surface effectively eliminated nonspecific platelet/cell adsorption and dramatically enhanced capture compared to RGD/AGD-modified planar surfaces. In all cases, adhesion was reversible. Platelets and cells underwent morphological change on capture, the extent of which depended on the topography of the underlying substrate. This work demonstrates that both the nature of the modified interface and its underlying topography influence the capture of cancer cells and platelets. These insights may be useful in developing cell-based cancer diagnostics as well as in identifying strategies for the disruption of platelet cloaks around circulating tumor cells.

Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth.

PubMed

Yang, Chengli; Xing, Jianmin; Guan, Yueping; Liu, Huizhou

2006-09-01

An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.

Modeling Surface Growth of Escherichia coli on Agar Plates

PubMed Central

Fujikawa, Hiroshi; Morozumi, Satoshi

2005-01-01

Surface growth of Escherichia coli cells on a membrane filter placed on a nutrient agar plate under various conditions was studied with a mathematical model. The surface growth of bacterial cells showed a sigmoidal curve with time on a semilogarithmic plot. To describe it, a new logistic model that we presented earlier (H. Fujikawa et al., Food Microbiol. 21:501-509, 2004) was modified. Growth curves at various constant temperatures (10 to 34°C) were successfully described with the modified model (model III). Model III gave better predictions of the rate constant of growth and the lag period than a modified Gompertz model and the Baranyi model. Using the parameter values of model III at the constant temperatures, surface growth at various temperatures was successfully predicted. Surface growth curves at various initial cell numbers were also sigmoidal and converged to the same maximum cell numbers at the stationary phase. Surface growth curves at various nutrient levels were also sigmoidal. The maximum cell number and the rate of growth were lower as the nutrient level decreased. The surface growth curve was the same as that in a liquid, except for the large curvature at the deceleration period. These curves were also well described with model III. The pattern of increase in the ATP content of cells grown on a surface was sigmoidal, similar to that for cell growth. We discovered several characteristics of the surface growth of bacterial cells under various growth conditions and examined the applicability of our model to describe these growth curves. PMID:16332768

Transcriptional transitions in Alphonso mango (Mangifera indica L.) during fruit development and ripening explain its distinct aroma and shelf life characteristics.

PubMed

Deshpande, Ashish B; Anamika, Krishanpal; Jha, Vineet; Chidley, Hemangi G; Oak, Pranjali S; Kadoo, Narendra Y; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

2017-08-18

Alphonso is known as the "King of mangos" due to its unique flavor, attractive color, low fiber pulp and long shelf life. We analyzed the transcriptome of Alphonso mango through Illumina sequencing from seven stages of fruit development and ripening as well as flower. Total transcriptome data from these stages ranged between 65 and 143 Mb. Importantly, 20,755 unique transcripts were annotated and 4,611 were assigned enzyme commission numbers, which encoded 142 biological pathways. These included ethylene and flavor related secondary metabolite biosynthesis pathways, as well as those involved in metabolism of starch, sucrose, amino acids and fatty acids. Differential regulation (p-value ≤ 0.05) of thousands of transcripts was evident in various stages of fruit development and ripening. Novel transcripts for biosynthesis of mono-terpenes, sesqui-terpenes, di-terpenes, lactones and furanones involved in flavor formation were identified. Large number of transcripts encoding cell wall modifying enzymes was found to be steady in their expression, while few were differentially regulated through these stages. Novel 79 transcripts of inhibitors of cell wall modifying enzymes were simultaneously detected throughout Alphonso fruit development and ripening, suggesting controlled activity of these enzymes involved in fruit softening.

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.

PubMed

Ananphongmanee, Vorawit; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Boonchird, Chuenchit

2015-01-01

Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral administration.

Surface-modified bacterial nanofibrillar PHB scaffolds for bladder tissue repair.

PubMed

Karahaliloğlu, Zeynep; Demirbilek, Murat; Şam, Mesut; Sağlam, Necdet; Mızrak, Alpay Koray; Denkbaş, Emir Baki

2016-01-01

The aim of the study is in vitro investigation of the feasibility of surface-modified bacterial nanofibrous poly [(R)-3-hydroxybutyrate] (PHB) graft for bladder reconstruction. In this study, the surface of electrospun bacterial PHB was modified with PEG- or EDA via radio frequency glow discharge method. After plasma modification, contact angle of EDA-modified PHB scaffolds decreased from 110 ± 1.50 to 23 ± 0.5 degree. Interestingly, less calcium oxalate stone deposition was observed on modified PHB scaffolds compared to that of non-modified group. Results of this study show that surface-modified scaffolds not only inhibited calcium oxalate growth but also enhanced the uroepithelial cell viability and proliferation.

A Role for SHIP in Stem Cell Biology and Transplantation

PubMed Central

Kerr, William G.

2008-01-01

Inositol phospholipid signaling pathways have begun to emerge as important players in stem cell biology and bone marrow transplantation [1–4]. The SH2-containing Inositol Phosphatase (SHIP) is among the enzymes that can modify endogenous mammalian phosphoinositides. SHIP encodes an isoform specific to pluripotent stem (PS) cells [5,6] plays a role in hematopoietic stem (HS) cell biology [7,8] and allogeneic bone marrow (BM) transplantation [1,2,9,10]. Here I discuss our current understanding of the cell and molecular pathways that SHIP regulates that influence PS/HS cell biology and BM transplantation. Genetic models of SHIP-deficiency indicate this enzyme is a potential molecular target to enhance both autologous and allogeneic BM transplantation. Thus, strategies to reversibly target SHIP expression and their potential application to stem cell therapies and allogeneic BMT are also discussed. PMID:18473876

Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL

PubMed Central

Ko, Hyeok-Jin; Park, Eunhye; Song, Joseph; Yang, Taek Ho; Lee, Hee Jong; Kim, Kyoung Heon

2012-01-01

Autotransporters have been employed as the anchoring scaffold for cell surface display by replacing their passenger domains with heterologous proteins to be displayed. We adopted an autotransporter (YfaL) of Escherichia coli for the cell surface display system. The critical regions in YfaL for surface display were identified for the construction of a ligation-independent cloning (LIC)-based display system. The designed system showed no detrimental effect on either the growth of the host cell or overexpressing heterologous proteins on the cell surface. We functionally displayed monomeric red fluorescent protein (mRFP1) as a reporter protein and diverse agarolytic enzymes from Saccharophagus degradans 2-40, including Aga86C and Aga86E, which previously had failed to be functional expressed. The system could display different sizes of proteins ranging from 25.3 to 143 kDa. We also attempted controlled release of the displayed proteins by incorporating a tobacco etch virus protease cleavage site into the C termini of the displayed proteins. The maximum level of the displayed protein was 6.1 × 104 molecules per a single cell, which corresponds to 5.6% of the entire cell surface of actively growing E. coli. PMID:22344647

DNA-cell conjugates

DOEpatents

Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

2016-05-03

The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

DNA-cell conjugates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn

The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

A Disposable Organophosphorus Pesticides Enzyme Biosensor Based on Magnetic Composite Nano-Particles Modified Screen Printed Carbon Electrode

PubMed Central

Gan, Ning; Yang, Xin; Xie, Donghua; Wu, Yuanzhao; Wen, Weigang

2010-01-01

A disposable organophosphorus pesticides (OPs) enzyme biosensor based on magnetic composite nanoparticle-modified screen printed carbon electrodes (SPCE) has been developed. Firstly, an acetylcholinesterase (AChE)-coated Fe3O4/Au (GMP) magnetic nanoparticulate (GMP-AChE) was synthesized. Then, GMP-AChE was absorbed on the surface of a SPCE modified by carbon nanotubes (CNTs)/nano-ZrO2/prussian blue (PB)/Nafion (Nf) composite membrane by an external magnetic field. Thus, the biosensor (SPCE│CNTs/ZrO2/PB/Nf│GMP-AChE) for OPs was fabricated. The surface of the biosensor was characterized by scanning electron micrography (SEM) and X-ray fluorescence spectrometery (XRFS) and its electrochemical properties were studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The degree of inhibition (A%) of the AChE by OPs was determined by measuring the reduction current of the PB generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh). In pH = 7.5 KNO3 solution, the A was related linearly to the concentration of dimethoate in the range from 1.0 × 10−3–10 ng·mL−1 with a detection limit of 5.6 × 10−4 ng·mL−1. The recovery rates in Chinese cabbage exhibited a range of 88%–105%. The results were consistent with the standard gas chromatography (GC) method. Compared with other enzyme biosensors the proposed biosensor exhibited high sensitivity, good selectivity with disposable, low consumption of sample. In particular its surface can be easily renewed by removal of the magnet. The convenient, fast and sensitive voltammetric measurement opens new opportunities for OPs analysis. PMID:22315558

A cascading activity-based probe sequentially targets E1–E2–E3 ubiquitin enzymes

PubMed Central

Mulder, Monique P.C.; Witting, Katharina; Berlin, Ilana; Pruneda, Jonathan N.; Wu, Kuen-Phon; Chang, Jer-Gung; Merkx, Remco; Bialas, Johanna; Groettrup, Marcus; Vertegaal, Alfred C.O.; Schulman, Brenda A.; Komander, David; Neefjes, Jacques; Oualid, Farid El; Ovaa, Huib

2016-01-01

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe ‘hops’ and ‘traps’ catalytically active ubiquitin-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades. PMID:27182664

Sustained release and permeation of timolol from surface-modified solid lipid nanoparticles through bioengineered human cornea.

PubMed

Attama, A A; Reichl, S; Müller-Goymann, C C

2009-08-01

The aim of the study was to formulate and evaluate surface-modified solid lipid nanoparticles sustained delivery system of timolol hydrogen maleate, a prototype ocular drug using a human cornea construct. Surface-modified solid lipid nanoparticles containing timolol with and without phospholipid were formulated by melt emulsification with high-pressure homogenization and characterized by particle size, wide-angle X-ray diffraction, encapsulation efficiency, and in vitro drug release. Drug transport studies through cornea bioengineered from human donor cornea cells were carried out using a modified Franz diffusion cell and drug concentration analyzed by high-performance liquid chromatography. Results show that surface-modified solid lipid nanoparticles possessed very small particles (42.9 +/- 0.3 nm, 47.2 +/- 0.3 nm, 42.7 +/- 0.7 nm, and 37.7 +/- 0.3 nm, respectively for SM-SLN 1, SM-SLN 2, SM-SLN 3, and SM-SLN 4) with low polydispersity indices, increased encapsulation efficiency (> 44%), and sustained in vitro release compared with unmodified lipid nanoparticles whose particles were greater than 160 nm. Permeation of timolol hydrogen maleate from the surface-modified lipid nanoparticles across the cornea construct was sustained compared with timolol hydrogen maleate solution in distilled water. Surface-modified solid lipid nanoparticles could provide an efficient way of improving ocular bioavailability of timolol hydrogen maleate.

Stability and activity of lactate dehydrogenase on biofunctional layers deposited by activated vapor silanization (AVS) and immersion silanization (IS)

NASA Astrophysics Data System (ADS)

Calvo, Jorge Nieto-Márquez; Elices, Manuel; Guinea, Gustavo V.; Pérez-Rigueiro, José; Arroyo-Hernández, María

2017-09-01

The interaction between surfaces and biological elements, in particular, proteins is critical for the performance of biomaterials and biosensors. This interaction can be controlled by modifying the surface in a process known as biofunctionalization. In this work, the enzyme lactate dehydrogenase (LDH) is used to study the stability of the interaction between a functional protein and amine-functionalized surfaces. Two different functionalization procedures were compared: Activated Vapor Silanization (AVS) and Immersion Silanization (IS). Adsorption kinetics is shown to follow the Langmuir model for AVS-functionalized samples, while IS-functionalized samples show a certain instability if immersed in an aqueous medium for several hours. In turn, the enzymatic activity of LDH is preserved for longer times by using glutaraldehyde as crosslinker between the AVS biofunctional surface and the enzyme.

Study on the fabrication of back surface reflectors in nano-crystalline silicon thin-film solar cells by using random texturing aluminum anodization

NASA Astrophysics Data System (ADS)

Shin, Kang Sik; Jang, Eunseok; Cho, Jun-Sik; Yoo, Jinsu; Park, Joo Hyung; Byungsung, O.

2015-09-01

In recent decades, researchers have improved the efficiency of amorphous silicon solar cells in many ways. One of the easiest and most practical methods to improve solar-cell efficiency is adopting a back surface reflector (BSR) as the bottom layer or as the substrate. The BSR reflects the incident light back to the absorber layer in a solar cell, thus elongating the light path and causing the so-called "light trapping effect". The elongation of the light path in certain wavelength ranges can be enhanced with the proper scale of BSR surface structure or morphology. An aluminum substrate with a surface modified by aluminum anodizing is used to improve the optical properties for applications in amorphous silicon solar cells as a BSR in this research due to the high reflectivity and the low material cost. The solar cells with a BSR were formed and analyzed by using the following procedures: First, the surface of the aluminum substrate was degreased by using acetone, ethanol and distilled water, and it was chemically polished in a dilute alkali solution. After the cleaning process, the aluminum surface's morphology was modified by using a controlled anodization in a dilute acid solution to form oxide on the surface. The oxidized film was etched off by using an alkali solution to leave an aluminum surface with randomly-ordered dimple-patterns of approximately one micrometer in size. The anodizing conditions and the anodized aluminum surfaces after the oxide layer had been removed were systematically investigated according to the applied voltage. Finally, amorphous silicon solar cells were deposited on a modified aluminum plate by using dc magnetron sputtering. The surfaces of the anodized aluminum were observed by using field-emission scanning electron microscopy. The total and the diffuse reflectances of the surface-modified aluminum sheets were measured by using UV spectroscopy. We observed that the diffuse reflectances increased with increasing anodizing voltage. The properties of the solar cells on anodized aluminum substrates were analyzed by using a solar simulator.

Enzyme release of phenolics from muscadine grape (Vitis rotundifolia Michx.) skins and seeds.

PubMed

Xu, Changmou; Yagiz, Yavuz; Borejsza-Wysocki, Wlodzimierz; Lu, Jiang; Gu, Liwei; Ramírez-Rodrigues, Milena M; Marshall, Maurice R

2014-08-15

Enzyme degradation of plant cell wall polysaccharides can potentially enhance the release of bioactive phenolics. The aim of this study was to evaluate various combinations of solvent and enzyme, enzyme type (cellulase, pectinase, ß-glucosidase), and hydrolysis time (1, 4, 8, 24 h) on the release of muscadine grape skin and seed phenolics, and their antioxidant activities. Results showed that pre-treated muscadine skins and seeds with enzymes decreased total phenolic yield compared with solvent (50% ethanol) alone. Enzyme release of phenolics from skins of different muscadine varieties was significantly different while release from seeds was similar. Enzyme hydrolysis was found to shorten extraction time. Most importantly, enzyme hydrolysis modified the galloylated form of polyphenols to low molecular weight phenolics, releasing phenolic acids (especially gallic acid), and enhancing antioxidant activity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Functional and structural characterization of a novel putative cysteine protease cell wall-modifying multi-domain enzyme selected from a microbial metagenome.

PubMed

Faheem, Muhammad; Martins-de-Sa, Diogo; Vidal, Julia F D; Álvares, Alice C M; Brandão-Neto, José; Bird, Louise E; Tully, Mark D; von Delft, Frank; Souto, Betulia M; Quirino, Betania F; Freitas, Sonia M; Barbosa, João Alexandre R G

2016-12-09

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a K b of 1.8 × 10 5  M -1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space R g of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.

Laser-modified titanium surfaces enhance the osteogenic differentiation of human mesenchymal stem cells.

PubMed

Bressel, Tatiana A B; de Queiroz, Jana Dara Freires; Gomes Moreira, Susana Margarida; da Fonseca, Jéssyca T; Filho, Edson A; Guastaldi, Antônio Carlos; Batistuzzo de Medeiros, Silvia Regina

2017-11-28

Titanium surfaces have been modified by various approaches with the aim of improving the stimulation of osseointegration. Laser beam (Yb-YAG) treatment is a controllable and flexible approach to modifying surfaces. It creates a complex surface topography with micro and nano-scaled patterns, and an oxide layer that can improve the osseointegration of implants, increasing their usefulness as bone implant materials. Laser beam irradiation at various fluences (132, 210, or 235 J/cm 2 ) was used to treat commercially pure titanium discs to create complex surface topographies. The titanium discs were investigated by scanning electron microscopy, X-ray diffraction, and measurement of contact angles. The surface generated at a fluence of 235 J/cm 2 was used in the biological assays. The behavior of mesenchymal stem cells from an umbilical cord vein was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a mineralization assay, and an alkaline phosphatase activity assay and by carrying out a quantitative real-time polymerase chain reaction for osteogenic markers. CHO-k1 cells were also exposed to titanium discs in the MTT assay. The best titanium surface was that produced by laser beam irradiation at 235 J/cm 2 fluence. Cell proliferation analysis revealed that the CHO-k1 and mesenchymal stem cells behaved differently. The laser-processed titanium surface increased the proliferation of CHO-k1 cells, reduced the proliferation of mesenchymal stem cells, upregulated the expression of the osteogenic markers, and enhanced alkaline phosphatase activity. The laser-treated titanium surface modulated cellular behavior depending on the cell type, and stimulated osteogenic differentiation. This evidence supports the potential use of laser-processed titanium surfaces as bone implant materials, and their use in regenerative medicine could promote better outcomes.

Effect of Atmospheric Plasma Treatment to Titanium Surface on Initial Osteoblast-Like Cell Spreading. .

PubMed

Kim, In-Hye; Son, Jun-Sik; Kwon, Tae-Yub; Kim, Kyo-Han

2015-01-01

Plasma treatments are becoming a popular method for modifying the characteristics of a range of substrate surfaces. Atmospheric pressure plasma is cost-efficient, safe and simple compared to high-pressure plasma. This study examined the effects of atmospheric pressure plasma to a titanium (Ti) surface on osteoblast-like cell (osteoblast) spreading and cellular networks. The characteristics of the Ti surface before and after the atmospheric plasma treatment were analyzed by X-ray photoemission spectroscopy (XPS), scanning electron microscopy (SEM), contact angle measurements, and an optical 3D profiling system. The morphology of osteoblasts attached to the Ti surfaces was observed by SEM and confocal laser scanning microscopy. The atmospheric pressure plasma made the Ti surfaces more hydrophilic. The osteoblasts that adhered to the untreated surface were round and spherical, whereas the cells covered a larger surface area on the plasma-treated surface. The plasma-treated Ti surface showed enhanced cell spreading and migration with more developed cellular networks. In conclusion, an atmospheric plasma treatment is a potential surface modifying method that can enhance the initial the cell affinity at the early stages in vitro.

The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms.

PubMed

Bannister, L H

1979-04-11

Intracellular genera are found in all the major groups of Protista, but are particularly common among the dinoflagellates, trypanosomatid zooflagellates and suctorian ciliates; the Sporozoa are nearly all intracellular at some stage of their life, and the Microspora entirely so. Intracellular forms can dwell in the nucleus, within phagosomal or other vacuoles or may lie free in the hyaloplasm of their host cells. Organisms tend to select their hosts from a restricted taxonomic range although there are some notable exceptions. There is also great variation in the types of host cell inhabited. There are various reasons for both host and cell selectivity including recognition phenomena at the cell surfaces. Invasion of host cells is usually preceded by surface interactions with the invader. Some organisms depend upon phagocytosis for entry, but others induce host cells to engulf them by non-phagocytic means or invade by microinjection through the host plasma membrane. Protista avoid lysosomal destruction by their resistance to enzyme attack, by surrounding themselves with lysosome-inhibiting vacuoles, by escaping from the phagosomal system into the hyaloplasm and by choosing host cells which lack lysosomes. Nutrition of intracellular heterotrophic organisms involves some degree of competition with the host cell's metabolism as well as erosion of host cell cytoplasm. In Plasmodium infections, red cells are made more permeable to required nutrients by the action of the parasite on the host cell membrane. The parasite is often dependent upon the host cell for complex nutrients which it cannot synthesize for itself. Intracellular forms often profoundly modify the structure and metabolism of the host cell or interfere with its growth and multiplication. This may result in the final lysis of the host cell at the end of the intracellular phase or before the infection of other cells. Certain types of intracellular organisms may have arisen initially as forms attached to the cell surface of digestive or other organs, but the intracellular habit appears to have arisen independently in several groups of Protista.

Surface modification of starch based blends using potassium permanganate-nitric acid system and its effect on the adhesion and proliferation of osteoblast-like cells.

PubMed

Pashkuleva, I; Marques, A P; Vaz, F; Reis, R L

2005-01-01

The surface modification of three starch based polymeric biomaterials, using a KMnO4/HNO3 oxidizing system, and the effect of that modification on the osteoblastic cell adhesion has been investigated. The rationale of this work is as follows--starch based polymers have been proposed for use as tissue engineering scaffolds in several publications. It is known that in biodegradable systems it is quite difficult to have both cell adhesion and proliferation. Starch based polymers have shown to perform better than poly-lactic acid based materials but there is still room for improvement. This particular work is aimed at enhancing cell adhesion and proliferation on the surface of several starch based polymer blends that are being proposed as tissue engineering scaffolds. The surface of the polymeric biomaterials was chemically modified using a KMnO4/HNO3 system. This treatment resulted in more hydrophilic surfaces, which was confirmed by contact angle measurements. The effect of the treatment on the bioactivity of the surface modified biomaterials was also studied. The bioactivity tests, performed in simulated body fluid after biomimetic coating, showed that a dense film of calcium phosphate was formed after 30 days. Finally, human osteoblast-like cells were cultured on unmodified (control) and modified materials in order to observe the effect of the presence of higher numbers of polar groups on the adhesion and proliferation of those cells. Two of the modified polymers presented changes in the adhesion behavior and a significant increase in the proliferation rate kinetics when compared to the unmodified controls.

Effects of a hybrid micro/nanorod topography-modified titanium implant on adhesion and osteogenic differentiation in rat bone marrow mesenchymal stem cells.

PubMed

Zhang, Wenjie; Li, Zihui; Huang, Qingfeng; Xu, Ling; Li, Jinhua; Jin, Yuqin; Wang, Guifang; Liu, Xuanyong; Jiang, Xinquan

2013-01-01

Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells. The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells. This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo.

Fabrication and characterization of electrochemically prepared bioanode (polyaniline/ferritin/glucose oxidase) for biofuel cell application

NASA Astrophysics Data System (ADS)

ul Haque, Sufia; Inamuddin; Nasar, Abu; Asiri, Abdullah M.

2018-01-01

Porous matrix of polyaniline (PANI) has been electrodeposited along with the entrapment of biocompatible redox mediator ferritin (Frt) and glucose oxidase (GOx) on the surface of glassy carbon (GC) electrode. The characterizations have been carried out by X-ray Diffraction (XRD) and Transmission electron microscopy (TEM). The enhanced electrochemical signal transfer rate from enzyme to the electrode surface was due to the intimate contact of the enzyme with the electrochemically polymerized conducting PANI matrix. The PANI/Frt/GOx modified GC bioanode was used to investigate the electrocatalytic activity as a function of the concentration of glucose in the range of 10-60 mM. It was confirmed by the electrochemical impedance spectroscopy (EIS), the thick deposition of PANI layer becomes more compact due to which the charge transfer resistance of PANI matrix becomes higher. All the electrochemical measurements of the electrode were carried out by using cyclic voltammetry (CV) and linear sweep voltammetry (LSV). CV curves were recorded at different scan rates (20-100 mV/s) at 50 mM of glucose in 0.3 M potassium ferrocyanide. A normalized saturation current density of 22.3 ± 2 mA/cm2 was observed for the oxidation of 50 mM glucose at a scan rate of 100 mV/s.

Influence of layer-by-layer assembled electrospun poly (L-lactic acid) nanofiber mats on the bioactivity of endothelial cells

NASA Astrophysics Data System (ADS)

Wu, Keke; Zhang, Xiazhi; Yang, Wufeng; Liu, Xiaoyan; Jiao, Yanpeng; Zhou, Changren

2016-12-01

Electrospun poly(L-lactic acid) (PLLA) nanofiber mats were successfully modified by deposition of multilayers with chitosan (CS), heparin (Hep) and graphene oxide (GO) through electrostatic layer-by-layer (LBL) self-assembly method. In this study, the surface properties of PLLA nanofiber mats before and after modification were investigated via scanning electron microscope (SEM), atomic force microscopy (AFM), attenuated total reflectance fourier transformation infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and water contact angle measurement. In addition, the cytocompatibility of the modified PLLA nanofiber mats were investigated by testing endothelial cells compatibility, including cell attachment, cell proliferation and cell cycle. The results revealed that the surfaces of modified PLLA nanofiber mats become much rougher, stifiness and the hydrophilicity of the LBL modified PLLA nanofiber mats were improved compared to original PLLA one. Moreover, the modified PLLA nanofiber mats had promoted the endothelial cells viability attachment significantly. Besides, we studied the PLLA nanofiber mats on the expression of necrosis factor (TNF-α), interleukine-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. The results showed that modified PLLA nanofiber mats had inhibited the inflammatory response to some extent.

Enzymatic measurement of free and esterified cholesterol levels in plasma and other biological preparations using the oxygen electrode in a modified glucose analyzer.

PubMed

Dietschy, J M; Weeks, L E; Delente, J J

1976-12-01

A method is described for assaying free and esterified cholesterol using the oxygen electrode in a modified glucose analyzer to measure the relative amount of oxygen utilization taking place during oxydation of free cholesterol by the enzyme, cholesterol oxidase. A second enzyme, cholesterol ester hydrolase, is utilized to generate free cholesterol from cholesterol esters. This assay procedure is rapid, specific, reproducible and applicable to the measurement of free and esterified cholesterol carried in the major plasma lipoprotein fractions of man and the rat and, in addition, it can be utilized for the assay of sterols in subcellular fractions of cells.

A sensitive label-free electrochemical immunosensor for detection of cytokeratin 19 fragment antigen 21-1 based on 3D graphene with gold nanopaticle modified electrode.

PubMed

Zeng, Yan; Bao, Jing; Zhao, Yanan; Huo, Danqun; Chen, Mei; Yang, Mei; Fa, Huanbao; Hou, Changjun

2018-02-01

Previous studies have confirmed that cytokeratin 19 fragment antigen 21-1 (CYFRA 21-1) serves as a powerful biomarker in non-small cell lung cancer (NSCLC). Herein, we report for the first time a label-free electrochemical immunosensor for sensitive and selective detection of tumor marker CYFRA21-1. In this work, three-dimensional graphene @ gold nanoparticles (3D-G@Au) nanocomposite was modified on the glassy carbon electrode (GCE) surface to enhance the conductivity of immunosensor. The anti-CYFRA21-1 captured and fixed on the modified GCE through the cross-linking of chitosan (CS), glutaraldehyde (GA) and anti-CYFRA21-1. The differential pulse voltammetry (DPV) peak current change due to the specific interaction between anti-CYFRA21-1 and CYFRA21-1 on the modified electrode surface was utilized to detect CYFRA21-1. Under optimized conditions, the proposed electrochemical immunosensor was employed to detect CYFRA21-1 and exhibited a wide linear range of 0.25-800ngmL -1 and low detection limit of 100pgmL -1 (S/N = 3). Moreover, the recovery rates of serum samples were in the range from 95.2% to 108.7% and the developed immunosensor also shows a good correlation (less than 6.6%) with enzyme-linked immunosorbent assay (ELISA) in the detection of clinical serum samples. Therefore, it is expected that the proposed immunosensor based on a 3D-G@Au has great potential in clinical medical diagnosis of CYFRA21-1. Copyright © 2017 Elsevier B.V. All rights reserved.

Technology Prospecting on Enzymes: Application, Marketing and Engineering

PubMed Central

Li, Shuang; Yang, Xiaofeng; Yang, Shuai; Zhu, Muzi; Wang, Xiaoning

2012-01-01

Enzymes are protein molecules functioning as specialized catalysts for chemical reactions. They have contributed greatly to the traditional and modern chemical industry by improving existing processes. In this article, we first give a survey of representative industrial applications of enzymes, focusing on the technical applications, feed industry, food processing and cosmetic products. The recent important developments and applications of enzymes in industry are reviewed. Then large efforts are dedicated to the worldwide enzyme market from the demand and production perspectives. Special attention is laid on the Chinese enzyme market. Although enzyme applications are being developed in full swing, breakthroughs are needed to overcome their weaknesses in maintaining activities during the catalytic processes. Strategies of metagomic analysis, cell surface display technology and cell-free system might give valuable solutions in novel enzyme exploiting and enzyme engineering. PMID:24688658

Surface modification of poly(L-lactic acid) to improve its cytocompatibility via assembly of polyelectrolytes and gelatin.

PubMed

Lin, Yuan; Wang, Luling; Zhang, Peibiao; Wang, Xin; Chen, Xuesi; Jing, Xiabin; Su, Zhaohui

2006-03-01

Poly(L-lactide) (PLLA) surface was modified via aminolysis by poly(allylamine hydrochloride) (PAH) at high pH and subsequent electrostatic self-assembly of poly(sodium styrenesulfonate) (PSS) and PAH, and the process was monitored by X-ray photoelectron spectroscopy (XPS) and contact angle measurement. These modified PLLAs were then used as charged substrates for further incorporation of gelatin to improve their cytocompatibility. The amphoteric nature of the gelatin was exploited and the gelatin was adsorbed to the negatively charged PLLA/PSS and positively charged PLLA/PAH at pH=3.4 and 7.4, respectively. XPS and water contact angle data indicated that the gelatin adsorption at pH=3.4 resulted in much higher surface coverage by gelatin than at pH=7.4. All the modified PLLA surfaces became more hydrophilic than the virgin PLLA. Chondrocyte culture was used to test the cell attachment, cell morphology and cell viability on the modified PLLA substrates. The results showed that the PAH and PSS modified PLLA exhibited better cytocompatibility than virgin PLLA, and the incorporation of the gelatin on these modified PLLA substrates further improved their cytocompatibility, with the PLLA/PSS substrate treated with the gelatin at pH=3.4 being the best, exceeding the chondrocyte compatibility of the tissue culture polystyrene.

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

PubMed

Wieczorek, Andrew S; Martin, Vincent J J

2012-12-15

The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli β-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli β-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study, chimeric protein scaffolds consisting of type 1 and type 2 cohesins anchored on the surface of L. lactis allowed for the controlled positioning of dockerin-fused reporter enzymes onto the scaffolds. By binding single enzymes or enzyme pairs to the scaffolds, our data also suggest that the size and relative positions of enzymes can affect the catalytic profiles of the resulting complexes. These insights will be of great value as we engineer more advanced scaffold-guided protein complexes to optimize biochemical pathways.

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2

PubMed Central

Tyurin, V A; Balasubramanian, K; Winnica, D; Tyurina, Y Y; Vikulina, A S; He, R R; Kapralov, A A; Macphee, C H; Kagan, V E

2014-01-01

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H2O2 was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A2 (Lp-PLA2), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA2 was blocked by a selective inhibitor of Lp-PLA2, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic ‘eat-me' signal that participates in the engulfment of cells undergoing intrinsic apoptosis. PMID:24464221

Design of a Papain Immobilized Antimicrobial Food Package with Curcumin as a Crosslinker

PubMed Central

Sivakumar, Ponnurengam Malliappan; Doble, Mukesh

2015-01-01

Contamination of food products by spoilage and pathogenic microorganisms during post process handling is one of the major causes for food spoilage and food borne illnesses. The present green sustainable approach describes the covalent immobilization of papain to LDPE (low density polyethylene), HDPE (high density polyethylene), LLDPE (linear low density polyethylene) and PCL (polycaprolactam) with curcumin as the photocrosslinker. About 50% of curcumin and 82-92% of papain were successfully immobilized on these polymers. After 30 days, the free enzyme retained 87% of its original activity, while the immobilized enzyme retained more than 90% of its activity on these polymers. Papain crosslinked to LLDPE exhibited the best antibiofilm properties against Acinetobacter sp. KC119137.1 and Staphylococcus aureus NCIM 5021 when compared to the other three polymers, because of the highest amount of enzyme immobilized on this surface. Papain acts by damaging the cell membrane. The enzyme is able to reduce the amount of carbohydrate and protein contents in the biofilms formed by these organisms. Meat wrapped with the modified LDPE and stored at 4°C showed 9 log reduction of these organisms at the end of the seventh day when compared to samples wrapped with the bare polymer. This method of crosslinking can be used on polymers with or without functional groups and can be adopted to bind any type of antimicrobial agent. PMID:25906061

Opposite Roles of Furin and PC5A in N-Cadherin Processing12

PubMed Central

Maret, Deborah; Sadr, Mohamad Seyed; Sadr, Emad Seyed; Colman, David R; Del Maestro, Rolando F; Seidah, Nabil G

2012-01-01

We recently demonstrated that lack of Furin-processing of the N-cadherin precursor (proNCAD) in highly invasive melanoma and brain tumor cells results in the cell-surface expression of a nonadhesive protein favoring cell migration and invasion in vitro. Quantitative polymerase chain reaction analysis of malignant human brain tumor cells revealed that of all proprotein convertases (PCs) only the levels of Furin and PC5A are modulated, being inversely (Furin) or directly (PC5A) correlated with brain tumor invasive capacity. Intriguingly, the N-terminal sequence following the Furin-activated NCAD site (RQKR↓DW161, mouse nomenclature) reveals a second putative PC-processing site (RIRSDR↓DK189) located in the first extracellular domain. Cleavage at this site would abolish the adhesive functions of NCAD because of the loss of the critical Trp161. This was confirmed upon analysis of the fate of the endogenous prosegment of proNCAD in human malignant glioma cells expressing high levels of Furin and low levels of PC5A (U343) or high levels of PC5A and negligible Furin levels (U251). Cellular analyses revealed that Furin is the best activating convertase releasing an ∼17-kDa prosegment, whereas PC5A is the major inactivating enzyme resulting in the secretion of an ∼20-kDa product. Like expression of proNCAD at the cell surface, cleavage of the NCAD molecule at RIRSDR↓DK189 renders the U251 cancer cells less adhesive to one another and more migratory. Our work modifies the present view on posttranslational processing and surface expression of classic cadherins and clarifies how NCAD possesses a range of adhesive potentials and plays a critical role in tumor progression. PMID:23097623

Chemical Modification in the Design of Immobilized Enzyme Biocatalysts: Drawbacks and Opportunities.

PubMed

Rueda, Nazzoly; Dos Santos, Jose C S; Ortiz, Claudia; Torres, Rodrigo; Barbosa, Oveimar; Rodrigues, Rafael C; Berenguer-Murcia, Ángel; Fernandez-Lafuente, Roberto

2016-06-01

Chemical modification of enzymes and immobilization used to be considered as separate ways to improve enzyme properties. This review shows how the coupled use of both tools may greatly improve the final biocatalyst performance. Chemical modification of a previously immobilized enzyme is far simpler and easier to control than the modification of the free enzyme. Moreover, if protein modification is performed to improve its immobilization (enriching the enzyme in reactive groups), the final features of the immobilized enzyme may be greatly improved. Chemical modification may be directed to improve enzyme stability, but also to improve selectivity, specificity, activity, and even cell penetrability. Coupling of immobilization and chemical modification with site-directed mutagenesis is a powerful instrument to obtain fully controlled modification. Some new ideas such as photoreceptive enzyme modifiers that change their physical properties under UV exposition are discussed. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

PubMed Central

Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

2013-01-01

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

ZnS nanoparticles electrodeposited onto ITO electrode as a platform for fabrication of enzyme-based biosensors of glucose.

PubMed

Du, Jian; Yu, Xiuping; Wu, Ying; Di, Junwei

2013-05-01

The electrochemical and photoelectrochemical biosensors based on glucose oxidase (GOD) and ZnS nanoparticles modified indium tin oxide (ITO) electrode were investigated. The ZnS nanoparticles were electrodeposited directly on the surface of ITO electrode. The enzyme was immobilized on ZnS/ITO electrode surface by sol-gel method to fabricate glucose biosensor. GOD could electrocatalyze the reduction of dissolved oxygen, which resulted in a great increase of the reduction peak current. The reduction peak current decreased linearly with the addition of glucose, which could be used for glucose detection. Moreover, ZnS nanoparticles deposited on ITO electrode surface showed good photocurrent response under illumination. A photoelectrochemical biosensor for the detection of glucose was also developed by monitoring the decreases in the cathodic peak photocurrent. The results indicated that ZnS nanoparticles deposited on ITO substrate were a good candidate material for the immobilization of enzyme in glucose biosensor construction. Copyright © 2013 Elsevier B.V. All rights reserved.

Allosteric regulation of epigenetic modifying enzymes.

PubMed

Zucconi, Beth E; Cole, Philip A

2017-08-01

Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

A sweet new role for LCP enzymes in protein glycosylation

DOE PAGES

Amer, Brendan R.; Clubb, Robert T.

2014-11-21

The peptidoglycan that surrounds Gram-positive bacteria is affixed with a range of macromolecules that enable the microbe to effectively interact with its environment. Distinct enzymes decorate the cell wall with proteins and glycopolymers. Sortase enzymes covalently attach proteins to the peptidoglycan, while LytRCpsA-Psr (LCP) proteins are thought to attach teichoic acid polymers and capsular polysaccharides. Ton-That and colleagues have discovered a new glycosylation pathway in the oral bacterium Actinomyces oris in which sortase and LCP enzymes operate on the same protein substrate. The A. oris LCP protein has a novel function, acting on the cell surface to transfer glycan macromoleculesmore » to a protein, which is then attached to the cell wall by a sortase. The reactions are tightly coupled, as elimination of the sortase causes the lethal accumulation of glycosylated protein in the membrane. Furthermore, since sortase enzymes are attractive drug targets, this novel finding may provide a convenient cell-based tool to discover inhibitors of this important enzyme family.« less

Preparation of glucose sensors using gold nanoparticles modified diamond electrode

NASA Astrophysics Data System (ADS)

Fachrurrazie; Ivandini, T. A.; Wibowo, W.

2017-04-01

A glucose sensor was successfully developed by immobilizing glucose oxidase (GOx) at boron-doped diamond (BDD) electrodes. Prior to GOx immobilization, the BDD was modified with gold nanoparticles (AuNPs). To immobilize AuNPs, the gold surface was modified to nitrogen termination. The characterization of the electrode surface was performed using an X-ray photoelectron spectroscopy and a scanning electron microscope, while the electrochemical properties of the enzyme electrode were characterized using cyclic voltammetry. Cyclic voltammograms of the prepared electrode for D-glucose in phosphate buffer solution pH 7 showed a new reduction peak at +0.16 V. The currents of the peak were linear in the concentration range of 0.1 M to 0.9 M, indicated that the GOx-AuNP-BDD can be applied for electrochemical glucose detection.

Enzymatic reactions in confined environments

NASA Astrophysics Data System (ADS)

Küchler, Andreas; Yoshimoto, Makoto; Luginbühl, Sandra; Mavelli, Fabio; Walde, Peter

2016-05-01

Within each biological cell, surface- and volume-confined enzymes control a highly complex network of chemical reactions. These reactions are efficient, timely, and spatially defined. Efforts to transfer such appealing features to in vitro systems have led to several successful examples of chemical reactions catalysed by isolated and immobilized enzymes. In most cases, these enzymes are either bound or adsorbed to an insoluble support, physically trapped in a macromolecular network, or encapsulated within compartments. Advanced applications of enzymatic cascade reactions with immobilized enzymes include enzymatic fuel cells and enzymatic nanoreactors, both for in vitro and possible in vivo applications. In this Review, we discuss some of the general principles of enzymatic reactions confined on surfaces, at interfaces, and inside small volumes. We also highlight the similarities and differences between the in vivo and in vitro cases and attempt to critically evaluate some of the necessary future steps to improve our fundamental understanding of these systems.

The use of magnetron sputtering for the deposition of thin titanium coatings on the surface of bioresorbable electrospun fibrous scaffolds for vascular tissue engineering: A pilot study

NASA Astrophysics Data System (ADS)

Bolbasov, E. N.; Antonova, L. V.; Stankevich, K. S.; Ashrafov, A.; Matveeva, V. G.; Velikanova, E. A.; Khodyrevskaya, Yu. I.; Kudryavtseva, Yu. A.; Anissimov, Y. G.; Tverdokhlebov, S. I.; Barbarash, L. S.

2017-03-01

The deposition of thin titanium coatings using magnetron spattering on the surface of bioresorbable fibrous scaffolds produced by electrospinning was investigated. Parameters that allow the surface modification without damaging the "macro" structure of scaffolds were determined. Physicochemical properties of the modified scaffolds were described using SEM, EDS, DSC, optical goniometry, and mechanical testing. It was shown that plasma treatment has a significant influence on the scaffolds' fiber surface relief. The modification process leads to a slight decrease of the scaffold mechanical performance mainly caused by polymer crystallization. Increasing the deposition time increases the amount of titanium on the surface. The biocompatibility of the modified scaffolds was studied using hybridoma of the endothelial cells of human umbilical vein and human lung carcinoma (EA.hy 926 cell line). Cell adhesion, viability, and secretion of interleukin-6 (IL6), interleukin-8 (IL8), and vascular endothelial growth factor (VEGF) were investigated. It was demonstrated that the deposition of thin titanium coatings on the fibrous scaffolds' surface enhances cell adhesion. Additionally, it was determined that modified scaffolds have proangiogenic activity.

Enhanced preferential cytotoxicity through surface modification: synthesis, characterization and comparative in vitro evaluation of TritonX-100 modified and unmodified zinc oxide nanoparticles in human breast cancer cell (MDA-MB-231).

PubMed

Kc, Biplab; Paudel, Siddhi Nath; Rayamajhi, Sagar; Karna, Deepak; Adhikari, Sandeep; Shrestha, Bhupal G; Bisht, Gunjan

2016-01-01

Nanoparticles (NPs) are receiving increasing interest in biomedical research owing to their comparable size with biomolecules, novel properties and easy surface engineering for targeted therapy, drug delivery and selective treatment making them a better substituent against traditional therapeutic agents. ZnO NPs, despite other applications, also show selective anticancer property which makes it good option over other metal oxide NPs. ZnO NPs were synthesized by chemical precipitation technique, and then surface modified using Triton X-100. Comparative study of cytotoxicity of these modified and unmodified NPs on breast cancer cell line (MDA-MB-231) and normal cell line (NIH 3T3) were carried out. ZnO NPsof average size 18.67 ± 2.2 nm and Triton-X modified ZnO NPs of size 13.45 ± 1.42 nm were synthesized and successful characterization of synthesized NPs was done by Fourier transform infrared spectroscopy (FT-IR), X-Ray diffraction (XRD), transmission electron microscopy (TEM) analysis. Surface modification of NPs was proved by FT-IR analysis whereas structure and size by XRD analysis. Morphological analysis was done by TEM. Cell viability assay showed concentration dependent cytotoxicity of ZnO NPs in breast cancer cell line (MDA-MB-231) whereas no positive correlation was found between cytotoxicity and increasing concentration of stress in normal cell line (NIH 3T3) within given concentration range. Half maximum effective concentration (EC50) value for ZnO NPs was found to be 38.44 µg/ml and that of modified ZnO NPs to be 55.24 µg/ml for MDA-MB-231. Crystal violet (CV) staining image showed reduction in number of viable cells in NPs treated cell lines further supporting this result. DNA fragmentation assay showed fragmented bands indicating that the mechanism of cytotoxicity is through apoptosis. Although use of surfactant decreases particle size, toxicity of modified ZnO NPs were still less than unmodified NPs on MDA-MB-231 contributed by biocompatible surface coating. Both samples show significantly less toxicity towards NIH 3T3 in concentration independent manner. But use of Triton-X, a biocompatible polymer, enhances this preferentiality effect. Since therapeutic significance should be analyzed through its comparative effect on both normal and cancer cells, possible application of biocompatible polymer modified nanoparticles as therapeutic agent holds better promise.Graphical abstractSurface coating, characterization and comparative in vitro cytotoxicity study on MDA-MB 231 and NIH 3T3 of ZnO NPs showing enhanced preferentiality by biocompatible surface modification.

Dendritic Cells: A Spot on Sialic Acid

PubMed Central

Crespo, Hélio J.; Lau, Joseph T. Y.; Videira, Paula A.

2013-01-01

Glycans decorating cell surface and secreted proteins and lipids occupy the juncture where critical host–host and host-pathogen interactions occur. The role of glycan epitopes in cell–cell and cell-pathogen adhesive events is already well-established, and cell surface glycan structures change rapidly in response to stimulus and inflammatory cues. Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how glycans and their changes contribute to the overall immune response remains poorly defined. Sialic acids are unique sugars that usually occupy the terminal position of the glycan chains and may be modified by external factors, such as pathogens, or upon specific physiological cellular events. At cell surface, sialic acid-modified structures form the key fundamental determinants for a number of receptors with known involvement in cellular adhesiveness and cell trafficking, such as the Selectins and the Siglec families of carbohydrate recognizing receptors. Dendritic cells (DCs) preside over the transition from innate to the adaptive immune repertoires, and no other cell has such relevant role in antigen screening, uptake, and its presentation to lymphocytes, ultimately triggering the adaptive immune response. Interestingly, sialic acid-modified structures are involved in all DC functions, such as antigen uptake, DC migration, and capacity to prime T cell responses. Sialic acid content changes along DC differentiation and activation and, while, not yet fully understood, these changes have important implications in DC functions. This review focuses on the developmental regulation of DC surface sialic acids and how manipulation of DC surface sialic acids can affect immune-critical DC functions by altering antigen endocytosis, pathogen and tumor cell recognition, cell recruitment, and capacity for T cell priming. The existing evidence points to a potential of DC surface sialylation as a therapeutic target to improve and diversify DC-based therapies. PMID:24409183

Biocompatible, smooth, plasma-treated nickel-titanium surface--an adequate platform for cell growth.

PubMed

Chrzanowski, W; Szade, J; Hart, A D; Knowles, J C; Dalby, M J

2012-02-01

High nickel content is believed to reduce the number of biomedical applications of nickel-titanium alloy due to the reported toxicity of nickel. The reduction in nickel release and minimized exposure of the cell to nickel can optimize the biocompatibility of the alloy and increase its use in the application where its shape memory effects and pseudoelasticity are particularly useful, e.g., spinal implants. Many treatments have been tried to improve the biocompatibility of Ni-Ti, and results suggest that a native, smooth surface could provide sufficient tolerance, biologically. We hypothesized that the native surface of nickel-titanium supports cell differentiation and insures good biocompatibility. Three types of surface modifications were investigated: thermal oxidation, alkali treatment, and plasma sputtering, and compared with smooth, ground surface. Thermal oxidation caused a drop in surface nickel content, while negligible chemistry changes were observed for plasma-modified samples when compared with control ground samples. In contrast, alkali treatment caused significant increase in surface nickel concentration and accelerated nickel release. Nickel release was also accelerated in thermally oxidized samples at 600 °C, while in other samples it remained at low level. Both thermal oxidation and alkali treatment increased the roughness of the surface, but mean roughness R(a) was significantly greater for the alkali-treated ones. Ground and plasma-modified samples had 'smooth' surfaces with R(a)=4 nm. Deformability tests showed that the adhesion of the surface layers on samples oxidized at 600 °C and alkali treatment samples was not sufficient; the layer delaminated upon deformation. It was observed that the cell cytoskeletons on the samples with a high nickel content or release were less developed, suggesting some negative effects of nickel on cell growth. These effects were observed primarily during initial cell contact with the surface. The most favorable cell responses were observed for ground and plasma-sputtered surfaces. These studies indicated that smooth, plasma-modified surfaces provide sufficient properties for cells to grow. © The Author(s), 2011.

Immobilization of Ag nanoparticles/FGF-2 on a modified titanium implant surface and improved human gingival fibroblasts behavior.

PubMed

Ma, Qianli; Mei, Shenglin; Ji, Kun; Zhang, Yumei; Chu, Paul K

2011-08-01

The objective of this study was to form a rapid and firm soft tissue sealing around dental implants that resists bacterial invasion. We present a novel approach to modify Ti surface by immobilizing Ag nanoparticles/FGF-2 compound bioactive factors onto a titania nanotubular surface. The titanium samples were anodized to form vertically organized TiO(2) nanotube arrays and Ag nanoparticles were electrodeposited onto the nanotubular surface, on which FGF-2 was immobilized with repeated lyophilization. A uniform distribution of Ag nanoparticles/FGF-2 was observed on the TiO(2) nanotubular surface. The L929 cell line was used for cytotoxicity assessment. Human gingival fibroblasts (HGFs) were cultured on the modified surface for cytocompatibility determination. The Ag/FGF-2 immobilized samples displayed excellent cytocompatibility, negligible cytotoxicity, and enhanced HGF functions such as cell attachment, proliferation, and ECM-related gene expression. The Ag nanoparticles also exhibit some bioactivity. In conclusion, this modified TiO(2) nanotubular surface has a large potential for use in dental implant abutment. Copyright © 2011 Wiley Periodicals, Inc.

Enhanced stability and catalytic activity of immobilized α-amylase on modified Fe3O4 nanoparticles for potential application in food industries

NASA Astrophysics Data System (ADS)

Hosseinipour, Seyyedeh Leila; Khiabani, Mahmoud Sowti; Hamishehkar, Hamed; Salehi, Roya

2015-09-01

Enzymes play an essential role in catalyzing various reactions. However, their instability upon repetitive/prolonged use, elevated temperature, acidic or alkaline pH remains an area of concern. α-Amylase, a widely used enzyme in food industries for starch hydrolysis, was covalently immobilized on the surface of two developed matrices, amino-functionalized silica-coated magnetite nanoparticles (AFSMNPs) alone and covered with chitosan. The synthesis steps and characterizations of NPs were examined by FT-IR, VSM, and SEM. Modified nanoparticles with average diameters of 20-80 nm were obtained. Enzyme immobilization efficiencies of 89 and 74 were obtained for AFSMNPs and chitosan-coated AFSMNPs, respectively. The optimum pH obtained was 6.5 and 8.0 for the enzyme immobilized on AFSMNPs and chitosan-coated AFSMNPs, respectively. Optimum temperature for the immobilized enzyme shifted toward higher temperatures. Considerable enhancements in thermal stabilities were observed for the immobilized enzyme at elevated temperatures up to 80 °C. A frequent use experiment demonstrated that the immobilized enzyme retained 74 and 85 % of its original activity even after 20 times of repeated use in AFSMNPs and chitosan-coated AFSMNPs, respectively. Storage stability demonstrated that free enzyme lost its activity completely within 30 days. But, immobilized enzyme on AFSMNPs and chitosan-coated AFSMNPs preserved 65.73 and 78.63 % of its initial activity, respectively, after 80 days of incubation. In conclusion, a substantial improvement in the performance of the immobilized enzyme with reference to the free enzyme was obtained. Furthermore, the relative activities of immobilized enzyme are superior than free enzyme over the broader pH and temperature ranges.

Carbon nanotube-based glucose oxidase nanocomposite anode materials for bio-fuel cells

NASA Astrophysics Data System (ADS)

Dudzik, Jonathan

The field of nanotechnology has benefited medicine, science, and engineering. The advent of Carbon Nanotubes (CNTs) and protein-inorganic interfacing have received much attention due to their unique nanostructures which can be modified to act as a scaffold to house proteins or create nanowires. The current trend incorporates the robustness and specificity characteristics of proteins to the mechanical strength, enlarged surface area, and conductive capabilities emblematic of their inorganic counterparts. Bio-Fuel Cells (BFCs) and Biosensors remain at the forefront and devices such as implantable glucose monitors are closer to realization than ever before. This research strives to exploit potential energy from the eukaryotic enzyme Glucose Oxidase (GOx) during oxidation of its substrate, glucose. During this process, a two-electron transfer occurs at its two FAD redox centres which can be harnessed via an electrochemical setup involving a Multi-Walled Carbon Nanotube (MWCNTs) modified electrode. The objective is to develop a MWCNT-GOx bionanocomposite capable of producing and sustaining a competitive power output. To help with this aim, investigation into a crosslinked enzyme cluster (CEC) immobilization technique is envisioned to amplify power output due to its highly concentrated, reusable, and thermally stable characteristics. Numerous CEC-GOx-MWCNT composites were fabricated with the highest initial output reaching 170 muW/cm 2. It was hypothesized that the carbohydrate moiety increased tunnelling distance and therefore hindered electron transfer. Efforts to produce a recombinant GOx without the encumbrance were unsuccessful. Two sub-clone constructs were explored and although a recombinant protein was identified, it was not confirmed to be GOx. BFC testing on bionanocomposites integrating non-glycosylated GOx could not be performed although there remains a strong contention that the recombinant would demonstrate superior power densities in comparison to its native form.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

PubMed Central

Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

1995-01-01

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. Images Fig. 2 Fig. 3 PMID:8618847

Intracellular implantation of enzymes in hollow silica nanospheres for protein therapy: cascade system of superoxide dismutase and catalase.

PubMed

Chang, Feng-Peng; Chen, Yi-Ping; Mou, Chung-Yuan

2014-11-01

An approach for enzyme therapeutics is elaborated with cell-implanted nanoreactors that are based on multiple enzymes encapsulated in hollow silica nanospheres (HSNs). The synthesis of HSNs is carried out by silica sol-gel templating of water-in-oil microemulsions so that polyethyleneimine (PEI) modified enzymes in aqueous phase are encapsulated inside the HSNs. PEI-grafted superoxide dismutase (PEI-SOD) and catalase (PEI-CAT) encapsulated in HSNs are prepared with quantitative control of the enzyme loadings. Excellent activities of superoxide dismutation by PEI-SOD@HSN are found and transformation of H2 O2 to water by PEI-CAT@HSN. When PEI-SOD and PEI-CAT are co-encapsulated, cascade transformation of superoxide through hydrogen peroxide to water was facile. Substantial fractions of HSNs exhibit endosome escape to cytosol after their delivery to cells. The production of downstream reactive oxygen species (ROS) and COX-2/p-p38 expression show that co-encapsulated SOD/CAT inside the HSNs renders the highest cell protection against the toxicant N,N'-dimethyl-4,4'-bipyridinium dichloride (paraquat). The rapid cell uptake and strong detoxification effect on superoxide radicals by the SOD/CAT-encapsulated hollow mesoporous silica nanoparticles demonstrate the general concept of implanting catalytic nanoreactors in biological cells with designed functions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improved mucoadhesion and cell uptake of chitosan and chitosan oligosaccharide surface-modified polymer nanoparticles for mucosal delivery of proteins.

PubMed

Dyawanapelly, Sathish; Koli, Uday; Dharamdasani, Vimisha; Jain, Ratnesh; Dandekar, Prajakta

2016-08-01

The main aim of the present study was to compare mucoadhesion and cellular uptake efficiency of chitosan (CS) and chitosan oligosaccharide (COS) surface-modified polymer nanoparticles (NPs) for mucosal delivery of proteins. We have developed poly (D, L-lactide-co-glycolide) (PLGA) NPs, surface-modified COS-PLGA NPs and CS-PLGA NPs, by using double emulsion solvent evaporation method, for encapsulating bovine serum albumin (BSA) as a model protein. Surface modification of NPs was confirmed using physicochemical characterization methods such as particle size and zeta potential, SEM, TEM and FTIR analysis. Both surface-modified PLGA NPs displayed a slow release of protein compared to PLGA NPs. Furthermore, we have explored the mucoadhesive property of COS as a material for modifying the surface of polymeric NPs. During in vitro mucoadhesion test, positively charged COS-PLGA NPs and CS-PLGA NPs exhibited enhanced mucoadhesion, compared to negatively charged PLGA NPs. This interaction was anticipated to improve the cell interaction and uptake of NPs, which is an important requirement for mucosal delivery of proteins. All nanoformulations were found to be safe for cellular delivery when evaluated in A549 cells. Moreover, intracellular uptake behaviour of FITC-BSA loaded NPs was extensively investigated by confocal laser scanning microscopy and flow cytometry. As we hypothesized, positively charged COS-PLGA NPs and CS-PLGA NPs displayed enhanced intracellular uptake compared to negatively charged PLGA NPs. Our results demonstrated that CS- and COS-modified polymer NPs could be promising carriers for proteins, drugs and nucleic acids via nasal, oral, buccal, ocular and vaginal mucosal routes.

High performance membrane-electrode assembly based on a surface-modified membrane

NASA Astrophysics Data System (ADS)

Han, Sangil; Lee, Jang Woo; Kwak, Chan; Chai, Geun Seok; Son, In Hyuk; Jang, Moon Yup; An, Sung Guk; Cho, Sung Yong; Kim, Jun Young; Kim, Hyung Wook; Serov, Alexey Alexandrovych; Yoo, Youngtai; Nam, Kie Hyun

A surface-modified membrane is prepared using a sputtering technique that deposits gold directly on a Nafion ® 115 membrane surface that is roughened with silicon carbide paper. The surface-modified membranes are characterized by means of a scanning electron microscope (SEM), differential scanning calorimetry (DSC), and water contact-angle analysis. A single direct methanol fuel cell (DMFC) with a surface-modified membrane exhibits enhanced performance (160 mW cm -2), while a bare Nafion ® 115 cell yields 113 mW cm -2 at 0.4 V and an operating temperature of 70 °C. From FE-SEM images and CO ad stripping voltammograms, it is also found that the gold layer is composed of clusters of porous nodule-like particles, which indicates that an anode with nodule-like gold leads to the preferential oxidation of carbon monoxide. These results suggest that the topology of gold in the interfacial area and its electrocatalytic nature may be the critical factors that affect DMFC performance.

Preparation of sumoylated substrates for biochemical analysis.

PubMed

Knipscheer, Puck; Klug, Helene; Sixma, Titia K; Pichler, Andrea

2009-01-01

Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

Novel Architectures for Achieving Direct Electron Transfer in Enzymatic Biofuel Cells

NASA Astrophysics Data System (ADS)

Blaik, Rita A.

Enzymatic biofuel cells are a promising source of alternative energy for small device applications, but still face the challenge of achieving direct electron transfer with high enzyme concentrations in a simple system. In this dissertation, methods of constructing electrodes consisting of enzymes attached to nanoparticle-enhanced substrates that serve as high surface area templates are evaluated. In the first method described, glucose oxidase is covalently attached to gold nanoparticles that are assembled onto genetically engineered M13 bacteriophage. The resulting anodes achieve a high peak current per area and a significant improvement in enzyme surface coverage. In the second system, fructose dehydrogenase, a membrane-bound enzyme that has the natural ability to achieve direct electron transfer, is immobilized into a matrix consisting of binders and carbon nanotubes to extend the lifetime of the anode. For the cathode, bilirubin oxidase is immobilized in a carbon nanotube and sol-gel matrix to achieve direct electron transfer. Finally, a full fuel cell consisting of both an anode and cathode is constructed and evaluated with each system described.

Changes in physicochemical properties and in vitro starch digestion of native and extruded maize flours subjected to branching enzyme and maltogenic α-amylase treatment.

PubMed

Román, Laura; Martínez, Mario M; Rosell, Cristina M; Gómez, Manuel

2017-08-01

Extrusion is an increasingly used type of processing which combined with enzymatic action could open extended possibilities for obtaining clean label modified flours. In this study, native and extruded maize flours were modified using branching enzyme (B) and a combination of branching enzyme and maltogenic α-amylase (BMA) in order to modulate their hydrolysis properties. The microstructure, pasting properties, in vitro starch hydrolysis and resistant starch content of the flours were investigated. Whereas BMA treatment led to greater number of holes on the granule surface in native samples, B and BMA extruded samples showed rougher surfaces with cavities. A reduction in the retrogradation trend was observed for B and BMA native flours, in opposition to the flat pasting profile of their extruded counterparts. The glucose release increased gradually for native flours as the time of reaction did, whereas for extruded flours a fast increase of glucose release was observed during the first minutes of reaction, and kept till the end, indicating a greater accessibility to their porous structure. These results suggested that, in enzymatically treated extruded samples, changes produced at larger hierarchical levels in their starch structure could have masked a slowdown in the starch digestion properties. Copyright © 2017 Elsevier B.V. All rights reserved.

Method of generating a surface mesh

DOEpatents

Shepherd, Jason F [Albuquerque, NM; Benzley, Steven [Provo, UT; Grover, Benjamin T [Tracy, CA

2008-03-04

A method and machine-readable medium provide a technique to generate and modify a quadrilateral finite element surface mesh using dual creation and modification. After generating a dual of a surface (mesh), a predetermined algorithm may be followed to generate and modify a surface mesh of quadrilateral elements. The predetermined algorithm may include the steps of generating two-dimensional cell regions in dual space, determining existing nodes in primal space, generating new nodes in the dual space, and connecting nodes to form the quadrilateral elements (faces) for the generated and modifiable surface mesh.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

NASA Astrophysics Data System (ADS)

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-02-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

PubMed Central

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

2016-01-01

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion.

PubMed

Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W; Liu, Yan; Walter, Nils G; Yan, Hao

2016-02-10

Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance.

PubMed

Zhang, Hua; Zhao, Zhen; Lei, Zhen; Wang, Zhenxin

2016-12-06

The phosphorylation of nucleic acid with 5'-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL -1 and lysate of about 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.

Chemical modification of polyvinyl chloride and silicone elastomer in inhibiting adhesion of Aeromonas hydrophila.

PubMed

Kregiel, Dorota; Berlowska, Joanna; Mizerska, Urszula; Fortuniak, Witold; Chojnowski, Julian; Ambroziak, Wojciech

2013-07-01

Disease-causing bacteria of the genus Aeromonas are able to adhere to pipe materials, colonizing the surfaces and forming biofilms in water distribution systems. The aim of our research was to study how the modification of materials used commonly in the water industry can reduce bacterial cell attachment. Polyvinyl chloride and silicone elastomer surfaces were activated and modified with reactive organo-silanes by coupling or co-crosslinking silanes with the native material. Both the native and modified surfaces were tested using the bacterial strain Aeromonas hydrophila, which was isolated from the Polish water distribution system. The surface tension of both the native and modified surfaces was measured. To determine cell viability and bacterial adhesion two methods were used, namely plate count and luminometry. Results were expressed in colony-forming units (c.f.u.) and in relative light units (RLU) per cm(2). Almost all the chemically modified surfaces exhibited higher anti-adhesive and anti-microbial properties in comparison to the native surfaces. Among the modifying agents examined, poly[dimethylsiloxane-co-(N,N-dimethyl-N-n-octylammoniopropyl chloride) methylsiloxane)] terminated with hydroxydimethylsilyl groups (20 %) in silicone elastomer gave the most desirable results. The surface tension of this modifier, was comparable to the non-polar native surface. However, almost half of this value was due to the result of polar forces. In this case, in an adhesion analysis, only 1 RLU cm(-2) and less than 1 c.f.u. cm(-2) were noted. For the native gumosil, the results were 9,375 RLU cm(-2) and 2.5 × 10(8) c.f.u. cm(-2), respectively. The antibacterial activity of active organo-silanes was associated only with the carrier surface because no antibacterial compounds were detected in liquid culture media, in concentrations that were able to inhibit cell growth.

CD10/NEP in non-small cell lung carcinomas. Relationship to cellular proliferation.

PubMed Central

Ganju, R K; Sunday, M; Tsarwhas, D G; Card, A; Shipp, M A

1994-01-01

The cell surface metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) hydrolyzes a variety of peptide substrates and reduces cellular responses to specific peptide hormones. Because CD10/NEP modulates peptide-mediated proliferation of small cell carcinomas of the lung (SCLC) and normal fetal bronchial epithelium, we evaluated the enzyme's expression in non-small cell lung carcinomas (NSCLC). Bronchoalveolar and large cell carcinoma cell lines had low levels of CD10/NEP expression whereas squamous, adenosquamous, and adenocarcinoma cell lines had higher and more variable levels of the cell surface enzyme. Regional variations in CD10/NEP immunostaining in primary NSCLC specimens prompted us to correlate CD10/NEP expression with cell growth. In primary carcinomas of the lung, clonal NSCLC cell lines and SV40-transformed fetal airway epithelium, subsets of cells expressed primarily CD10/NEP or the proliferating cell nuclear antigen (PCNA). Cultured airway epithelial cells had the lowest levels of CD10/NEP expression when the highest percentage of cells were actively dividing; in addition, these cells grew more rapidly when cell surface CD10/NEP was inhibited. NSCLC cell lines had receptors for a variety of mitogenic peptides known to be CD10/NEP substrates, underscoring the functional significance of growth-related variability in CD10/NEP expression. Images PMID:7962523

Synthesis and effect of modification on methacylate - acrylate microspheres for Trametes versicolor laccase enzyme immobilization

NASA Astrophysics Data System (ADS)

Mazlan, Siti Zulaikha; Hanifah, Sharina Abu

2014-09-01

Immobilization of laccase on the modified copolymer methacrylate-acrylate microspheres was studied. A poly (glycidyl methacrylate-co-n-butyl acrylate) microsphere consists of epoxy groups were synthesized using suspension photocuring technique. The epoxy group in poly (GMA-nBA) microspheres were converted into amino groups with aldehyde group. Laccase immobilization is based on having the amino groups on the enzyme surface and aldehyde group on the microspheres via covalent binding. Fourier transform infrared spectroscopy (FT-IR) analysis proved the successful surface modification on microspheres. The FTIR spectrum shows the characteristic peaks at 1646 cm-1 assigned to the conformation of the polymerization that took place between monomer GMA and nBA respectively. In addition, after modification, FTIR peaks that assigned to the epoxy ring (844 cm-1 and 904 cm-1) were decreased. The results obtained from FTIR method signify good agreement with the epoxy content method. Hence, the activity of the laccase-immobilized microspheres increased upon increasing the epoxy content. Furthermore, poly (GMA-nBA) exhibited uniform microspheres with below 2 μm surface. Immobilized enzyme showed a broader pH profile and higher temperature compared native enzyme.

Biosensors Fabricated through Electrostatic Assembly of Enzymes/Polyelectrolyte Hybrid Layers on Carbon Nanotubes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Yuehe; Liu, Guodong; Wang, Jun

2006-06-01

Carbon nanotubes (CNTs) have emerged as new class of nanomaterials that is receiving considerable interest because of their unique structure, mechanical, and electronic properties. One promising application of CNTs is to fabricate highly sensitive chemo/biosensors.1-4 For construction of these CNT-based sensors, the CNTs first have to be modified with some molecules specific to the interests. Generally, covalent binding, affinity, and electrostatic interaction have been utilized for the modification of CNTs. Among them, the electrostatic method is attractive due to its simplicity and high efficiency. In present work, we have developed highly sensitively amperometric biosensors for glucose, choline, organophosphate pesticide (OPP)more » and nerve agents (NAs) based on electrostatically assembling enzymes on the surface of CNTs. All these biosensors were fabricated by immobilization of enzymes on the negatively charged CNTs surface through alternately assembling a cationic poly(diallydimethylammonium chloride) (PDDA) layer and an enzyme layer. Using this layer-by-layer (LBL) technique, a bioactive nanocomposite film was fabricated on the electrode surface. Owing to the electrocatalytic effect of CNTs, an amplified electrochemical signal was achieved, which leads to low detections limits for glucose, choline, and OPP and NAs.« less

Optimization of the Synthesis of Structured Phosphatidylcholine with Medium Chain Fatty Acid.

PubMed

Ochoa-Flores, Angélica A; Hernández-Becerra, Josafat A; Cavazos-Garduño, Adriana; Vernon-Carter, Eduardo J; García, Hugo S

2017-11-01

Structured phosphatidylcholine was successfully produced by acidolysis between phosphatidylcholine and free medium chain fatty acid, using phospholipase A 1 immobilized on Duolite A568. Response surface methodology was applied to optimize the reaction system using three process parameters: molar ratio of substrates (phosphatidylcholine to free medium chain fatty acid), enzyme loading, and reaction temperature. All parameters evaluated showed linear and quadratic significant effects on the production of modified phosphatidylcholine; molar ratio of substrates contributed positively, but temperature influenced negatively. Increased enzyme loading also led to increased production of modified phosphatidylcholine but only during the first 9 hours of the acidolysis reaction. Optimal conditions obtained from the model were a ratio of phosphatidylcholine to free medium chain fatty acid of 1:15, an enzyme loading of 12%, and a temperature of 45°C. Under these conditions a production of modified phosphatidylcholine of 52.98 % were obtained after 24 h of reaction. The prediction was confirmed from the verification experiments; the production of modified phosphatidylcholine was 53.02%, the total yield of phosphatidylcholine 64.28% and the molar incorporation of medium chain fatty acid was 42.31%. The acidolysis reaction was scaled-up in a batch reactor with a similar production of modified phosphatidylcholine, total yield of phosphatidylcholine and molar incorporation of medium chain fatty acid. Purification by column chromatography of the structured phosphatidylcholine yielded 62.53% of phosphatidylcholine enriched with 42.52% of medium chain fatty acid.

Extracellular enzymes facilitate electron uptake in biocorrosion and bioelectrosynthesis.

PubMed

Deutzmann, Jörg S; Sahin, Merve; Spormann, Alfred M

2015-04-21

Direct, mediator-free transfer of electrons between a microbial cell and a solid phase in its surrounding environment has been suggested to be a widespread and ecologically significant process. The high rates of microbial electron uptake observed during microbially influenced corrosion of iron [Fe(0)] and during microbial electrosynthesis have been considered support for a direct electron uptake in these microbial processes. However, the underlying molecular mechanisms of direct electron uptake are unknown. We investigated the electron uptake characteristics of the Fe(0)-corroding and electromethanogenic archaeon Methanococcus maripaludis and discovered that free, surface-associated redox enzymes, such as hydrogenases and presumably formate dehydrogenases, are sufficient to mediate an apparent direct electron uptake. In genetic and biochemical experiments, we showed that these enzymes, which are released from cells during routine culturing, catalyze the formation of H2 or formate when sorbed to an appropriate redox-active surface. These low-molecular-weight products are rapidly consumed by M. maripaludis cells when present, thereby preventing their accumulation to any appreciable or even detectable level. Rates of H2 and formate formation by cell-free spent culture medium were sufficient to explain the observed rates of methane formation from Fe(0) and cathode-derived electrons by wild-type M. maripaludis as well as by a mutant strain carrying deletions in all catabolic hydrogenases. Our data collectively show that cell-derived free enzymes can mimic direct extracellular electron transfer during Fe(0) corrosion and microbial electrosynthesis and may represent an ecologically important but so far overlooked mechanism in biological electron transfer. The intriguing trait of some microbial organisms to engage in direct electron transfer is thought to be widespread in nature. Consequently, direct uptake of electrons into microbial cells from solid surfaces is assumed to have a significant impact not only on fundamental microbial and biogeochemical processes but also on applied bioelectrochemical systems, such as microbial electrosynthesis and biocorrosion. This study provides a simple mechanistic explanation for frequently observed fast electron uptake kinetics in microbiological systems without a direct transfer: free, cell-derived enzymes can interact with cathodic surfaces and catalyze the formation of intermediates that are rapidly consumed by microbial cells. This electron transfer mechanism likely plays a significant role in various microbial electron transfer reactions in the environment. Copyright © 2015 Deutzmann et al.

A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format.

PubMed

Beer, Meike V; Rech, Claudia; Diederichs, Sylvia; Hahn, Kathrin; Bruellhoff, Kristina; Möller, Martin; Elling, Lothar; Groll, Jürgen

2012-04-01

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.

Cell-Adhesive and Cell-Repulsive Zwitterionic Oligopeptides for Micropatterning and Rapid Electrochemical Detachment of Cells

PubMed Central

Kakegawa, Takahiro; Mochizuki, Naoto; Sadr, Nasser; Suzuki, Hiroaki

2013-01-01

In this study, we describe the development of oligopeptide-modified cell culture surfaces from which adherent cells can be rapidly detached by application of an electrical stimulus. An oligopeptide, CGGGKEKEKEK, was designed with a terminal cysteine residue to mediate binding to a gold surface via a gold–thiolate bond. The peptide forms a self-assembled monolayer through the electrostatic force between the sequence of alternating charged glutamic acid (E) and lysine (K) residues. The dense and electrically neutral oligopeptide zwitterionic layer of the modified surface was resistant to nonspecific adsorption of proteins and adhesion of cells, while the surface was altered to cell adhesive by the addition of a second oligopeptide (CGGGKEKEKEKGRGDSP) containing the RGD cell adhesion motif. Application of a negative electrical potential to this gold surface cleaved the gold–thiolate bond, leading to desorption of the oligopeptide layer, and rapid (within 2 min) detachment of virtually all cells. This approach was applicable not only to detachment of cell sheets but also for transfer of cell micropatterns to a hydrogel. This electrochemical approach of cell detachment may be a useful tool for tissue-engineering applications. PMID:22853640

Enzymatic aspects in ENT cancer-Matrix metalloproteinases

PubMed Central

Zamfir Chiru, AA; Popescu, CR; Gheorghe, DC

2014-01-01

Abstract The study of ENT cancer allows the implementation of molecular biology methods in diagnosis, predicting the evolution of the disease and suggesting a certain treatment. MMPs are proteolytic enzymes, zinc dependent endopeptidases, secreted by tissues and proinflammatory cells that play a role in the clearance of cell surface receptors. They are expressed as zymogens (inactive forms). Proteolytic enzymes cleave zymogens generating active forms. They are involved in cell proliferation, adhesion, differentiation, migration, angiogenesis, apoptosis and host defense. PMID:25408759

Study of zeolite influence on analytical characteristics of urea biosensor based on ion-selective field-effect transistors

PubMed Central

2014-01-01

A possibility of the creation of potentiometric biosensor by adsorption of enzyme urease on zeolite was investigated. Several variants of zeolites (nano beta, calcinated nano beta, silicalite, and nano L) were chosen for experiments. The surface of pH-sensitive field-effect transistors was modified with particles of zeolites, and then the enzyme was adsorbed. As a control, we used the method of enzyme immobilization in glutaraldehyde vapour (without zeolites). It was shown that all used zeolites can serve as adsorbents (with different effectiveness). The biosensors obtained by urease adsorption on zeolites were characterized by good analytical parameters (signal reproducibility, linear range, detection limit and the minimal drift factor of a baseline). In this work, it was shown that modification of the surface of pH-sensitive field-effect transistors with zeolites can improve some characteristics of biosensors. PMID:24636423

Influence of physicochemical properties of laser-modified polystyrene on bovine serum albumin adsorption and rat C6 glioma cell behavior.

PubMed

Wang, Xuefeng; Ohlin, C André; Lu, Qinghua; Hu, Jun

2006-09-15

Biomaterial surface modification is an efficient way of improving cell-material interactions. In this study, sub-micrometer laser-induced periodic surface structures (LIPSS) were produced on polystyrene by laser irradiation. FT-IR analysis confirmed that this treatment also led to surface oxidation and anisotropic orientation of the produced carbonyl groups. As a consequence, the surface energy of the laser-treated polystyrene was 1.45 times that of the untreated polystyrene, as measured by contact-angle goniometry. Protein adsorption and rat C6 glioma cell behavior on the two substrates were investigated, showing that the changed physicochemical properties of laser-modified polystyrene surface led to an increase in the quantity of adsorbed bovine serum albumin and significantly affected the behavior of rat C6 glioma cells. In the early stages of cell spreading, cells explored their microenvironment using filopodium as the main sensor. Moreover, cells actively aligned themselves along the direction of LIPSS gradually and cell attachment and proliferation were significantly enhanced. 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006.

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Curbo, Sophie; Gaudin, Raphael; Carlsten, Mattias

2009-12-25

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4R{alpha} receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown tomore » be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.« less

SUMO chain formation relies on the amino-terminal region of SUMO-conjugating enzyme and has dedicated substrates in plants

PubMed Central

Tomanov, Konstantin; Nehlin, Lilian; Ziba, Ionida

2018-01-01

The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation. PMID:29133528

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

NASA Astrophysics Data System (ADS)

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.

Horseradish peroxidase-nanoclay hybrid particles of high functional and colloidal stability.

PubMed

Pavlovic, Marko; Rouster, Paul; Somosi, Zoltan; Szilagyi, Istvan

2018-08-15

Highly stable dispersions of enzyme-clay nanohybrids of excellent horseradish peroxidase activity were developed. Layered double hydroxide nanoclay was synthesized and functionalized with heparin polyelectrolyte to immobilize the horseradish peroxidase enzyme. The formation of a saturated heparin layer on the platelets led to charge inversion of the positively charged bare nanoclay and to highly stable aqueous dispersions. Great affinity of the enzyme to the surface modified platelets resulted in strong horseradish peroxidase adsorption through electrostatic and hydrophobic interactions as well as hydrogen bonding network and prevented enzyme leakage from the obtained material. The enzyme kept its functional integrity upon immobilization and showed excellent activity in decomposition of hydrogen peroxide and oxidation of an aromatic compound in the test reactions. In addition, remarkable long term functional stability of the enzyme-nanoclay hybrid was observed making the developed colloidal system a promising antioxidant candidate in biomedical treatments and industrial processes. Copyright © 2018 Elsevier Inc. All rights reserved.

Influence of local anesthetics upon human polymorphonuclear leukocyte function in vitro. Reduction of lysosomal enzyme release and superoxide anion production

PubMed Central

1977-01-01

Cationic local anesthetics have been reported to influence cellular responses to surface stimuli by interfering with the function of microtubules and microfilaments. Since unimpaired microtubule and microfilament functions are required by human polymorphonuclear leukocytes in order to respond normally to surface stimulation, we have studied effects of the local anesthetic, tetracaine on the function and morphology of these cells in vitro. Tetracaine (0.25--1.0 mM) significantly reduced extracellular release of the lysosomal enzymes, beta-glucuronidase and lysozyme from polymorphonuclear leukocytes exposed to serum-treated zymosan (a particulate stimulus), zymosan- treated serum (a soluble stimulus), and to the surface-active lectin, concanavalin A. Tetracaine also significantly reduced superoixde anion production (superoxide dismutase-inhibitable cytochrome c reduction) by these cells. Tetrancaine was not cytotoxic and its effects could be reversed completely by washing cells once with buffer. Electron microscope examination of tetracaine-treated cells revealed marked alterations of surface membranes. Microtubules and microfilaments appeared normal in "resting" polymorphonuclear leukocytes, but the increase in microtubules normally observed in stimulated cells was not seen after tetracaine treatment. These results suggest that tetracaine interferes with those interactions between immune reactants and the polymorphonuclear leukocyte cell surface which provoke exocytosis and increased oxidative metabolism. PMID:195003

Designing the Sniper: Improving Targeted Human Cytolytic Fusion Proteins for Anti-Cancer Therapy via Molecular Simulation.

PubMed

Bochicchio, Anna; Jordaan, Sandra; Losasso, Valeria; Chetty, Shivan; Perera, Rodrigo Casasnovas; Ippoliti, Emiliano; Barth, Stefan; Carloni, Paolo

2017-02-17

Targeted human cytolytic fusion proteins (hCFPs) are humanized immunotoxins for selective treatment of different diseases including cancer. They are composed of a ligand specifically binding to target cells genetically linked to a human apoptosis-inducing enzyme. hCFPs target cancer cells via an antibody or derivative (scFv) specifically binding to e.g., tumor associated antigens (TAAs). After internalization and translocation of the enzyme from endocytosed endosomes, the human enzymes introduced into the cytosol are efficiently inducing apoptosis. Under in vivo conditions such enzymes are subject to tight regulation by native inhibitors in order to prevent inappropriate induction of cell death in healthy cells. Tumor cells are known to upregulate these inhibitors as a survival mechanism resulting in escape of malignant cells from elimination by immune effector cells. Cytosolic inhibitors of Granzyme B and Angiogenin (Serpin P9 and RNH1, respectively), reduce the efficacy of hCFPs with these enzymes as effector domains, requiring detrimentally high doses in order to saturate inhibitor binding and rescue cytolytic activity. Variants of Granzyme B and Angiogenin might feature reduced affinity for their respective inhibitors, while retaining or even enhancing their catalytic activity. A powerful tool to design hCFPs mutants with improved potency is given by in silico methods. These include molecular dynamics (MD) simulations and enhanced sampling methods (ESM). MD and ESM allow predicting the enzyme-protein inhibitor binding stability and the associated conformational changes, provided that structural information is available. Such "high-resolution" detailed description enables the elucidation of interaction domains and the identification of sites where particular point mutations may modify those interactions. This review discusses recent advances in the use of MD and ESM for hCFP development from the viewpoints of scientists involved in both fields.

Designing the Sniper: Improving Targeted Human Cytolytic Fusion Proteins for Anti-Cancer Therapy via Molecular Simulation

PubMed Central

Bochicchio, Anna; Jordaan, Sandra; Losasso, Valeria; Chetty, Shivan; Casasnovas Perera, Rodrigo; Ippoliti, Emiliano; Barth, Stefan; Carloni, Paolo

2017-01-01

Targeted human cytolytic fusion proteins (hCFPs) are humanized immunotoxins for selective treatment of different diseases including cancer. They are composed of a ligand specifically binding to target cells genetically linked to a human apoptosis-inducing enzyme. hCFPs target cancer cells via an antibody or derivative (scFv) specifically binding to e.g., tumor associated antigens (TAAs). After internalization and translocation of the enzyme from endocytosed endosomes, the human enzymes introduced into the cytosol are efficiently inducing apoptosis. Under in vivo conditions such enzymes are subject to tight regulation by native inhibitors in order to prevent inappropriate induction of cell death in healthy cells. Tumor cells are known to up-regulate these inhibitors as a survival mechanism resulting in escape of malignant cells from elimination by immune effector cells. Cytosolic inhibitors of Granzyme B and Angiogenin (Serpin P9 and RNH1, respectively), reduce the efficacy of hCFPs with these enzymes as effector domains, requiring detrimentally high doses in order to saturate inhibitor binding and rescue cytolytic activity. Variants of Granzyme B and Angiogenin might feature reduced affinity for their respective inhibitors, while retaining or even enhancing their catalytic activity. A powerful tool to design hCFPs mutants with improved potency is given by in silico methods. These include molecular dynamics (MD) simulations and enhanced sampling methods (ESM). MD and ESM allow predicting the enzyme-protein inhibitor binding stability and the associated conformational changes, provided that structural information is available. Such “high-resolution” detailed description enables the elucidation of interaction domains and the identification of sites where particular point mutations may modify those interactions. This review discusses recent advances in the use of MD and ESM for hCFP development from the viewpoints of scientists involved in both fields. PMID:28536352

Modification of erythrocyte membrane proteins, enzymes and transport mechanisms in chronic alcoholics: an in vivo and in vitro study.

PubMed

Maturu, Paramahamsa; Vaddi, Damodara Reddy; Pannuru, Padmavathi; Nallanchakravarthula, Varadacharyulu

2013-01-01

The aim of the study was to elucidate the molecular mechanisms underlying the alcohol perturbation leading to deleterious effects on erythrocyte membrane transport in chronic alcoholics. Membrane bound enzyme activities such as Na(+), K(+)-ATPase, Ca(2+),Mg(2+)-ATPase and acetylcholine esterase and membrane transport analysis by in vitro and erythrocyte membrane profile analysis in controls and chronic alcoholic red cells were analyzed. It was observed that decreased Na(+), K(+)-ATPase enzyme activity and increased activities of Ca(2+),Mg(2+)-ATPase and acetylcholine esterase in chronic alcoholics compared to controls. The in vitro studies of erythrocytes suggested that there is an increased uptake of glucose through chronic alcoholic red cells. However, glucose utilization by chronic alcoholic red cells was decreased. An increased sensitivity of ouabain for its binding site on Na(+), K(+)-ATPase in chronic alcoholic erythrocyte membrane was evident from this study. Though there appears to be an increased Na(+) influx in chronic alcoholic cells, the status of Na(+) transport is not altered much. However, ouabain caused slight disturbances in the transport of sodium, similar disturbances in the potassium transport resulting in much accumulation of potassium in red cells. It was concluded that chronic alcohol consumption modified certain membrane bound proteins, enzymes and transport mechanisms in chronic alcoholics.

Osseointegration improvement by plasma electrolytic oxidation of modified titanium alloys surfaces.

PubMed

Echeverry-Rendón, Mónica; Galvis, Oscar; Quintero Giraldo, David; Pavón, Juan; López-Lacomba, José Luis; Jiménez-Piqué, Emilio; Anglada, Marc; Robledo, Sara M; Castaño, Juan G; Echeverría, Félix

2015-02-01

Titanium (Ti) is a material frequently used in orthopedic applications, due to its good mechanical properties and high corrosion resistance. However, formation of a non-adherent fibrous tissue between material and bone drastically could affect the osseointegration process and, therefore, the mechanical stability of the implant. Modifications of topography and configuration of the tissue/material interface is one of the mechanisms to improve that process by manipulating parameters such as morphology and roughness. There are different techniques that can be used to modify the titanium surface; plasma electrolytic oxidation (PEO) is one of those alternatives, which consists of obtaining porous anodic coatings by controlling parameters such as voltage, current, anodizing solution and time of the reaction. From all of the above factors, and based on previous studies that demonstrated that bone cells sense substrates features to grow new tissue, in this work commercially pure Ti (c.p Ti) and Ti6Al4V alloy samples were modified at their surface by PEO in different anodizing solutions composed of H2SO4 and H3PO4 mixtures. Treated surfaces were characterized and used as platforms to grow osteoblasts; subsequently, cell behavior parameters like adhesion, proliferation and differentiation were also studied. Although the results showed no significant differences in proliferation, differentiation and cell biological activity, overall results showed an important influence of topography of the modified surfaces compared with polished untreated surfaces. Finally, this study offers an alternative protocol to modify surfaces of Ti and their alloys in a controlled and reproducible way in which biocompatibility of the material is not compromised and osseointegration would be improved.

Enzymatic degradation of cell wall and related plant polysaccharides.

PubMed

Ward, O P; Moo-Young, M

1989-01-01

Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.

Nrf2 Expression Modifies Influenza A Entry and Replication inNasal Epithelial Cells

EPA Science Inventory

Influenza infection is a major cause of morbidity and mortality worldwide, especially during pandemics outbreaks. Emerging data indicate that phase II antioxidant enzyme pathways could playa role in virus-associated inflammation and immune clearance. While Nrf2-dependent gene exp...

Cell adhesion and growth on ultrananocrystalline diamond and diamond-like carbon films after different surface modifications

NASA Astrophysics Data System (ADS)

Miksovsky, J.; Voss, A.; Kozarova, R.; Kocourek, T.; Pisarik, P.; Ceccone, G.; Kulisch, W.; Jelinek, M.; Apostolova, M. D.; Reithmaier, J. P.; Popov, C.

2014-04-01

Diamond and diamond-like carbon (DLC) films possess a set of excellent physical and chemical properties which together with a high biocompatibility make them attractive candidates for a number of medical and biotechnological applications. In the current work thin ultrananocrystalline diamond (UNCD) and DLC films were comparatively investigated with respect to cell attachment and proliferation after different surface modifications. The UNCD films were prepared by microwave plasma enhanced chemical vapor deposition, the DLC films by pulsed laser deposition (PLD). The films were comprehensively characterized with respect to their basic properties, e.g. crystallinity, morphology, chemical bonding nature, etc. Afterwards the UNCD and DLC films were modified applying O2 or NH3/N2 plasmas and UV/O3 treatments to alter their surface termination. The surface composition of as-grown and modified samples was studied by X-ray photoelectron spectroscopy (XPS). Furthermore the films were characterized by contact angle measurements with water, formamide, 1-decanol and diiodomethane; from the results obtained the surface energy with its dispersive and polar components was calculated. The adhesion and proliferation of MG63 osteosarcoma cells on the different UNCD and DLC samples were assessed by measurement of the cell attachment efficiency and MTT assays. The determined cell densities were compared and correlated with the surface properties of as-deposited and modified UNCD and DLC films.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components

PubMed Central

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, UA

2014-01-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and Impact of the Study Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. PMID:24935714

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amer, Brendan R.; Clubb, Robert T.

The peptidoglycan that surrounds Gram-positive bacteria is affixed with a range of macromolecules that enable the microbe to effectively interact with its environment. Distinct enzymes decorate the cell wall with proteins and glycopolymers. Sortase enzymes covalently attach proteins to the peptidoglycan, while LytRCpsA-Psr (LCP) proteins are thought to attach teichoic acid polymers and capsular polysaccharides. Ton-That and colleagues have discovered a new glycosylation pathway in the oral bacterium Actinomyces oris in which sortase and LCP enzymes operate on the same protein substrate. The A. oris LCP protein has a novel function, acting on the cell surface to transfer glycan macromoleculesmore » to a protein, which is then attached to the cell wall by a sortase. The reactions are tightly coupled, as elimination of the sortase causes the lethal accumulation of glycosylated protein in the membrane. Furthermore, since sortase enzymes are attractive drug targets, this novel finding may provide a convenient cell-based tool to discover inhibitors of this important enzyme family.« less

Alteration and modulation of protein activity by varying post-translational modification

DOEpatents

Thompson, David N; Reed, David W; Thompson, Vicki S; Lacey, Jeffrey A; Apel, William A

2015-03-03

Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

Alteration and modulation of protein activity by varying post-translational modification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, David N.; Reed, David W.; Thompson, Vicki S.

Embodiments of the invention include methods of altering the enzymatic activity or solubility of an extremophilic enzyme or post-translationally modifying a protein of interest via using isolated or partially purified glycosyltransferases and/or post-translational modification proteins, extracts of cells comprising glycosyltransferases and/or post-translational modification proteins, and/or in cells comprising one or more glycosyltransferases and/or post-translational modification proteins.

Genetically Engineered Theranostic Mesenchymal Stem Cells for the Evaluation of the Anticancer Efficacy of Enzyme/Prodrug Systems

PubMed Central

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-01-01

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TKSR39 mutants), yeast cytosine deaminase: uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs’ bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. PMID:25575867

Synthesization, Characterization, and in Vitro Evaluation of Cytotoxicity of Biomaterials Based on Halloysite Nanotubes.

PubMed

Sánchez-Fernández, Antonio; Peña-Parás, Laura; Vidaltamayo, Román; Cué-Sampedro, Rodrigo; Mendoza-Martínez, Ana; Zomosa-Signoret, Viviana C; Rivas-Estilla, Ana M; Riojas, Paulina

2014-12-04

Halloysite is an aluminosilicate clay that has been widely used for controlled drug delivery, immobilization of enzymes, and for the capture of circulating tumor cells (CTCs). Surface modification of halloysite by organosilanes has been explored to improve their properties. In this study halloysite clay nanotubes (HNTs) were functionalized by two different organosilanes: Trimethoxy(propyl)silane (TMPS), and Triethoxy(octyl)silane (EOS). Untreated and modified samples were characterized by scanning electron microscopy (SEM), X-ray diffractometry (XRD), thermogravimetrical analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). Results showed a strong interaction of organosilanes with the chemical groups present in HNTs. Biocompatibility and cytotoxicity of these nanomaterials were determined using C6 rat glioblastoma cells. Our results indicate that prior to functionalization, HNTs show a high biocompatibility and low cytotoxicity. However, HNTs functionalized with EOS and TMPS showed high cytotoxicity by inducing apoptosis. These results allow the identification of potential applications in biomedical areas for HNTs.

Synthesization, Characterization, and in Vitro Evaluation of Cytotoxicity of Biomaterials Based on Halloysite Nanotubes

PubMed Central

Sánchez-Fernández, Antonio; Peña-Parás, Laura; Vidaltamayo, Román; Cué-Sampedro, Rodrigo; Mendoza-Martínez, Ana; Zomosa-Signoret, Viviana C.; Rivas-Estilla, Ana M.; Riojas, Paulina

2014-01-01

Halloysite is an aluminosilicate clay that has been widely used for controlled drug delivery, immobilization of enzymes, and for the capture of circulating tumor cells (CTCs). Surface modification of halloysite by organosilanes has been explored to improve their properties. In this study halloysite clay nanotubes (HNTs) were functionalized by two different organosilanes: Trimethoxy(propyl)silane (TMPS), and Triethoxy(octyl)silane (EOS). Untreated and modified samples were characterized by scanning electron microscopy (SEM), X-ray diffractometry (XRD), thermogravimetrical analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). Results showed a strong interaction of organosilanes with the chemical groups present in HNTs. Biocompatibility and cytotoxicity of these nanomaterials were determined using C6 rat glioblastoma cells. Our results indicate that prior to functionalization, HNTs show a high biocompatibility and low cytotoxicity. However, HNTs functionalized with EOS and TMPS showed high cytotoxicity by inducing apoptosis. These results allow the identification of potential applications in biomedical areas for HNTs. PMID:28788274

Characterization of an anti-glucosyltransferase serum specific for insoluble glucan synthesis by Streptococcus mutans.

PubMed

Linzer, R; Slade, H D

1976-02-01

An anti-glucosyltransferase serum, which synthesized 96% insoluble glucans, was prepared against a purified enzyme preparation from Streptococcus mutans strain HS6 (serotype a). This serum was examined for its effects on glucan synthesis by crude enzyme preparations from eight strains (four serotypes) of S. mutans and for the ability of these preparations to promote adherence of S. mutans to a smooth surface. Glucosyltransferase activity was assayed by measuring the incorporation of glucose from [14C]glucose-labeled sucrose into water-insoluble and water-soluble (ethanol-insoluble) glucans. Anti-glucosyltransferase serum inhibited insoluble glucan synthesis by crude enzyme preparations from cells of the four serotypes of S. mutans. Enzymes from strains of types a, b, and d were inhibited between 70 to 90%; enzymes from type c strains were inhibited from 45 to 60%. The adherence to a glass surface of heat-killed cells from these four serotypes was likewise inhibited. Soluble glucan synthesis was not inhibited by the serum, and in some cases its synthesis increased as insoluble glucan synthesis decreased.

Electrospun fiber membranes enable proliferation of genetically modified cells

PubMed Central

Borjigin, Mandula; Eskridge, Chris; Niamat, Rohina; Strouse, Bryan; Bialk, Pawel; Kmiec, Eric B

2013-01-01

Polycaprolactone (PCL) and its blended composites (chitosan, gelatin, and lecithin) are well-established biomaterials that can enrich cell growth and enable tissue engineering. However, their application in the recovery and proliferation of genetically modified cells has not been studied. In the study reported here, we fabricated PCL-biomaterial blended fiber membranes, characterized them using physicochemical techniques, and used them as templates for the growth of genetically modified HCT116-19 colon cancer cells. Our data show that the blended polymers are highly miscible and form homogenous electrospun fiber membranes of uniform texture. The aligned PCL nanofibers support robust cell growth, yielding a 2.5-fold higher proliferation rate than cells plated on standard plastic plate surfaces. PCL-lecithin fiber membranes yielded a 2.7-fold higher rate of proliferation, while PCL-chitosan supported a more modest growth rate (1.5-fold higher). Surprisingly, PCL-gelatin did not enhance cell proliferation when compared to the rate of cell growth on plastic surfaces. PMID:23467983

Capture and release of cells using a temperature-responsive surface that immobilizes an antibody through DNA duplex formation.

PubMed

Kimura, Tsuyoshi; Nakamura, Naoko; Umeda, Kanji; Hashimoto, Yoshihide; Kishida, Akio

We synthesized a temperature-responsive surface that immobilized an antibody via DNA duplex formation for selective capture and release of target cells. Polyethylene films were modified by grafting poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), which were prepared at various ratios of NIPAAm/AAc. The increased hydrophilicity of P(NIPAAm-co-PAA) film with decreased temperature was confirmed by water contact angle measurement. Single strand DNA (20mer) was chemically immobilized on the surface and then antibody (anti-mouse CD45, mCD45) modified with the complementary single strand DNA was immobilized on the surface through DNA duplex formation. The mCD45 antibody immobilization was confirmed by immunostaining. HeLa cells (mCD45 negative) and mouse bone marrow (BM) cells (mCD45 positive) were adhered on the surfaces at 37 °C. Although HeLa cells were detached by 4 °C incubation, BM cells were still adhered on the surface and then the adhered cells were released by DNase treatment. From these results, it was suggested that cells could be selectively captured and collected by using a film having surface that immobilizes an antibody via DNA duplex formation.

Catalytic performance of subtilisin immobilized without covalently attachment on surface-functionalized mesoporous silica materials

NASA Astrophysics Data System (ADS)

Murai, K.; Nonoyama, T.; Ando, F.; Kato, K.

2011-10-01

Mesoporous silica (MPS) materials were synthesized using cetyltrimethylammonium bromide or amphiphilic pluronic polymer P123 (EO20PO70EO20) as structure-directing agent. MPS samples were characterized by FE-SEM and N2 adsorption-desorption isotherms, respectively. Subtilisin from Bacillus licheiformis (4.1 × 7.8 × 3.7 nm) was easily immobilized by a direct one-step immobilization process onto MPS with different organo-functinalized surfaces. However, enzyme immobilized on MPS modified with 3-mercaptopropyl group strongly reduced its enantioselectivity. Denaturation temperature of immobilized subtilisin shifted to a high temperature compared to free-enzyme. These biocatalysts on MPS particles retained about 30% of original activity even after 5 cycles of recycle use.

In vitro mesenchymal stem cell response to a CO2 laser modified polymeric material.

PubMed

Waugh, D G; Hussain, I; Lawrence, J; Smith, G C; Cosgrove, D; Toccaceli, C

2016-10-01

With an ageing world population it is becoming significantly apparent that there is a need to produce implants and platforms to manipulate stem cell growth on a pharmaceutical scale. This is needed to meet the socio-economic demands of many countries worldwide. This paper details one of the first ever studies in to the manipulation of stem cell growth on CO2 laser surface treated nylon 6,6 highlighting its potential as an inexpensive platform to manipulate stem cell growth on a pharmaceutical scale. Through CO2 laser surface treatment discrete changes to the surfaces were made. That is, the surface roughness of the nylon 6,6 was increased by up to 4.3μm, the contact angle was modulated by up to 5° and the surface oxygen content increased by up to 1atom %. Following mesenchymal stem cell growth on the laser treated samples, it was identified that CO2 laser surface treatment gave rise to an enhanced response with an increase in viable cell count of up to 60,000cells/ml when compared to the as-received sample. The effect of surface parameters modified by the CO2 laser surface treatment on the mesenchymal stem cell response is also discussed along with potential trends that could be identified to govern the mesenchymal stem cell response. Copyright © 2016. Published by Elsevier B.V.

Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes.

PubMed

Cosgrove, Daniel J

2016-01-01

The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the 'Young's modulus' of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Systems Approaches to Predict the Functions of Glycoside Hydrolases during the Life Cycle of Aspergillus niger Using Developmental Mutants ∆brlA and ∆flbA

PubMed Central

van Munster, Jolanda M.; Nitsche, Benjamin M.; Akeroyd, Michiel; Dijkhuizen, Lubbert; van der Maarel, Marc J. E. C.; Ram, Arthur F. J.

2015-01-01

Background The filamentous fungus Aspergillus niger encounters carbon starvation in nature as well as during industrial fermentations. In response, regulatory networks initiate and control autolysis and sporulation. Carbohydrate-active enzymes play an important role in these processes, for example by modifying cell walls during spore cell wall biogenesis or in cell wall degradation connected to autolysis. Results In this study, we used developmental mutants (ΔflbA and ΔbrlA) which are characterized by an aconidial phenotype when grown on a plate, but also in bioreactor-controlled submerged cultivations during carbon starvation. By comparing the transcriptomes, proteomes, enzyme activities and the fungal cell wall compositions of a wild type A. niger strain and these developmental mutants during carbon starvation, a global overview of the function of carbohydrate-active enzymes is provided. Seven genes encoding carbohydrate-active enzymes, including cfcA, were expressed during starvation in all strains; they may encode enzymes involved in cell wall recycling. Genes expressed in the wild-type during starvation, but not in the developmental mutants are likely involved in conidiogenesis. Eighteen of such genes were identified, including characterized sporulation-specific chitinases and An15g02350, member of the recently identified carbohydrate-active enzyme family AA11. Eight of the eighteen genes were also expressed, independent of FlbA or BrlA, in vegetative mycelium, indicating that they also have a role during vegetative growth. The ΔflbA strain had a reduced specific growth rate, an increased chitin content of the cell wall and specific expression of genes that are induced in response to cell wall stress, indicating that integrity of the cell wall of strain ΔflbA is reduced. Conclusion The combination of the developmental mutants ΔflbA and ΔbrlA resulted in the identification of enzymes involved in cell wall recycling and sporulation-specific cell wall modification, which contributes to understanding cell wall remodeling mechanisms during development. PMID:25629352

Accelerated cell-surface interlocking on plasma polymer-modified porous ceramics.

PubMed

Rebl, Henrike; Finke, Birgit; Schmidt, Jürgen; Mohamad, Heba S; Ihrke, Roland; Helm, Christiane A; Nebe, J Barbara

2016-12-01

Excellent osseointegration of permanent implants is crucial for the long lasting success of the implantation. To improve the osseointegrative potential, bio-inert titanium alloy surfaces (Ti6Al4V) are modified by plasma chemical oxidation (PCO®) of the titanium-oxide layer to a non-stoichiometric, amorphous calcium phosphate layer. The native titanium-oxide film measuring only a few nanometers is converted by PCO® to a thick porous calcium phosphate layer of about 10μm. In a second step the PCO surface is combined with a cell adhesive plasma-polymerized allylamine (PPAAm) nano film (5 and 50nm). Independent of the PPAAm coating homogeneity, the human osteoblast-like MG-63 cells show a remarkable increase in cell size and well-developed filopodia. Analyses of the actin cytoskeleton reveal that the cells mold to the pore shape of the PPAAm-covered PCO, thereby establishing a strong attachment to the surface. Interestingly, we could demonstrate that even though our untreated PCO shows excellent hydrophilicity, this alone is not sufficient to facilitate fast cell spreading, but the positive surface charges mediated by PPAAm. This multilayer composite material guarantees enhanced interlocking of the cells with the porous surface. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

NASA Astrophysics Data System (ADS)

Balaev, V. V.; Lashkov, A. A.; Gabdoulkhakov, A. G.; Seregina, T. A.; Dontsova, M. V.; Mikhailov, A. M.

2015-03-01

Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis ( YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability ( R work = 16.2, R free = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.

Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

NASA Astrophysics Data System (ADS)

Wilson, James M.; Birinyi, Louis K.; Salomon, Robert N.; Libby, Peter; Callow, Allan D.; Mulligan, Richard C.

1989-06-01

The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution.

PubMed

Kwan, David H; Constantinescu, Iren; Chapanian, Rafi; Higgins, Melanie A; Kötzler, Miriam P; Samain, Eric; Boraston, Alisdair B; Kizhakkedathu, Jayachandran N; Withers, Stephen G

2015-05-06

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.

Towards improved artificial lungs through biocatalysis.

PubMed

Kaar, Joel L; Oh, Heung-Il; Russell, Alan J; Federspiel, William J

2007-07-01

Inefficient CO(2) removal due to limited diffusion represents a significant barrier in the development of artificial lungs and respiratory assist devices, which use hollow fiber membranes (HFMs) as the blood-gas interface and can require large blood-contacting membrane area. To offset the underlying diffusional challenge, "bioactive" HFMs that facilitate CO(2) diffusion were prepared via covalent immobilization of carbonic anhydrase (CA), an enzyme which catalyzes the conversion of bicarbonate in blood to CO(2), onto the surface of plasma-modified conventional HFMs. This study examines the impact of enzyme attachment on the diffusional properties and the rate of CO(2) removal of the bioactive membranes. Plasma deposition of surface reactive hydroxyls, to which CA could be attached, did not change gas permeance of the HFMs or generate membrane defects, as determined by scanning electron microscopy, when low plasma discharge power and short exposure times were employed. Cyanogen bromide activation of the surface hydroxyls and subsequent modification with CA resulted in near monolayer enzyme coverage (88%) on the membrane. The effect of increased plasma discharge power and exposure time on enzyme loading was negligible while gas permeance studies showed enzyme attachment did not impede CO(2) or O(2) diffusion. Furthermore, when employed in a model respiratory assist device, the bioactive membranes improved CO(2) removal rates by as much as 75% from physiological bicarbonate solutions with no enzyme leaching. These results demonstrate the potential of bioactive HFMs with immobilized CA to enhance CO(2) exchange in respiratory devices.

Production of multi-fiber modifying enzyme from Mamillisphaeria sp. for refining of recycled paper pulp.

PubMed

Laothanachareon, Thanaporn; Khonzue, Parichart; Rattanaphan, Nakul; Tinnasulanon, Phungjai; Apawasin, Saowanee; Paemanee, Atchara; Ruanglek, Vasimon; Tanapongpipat, Sutipa; Champreda, Verawat; Eurwilaichitr, Lily

2011-01-01

Enzymatic modification of pulp is receiving increasing interest for energy reduction at the refining step of the paper-making process. In this study, the production of a multi-fiber modifying enzyme from Mamillisphaeria sp. BCC8893 was optimized in submerged fermentation using a response-surface methodology. Maximal production was obtained in a complex medium comprising wheat bran, soybean, and rice bran supplemented with yeast extract at pH 6.0 and a harvest time of 7 d, resulting in 9.2 IU/mL of carboxymethyl cellulase (CMCase), 14.9 IU/mL of filter paper activity (FPase), and 242.7 IU/mL of xylanase. Treatment of old corrugated container pulp at 0.2-0.3 IU of CMCase/g of pulp led to reductions in refining energy of 8.5-14.8%. The major physical properties were retained, including tensile and compression strength. Proteomic analysis showed that the enzyme was a complex composite of endo-glucanases, cellobiohydrolases, beta-1,4-xylanases, and beta-glucanases belonging to various glycosyl hydrolase families, suggestive of cooperative enzyme action in fiber modification, providing the basis for refining efficiency.

Enhancing the efficiency of direct reprogramming of human mesenchymal stem cells into mature neuronal-like cells with the combination of small molecule modulators of chromatin modifying enzymes, SMAD signaling and cyclic adenosine monophosphate levels.

PubMed

Alexanian, Arshak R; Liu, Qing-song; Zhang, Zhiying

2013-08-01

Advances in cell reprogramming technologies to generate patient-specific cells of a desired type will revolutionize the field of regenerative medicine. While several cell reprogramming methods have been developed over the last decades, the majority of these technologies require the exposure of cell nuclei to reprogramming large molecules via transfection, transduction, cell fusion or nuclear transfer. This raises several technical, safety and ethical issues. Chemical genetics is an alternative approach for cell reprogramming that uses small, cell membrane penetrable substances to regulate multiple cellular processes including cell plasticity. Recently, using the combination of small molecules that are involved in the regulation chromatin structure and function and agents that favor neural differentiation we have been able to generate neural-like cells from human mesenchymal stem cells. In this study, to improve the efficiency of neuronal differentiation and maturation, two specific inhibitors of SMAD signaling (SMAD1/3 and SMAD3/5/8) that play an important role in neuronal differentiation of embryonic stem cells, were added to our previous neural induction recipe. Results demonstrated that human mesenchymal stem cells grown in this culture conditions exhibited higher expression of several mature neuronal genes, formed synapse-like structures and exerted electrophysiological properties of differentiating neural stem cells. Thus, an efficient method for production of mature neuronal-like cells from human adult bone marrow derived mesenchymal stem cells has been developed. We concluded that specific combinations of small molecules that target specific cell signaling pathways and chromatin modifying enzymes could be a promising approach for manipulation of adult stem cell plasticity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilsbacher, Julie L.; Cheng, Min; Cheng, Dong

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell deathmore » when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236–45.« less

THE UPTAKE OF RADIOCOLLOIDS BY MACROPHAGES IN VITRO

PubMed Central

Gosselin, Robert E.

1956-01-01

Macrophages isolated from the rabbit peritoneal cavity extract radioactive colloidal gold from solutions in vitro. This reaction (ultraphagocytosis) involves two phases: the reversible adsorption of gold on the cell surface and the subsequent irreversible removal of surface-bound colloid into the cell. The latter process (called ingestion) appears to proceed at a rate which is proportional at any moment to the amount of gold attached to the cell surface; the latter in turn can be related to the concentration in extracellular fluid by a simple adsorption isotherm. In terms of rate, therefore, ingestion is related to the extracellular gold concentration in the same way that many enzyme reactions are related to the substrate concentration. Although enzyme kinetics are useful in describing rates of ultraphagocytosis, there is no evidence that enzymes participate in either adsorption or ingestion or that metabolic energy is required of the macrophage. Exudative leucocytes of the heterophilic series show little or no interaction with these finely dispersed gold sols (mean particle diameter 6 to 9 millimicrons). 37°C. three parameters are sufficient to characterize the reaction between gold and a suspension of macrophages, namely an affinity constant (1/Ks), an adsorption maximum (L), and a rate constant of ingestion (k 3). Although numerical values differed markedly among cells of different exudates, all three parameters were estimated in three instances. In these suspensions between 2 and 20 per cent of the surface-bound gold was ingested each minute (37°C., pH 7.4). Under conditions of surface saturation, it was estimated that tens of thousands of gold particles were attached to the surface of an average macrophage; this amount of colloid, however, occupied less than 1 per cent of the geometric area of the cell surface. Although surface saturation imposed an upper limit on the rate of ingestion, no practical limit was noted in the capacity of macrophages to continue the reaction. Optical measurements imply that within the cell agglutination of colloidal gold began promptly after its ingestion. These data are compared with published kinetic studies on the phagocytosis of microscopic particulates and on the parasitism of bacteria by virus. PMID:13319653

Synthesis and patterning of polymers for biomedical applications

NASA Astrophysics Data System (ADS)

He, Wei

The goal of this dissertation is to synthesize and characterize novel polymers, as well as to explore alternative techniques for biomedical applications. Although significant progress has been achieved in the design and preparation of new biomaterials over the past years, much remains to be accomplished. The interactions between biomaterials and cells are very important, especially in the emerging field of tissue engineering. The focus of this research is to improve such interactions via several different approaches. One way to engineer cellular interaction is by modifying surface topography through micro-patterning. Although photolithography is widely used for patterning, it is not suitable for direct cell and protein patterning because of the usage of organic solvent for feature development. To address this issue, a biocompatible chemically amplified resist derived from N-vinyl-2-pyrrolidone (NVP) was prepared. The results have shown that no organic solvent development was required to reveal the patterns and cells can be cultured on these patterned surfaces directly. Strong cell alignment was observed. The other issue addressed in this research is to develop a technique that can modify surface morphology and surface chemistry simultaneously. Such a technique is called masked ion beam lithography (MIBL). By implanting phosphorous ions on polymeric substrates through masks, not only micron/nano size patterns were generated on the surface, but also the phosphorous ions were incorporated. Incubation of bone forming osteoblast cells on these ion beam processed samples has shown that osteoblast cell attachment to the substrate was enhanced, as a consequence of the increased surface roughness as well as the implanted phosphorous ions. This indicates that MIBL can not only generate micro/nanostructures on the surface of a biocompatible polymer, but can also selectively modify the surface chemistry by implanting with specific ions. These factors can contribute to an osteogenic environment.

Surface modification of polyvinyl alcohol/malonic acid nanofibers by gaseous dielectric barrier discharge plasma for glucose oxidase immobilization

NASA Astrophysics Data System (ADS)

Afshari, Esmail; Mazinani, Saeedeh; Ranaei-Siadat, Seyed-Omid; Ghomi, Hamid

2016-11-01

Polymeric nanofiber prepares a suitable situation for enzyme immobilization for variety of applications. In this research, we have fabricated polyvinyl alcohol (PVA)/malonic acid nanofibers using electrospinning. After fabrication of nanofibers, the effect of air, nitrogen, CO2, and argon DBD (dielectric barrier discharge) plasmas on PVA/malonic acid nanofibers were analysed. Among them, air plasma had the most significant effect on glucose oxidase (GOx) immobilization. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectrum analysis and X-ray photoelectron spectroscopy (XPS) results revealed that in case of air plasma modified nanofibers, the carboxyl groups on the surface are increased. The scanning electron microscopy (SEM) images showed that, after GOx immobilization, the modified nanofibers with plasma has retained its nanofiber structure. Finally, we analysed reusability and storage stability of GOx immobilized on plasma modified and unmodified nanofibers. The results were more satisfactory for modified nanofibers with respect to unmodified ones.

Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response

PubMed Central

Tsai, Alex Yi-Lin; Chan, Kin; Ho, Chi-Yip; Canam, Thomas; Capron, Resmi; Master, Emma R.; Bräutigam, Katharina

2017-01-01

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to the hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses. PMID:28253318

The role of surface charge on the uptake and biocompatibility of hydroxyapatite nanoparticles with osteoblast cells

PubMed Central

Chen, Liang; Mccrate, Joseph M.; Lee, James C-M.; Li, Hao

2011-01-01

The objective of this study is to evaluate the effect of hydroxyapatite (HAP) nanoparticles with different surface charges on the cellular uptake behavior and in vitro cell viability and proliferation of MC3T3-E1 cell lines (osteoblast). The nanoparticles surface charge was varied by the surface modification with two carboxylic acids: 12-aminododecanoic acid (positive) and dodecanedioic acid (negative). The untreated HAP nanoparticles and dodecanoic acid modified HAP nanoparticles (neutral) were used as the control. X-ray diffraction (XRD) revealed that surface modifications by the three carboxylic acids did not change the crystal structure of HAP nanoparticles; Fourier transform infrared spectroscopy (FTIR) confirmed the adsorption and binding of the carboxylic acids on HAP nanoparticle surface; and zeta potential measurement confirmed that the chemicals successfully modified the surface charge of HAP nanoparticles in water based solution. Transmission electron microscopy (TEM) images showed that positively charged, negatively charged and untreated HAP nanoparticles, with similar size and shape, all penetrated into the cells and cells had more uptake of HAP nanoparticles with positive charge compared to those with negative charge, which might be attributed to the attractive or repulsive interaction between the negatively charged cell membrane and positively/negatively charged HAP nanoparticles. The neutral HAP nanoparticles could not penetrate cell membrane due to the larger size. MTT assay and LDH assay results indicated that as compared with the polystyrene control, greater cell viability and cell proliferation were measured on MC3T3-E1 cells treated with the three kinds of the HAP nanoparticles (neutral, positive, and untreated), among which positively charged HAP nanoparticles shows strongest improvement for cell viability and cell proliferation. In summary, the surface charge of HAP nanoparticles can be modified to influence the cellular uptake of HAP nanoparticles and the different uptake also influence the behavior of cells. These in-vitro results may also provide useful information for investigations of HAP nanoparticles applications in the gene delivery and intracellular drug delivery. PMID:21289408

Application of multifunctional targeting epirubicin liposomes in the treatment of non-small-cell lung cancer

PubMed Central

Song, Xiao-li; Ju, Rui-jun; Xiao, Yao; Wang, Xin; Liu, Shuang; Fu, Min; Liu, Jing-jing; Gu, Li-yan; Li, Xue-tao; Cheng, Lan

2017-01-01

Chemotherapy for aggressive non-small-cell lung cancer (NSCLC) usually results in a poor prognosis due to tumor metastasis, vasculogenic mimicry (VM) channels, limited killing of tumor cells, and severe systemic toxicity. Herein, we developed a kind of multifunctional targeting epirubicin liposomes to enhance antitumor efficacy for NSCLC. In the liposomes, octreotide was modified on liposomal surface for obtaining a receptor-mediated targeting effect, and honokiol was incorporated into the lipid bilayer for inhibiting tumor metastasis and eliminating VM channels. In vitro cellular assays showed that multifunctional targeting epirubicin liposomes not only exhibited the strongest cytotoxic effect on Lewis lung tumor cells but also showed the most efficient inhibition on VM channels. Action mechanism studies showed that multifunctional targeting epirubicin liposomes could downregulate PI3K, MMP-2, MMP-9, VE-Cadherin, and FAK and activate apoptotic enzyme caspase 3. In vivo results exhibited that multifunctional targeting epirubicin liposomes could accumulate selectively in tumor site and display an obvious antitumor efficacy. In addition, no significant toxicity of blood system and major organs was observed at a test dose. Therefore, multifunctional targeting epirubicin liposomes may provide a safe and efficient therapy strategy for NSCLC. PMID:29066893

Nanosized zeolites as a perspective material for conductometric biosensors creation

NASA Astrophysics Data System (ADS)

Kucherenko, Ivan; Soldatkin, Oleksandr; Kasap, Berna Ozansoy; Kirdeciler, Salih Kaan; Kurc, Burcu Akata; Jaffrezic-Renault, Nicole; Soldatkin, Alexei; Lagarde, Florence; Dzyadevych, Sergei

2015-05-01

In this work, the method of enzyme adsorption on different zeolites and mesoporous silica spheres (MSS) was investigated for the creation of conductometric biosensors. The conductometric transducers consisted of gold interdigitated electrodes were placed on the ceramic support. The transducers were modified with zeolites and MSS, and then the enzymes were adsorbed on the transducer surface. Different methods of zeolite attachment to the transducer surface were used; drop coating with heating to 200°C turned out to be the best one. Nanozeolites beta and L, zeolite L, MSS, and silicalite-1 (80 to 450 nm) were tested as the adsorbents for enzyme urease. The biosensors with all tested particles except zeolite L had good analytical characteristics. Silicalite-1 (450 nm) was also used for adsorption of glucose oxidase, acetylcholinesterase, and butyrylcholinesterase. The glucose and acetylcholine biosensors were successfully created, whereas butyrylcholinesterase was not adsorbed on silicalite-1. The enzyme adsorption on zeolites and MSS is simple, quick, well reproducible, does not require use of toxic compounds, and therefore can be recommended for the development of biosensors when these advantages are especially important.

Discovery of 2-methylpyridine-based biaryl amides as γ-secretase modulators for the treatment of Alzheimer's disease.

PubMed

Chen, Jian Jeffrey; Qian, Wenyuan; Biswas, Kaustav; Yuan, Chester; Amegadzie, Albert; Liu, Qingyian; Nixey, Thomas; Zhu, Joe; Ncube, Mqhele; Rzasa, Robert M; Chavez, Frank; Chen, Ning; DeMorin, Frenel; Rumfelt, Shannon; Tegley, Christopher M; Allen, Jennifer R; Hitchcock, Stephen; Hungate, Randy; Bartberger, Michael D; Zalameda, Leeanne; Liu, Yichin; McCarter, John D; Zhang, Jianhua; Zhu, Li; Babu-Khan, Safura; Luo, Yi; Bradley, Jodi; Wen, Paul H; Reid, Darren L; Koegler, Frank; Dean, Charles; Hickman, Dean; Correll, Tiffany L; Williamson, Toni; Wood, Stephen

2013-12-01

γ-Secretase modulators (GSMs) are potentially disease-modifying treatments for Alzheimer's disease. They selectively lower pathogenic Aβ42 levels by shifting the enzyme cleavage sites without inhibiting γ-secretase activity, possibly avoiding known adverse effects observed with complete inhibition of the enzyme complex. A cell-based HTS effort identified the sulfonamide 1 as a GSM lead. Lead optimization studies identified compound 25 with improved cell potency, PKDM properties, and it lowered Aβ42 levels in the cerebrospinal fluid (CSF) of Sprague-Dawley rats following oral administration. Further optimization of 25 to improve cellular potency is described. Copyright © 2013 Elsevier Ltd. All rights reserved.

Notch Signaling Is Involved in Neurogenic Commitment of Human Periodontal Ligament-Derived Mesenchymal Stem Cells

PubMed Central

Osathanon, Thanaphum; Manokawinchoke, Jeeranan; Nowwarote, Nunthawan; Aguilar, Panuroot; Palaga, Tanapat

2013-01-01

Notch signaling plays critical roles in stem cells by regulating cell fate determination and differentiation. The aim of this study was to evaluate the participation of Notch signaling in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells (hPDLSCs) and to examine the ability to control differentiation of these cells using modified surfaces containing affinity immobilized Notch ligands. Neurogenic induction of hPDLSCs was performed via neurosphere formation. Cells were aggregated and form spheres as early 1 day in culture. In addition, the induced cells exhibited increased mRNA and protein expression of neuronal markers that is, β3-tubulin and neurofilament. During neuronal differentiation, a significant increase of Hes1 and Hey1 mRNA expression was noted. Using pharmacological inhibition (γ-secretase inhibitor) or genetic manipulation (overexpression of dominant negative mastermind-like transcription co-activators), neurosphere formation was attenuated and a marked decrease in neurogenic mRNA expression was observed. To confirm the role of Notch signaling in neuronal differentiation of hPDLSCs, the Notch ligand, Jagged-1, is bound to the surface using an affinity immobilization technique. The hPDLSC cultured on a Jagged-1-modified surface had increased expression of Notch signaling target genes, Hes-1 and Hey-1, confirming the activity and potency of surface-bound Jagged-1. Further, hPDLSC on surface-bound Jagged-1 under serum-free conditions showed multiple long and thin neurite-like extensions, and an increase in the expression of neurogenic mRNA markers was observed. Pretreatment of the cells with γ-secretase inhibitor, DAPT, before seeding on the Jagged-1-modified surface blocked development of the neurite-like morphology. Together, the results in this study suggest the involvement of Notch signaling in neurogenic commitment of hPDLSCs. PMID:23379739

The role of arginine and the modified arginine deiminase enzyme ADI-PEG 20 in cancer therapy with special emphasis on Phase I/II clinical trials.

PubMed

Synakiewicz, Anna; Stachowicz-Stencel, Teresa; Adamkiewicz-Drozynska, Elzbieta

2014-11-01

The metabolic differences between normal, healthy cells and neoplastic cells have been exploited by anticancer therapies targeting metabolic pathways. Various studies of malignant processes have demonstrated disturbances in both arginine synthesis and metabolism that enhance or inhibit tumor cell growth. Consequently, there has been an increased interest in the arginine-depleting enzyme arginine deiminase (ADI) as a potential antineoplastic therapy. This review summarizes the literature on the potential anti-cancer therapeutics arginine and ADI, an arginine-catabolizing enzyme. The authors searched the MEDLINE database PubMed using the key words: 'arginine, 'ADI', 'arginine in cancer' and 'ADI and cancer'. The authors evaluate prospective randomized studies on cancer patients between 2004 and 2013 as well as ongoing research found through the US National Institutes of Health trial database. The results of current studies are promising but do not give unequivocal answers and so it is impossible to recommend arginine or its enzyme ADI as a therapeutic. In the opinion of the authors, further identification of arginine-dependent malignant tumors and their metabolism should be investigated. Furthermore, the use of these chemicals, in combination with other chemotherapeutics drugs, should be investigated and indeed may improve the success of arginine-depleting enzymes such as pegylated ADI (ADI-PEG20).

Chinese Medicine Amygdalin and β-Glucosidase Combined with Antibody Enzymatic Prodrug System As A Feasible Antitumor Therapy.

PubMed

Li, Yun-Long; Li, Qiao-Xing; Liu, Rui-Jiang; Shen, Xiang-Qian

2018-03-01

Amarogentin is an efficacious Chinese herbal medicine and a component of the bitter apricot kernel. It is commonly used as an expectorant and supplementary anti-cancer drug. β-Glucosidase is an enzyme that hydrolyzes the glycosidic bond between aryl and saccharide groups to release glucose. Upon their interaction, β-glucosidase catalyzes amarogentin to produce considerable amounts of hydrocyanic acid, which inhibits cytochrome C oxidase, the terminal enzyme in the mitochondrial respiration chain, and suspends adenosine triphosphate synthesis, resulting in cell death. Hydrocyanic acid is a cell-cycle-stage-nonspecific agent that kills cancer cells. Thus, β-glucosidase can be coupled with a tumor-specific monoclonal antibody. β-Glucosidase can combine with cancer-cell-surface antigens and specifically convert amarogentin to an active drug that acts on cancer cells and the surrounding antibodies to achieve a killing effect. β-Glucosidase is injected intravenously and recognizes cancer-cell-surface antigens with the help of an antibody. The prodrug amarogentin is infused after β-glucosidase has reached the target position. Coupling of cell membrane peptides with β-glucosidase allows the enzyme to penetrate capillary endothelial cells and clear extracellular deep solid tumors to kill the cells therein. The Chinese medicine amarogentin and β-glucosidase will become an important treatment for various tumors when an appropriate monoclonal antibody is developed.

Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mobasseri, Rezvan; Center for Nanofibers & Nanotechnology, Department of Mechanical Engineering, National University of Singapore, 117576; Tian, Lingling

Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on differentmore » substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation. - Highlights: • Bioactive molecules modified surface is a strategy to design biomimicry scaffold. • Bi-functional Tat-derived peptide (R-pept) enhanced MSCs adhesion and proliferation. • R-pept showed similar influences to fibronectin on FA formation and attachment.« less

Maintaining Genome Stability: The Role of Helicases and Deaminases

DTIC Science & Technology

2008-07-01

deaminases. The cells response to different forms of damage is fundamental to its ability to repair itself when challenged by environmental or...Page 4 of 12 INTRODUCTION Genomic DNA stores all the information for living organisms. The faithful duplication the maintanance of DNA are...maturation of the immune system by modifying enzymes called deaminases. The cells response to different forms of damage is fundamental to its ability to

Characteristics of alpha-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods.

PubMed

Morita, Masakazu; Shimamura, Hiroko; Ishida, Natsuko; Imamura, Koreyoshi; Sakiyama, Takaharu; Nakanishi, Kazuhiro

2004-01-01

alpha-Glucosidase was produced using recombinant Aspergillus oryzae by membrane-surface liquid culture (MSLC), a method previously developed by the authors and the results compared with other methods, including shaking flask culture (SFC), agar-plate culture (APC), culture on urethane sponge supports (USC), and liquid surface culture (LSC) to determine possible reasons for the advantageous features of MSLC. When yeast extract was used as a nitrogen source, the amount of enzyme produced by MSLC was 5 or more times higher than those for SFC and LSC, but similar to that using APC. Enzyme production in USC was slightly lower than in MSLC and APC. Cell growth was similar irrespective of the cultivation method used. When NaNO3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO3, the amount of the enzyme produced by MSLC was 8 to 20 times higher than those by SFC, APC, USC, and LSC. Although cell growth using NaNO3 was similar to that for yeast extract in MSLC, it was markedly decreased in SFC, APC, and LSC. The reason for the difference in enzyme productivity for various cultivation methods using yeast extract and NaNO3 as a nitrogen source is discussed, on the basis of the experimental findings. The role of the oxygen transfer effect and gene expression levels in enzyme production were also examined.

Development of Microbial and Enzymatic Fuel Cells for Bio-Inspired Power Sources

DTIC Science & Technology

2009-03-01

that of the oxidation of NADH as possible.[30] A variety of organic mediators have been studied for the anode, including phenazines ,[38] dyes,[39,40...glucose-6-phosphate dehydrogenase on the rotating graphite disc electrode modified with phenazine methosulfate. Enzyme Microb. Technol. 1993, 15 (6), 525

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

PubMed

Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

2014-10-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

A chitin deacetylase from the endophytic fungus Pestalotiopsis sp. efficiently inactivates the elicitor activity of chitin oligomers in rice cells.

PubMed

Cord-Landwehr, Stefan; Melcher, Rebecca L J; Kolkenbrock, Stephan; Moerschbacher, Bruno M

2016-11-30

To successfully survive in plants, endophytes need strategies to avoid being detected by the plant immune system, as the cell walls of endophytes contain easily detectible chitin. It is possible that endophytes "hide" this chitin from the plant immune system by modifying it, or oligomers derived from it, using chitin deacetylases (CDA). To explore this hypothesis, we identified and expressed a CDA from Pestalotiopsis sp. (PesCDA), an endophytic fungus, in E. coli and characterized this enzyme and its chitosan oligomer products. We found that when PesCDA modifies chitin oligomers, the products are partially deacetylated chitosan oligomers with a specific acetylation pattern: GlcNAc-GlcNAc-(GlcN) n -GlcNAc (n ≥ 1). Then, in a bioactivity assay where suspension-cultured rice cells were incubated with the PesCDA products (processed chitin hexamers), we found that, unlike the substrate hexamers, chitosan oligomer products no longer elicited the plant immune system. Thus, this endophytic enzyme can prevent the endophyte from being recognized by the plant immune system; this might represent a more general hypothesis for how certain fungi are able to live in or on their hosts.

A chitin deacetylase from the endophytic fungus Pestalotiopsis sp. efficiently inactivates the elicitor activity of chitin oligomers in rice cells

PubMed Central

Cord-Landwehr, Stefan; Melcher, Rebecca L. J.; Kolkenbrock, Stephan; Moerschbacher, Bruno M.

2016-01-01

To successfully survive in plants, endophytes need strategies to avoid being detected by the plant immune system, as the cell walls of endophytes contain easily detectible chitin. It is possible that endophytes “hide” this chitin from the plant immune system by modifying it, or oligomers derived from it, using chitin deacetylases (CDA). To explore this hypothesis, we identified and expressed a CDA from Pestalotiopsis sp. (PesCDA), an endophytic fungus, in E. coli and characterized this enzyme and its chitosan oligomer products. We found that when PesCDA modifies chitin oligomers, the products are partially deacetylated chitosan oligomers with a specific acetylation pattern: GlcNAc-GlcNAc-(GlcN)n-GlcNAc (n ≥ 1). Then, in a bioactivity assay where suspension-cultured rice cells were incubated with the PesCDA products (processed chitin hexamers), we found that, unlike the substrate hexamers, chitosan oligomer products no longer elicited the plant immune system. Thus, this endophytic enzyme can prevent the endophyte from being recognized by the plant immune system; this might represent a more general hypothesis for how certain fungi are able to live in or on their hosts. PMID:27901067

Surface modification of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer films for promoting interaction with bladder urothelial cells.

PubMed

García-García, José M; López, Laura; París, Rodrigo; Núñez-López, María Teresa; Quijada-Garrido, Isabel; de la Peña Zarzuelo, Enrique; Garrido, Leoncio

2012-01-01

Often bladder dysfunction and diseases lead to therapeutic interventions that require partial or complete replacement of damaged tissue. For this reason, the development of biomaterials to repair the bladder by promoting the adhesion and growth of urothelial cells is of interest. With this aim, a modified copolyester of biocompatible and biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] was used as scaffold for porcine urothelial cell culture. In addition to good biocompatibility, the surface of P(HB-co-HV) substrates was modified to provide both, higher hydrophilicity and a better interaction with urothelial cells. Chemical treatments with ethylenediamine (ED) and sodium hydroxide (NaOH) led to substrate surfaces with decreasing hydrophobicity and provided functional groups that enable the grafting of bioactive molecules, such as a laminin derived YIGSR sequence. Physico-chemical properties of modified substrates were studied and compared with those of the pristine P(HB-co-HV). Urothelial cell morphology on treated substrates was studied. The results showed that focal attachment and cell-related properties were improved for peptide grafted polymer compared with both, the unmodified and functionalized copolyester. Copyright © 2011 Wiley Periodicals, Inc.

Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus

PubMed Central

2013-01-01

Background Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. Results In this study, genes encoding putative enzymes from carbon metabolism were identified and their expression was studied in different growth stages of A. bisporus. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies. Conclusions The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples. PMID:24074284

Detailed expression profile of the six Glypicans and their modifying enzyme, Notum during chick limb and feather development.

PubMed

Saad, Kawakeb; Theis, Susanne; Otto, Anthony; Luke, Graham; Patel, Ketan

2017-04-30

The development of vertebrate appendages, especially the limb and feather buds are orchestrated by numerous secreted signalling molecules including Sonic Hedgehog, Bone Morphogenetic Proteins, Fibroblast Growth Factors and Wnts. These proteins coordinate the growth and patterning of ectodermal and mesenchymal cells. The influence of signalling molecules is affected over large distances by their concentration (morphogen activity) but also at local levels by the presence of proteins that either attenuate or promote their activity. Glypicans are cell surface molecules that regulate the activity of the major secreted signalling molecules expressed in the limb and feather bud. Here we investigated the expression of all Glypicans during chick limb and feather development. In addition we profiled the expression of Notum, an enzyme that regulates Glypican activity. We show that five of the six Glypicans and Notum are expressed in a dynamic manner during the development of limbs and feathers. We also investigated the expression of key Glypicans and show that they are controlled by signalling molecules highlighting the presence of feedback loops. Lastly we show that Glypicans and Notum are expressed in a tissue specific manner in adult chicken tissues. Our results strongly suggest that the Glypicans and Notum have many as yet undiscovered roles to play during the development of vertebrate appendages. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure of a bacterial toxin-activating acyltransferase.

PubMed

Greene, Nicholas P; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2015-06-09

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host-cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.

In vivo modulation of foreign body response on polyurethane by surface entrapment technique.

PubMed

Khandwekar, Anand P; Patil, Deepak P; Hardikar, Anand A; Shouche, Yogesh S; Doble, Mukesh

2010-11-01

Implanted polymeric materials, such as medical devices, provoke the body to initiate an inflammatory reaction, known as the foreign body response (FBR), which causes several complications. In this study, polyurethane (Tecoflex®, PU) surface modified with the nonionic surfactant Tween80® (PU/T80) and the cell adhesive PLL-RGD peptide (PU/PLL-RGD) by a previously described entrapment technique were implanted in the peritoneal cavity of Wistar rats for 30 days. Implants were retrieved and examined for tissue reactivity and cellular adherence by various microscopic and analytical techniques. Surface-induced inflammatory response was assessed by real-time PCR based quantification of proinflammatory cytokine transcripts, namely, TNF-α and IL-1β, normalized to housekeeping gene GAPDH. Cellular adherence and their distribution profile were assessed by microscopic examination of H&E stained implant sections. It was observed that PU/PLL-RGD followed by the bare PU surface exhibited severe inflammatory and fibrotic response with an average mean thickness of 19 and 12 μm, respectively, in 30 days. In contrast, PU/T80 surface showed only a cellular monolayer of 2-3 μm in thickness, with a mild inflammatory response and no fibrotic encapsulation. The PU/PLL-RGD peptide-modified substrate promoted an enhanced rate of macrophage cell fusion to form foreign body giant cell (FBGCs), whereas FBGCs were rarely observed on Tween80®-modified substrate. The expression levels of proinflammatory cytokines (TNF-α and IL-1β) were upregulated on PU/PLL-RGD surface followed by bare PU, whereas the cytokine expressions were significantly suppressed on PU/T80 surface. Thus, our study highlights modulation of foreign body response on polyurethane surfaces through surface entrapment technique in the form of differential responses observed on PLL-RGD and Tween80® modified surfaces with the former effective in triggering tissue cell adhesion thereby fibrous encapsulation, while the later being mostly resistant to this phenomenon.

Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes

PubMed Central

Koch, Claudia; Wabbel, Katrin; Eber, Fabian J.; Krolla-Sidenstein, Peter; Azucena, Carlos; Gliemann, Hartmut; Eiben, Sabine; Geiger, Fania; Wege, Christina

2015-01-01

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins. PMID:26734040

Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a Dynamic Interaction between Soluble and Insoluble Grape Cell Wall Components.

PubMed

Bindon, Keren A; Li, Sijing; Kassara, Stella; Smith, Paul A

2016-11-09

For better understanding of the factors that impact proanthocyanidin (PA) adsorption by insoluble cell walls or interaction with soluble cell wall-derived components, application of a commercial polygalacturonase enzyme preparation was investigated to modify grape cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and mesocarp components of Vitis vinifera Shiraz grapes. It was observed that significant depolymerization of the insoluble grape cell wall occurred following enzyme application to both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched, low molecular weight polysaccharides. However, in the case of grape mesocarp, the solubilization of protein from cell walls (in buffer) was significant and increased only slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of PA by insoluble cell walls, but this effect was observed only when material solubilized from grape cell walls had been removed. The loss of PA through interaction with the soluble cell wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the precipitation of PA. This identified a potential mechanism by which extracted grape PA may be lost from wine during vinification, as a precipitate with solubilized grape mesocarp proteins. Although protein was a minor component in terms of total concentration, losses of PA via precipitation with proteins were in the order of 50% of available PA. PA-induced precipitation could proceed until all protein was removed from solution and may account for the very low levels of residual protein observed in red wines. The results point to a dynamic interaction of grape insoluble and soluble components in modulating PA retention in wine.

P7C3 neuroprotective chemicals function by activating the rate-limiting enzyme in NAD salvage.

PubMed

Wang, Gelin; Han, Ting; Nijhawan, Deepak; Theodoropoulos, Pano; Naidoo, Jacinth; Yadavalli, Sivaramakrishnan; Mirzaei, Hamid; Pieper, Andrew A; Ready, Joseph M; McKnight, Steven L

2014-09-11

The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage. Copyright © 2014 Elsevier Inc. All rights reserved.

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

NASA Astrophysics Data System (ADS)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

2016-11-01

Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

Pummelo Protects Doxorubicin-Induced Cardiac Cell Death by Reducing Oxidative Stress, Modifying Glutathione Transferase Expression, and Preventing Cellular Senescence

PubMed Central

Chularojmontri, L.; Gerdprasert, O.; Wattanapitayakul, S. K.

2013-01-01

Citrus flavonoids have been shown to reduce cardiovascular disease (CVD) risks prominently due to their antioxidant effects. Here we investigated the protective effect of pummelo (Citrus maxima, CM) fruit juice in rat cardiac H9c2 cells against doxorubicin (DOX-) induced cytotoxicity. Four antioxidant compositions (ascorbic acid, hesperidin, naringin, and gallic acid) were determined by HPLC. CM significantly increased cardiac cell survival from DOX toxicity as evaluated by MTT assay. Reduction of cellular oxidative stress was monitored by the formation of DCF fluorescent product and total glutathione (GSH) levels. The changes in glutathione-S-transferase (GST) activity and expression were determined by enzyme activity assay and Western blot analysis, respectively. Influence of CM on senescence-associated β-galactosidase activity (SA-β-gal) was also determined. The mechanisms of cytoprotection involved reduction of intracellular oxidative stress, maintaining GSH availability, and enhanced GST enzyme activity and expression. DOX-induced cellular senescence was also attenuated by long-term CM treatment. Thus, CM fruit juice can be promoted as functional fruit to protect cells from oxidative cell death, enhance the phase II GSTP enzyme activity, and decrease senescence phenotype population induced by cardiotoxic agent such as DOX. PMID:23401708

Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential.

PubMed

Sultan, Abida; Frisvad, Jens C; Andersen, Birgit; Svensson, Birte; Finnie, Christine

2017-10-03

The indigenous fungal species populating cereal grains produce numerous plant cell wall-degrading enzymes including xylanases, which could play important role in plant-pathogen interactions and in adaptation of the fungi to varying carbon sources. To gain more insight into the grain surface-associated enzyme activity, members of the populating fungal community were isolated, and their secretomes and xylanolytic activities assessed. Twenty-seven different fungal species were isolated from grains of six barley cultivars over different harvest years and growing sites. The isolated fungi were grown on medium containing barley flour or wheat arabinoxylan as sole carbon source. Their secretomes and xylanase activities were analyzed using SDS-PAGE and enzyme assays and were found to vary according to species and carbon source. Secretomes were dominated by cell wall degrading enzymes with xylanases and xylanolytic enzymes being the most abundant. A 2-DE-based secretome analysis of Aspergillus niger and the less-studied pathogenic fungus Fusarium poae grown on barley flour and wheat arabinoxylan resulted in identification of 82 A. niger and 31 F. poae proteins many of which were hydrolytic enzymes, including xylanases. The microorganisms that inhabit the surface of cereal grains are specialized in production of enzymes such as xylanases, which depolymerize plant cell walls. Integration of gel-based proteomics approach with activity assays is a powerful tool for analysis and characterization of fungal secretomes and xylanolytic activities which can lead to identification of new enzymes with interesting properties, as well as provide insight into plant-fungal interactions, fungal pathogenicity and adaptation. Understanding the fungal response to host niche is of importance to uncover novel targets for potential symbionts, anti-fungal agents and biotechnical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs).

PubMed

Gallagher, Erin; Minn, Il; Chambers, Janice E; Searson, Peter C

2016-07-11

Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection. To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-PAM is a substrate for common brain efflux pumps, experiments were performed in the MDCKII-MDR1 cell line, transfected to overexpress the P-gp efflux pump, and the MDCKII-FLuc-ABCG2 cell line, transfected to overexpress the BCRP efflux pump. To determine how transcellular transport influences enzyme reactivation, we developed a modified transwell assay where the inhibited acetylcholinesterase enzyme, substrate, and reporter are introduced into the basolateral chamber. Enzymatic activity was inhibited using paraoxon and parathion. The permeability of 2-PAM is about 2 × 10(-6) cm s(-1) in MDCK cells and about 1 × 10(-6) cm s(-1) in BC1-hBMECs. Permeability is not influenced by pre-treatment with atropine. In addition, 2-PAM is not a substrate for the P-gp or BCRP efflux pumps. The low permeability explains poor brain penetration of 2-PAM and therefore the slow enzyme reactivation. This elucidates one of the reasons for the necessity of sustained intravascular (IV) infusion in response to organophosphate poisoning.

Enzymatic biofuel cell based on electrodes modified with lipid liquid-crystalline cubic phases

NASA Astrophysics Data System (ADS)

Nazaruk, Ewa; Smoliński, Sławomir; Swatko-Ossor, Marta; Ginalska, Grażyna; Fiedurek, Jan; Rogalski, Jerzy; Bilewicz, Renata

Two glassy carbon electrodes modified with enzymes embedded in lyotropic liquid-crystalline cubic phase were used for the biofuel cell construction. The monoolein liquid-crystalline film allowed to avoid separators in the biofuel cell. Glucose and oxygen as fuels, and glucose oxidase and laccase as anode and cathode biocatalysts, respectively were used. The biofuel cell parameters were examined in McIlvaine buffer, pH 7 solution containing 15 mM of glucose and saturated with dioxygen. A series of mediators were tested taking into account their formal potentials, stability in the cubic phase and efficiency of mediation. Most stable was the biofuel cell based on tetrathiafulvalene (TTF) and 2,2‧-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as anode and cathode mediators, respectively. The open-circuit voltage was equal to 450 ± 40 mV. The power densities and current densities were measured for all the systems studied.

Biological Functionalization of Drug Delivery Carriers to Bypass Size Restrictions of Receptor-Mediated Endocytosis Independently from Receptor Targeting

PubMed Central

Ansar, Maria; Serrano, Daniel; Papademetriou, Iason; Bhowmick, Tridib Kumar; Muro, Silvia

2014-01-01

Targeting of drug carriers to cell-surface receptors involved in endocytosis is commonly used for intracellular drug delivery. However, most endocytic receptors mediate uptake via clathrin or caveolar pathways associated with ≤200-nm vesicles, restricting carrier design. We recently showed that endocytosis mediated by intercellular adhesion molecule 1 (ICAM-1), which differs from clathrin- and caveolar-mediated pathways, allows uptake of nano- and micro-carriers in cell culture and in vivo due to recruitment of cellular sphingomyelinases to the plasmalemma. This leads to ceramide generation at carrier binding sites and formation of actin stress-fibers, enabling engulfment and uptake of a wide size-range of carriers. Here we adapted this paradigm to enhance uptake of drug carriers targeted to receptors associated with size-restricted pathways. We coated sphingomyelinase onto model (polystyrene) submicro- and micro-carriers targeted to clathrin-associated mannose-6-phosphate receptor. In endothelial cells, this provided ceramide enrichment at the cell surface and actin stress-fiber formation, modifying the uptake pathway and enhancing carrier endocytosis without affecting targeting, endosomal transport, cell-associated degradation, or cell viability. This improvement depended on the carrier size and enzyme dose, and similar results were observed for other receptors (transferrin receptor) and cell types (epithelial cells). This phenomenon also enhanced tissue accumulation of carriers after intravenous injection in mice. Hence, it is possible to maintain targeting toward a selected receptor while bypassing natural size-restrictions of its associated endocytic route by functionalization of drug carriers with biological elements mimicking the ICAM-1 pathway. This strategy holds considerable promise to enhance flexibility of design of targeted drug delivery systems. PMID:24237309

Biological functionalization of drug delivery carriers to bypass size restrictions of receptor-mediated endocytosis independently from receptor targeting.

PubMed

Ansar, Maria; Serrano, Daniel; Papademetriou, Iason; Bhowmick, Tridib Kumar; Muro, Silvia

2013-12-23

Targeting of drug carriers to cell-surface receptors involved in endocytosis is commonly used for intracellular drug delivery. However, most endocytic receptors mediate uptake via clathrin or caveolar pathways associated with ≤200-nm vesicles, restricting carrier design. We recently showed that endocytosis mediated by intercellular adhesion molecule 1 (ICAM-1), which differs from clathrin- and caveolae-mediated pathways, allows uptake of nano- and microcarriers in cell culture and in vivo due to recruitment of cellular sphingomyelinases to the plasmalemma. This leads to ceramide generation at carrier binding sites and formation of actin stress-fibers, enabling engulfment and uptake of a wide size-range of carriers. Here we adapted this paradigm to enhance uptake of drug carriers targeted to receptors associated with size-restricted pathways. We coated sphingomyelinase onto model (polystyrene) submicro- and microcarriers targeted to clathrin-associated mannose-6-phosphate receptor. In endothelial cells, this provided ceramide enrichment at the cell surface and actin stress-fiber formation, modifying the uptake pathway and enhancing carrier endocytosis without affecting targeting, endosomal transport, cell-associated degradation, or cell viability. This improvement depended on the carrier size and enzyme dose, and similar results were observed for other receptors (transferrin receptor) and cell types (epithelial cells). This phenomenon also enhanced tissue accumulation of carriers after intravenous injection in mice. Hence, it is possible to maintain targeting toward a selected receptor while bypassing natural size restrictions of its associated endocytic route by functionalization of drug carriers with biological elements mimicking the ICAM-1 pathway. This strategy holds considerable promise to enhance flexibility of design of targeted drug delivery systems.

[Improvement of thermostability of beta-1,3-1,4-glucanase from Bacillus amyloliquefaciens BS5582 through in vitro evolution].

PubMed

Qin, Jiufu; Gao, Weiwei; Li, Qi; Li, Yongxian; Zheng, Feiyun; Liu, Chunfeng; Gu, Guoxian

2010-09-01

In vitro evolution methods are often used to modify protein with improved characteristics. We developed a directed evolution protocol to enhance the thermostability of the beta-1,3-1,4-glucanase. The thermostability of the enzyme was significantly improved after two rounds of directed evolution. Three variants with higher thermostability were obtained. The mutant enzymes were further analyzed by their melting temperature, halftime and kinetic parameters. Comparing to intact enzyme, the T50 of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were increased by 2.2 degrees C, 5.5 degrees C and 3.5 degrees C, respectively, the halftime (t1/2, 60 degrees C) of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were shortened by 4,13 and 17 min, respectively, the V(max) of mutant enzymes were decreased by 8.3%, 2.6% and 10.6%, respectively, while K(m) of mutant enzymes were nearly unchanged. Sequence analysis revealed seven single amino acid mutant happened among three mutant enzymes, such as 2-JF-01 (N36S, G213R), 2-JF-02 (C86R, S115I, N150G) and 2-JF-03 (E156V, K105R). Homology-modeling showed that five of seven substituted amino acids were located on the surface of or in hole of protein. 42.8% of substituted amino acids were arginine, which indicated that arginine may play a role in the improvement of the thermostability of the beta-1,3-1,4-glucanase.This study provide some intresting results of the structural basis of the thermostability of beta-1,3-1,4-glucanase,and provide some new point of view in modifying enzyme for future industrial use.

Biocompatible patterning of proteins on wettability gradient surface by thermo-transfer printing.

PubMed

Kim, Sungho; Ryu, Yong-Sang; Suh, Jeng-Hun; Keum, Chang-Min; Sohn, Youngjoo; Lee, Sin-Doo

2014-08-01

We develop a simple and biocompatible method of patterning proteins on a wettability gradient surface by thermo-transfer printing. The wettability gradient is produced on a poly(dimethylsiloxane) (PDMS)-modified glass substrate through the temperature gradient during thermo-transfer printing. The water contact angle on the PDMS-modified surface is found to gradually increase along the direction of the temperature gradient from a low to a high temperature region. Based on the wettability gradient, the gradual change in the adsorption and immobilization of proteins (cholera toxin B subunit) is achieved in a microfluidic cell with the PDMS-modified surface.

Quantification of confocal images of biofilms grown on irregular surfaces

PubMed Central

Ross, Stacy Sommerfeld; Tu, Mai Han; Falsetta, Megan L.; Ketterer, Margaret R.; Kiedrowski, Megan R.; Horswill, Alexander R.; Apicella, Michael A.; Reinhardt, Joseph M.; Fiegel, Jennifer

2014-01-01

Bacterial biofilms grow on many types of surfaces, including flat surfaces such as glass and metal and irregular surfaces such as rocks, biological tissues and polymers. While laser scanning confocal microscopy can provide high-resolution images of biofilms grown on any surface, quantification of biofilm-associated bacteria is currently limited to bacteria grown on flat surfaces. This can limit researchers studying irregular surfaces to qualitative analysis or quantification of only the total bacteria in an image. In this work, we introduce a new algorithm called modified connected volume filtration (MCVF) to quantify bacteria grown on top of an irregular surface that is fluorescently labeled or reflective. Using the MCVF algorithm, two new quantification parameters are introduced. The modified substratum coverage parameter enables quantification of the connected-biofilm bacteria on top of the surface and on the imaging substratum. The utility of MCVF and the modified substratum coverage parameter were shown with Pseudomonas aeruginosa and Staphylococcus aureus biofilms grown on human airway epithelial cells. A second parameter, the percent association, provides quantified data on the colocalization of the bacteria with a labeled component, including bacteria within a labeled tissue. The utility of quantifying the bacteria associated with the cell cytoplasm was demonstrated with Neisseria gonorrhoeae biofilms grown on cervical epithelial cells. This algorithm provides more flexibility and quantitative ability to researchers studying biofilms grown on a variety of irregular substrata. PMID:24632515

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel.

PubMed

Huang, Jinjin; Xia, Ji; Yang, Zhen; Guan, Feifei; Cui, Di; Guan, Guohua; Jiang, Wei; Li, Ying

2014-01-01

We previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel. Several new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%. Our experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.

Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Makarova, Olga V.; Adams, Daniel L.; Divan, Ralu

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and withoutmore » an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. Copyright 2016 The Authors. Published by Elsevier B.V. All rights reserved.« less

Mucin glycosylating enzyme GALNT2 suppresses malignancy in gastric adenocarcinoma by reducing MET phosphorylation

PubMed Central

Liu, Shin-Yun; Shun, Chia-Tung; Hung, Kuan-Yu; Juan, Hsueh-Fen; Hsu, Chia-Lang; Huang, Min-Chuan; Lai, I-Rue

2016-01-01

Glycosylation affects malignancy in cancer. Here, we report that N- acetylgalactosaminyltransferase 2 (GALNT2), an enzyme that mediates the initial step of mucin type-O glycosylation, suppresses malignant phenotypes in gastric adenocarcinoma (GCA) by modifying MET (Hepatocyte growth factor receptor) activity. GALNT2 mRNA and protein were downregulated in GCAs, and this reduction was associated with more advanced disease stage and shorter recurrence-free survival. Suppressing GALNT2 expression in GCA cells increased cell growth, migration, and invasion in vitro, and tumor metastasis in vivo. GALNT2 knockdown enhanced phosphorylation of MET and decreased expression of the Tn antigen on MET. Inhibiting MET activity with PHA665752 decreased the malignant phenotypes caused by GALNT2 knockdown in GCA cells. Our results indicate that GALNT2 suppresses the malignant potential of GCA cells and provide novel insights into the significance of O-glycosylation in MET activity and GCA progression. PMID:26848976

The role of protein methyltransferases as potential novel therapeutic targets in squamous cell carcinoma of the head and neck.

PubMed

Saloura, Vassiliki; Vougiouklakis, Theodore; Sievers, Cem; Burkitt, Kyunghee; Nakamura, Yusuke; Hager, Gordon; van Waes, Carter

2018-06-01

Squamous cell carcinoma of the head and neck is a lethal disease with suboptimal survival outcomes and standard therapies with significant comorbidities. Whole exome sequencing data recently revealed an abundance of genetic and expression alterations in a family of enzymes known as protein methyltransferases in a variety of cancer types, including squamous cell carcinoma of the head and neck. These enzymes are mostly known for their chromatin-modifying functions through methylation of various histone substrates, though evidence supports their function also through methylation of non-histone substrates. This review summarizes the current knowledge on the function of protein methyltransferases in squamous cell carcinoma of the head and neck and highlights their promising potential as the next generation of therapeutic targets in this disease. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

A flexible thermoresponsive cell culture substrate for direct transfer of keratinocyte cell sheets.

PubMed

Praveen, Wulligundam; Madathil, Bernadette K; Sajin Raj, R S; Kumary, T V; Anil Kumar, P R

2017-10-25

Most cell sheet engineering systems require a support or carrier to handle the harvested cell sheets. In this study, polyethylene terephthalate-based overhead projection transparency sheets (OHPS) were subjected to surface hydrolysis by alkali treatment to increase pliability and hydrophilicity and enable poly(N-isopropylacrylamide-co-glycidylmethacrylate) copolymer (NGMA) coating to impart thermoresponsiveness. NGMA was applied on the modified OHPS by the technique of spin coating using an indigenously designed spin coater. The spin coating had the advantage of using low volumes of the polymer and a reduced coating time. The surface chemistry and thermoresponsive coating was analyzed by Fourier transform infrared spectroscopy and water contact angle. Human keratinocyte cells were cultured on the spin coated surface and scaffold-free cell sheets were successfully harvested by simple variation of temperature. These cell sheets were found to be viable, exhibited epithelial characteristic and cell-cell contact as confirmed by positive immunostaining for ZO-1. The integrity and morphology of the cell sheet was confirmed by stereomicroscopy and E-SEM. These results highlight the potential of the NGMA spin coated modified OHPS to serve as a thermoresponsive culture surface-cum-flexible transfer tool.

Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and β-glucosidase.

PubMed

Tozakidis, Iasson E P; Brossette, Tatjana; Lenz, Florian; Maas, Ruth M; Jose, Joachim

2016-06-10

The production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities. This process could be simplified by displaying the necessary enzymes on a microbial cell surface. Such an approach, however, requires an appropriate host organism which on the one hand can withstand the rough environment coming along with lignocellulose hydrolysis, and on the other hand does not consume the generated glucose so that it remains available for subsequent fermentation steps. The robust soil bacterium Pseudomonas putida showed a strongly reduced uptake of glucose above a temperature of 50 °C, while remaining structurally intact hence recyclable, which makes it suitable for cellulose hydrolysis at elevated temperatures. Consequently, three complementary, thermophilic cellulases from Ruminiclostridium thermocellum were displayed on the surface of the bacterium. All three enzymes retained their activity on the cell surface. A mixture of three strains displaying each one of these enzymes was able to synergistically hydrolyze filter paper at 55 °C, producing 20 μg glucose per mL cell suspension in 24 h. We could establish Pseudomonas putida as host for the surface display of cellulases, and provided proof-of-concept for a fast and simple cellulose breakdown process at elevated temperatures. This study opens up new perspectives for the application of P. putida in the production of biofuels and other biotechnological products.

Metabolic signature of breast cancer cell line MCF-7: profiling of modified nucleosides via LC-IT MS coupling.

PubMed

Bullinger, Dino; Neubauer, Hans; Fehm, Tanja; Laufer, Stefan; Gleiter, Christoph H; Kammerer, Bernd

2007-11-29

Cancer, like other diseases accompanied by strong metabolic disorders, shows characteristic effects on cell turnover rate, activity of modifying enzymes and DNA/RNA modifications, resulting also in elevated amounts of excreted modified nucleosides. For a better understanding of the impaired RNA metabolism in breast cancer cells, we screened these metabolites in the cell culture supernatants of the breast cancer cell line MCF-7 and compared it to the human mammary epithelial cells MCF-10A. The nucleosides were isolated and analyzed via 2D-chromatographic techniques: In the first dimension by cis-diol specific boronate affinity extraction and subsequently by reversed phase chromatography coupled to an ion trap mass spectrometer. Besides the determination of ribonucleosides, additional compounds with cis-diol structure, deriving from cross-linked biochemical pathways, like purine-, histidine- and polyamine metabolism were detected. In total, 36 metabolites were identified by comparison of fragmentation patterns and retention time. Relation to the internal standard isoguanosine yielded normalized area ratios for each identified compound and enabled a semi-quantitative metabolic signature of both analyzed cell lines.13 of the identified 26 modified ribonucleosides were elevated in the cell culture supernatants of MCF-7 cells, with 5-methyluridine, N2,N2,7-trimethylguanosine, N6-methyl-N6-threonylcarbamoyladenosine and 3-(3-aminocarboxypropyl)-uridine showing the most significant differences. 1-ribosylimidazole-4-acetic acid, a histamine metabolite, was solely found in the supernatants of MCF-10A cells, whereas 1-ribosyl-4-carboxamido-5-aminoimidazole and S-adenosylmethionine occurred only in supernatants of MCF-7 cells. The obtained results are discussed against the background of pathological changes in cell metabolism, resulting in new perspectives for modified nucleosides and related metabolites as possible biomedical markers for breast carcinoma in vivo.

Three-dimensional plotter technology for fabricating polymeric scaffolds with micro-grooved surfaces.

PubMed

Son, JoonGon; Kim, GeunHyung

2009-01-01

Various mechanical techniques have been used to fabricate biomedical scaffolds, including rapid prototyping (RP) devices that operate from CAD files of the target feature information. The three-dimensional (3-D) bio-plotter is one RP system that can produce design-based scaffolds with good mechanical properties for mimicking cartilage and bones. However, the scaffolds fabricated by RP have very smooth surfaces, which tend to discourage initial cell attachment. Initial cell attachment, migration, differentiation and proliferation are strongly dependent on the chemical and physical characteristics of the scaffold surface. In this study, we propose a new 3-D plotting method supplemented with a piezoelectric system for fabricating surface-modified scaffolds. The effects of the physically-modified surface on the mechanical and hydrophilic properties were investigated, and the results of cell culturing of chondrocytes indicate that this technique is a feasible new method for fabricating high-quality 3-D polymeric scaffolds.

Voltammetric enzyme sensor for urea using mercaptohydroquinone-modified gold electrode as the base transducer.

PubMed

Mizutani, F; Yabuki, S; Sato, Y

1997-01-01

A voltammetric urea-sensing electrode was prepared by combining a lipid-attached urease layer with a 2,5-dihydroxythiophenol-modified gold electrode. A self-assembled monolayer of dihydroxythiophenol was prepared on the gold surface by soaking the electrode into an ethanolic solution containing the modifier. A layer of the lipid-attached enzyme and that of acetyl cellulose overcoat were successively made on the dihydroxythiophenol-modified electrode by applying a dip-coating procedure. The addition of urea in a test solution (10 mM phosphate buffer, pH 7.0) brought about an increase of pH near the urease layer. The pH shift accompanied a negative shift of the anodic peak, which corresponded to the electro-oxidation of dihydroxyphenol moiety to form quinone, on the linear sweep voltammograms for the urease/dihydroxythiophenol electrode. The concentration of urea (0.2-5 mM) could be determined by measuring the electrode current at -0.05 V versus Ag/AgCl from the voltammogram. The electrode was applied to the determination of urea in human urine; the measurement of electrode current at such a low potential provided the urea determination without any electrochemical interference from L-ascorbic acid and uric acid.

Confocal Raman Microscopy for the Determination of Protein and Quaternary Ammonium Ion Loadings in Biocatalytic Membranes for Electrochemical Energy Conversion and Storage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Rong; Abdellaoui, Sofiene; Kitt, Jay P.

Here, the need to immobilize active enzyme, while ensuring high rates of substrate turnover and electronic charge transfer with an electrode, is a centrally important challenge in the field of bioelectrocatalysis. In this work, we demonstrate the use of confocal Raman microscopy as a tool for quantitation and molecular-scale structural characterization of ionomers and proteins within biocatalytic membranes to aid in the development of energy efficient biofuel cells. A set of recently available short side chain Aquivion ionomers spanning a range of equivalent weight (EW) suitable for enzyme immobilization was investigated. Aquivion ionomers (790 EW, 830 EW and 980 EW)more » received in the proton-exchanged (SO 3H) form were treated with tetra-n-butylammonium bromide (TBAB) to neutralize the ionomer and expand the size of ionic domains for enzyme incorporation. Through the use of confocal Raman microscopy, membrane TBA+ ion content was predicted in calibration studies to within a few percent of the conventional titrimetric method across the full range of TBA + : SO 3 - ratios of practical interest (0.1 to 1.7). Protein incorporation into membranes was quantified at the levels expected in biofuel cell electrodes. Furthermore, features associated with the catalytically active, enzyme-coordinated copper center were evident between 400 cm -1 - 500 cm -1 in spectra of laccase catalytic membranes, demonstrating the potential to interrogate mechanistic chemistry at the enzyme active site of biocathodes under fuel cell reaction conditions. When benchmarked against the 1100 EW Nafion ionomer in glucose/air enzymatic fuel cells (EFCs), EFCs with laccase air-breathing cathodes prepared from TBA + modified Aquivion ionomers were able to reach maximum power densities (P max) up to 1.5 times higher than EFCs constructed with cathodes prepared from TBA + modified Nafion. The improved performance of EFCs containing the short side chain Aquivion ionomers relative to Nafion is traced to effects of ionomer ion-exchange capacity (IEC, where IEC = EW -1), where the greater density of SO 3 - moieties in the Aquivion materials produces an environment more favourable to mass transport and higher TBA + concentrations.« less

Confocal Raman Microscopy for the Determination of Protein and Quaternary Ammonium Ion Loadings in Biocatalytic Membranes for Electrochemical Energy Conversion and Storage

DOE PAGES

Cai, Rong; Abdellaoui, Sofiene; Kitt, Jay P.; ...

2017-11-14

Here, the need to immobilize active enzyme, while ensuring high rates of substrate turnover and electronic charge transfer with an electrode, is a centrally important challenge in the field of bioelectrocatalysis. In this work, we demonstrate the use of confocal Raman microscopy as a tool for quantitation and molecular-scale structural characterization of ionomers and proteins within biocatalytic membranes to aid in the development of energy efficient biofuel cells. A set of recently available short side chain Aquivion ionomers spanning a range of equivalent weight (EW) suitable for enzyme immobilization was investigated. Aquivion ionomers (790 EW, 830 EW and 980 EW)more » received in the proton-exchanged (SO 3H) form were treated with tetra-n-butylammonium bromide (TBAB) to neutralize the ionomer and expand the size of ionic domains for enzyme incorporation. Through the use of confocal Raman microscopy, membrane TBA+ ion content was predicted in calibration studies to within a few percent of the conventional titrimetric method across the full range of TBA + : SO 3 - ratios of practical interest (0.1 to 1.7). Protein incorporation into membranes was quantified at the levels expected in biofuel cell electrodes. Furthermore, features associated with the catalytically active, enzyme-coordinated copper center were evident between 400 cm -1 - 500 cm -1 in spectra of laccase catalytic membranes, demonstrating the potential to interrogate mechanistic chemistry at the enzyme active site of biocathodes under fuel cell reaction conditions. When benchmarked against the 1100 EW Nafion ionomer in glucose/air enzymatic fuel cells (EFCs), EFCs with laccase air-breathing cathodes prepared from TBA + modified Aquivion ionomers were able to reach maximum power densities (P max) up to 1.5 times higher than EFCs constructed with cathodes prepared from TBA + modified Nafion. The improved performance of EFCs containing the short side chain Aquivion ionomers relative to Nafion is traced to effects of ionomer ion-exchange capacity (IEC, where IEC = EW -1), where the greater density of SO 3 - moieties in the Aquivion materials produces an environment more favourable to mass transport and higher TBA + concentrations.« less

Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Tadanobu; Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST, JST, and COE Program in the 21st Century, Shizuoka 422-8526; Moriyama, Yusuke

Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism.

Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

PubMed Central

Hagen, Sven; Baumann, Tobias; Wagner, Hanna J.; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M.; Müller, Kristian M.

2014-01-01

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs

PubMed Central

2014-01-01

Background Lipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli. Results The lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents. Conclusions Co-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic activity appears not to be hindered by surface display. Furthermore, the cells and the membrane preparations appeared to be stable enough to endure a European standard laundry test and show efficient fat removal properties herein. PMID:24476025

Development of a biomimetic enzyme-linked immunosorbent assay based on molecularly imprinted polymers on paper for the detection of carbaryl.

PubMed

Zhang, Can; Cui, Hanyu; Han, Yufeng; Yu, Fangfang; Shi, Xiaoman

2018-02-01

A biomimetic enzyme-linked immunosorbent assay (BELISA) which was based on molecularly imprinted polymers on paper (MIPs-paper) with specific recognition was developed. As a detector, the surface of paper was modified with γ-MAPS by hydrolytic action and anchored the MIP layer on γ-MAPS modified-paper by copolymerization to construct the artificial antibody Through a series of experimentation and verification, we successful got the MIPs-paper and established BELISA for the detection of carbaryl. The development of MIPs-paper based on BELISA was applied to detect carbaryl in real samples and validated by an enzyme-linked immunosorbent assay (ELISA) based on anti-carbaryl biological antibody. The results of these two methods (BELISA and ELISA) were well correlated (R 2 =0.944). The established method of MIPs-paper BELISA exhibits the advantages of low cost, higher stability and being re-generable, which can be applied as a convenient tool for the fast and efficient detection of carbaryl. Copyright © 2017. Published by Elsevier Ltd.

A novel design of combining the angiotensin converting enzyme (ACE) inhibitor captopril with the angiotensin receptor blocker (ARB) losartan using homo coupling via PEG diacid linker.

PubMed

Hashemzadeh, Mehrnoosh; Park, Shery; Ju, Hee; Movahed, Mohammad R

2013-12-01

Cardiovascular disease is the leading cause of death in American adults. Furthermore, the incidence of congestive heart failure is on the rise as a major cause of hospitalization and mortality in this population. Angiotensin Converting Enzyme (ACE) inhibitors prevent the production of angiotensin II, which has been shown to reduce mortality in patients with congestive heart failure. Angiotensin II receptor blockers (ARB) were developed as a direct inhibitor of angiotensin II. ARBs have been shown to be effective in the treatment of patients with systolic heart failure but do not cause chronic coughing which is a common side effect of ACE inhibitors. In theory, a compound that has the combined effect of an ACE inhibitor and an ARB should be more effective in treating heart failure patients than either agents alone. Therefore, the purpose of this manuscript is to design and discuss the benefits of a new molecule, which combines captopril, an ACE inhibitor, with losartan, an ARB. In this experiment Captopril and Losartan were modified and synthesized separately and combined by homo or mono coupling. This was achieved by taking advantage of PEG (Polyethylene glycol) as a linker. It is expected that this molecule will have the combined modes of action of both ACEs and ARBs. Benefits from combination therapy include; increased efficacy, reduced adverse effects, convenience, compliance, and prolonged duration. Consequently, this combined molecule is expected to block the production of angiotensin II more efficiently and effectively. Although captopril and losartan work in the same system by blocking the effect of angiotensin II they have different action sites and mechanisms some patents are also discussed. Losartan blocks the AT1 receptor which is expressed on the cell surface, while captopril inhibits ACE, preventing production of angiotensin II, which is present in both the plasma and on the cell surface, especially on endothelial and smooth muscle cells.

Inducible colitis-associated glycome capable of stimulating the proliferation of memory CD4+ T cells.

PubMed

Nishida, Atsushi; Nagahama, Kiyotaka; Imaeda, Hirotsugu; Ogawa, Atsuhiro; Lau, Cindy W; Kobayashi, Taku; Hisamatsu, Tadakazu; Preffer, Frederic I; Mizoguchi, Emiko; Ikeuchi, Hiroki; Hibi, Toshifumi; Fukuda, Minoru; Andoh, Akira; Blumberg, Richard S; Mizoguchi, Atsushi

2012-12-17

Immune responses are modified by a diverse and abundant repertoire of carbohydrate structures on the cell surface, which is known as the glycome. In this study, we propose that a unique glycome that can be identified through the binding of galectin-4 is created on local, but not systemic, memory CD4+ T cells under diverse intestinal inflammatory conditions, but not in the healthy state. The colitis-associated glycome (CAG) represents an immature core 1-expressing O-glycan. Development of CAG may be mediated by down-regulation of the expression of core-2 β1,6-N-acetylglucosaminyltransferase (C2GnT) 1, a key enzyme responsible for the production of core-2 O-glycan branch through addition of N-acetylglucosamine (GlcNAc) to a core-1 O-glycan structure. Mechanistically, the CAG seems to contribute to super raft formation associated with the immunological synapse on colonic memory CD4+ T cells and to the consequent stabilization of protein kinase C θ activation, resulting in the stimulation of memory CD4+ T cell expansion in the inflamed intestine. Functionally, CAG-mediated CD4+ T cell expansion contributes to the exacerbation of T cell-mediated experimental intestinal inflammations. Therefore, the CAG may be an attractive therapeutic target to specifically suppress the expansion of effector memory CD4+ T cells in intestinal inflammation such as that seen in inflammatory bowel disease.

Water at Biological Phase Boundaries: Its Role in Interfacial Activation of Enzymes and Metabolic Pathways.

PubMed

Damodaran, Srinivasan

2015-01-01

Many life-sustaining activities in living cells occur at the membrane-water interface. The pertinent questions that we need to ask are, what are the evolutionary reasons in biology for choosing the membrane-water interface as the site for performing and/or controlling crucial biological reactions, and what is the key physical principle that is very singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this chapter, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes through manipulation of the thermodynamic activity of water in the lipid-water interfacial region. The hypothesis is based on the fact that the surface pressure of a lipid monolayer is a direct measure of the thermodynamic activity of water at the lipid-water interface. Accordingly, the surface pressure-dependent activation or inactivation of interfacial enzymes is directly related to changes in the thermodynamic activity of interfacial water. Extension of this argument suggests that cells may manipulate conformations (and activities) of membrane-bound enzymes by manipulating the (re)activity of interfacial water at various locations in the membrane by localized compression or expansion of the interface. In this respect, cells may use the membrane-bound hormone receptors, lipid phase transition, and local variations in membrane lipid composition as effectors of local compression and/or expansion of membrane, and thereby local water activity. Several experimental data in the literature will be reexamined in the light of this hypothesis.

Effect of C-implantation on Nerve-Cell Attachment to Polystyrene Films

NASA Astrophysics Data System (ADS)

Sommani, Piyanuch; Tsuji, Hiroshi; Kitamura, Tsuyoshi; Hattori, Mitsutaka; Yamada, Tetsuya; Sato, Hiroko; Gotoh, Yasuhito; Ishikawa, Junzo

The surfaces of the polystyrene films spin-coated on glass were modified by carbon negative-ion implantation with various ion doses from 1×1014 to 3×1016 ions/cm2 at 5 and 10 keV. The implantation conditions with and without a pattering mask were for investigation of the cell-attachment properties and for evaluation of surface physical properties of contact angle, respectively. The contact angles of modified surface were investigated by pure water drop and air bubble method. The lowest angle value of the implanted films at 5 and 10 keV were approximately 72° at 3×1015 ions/cm2 after dipping in the de-ionized water for 2 hours. The lowering of contact angles on C-implanted surfaces when increase the ion dose is due to formation of the OH and C-O bonds. Nerve-cell-attachment properties of modified surface were investigated by the nerve-like cell of rat adrenal pheochromocytoma (PC12h) in vitro. After 2 days culture of the PC12h cells, no cells attached on the polystyrene films implanted with low ion dose from 1×1014 to 3×1014 ions/cm2. On the polystyrene films implanted with the dose order of 1015 ions/cm2, the cells selectively attached only on the implanted region. Whereas on the surfaces implanted with high dose such as 1×1016 and 3×1016 ions/cm2 mostly cells attached on the implanted region, and some attached on the unimplanted region, as well as cells were abnormal in shape and large size. Therefore, the suitable dose implantation for the selective-attachment of nerve-cells on the polystyrene films implanted at 5 and 10 keV were obtained around the dose order of 1015 ions/cm2, and the best condition for the selective attachment properties was at 3×1015 ions/cm2 corresponding to the lowest contact angle.

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

PubMed

Smith, M Ryan; Vayalil, Praveen K; Zhou, Fen; Benavides, Gloria A; Beggs, Reena R; Golzarian, Hafez; Nijampatnam, Bhavitavya; Oliver, Patsy G; Smith, Robin A J; Murphy, Michael P; Velu, Sadanandan E; Landar, Aimee

2016-08-01

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Construction of a multifunctional coating consisting of phospholipids and endothelial progenitor cell-specific peptides on titanium substrates

NASA Astrophysics Data System (ADS)

Chen, Huiqing; Li, Xiaojing; Zhao, Yuancong; Li, Jingan; Chen, Jiang; Yang, Ping; Maitz, Manfred F.; Huang, Nan

2015-08-01

A phospholipid/peptide polymer (PMMDP) with phosphorylcholine groups, endothelial progenitor cell (EPC)-specific peptides and catechol groups was anchored onto a titanium (Ti) surface to fabricate a biomimetic multifunctional surface. The PMMDP coating was characterized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements and atomic force microscopy (AFM), respectively. The amount of PMMDP coating on the Ti surface was quantified by using the quartz crystal microbalance with dissipation (QCM-D). Interactions between blood components and the coated and bare Ti substrates were evaluated by platelet adhesion and activation assays and fibrinogen denaturation test using platelet rich plasma (PRP). The results revealed that the PMMDP-modified surface inhibited fibrinogen denaturation and reduced platelet adhesion and activation. EPC cell culture on the PMMDP-modified surface showed increased adhesion and proliferation of EPCs when compared to the cells cultured on untreated Ti surface. The inhibition of fibrinogen denaturation and platelet adhesion and support of EPCs attachment and proliferation indicated that this coating might be beneficial for future applications in blood-contacting implants, such as vascular stents.

Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition.

PubMed

Woodcock, Clayton B; Yakubov, Aziz B; Reich, Norbert O

2017-08-01

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K m , k cat , k methylation , and K d for single-stranded and hemimethylated substrates, revealing discrimination of 10 7 -fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

Co-immobilization of cellulase and lysozyme on amino-functionalized magnetic nanoparticles: An activity-tunable biocatalyst for extraction of lipids from microalgae.

PubMed

Chen, Qingtai; Liu, Dong; Wu, Chongchong; Yao, Kaisheng; Li, Zhiheng; Shi, Nan; Wen, Fushan; Gates, Ian D

2018-05-03

An activity-tunable biocatalyst for Nannochloropsis sp. cell-walls degradation was prepared by co-immobilization of cellulase and lysozyme on the surface of amino-functionalized magnetic nanoparticles (MNPs) employing glutaraldehyde. The competition between cellulase and lysozyme during immobilization was caused by the limited active sites of the MNPs. The maximum recovery of activities (cellulase: 78.9% and lysozyme: 69.6%) were achieved due to synergistic effects during dual-enzyme co-immobilization. The thermal stability in terms of half-life of the co-immobilized enzymes was three times higher than that in free form and had higher catalytic efficiency for hydrolysis of cell walls. Moreover, the co-immobilized enzymes showed greater thermal stability and wider pH tolerance than free enzymes under harsh conditions. Furthermore, the co-immobilized enzymes retained up to 60% of the residual activity after being recycled 6 times. This study provides a feasible approach for the industrialization of enzyme during cell-walls disruption and lipids extraction from Nannochloropsis sp. Copyright © 2018. Published by Elsevier Ltd.

Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis.

PubMed

Tsuji, Daisuke; Akeboshi, Hiromi; Matsuoka, Kazuhiko; Yasuoka, Hiroko; Miyasaki, Eri; Kasahara, Yoshiko; Kawashima, Ikuo; Chiba, Yasunori; Jigami, Yoshifumi; Taki, Takao; Sakuraba, Hitoshi; Itoh, Kohji

2011-04-01

Novel recombinant human lysosomal β-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases. Copyright © 2010 American Neurological Association.

Adenovirus receptors and their implications in gene delivery

PubMed Central

Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

2010-01-01

Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

Phosphatidylserine metabolism modification precedes manganese-induced apoptosis and phosphatidylserine exposure in PC12 cells.

PubMed

Ferrara, G; Gambelunghe, A; Mozzi, R; Marchetti, M C; Migliorati, G; Muzi, G; Buratta, S

2013-12-01

Long-term exposure to high manganese (Mn) levels can lead to Parkinson-like neurological disorders. Molecular mechanisms underlying Mn cytotoxicity have been not defined. It is known that Mn induces apoptosis in PC12 cells and that this involves the activation of some signal transduction pathways. Although the role of phospholipids in apoptosis and signal transduction is well-known, the membrane phospholipid component in Mn-related damage has not yet been investigated. Phosphatidylserine (PS) facilitates protein translocation from cytosol to plasma membrane and PS exposure on the cell surface allows macrophage recognition of apoptotic cells. This study investigates the effects of MnCl2 on PS metabolism in PC12 cells, relating them to those on cell apoptosis. Apoptosis induction decreased PS radioactivity of PC12 cells incubated with radioactive serine. MnCl2 reduced PS radioactivity even under conditions that did not affect cell viability or PS exposure, suggesting that the effects on PS metabolism may represent an early event in cell apoptosis. Thus the latter conditions that also induced a greater PS decarboxylation were utilized for further investigating on the effects on PS synthesis, by measuring the activity and expression of PS-synthesizing enzymes, in cell lysates and in total cellular membranes (TM). Compared with corresponding controls, enzyme activity of MnCl2-treated cells was lower in cell lysates and greater in TM. Evaluating the expression of two isoforms of PS-synthesizing enzyme (PSS), PSSII was increased both in cell lysate and TM, while PSSI was unchanged. MnCl2 addition to control cell lysate reduced enzyme activity. These results suggest Mn plays a dual role on PS synthesis. Once inside the cell, Mn inhibits the enzyme/s, thus accounting for reduced PS synthesis in lysates and intact cells. On the other hand, it increases PSSII expression in cell membranes. The possibility that this occurs to counteract the direct effects of Mn ions on enzyme activity cannot be excluded. The effects on membrane enzyme activity and expression may also participate to PS exposure, observed at longer periods of treatment, by increasing membrane PS content. Copyright © 2013 Elsevier Inc. All rights reserved.

Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

PubMed Central

Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

2002-01-01

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID:11772392

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay.

PubMed

Yoo, Gu; Park, Min; Lee, Eun-Hang; Jose, Joachim; Pyun, Jae-Chul

2011-11-30

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

Improved bread-baking process using Saccharomyces cerevisiae displayed with engineered cyclodextrin glucanotransferase.

PubMed

Shim, Jae-Hoon; Seo, Nam-Seok; Roh, Sun-Ah; Kim, Jung-Wan; Cha, Hyunju; Park, Kwan-Hwa

2007-06-13

A bread-baking process was developed using a potential novel enzyme, cyclodextrin glucanotransferase[3-18] (CGTase[3-18]), that had previously been engineered to have enhanced hydrolyzing activity with little cyclodextrin (CD) formation activity toward starch. CGTase[3-18] was primarily manipulated to be displayed on the cell surface of Saccharomyces cerevisiae. S. cerevisiae carrying pdeltaCGT integrated into the chromosome exhibited starch-hydrolyzing activity at the same optimal pH and temperature as the free enzyme. Volumes of the bread loaves and rice cakes prepared using S. cerevisiae/pdeltaCGT increased by 20% and 45%, respectively, with no detectable CD. Retrogradation rates of the bread and rice cakes decreased significantly during storage. In comparison to the wild type, S. cerevisiae/pdeltaCGT showed improved viability during four freeze-thaw cycles. The results indicated that CGTase[3-18] displayed on the surface of yeast hydrolyzed starch to glucose and maltose that can be used more efficiently for yeast fermentation. Therefore, display of an antistaling enzyme on the cell surface of yeast has potential for enhancing the baking process.

Structure of a complex of uridine phosphorylase from Yersinia pseudotuberculosis with the modified bacteriostatic antibacterial drug determined by X-ray crystallography and computer analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balaev, V. V.; Lashkov, A. A., E-mail: alashkov83@gmail.com; Gabdoulkhakov, A. G.

2015-03-15

Pseudotuberculosis and bubonic plague are acute infectious diseases caused by the bacteria Yersinia pseudotuberculosis and Yersinia pestis. These diseases are treated, in particular, with trimethoprim and its modified analogues. However, uridine phosphorylases (pyrimidine nucleoside phosphorylases) that are present in bacterial cells neutralize the action of trimethoprim and its modified analogues on the cells. In order to reveal the character of the interaction of the drug with bacterial uridine phosphorylase, the atomic structure of the unligated molecule of uridine-specific pyrimidine nucleoside phosphorylase from Yersinia pseudotuberculosis (YptUPh) was determined by X-ray diffraction at 1.7 Å resolution with high reliability (R{sub work} =more » 16.2, R{sub free} = 19.4%; r.m.s.d. of bond lengths and bond angles are 0.006 Å and 1.005°, respectively; DPI = 0.107 Å). The atoms of the amino acid residues of the functionally important secondary-structure elements—the loop L9 and the helix H8—of the enzyme YptUPh were located. The three-dimensional structure of the complex of YptUPh with modified trimethoprim—referred to as 53I—was determined by the computer simulation. It was shown that 53I is a pseudosubstrate of uridine phosphorylases, and its pyrimidine-2,4-diamine group is located in the phosphate-binding site of the enzyme YptUPh.« less

Degradation of phagocytosed lysosomes by Kupffer cell lysosomes.

PubMed

Henell, F; Ericsson, J L; Glaumann, H

1983-05-01

Lysosomal membranes are apparently resistant to hydrolytic attack from their own enzymes. Alternatively, degradation occurs but is compensated for by continuous insertion of new membrane components. It may be hypothesized that a mechanism operating exclusively on the luminal side of the lysosomal membrane serves to protect the membrane from being degraded. To evaluate this notion the cytoplasmic side of the lysosomal membrane has been exposed to lysosomal enzymes in vivo. Lysosomes were isolated and subsequently injected into the portal vein of a series of rats. The uptake of the injected organelles by Kupffer cells and their subsequent degradation in lysosomes were monitored by means of electron microscopy. Four minutes after injection lysosomes were seen attached to the surface of the Kupffer cells. After 30 minutes the injected material was present in Kupffer cell phagolysosomes, and signs of degradation of the phagocytosed lysosomes were seen. By 2 hours only a few distinct membranes were left, and by 12 hours the injected lysosomes were no longer recognizable. Instead, the phagolysosomes of Kupffer cells were laden with lipid-like droplets and irregular membranous structures. Acid phosphatase histochemistry and labeling of preexisting Kupffer cell lysosomes with marker particles indicated that the phagosomes engulfing the injected lysosomes acquired hydrolytic enzymes within 30 minutes after their formation. The degradation rate of injected lysosomes was estimated by measuring the decay of radioactivity from a rat liver mitochondrial lysosomal fraction after administration of lysosomes isotopically prelabeled with 14C-leucine and 14C-glycerol. The half-life of the lysosomal membrane proteins varied between 1.5 and 2.0 hours, whereas that of the lipid component was in the range of 2.0 to 3.5 hours. These findings demonstrate that lysosomal membranes are degraded if their outer surface is exposed to lysosomal enzymes. Both the ultrastructural analysis and the isotopic studies indicate that proteins are degraded faster than lipids. Apparently, the cytoplasmic surface of the lysosomes is susceptible to lysosomal hydrolytic attack.

Nanomedicine strategy for optimizing delivery to outer hair cells by surface-modified poly(lactic/glycolic acid) nanoparticles with hydrophilic molecules

PubMed Central

Wen, Xingxing; Ding, Shan; Cai, Hui; Wang, Junyi; Wen, Lu; Yang, Fan; Chen, Gang

2016-01-01

Targeted drug delivery to outer hair cells (OHCs) in the cochlea by nanomedicine strategies forms an effective therapeutic approach for treating hearing loss. Surface chemistry plays a deciding role in nanoparticle (NP) biodistribution, but its influence on such distribution in the cochlea remains largely unknown. Herein, we report the first systematic comparison of poly(lactic/glycolic acid) nanoparticles (PLGA NPs) with or without surface modification of hydrophilic molecules for optimizing the delivery to OHCs both in vitro and in vivo. NPs that were surface modified with poloxamer 407 (P407), chitosan, or methoxy poly(ethylene glycol) and the unmodified NPs were highly biocompatible with L929 and House Ear Institute-organ of Corti 1 cells as well as cochlear tissues. Interestingly, among all the examined NPs, P407-PLGA NPs showed the greatest cellular uptake and prominent fluorescence in cochlear imaging. More importantly, we provide novel evidence that the surface-modified NPs reached the organ of Corti and were transported into the OHCs at a higher level. Together, these observations suggest that surface modification with hydrophilic molecules will allow future clinical applications of PLGA NPs, especially P407-PLGA NPs, in efficient hearing loss therapy. PMID:27877041

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta.

PubMed

Wakayama, Masataka; Ohnishi, Jun-ichi; Ueno, Osamu

2006-05-01

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades--namely, the leaf sheath, stem, scale leaf, and constituents of the spike--also have this unique anatomy and the C4 pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C4-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C4 anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C3 and C4 enzymes revealed that all the organs exhibited essentially the same C4 pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C4 pathway operation.

Influence of hesperidin on renal cell surface glycoprotein content, nucleic acids, lysosomal enzymes and macromolecules against 7, 12-dimethylbenz [a] anthracene induced experimental breast carcinoma.

PubMed

Nandakumar, Natarajan; Jayaprakash, Ramachandran; Balasubramanian, Maruthaiveeran Periyasamy

2012-01-01

Therapeutic substances may reduce the risk of developing cancer by modulating the factors responsible for carcinogenesis. To evaluate these hypotheses, the present study was designed to investigate the modulatory effect of bioflavonoid "Hesperidin" against DMBA induced experimental breast cancer with reference to renal cell surface glycoproteins, nucleic acids, protein content, lipid profile and lysosomal enzymes. The female sprague-dawley rats were orally administered with single dose of 7, 12-DMBA to induce breast cancer and were treated with hesperidin [30 mg/kg/body weight] for a consecutive 45 days. The results revealed that there was a significant elevation in the levels of glycoproteins, nucleic acids, lysosomal enzymes and also significant alterations in macromolecules in renal tissues of cancer bearing animals. Interestingly, the altered levels of these parameters were remarkably reverted back to near normal in hesperidin treatment. The histopathological analysis of liver and kidney tissues were well supported the biochemical alterations and inevitably proves the protective role of hesperidin. It is proposed that, the effect of hesperidin during DMBA induced breast cancer could be due to the intervention strategies of hesperidin in the protein, nucleic acid biosynthesis, membrane stabilizing potentials on lysosomal compartment and inhibitory effect on cell surface glycoproteins and bio-fuel such as lipids.

Carbon Dots as Versatile Photosensitizers for Solar-Driven Catalysis with Redox Enzymes.

PubMed

Hutton, Georgina A M; Reuillard, Bertrand; Martindale, Benjamin C M; Caputo, Christine A; Lockwood, Colin W J; Butt, Julea N; Reisner, Erwin

2016-12-28

Light-driven enzymatic catalysis is enabled by the productive coupling of a protein to a photosensitizer. Photosensitizers used in such hybrid systems are typically costly, toxic, and/or fragile, with limited chemical versatility. Carbon dots (CDs) are low-cost, nanosized light-harvesters that are attractive photosensitizers for biological systems as they are water-soluble, photostable, nontoxic, and their surface chemistry can be easily modified. We demonstrate here that CDs act as excellent light-absorbers in two semibiological photosynthetic systems utilizing either a fumarate reductase (FccA) for the solar-driven hydrogenation of fumarate to succinate or a hydrogenase (H 2 ase) for reduction of protons to H 2 . The tunable surface chemistry of the CDs was exploited to synthesize positively charged ammonium-terminated CDs (CD-NHMe 2 + ), which were capable of transferring photoexcited electrons directly to the negatively charged enzymes with high efficiency and stability. Enzyme-based turnover numbers of 6000 mol succinate (mol FccA) -1 and 43,000 mol H 2 (mol H 2 ase) -1 were reached after 24 h. Negatively charged carboxylate-terminated CDs (CD-CO 2 - ) displayed little or no activity, and the electrostatic interactions at the CD-enzyme interface were determined to be essential to the high photocatalytic activity observed with CD-NHMe 2 + . The modular surface chemistry of CDs together with their photostability and aqueous solubility make CDs versatile photosensitizers for redox enzymes with great scope for their utilization in photobiocatalysis.

Interaction of multi-functional silver nanoparticles with living cells

NASA Astrophysics Data System (ADS)

Sur, Ilknur; Cam, Dilek; Kahraman, Mehmet; Baysal, Asli; Culha, Mustafa

2010-04-01

Silver nanoparticles (AgNPs) are widely used in household products and in medicine due to their antibacterial and to wound healing properties. In recent years, there is also an effort for their use in biomedical imaging and photothermal therapy. The primary reason behind the effort for their utility in biomedicine and therapy is their unique plasmonic properties and easy surface chemistry for a variety of functionalizations. In this study, AgNPs modified with glucose, lactose, oligonucleotides and combinations of these ligands are investigated for their cytotoxicity and cellular uptake in living non-cancer (L929) and cancer (A549) cells. It is found that the chemical nature of the ligand strongly influences the toxicity and cellular uptake into the model cells. While the lactose-and glucose-modified AgNPs enter the L929 cells at about the same rate, a significant increase in the rate of lactose-modified AgNPs into the A549 cells is observed. The binding of oligonucleotides along with the carbohydrate on the AgNP surfaces influences the differential uptake rate pattern into the cells. The cytotoxicity study with the modified AgNPs reveals that only naked AgNPs influence the viability of the A549 cells. The findings of this study may provide the key to developing effective applications in medicine such as cancer therapy.

Development and application of test methods for the detection of dietary constituents which protect against heterocyclic aromatic amines.

PubMed

Kassie, Fekadu; Sundermann, Volker Mersch; Edenharder, R; Platt, Karl L; Darroudi, F; Lhoste, Evelyne; Humbolt, C; Muckel, Eva; Uhl, Maria; Kundi, Michael; Knasmüller, Siegfried

2003-01-01

This article describes the development and use of assay models in vitro (genotoxicity assay with genetically engineered cells and human hepatoma (HepG2) cells) and in vivo (genotoxicity and short-term carcinogenicity assays with rodents) for the identification of dietary constituents which protect against the genotoxic and carcinogenic effects of heterocyclic aromatic amines (HAs). The use of genetically engineered cells expressing enzymes responsible for the bioactivation of HAs enables the detection of dietary factors that inhibit the metabolic activation of HAs. Human derived hepatoma (HepG2) cells are sensitive towards HAs and express several enzymes [glutathione S-transferase (GST), N-acetyltransferase (NAT), sulfotransferase (SULT), UDP-glucuronosyltransferase (UDPGT), and cytochrome P450 isozymes] involved in the biotransformation of HAs. Hence these cells may reflect protective effects, which are due to inhibition of activating enzymes and/or induction of detoxifying enzymes. The SCGE assay with rodent cells has the advantage that HA-induced DNA damage can be monitored in a variety of organs which are targets for tumor induction by HAs. ACF and GST-P(+) foci constitute preneoplastic lesions that may develop into tumors. Therefore, agents that prevent the formation of these lesions may be anticarcinogens. The foci yield and the sensitivity of the system could be substantially increased by using a modified diet. The predictive value of the different in vitro and in vivo assays described here for the identification of HA-protective dietary substances relevant for humans is probably better than that of conventional in vitro test methods with enzyme homogenates. Nevertheless, the new test methods are not without shortcomings and these issues are critically discussed in the present article. Copyright 2002 Elsevier Science B.V.

Aptamer modification improves the adenoviral transduction of malignant glioma cells.

PubMed

Chen, Hao; Zheng, Xiaojing; Di, BingYan; Wang, Dongyang; Zhang, Yaling; Xia, Haibin; Mao, Qinwen

2013-12-01

Adenovirus has shown increasing promise in the gene-viral therapy for glioblastoma, a treatment strategy that relies on the delivery of viruses or transgenes into tumor cells. However, targeting of adenovirus to human glioblastoma remains a challenge due to the low expression level of coxsackie and adenovirus receptor (CAR) in glioma cells. Aptamers are small and highly structured single-stranded oligonucleotides that bind at high affinity to a target molecule, and are good candidates for targeted imaging and therapy. In this study, to construct an aptamer-modified Ad5, we first genetically modified the HVR5 of Ad hexon by biotin acceptor peptide (BAP), which would be metabolically biotinylated during production in HEK293 cells, and then attached the biotin labeled aptamer to the modified Ad through avidin–biotin binding. The aptamers used in this study includes AS1411 and GBI-10. The former is a DNA aptamer that can bind to nucleolin, a nuclear matrix protein found on the surface of cancer cells. The latter is a DNA aptamer that can recognize the extracellular matrix protein tenascin-C on the surface of human glioblastoma cells. To examine if aptamer-modification of the hexon protein could improve the adenoviral transduction efficiency, a glioblastoma cell line, U251, was transduced with aptamer-modified Ads. The transduction efficiency of AS1411- or GBI-10-modified Ad was approximately 4.1-fold or 5.2-fold higher than that of the control. The data indicated that aptamer modified adenovirus would be a useful tool for cancer gene therapy. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Surface modified stainless steels for PEM fuel cell bipolar plates

DOEpatents

Brady, Michael P [Oak Ridge, TN; Wang, Heli [Littleton, CO; Turner, John A [Littleton, CO

2007-07-24

A nitridation treated stainless steel article (such as a bipolar plate for a proton exchange membrane fuel cell) having lower interfacial contact electrical resistance and better corrosion resistance than an untreated stainless steel article is disclosed. The treated stainless steel article has a surface layer including nitrogen-modified chromium-base oxide and precipitates of chromium nitride formed during nitridation wherein oxygen is present in the surface layer at a greater concentration than nitrogen. The surface layer may further include precipitates of titanium nitride and/or aluminum oxide. The surface layer in the treated article is chemically heterogeneous surface rather than a uniform or semi-uniform surface layer exclusively rich in chromium, titanium or aluminum. The precipitates of titanium nitride and/or aluminum oxide are formed by the nitriding treatment wherein titanium and/or aluminum in the stainless steel are segregated to the surface layer in forms that exhibit a low contact resistance and good corrosion resistance.

Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology

PubMed Central

Hynninen, Ville; Vuori, Leena; Hannula, Markku; Tapio, Kosti; Lahtonen, Kimmo; Isoniemi, Tommi; Lehtonen, Elina; Hirsimäki, Mika; Toppari, J. Jussi; Valden, Mika; Hytönen, Vesa P.

2016-01-01

A straightforward solution-based method to modify the biofunctionality of stainless steel (SS) using heterobifunctional silane-polyethylene glycol (silane-PEG) overlayers is reported. Reduced nonspecific biofouling of both proteins and bacteria onto SS and further selective biofunctionalization of the modified surface were achieved. According to photoelectron spectroscopy analyses, the silane-PEGs formed less than 10 Å thick overlayers with close to 90% surface coverage and reproducible chemical compositions. Consequently, the surfaces also became more hydrophilic, and the observed non-specific biofouling of proteins was reduced by approximately 70%. In addition, the attachment of E. coli was reduced by more than 65%. Moreover, the potential of the overlayer to be further modified was demonstrated by successfully coupling biotinylated alkaline phosphatase (bAP) to a silane-PEG-biotin overlayer via avidin-biotin bridges. The activity of the immobilized enzyme was shown to be well preserved without compromising the achieved antifouling properties. Overall, the simple solution-based approach enables the tailoring of SS to enhance its activity for biomedical and biotechnological applications. PMID:27381834

Improved antifouling properties and selective biofunctionalization of stainless steel by employing heterobifunctional silane-polyethylene glycol overlayers and avidin-biotin technology

NASA Astrophysics Data System (ADS)

Hynninen, Ville; Vuori, Leena; Hannula, Markku; Tapio, Kosti; Lahtonen, Kimmo; Isoniemi, Tommi; Lehtonen, Elina; Hirsimäki, Mika; Toppari, J. Jussi; Valden, Mika; Hytönen, Vesa P.

2016-07-01

A straightforward solution-based method to modify the biofunctionality of stainless steel (SS) using heterobifunctional silane-polyethylene glycol (silane-PEG) overlayers is reported. Reduced nonspecific biofouling of both proteins and bacteria onto SS and further selective biofunctionalization of the modified surface were achieved. According to photoelectron spectroscopy analyses, the silane-PEGs formed less than 10 Å thick overlayers with close to 90% surface coverage and reproducible chemical compositions. Consequently, the surfaces also became more hydrophilic, and the observed non-specific biofouling of proteins was reduced by approximately 70%. In addition, the attachment of E. coli was reduced by more than 65%. Moreover, the potential of the overlayer to be further modified was demonstrated by successfully coupling biotinylated alkaline phosphatase (bAP) to a silane-PEG-biotin overlayer via avidin-biotin bridges. The activity of the immobilized enzyme was shown to be well preserved without compromising the achieved antifouling properties. Overall, the simple solution-based approach enables the tailoring of SS to enhance its activity for biomedical and biotechnological applications.

Protic ionic liquid modified electrocatalyst enables robust anode under cell reversal condition

NASA Astrophysics Data System (ADS)

Zhu, Zhengyu; Yan, Xiaocong; Tang, Haolin; Cai, Haopeng; Pan, Mu; Zhang, Haining; Luo, Jiangshui

2017-05-01

Pt/C has been commercially used as anode electrocatalyst for fuel cells but generally exhibits limited durability under conditions of fuel starvation and subsequent cell reversal. Herein we report an improved scaffold concept to simultaneously stabilize the catalyst against particle growth and reduce the adverse effects of cell reversal by modifying Pt/C with suitable protic ionic liquids (PILs). The modified Pt/C catalysts show enhanced cell reversal tolerance because of their high activity towards oxygen evolution reaction (OER), up to 300 mV lower overpotential compared to the unmodified Pt/C. Moreover, the PIL modified catalysts show better resistance to the loss of electrochemical surface area (ECSA) under simulated cell reversal conditions. The results indicate that modification of Pt/C catalysts with PILs is a promising strategy to enhance the stability and durability of electrocatalysts in fuel cell applications with the risk of frequent fuel starvation events, such as automotive fuel cells.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, F.H.; Moore, J.C.

1998-04-21

A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases. These enzymes exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

Preparation and antifouling properties of 2-(meth-acryloyloxy)ethyl cholinephosphate based polymers modified surface with different molecular architectures by ATRP.

PubMed

Jiang, Yuchen; Su, Yuling; Zhao, Lili; Meng, Fancui; Wang, Quanxin; Ding, Chunmei; Luo, Jianbin; Li, Jianshu

2017-08-01

Choline phosphate (CP) containing polymers modified surfaces have been shown good resist to the adhesion of proteins while prompt the attaching of mammalian cells due to the dipole pairing between the CP groups of the polymer and the phosphorylcholine (PC) groups on the cell membrane. However, the antifouling activities of CP modified surface against microbes have not been investigated at present. In addition, CP containing polymers modified surface with different molecular architectures has not been prepared and studied. To this end, glass slides surface modified with two different 2-(meth-acryloyloxy)ethyl cholinephosphate (MCP) containing polymer (PMCP) structures, i.e. brush-like (Glass-PMCP) and bottle brush-like (Glass-PHEMA-g-PMCP) architectures, were prepared in this work by surface-initiated atom transfer radical polymerization (SI-ATRP). The surface physichemical and antifouling properties of the prepared surfaces were characterized and studied. The Glass-PMCP shows improved antifouling properties against proteins and bacteria as compared to pristine glass slides (Glass-OH) and glass slides grafted with poly(2-hydroxyethyl methacrylate) (Glass-PHEMA). Notably, a synergetic fouling resistant properties of PHEMA and PMCP is presented for Glass-PHEMA-g-PMCP, which shows superior antifouling activities over Glass-PHEMA and Glass-PMCP. Furthermore, glass slides containing PMCP, i.e. Glass-PMCP and Glas-PHEMA-g-PMCP, decrease platelet adhesion and prevent their activation significantly. Therefore, the combination of antifouling PHEMA and PMCP into one system holds potential for prevention of bacterial fouling and biomaterial-centered infections. Copyright © 2017 Elsevier B.V. All rights reserved.

Performance enhancement in organic photovoltaic solar cells using iridium (Ir) ultra-thin surface modifier (USM)

NASA Astrophysics Data System (ADS)

Pandey, Rina; Lim, Ju Won; Kim, Jung Hyuk; Angadi, Basavaraj; Choi, Ji Won; Choi, Won Kook

2018-06-01

In this study, Iridium (Ir) metallic layer as an ultra-thin surface modifier (USM) was deposited on ITO coated glass substrate using radio frequency magnetron sputtering for improving the photo-conversion efficiency of organic photovoltaic cells. Ultra-thin Ir acts as a surface modifier replacing the conventional hole transport layer (HTL) PEDOT:PSS in organic photovoltaic (OPV) cells with two different active layers P3HT:PC60BM and PTB7:PC70BM. The Ir USM (1.0 nm) coated on ITO glass substrate showed transmittance of 84.1% and work function of >5.0 eV, which is higher than that of ITO (4.5-4.7 eV). The OPV cells with Ir USM (1.0 nm) exhibits increased power conversion efficiency of 3.70% (for P3HT:PC60BM active layer) and 7.28% (for PTB7:PC70BM active layer) under 100 mW/cm2 illumination (AM 1.5G) which are higher than those of 3.26% and 6.95% for the same OPV cells but with PEDOT:PSS as HTL instead of Ir USM. The results reveal that the chemically stable Ir USM layer could be used as an alternative material for PEDOT:PSS in organic photovoltaic cells.

Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture.

PubMed

Yang, Jian; Shi, Guixin; Bei, Jianzhong; Wang, Shenguo; Cao, Yilin; Shang, Qingxin; Yang, Guanghui; Wang, Wenjing

2002-12-05

The fabrication and surface modification of a porous cell scaffold are very important in tissue engineering. Of most concern are high-density cell seeding, nutrient and oxygen supply, and cell affinity. In the present study, poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds with different pore structures were fabricated. An improved method based on Archimedes' Principle for measuring the porosity of scaffolds, using a density bottle, was developed. Anhydrous ammonia plasma treatment was used to modify surface properties to improve the cell affinity of the scaffolds. The results show that hydrophilicity and surface energy were improved. The polar N-containing groups and positive charged groups also were incorporated into the sample surface. A low-temperature treatment was used to maintain the plasma-modified surface properties effectively. It would do help to the further application of plasma treatment technique. Cell culture results showed that pores smaller than 160 microm are suitable for human skin fibroblast cell growth. Cell seeding efficiency was maintained at above 99%, which is better than the efficiency achieved with the common method of prewetting by ethanol. The plasma-treatment method also helped to resolve the problem of cell loss during cell seeding, and the negative effects of the ethanol trace on cell culture were avoided. The results suggest that anhydrous ammonia plasma treatment enhances the cell affinity of porous scaffolds. Mass transport issues also have been considered. Copyright 2002 Wiley Periodicals, Inc.

Cystic fibrosis gene modifier SLC26A9 modulates airway response to CFTR-directed therapeutics.

PubMed

Strug, Lisa J; Gonska, Tanja; He, Gengming; Keenan, Katherine; Ip, Wan; Boëlle, Pierre-Yves; Lin, Fan; Panjwani, Naim; Gong, Jiafen; Li, Weili; Soave, David; Xiao, Bowei; Tullis, Elizabeth; Rabin, Harvey; Parkins, Michael D; Price, April; Zuberbuhler, Peter C; Corvol, Harriet; Ratjen, Felix; Sun, Lei; Bear, Christine E; Rommens, Johanna M

2016-10-15

Cystic fibrosis is realizing the promise of personalized medicine. Recent advances in drug development that target the causal CFTR directly result in lung function improvement, but variability in response is demanding better prediction of outcomes to improve management decisions. The genetic modifier SLC26A9 contributes to disease severity in the CF pancreas and intestine at birth and here we assess its relationship with disease severity and therapeutic response in the airways. SLC26A9 association with lung disease was assessed in individuals from the Canadian and French CF Gene Modifier consortia with CFTR-gating mutations and in those homozygous for the common Phe508del mutation. Variability in response to a CFTR-directed therapy attributed to SLC26A9 genotype was assessed in Canadian patients with gating mutations. A primary airway model system determined if SLC26A9 shows modification of Phe508del CFTR function upon treatment with a CFTR corrector. In those with gating mutations that retain cell surface-localized CFTR we show that SLC26A9 modifies lung function while this is not the case in individuals homozygous for Phe508del where cell surface expression is lacking. Treatment response to ivacaftor, which aims to improve CFTR-channel opening probability in patients with gating mutations, shows substantial variability in response, 28% of which can be explained by rs7512462 in SLC26A9 (P = 0.0006). When homozygous Phe508del primary bronchial cells are treated to restore surface CFTR, SLC26A9 likewise modifies treatment response (P = 0.02). Our findings indicate that SLC26A9 airway modification requires CFTR at the cell surface, and that a common variant in SLC26A9 may predict response to CFTR-directed therapeutics.

Kinetics of biochemical sensing by single cells and populations of cells

NASA Astrophysics Data System (ADS)

Saakian, David B.

2017-10-01

We investigate the collective stationary sensing using N communicative cells, which involves surface receptors, diffusive signaling molecules, and cell-cell communication messengers. We restrict the scenarios to the signal-to-noise ratios (SNRs) for both strong communication and extrinsic noise only. We modified a previous model [Bialek and Setayeshgar, Proc. Natl. Acad. Sci. USA 102, 10040 (2005), 10.1073/pnas.0504321102] to eliminate the singularities in the fluctuation correlations by considering a uniform receptor distribution over the surface of each cell with a finite radius a . The modified model enables a simple and rigorous mathematical treatment of the collective sensing phenomenon. We then derive the scaling of the SNR for both juxtacrine and autocrine cases in all dimensions. For the optimal locations of the cells in the autocrine case, we find identical scaling for both two and three dimensions.

Citrobacter amalonaticus Phytase on the Cell Surface of Pichia pastoris Exhibits High pH Stability as a Promising Potential Feed Supplement

PubMed Central

Li, Cheng; Lin, Ying; Huang, Yuanyuan; Liu, Xiaoxiao; Liang, Shuli

2014-01-01

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase. PMID:25490768

Citrobacter amalonaticus phytase on the cell surface of Pichia pastoris exhibits high pH stability as a promising potential feed supplement.

PubMed

Li, Cheng; Lin, Ying; Huang, Yuanyuan; Liu, Xiaoxiao; Liang, Shuli

2014-01-01

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.

Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems.

PubMed

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-02-28

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TK(SR39) mutants), yeast cytosine deaminase:uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs' bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Computational optimization of AG18051 inhibitor for amyloid-beta binding alcohol dehydrogenase enzyme

NASA Astrophysics Data System (ADS)

Marques, Alexandra T.; Antunes, Agostinho; Fernandes, Pedro A.; Ramos, Maria J.

Amyloid-beta (Abeta) binding alcohol dehydrogenase (ABAD) is a multifunctional enzyme involved in maintaining the homeostasis. The enzyme can also mediate some diseases, including genetic diseases, Alzheimer's disease, and possibly some prostate cancers. Potent inhibitors of ABAD might facilitate a better clarification of the functions of the enzyme under normal and pathogenic conditions and might also be used for therapeutic intervention in disease conditions mediated by the enzyme. The AG18051 is the only presently available inhibitor of ABAD. It binds in the active-site cavity of the enzyme and reacts with the NAD+ cofactor to form a covalent adduct. In this work, we use computational methods to perform a rational optimization of the AG18051 inhibitor, through the introduction of chemical substitutions directed to improve the affinity of the inhibitor to the enzyme. The molecular mechanics-Poisson-Boltzmann surface area methodology was used to predict the relative free binding energy of the different modified inhibitor-NAD-enzyme complexes. We show that it is possible to increase significantly the affinity of the inhibitor to the enzyme with small modifications, without changing the overall structure and ADME (absorption, distribution, metabolism, and excretion) properties of the original inhibitor.

Distinct contributions of the nisin biosynthesis enzymes NisB and NisC and transporter NisT to prenisin production by Lactococcus lactis.

PubMed

van den Berg van Saparoea, H Bart; Bakkes, Patrick J; Moll, Gert N; Driessen, Arnold J M

2008-09-01

Several Lactococcus lactis strains produce the lantibiotic nisin. The dedicated enzymes NisB and NisC and the transporter NisT modify and secrete the ribosomally synthesized nisin precursor peptide. NisB can function in the absence of the cyclase NisC, yielding the dehydrated prenisin that lacks the thioether rings. A kinetic analysis of nisin production by L. lactis NZ9700 demonstrated that the prenisin was released from the cell into the medium before the processing of the leader sequence occurred. Upon the deletion of nisC, the production of prenisin was reduced by 70%, while in the absence of nisB, the production of prenisin was nearly completely abolished. In cells lacking nisT, no secretion was observed, while the expression of nisABC in these cells resulted in considerable growth rate inhibition caused by the intracellular accumulation of active nisin. Overall, these data indicate that the efficiency of prenisin transport by NisT is markedly enhanced by NisB, suggesting a channeling mechanism of prenisin transfer between the nisin modification enzymes and the transporter.

Inflammatory cytokine response to titanium chemical composition and nanoscale calcium phosphate surface modification.

PubMed

Hamlet, Stephen; Ivanovski, Saso

2011-05-01

Nanoscale surface modification of titanium dental implants with calcium phosphate (CaP) has been shown to achieve superior bone wound healing and osseointegration compared with smooth or microrough titanium surfaces alone. As bone healing has been shown to be influenced by the action of cytokines, this study examined whether changes in cytokine gene expression from RAW 264.7 cells cultured on commercially pure and titanium alloy (Ti-6Al-4V) microrough or nanoscale crystalline CaP-modified surfaces, may influence downstream events in bone wound healing and osseointegration. Whilst no significant difference in the attachment or proliferation of RAW 264.7 cells was observed, the nanoscale CaP-modified surface elicited a gene expression profile with marked down-regulation of a number of pro-inflammatory cytokines and chemokines. Inflammatory cytokine gene expression was further influenced by chemical composition, with lower levels of pro-inflammatory markers noted following exposure of the macrophage-like cells to titanium alloy (Ti-6Al-4V) compared with the commercially pure titanium surface. Down-regulation of pro-inflammatory cytokine gene expression (confirmed at the protein level for TNFα and CCL5), may thus facilitate the enhanced bone wound healing and osseointegration observed clinically with nanoscale calcium phosphate-modified implant surfaces. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

A rapid generation of adenovirus vector with a genetic modification in hexon protein.

PubMed

Di, Bingyan; Mao, Qinwen; Zhao, Junli; Li, Xing; Wang, Dongyang; Xia, Haibin

2012-02-10

The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair.

PubMed

Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

2016-08-31

DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell's genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

Development of Low-cost, High Energy-per-unit-area Solar Cell Modules

NASA Technical Reports Server (NTRS)

Jones, G. T.; Chitre, S.; Rhee, S. S.

1978-01-01

The development of two hexagonal solar cell process sequences, a laserscribing process technique for scribing hexagonal and modified hexagonal solar cells, a large through-put diffusion process, and two surface macrostructure processes suitable for large scale production is reported. Experimental analysis was made on automated spin-on anti-reflective coating equipment and high pressure wafer cleaning equipment. Six hexagonal solar cell modules were fabricated. Also covered is a detailed theoretical analysis on the optimum silicon utilization by modified hexagonal solar cells.

DNA-mediated engineering of multicomponent enzyme crystals

PubMed Central

Brodin, Jeffrey D.; Auyeung, Evelyn; Mirkin, Chad A.

2015-01-01

The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. Herein, we explore the concept of trading protein–protein interactions for DNA–DNA interactions to direct the assembly of two nucleic-acid–functionalized proteins with distinct surface chemistries into six unique lattices composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA–DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. This study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials. PMID:25831510

Molecular and functional assessment of multicellular cancer spheroids produced in double emulsions enabled by efficient airway resistance based selective surface treatment

NASA Astrophysics Data System (ADS)

Ma, Xiao; Leth Jepsen, Morten; Ivarsen, Anne Kathrine R.; Knudsen, Birgitta R.; Ho, Yi-Ping

2017-09-01

Multicellular spheroids have garnered significant attention as an in vitro three-dimensional cancer model which can mimick the in vivo microenvironmental features. While microfluidics generated double emulsions have become a potential method to generate spheroids, challenges remain on the tedious procedures. Enabled by a novel ‘airway resistance’ based selective surface treatment, this study presents an easy and facile generation of double emulsions for the initiation and cultivation of multicellular spheroids in a scaffold-free format. Combining with our previously developed DNA nanosensors, intestinal spheroids produced in the double emulsions have shown an elevated activities of an essential DNA modifying enzyme, the topoisomerase I. The observed molecular and functional characteristics of spheroids produced in double emulsions are similar to the counterparts produced by the commercially available ultra-low attachment plates. However, the double emulsions excel for their improved uniformity, and the consistency of the results obtained by subsequent analysis of the spheroids. The presented technique is expected to ease the burden of producing spheroids and to promote the spheroids model for cancer or stem cell study.

DNA-mediated engineering of multicomponent enzyme crystals

DOE PAGES

Brodin, Jeffrey D.; Auyeung, Evelyn; Mirkin, Chad A.

2015-03-23

The ability to predictably control the coassembly of multiple nanoscale building blocks, especially those with disparate chemical and physical properties such as biomolecules and inorganic nanoparticles, has far-reaching implications in catalysis, sensing, and photonics, but a generalizable strategy for engineering specific contacts between these particles is an outstanding challenge. This is especially true in the case of proteins, where the types of possible interparticle interactions are numerous, diverse, and complex. In this paper, we explore the concept of trading protein–protein interactions for DNA–DNA interactions to direct the assembly of two nucleic-acid–functionalized proteins with distinct surface chemistries into six unique latticesmore » composed of catalytically active proteins, or of a combination of proteins and DNA-modified gold nanoparticles. The programmable nature of DNA–DNA interactions used in this strategy allows us to control the lattice symmetries and unit cell constants, as well as the compositions and habit, of the resulting crystals. Finally, this study provides a potentially generalizable strategy for constructing a unique class of materials that take advantage of the diverse morphologies, surface chemistries, and functionalities of proteins for assembling functional crystalline materials.« less

Biosilica from Living Diatoms: Investigations on Biocompatibility of Bare and Chemically Modified Thalassiosira weissflogii Silica Shells

PubMed Central

Cicco, Stefania Roberta; Vona, Danilo; Gristina, Roberto; Sardella, Eloisa; Ragni, Roberta; Lo Presti, Marco; Farinola, Gianluca Maria

2016-01-01

In the past decade, mesoporous silica nanoparticles (MSNs) with a large surface area and pore volume have attracted considerable attention for their application in drug delivery and biomedicine. Here we propose biosilica from diatoms as an alternative source of mesoporous materials in the field of multifunctional supports for cell growth: the biosilica surfaces were chemically modified by traditional silanization methods resulting in diatom silica microparticles functionalized with 3-mercaptopropyl-trimethoxysilane (MPTMS) and 3-aminopropyl-triethoxysilane (APTES). Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses revealed that the –SH or –NH2 were successfully grafted onto the biosilica surface. The relationship among the type of functional groups and the cell viability was established as well as the interaction of the cells with the nanoporosity of frustules. These results show that diatom microparticles are promising natural biomaterials suitable for cell growth, and that the surfaces, owing to the mercapto groups, exhibit good biocompatibility. PMID:28952597

Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

NASA Astrophysics Data System (ADS)

Feuser, Paulo Emilio; Jacques, Amanda Virtuoso; Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin; dos Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H. Hermes

2016-04-01

The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

Magnetic biocatalysts and their uses to obtain bioproducts

NASA Astrophysics Data System (ADS)

López, Carmen; Cruz-Izquierdo, Álvaro; Picó, Enrique; García-Bárcena, Teresa; Villarroel, Noelia; Llama, María; Serra, Juan

2014-08-01

Nanobiocatalysis, as the synergistic combination of nanotechnology and biocatalysis, is rapidly emerging as a new frontier of biotechnology. The use of immobilized enzymes in industrial applications often presents advantages over their soluble counterparts, mainly in view of stability, reusability and simpler operational processing. Because of their singular properties, such as biocompatibility, large and modifiable surface and easy recovery, iron oxide magnetic nanoparticles (MNPs) are attractive super-paramagnetic materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field. Cross-linked enzyme aggregates (CLEAs) have several benefits in the context of industrial applications since they can be cheaply and easily prepared from unpurified enzyme extracts and show improved storage and operational stability against denaturation by heat and organic solvents. In this work, by using the aforementioned advantages of MNPs of magnetite and CLEAs, we prepared two robust magnetically-separable types of nanobiocatalysts by binding either soluble enzyme onto the surface of MNPs functionalized with amino groups or by cross-linking aggregates of enzyme among them and to MNPs to obtain magnetic CLEAs. For this purpose the lipase B of Candida antarctica (CALB) was used. The hydrolytic and biosynthetic activities of the resulting magnetic nanobiocatalysts were assessed in aqueous and organic media and compared between them and to those showed by the corresponding soluble enzyme. Thus, the hydrolysis of triglycerides or the transesterification reactions to synthesize biodiesel and biosurfactants were studied using magnetic CLEAs of CALB.

Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B

PubMed Central

Wang, Pan; He, Jie; Sun, Yufei; Reynolds, Matthew; Zhang, Li; Han, Shuangyan; Liang, Shuli; Sui, Haixin; Lin, Ying

2016-01-01

To modify the Pichia pastoris cell surface, two classes of hydrophobins, SC3 from Schizophyllum commune and HFBI from Trichoderma reesei, were separately displayed on the cell wall. There was an observable increase in the hydrophobicity of recombinant strains. Candida antarctica lipase B (CALB) was then co-displayed on the modified cells, generating strains GS115/SC3-61/CALB-51 and GS115/HFBI-61/CALB-51. Interestingly, the hydrolytic and synthetic activities of strain GS115/HFBI-61/CALB-51 increased by 37% and 109%, respectively, but decreased by 26% and 43%, respectively, in strain GS115/SC3-61/CALB-51 compared with the hydrophobin-minus recombinant strain GS115/CALB-GCW51. The amount of glycerol by-product from the transesterification reaction adsorbed on the cell surface was significantly decreased following hydrophobin modification, removing the glycerol barrier and allowing substrates to access the active sites of lipases. Electron micrographs indicated that the cell wall structures of both recombinant strains appeared altered, including changes to the inner glucan layer and outer mannan layer. These results suggest that the display of hydrophobins can change the surface structure and hydrophobic properties of P. pastoris, and affect the catalytic activities of CALB displayed on the surface of P. pastoris cells. PMID:26969039

Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity.

PubMed

Merhi, Maysaloun; Dombu, Christophe Youta; Brient, Alizée; Chang, Jiang; Platel, Anne; Le Curieux, Frank; Marzin, Daniel; Nesslany, Fabrice; Betbeder, Didier

2012-02-14

We used well-characterized and positively charged nanoparticles (NP(+)) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP(+) physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP(+) surface charge was significantly reduced, proportionally to NP(+)/serum and NP(+)/BSA ratios, while NP(+) size was not modified. Microscopy studies showed high endocytosis of NP(+) in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP(+). No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP(+) genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP(+) might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity. Copyright © 2011 Elsevier B.V. All rights reserved.

Epigenetic developmental programs and adipogenesis: implications for psychotropic induced obesity.

PubMed

Chase, Kayla; Sharma, Rajiv P

2013-11-01

Psychotropic agents are notorious for their ability to increase fat mass in psychiatric patients. The two determinants of fat mass are the production of newly differentiated adipocytes (adipogenesis), and the volume of lipid accumulation. Epigenetic programs have a prominent role in cell fate commitments and differentiation required for adipogenesis. In parallel, epigenetic effects on energy metabolism are well supported by several genetic models. Consequently, a variety of psychotropics, often prescribed in combinations and for long periods, may utilize a common epigenetic effector path causing an increase in adipogenesis or reduction in energy metabolism. In particular, the recent discovery that G protein coupled signaling cascades can directly modify epigenetic regulatory enzymes implicates surface receptor activity by psychotropic medications. The potential therapeutic implications are also suggested by the effects of the clinically approved antidepressant tranylcypromine, also a histone demethylase inhibitor, which has impressive therapeutic effects on metabolism in the obese phenotype.

Hybrid biobattery based on arylated carbon nanotubes and laccase.

PubMed

Stolarczyk, Krzysztof; Sepelowska, Małgorzata; Lyp, Dominika; Zelechowska, Kamila; Biernat, Jan F; Rogalski, Jerzy; Farmer, Kevin D; Roberts, Ken N; Bilewicz, Renata

2012-10-01

Single-walled carbon nanotubes (SWCNT) were covalently modified with anthracene and anthraquinone and used for the construction of cathodes for biocatalytic reduction of dioxygen. The nanotubes with aromatic groups casted onto the electrode increased the working surface of the electrode and enabled efficient direct electron transfer (DET) between the enzyme and the electrode. The aryl groups enter the hydrophobic pocket of the T1 center of laccase responsible for exchanging electrons with the substrate. Glassy carbon electrode covered with arylated SWCNT and coated with a layer of neutralized Nafion containing laccase was found to be a very efficient cathode in the hybrid battery. Zn wire covered with a Nafion film served as the anode. The cell parameters were determined: power density was 2 mW/cm(2) and the open circuit potential was 1.5 V. Copyright © 2011 Elsevier B.V. All rights reserved.

A novel helper phage for HaloTag-mediated co-display of enzyme and substrate on phage.

PubMed

Delespaul, Wouter; Peeters, Yves; Herdewijn, Piet; Robben, Johan

2015-05-01

Phage display is an established technique for the molecular evolution of peptides and proteins. For the selection of enzymes based on catalytic activity however, simultaneous coupling of an enzyme and its substrate to the phage surface is required. To facilitate this process of co-display, we developed a new helper phage displaying HaloTag, a modified haloalkane dehalogenase that binds specifically and covalently to functionalized haloalkane ligands. The display of functional HaloTag was demonstrated by capture on streptavidin-coated magnetic beads, after coupling a biotinylated haloalkane ligand, or after on-phage extension of a DNA oligonucleotide primer with a biotinylated nucleotide by phi29 DNA polymerase. We also achieved co-display of HaloTag and phi29 DNA polymerase, thereby opening perspectives for the molecular evolution of this enzyme (and others) towards new substrate specificities. Copyright © 2015 Elsevier Inc. All rights reserved.

Biomimetic Multispiked Connecting Ti-Alloy Scaffold Prototype for Entirely-Cementless Resurfacing Arthroplasty Endoprostheses-Exemplary Results of Implantation of the Ca-P Surface-Modified Scaffold Prototypes in Animal Model and Osteoblast Culture Evaluation.

PubMed

Uklejewski, Ryszard; Rogala, Piotr; Winiecki, Mariusz; Tokłowicz, Renata; Ruszkowski, Piotr; Wołuń-Cholewa, Maria

2016-06-29

We present here-designed, manufactured, and tested by our research team-the Ti-alloy prototype of the multispiked connecting scaffold (MSC-Scaffold) interfacing the components of resurfacing arthroplasty (RA) endoprostheses with bone. The spikes of the MSC-Scaffold prototype mimic the interdigitations of the articular subchondral bone, which is the natural biostructure interfacing the articular cartilage with the periarticular trabecular bone. To enhance the osteoinduction/osteointegration potential of the MSC-Scaffold, the attempts to modify its bone contacting surfaces by the process of electrochemical cathodic deposition of Ca-P was performed with further immersion of the MSC-Scaffold prototypes in SBF in order to transform the amorphous calcium-phosphate coating in hydroxyapatite-like (HA-like) coating. The pilot experimental study of biointegration of unmodified and Ca-P surface-modified MSC-Scaffold prototypes was conducted in an animal model (swine) and in osteoblast cell culture. On the basis of a microscope-histological method the biointegration was proven by the presence of trabeculae in the interspike spaces of the MSC-Scaffold prototype on longitudinal and cross-sections of bone-implant specimens. The percentage of trabeculae in the area between the spikes of specimen containing Ca-P surface modified scaffold prototype observed in microCT reconstructions of the explanted joints was visibly higher than in the case of unmodified MSC-Scaffold prototypes. Significantly higher Alkaline Phosphatase (ALP) activity and the cellular proliferation in the case of Ca-P-modified MSC-Scaffold pre-prototypes, in comparison with unmodified pre-prototypes, was found in osteoblast cell cultures. The obtained results of experimental implantation in an animal model and osteoblast cell culture evaluations of Ca-P surface-modified and non-modified biomimetic MSC-Scaffold prototypes for biomimetic entirely-cementless RA endoprostheses indicate the enhancement of the osteoinduction/osteointegration potential by the Ca-P surface modification of the Ti-alloy MSC-Scaffold prototype. Planned further research on the prototype of this biomimetic MSC-Scaffold for a new generation of RA endoprostheses is also given.

Biomimetic Multispiked Connecting Ti-Alloy Scaffold Prototype for Entirely-Cementless Resurfacing Arthroplasty Endoprostheses—Exemplary Results of Implantation of the Ca-P Surface-Modified Scaffold Prototypes in Animal Model and Osteoblast Culture Evaluation

PubMed Central

Uklejewski, Ryszard; Rogala, Piotr; Winiecki, Mariusz; Tokłowicz, Renata; Ruszkowski, Piotr; Wołuń-Cholewa, Maria

2016-01-01

We present here—designed, manufactured, and tested by our research team—the Ti-alloy prototype of the multispiked connecting scaffold (MSC-Scaffold) interfacing the components of resurfacing arthroplasty (RA) endoprostheses with bone. The spikes of the MSC-Scaffold prototype mimic the interdigitations of the articular subchondral bone, which is the natural biostructure interfacing the articular cartilage with the periarticular trabecular bone. To enhance the osteoinduction/osteointegration potential of the MSC-Scaffold, the attempts to modify its bone contacting surfaces by the process of electrochemical cathodic deposition of Ca-P was performed with further immersion of the MSC-Scaffold prototypes in SBF in order to transform the amorphous calcium-phosphate coating in hydroxyapatite-like (HA-like) coating. The pilot experimental study of biointegration of unmodified and Ca-P surface-modified MSC-Scaffold prototypes was conducted in an animal model (swine) and in osteoblast cell culture. On the basis of a microscope-histological method the biointegration was proven by the presence of trabeculae in the interspike spaces of the MSC-Scaffold prototype on longitudinal and cross-sections of bone-implant specimens. The percentage of trabeculae in the area between the spikes of specimen containing Ca-P surface modified scaffold prototype observed in microCT reconstructions of the explanted joints was visibly higher than in the case of unmodified MSC-Scaffold prototypes. Significantly higher Alkaline Phosphatase (ALP) activity and the cellular proliferation in the case of Ca-P-modified MSC-Scaffold pre-prototypes, in comparison with unmodified pre-prototypes, was found in osteoblast cell cultures. The obtained results of experimental implantation in an animal model and osteoblast cell culture evaluations of Ca-P surface-modified and non-modified biomimetic MSC-Scaffold prototypes for biomimetic entirely-cementless RA endoprostheses indicate the enhancement of the osteoinduction/osteointegration potential by the Ca-P surface modification of the Ti-alloy MSC-Scaffold prototype. Planned further research on the prototype of this biomimetic MSC-Scaffold for a new generation of RA endoprostheses is also given. PMID:28773652

Commentary on Some Recent Theses Relevant to Combating Aging: August 2017.

PubMed

Zealley, Benjamin; de Grey, Aubrey D N J

2017-08-01

Theses reviewed in this issue include "Engineering Cellular Input-Output for the Robust Control of Mammalian Cell-Based Therapies"; "Enzyme-Instructed Self-Assembly (EISA) Selectively Targets Cancer Cells"; "Exploration of helminth-derived immunoregulatory molecules as options for therapeutic intervention in allograft rejection and autoimmune disease"; "Expression of the Medial HOXA genes is indispensible for self-renewal in human hematopoietic stem cells"; "Gamma frequency entrainment attenuates amyloid load and modifies microglia"; and "Heterogeneous Distribution of Microvascular Blood Flow Contributes to Impaired Skeletal Muscle Oxygenation in Diabetes".

Surface Proteins of Gram-Positive Pathogens: Using Crystallography to Uncover Novel Features in Drug and Vaccine Candidates

NASA Astrophysics Data System (ADS)

Baker, Edward N.; Proft, Thomas; Kang, Haejoo

Proteins displayed on the cell surfaces of pathogenic organisms are the front-line troops of bacterial attack, playing critical roles in colonization, infection and virulence. Although such proteins can often be recognized from genome sequence data, through characteristic sequence motifs, their functions are often unknown. One such group of surface proteins is attached to the cell surface of Gram-positive pathogens through the action of sortase enzymes. Some of these proteins are now known to form pili: long filamentous structures that mediate attachment to human cells. Crystallographic analyses of these and other cell surface proteins have uncovered novel features in their structure, assembly and stability, including the presence of inter- and intramolecular isopeptide crosslinks. This improved understanding of structures on the bacterial cell surface offers opportunities for the development of some new drug targets and for novel approaches to vaccine design.

High performance dye-sensitized solar cells using graphene modified fluorine-doped tin oxide glass by Langmuir–Blodgett technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roh, Ki-Min; Jo, Eun-Hee; Chang, Hankwon

Since the introduction of dye-sensitized solar cells (DSSCs) with low fabrication cost and high power conversion efficiency, extensive studies have been carried out to improve the charge transfer rate and performance of DSSCs. In this paper, we present DSSCs that use surface modified fluorine-doped tin oxide (FTO) substrates with reduced graphene oxide (r-GO) sheets prepared using the Langmuir–Blodgett (LB) technique to decrease the charge recombination at the TiO{sub 2}/FTO interface. R-GO sheets were excellently attached on FTO surface without physical deformations such as wrinkles; effects of the surface coverage of r-GO on the DSSC performance were also investigated. By usingmore » graphene modified FTO substrates, the resistance at the interface of TiO{sub 2}/FTO was reduced and the power conversion efficiency was increased to 8.44%. - Graphical abstract: DSSCs with graphene modified FTO glass were fabricated with the Langmuir Blodgett technique. GO sheets were transferred to FTO at various surface pressures in order to change the surface density of graphene and the highest power conversion efficiency of the DSSC was 8.44%. - Highlights: • By LB technique, r-GO sheets were coated on FTO without physical deformation. • DSSCs were fabricated with, r-GO modified FTO substrates. • With surface modification by r-GO, the interface resistance of DSSC decreased. • Maximum PCE of the DSSC was increased up to 8.44%.« less

Migration of lymphocytes on fibronectin-coated surfaces: temporal evolution of migratory parameters

NASA Technical Reports Server (NTRS)

Bergman, A. J.; Zygourakis, K.; McIntire, L. V. (Principal Investigator)

1999-01-01

Lymphocytes typically interact with implanted biomaterials through adsorbed exogenous proteins. To provide a more complete characterization of these interactions, analysis of lymphocyte migration on adsorbed extracellular matrix proteins must accompany the commonly performed adhesion studies. We report here a comparison of the migratory and adhesion behavior of Jurkat cells (a T lymphoblastoid cell line) on tissue culture treated and untreated polystyrene surfaces coated with various concentrations of fibronectin. The average speed of cell locomotion showed a biphasic response to substrate adhesiveness for cells migrating on untreated polystyrene and a monotonic decrease for cells migrating on tissue culture-treated polystyrene. A modified approach to the persistent random walk model was implemented to determine the time dependence of cell migration parameters. The random motility coefficient showed significant increases with time when cells migrated on tissue culture-treated polystyrene surfaces, while it remained relatively constant for experiments with untreated polystyrene plates. Finally, a cell migration computer model was developed to verify our modified persistent random walk analysis. Simulation results suggest that our experimental data were consistent with temporally increasing random motility coefficients.

Hydrophilic group formation and cell culturing on polystyrene Petri-dish modified by ion-assisted reaction

NASA Astrophysics Data System (ADS)

Kim, Ki-Hwan; Cho, Jun-Sik; Choi, Doo-Jin; Koh, Seok-Keun

2001-04-01

Polystyrene (PS) Petri-dishes were modified by an ion-assisted reaction (IAR) to improve wettability and to supply a suitable surface for cell culturing. Low energy Ar + ions with 1000 eV were irradiated on the surface of PS in oxygen gas environment. Water contact angles of PS were not reduced much by ion irradiation without oxygen gas and had a value of 40°. In the case of ion irradiation with flowing oxygen gas, however, the water contact angles were dropped significantly from 73° to 19°. X-ray photoelectron spectroscopy analysis showed that the hydrophilic groups were formed on the surface of PS by a chemical reaction between unstable chains induced by ion irradiation and the oxygen gas. Newly formed hydrophilic groups were identified as -(C-O)-, -(CO)- and -(CO)-O- bonds. The influence of the surface modification on growth of the rat pheochromocytoma (PC12) cells was investigated. The IAR-treated PS surfaces showed enhanced attachment and growth in PC12 cell culture test.

Alterations in the skin of Labeo rohita exposed to an azo dye, Eriochrome black T: a histopathological and enzyme biochemical investigation.

PubMed

Srivastava, Ayan; Verma, Neeraj; Mistri, Arup; Ranjan, Brijesh; Nigam, Ashwini Kumar; Kumari, Usha; Mittal, Swati; Mittal, Ajay Kumar

2017-03-01

Histopathological changes and alterations in the activity of certain metabolic and antioxidant enzymes were analyzed in the head skin of Labeo rohita, exposed to sublethal test concentrations of the azo dye, Eriochrome black T for 4 days, using 24 h renewal bioassay method. Hypertrophied epithelial cells, increased density of mucous goblet cells, and profuse mucous secretion at the surface were considered to protect the skin from toxic impact of the azo dye. Degenerative changes including vacuolization, shrinkage, decrease in dimension, and density of club cells with simultaneous release of their contents in the intercellular spaces were associated to plug them, preventing indiscriminate entry of foreign matter. On exposure of fish to the dye, significant decline in the activity of enzymes-alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase-was associated with the binding of dye to the enzymes. Gradual increase in the activity of lactate dehydrogenase was considered to reflect a shift from aerobic to anaerobic metabolism. On transfer of azo dye exposed fish to freshwater, skin gradually recovers and, by 8 days, density and area of mucous goblet cells, club cells, and activity of the enzymes appear similar to that of controls. Alteration in histopathology and enzyme activity could be considered beneficial tool in monitoring environmental toxicity, valuable in the sustenance of fish populations.

A review study of (bio)sensor systems based on conducting polymers.

PubMed

Ates, Murat

2013-05-01

This review article concentrates on the electrochemical biosensor systems with conducting polymers. The area of electro-active polymers confined to different electrode surfaces has attracted great attention. Polymer modified carbon substrate electrodes can be designed through polymer screening to provide tremendous improvements in sensitivity, selectivity, stability and reproducibility of the electrode response to detect a variety of analytes. The electro-active films have been used to entrap different enzymes and/or proteins at the electrode surface, but without obvious loss of their bioactivity for the development of biosensors. Electropolymerization is a well-known technique used to immobilize biomaterials to the modified electrode surface. Polymers might be covalently bonding to enzymes or proteins; therefore, thickness, permeation and charge transport characteristics of the polymeric films can be easily and precisely controlled by modulating the electrochemical parameters for various electrochemical techniques, such as chronoamperometry, chronopotentiometry, cyclic voltammetry, and differential pulse voltammetry. This review article is divided into three main parts as given in the table of contents related to the immobilization process of some important conducting polymers, polypyrrole, polythiophene, poly(3,4-ethylenedioxythiophene), polycarbazole, polyaniline, polyphenol, poly(o-phenylenediamine), polyacetylene, polyfuran and their derivatives. A total of 216 references are cited in this review article. The literature reviewed covers a 7 year period beginning from 2005. Copyright © 2013 Elsevier B.V. All rights reserved.

Tailored functionalization of iron oxide nanoparticles for MRI, drug delivery, magnetic separation and immobilization of biosubstances.

PubMed

Hola, Katerina; Markova, Zdenka; Zoppellaro, Giorgio; Tucek, Jiri; Zboril, Radek

2015-11-01

In this critical review, we outline various covalent and non-covalent approaches for the functionalization of iron oxide nanoparticles (IONPs). Tuning the surface chemistry and design of magnetic nanoparticles are described in relation to their applicability in advanced medical technologies and biotechnologies including magnetic resonance imaging (MRI) contrast agents, targeted drug delivery, magnetic separations and immobilizations of proteins, enzymes, antibodies, targeting agents and other biosubstances. We review synthetic strategies for the controlled preparation of IONPs modified with frequently used functional groups including amine, carboxyl and hydroxyl groups as well as the preparation of IONPs functionalized with other species, e.g., epoxy, thiol, alkane, azide, and alkyne groups. Three main coupling strategies for linking IONPs with active agents are presented: (i) chemical modification of amine groups on the surface of IONPs, (ii) chemical modification of bioactive substances (e.g. with fluorescent dyes), and (iii) the activation of carboxyl groups mainly for enzyme immobilization. Applications for drug delivery using click chemistry linking or biodegradable bonds are compared to non-covalent methods based on polymer modified condensed magnetic nanoclusters. Among many challenges, we highlight the specific surface engineering allowing both therapeutic and diagnostic applications (theranostics) of IONPs and magnetic/metallic hybrid nanostructures possessing a huge potential in biocatalysis, green chemistry, magnetic bioseparations and bioimaging. Copyright © 2015 Elsevier Inc. All rights reserved.

Efficient inverted polymer solar cells based on conjugated polyelectrolyte and zinc oxide modified ITO electrode

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Tao; Zhu, Xiaoguang; Tu, Guoli, E-mail: tgl@hust.edu.cn

Efficient inverted polymer solar cells (PSCs) were constructed by utilizing a conjugated polyelectrolyte PF{sub EO}SO{sub 3}Na and zinc oxide to modify the indium tin oxide (ITO) electrode. The ITO electrode modified by PF{sub EO}SO{sub 3}Na and zinc oxide possesses high transparency, increased electron mobility, smoothened surface, and lower work function. PTB7:PC{sub 71}BM inverted PSCs containing the modified ITO electrode achieved a high power conversion efficiency (PCE) of 8.49%, exceeding that of the control device containing a ZnO modified ITO electrode (7.48%). Especially, PCE-10:PC{sub 71}BM inverted polymer solar cells achieved a high PCE up to 9.4%. These results demonstrate a usefulmore » approach to improve the performance of inverted polymer solar cells.« less

Detection of thiopurine methyltransferase activity in lysed red blood cells by means of lab-on-a-chip surface enhanced Raman spectroscopy (LOC-SERS).

PubMed

März, Anne; Mönch, Bettina; Rösch, Petra; Kiehntopf, Michael; Henkel, Thomas; Popp, Jürgen

2011-07-01

In this contribution, the great potential of surface enhanced Raman spectroscopy (SERS) in a lab-on-a-chip (LOC) device for the detection of analyte molecules in a complex environment is demonstrated. Using LOC-SERS, the enzyme activity of thiopurine S-methyltransferase (TPMT) is analysed and identified in lysed red blood cells. The conversion of 6-mercaptopurine to 6-methylmercaptopurine catalysed by TPMT is observed as it gives evidence for the enzyme activity. Being able to determine the TPMT activity before starting a treatment using 6-mercaptopurine, an optimized dosage can be applied to each patient and serious toxicity appearing within thiopurine treatment will be prevented.

Characterization of the receptor-destroying enzyme activity from infectious salmon anaemia virus.

PubMed

Kristiansen, Marianne; Frøystad, Marianne K; Rishovd, Anne Lise; Gjøen, Tor

2002-11-01

Infectious salmon anaemia virus (ISAV) infects cells via the endocytic pathway and, like many other enveloped viruses, ISAV contains a receptor-destroying enzyme. We have analysed this acetylesterase activity with respect to substrate specificity, enzyme kinetics, inhibitors, temperature and pH stability. The ISAV acetylesterase was inhibited by di-isopropyl fluorophosphate (DFP) in a dose-dependent fashion but not by other known hydrolase inhibitors, suggesting that a serine residue is part of the active site. The pH optimum of the enzyme was in the range 7.5-8.0 and the enzymatic activity was lessened at temperatures above 40 degrees C. The effect of DFP on agglutination/elution of erythrocytes by ISAV demonstrated that the acetylesterase activity is the bona fide receptor-destroying enzyme. A haemadsorption assay was used to analyse whether the esterase was active on the surface of infected cells or not.

On-line observation of cell growth in a three-dimensional matrix on surface-modified microelectrode arrays.

PubMed

Lin, Shu-Ping; Kyriakides, Themis R; Chen, Jia-Jin J

2009-06-01

Despite many successful applications of microelectrode arrays (MEAs), typical two-dimensional in-vitro cultures do not project the full scale of the cell growth environment in the three-dimensional (3D) in-vivo setting. This study aims to on-line monitor in-vitro cell growth in a 3D matrix on the surface-modified MEAs with a dynamic perfusion culture system. A 3D matrix consisting of poly(ethylene glycol) hydrogel supplemented with poly-D-lysine was subsequently synthesized in situ on the self-assembled monolayer modified MEAs. FTIR spectrum analysis revealed a peak at 2100 cm(-1) due to the degradation of the structure of the 3D matrix. After 2 wks, microscopic examination revealed that the non-degraded area was around 1500 microm(2) and provided enough space for cell growth. Fluorescence microscopy revealed that the degraded 3D matrix was non-cytotoxic allowing the growth of NIH3T3 fibroblasts and cortical neurons in vitro. Time-course changes of total impedance including resistance and reactance were recorded for 8 days to evaluate the cell growth in the 3D matrix on the MEA. A consistent trend reflecting changes of reactance and total impedance was observed. These in-vitro assays demonstrate that our 3D matrix can construct a biomimetic system for cell growth and analysis of cell surface interactions.

Effect of Surfaces on Amyloid Fibril Formation

PubMed Central

Moores, Bradley; Drolle, Elizabeth; Attwood, Simon J.; Simons, Janet; Leonenko, Zoya

2011-01-01

Using atomic force microscopy (AFM) we investigated the interaction of amyloid beta (Aβ) (1–42) peptide with chemically modified surfaces in order to better understand the mechanism of amyloid toxicity, which involves interaction of amyloid with cell membrane surfaces. We compared the structure and density of Aβ fibrils on positively and negatively charged as well as hydrophobic chemically-modified surfaces at physiologically relevant conditions. We report that due to the complex distribution of charge and hydrophobicity amyloid oligomers bind to all types of surfaces investigated (CH3, COOH, and NH2) although the charge and hydrophobicity of surfaces affected the structure and size of amyloid deposits as well as surface coverage. Hydrophobic surfaces promote formation of spherical amorphous clusters, while charged surfaces promote protofibril formation. We used the nonlinear Poisson-Boltzmann equation (PBE) approach to analyze the electrostatic interactions of amyloid monomers and oligomers with modified surfaces to complement our AFM data. PMID:22016789

Analysis of Lactobacillus sakei Mutants Selected after Adaptation to the Gastrointestinal Tracts of Axenic Mice▿ †

PubMed Central

Chiaramonte, Fabrizio; Anglade, Patricia; Baraige, Fabienne; Gratadoux, Jean-Jacques; Langella, Philippe; Champomier-Vergès, Marie-Christine; Zagorec, Monique

2010-01-01

We recently showed that Lactobacillus sakei, a natural meat-borne lactic acid bacterium, can colonize the gastrointestinal tracts (GIT) of axenic mice but that this colonization in the intestinal environment selects L. sakei mutants showing modified colony morphology (small and rough) and cell shape, most probably resulting from the accumulation of various mutations that confer a selective advantage for persistence in the GIT. In the present study, we analyzed such clones, issued from three different L. sakei strains, in order to determine which functions were modified in the mutants. In the elongated filamentous cells of the rough clones, transmission electron microscopy (TEM) analysis showed a septation defect and dotted and slanted black bands, suggesting the presence of a helical structure around the cells. Comparison of the cytoplasmic and cell wall/membrane proteomes of the meat isolate L. sakei 23K and of one of its rough derivatives revealed a modified expression for 38 spots. The expression of six oxidoreductases, several stress proteins, and four ABC transporters was strongly reduced in the GIT-adapted strain, while the actin-like MreB protein responsible for cell shaping was upregulated. In addition, the expression of several enzymes involved in carbohydrate metabolism was modified, which may correlate with the observation of modified growth of mutants on various carbon sources. These results suggest that the modifications leading to a better adaptation to the GIT are pleiotropic and are characterized in a rough mutant by a different stress status, a cell wall modification, and modified use of energy sources, leading to an improved fitness for the colonization of the GIT. PMID:20208026

A highly sensitive electrochemical biosensor for catechol using conducting polymer reduced graphene oxide-metal oxide enzyme modified electrode.

PubMed

Sethuraman, V; Muthuraja, P; Anandha Raj, J; Manisankar, P

2016-10-15

The fabrication, characterization and analytical performances were investigated for a catechol biosensor, based on the PEDOT-rGO-Fe2O3-PPO composite modified glassy carbon (GC) electrode. The graphene oxide (GO) doped conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT) was prepared through electrochemical polymerization by potential cycling. Reduction of PEDOT-GO was carried out by amperometric method. Fe2O3 nanoparticles were synthesized in ethanol by hydrothermal method. The mixture of Fe2O3, PPO and glutaraldehyde was casted on the PEDOT-rGO electrode. The surface morphology of the modified electrodes was studied by FE-SEM and AFM. Cyclic voltammetric studies of catechol on the enzyme modified electrode revealed higher reduction peak current. Determination of catechol was carried out successfully by Differential Pulse Voltammetry (DPV) technique. The fabricated biosensor investigated shows a maximum current response at pH 6.5. The catechol biosensor exhibited wide sensing linear range from 4×10(-8) to 6.20×10(-5)M, lower detection limit of 7×10(-9)M, current maxima (Imax) of 92.55µA and Michaelis-Menten (Km) constant of 30.48µM. The activation energy (Ea) of enzyme electrode is 35.93KJmol(-1) at 50°C. There is no interference from d-glucose and l-glutamic acid, ascorbic acid and o-nitrophenol. The PEDOT-rGO-Fe2O3-PPO biosensor was stable for at least 75 days when stored in a buffer at about 4°C. Copyright © 2015 Elsevier B.V. All rights reserved.

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

PubMed Central

Chen, Chihchen; Lin, Bo-Ren; Hsu, Min-Yen; Cheng, Chao-Min

2015-01-01

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 μl. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease. PMID:25867034

Explorations in the application of nanotechnology to improve the mechanical properties of composite materials

NASA Astrophysics Data System (ADS)

Yang, Cheng

2007-12-01

This thesis presents the research achievements on the design, preparation, characterization, and analysis of a series of composite materials. By studying the interface interaction of the composite materials using nanotechnology, we developed composite materials that achieve satisfactory mechanical properties in two classes of materials. Durable press (DP) natural textiles are important consumer products usually achieved by erosslinking the molecules in the textiles to achieve long-term wrinkle resistance, which, however, also leads to the simultaneous significant drop of mechanical properties. Herein, a series of polymeric nanoparticl es were investigated, the application of as little as ˜0.14 wt% addition of the nanoparticles improved the mechanical property of the DP cotton fabric by 56% in tearing resistance and 100% in abrasion resistance; the loss in recovery angle is negligible. The author also studied the enzyme-triggered DP treatments of silk fabrics, as a green process method. After the treatment of enzymes, excellent DP property was achieved with improved strain property. Injectable calcium phosphate powder containing acrylic bone cements are widely used in orthopedic surgery to fix artificial prostheses. However, the bending strength is still unsatisfactory. The author modified the surface of the strontium (Sr) containing hydroxyapatite (HA) filler powders with acrylolpamidronate in order to improve the overall mechanical performance of the bone cement composites. By adding 0.25 wt% of acrylolpamidronate to the Sr-HA nanopowders, more than 19% of the bending strength and more than 23% compression strength of the Sr-HA bone cement were improved. Biological evaluations revealed that these bone cement composites were biocompatible and bioactive in cell culture. The results obtained in this thesis work show an effective method to significantly enhance the mechanical properties of composite materials. Different from other available methods, by developing a new series of chemical compounds and nanoparticles, we successfully bound them to the surface or to the constitutional components of the materials through covalent bond. The treatment can enhance and modulate the interface-bonding of the filler materials and enhances the mechanical property of the surface through grafting a thin nano-layer. Since only surface reaction is involved, very small amount of the new material is needed, and the treatment can be readily integrated to the existing processes. The work is instructive in modifying available composite materials to acquire ultra-high mechanical performance.

Oral Delivery of DMAB-Modified Docetaxel-Loaded PLGA-TPGS Nanoparticles for Cancer Chemotherapy

NASA Astrophysics Data System (ADS)

Chen, Hongbo; Zheng, Yi; Tian, Ge; Tian, Yan; Zeng, Xiaowei; Liu, Gan; Liu, Kexin; Li, Lei; Li, Zhen; Mei, Lin; Huang, Laiqiang

2011-12-01

Three types of nanoparticle formulation from biodegradable PLGA-TPGS random copolymer were developed in this research for oral administration of anticancer drugs, which include DMAB-modified PLGA nanoparticles, unmodified PLGA-TPGS nanoparticles and DMAB-modified PLGA-TPGS nanoparticles. Firstly, the PLGA-TPGS random copolymer was synthesized and characterized. DMAB was used to increase retention time at the cell surface, thus increasing the chances of particle uptake and improving oral drug bioavailability. Nanoparticles were found to be of spherical shape with an average particle diameter of around 250 nm. The surface charge of PLGA-TPGS nanoparticles was changed to positive after DMAB modification. The results also showed that the DMAB-modified PLGA-TPGS nanoparticles have significantly higher level of the cellular uptake than that of DMAB-modified PLGA nanoparticles and unmodified PLGA-TPGS nanoparticles. In vitro, cytotoxicity experiment showed advantages of the DMAB-modified PLGA-TPGS nanoparticle formulation over commercial Taxotere® in terms of cytotoxicity against MCF-7 cells. In conclusion, oral chemotherapy by DMAB-modified PLGA-TPGS nanoparticle formulation is an attractive and promising treatment option for patients.

Extracellular vesicles are independent metabolic units with asparaginase activity

PubMed Central

Leonardi, Tommaso; Costa, Ana S. H.; Cossetti, Chiara; Peruzzotti-Jametti, Luca; Bernstock, Joshua D.; Saini, Harpreet K.; Gelati, Maurizio; Vescovi, Angelo Luigi; Bastos, Carlos; Faria, Nuno; Occhipinti, Luigi G.; Enright, Anton J.; Frezza, Christian; Pluchino, Stefano

2017-01-01

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. While it has been shown that cells can traffic metabolic enzymes via EVs much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Both our metabolomics and functional analyses revealed that EVs harbour L-asparaginase activity catalysed by the enzyme Asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC-derived EVs traffic ASRGL1. Our results demonstrate for the first time that NSC EVs function as independent, extracellular metabolic units able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment. PMID:28671681

Extracellular Enzymes Facilitate Electron Uptake in Biocorrosion and Bioelectrosynthesis

PubMed Central

Deutzmann, Jörg S.; Sahin, Merve

2015-01-01

ABSTRACT Direct, mediator-free transfer of electrons between a microbial cell and a solid phase in its surrounding environment has been suggested to be a widespread and ecologically significant process. The high rates of microbial electron uptake observed during microbially influenced corrosion of iron [Fe(0)] and during microbial electrosynthesis have been considered support for a direct electron uptake in these microbial processes. However, the underlying molecular mechanisms of direct electron uptake are unknown. We investigated the electron uptake characteristics of the Fe(0)-corroding and electromethanogenic archaeon Methanococcus maripaludis and discovered that free, surface-associated redox enzymes, such as hydrogenases and presumably formate dehydrogenases, are sufficient to mediate an apparent direct electron uptake. In genetic and biochemical experiments, we showed that these enzymes, which are released from cells during routine culturing, catalyze the formation of H2 or formate when sorbed to an appropriate redox-active surface. These low-molecular-weight products are rapidly consumed by M. maripaludis cells when present, thereby preventing their accumulation to any appreciable or even detectable level. Rates of H2 and formate formation by cell-free spent culture medium were sufficient to explain the observed rates of methane formation from Fe(0) and cathode-derived electrons by wild-type M. maripaludis as well as by a mutant strain carrying deletions in all catabolic hydrogenases. Our data collectively show that cell-derived free enzymes can mimic direct extracellular electron transfer during Fe(0) corrosion and microbial electrosynthesis and may represent an ecologically important but so far overlooked mechanism in biological electron transfer. PMID:25900658

Bio-inspired nanocatalysts for the oxygen reduction reaction.

PubMed

Grumelli, Doris; Wurster, Benjamin; Stepanow, Sebastian; Kern, Klaus

2013-01-01

Electrochemical conversions at fuel cell electrodes are complex processes. In particular, the oxygen reduction reaction has substantial overpotential limiting the electrical power output efficiency. Effective and inexpensive catalytic interfaces are therefore essential for increased performance. Taking inspiration from enzymes, earth-abundant metal centres embedded in organic environments present remarkable catalytic active sites. Here we show that these enzyme-inspired centres can be effectively mimicked in two-dimensional metal-organic coordination networks self-assembled on electrode surfaces. Networks consisting of trimesic acid and bis-pyridyl-bispyrimidine coordinating to single iron and manganese atoms on Au(111) effectively catalyse the oxygen reduction and reveal distinctive catalytic activity in alkaline media. These results demonstrate the potential of surface-engineered metal-organic networks for electrocatalytic conversions. Specifically designed coordination complexes at surfaces inspired by enzyme cofactors represent a new class of nanocatalysts with promising applications in electrocatalysis.

Corrosion resistance and biocompatibility of magnesium alloy modified by alkali heating treatment followed by the immobilization of poly (ethylene glycol), fibronectin and heparin.

PubMed

Pan, Changjiang; Hu, Youdong; Hou, Yu; Liu, Tao; Lin, Yuebin; Ye, Wei; Hou, Yanhua; Gong, Tao

2017-01-01

In recent years, magnesium alloys are attracting more and more attention as a kind of biodegradable metallic biomaterials, however, their uncontrollable biodegradation speed in vivo and the limited surface biocompatibility hinder their clinical applications. In the present study, with the aim of improving the corrosion resistance and biocompatibility, the magnesium alloy (AZ31B) surface was modified by alkali heating treatment followed by the self-assembly of 3-aminopropyltrimethoxysilane (APTMS). Subsequently, poly (ethylene glycol) (PEG) and fibronectin or fibronectin/heparin complex were sequentially immobilized on the modified surface. The results of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed that the above molecules were successfully immobilized on the magnesium alloy surface. An excellent hydrophilic surface was obtained after the alkali heating treatment while the hydrophilicity decreased to some degree after the self-assembly of APTMS, the surface hydrophilicity was gradually improved again after the immobilization of PEG, fibronectin or fibronectin/heparin complex. The corrosion resistance of the control magnesium alloy was significantly improved by the alkali heating treatment. The self-assembly of APTMS and the following immobilization of PEG further enhanced the corrosion resistance of the substrates, however, the grafting of fibronectin or fibronectin/heparin complex slightly lowered the corrosion resistance. As compared to the pristine magnesium alloy, the samples modified by the immobilization of PEG and fibronectin/heparin complex presented better blood compatibility according to the results of hemolysis assay and platelet adhesion as well as the activated partial thromboplastin time (APTT). In addition, the modified substrates had better cytocompatibility to endothelial cells due to the improved anticorrosion and the introduction of fibronectin. The substrates modified by fibronectin or fibronectin/heparin complex can significantly promote endothelial cell adhesion and proliferation. Taking all these results into consideration, the method of the present study can be used for the surface modification of the magnesium alloy to simultaneously impart it better corrosion resistance, favorable blood compatibility and good cytocompatibility to endothelial cells. Copyright © 2016 Elsevier B.V. All rights reserved.

A novel electrochemical biosensor based on polyadenine modified aptamer for label-free and ultrasensitive detection of human breast cancer cells.

PubMed

Wang, Kun; He, Meng-Qi; Zhai, Fu-Heng; He, Rong-Huan; Yu, Yong-Liang

2017-05-01

Simple, rapid, sensitive, and specific detection of cancer cells plays a pivotal role in the diagnosis and prognosis of cancer. A sandwich electrochemical biosensor was developed based on polyadenine (polydA)-aptamer modified gold electrode (GE) and polydA-aptamer functionalized gold nanoparticles/graphene oxide (AuNPs/GO) hybrid for the label-free and selective detection of breast cancer cells (MCF-7) via a differential pulse voltammetry (DPV) technique. Due to the intrinsic affinity between multiple consecutive adenines of polydA sequences and gold, polydA modified aptamer instead of thiol terminated aptamer was immobilized on the surface of GE and AuNPs/GO. The label-free MCF-7 cells could be recognized by polydA-aptamer and self-assembled onto the surface of GE. The polydA-aptamer functionalized AuNPs/GO hybrid could further bind to MCF-7 cells to form a sandwich sensing system. Characterization of the surface modified GE was carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using Fe(CN) 6 3-/4- as a redox probe. Under the optimized experimental conditions, a detection limit of 8 cellsmL -1 (3σ/slope) was obtained for MCF-7 cells by the present electrochemical biosensor, along with a linear range of 10-10 5 cellsmL -1 . By virtue of excellent sensitivity, specificity and repeatability, the present electrochemical biosensor provides a potential application in point-of-care cancer diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

A facile way to fabricate manganese phosphate self-assembled carbon networks as efficient electrochemical catalysts for real-time monitoring of superoxide anions released from HepG2 cells.

PubMed

Cai, Xuan; Shi, Libo; Sun, Wenqian; Zhao, Hongli; Li, Hong; He, Haiyan; Lan, Minbo

2018-04-15

Quantification of superoxide anions (O 2 •- ) is significant in the monitoring of many serious diseases and the design of enzyme-mimic catalysts plays the main role in the development of non-enzymatic O 2 •- sensors. Herein, we proposed a facile self-assembly process to synthesize manganese phosphate modified carbon networks using three kinds of widely-used carbon materials (MWCNTs, NGS and GO) as pillar connectors. Characterizations demonstrate that manganese phosphate is widely dispersed inside and on the surface of carbon networks without visible morphology. Meanwhile, all three kinds of synthesized catalysts were successfully immobilized on the screen-printed carbon electrodes to evaluate the electrochemical performance of fabricated sensors. The results indicate that sensors based on Mn x (PO 4 ) y modified MWCNTs exhibit high sensitivity with an extremely low detection limit of 0.127μM (S/N = 3) and a wide liner range of 0-1.817mM (R 2 = 0.998). We further employed the recommended sensors in the real-time monitoring of HepG2 cells released O 2 •- under the stimulating of Zymosan (20mg/mL). Noticeably, the proposed sensors exhibit not only sensitive response but also stable current steps upon different addition of Zymosan. The calculated concentrations of cell-released O 2 •- vary from 6.772 to 24.652pM cell -1 for the Zymosan amount used in this work. The established novel sensors display low background current and signal noises, thus holding unique advantages in the trace analysis of O 2 •- in biological samples and in vivo environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, Frances H.; Moore, Jeffrey C.

1998-01-01

A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

DOEpatents

Arnold, Frances H.; Moore, Jeffrey C.

1999-01-01

A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

Glycobiology of the cell surface: Its debt to cell electrophoresis 1940-65.

PubMed

Cook, Geoffrey M W

2016-06-01

This Review describes how in the period 1940-1959 cell electrophoresis (in the earlier literature often referred to as 'microelectrophoresis') was used to explore the surface chemistry of cells. Using the erythrocyte as a suitable model for the study of biological membranes, the early investigators were agreed on the presence of negatively charged groups at the surface of this cell. The contemporary dogma was that these were phosphate groups associated with phospholipids. Work in the 1960s, particularly on changes in the electrokinetic properties of erythrocytes following treatment with proteolytic enzymes, lead to the realization that the negatively charged groups at the red cell surface are predominantly due to sialic acids carried on glycoproteins. It quickly became apparent from cell electrophoresis that sialic acids have a ubiquitous presence on the surface of animal cells. This finding required that any complete model of the plasma membrane must include glycosylated molecules at the cell periphery, thus laying the foundations for the field termed 'Glycobiology of the Cell Surface'. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

PubMed

Bi, Xiaodong; Liu, Zhen

2014-12-16

Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

A Strontium-Modified Titanium Surface Produced by a New Method and Its Biocompatibility In Vitro

PubMed Central

Liu, Chundong; Zhang, Yanli; Wang, Lichao; Zhang, Xinhua; Chen, Qiuyue; Wu, Buling

2015-01-01

Objective To present a new and effective method of producing titanium surfaces modified with strontium and to investigate the surface characteristics and in vitro biocompatibility of titanium (Ti) surfaces modified with strontium (Sr) for bone implant applications. Materials and Methods Sr-modified Ti surfaces were produced by sequential treatments with NaOH, strontium acetate, heat and water. The surface characteristics and the concentration of the Sr ions released from the samples were examined. Cell adhesion, morphology and growth were investigated using osteoblasts isolated from the calvaria of neonatal Sprague-Dawley rats. Expression of osteogenesis-related genes and proteins was examined to assess the effect of the Sr-modified Ti surfaces on osteoblasts. Results The modified titanium surface had a mesh structure with significantly greater porosity, and approximately5.37±0.35at.% of Sr was incorporated into the surface. The hydrophilicity was enhanced by the incorporation of Sr ions and water treatment. The average amounts of Sr released from the Sr-modified plates subjected to water treatment were slight higher than the plates without water treatment. Sr promoted cellular adhesion, spreading and growth compared with untreated Ti surfaces. The Sr-modified Ti plates also promoted expression of osteogenesis-related genes,and expression of OPN and COL-І by osteoblasts. Ti plates heat treated at 700°C showed increased bioactivity in comparison with those treated at 600°C. Water treatment upregulated the expression of osteogenesis-related genes. Conclusions These results show that Sr-modification of Ti surfaces may improve bioactivity in vitro. Water treatment has enhanced the response of osteoblasts. The Sr-modified Ti heat-treated at 700°C exhibited better bioactivity compared with that heated at 600°C. PMID:26529234

A Strontium-Modified Titanium Surface Produced by a New Method and Its Biocompatibility In Vitro.

PubMed

Liu, Chundong; Zhang, Yanli; Wang, Lichao; Zhang, Xinhua; Chen, Qiuyue; Wu, Buling

2015-01-01

To present a new and effective method of producing titanium surfaces modified with strontium and to investigate the surface characteristics and in vitro biocompatibility of titanium (Ti) surfaces modified with strontium (Sr) for bone implant applications. Sr-modified Ti surfaces were produced by sequential treatments with NaOH, strontium acetate, heat and water. The surface characteristics and the concentration of the Sr ions released from the samples were examined. Cell adhesion, morphology and growth were investigated using osteoblasts isolated from the calvaria of neonatal Sprague-Dawley rats. Expression of osteogenesis-related genes and proteins was examined to assess the effect of the Sr-modified Ti surfaces on osteoblasts. The modified titanium surface had a mesh structure with significantly greater porosity, and approximately5.37±0.35at.% of Sr was incorporated into the surface. The hydrophilicity was enhanced by the incorporation of Sr ions and water treatment. The average amounts of Sr released from the Sr-modified plates subjected to water treatment were slight higher than the plates without water treatment. Sr promoted cellular adhesion, spreading and growth compared with untreated Ti surfaces. The Sr-modified Ti plates also promoted expression of osteogenesis-related genes,and expression of OPN and COL-І by osteoblasts. Ti plates heat treated at 700°C showed increased bioactivity in comparison with those treated at 600°C. Water treatment upregulated the expression of osteogenesis-related genes. These results show that Sr-modification of Ti surfaces may improve bioactivity in vitro. Water treatment has enhanced the response of osteoblasts. The Sr-modified Ti heat-treated at 700°C exhibited better bioactivity compared with that heated at 600°C.

Enhancing Antitumor Efficacy of Chimeric Antigen Receptor T Cells Through Constitutive CD40L Expression

PubMed Central

Curran, Kevin J; Seinstra, Beatrijs A; Nikhamin, Yan; Yeh, Raymond; Usachenko, Yelena; van Leeuwen, Dayenne G; Purdon, Terence; Pegram, Hollie J; Brentjens, Renier J

2015-01-01

Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40+ tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40+ tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19+ systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy. PMID:25582824

Biodegradable polyester-based microcarriers with modified surface tailored for tissue engineering.

PubMed

Privalova, A; Markvicheva, E; Sevrin, Ch; Drozdova, M; Kottgen, C; Gilbert, B; Ortiz, M; Grandfils, Ch

2015-03-01

Microcarriers have been proposed in tissue engineering, namely for bone, cartilage, skin, vascular, and central nervous system. Although polyester-based microcarriers have been already used for this purpose, their surface properties should be improved to provide better cell growth. The goal of this study was to prepare microbeads based on poly(D,L-lactide) acid, poly(L-lactide) acid, and to study cell behavior (adhesion, spreading, growth, and proliferation) in function of microbead topography and surface chemistry. To improve L-929 fibroblasts adhesion, microbead surface has been modified with three polycations: chitosan, poly(2-dimethylamino ethylmethacrylate) (PDMAEMA), or chitosan-g-oligolactide copolymer (chit-g-OLA). Although modification of the microbead surface with chitosan and PDMAEMA was performed through physical adsorption on the previously prepared microbeads, chit-g-OLA copolymer was introduced directly during microbead processing. This simple approach (1) bypass the use of an emulsifier (polyvinyl alcohol, PVA); (2) avoid surface "contamination" with PVA molecules limiting a control of the surface characteristics. In vitro study of the growth of mouse fibroblasts on the microbeads showed that both surface topography and chemistry affected cell attachment, spreading, and proliferation. Cultivation of L-929 fibroblasts for 7 days resulted in the formation of a 3D cell-scaffold network. © 2014 Wiley Periodicals, Inc.

Altered Calcium Dynamics in Cardiac Cells Grown on Silane-Modified Surfaces

PubMed Central

Ravenscroft-Chang, Melissa S.; Stohlman, Jayna; Molnar, Peter; Natarajan, Anupama; Canavan, Heather E.; Teliska, Maggie; Stancescu, Maria; Krauthamer, Victor; Hickman, J.J.

2013-01-01

Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca2+ because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca2+ imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca2+ concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca2+ changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca2+ dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell-surface interactions in cardiac systems. PMID:19828193

Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle.

PubMed

Gao, Hua-De; Thanasekaran, Pounraj; Chiang, Chao-Wei; Hong, Jia-Lin; Liu, Yen-Chun; Chang, Yu-Hsu; Lee, Hsien-Ming

2015-07-28

Photoactivatable (caged) bioeffectors provide a way to remotely trigger or disable biochemical pathways in living organisms at a desired time and location with a pulse of light (uncaging), but the phototoxicity of ultraviolet (UV) often limits its application. In this study, we have demonstrated the near-infrared (NIR) photoactivatable enzyme platform using protein kinase A (PKA), an important enzyme in cell biology. We successfully photoactivated PKA using NIR to phosphorylate its substrate, and this induced a downstream cellular response in living cells with high spatiotemporal resolution. In addition, this system allows NIR to selectively activate the caged enzyme immobilized on the nanoparticle surface without activating other caged proteins in the cytosol. This NIR-responsive enzyme-nanoparticle system provides an innovative approach to remote-control proteins and enzymes, which can be used by researchers who need to avoid direct UV irradiation or use UV as a secondary channel to turn on a bioeffector.

Proteinase-activated receptor 2 (PAR-2) in gastrointestinal and pancreatic pathophysiology, inflammation and neoplasia.

PubMed

Søreide, Kjetil

2008-08-01

Of all the body systems, the gastrointestinal (GI) tract is the most exposed to proteinases. Proteolytic activity must thus be tightly regulated in the face of diverse environmental challenges, because unrestrained or excessive proteolysis leads to pathological GI conditions. The protease-activated receptor-2 (PAR-2) is expressed in numerous cell types within the GI tract, suggesting both multiple functions and numerous modes of receptor activation. Although best known as a pancreatic digestive enzyme, trypsin has also been found in other tissues and various cancers. Of interest, trypsin and PAR-2 act together in an autocrine loop that promotes proliferation, invasion and metastasis in neoplasia through various mechanisms. Trypsin and PAR-2 seem to act both directly and indirectly through activation of other proteinase cascades, including metalloproteinases. PAR-2 activation can participate in inflammatory reactions, be protective to mucosal surfaces, send or inhibit nociceptive messages, modify gut motility or secretory functions, and stimulate cell proliferation and motility. Several studies point to a role for the PARs in disease processes of the GI tract and pancreas ranging from inflammatory bowel disease, symptoms associated with irritable bowel syndrome, pain in pancreatitis, development of colon and other GI cancers, and even infectious colitis. Proteinases should not only be considered from the traditional view as digestive or degradative enzymes in the gut, but additionally as signalling molecules that actively participate in the spectrum of physiology and diseased states of the GI tract.

Cell-polymer interactions of fluorescent polystyrene latex particles coated with thermosensitive poly(N-isopropylacrylamide) and poly(N-vinylcaprolactam) or grafted with poly(ethylene oxide)-macromonomer.

PubMed

Vihola, Henna; Marttila, Anna-Kaisa; Pakkanen, Jukka S; Andersson, Mirja; Laukkanen, Antti; Kaukonen, Ann Marie; Tenhu, Heikki; Hirvonen, Jouni

2007-10-01

Cell-polymer interactions of thermosensitive poly(N-isopropylacrylamide) (PNIPAM) or poly(N-vinylcaprolactam) (PVCL) coated particles with RAW264.7 macrophages and intestinal Caco-2 cells were evaluated. Nanosized particles were prepared by modifying the surface of fluorescent polystyrene (FPS) particles with the thermosensitive polymer gels or with poly(ethylene oxide) (PEO)-macromonomer grafts. The particles were characterized by IR-spectroscopy for functional groups, light scattering for size distribution and zeta-potential for surface charge. Effects of temperature and polymer coating/grafting on the cellular interactions were evaluated by cell association/uptake and visualized by confocal scanning microscope. PEO and PNIPAM inhibited the polymer-cell contact by steric repulsion, evidenced by weak attachment of the particles. PVCL-coated FPS was adsorbed on the cells more strongly, especially at 37 degrees C, because of more hydrophobic nature at higher temperatures. The results suggest feasibility of the PNIPAM and PVCL for biotechnological/pharmaceutical applications, as the cell-particle interactions may be modified by size, surface charge, hydrophobicity, steric repulsion and temperature.

Nuclear Shield: A Multi-Enzyme Task-Force for Nucleus Protection

PubMed Central

Pallottini, Valentina; Canuti, Lorena; De Canio, Michele; Urbani, Andrea; Marzano, Valeria; Cornetta, Tommaso; Stano, Pasquale; Giovanetti, Anna; Stella, Lorenzo; Canini, Antonella; Federici, Giorgio; Ricci, Giorgio

2010-01-01

Background In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions. Methodology/Principal Findings Electron spectroscopic imaging, zeta potential measurements, isoelectrofocusing, comet assay and mass spectrometry have been used to characterize this surprising structure that is present in the cells of all rat tissues examined (liver, kidney, heart, lung and brain), and that behaves as a “nuclear shield”. In hepatocytes, this hyper-crowding structure is about 300 nm thick, it is mainly formed by cationic enzymes and the local concentration of key protection enzymes, such as glutathione transferase, catalase and glutathione peroxidase is up to seven times higher than in the cytosol. The catalytic activity of these enzymes, when packed in the shield, is not modified and their relative concentrations vary remarkably in different tissues. Removal of this protective shield renders chromosomes more sensitive to damage by oxidative stress. Specific nuclear proteins anchored to the outer nuclear envelope are likely involved in the shield formation and stabilization. Conclusions/Significance The characterization of this previously unrecognized nuclear shield in different tissues opens a new interesting scenario for physiological and protection processes in eukaryotic cells. Selection and accumulation of protection enzymes near sensitive targets represents a new safeguard modality which deeply differs from the adaptive response which is based on expression of specific enzymes. PMID:21170318

Silver-nanoparticles-modified biomaterial surface resistant to staphylococcus: new insight into the antimicrobial action of silver

PubMed Central

Wang, Jiaxing; Li, Jinhua; Guo, Geyong; Wang, Qiaojie; Tang, Jin; Zhao, Yaochao; Qin, Hui; Wahafu, Tuerhongjiang; Shen, Hao; Liu, Xuanyong; Zhang, Xianlong

2016-01-01

Titanium implants are widely used clinically, but postoperative implant infection remains a potential severe complication. The purpose of this study was to investigate the antibacterial activity of nano-silver(Ag)-functionalized Ti surfaces against epidemic Staphylococcus from the perspective of the regulation of biofilm-related genes and based on a bacteria-cell co-culture study. To achieve this goal, two representative epidemic Staphylococcus strains, Staphylococcus epidermidis (S. epidermidis, RP62A) and Staphylococcus aureus (S. aureus, USA 300), were used, and it was found that an Ag-nanoparticle-modified Ti surface could regulate the expression levels of biofilm-related genes (icaA and icaR for S. epidermidis; fnbA and fnbB for S. aureus) to inhibit bacterial adhesion and biofilm formation. Moreover, a novel bacteria-fibroblast co-culture study revealed that the incorporation of Ag nanoparticles on such a surface can help mammalian cells to survive, adhere and spread more successfully than Staphylococcus. Therefore, the modified surface was demonstrated to possess a good anti-infective capability against both sessile bacteria and planktonic bacteria through synergy between the effects of Ag nanoparticles and ion release. This work provides new insight into the antimicrobial action and mechanism of Ag-nanoparticle-functionalized Ti surfaces with bacteria-killing and cell-assisting capabilities and paves the way towards better satisfying the clinical needs. PMID:27599568

A glucose biosensor based on direct electrochemistry of glucose oxidase immobilized on nitrogen-doped carbon nanotubes.

PubMed

Deng, Shengyuan; Jian, Guoqiang; Lei, Jianping; Hu, Zheng; Ju, Huangxian

2009-10-15

A novel biosensor for glucose was prepared by immobilizing glucose oxidase (GOx) on nitrogen-doped carbon nanotubes (CNx-MWNTs) modified electrode. The CNx-MWNTs membrane showed an excellent electrocatalytic activity toward the reduction of O(2) due to its diatomic side-on adsorption on CNx-MWNTs. The nitrogen doping accelerated the electron transfer from electrode surface to the immobilized GOx, leading to the direct electrochemistry of GOx. The biofunctional surface showed good biocompatibility, excellent electron-conductive network and large surface-to-volume ratio, which were characterized by scanning electron microscopy, contact angle and electrochemical impedance technique. The direct electron transfer of immobilized GOx led to stable amperometric biosensing for glucose with a linear range from 0.02 to 1.02 mM and a detection limit of 0.01 mM (S/N=3). These results indicated that CNx-MWNTs are good candidate material for construction of the third-generation enzyme biosensors based on the direct electrochemistry of immobilized enzymes.

Effects of self-assembly of 3-phosphonopropionic acid, 3-aminopropyltrimethoxysilane and dopamine on the corrosion behaviors and biocompatibility of a magnesium alloy.

PubMed

Pan, Chang-Jiang; Hou, Yu; Wang, Ya-Nan; Gao, Fei; Liu, Tao; Hou, Yan-Hua; Zhu, Yu-Fu; Ye, Wei; Wang, Ling-Ren

2016-10-01

Magnesium based alloys are attracting tremendous interests as the novel biodegradable metallic biomaterials. However, the rapid in vivo degradation and the limited surface biocompatibility restrict their clinical applications. Surface modification represents one of the important approaches to control the corrosion rate of Mg based alloys and to enhance the biocompatibility. In the present study, in order to improve the corrosion resistance and surface biocompatibility, magnesium alloy (AZ31B) was modified by the alkali heating treatment followed by the self-assembly of 3-phosphonopropionic acid, 3-aminopropyltrimethoxysilane (APTMS) and dopamine, respectively. The results of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectra (XPS) indicated that the molecules were successfully immobilized on the magnesium alloy surface by the self-assembly. An excellent hydrophilic surface was obtained after the alkali heating treatment and the water contact angle increased to some degree after the self-assembly of dopamine, APTMS and 3-phosphonopropionic acid, however, the hydrophilicity of the modified samples was better than that of the pristine magnesium substrate. Due to the formation of the passivation layer after the alkali heating treatment, the corrosion resistance of the magnesium alloy was obviously improved. The corrosion rate further decreased to varying degrees after the self-assembly surface modification. The blood compatibility of the pristine magnesium was significantly improved after the surface modification. The hemolysis rate was reduced from 56% of the blank magnesium alloy to 18% of the alkali heating treated sample and the values were further reduced to about 10% of dopamine-modified sample and 7% of APTMS-modified sample. The hemolysis rate was below 5% for the 3-phosphonopropionic acid modified sample. As compared to the pristine magnesium alloy, fewer platelets were attached and activated on the modified surfaces and the activated partial thromboplastin times (APTT) were prolonged to some degree. Furthermore, the modified samples showed good cytocompatibility. Endothelial cells exhibited the improved proliferative profiles in terms of CCK-8 assay as compared to those on the pristine magnesium alloy. The modified samples showed better endothelial cell adhesion and spreading than the pristine magnesium alloy. Taking all these results into consideration, the method of this study can be used to modify the magnesium alloy surface to improve the corrosion resistance and biocompatibility simultaneously. Copyright © 2016 Elsevier B.V. All rights reserved.

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses.

PubMed

Pardi, Norbert; Hogan, Michael J; Naradikian, Martin S; Parkhouse, Kaela; Cain, Derek W; Jones, Letitia; Moody, M Anthony; Verkerke, Hans P; Myles, Arpita; Willis, Elinor; LaBranche, Celia C; Montefiori, David C; Lobby, Jenna L; Saunders, Kevin O; Liao, Hua-Xin; Korber, Bette T; Sutherland, Laura L; Scearce, Richard M; Hraber, Peter T; Tombácz, István; Muramatsu, Hiromi; Ni, Houping; Balikov, Daniel A; Li, Charles; Mui, Barbara L; Tam, Ying K; Krammer, Florian; Karikó, Katalin; Polacino, Patricia; Eisenlohr, Laurence C; Madden, Thomas D; Hope, Michael J; Lewis, Mark G; Lee, Kelly K; Hu, Shiu-Lok; Hensley, Scott E; Cancro, Michael P; Haynes, Barton F; Weissman, Drew

2018-06-04

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4 + T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses. © 2018 Pardi et al.

THE E1 PROTEINS

PubMed Central

Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

2013-01-01

E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner. PMID:24029589

Cellular redox homeostasis in endothelial cells treated with nonmodified and Fenton-modified nanodiamond powders.

PubMed

Solarska-Ściuk, K; Gajewska, A; Skolimowski, J; Gajek, A; Bartosz, G

2014-01-01

Diamond nanoparticles find numerous applications in pharmacy, medicine, cosmetics, and biotechnology. However, possible adverse cellular effects of diamond nanoparticle cells have been reported, which may limit their use. The aim of this study was to compare the effect of nonmodified diamond nanoparticles (D) and diamond nanoparticles modified by the Fenton reaction (D+OH) on human umbilical cord endothelial cells (HUVEC-ST). We found that both D and D+OH show time- and concentration-dependent cytotoxicity, inducing apoptosis and necrosis of HUVEC-ST. Interaction with D and D+OH also induced changes in the production of reactive oxygen and nitrogen species and changes in the level of glutathione and activities of antioxidant enzymes in the cells. These data demonstrate that diamond nanoparticles may induce oxidative stress in human endothelial cells, which contributes to their cytotoxic effects seen at higher concentrations of D and D+OH. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

PREditOR: a synthetic biology approach to removing heterochromatin from cells.

PubMed

Molina, Oscar; Carmena, Mar; Maudlin, Isabella E; Earnshaw, William C

2016-12-01

It is widely accepted that heterochromatin is necessary to maintain genomic stability. However, direct experimental evidence supporting this is slim. Previous studies using either enzyme inhibitors, gene knockout or knockdown studies all are subject to the caveat that drugs may have off-target effects and enzymes that modify chromatin proteins to support heterochromatin formation may also have numerous other cellular targets as well. Here, we describe PREditOR (protein reading and editing of residues), a synthetic biology approach that allows us to directly remove heterochromatin from cells without either drugs or global interference with gene function. We find that removal of heterochromatin perturbs mitotic progression and causes a dramatic increase in chromosome segregation defects, possibly as a result of interfering with the normal centromeric localization of the chromosomal passenger complex.

[Modification of L-asparaginase with colominic acid and the new characteristics of the modified enzyme].

PubMed

Wang, Y D; Guo, L; Qian, S J; Meng, G Z; Zhang, S Z

2000-07-01

The colominic acid was covalently coupled to L-asparaginase molecule by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (30:1, 50:1 and 100:1), a modified enzyme molecule contained 4.7, 7.2 and 12 colominic acid molecule, they retained 58%, 56% and 33.2% of the initial asparaginase activity, respectively. In comparison with the native enzyme, modified enzyme had lower immunogenicity and antigenicity, longer half-life time (in vitro), more resistance ability to trypsin proteolysis, and similar Km value for L-asparagine.

Microbial surface displayed enzymes based biofuel cell utilizing degradation products of lignocellulosic biomass for direct electrical energy.

PubMed

Fan, Shuqin; Hou, Chuantao; Liang, Bo; Feng, Ruirui; Liu, Aihua

2015-09-01

In this work, a bacterial surface displaying enzyme based two-compartment biofuel cell for the direct electrical energy conversion from degradation products of lignocellulosic biomass is reported. Considering that the main degradation products of the lignocellulose are glucose and xylose, xylose dehydrogenase (XDH) displayed bacteria (XDH-bacteria) and glucose dehydrogenase (GDH) displayed bacteria (GDH-bacteria) were used as anode catalysts in anode chamber with methylene blue as electron transfer mediator. While the cathode chamber was constructed with laccase/multi-walled-carbon nanotube/glassy-carbon-electrode. XDH-bacteria exhibited 1.75 times higher catalytic efficiency than GDH-bacteria. This assembled enzymatic fuel cell exhibited a high open-circuit potential of 0.80 V, acceptable stability and energy conversion efficiency. Moreover, the maximum power density of the cell could reach 53 μW cm(-2) when fueled with degradation products of corn stalk. Thus, this finding holds great potential to directly convert degradation products of biomass into electrical energy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response

DOE PAGES

Tsai, Alex Yi-Lin; Chan, Kin; Ho, Chi-Yip; ...

2017-03-02

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to themore » hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses.« less

Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers transcriptional patterns related to biotic stress and defense response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsai, Alex Yi-Lin; Chan, Kin; Ho, Chi-Yip

The plant cell wall is an abundant and renewable resource for lignocellulosic applications such as the production of biofuel. Due to structural and compositional complexities, the plant cell wall is, however, recalcitrant to hydrolysis and extraction of platform sugars. A cell wall engineering strategy to reduce this recalcitrance makes use of microbial cell wall modifying enzymes that are expressed directly in plants themselves. Previously, we constructed transgenic Arabidopsis thaliana constitutively expressing the fungal hemicellulases: Phanerochaete carnosa glucurnoyl esterase (PcGCE) and Aspergillus nidulans α-arabinofuranosidase (AnAF54). While the PcGCE lines demonstrated improved xylan extractability, they also displayed chlorotic leaves leading to themore » hypothesis that expression of such enzymes in planta resulted in plant stress. The objective of this study is to investigate the impact of transgenic expression of the aforementioned microbial hemicellulases in planta on the host arabidopsis. More specifically, we investigated transcriptome profiles by short read high throughput sequencing (RNAseq) from developmentally distinct parts of the plant stem. When compared to non-transformed wild-type plants, a subset of genes was identified that showed differential transcript abundance in all transgenic lines and tissues investigated. Intriguingly, this core set of genes was significantly enriched for those involved in plant defense and biotic stress responses. While stress and defense-related genes showed increased transcript abundance in the transgenic plants regardless of tissue or genotype, genes involved in photosynthesis (light harvesting) were decreased in their transcript abundance potentially reflecting wide-spread effects of heterologous microbial transgene expression and the maintenance of plant homeostasis. Additionally, an increase in transcript abundance for genes involved in salicylic acid signaling further substantiates our finding that transgenic expression of microbial cell wall modifying enzymes induces transcriptome responses similar to those observed in defense responses.« less

Chitosan Mediates Germling Adhesion in Magnaporthe oryzae and Is Required for Surface Sensing and Germling Morphogenesis

PubMed Central

Geoghegan, Ivey A.; Gurr, Sarah J.

2016-01-01

The fungal cell wall not only plays a critical role in maintaining cellular integrity, but also forms the interface between fungi and their environment. The composition of the cell wall can therefore influence the interactions of fungi with their physical and biological environments. Chitin, one of the main polysaccharide components of the wall, can be chemically modified by deacetylation. This reaction is catalyzed by a family of enzymes known as chitin deacetylases (CDAs), and results in the formation of chitosan, a polymer of β1,4-glucosamine. Chitosan has previously been shown to accumulate in the cell wall of infection structures in phytopathogenic fungi. Here, it has long been hypothesized to act as a 'stealth' molecule, necessary for full pathogenesis. In this study, we used the crop pathogen and model organism Magnaporthe oryzae to test this hypothesis. We first confirmed that chitosan localizes to the germ tube and appressorium, then deleted CDA genes on the basis of their elevated transcript levels during appressorium differentiation. Germlings of the deletion strains showed loss of chitin deacetylation, and were compromised in their ability to adhere and form appressoria on artificial hydrophobic surfaces. Surprisingly, the addition of exogenous chitosan fully restored germling adhesion and appressorium development. Despite the lack of appressorium development on artificial surfaces, pathogenicity was unaffected in the mutant strains. Further analyses demonstrated that cuticular waxes are sufficient to over-ride the requirement for chitosan during appressorium development on the plant surface. Thus, chitosan does not have a role as a 'stealth' molecule, but instead mediates the adhesion of germlings to surfaces, thereby allowing the perception of the physical stimuli necessary to promote appressorium development. This study thus reveals a novel role for chitosan in phytopathogenic fungi, and gives further insight into the mechanisms governing appressorium development in M.oryzae. PMID:27315248

A synthetic biochemistry platform for cell free production of monoterpenes from glucose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korman, Tyler P.; Opgenorth, Paul H.; Bowie, James U.

Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. We design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose.more » We also obtain conversion yields 495% and titres 415 g l -1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals.« less

A synthetic biochemistry platform for cell free production of monoterpenes from glucose

DOE PAGES

Korman, Tyler P.; Opgenorth, Paul H.; Bowie, James U.

2017-05-24

Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. We design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose.more » We also obtain conversion yields 495% and titres 415 g l -1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals.« less

Mold Pectinase Modified with Dialdehyde Derivatives of Dextran and Cellulose.

PubMed

Kobayashi, M; Chiba, Y; Funane, K; Ohya, S; Kato, Y

1996-01-01

Chemical modification of mold pectinase with dextran- and cellulose-dialdehydes was examined to improve the enzyme characteristics. The modified pectinase with dextran-dialdehyde retained about 50% of the original activity, and more than 80% of the total amino groups were modified. HPLC gel filtration analysis showed an increase in molecular weight of the reaction product. Reaction with cellulose-dialdehyde provided an immobilized form of pectinase. The immobilized pectinase was resistant to both acidic and alkaline pHs, and also acquired heat stability at 60°C. The optimum pH of the modified enzyme shifted from pH 4.5 to 5.0-5.5, and this enzyme had higher activity at neutral pH regions than the native enzyme. A rather low recovery of immobilized enzyme (14.5%) should be improved by the combination with various methods hitherto established.

Protective effects of melatonin-loaded lipid-core nanocapsules on paraquat-induced cytotoxicity and genotoxicity in a pulmonary cell line.

PubMed

Charão, Mariele F; Baierle, Marília; Gauer, Bruna; Goethel, Gabriela; Fracasso, Rafael; Paese, Karina; Brucker, Natália; Moro, Angela M; Bubols, Guilherme B; Dias, Bruna B; Matte, Ursula S; Guterres, Silvia S; Pohlmann, Adriana R; Garcia, Solange C

2015-06-01

Many acute poisonings lack effective and specific antidotes. Due to both intentional and accidental exposures, paraquat (PQ) causes thousands of deaths annually, especially by pulmonary fibrosis. Melatonin (Mel), when incorporated into lipid-core nanocapsules (Mel-LNC), has enhanced antioxidant properties. The effects of such a formulation have not yet been studied with respect to mitigation of PQ- induced cytotoxicity and DNA damage. Here, we have tested whether Mel-LNC can ameliorate PQ-induced toxicity in the A549 alveolar epithelial cell line. Physicochemical characterization of the formulations was performed. Cellular uptake was measured using nanocapsules marked with rhodamine B. Cell viability was determined by the MTT assay and DNA damage was assessed by the comet assay. The enzyme-modified comet assay with endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) were used to investigate oxidative DNA damage. Incubation with culture medium for 24h did not alter the granulometric profile of Mel-LNC formulations. Following treatment (3 and 24h), red fluorescence was detected around the cell nucleus, indicating internalization of the formulation. Melatonin solution (Mel), Mel-LNC, and LNC did not have significant effects on cell viability or DNA damage. Pre-treatment with Mel-LNC enhanced cell viability and showed a remarkable reduction in % DNA in tail compared to the PQ group; this was not observed in cells pre-treated with Mel. PQ induces oxidative DNA damage detected with the enzyme-modified comet assay. Mel-LNC reduced this damage more effectively than did Mel. In summary, Mel-LNC is better than Mel at protecting A549 cells from the cytotoxic and genotoxic effects of PQ. Copyright © 2015 Elsevier B.V. All rights reserved.

Accumulation of N-Acetylglucosamine Oligomers in the Plant Cell Wall Affects Plant Architecture in a Dose-Dependent and Conditional Manner1[W][OPEN

PubMed Central

Vanholme, Bartel; Vanholme, Ruben; Turumtay, Halbay; Goeminne, Geert; Cesarino, Igor; Goubet, Florence; Morreel, Kris; Rencoret, Jorge; Bulone, Vincent; Hooijmaijers, Cortwa; De Rycke, Riet; Gheysen, Godelieve; Ralph, John; De Block, Marc; Meulewaeter, Frank; Boerjan, Wout

2014-01-01

To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of plants, N-ACETYLGLUCOSAMINYLTRANSFERASE (NodC) of Azorhizobium caulinodans was expressed in Arabidopsis (Arabidopsis thaliana). The corresponding enzyme catalyzes the polymerization of GlcNAc and, accordingly, β-1,4-GlcNAc oligomers accumulated in the plant. A phenotype characterized by difficulties in developing an inflorescence stem was visible when plants were grown for several weeks under short-day conditions before transfer to long-day conditions. In addition, a positive correlation between the oligomer concentration and the penetrance of the phenotype was demonstrated. Although NodC overexpression lines produced less cell wall compared with wild-type plants under nonpermissive conditions, no indications were found for changes in the amount of the major cell wall polymers. The effect on the cell wall was reflected at the transcriptome level. In addition to genes encoding cell wall-modifying enzymes, a whole set of genes encoding membrane-coupled receptor-like kinases were differentially expressed upon GlcNAc accumulation, many of which encoded proteins with an extracellular Domain of Unknown Function26. Although stress-related genes were also differentially expressed, the observed response differed from that of a classical chitin response. This is in line with the fact that the produced chitin oligomers were too small to activate the chitin receptor-mediated signal cascade. Based on our observations, we propose a model in which the oligosaccharides modify the architecture of the cell wall by acting as competitors in carbohydrate-carbohydrate or carbohydrate-protein interactions, thereby affecting noncovalent interactions in the cell wall or at the interface between the cell wall and the plasma membrane. PMID:24664205

Simple route for nano-hydroxyapatite properties expansion.

PubMed

Rojas, L; Olmedo, H; García-Piñeres, A J; Silveira, C; Tasic, L; Fraga, F; Montero, M L

2015-10-20

Simple surface modification of nano-hydroxyapatite, through acid-basic reactions, allows expanding the properties of this material. Introduction of organic groups such as hydrophobic alkyl chains, carboxylic acid, and amide or amine basic groups on the hydroxyapatite surface systematically change the polarity, surface area, and reactivity of hydroxyapatite without modifying its phase. Physical and chemical properties of the new derivative particles were analyzed. The biocompatibility of modified Nano-Hap on Raw 264.7 cells was also assessed.

Morphological investigations of cells that adhered to the irregular patterned polydimethylsiloxane (PDMS) surface without reagents.

PubMed

Chung, Sung Hee; Min, Junhong

2009-07-01

Polydimethylsiloxane (PDMS) surface consisting irregular pattern was investigated to develop cell-based biochip using PDMS. PDMS surface was modified with nano- and micro-combined patterns using surface deformation technology. Hydrophobicity of nano-patterned PDMS surface was sustained. Nevertheless it has irregular patterns consisting of micro- and nano-patterns. According to atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy results by immunostaining method, human mammary epithelial cells (HMEC) adhered well on irregularly patterned surface without any reagents such as gelatin and collagen, compared to commercial culture dish. It implies PDMS material can be utilized as template for cell-based biochip without any reagents.

Effect of the cancer specific shorter form of human 6-phosphofructo-1-kinase on the metabolism of the yeast Saccharomyces cerevisiae.

PubMed

Andrejc, Darjan; Možir, Alenka; Legiša, Matic

2017-05-08

At first glance, there appears to be a high degree of similarity between the metabolism of yeast (the Crabtree effect) and human cancer cells (the Warburg effect). At the root of both effects is accelerated metabolic flow through glycolysis which leads to overflows of ethanol and lactic acid, respectively. It has been proposed that enhanced glycolytic flow in cancer cells is triggered by the altered kinetic characteristics of the key glycolytic regulatory enzyme 6-phosphofructo-1-kinase (Pfk). Through a posttranslational modification, highly active shorter Pfk-M fragments, which are resistant to feedback inhibition, are formed after the proteolytic cleavage of the C-terminus of the native human Pfk-M. Alternatively, enhanced glycolysis is triggered by optimal growth conditions in the yeast Saccharomyces cerevisiae. To assess the deregulation of glycolysis in yeast cells, the sfPFKM gene encoding highly active human shorter Pfk-M fragments was introduced into pfk-null S. cerevisiae. No growth of the transformants with the sfPFKM gene was observed on glucose and fructose. Glucose even induced rapid deactivation of Pfk1 activities in such transformants. However, Pfk1 activities of the sfPFKM transformants were detected in maltose medium, but the growth in maltose was possible only after the addition of 10 mM of ethanol to the medium. Ethanol seemed to alleviate the severely unbalanced NADH/NADPH ratio in the sfPFKM cells. However, the transformants carrying modified Pfk-M enzymes grew faster than the transformants with the human native human Pfk-M enzyme in a narrow ecological niche with a low maltose concentration medium that was further improved by additional modifications. Interestingly, periodic extracellular accumulation of phenylacetaldehyde was detected during the growth of the strain with modified Pfk-M but not with the strain encoding the human native enzyme. Highly active cancer-specific shorter Pfk-M fragments appear to trigger several controlling mechanisms in the primary metabolism of yeast S. cerevisiae cells. These results suggest more complex metabolic regulation is present in S. cerevisiae as free living unicellular eukaryotic organisms in comparison to metazoan human cells. However, increased productivity under broader growth conditions may be achieved if more gene engineering is performed to reduce or omit several controlling mechanisms.

Cell surface engineering of Saccharomyces cerevisiae combined with membrane separation technology for xylitol production from rice straw hydrolysate.

PubMed

Guirimand, Gregory; Sasaki, Kengo; Inokuma, Kentaro; Bamba, Takahiro; Hasunuma, Tomohisa; Kondo, Akihiko

2016-04-01

Xylitol, a value-added polyol deriving from D-xylose, is widely used in both the food and pharmaceutical industries. Despite extensive studies aiming to streamline the production of xylitol, the manufacturing cost of this product remains high while demand is constantly growing worldwide. Biotechnological production of xylitol from lignocellulosic waste may constitute an advantageous and sustainable option to address this issue. However, to date, there have been few reports of biomass conversion to xylitol. In the present study, xylitol was directly produced from rice straw hydrolysate using a recombinant Saccharomyces cerevisiae YPH499 strain expressing cytosolic xylose reductase (XR), along with β-glucosidase (BGL), xylosidase (XYL), and xylanase (XYN) enzymes (co-)displayed on the cell surface; xylitol production by this strain did not require addition of any commercial enzymes. All of these enzymes contributed to the consolidated bioprocessing (CBP) of the lignocellulosic hydrolysate to xylitol to produce 5.8 g/L xylitol with 79.5 % of theoretical yield from xylose contained in the biomass. Furthermore, nanofiltration of the rice straw hydrolysate provided removal of fermentation inhibitors while simultaneously increasing sugar concentrations, facilitating high concentration xylitol production (37.9 g/L) in the CBP. This study is the first report (to our knowledge) of the combination of cell surface engineering approach and membrane separation technology for xylitol production, which could be extended to further industrial applications.

Mapping the conformational space accessible to BACE2 using surface mutants and cocrystals with Fab fragments, Fynomers and Xaperones.

PubMed

Banner, David W; Gsell, Bernard; Benz, Jörg; Bertschinger, Julian; Burger, Dominique; Brack, Simon; Cuppuleri, Simon; Debulpaep, Maja; Gast, Alain; Grabulovski, Dragan; Hennig, Michael; Hilpert, Hans; Huber, Walter; Kuglstatter, Andreas; Kusznir, Eric; Laeremans, Toon; Matile, Hugues; Miscenic, Christian; Rufer, Arne C; Schlatter, Daniel; Steyaert, Jan; Stihle, Martine; Thoma, Ralf; Weber, Martin; Ruf, Armin

2013-06-01

The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic β-cells, which leads to inactivation of the β-cell proliferating activity of Tmem27. This role of BACE2 in the control of β-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme β-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.

Fundamental and Applied Studies of Polymer Membranes

NASA Astrophysics Data System (ADS)

Imbrogno, Joseph

Four major areas have been studied in this research: 1) synthesizing novel monomers, e.g. chiral monomers, to produce new types of functionalized membranes for the biotechnology and pharmaceutical industries, 2) hydrophobic brush membranes for desalinating brackish water, sea water, and separating organics, 3) fundamental studies of water interactions at surfaces using sum frequency generation (SFG), and 4) discovering new surface chemistries that will control the growth and differentiation of stem cells. We have developed a novel synthesis method in order to increase the breadth of our high throughput screening library. This library was generated using maleimide chemistry to react a common methacrylate linker with a variety of different functions groups (R groups) in order to form new monomers that were grafted from the surface of PES ultrafiltration membranes. From this work, we discovered that the chirality of a membrane can affect performance when separating chiral feed streams. This effect was observed when filtering bovine serum albumin (BSA) and ovalbumin in a high salt phosphate buffered saline (PBS, 150 mM salt). The Phe grafted membranes showed a large difference in performance when filtering BSA with selectivity of 1.13 and 1.00 for (S) and (R) Phe, respectively. However, when filtering ovalbumin, the (S) and (R) modified surfaces showed selectivity of 2.06 and 2.31, respectively. The higher selectivity enantiomer switched for the two different proteins. Permeability when filtering BSA was 3.06 LMH kPa-1 and 4.31 LMH kPa -1 for (S)- and (R)- Phe, respectively, and 2.65 LMH kPa -1 and 2.10 LMH kPa-1 when filtering ovalbumin for (S)- and (R)- Phe, respectively. Additionally, these effects were no longer present when using a low salt phosphate buffer (PB, 10 mM salt). Since, to our knowledge, membrane chirality is not considered in current industrial systems, this discovery could have a large impact on the pharmaceutical and biotechnology industries. We have developed hydrophobic brush membranes that were able to selectively separate valuable organics (isobutanol) from water, while rejecting other undesirable species, such as enzymes, using pervaporation (PV). These membranes (grafted from nanofiltration (NF) support membranes) had a selectivity ˜1.5x higher than the current industrial standard, polydimethylsiloxane (PDMS), with alpha = 10.1 +/- 0.9 for our brush membranes and alpha = 6.7 +/- 0.1 for PDMS membranes. Since the mechanism of pervaporation is based on the solution diffusion (SD) model, these membranes may be used to desalinate water or fractionate gases since they are also based on the SD mechanism. We have discovered that hydrophobic brush membranes are able to reject monovalent salt ions. This type of membrane is analogous to carbon nanotubes (CNTs), which are believed to have extremely high water fluxes through them due to near frictionless flow caused by a lack of hydrogen bonding. Using these brush membranes we were able to achieve 42% monovalent (NaCl) salt rejection of simulated seawater (32,000 ppm salt). These membranes are easier to scale-up than current composite membranes produced using interfacial polymerization. We have been using SFG to study interfacial water on membrane surfaces. We believe that water interactions with the membrane surface and with the feed species, e.g. proteins, play a critical role during the fouling process. Relevant buffers, such as phosphate buffered saline (PBS) and phosphate buffer, contain ions that are known to restructure water at interfaces. Sum frequency generation spectroscopy (SFG) was used to characterize interfacial water structure at poly(ether sulfone) (PES) thin films in the presence of 0.01 M phosphate buffer (low salt) and 0.01 M phosphate buffered saline (high salt). Three model surfaces were studied: unmodified PES, hydrophobic alkane (C18) modified PES, and poly(ethylene glycol) (PEG) modified PES. In the presence of the low salt phosphate buffer (10 mM salt), phosphate anions were excluded from the PEG-modified PES film. This led to a charge separation between the phosphate anions and sodium cations, creating a surface potential which strongly ordered water molecules into the bulk. When using high salt PBS (138 mM salt) the sodium chloride ions screened this charge and reduced water ordering. Interestingly, this effect was the greatest for the PEG modified surface, with minor or no effects observed for the C18 modified PES and unmodified PES, respectively. Using our high throughput screening platform, we were able to determine that (N-[3-(dimethylamino)propyl] methacrylamide), DMAPMA, supported strong attachment and long-term self-renewal of mouse embryonic stem (ES) cells while preventing differentiation (maintaining pluripotency). After developing this platform, it was used to screen for a surface that could instead induce differentiation of bovine and human retinal pigment epithelium (RPE) cells while promoting cell growth. Several PEG based surfaces were able to induce cobblestone morphology of the RPE cells, which is indicative of differentiation. (Abstract shortened by UMI.).

The ROS-sensitive microRNA-9/9* controls the expression of mitochondrial tRNA-modifying enzymes and is involved in the molecular mechanism of MELAS syndrome.

PubMed

Meseguer, Salvador; Martínez-Zamora, Ana; García-Arumí, Elena; Andreu, Antonio L; Armengod, M-Eugenia

2015-01-01

Mitochondrial dysfunction activates mitochondria-to-nucleus signaling pathways whose components are mostly unknown. Identification of these components is important to understand the molecular mechanisms underlying mitochondrial diseases and to discover putative therapeutic targets. MELAS syndrome is a rare neurodegenerative disease caused by mutations in mitochondrial (mt) DNA affecting mt-tRNA(Leu(UUR)). Patient and cybrid cells exhibit elevated oxidative stress. Moreover, mutant mt-tRNAs(Leu(UUR)) lack the taurine-containing modification normally present at the wobble uridine (U34) of wild-type mt-tRNA(Leu(UUR)), which is considered an etiology of MELAS. However, the molecular mechanism is still unclear. We found that MELAS cybrids exhibit a significant decrease in the steady-state levels of several mt-tRNA-modification enzymes, which is not due to transcriptional regulation. We demonstrated that oxidative stress mediates an NFkB-dependent induction of microRNA-9/9*, which acts as a post-transcriptional negative regulator of the mt-tRNA-modification enzymes GTPBP3, MTO1 and TRMU. Down-regulation of these enzymes by microRNA-9/9* affects the U34 modification status of non-mutant tRNAs and contributes to the MELAS phenotype. Anti-microRNA-9 treatments of MELAS cybrids reverse the phenotype, whereas miR-9 transfection of wild-type cells mimics the effects of siRNA-mediated down-regulation of GTPBP3, MTO1 and TRMU. Our data represent the first evidence that an mt-DNA disease can directly affect microRNA expression. Moreover, we demonstrate that the modification status of mt-tRNAs is dynamic and that cells respond to stress by modulating the expression of mt-tRNA-modifying enzymes. microRNA-9/9* is a crucial player in mitochondria-to-nucleus signaling as it regulates expression of nuclear genes in response to changes in the functional state of mitochondria. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Biological Behavior of Osteoblast Cell and Apatite Forming Ability of the Surface Modified Ti Alloys.

PubMed

Zhao, Jingming; Hwang, K H; Choi, W S; Shin, S J; Lee, J K

2016-02-01

Titanium as one kind of biomaterials comes in direct contact with the body, making evaluation of biocompatibility an important aspect to biomaterials development. Surface chemistry of titanium plays an important role in osseointegration. Different surface modification alters the surface chemistry and result in different biological response. In this study, three kinds of mixed acid solutions were used to treat Ti specimens to induce Ca-P formation. Following a strong mixed acid activation process, Ca-P coating successfully formed on the Ti surfaces in simulated body fluid. Strong mixed acid increased the roughness of the metal surface, because the porous and rough surface allows better adhesion between Ca-P coatings and substrates. After modification of titanium surface by mixed acidic solution and subsequently H2O2/HCL treatment evaluation of biocompatibility was conducted from hydroxyapatite formation by biomimetic process and cell viability on modified titanium surface. Nano-scale modification of titanium surfaces can alter cellular and tissue responses, which may benefit osseointegration and dental implant therapy. Results from this study indicated that surface treatment methods affect the surface morphology, type of TiO2 layer formed and subsequent apatite deposition and biological responses. The thermo scientific alamarblue cell viability assay reagent is used to quantitatively measure the viability of mammalian cell lines, bacteria and fungi by incorporating a rapid, sensitive and reliable fluorometric/colorimetric growth indicator, without any toxic and side effect to cell line. In addition, mixed acid treatment uses a lower temperature and shorter time period than widely used alkali treatment.

Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

NASA Technical Reports Server (NTRS)

Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

2003-01-01

Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

PubMed

Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

2014-03-20

Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of method of detachment on electrophoretic mobility of mammalian cells grown in monolayer culture

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

A variety of proteolytic and micolytic enzumes, mechanical procedures, and changes in the ionic environment, especially Ca chelation, are used for dispersal of monolayer grown cells. If either chelating agents or mechanical dispersion are used alone, the cell yield is often low and suspensions of single cells are difficult to obtain. Confluent monolayers treated with EDTA tend to be released from their surfaces in sheets, and clumps of cells remain even after further incubation in EDTA. Crude trypsin is the most popular dispersal agent and is known to contain a variety of contaminating enzymes which contribute to the dispersal of cells. A variety of cell injuries resulting from the activity of proteolytic enzymes are reported. It is shown that crystalline trypsin is least harmful to cell integrity as judged by trypan blue uptake.

Modification of a single tryptophan residue in human Cu,Zn-superoxide dismutase by peroxynitrite in the presence of bicarbonate.

PubMed

Yamakura, F; Matsumoto, T; Fujimura, T; Taka, H; Murayama, K; Imai, T; Uchida, K

2001-07-09

Human recombinant Cu,Zn-SOD was reacted with peroxynitrite in a reaction mixture containing 150 mM potassium phosphate buffer (pH 7.4) 25 mM sodium bicarbonate, and 0.1 mM diethylenetriamine pentaacetic acid. Disappearance of fluorescence emission at 350 nm, which could be attributed to modification of a single tryptophan residue, was observed in the modified enzyme with a pH optimum of around 8.4. A fluorescence decrease with the same pH optimum was also observed without sodium bicarbonate, but with less efficiency. Amino acid contents of the modified enzyme showed no significant difference in all amino acids except the loss of a single tryptophan residue of the enzyme. The peroxynitrite-modified enzyme showed an increase in optical absorption around 350 nm and 30% reduced enzyme activity based on the copper contents. The modified enzyme showed the same electron paramagnetic resonance spectrum as that of the control enzyme. The modified Cu,Zn-SOD showed a single protein band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and five protein bands in non-denaturing PAGE. From this evidence, we conclude that nitration and/or oxidation of the single tryptophan 32 and partial inactivation of the enzyme activity of Cu,Zn-SOD is caused by a peroxynitrite-carbon dioxide adduct without perturbation of the active site copper integrity.

Rod outer segment-associated N-acetylgalactosaminylphosphotransferase.

PubMed

Sweatt, A J; Balsamo, J; Lilien, J

1995-01-01

To determine the exact location of a cell surface glycosyltransferase (N-acetylgalactosaminylphosphotransferase, (GalNAcPTase) immunochemically identified in mammalian rod outer segments (ROS), to determine whether anti-GalNAcPTase antibody recognizes retinal molecules that possess transferase activity and to characterize ROS transferase enzyme activity and acceptors. The GalNAcPTase is known to be associated with the adhesion molecule N-cadherin in embryonic avian retinas and with E-cadherin in mammalian pancreatic islet cells. Purified, fixed ROS were reacted with anti-chick GalNAcPTase antibody followed by secondary antibody conjugated to colloidal gold and were examined by electron microscopy. Fractions of retinal and ROS proteins enriched in the transferase were obtained through batch adsorption on Sepharose, separated by gel electrophoresis, transferred to nitrocellulose, and either reacted with anti-GalNAcPTase antibody or assayed for transferase activity. Interphotoreceptor matrix (IPM) was examined for the presence of immunoreactive GalNAcPTase by gel electrophoresis and immunoblot. The kinetics and endogenous acceptors of the cow ROS transferase were characterized. ROS are specifically labeled by anti-GalNAcPTase antibody at the cell surface. The immunogold label was associated with the cell surface and with flocculent material adherent to the cell surface. In addition, soluble and particulate fractions of the IPM showed GalNAcPTase-like immunoreactivity. The transferase appears as single immunoreactive band at or near 220 kd. Transferase enzyme activity was present at this position on Western transfers of retinal and ROS proteins. In whole ROS, transferase activity was directed toward endogenous acceptors of very high molecular mass. The GalNAcPTase is localized on ROS in association with the cell surface and with components of the IPM. The molecule recognized by the anti-GalNAcPTase antibody possesses transferase activity toward itself and a few other proteins, but mostly toward very large molecules that may be IPM proteoglycans. It is not yet known whether the enzyme of the adult retina specifically transfers sugar or sugar-phosphate groups to its acceptors. It is proposed that the ROS GalNAcPTase is involved in the modulation of adhesive phenomena between or within photoreceptors or between photoreceptors and the interphotoreceptor matrix.

Surface modification for interaction study with bacteria and preosteoblast cells

NASA Astrophysics Data System (ADS)

Song, Qing

Surface modification plays a pivotal role in bioengineering. Polymer coatings can provide biocompatibility and biofunctionalities to biomaterials through surface modification. In this dissertation, initiated chemical vapor deposition (iCVD) was utilized to coat two-dimensional (2D) and three-dimensional (3D) substrates with differently charged polyelectrolytes in order to generate antimicrobial and osteocompatible biomaterials. ICVD is a modified CVD technique that enables surface modification in an all-dry condition without substrate damage and solvent contamination. The free-radical polymerization allows the vinyl polymers to conformally coat on various micro- and nano-structured substrates and maintains the delicate structure of the functional groups. The vapor deposition of polycations provided antimicrobial activity to planar and porous substrates through destroying the negatively charged bacterial membrane and brought about high contact-killing efficiency (99.99%) against Gram-positive Bacillus subtilis and Gram-negative Escherichia coli. Additionally, the polyampholytes synthesized by iCVD exhibited excellent antifouling performance against the adhesion of Gram-positive Listeria innocua and Gram-negative E. coli in phosphate buffered saline (PBS). Their antifouling activities were attributed to the electrostatic interaction and hydration layers that served as physical and energetic barriers to prevent bacterial adhesion. The contact-killing and antifouling polymers synthesized by iCVD can be applied to surface modification of food processing equipment and medical devices with the aim of reducing foodborne diseases and medical infections. Moreover, the charged polyelectrolyte modified 2D polystyrene surfaces displayed good osteocompatibility and enhanced osteogenesis of preosteoblast cells than the un-modified polystyrene surface. In order to promote osteoinduction of hydroxyapatite (HA) scaffolds, bioinspired polymer-controlled mineralization was conducted on the polyelectrolyte modified HA scaffolds. The mineralized scaffolds stimulated osteogenesis of preosteoblast cells compared with the control HA scaffolds. Therefore, the surface modification through vapor deposition of polyelectrolytes and polymer-controlled mineralization can improve osteoinduction of bone materials. In summary, the iCVD-mediated surface modification is a simple and promising approach to biofunctionalizing various structured substrates and generating antimicrobial and biocompatible biomaterials.

Biocompatibility of modified ultra-high-molecular-weight polyethylene

NASA Astrophysics Data System (ADS)

Novotná, Z.; Lacmanová, V.; Rimpelová, S.; Juřik, P.; Polívková, M.; Å vorčik, V.

2016-09-01

Ultra-high-molecular-weight polyethylene (UHMWPE, PE) is a synthetic polymer used for biomedical applications because of its high impact resistance, ductility and stability in contact with physiological fluids. Therefore this material is being used in human orthopedic implants such as total joint replacements. Surface modification of this material relates to changes of its surface hydrophilicity, energy, microstructure, roughness, and morphology, all influencing its biological response. In our recent work, PE was treated by an Ar+ plasma discharge and then grafted with biologically active polyethylene glycol in order to enhance adhesion and proliferation of mouse fibroblast (L929). The surface properties of pristine PE and its grafted counterparts were studied by goniometry (surface wettability). Furthermore, Atomic Force Microscopy was used to determine the surface morphology and roughness. The biological response of the L929 cell lines seeded on untreated and plasma treated PE matrices was quantified in terms of the cell adhesion, density, and metabolic activity. Plasma treatment leads to the ablation of the polymer surface layers. Plasma treatment and subsequent poly(ethylene glycol) grafting lead to dramatic changes in the polymer surface morphology and roughness. Biological tests, performed in vitro, show increased adhesion and proliferation of cells on modified polymers. Grafting with poly(ethylene glycol) increases cell proliferation compared to plasma treatment.

Enzymes and other agents that enhance cell wall extensibility

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.

1999-01-01

Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

Interaction and cellular uptake of surface-modified carbon dot nanoparticles by J774.1 macrophages

PubMed Central

Thoo, Lester; Fahmi, Mochamad Z; Zulkipli, Ihsan N; Keasberry, Natasha

2017-01-01

Carbon dot (Cdot) nanoparticles are an emerging class of carbon nanomaterials with a promising potential for drug delivery and bio imaging applications. Although the interaction between Cdots and non-immune cell types has been well studied, Cdot interactions with macrophages have not been investigated. Exposure of Cdot nanoparticles to J774.1 cells, a murine macrophage cell line, resulted in minimal toxicity, where notable toxicity was only seen with Cdot concentrations higher than 0.5 mg/ml. Flow cytometric analysis revealed that Cdots prepared from citric acid were internalized at significantly higher levels by macrophages compared with those prepared from bamboo leaves. Interestingly, macrophages preferentially took up phenylboronic acid (PB)-modified nanoparticles. By fluorescence microscopy, strong blue light-specific punctate Cdot fluorescence resembling Cdot structures in the cytosolic space was mostly observed in J774.1 macrophages exposed to PB-modified nanoparticles and not unmodified Cdot nanoparticles. PB binds to sialic acid residues that are overexpressed on diseased cell surfaces. Our findings demonstrate that PB-conjugated Cdots can be taken up by macrophages with low toxicity and high efficiency. These modified Cdots can be used to deliver drugs to suppress or eliminate aberrant immune cells such as macrophages associated with tumors such as tumor-associated macrophages. PMID:29204100

Probing the Role of N-Linked Glycans in the Stability and Activity of Fungal Cellobiohydrolases by Mutational Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adney, W. S.; Jeoh, T.; Beckham, G. T.

2009-01-01

The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10, 2008) that expression of T. reesei cellobiohydrolase I in a commonlymore » used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120 h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that 'tuning' enzyme glycosylation for expression from heterologous expression hosts is essential for generating engineered enzymes with optimal stability and activity.« less

Versatile microbial surface-display for environmental remediation and biofuels production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Cindy H.; Mulchandani, Ashok; Chen, wilfred

2008-02-14

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.

Oxide modified air electrode surface for high temperature electrochemical cells

DOEpatents

Singh, Prabhakar; Ruka, Roswell J.

1992-01-01

An electrochemical cell is made having a porous cermet electrode (16) and a porous lanthanum manganite electrode (14), with solid oxide electrolyte (15) between them, where the lanthanum manganite surface next to the electrolyte contains a thin discontinuous layer of high surface area cerium oxide and/or praseodymium oxide, preferably as discrete particles (30) in contact with the air electrode and electrolyte.

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development.

PubMed

Lee, Chris; Chronis, Demosthenis; Kenning, Charlotte; Peret, Benjamin; Hewezi, Tarek; Davis, Eric L; Baum, Thomas J; Hussey, Richard; Bennett, Malcolm; Mitchum, Melissa G

2011-02-01

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.

Evaluation of the Antimicrobial Activity of Lysostaphin-Coated Hernia Repair Meshes▿

PubMed Central

Satishkumar, Rohan; Sankar, Sriram; Yurko, Yuliya; Lincourt, Amy; Shipp, John; Heniford, B. Todd; Vertegel, Alexey

2011-01-01

Bacterial infections by antibiotic-resistant Staphylococcus aureus strains are among the most common postoperative complications in surgical hernia repair with synthetic mesh. Surface coating of medical devices/implants using antibacterial peptides and enzymes has recently emerged as a potentially effective method for preventing infections. The objective of this study was to evaluate the in vitro antimicrobial activity of hernia repair meshes coated by the antimicrobial enzyme lysostaphin at different initial concentrations. Lysostaphin was adsorbed on pieces of polypropylene (Ultrapro) mesh with binding yields of ∼10 to 40% at different coating concentrations of between 10 and 500 μg/ml. Leaching of enzyme from the surface of all the samples was studied in 2% (wt/vol) bovine serum albumin in phosphate-buffered saline buffer at 37°C, and it was found that less than 3% of adsorbed enzyme desorbed from the surface after 24 h of incubation. Studies of antibacterial activity against a cell suspension of S. aureus were performed using turbidity assay and demonstrated that the small amount of enzyme leaching from the mesh surface contributes to the lytic activity of the lysostaphin-coated samples. Colony counting data from the broth count (model for bacteria in wound fluid) and wash count (model for colonized bacteria) for the enzyme-coated samples showed significantly decreased numbers of CFU compared to uncoated samples (P < 0.05). A pilot in vivo study showed a dose-dependent efficacy of lysostaphin-coated meshes in a rat model of S. aureus infection. The antimicrobial activity of the lysostaphin-coated meshes suggests that such enzyme-leaching surfaces could be efficient at actively resisting initial bacterial adhesion and preventing subsequent colonization of hernia repair meshes. PMID:21709102