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

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.

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

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.

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.

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.

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

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.

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.

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

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.

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.

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.

Effect of surface modifiers on an ectoenzyme: granulocyte 5'-nucleotidase.

PubMed

Smolen, J E; Karnovsky, M L

1980-05-01

Several agents that react with plasma membranes, namely the native lectins concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin, the modified lectin succinyl concanavalin A, and sodium meta-periodate, inhibited the ecto-5'-nucleotidase of intact guinea pig granulocytes. Stimulation of the enzyme was not observed at any lectin concentration. Inhibition by native lectins could be blocked or reversed by appropriate competing hapten sugars. In the case of concanavalin A, reversal could be achieved at 37 degrees C, but not at 5 degrees C. When lectins were used in combination with each other, the effects were found to be largely independent. However, when concanavalin A and R. communis agglutinin were applied together, complications arose because the former lectin binds to the latter as well as to the cell surface. To avoid some of the complexities inherent in studying intact cell 5'-nucleotidase and to gain additional information about the system, two broken cell enzyme preparations were also examined. The enzyme of plasma membrane-enriched fractions was inhibited by all five agents mentioned above. 5'-Nucleotidase solubilized in sodium deoxycholate was inhibited by the four lectins but stimulated by periodate. The effects of the surface modifiers on kinetic data for all three enzyme preparations are consistent with the hypothesis that direct interactions with the enzyme molecule give rise to changes in Vmax; interactions at membrane sites other than 5'-nucleotidase itself could cause increases in apparent Km values. Effects of interactions of ectoenzymes with plant lectins may serve as models for phenomena that result from cell-cell interactions or from interactions of animal cells with lectin-like components of the cellular environment.

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.

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

Determination of glutamine and glutamic acid in mammalian cell cultures using tetrathiafulvalene modified enzyme electrodes.

PubMed

Mulchandani, A; Bassi, A S

1996-01-01

Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures. The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase. The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol. Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system. When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.

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

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.

Yeast surface displaying glucose oxidase as whole-cell biocatalyst: construction, characterization, and its electrochemical glucose sensing application.

PubMed

Wang, Hongwei; Lang, Qiaolin; Li, Liang; Liang, Bo; Tang, Xiangjiang; Kong, Lingrang; Mascini, Marco; Liu, Aihua

2013-06-18

The display of glucose oxidase (GOx) on yeast cell surface using a-agglutinin as an anchor motif was successfully developed. Both the immunochemical analysis and enzymatic assay showed that active GOx was efficiently expressed and translocated on the cell surface. Compared with conventional GOx, the yeast cell surface that displayed GOx (GOx-yeast) demonstrated excellent enzyme properties, such as good stability within a wide pH range (pH 3.5-11.5), good thermostability (retaining over 94.8% enzyme activity at 52 °C and 84.2% enzyme activity at 56 °C), and high d-glucose specificity. In addition, direct electrochemistry was achieved at a GOx-yeast/multiwalled-carbon-nanotube modified electrode, suggesting that the host cell of yeast did not have any adverse effect on the electrocatalytic property of the recombinant GOx. Thus, a novel electrochemical glucose biosensor based on this GOx-yeast was developed. The as-prepared biosensor was linear with the concentration of d-glucose within the range of 0.1-14 mM and a low detection limit of 0.05 mM (signal-to-noise ratio of S/N = 3). Moreover, the as-prepared biosensor is stable, specific, reproducible, simple, and cost-effective, which can be applicable for real sample detection. The proposed strategy to construct robust GOx-yeast may be applied to explore other oxidase-displaying-system-based whole-cell biocatalysts, which can find broad potential application in biosensors, bioenergy, and industrial catalysis.

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

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.

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

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.

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.

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

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.

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

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

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

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

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.

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.

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

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.

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.

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.

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.

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

Construction of a laccase chimerical gene: recombinant protein characterization and gene expression via yeast surface display.

PubMed

Bleve, G; Lezzi, C; Spagnolo, S; Rampino, P; Perrotta, C; Mita, G; Grieco, Francesco

2014-03-01

The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N- and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N- and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 or the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.

Targeted modulation of reactive oxygen species in the vascular endothelium.

PubMed

Shuvaev, Vladimir V; Muzykantov, Vladimir R

2011-07-15

'Endothelial cells lining vascular luminal surface represent an important site of signaling and injurious effects of reactive oxygen species (ROS) produced by other cells and endothelium itself in ischemia, inflammation and other pathological conditions. Targeted delivery of ROS modulating enzymes conjugated with antibodies to endothelial surface molecules (vascular immunotargeting) provides site-specific interventions in the endothelial ROS, unattainable by other formulations including PEG-modified enzymes. Targeting of ROS generating enzymes (e.g., glucose oxidase) provides ROS- and site-specific models of endothelial oxidative stress, whereas targeting of antioxidant enzymes SOD and catalase offers site-specific quenching of superoxide anion and H(2)O(2). These targeted antioxidant interventions help to clarify specific role of endothelial ROS in vascular and pulmonary pathologies and provide basis for design of targeted therapeutics for treatment of these pathologies. In particular, antibody/catalase conjugates alleviate acute lung ischemia/reperfusion injury, whereas antibody/SOD conjugates inhibit ROS-mediated vasoconstriction and inflammatory endothelial signaling. Encapsulation in protease-resistant, ROS-permeable carriers targeted to endothelium prolongs protective effects of antioxidant enzymes, further diversifying the means for targeted modulation of endothelial ROS. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

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.

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

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.

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.

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.

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.

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.

Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces.

PubMed

Voss, Alexandra; Wei, HongYing; Zhang, Yi; Turner, Stuart; Ceccone, Giacomo; Reithmaier, Johann Peter; Stengl, Monika; Popov, Cyril

2016-07-01

Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O3 treatment or NH3 plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H-, O- and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability. Copyright © 2016 Elsevier B.V. All rights reserved.

Photo-switchable microbial fuel-cells.

PubMed

Schlesinger, Orr; Dandela, Rambabu; Bhagat, Ashok; Adepu, Raju; Meijler, Michael M; Xia, Lin; Alfonta, Lital

2018-05-01

Regulation of Bio-systems in a clean, simple, and efficient way is important for the design of smart bio-interfaces and bioelectronic devices. Light as a non-invasive mean to control the activity of a protein enables spatial and temporal control far superior to other chemical and physical methods. The ability to regulate the activity of a catalytic enzyme in a biofuel-cell reduces the waste of resources and energy and turns the fuel-cell into a smart and more efficient device for power generation. Here we present a microbial-fuel-cell based on a surface displayed, photo-switchable alcohol dehydrogenase. The enzyme was modified near the active site using non-canonical amino acids and a small photo-reactive molecule, which enables reversible control of enzymatic activity. Depending on the modification site, the enzyme exhibits reversible behavior upon irradiation with UV and visible light, in both biochemical, and electrochemical assays. The change observed in power output of a microbial fuel cell utilizing the modified enzyme was almost five-fold, between inactive and active states. © 2018 Wiley Periodicals, Inc.

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.

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.

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.

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.

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.

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.

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.

Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers.

PubMed

Botyanszki, Zsofia; Tay, Pei Kun R; Nguyen, Peter Q; Nussbaumer, Martin G; Joshi, Neel S

2015-10-01

Biocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long-term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site-specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site-specifically immobilized a recombinant α-amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α-amylase in unfavorable conditions. This enzyme-modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm-based catalysts, which rely on high cellular metabolism, the modified curli-based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces. © 2015 Wiley Periodicals, Inc.

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.

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.

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.

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.

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.

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

Optimizing immobilized enzyme performance in cell-free environments to produce liquid fuels.

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

Kumar, Sanat

The overall goal of this project was to optimize enzyme performance for the production of bio-diesel fuel. Enzyme immobilization has attracted much attention as a means to increase productivity. Mesorporous silica materials have been known to be best suited for immobilizing enzymes. A major challenge is to ensure that the enzymatic activity is retained after immobilization. Two major factors which drive enzymatic deactivation are protein-surface and inter-protein interactions. Previously, we studied protein stability inside pores and how to optimize protein-surface interactions to minimize protein denaturation. In this work we studied eh effect of surface curvature and chemistry on inter-protein interactions.more » Our goal was to find suitable immobilization supports which minimize these inter-protein interactions. Our studies carried out in the frame work of Hydrophobic-Polar (HP) model showed that enzymes immobilized inside hydrophobic pores of optimal sizes are best suited to minimize these inter-protein interactions. Besides, this study is also of biological importance to understand the role of chaperonins in protein disaggregation. Both of these aspects profited immensely with collaborations with our experimental colleague, Prof. Georges Belfort (RPI), who performed the experimental analog of our theoretical works.« less

Fibroblast adhesion and activation onto micro-machined titanium surfaces.

PubMed

Guillem-Marti, J; Delgado, L; Godoy-Gallardo, M; Pegueroles, M; Herrero, M; Gil, F J

2013-07-01

Surface modifications performed at the neck of dental implants, in the manner of micro-grooved surfaces, can reduce fibrous tissue encapsulation and prevent bacterial colonization, thereby improving fibrointegration and the formation of a biological seal. However, the applied procedures are technically complex and/or time consuming methods. The aim of this study was to analyse the fibroblast behaviour on modified titanium surfaces obtained, applying a simple and low-cost method. An array of titanium surfaces was obtained using a commercial computerized numerical control lathe, modifying the feed rate and the cutting depth. To elucidate the potential ability of the generated surfaces to activate connective tissue cells, a thorough gene (by real time - qPCR) and protein (by western blot or zymography) expression and cellular response characterization (cell morphology, cell adhesion and cell activation by secreting extracellular matrix (ECM) components and their enzyme regulators) was performed. Micro-grooved surfaces have statistically significant differences in the groove's width (approximately 10, 50 and 100 μm) depending on the applied advancing fixed speed. Field emission scanning electron microscopy images showed that fibroblasts oriented along the generated grooves, but they were only entirely accommodated on the wider grooves (≥50 μm). Micro-grooved surfaces exhibited an earlier cell attachment and activation, as seen by collagen Iα1 and fibronectin deposition and activation of ECM remodelling enzymes, compared with the other surfaces. However, fibroblasts could remain in an activated state on narrower surfaces (<50 μm) at later stages. The use of micro-grooved surfaces could improve implant integration at the gingival site with respect to polished surfaces. Micro-grooved surfaces enhance early fibroblast adhesion and activation, which could be critical for the formation of a biological seal and finally promote tissue integration. Surfaces with wider grooves (≥50 μm) seem to be more appropriate than surfaces with narrow grooves (<50 μm), as fibroblasts could persist in an activated state on narrower grooved surfaces, increasing the probability of producing a fibrotic response. © 2012 John Wiley & Sons A/S.

Carbohydrates, proteins, cell surfaces, and the biochemistry of pathogenesis

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

Albersheim, P.; Anderson-Prouty, A.J.

1975-01-01

General plant resistance to pathogenic attack by a myriad of microorganisms, viruses, nematodes, and insects are reviewed. Specifically discussed are: The role of the cell wall and wall-degrading enzymes in infective processes; an hypothesis to account for varietal specificity in gene-for-gene host-pathogen systems; examples which demonstrate that cell surface recognition phenomena are mediated through the interaction of carbohydrate-containing macromolecules and proteins; elicitors of phytoalexin production; and further consideration of the hypothesis and how the gene-for-gene relationship may have evolved. (JWP)

Virus scaffolds as enzyme nano-carriers.

PubMed

Cardinale, Daniela; Carette, Noëlle; Michon, Thierry

2012-07-01

The cooperative organization of enzymes by cells is a key feature for the efficiency of living systems. In the field of nanotechnologies, effort currently aims at mimicking this natural organization. Nanoscale resolution and high-registration alignment are necessary to control enzyme distribution in nano-containers or on the surface of solid supports. Virus capsid self-assembly is driven by precise supramolecular combinations of protein monomers, which have made them attractive building blocks to engineer enzyme nano-carriers (ENCs). We discuss some examples of what in our opinion constitute the latest advances in the use of plant viruses, bacteriophages and virus-like particles (VLPs) as nano-scaffolds for enzyme selection, enzyme confinement and patterning, phage therapy, raw material processing, and single molecule enzyme kinetics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Polymer-Based Surfaces Designed to Reduce Biofilm Formation: From Antimicrobial Polymers to Strategies for Long-Term Applications.

PubMed

Riga, Esther K; Vöhringer, Maria; Widyaya, Vania Tanda; Lienkamp, Karen

2017-10-01

Contact-active antimicrobial polymer surfaces bear cationic charges and kill or deactivate bacteria by interaction with the negatively charged parts of their cell envelope (lipopolysaccharides, peptidoglycan, and membrane lipids). The exact mechanism of this interaction is still under debate. While cationic antimicrobial polymer surfaces can be very useful for short-term applications, they lose their activity once they are contaminated by a sufficiently thick layer of adhering biomolecules or bacterial cell debris. This layer shields incoming bacteria from the antimicrobially active cationic surface moieties. Besides discussing antimicrobial surfaces, this feature article focuses on recent strategies that were developed to overcome the contamination problem. This includes bifunctional materials with simultaneously presented antimicrobial and protein-repellent moieties; polymer surfaces that can be switched from an antimicrobial, cell-attractive to a cell-repellent state; polymer surfaces that can be regenerated by enzyme action; degradable antimicrobial polymers; and antimicrobial polymer surfaces with removable top layers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Harnessing surface-bound enzymatic reactions to organize microcapsules in solution

PubMed Central

Shklyaev, Oleg E.; Shum, Henry; Sen, Ayusman; Balazs, Anna C.

2016-01-01

By developing new computational models, we examine how enzymatic reactions on an underlying surface can be harnessed to direct the motion and organization of reagent-laden microcapsules in a fluid-filled microchannel. In the presence of appropriate reagents, surface-bound enzymes can act as pumps, which drive large-scale fluid flows. When the reagents diffuse through the capsules’ porous shells, they can react with enzymatic sites on the bottom surface. The ensuing reaction generates fluid density variations, which result in fluid flows. These flows carry the suspended microcapsules and drive them to aggregate into “colonies” on and near the enzyme-covered sites. This aggregation continues until the reagent has been depleted and the convection stops. We show that the shape of the assembled colonies can be tailored by patterning the distribution of enzymes on the surface. This fundamental physicochemical mechanism could have played a role in the self-organization of early biological cells (protocells) and can be used to regulate the autonomous motion and targeted delivery of microcarriers in microfluidic devices. PMID:27034990

Functional cell-surface display of a lipase-specific chaperone.

PubMed

Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich

2007-01-02

Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen

PubMed Central

Gründel, Anne; Friedrich, Kathleen; Pfeiffer, Melanie; Jacobs, Enno; Dumke, Roger

2015-01-01

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen. PMID:25978044

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

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

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less

Bacillus cereus strain MCN as a debriding agent

NASA Technical Reports Server (NTRS)

Dalton, H. P.; Haynes, B. W.; Stone, L. L.

1978-01-01

Biologically active means are effective for rapidly removing scar tissue caused by burns or corrosive agents. Specially selected strain of bacteria applied to injury site releases enzymes which are active against eschar. These bacteria tend to locate between eschar and unburned tissue, thus providing optimal cell surface area arrangement for enzyme dispersal. Procedure may prove especially useful in treatment of disaster casualties under relatively primitive conditions.

Antibody Therapeutics in Oncology.

PubMed

Wold, Erik D; Smider, Vaughn V; Felding, Brunhilde H

2016-03-01

One of the newer classes of targeted cancer therapeutics is monoclonal antibodies. Monoclonal antibody therapeutics are a successful and rapidly expanding drug class due to their high specificity, activity, favourable pharmacokinetics, and standardized manufacturing processes. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody dependent cellular cytotoxicity. In an ideal scenario the initial tumor cell destruction induced by administration of a therapeutic antibody can result in uptake of tumor associated antigens by antigen-presenting cells, establishing a prolonged memory effect. Mechanisms of direct tumor cell killing by antibodies include antibody recognition of cell surface bound enzymes to neutralize enzyme activity and signaling, or induction of receptor agonist or antagonist activity. Both approaches result in cellular apoptosis. In another and very direct approach, antibodies are used to deliver drugs to target cells and cause cell death. Such antibody drug conjugates (ADCs) direct cytotoxic compounds to tumor cells, after selective binding to cell surface antigens, internalization, and intracellular drug release. Efficacy and safety of ADCs for cancer therapy has recently been greatly advanced based on innovative approaches for site-specific drug conjugation to the antibody structure. This technology enabled rational optimization of function and pharmacokinetics of the resulting conjugates, and is now beginning to yield therapeutics with defined, uniform molecular characteristics, and unprecedented promise to advance cancer treatment.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes.

PubMed

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø; de Souza, Gustavo A; Sollid, Ludvig M

2016-05-05

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

PubMed Central

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

2016-01-01

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

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.

Glucose oxidase/cellulose-carbon nanotube composite paper as a biocompatible bioelectrode for biofuel cells.

PubMed

Won, Keehoon; Kim, Young-Hoo; An, Seulji; Lee, Hye Jung; Park, Saerom; Choi, Yong-Keun; Kim, Ji Hyeon; Hwang, Hak-In; Kim, Hyung Joo; Kim, Hyungsup; Lee, Sang Hyun

2013-11-01

Biofuel cells are devices for generating electrical energy directly from chemical energy of renewable biomass using biocatalysts such as enzymes. Efficient electrical communication between redox enzymes and electrodes is essential for enzymatic biofuel cells. Carbon nanotubes (CNTs) have been recognized as ideal electrode materials because of their high electrical conductivity, large surface area, and inertness. Electrodes consisting entirely of CNTs, which are known as CNT paper, have high surface areas but are typically weak in mechanical strength. In this study, cellulose (CL)-CNT composite paper was fabricated as electrodes for enzymatic biofuel cells. This composite electrode was prepared by vacuum filtration of CNTs followed by reconstitution of cellulose dissolved in ionic liquid, 1-ethyl-3-methylimidazolium acetate. Glucose oxidase (GOx), which is a redox enzyme capable of oxidizing glucose as a renewable fuel using oxygen, was immobilized on the CL-CNT composite paper. Cyclic voltammograms revealed that the GOx/CL-CNT paper electrode showed a pair of well-defined peaks, which agreed well with that of FAD/FADH2, the redox center of GOx. This result clearly shows that the direct electron transfer (DET) between the GOx and the composite electrode was achieved. However, this DET was dependent on the type of CNTs. It was also found that the GOx immobilized on the composite electrode retained catalytic activity for the oxidation of glucose.

Mannose phosphate isomerase regulates fibroblast growth factor receptor family signaling and glioma radiosensitivity.

PubMed

Cazet, Aurélie; Charest, Jonathan; Bennett, Daniel C; Sambrooks, Cecilia Lopez; Contessa, Joseph N

2014-01-01

Asparagine-linked glycosylation is an endoplasmic reticulum co- and post-translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.

Effects of ribonuclease A on amino acid transport in Neurospora crassa.

PubMed

Stuart, W D; Woodward, D O

1975-04-01

Incubation of Neurospora crassa conidia with ribonuclease (RNase) A reduces transport of L-phenylalanine by those cells. Under similar conditions, oxidized RNase A, RNase T1, and RNase T2 do not have this effect. Incubation of conidia with active RNase covalently attached to polyacrylamide beads reduces L-phenylalanine transport. This indicates that the site of enzymatic action is at the cell surface. At the lower concentration of enzyme used in this study, incubation with RNase A reduces transport of L-phenylalanine by the general (G) amino acid permease. Increasing the enzyme concentration results in reduction of transport by the neutral aromatic (N)-specific permease. The increased transport activity that accompanies onset of conidial germination is also sensitive to incubation with RNase A. Application of the enzyme to actively transporting cells does not release amino acid transported prior to enzyme addition. Cells cultured on media supplemented with [2-14C] uridine release isotopic activity after RNase A incubation. Analogous treatments with Pronase, RNase T1, RNase T2, or deoxyribonuclease I do not release isotope activity. Pronase treatment does reduce L-phenylalanine transport. Incubation of conidia with RNase A also inhibits germination of those conidia.

Toward reducing immunogenicity of enzyme replacement therapy: altering the specificity of human β-glucuronidase to compensate for α-iduronidase deficiency.

PubMed

Chuang, Huai-Yao; Suen, Ching-Shu; Hwang, Ming-Jing; Roffler, Steve R

2015-11-01

Enzyme replacement therapy (ERT) is an effective treatment for many patients with lysosomal storage disorders caused by deficiency in enzymes involved in cell metabolism. However, immune responses that develop against the administered enzyme in some patients can hinder therapeutic efficacy and cause serious side effects. Here we investigated the feasibility of a general approach to decrease ERT immunogenicity by altering the specificity of a normal endogenous enzyme to compensate for a defective enzyme. We sought to identify human β-glucuronidase variants that display α-iduronidase activity, which is defective in mucopolysaccharidosis (MPS) type I patients. A human β-glucuronidase library was screened by the Enzyme Cleavable Surface-Tethered All-purpose Screen sYstem to isolate variants that exhibited 100-290-fold greater activity against the α-iduronidase substrate 4-methylumbelliferyl α-l-iduronide and 7900-24 500-fold enzymatic specificity shift when compared with wild-type β-glucuronidase. In vitro treatment of MPS I cells with the β-glucuronidase variants significantly restored lysosome appearance similar to treatment with α-iduronidase. Our study suggests that β-glucuronidase variants can be isolated to display α-iduronidase activity and produce significant phenotype improvement of MPS I cells. This strategy may represent a possible approach to produce enzymes that display therapeutic benefits with potentially less immunogenicity. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

Metal organic frameworks for enzyme immobilization in biofuel cells

NASA Astrophysics Data System (ADS)

Bodell, JaDee

Interest in biofuel cells has been rapidly expanding as an ever-growing segment of the population gains access to electronic devices. The largest areas of growth for new populations using electronic devices are often in communities without electrical infrastructure. This lack of infrastructure in remote environments is one of the key driving factors behind the development of biofuel cells. Biofuel cells employ biological catalysts such as enzymes to catalyze oxidation and reduction reactions of select fuels to generate power. There are several benefits to using enzymes to catalyze reactions as compared to traditional fuel cells which use metal catalysts. First, enzymes are able to catalyze reactions at or near room temperature, whereas traditional metal catalysts are only efficient at very high temperatures. Second, biofuel cells can operate under mild pH conditions which is important for the eventual design of safe, commercially viable devices. Also, biofuel cells allow for implantable and flexible technologies. Finally, enzymes exhibit high selectivity and can be combined to fully oxidize or reduce the fuel which can generate several electrons from a single molecule of fuel, increasing the overall device efficiency. One of the main challenges which persist in biofuel cells is the instability of enzymes over time which tend to denature after hours or days. For a viable commercial biofuel cell to be produced, the stability of enzymes must be extended to months or years. Enzymes have been shown to have improved stability after being immobilized. The focus of this research was to find a metal organic framework (MOF) structure which could successfully immobilize enzymes while still allowing for electron transport to occur between the catalytic center of the enzyme and the electrode surface within a biofuel cell for power generation. Four MOF structures were successfully synthesized and were subsequently tested to determine the MOF's ability to immobilize the following enzymes: nicotinamide adenine dinucleotide (NAD)-dependent alcohol and aldehyde dehydrogenases, and pyrroloquinoline quinone (PQQ)-dependent alcohol and aldehyde dehydrogenases, as well as flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase. Tb-meso MOF was shown to immobilize PQQ-dependent enzymes through ? stacking interactions of the heme in the enzyme and the triazine molecules in the ligand of the MOF. However, the PQQ-dependent dehydrogenases did not have enough catalytic activity present to be measured electrochemically. Finally, ZIF-90 was synthesized under aqueous conditions in the presence of FAD-dependent glucose dehydrogenase (GDH) which led to size selective sheltering of FAD-GDH. FAD-GDH had activity an order of magnitude larger than any of the alcohol dehydrogenases, which provided sufficient catalytic activity to measure electrochemically. The FAD-GDH bound within ZIF-90 was used to build a full biofuel cell resulting in an open circuit voltage of 708 +/- 16 mV and a maximum power density of 2.75 +/- 0.40 microW/cm2.

Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering

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

Hang, Howard C.; Bertozzi, Carolyn R.

2000-08-22

Novel chemical reactivity can be engendered on cell surfaces by the metabolic incorporation of unnatural sugars into cell surface glycoconjuagtes. 2-N-Acetamido sugars such as GalNAc and GlcNAc are abundant components of cell surface glycoconjugates, and hence attractive targets for metabolic cell surface engineering. Here we report (1) the synthesis of isosteric analogs bearing a ketone group in place of the N-acetamido group, and (2) evaluation of their metabolic incorporation into mammalian cell surface glycans. A ketone isostere of GalNAc was metabolized by CHO cells through the salvage pathway and delivered to O-linked glycoproteins on the cell surface. Its residence atmore » the core position of O-linked glycans is suggested by studies with a-benzyl GalNAc, an inhibitor of O-linked oligosaccharide extension. A mutant CHO cell line lacking endogenous UDP-GalNAc demonstrated enhanced metabolism of the GalNAc analog, suggesting that competition with native intermediates might limits enzymatic transformation in mammalian cells. A ketone isostere of GlcNAc could not be detected on CHO or human cell surfaces after incubation. Thus, the enzymes in the GlcNAc salvage pathway might be less permissive of unnatural substrates than those comprising the GalNAc salvage pathway. Alternatively, high levels of endogenous GlcNAc derivatives might compete with the ketone isostere and prevent its incorporation into oligosaccharides.« less

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.

Proteoglycans and neuronal migration in the cerebral cortex during development and disease

PubMed Central

Maeda, Nobuaki

2015-01-01

Chondroitin sulfate proteoglycans and heparan sulfate proteoglycans are major constituents of the extracellular matrix and the cell surface in the brain. Proteoglycans bind with many proteins including growth factors, chemokines, axon guidance molecules, and cell adhesion molecules through both the glycosaminoglycan and the core protein portions. The functions of proteoglycans are flexibly regulated due to the structural variability of glycosaminoglycans, which are generated by multiple glycosaminoglycan synthesis and modifying enzymes. Neuronal cell surface proteoglycans such as PTPζ, neuroglycan C and syndecan-3 function as direct receptors for heparin-binding growth factors that induce neuronal migration. The lectican family, secreted chondroitin sulfate proteoglycans, forms large aggregates with hyaluronic acid and tenascins, in which many signaling molecules and enzymes including matrix proteases are preserved. In the developing cerebrum, secreted chondroitin sulfate proteoglycans such as neurocan, versican and phosphacan are richly expressed in the areas that are strategically important for neuronal migration such as the striatum, marginal zone, subplate and subventricular zone in the neocortex. These proteoglycans may anchor various attractive and/or repulsive cues, regulating the migration routes of inhibitory neurons. Recent studies demonstrated that the genes encoding proteoglycan core proteins and glycosaminoglycan synthesis and modifying enzymes are associated with various psychiatric and intellectual disorders, which may be related to the defects of neuronal migration. PMID:25852466

IONIC EFFECTS ON LIGNIFICATION AND PEROXIDASE IN TISSUE CULTURES

PubMed Central

Lipetz, Jacques; Garro, Anthony J.

1965-01-01

Crown-gall tumor tissue cultures release peroxidase into the medium in response to the concentration of specific ions in the medium. This release is not due to diffusion from cut surfaces or injured cells. Calcium, magnesium, and ammonium were, in that order, most effective in increasing peroxidase release. The enzyme was demonstrated cytochemically on the cell walls and in the cytoplasm. Cell wall fractions, exhaustively washed in buffer, still contained bound peroxidase. This bound peroxidase could be released by treating the wall fractions with certain divalent cations or ammonium. The order of effectiveness for removing the enzyme from the washed cell walls is: Ca++ ≈ Sr++ > Ba++ > Mg++ > NH4 +. These data support the thesis presented that specific ions can control the deposition of lignin on cell walls by affecting the peroxidase levels on these walls. PMID:19866650

Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling

PubMed Central

Jeong, Hee-Jin; Abhiraman, Gita C.; Story, Craig M.

2017-01-01

Sortase A, a calcium-dependent transpeptidase derived from Staphylococcus aureus, is used in a broad range of applications, such as the conjugation of fluorescent dyes and other moieties to proteins or to the surface of eukaryotic cells. In vivo and cell-based applications of sortase have been somewhat limited by the large range of calcium concentrations, as well as by the often transient nature of protein-protein interactions in living systems. In order to use sortase A for cell labeling applications, we generated a new sortase A variant by combining multiple mutations to yield an enzyme that was both calcium-independent and highly active. This variant has enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions. PMID:29200433

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

Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

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

Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@med.monash.edu.au; Tochon-Danguy, Natalie; Ian Smith, A.

2010-07-23

Research highlights: {yields} PKC activation increases the trafficking of ECE-1 to the cell surface. {yields} This in turn leads to an increase in the amount of ECE-1 shed. {yields} Only the catalytically active C-terminal region is shed from the cell surface. -- Abstract: This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 {mu}M phorbol 12-myristate 13-acetate (PMA) which activates PKC. Themore » biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 {+-} 32.3% of control, n = 5). The ECE-1 activity (expressed as {mu}M substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor (CGS35066). The stimulation of cells by PMA (1 {mu}M, 6 h) significantly increased the ECE-1 activity (0.28 {+-} 0.02; n = 3) compared to the control (0.07 {+-} 0.02; n = 3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 {mu}M for 1 h; 0.10 {+-} 0.01; n = 3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 {+-} 0.01; n = 3) compared to control (0.08 {+-} 0.01; n = 3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface, respectively.« less

Tissue Printing to Visualize Polyphenol Oxidase and Peroxidase in Vegetables, Fruits, and Mushrooms

ERIC Educational Resources Information Center

Melberg, Amanda R.; Flurkey, William H.; Inlow, Jennifer K.

2009-01-01

A simple tissue-printing procedure to determine the tissue location of the endogenous enzymes polyphenol oxidase and peroxidase in a variety of vegetables, fruits, and mushrooms is described. In tissue printing, cell contents from the surface of a cut section of the tissue are transferred to an adsorptive surface, commonly a nitrocellulose…

Macrophage Biochemistry, Activation and Function

DTIC Science & Technology

1981-01-01

vacuolar apparatus become more abundant. Functional capabilities, including phagocytic activity, protein synthesis and surface receptors, also increase...properties of cell components of other tissues has led to the following assignment of marker enzymes to specific macrophage components. This assessment is...subfractions. The surface area of each histogram bar then gives the frac- tional amount of constituent present within each normalized fraction. Distribution

Immunological relationships between glucosyltransferases from Streptococcus mutans serotypes.

PubMed

Kuramitsu, H; Ingersoll, L

1976-09-01

Partially purified glycosyltransferase enzymes for Streptococcus mutans GS-5 (serotype c) have been utilized to prepare antibodies directed against the soluble glucan-synthesizing activity, GTF-B, and the insoluble-soluble glucan synthetic activity, GTF-A. Anti-GTF-A inhibited insoluble glucan formation catalyzed by the extracellular enzymes from strains GS-5 and FA-1 (serotype b) to a much greater extent than that of strains HS-6 (serotype a) or OMZ-176 (serotype d). This antibody fraction also inhibited both the cell-associated glucosyltransferase activities as well as the sucrose-mediated adherence of cells to glass surfaces by strains GS-5 and FA-1 but not that of strains HS-6 and OMZ-176. Anti-GTF-B inhibited soluble glucan formation catalyzed by the extracellular enzymes of strains GS-5 but not that of strain HS-6, FA-1, or OMZ-176. However, this antibody fraction did not strongly inhibit either the cell-associated glycosyltransferase activity or cellular adherence of any of the four strains. These results with body antibody fractions were also correlated with the ability of the antibodies to agglutinate the cells and form precipitin bands after immunodiffusion with the extracellular enzymes. Antibody prepared against the homogeneous soluble glucan-synthesizing enzyme demonstrated similar effects to the anti-GTF-B fraction. These results are discussed in terms of the antigenic relationships existing between the glucosyltransferases from different serotypes of S. mutans.

A Dual-Action Armed Replicating Adenovirus for the Treatment of Osteoblastic Bone Metastases of Prostate Cancer

DTIC Science & Technology

2007-03-01

ligand), a membrane-bound cytokine expressed in osteoblasts/stromal cells, which binds to RANK, a cell- surface protein present on osteoclast...cocultures were incubated for 10 days and then the culture medium was employed in an ELISA to detect the osteoclast marker , tartrate- resistant acid...bone marrow stromal cells and osteoclast precursor cells. Ten days post-infection, expression of the osteoclast marker enzyme TRAP was determined

Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV-positive and HIV-negative isolates.

PubMed

Portela, M B; Kneipp, L F; Ribeiro de Souza, I P; Holandino, C; Alviano, C S; Meyer-Fernandes, J R; de Araújo Soares, R M

2010-07-01

This study describes the expression of acidic ectophosphatase activity on twenty isolates of C. albicans from oral cavities of HIV-infected children (HIV+) and compares them with fifteen isolates from HIV-negative children (HIV-), as well as the fungal adhesion to epithelial cells and medical records. The activities were measured in intact cells grown in BHI medium for 48 h at 37 degrees C. Phosphatase activity was assayed at pH 5.5 using 4-methylumbelliferyl phosphate. Yeast adhesion was measured using the MA 104 epithelial cell line. Mean values of ectophosphatase activity were 610.27 +/- 166.36 and 241.25 +/- 78.96 picomoles 4-methylumbelliferone/h/10(7) cells for HIV+ and HIV- group, respectively (P = 0.049). No correlation between C. albicans enzyme activity from HIV children with viral load and CD4 percentual was observed. Yeasts with high enzyme activity, isolated from HIV+ children showed greater adherence than yeasts with basal levels of ectophosphatases from HIV- (Spearman correlation, r = 0.8). Surface phosphatase activity was apparently involved in the adhesion to host cells, as the enhanced attachment of C. albicans to host epithelial cells was reversed by pretreatment of yeast with sodium orthovanadate (1 mM), an acid phosphatase inhibitor. These results show that C. albicans from HIV+ has an ectophosphatase activity significantly higher than the other isolates. Yeasts expressing higher levels of surface phosphatase activity showed greater adhesion to epithelial cells. So, the activity of acidic surface phosphatases on these cells may contribute to the early mechanisms required for disease establishment.

Engineering yeast consortia for surface-display of complex cellulosome structures

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

Chen, Wilfred

As our society marches toward a more technologically advanced future, energy and environmental sustainability are some of the most challenging problems we face today. Biomass is one of the most abundant renewable-feedstock for sustainable production of biofuels. However, the main technological obstacle to more widespread uses of this resource is the lack of low-cost technologies to overcome the recalcitrant nature of the cellulosic structure, especially the hydrolysis step on highly ordered celluloses. In this proposal, we successfully engineered several efficient and inexpensive whole-cell biocatalysts in an effort to produce economically compatible and sustainable biofuels, namely cellulosic ethanol. Our approach wasmore » to display of a highly efficient cellulolytic enzyme complex, named cellulosome, on the surface of a historical ethanol producer Saccharomyces cerevisiae for the simultaneous and synergistic saccharification and fermentation of cellulose to ethanol. We first demonstrated the feasibility of assembling a mini-cellulosome by incubating E. coli lysates expressing three different cellulases. Resting cells displaying mini-cellulosomes produced 4-fold more ethanol from phosphoric acid-swollen cellulose (PASC) than cultures with only added enzymes. The flexibility to assemble the mini-cellulosome structure was further demonstrated using a synthetic yeast consortium through intracellular complementation. Direct ethanol production from PASC was demonstrated with resting cell cultures. To create a microorganism suitable for a more cost-effective process, called consolidated bioprocessing (CBP), a synthetic consortium capable of displaying mini-cellulosomes on the cell surface via intercellular complementation was created. To further improve the efficiency, a new adaptive strategy of employing anchoring and adaptor scaffoldins to amplify the number of enzymatic subunits was developed, resulting in the creation of an artificial tetravalent cellulosome on the yeast surface and a significant improvement in cellulosic ethanol production. Although this adaptive strategy is ideal for assembling more complex cellulosome for large-scale production of cellulosic ethanol, a substantially larger number of enzymes (up to 10 to 12) is needed to better mimic the natural cellulosome structures for practical usage of the technology.« less

An NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles for tumor targeted drug delivery in vitro and in vivo

NASA Astrophysics Data System (ADS)

Gayam, Srivardhan Reddy; Venkatesan, Parthiban; Sung, Yi-Ming; Sung, Shuo-Yuan; Hu, Shang-Hsiu; Hsu, Hsin-Yun; Wu, Shu-Pao

2016-06-01

The synthesis and characterization of an NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles (MSNPs) for on-command delivery applications has been described in this paper. Gatekeeping of MSNPs is achieved by the integration of mechanically interlocked rotaxane nanovalves on the surface of MSNPs. The rotaxane nanovalve system is composed of a linear stalk anchoring on the surface of MSNPs, an α-cyclodextrin ring that encircles it and locks the payload ``cargo'' molecules in the mesopores, and a benzoquinone stopper incorporated at the end of the stalk. The gate opening and controlled release of the cargo are triggered by cleavage of the benzoquinone stopper using an endogenous NQO1 enzyme. In addition to having efficient drug loading and controlled release mechanisms, this smart biocompatible carrier system showed obvious uptake and consequent release of the drug in tumor cells, could selectively induce the tumor cell death and enhance the capability of inhibition of tumor growth in vivo. The controlled drug delivery system demonstrated its use as a potential theranostic material.The synthesis and characterization of an NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles (MSNPs) for on-command delivery applications has been described in this paper. Gatekeeping of MSNPs is achieved by the integration of mechanically interlocked rotaxane nanovalves on the surface of MSNPs. The rotaxane nanovalve system is composed of a linear stalk anchoring on the surface of MSNPs, an α-cyclodextrin ring that encircles it and locks the payload ``cargo'' molecules in the mesopores, and a benzoquinone stopper incorporated at the end of the stalk. The gate opening and controlled release of the cargo are triggered by cleavage of the benzoquinone stopper using an endogenous NQO1 enzyme. In addition to having efficient drug loading and controlled release mechanisms, this smart biocompatible carrier system showed obvious uptake and consequent release of the drug in tumor cells, could selectively induce the tumor cell death and enhance the capability of inhibition of tumor growth in vivo. The controlled drug delivery system demonstrated its use as a potential theranostic material. Electronic supplementary information (ESI) available: Synthesis and characterization of the functional molecules and MSNPs is available in the ESI. See DOI: 10.1039/c6nr03525f

Mycoparasitism studies of Trichoderma harzianum against Sclerotinia sclerotiorum: evaluation of antagonism and expression of cell wall-degrading enzymes genes.

PubMed

Troian, Rogério Fraga; Steindorff, Andrei Stecca; Ramada, Marcelo Henrique Soller; Arruda, Walquiria; Ulhoa, Cirano José

2014-10-01

Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.

Crystal Structure of LAAO from Calloselasma rhodostoma with L-Phenylalanine Substrate: Insights into Structure and Mechanism

PubMed Central

Moustafa, Ibrahim M.; Foster, Scott; Lyubimov, Artem Y.; Vrielink, Alice

2007-01-01

L-amino acid oxidase (LAAO) is a dimeric glycosylated flavoenzyme, a major constituent of the snake-venom from Calloselasma rhodostoma. The enzyme exhibits apoptosis-inducing effects as well as antibacterial and anti-HIV activities. The structure of LAAO with its substrate (L-phenylalanine) has been refined to a resolution of 1.8 Å. The complex structure reveals the substrate bound to the reduced flavin (FADred). Alternate conformations for the key residues: His223 and Arg322 are evident, suggesting a dynamic active site. Furthermore conformational changes are also apparent for the isoalloxazine ring; the three ring system exhibits more bending around the N5—N10 axis compared to the oxidized flavin. The implications of the observed dynamics on the mechanism of catalysis are discussed. Inspection of buried surfaces in the enzyme reveals a Y shaped channel system extending from the external surface of the protein to the active site. One portion of this channel may serve as the entry path for O2 during the oxidative half reaction. The second region, separated from the proposed O2 channel by the N-terminus (residues 8 – 16) of the protein, may play a role in H2O2 release. Interestingly, the later portion of the channel would direct the H2O2 product to the exterior surface of the protein, near to the glycan moiety, thought to anchor the enzyme to the host cell. This channel location may explain the ability of the enzyme to localize H2O2 to the targeted cell inducing the apoptotic effect. PMID:17046020

A general strategy for the evolution of bond-forming enzymes using yeast display

PubMed Central

Chen, Irwin; Dorr, Brent M.; Liu, David R.

2011-01-01

The ability to routinely generate efficient protein catalysts of bond-forming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A–catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods. PMID:21697512

A little sugar goes a long way: The cell biology of O-GlcNAc

PubMed Central

2015-01-01

Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport. PMID:25825515

Surface-enhanced Raman scattering (SERS) imaging of alkyne-tagged small molecule drug in live cells with endocytosed gold nanoparticles

NASA Astrophysics Data System (ADS)

Ando, Jun; Sekiya, Takumasa; Ka, Den; Yamakoshi, Hiroyuki; Dodo, Kosuke; Sodeoka, Mikiko; Kawata, Satoshi; Fujita, Katsumasa

2017-02-01

We propose the combination of alkyne-tag and surface-enhanced Raman scattering (SERS) spectroscopy to perform highly-sensitive and selective drug imaging in live cells. Gold nanoparticles are introduced in lysosomes through endocytosis as SERS agents, and the alkyne-tagged drugs are subsequently administered in cells. Raman microscopic observation reveals the arrival of drug in lysosome through enhanced Raman signal of alkyne. Since the peak of alkyne appears in Raman-silent region of biomolecules, selective detection of drugs is possible without background signal of endogenous molecules. From endocytosed gold nanoparticles in living HeLa cells, we observed distinct Raman signal from alkyne-tagged inhibitor of lysosomal enzyme.

Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System*

PubMed Central

Campeotto, Ivan; Percy, Matthew G.; MacDonald, James T.; Förster, Andreas; Freemont, Paul S.; Gründling, Angelika

2014-01-01

Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaPLm initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaSLm extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaPLm and LtaSLm. Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaSLm structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing chain. PMID:25128528

Recent advances in yeast cell-surface display technologies for waste biorefineries.

PubMed

Liu, Zhuo; Ho, Shih-Hsin; Hasunuma, Tomohisa; Chang, Jo-Shu; Ren, Nan-Qi; Kondo, Akihiko

2016-09-01

Waste biorefinery aims to maximize the output of value-added products from various artificial/agricultural wastes by using integrated bioprocesses. To make waste biorefinery economically feasible, it is thus necessary to develop a low-cost, environment-friendly technique to perform simultaneous biodegradation and bioconversion of waste materials. Cell-surface display engineering is a novel, cost-effective technique that can auto-immobilize proteins on the cell exterior of microorganisms, and has been applied for use with waste biofinery. Through tethering different enzymes (e.g., cellulase, lipase, and protease) or metal-binding peptides on cell surfaces, various yeast strains can effectively produce biofuels and biochemicals from sugar/protein-rich waste materials, catalyze waste oils into biodiesels, or retrieve heavy metals from wastewater. This review critically summarizes recent applications of yeast cell-surface display on various types of waste biorefineries, highlighting its potential and future challenges with regard to commercializing this technology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan, inhibits type I-IV allergic inflammation and pro-inflammatory enzymes.

PubMed

Lee, Ji Yun; Kim, Chang Jong

2010-06-01

We previously reported that arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan isolated from Forsythia koreana, exhibits anti-inflammatory, antioxidant, and analgesic effects in animal models. In addition, arctigenin inhibited eosinophil peroxidase and activated myeloperoxidase in inflamed tissues. In this study, we tested the effects of arctigenin on type I-IV allergic inflammation and pro-inflammatory enzymes in vitro and in vivo. Arctigenin significantly inhibited the heterologous passive cutaneous anaphylaxis induced by ovalbumin in mice at 15 mg/kg, p.o., and compound 48/80-induced histamine release from rat peritoneal mast cells at 10 microM. Arctigenin (15 mg/kg, p.o.) significantly inhibited reversed cutaneous anaphylaxis. Further, arctigenin (15 mg/kg, p.o.) significantly inhibited the Arthus reaction to sheep's red blood cells, decreasing the hemolysis titer, the hemagglutination titer, and the plaque-forming cell number for SRBCs. In addition, arctigenin significantly inhibited delayed type hypersensitivity at 15 mg/kg, p.o. and the formation of rosette-forming cells at 45 mg/kg, p.o. Contact dermatitis induced by picrylchloride and dinitrofluorobenzene was significantly (p < 0.05) inhibited by surface treatment with arctigenin (0.3 mg/ear). Furthermore, arctigenin dose-dependently inhibited pro-inflammatory enzymes, such as cyclooxygenase-1 and 2, 5-lipoxygenase, phospholipase A2, and phosphodiesterase. Our results show that arctigenin significantly inhibited B- and T-cell mediated allergic inflammation as well as pro-inflammatory enzymes.

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.

The toxicology and immunology of detergent enzymes.

PubMed

Basketter, David; Berg, Ninna; Kruszewski, Francis H; Sarlo, Katherine; Concoby, Beth

2012-01-01

Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T(H))-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.

Sensitive electrochemical aptamer cytosensor for highly specific detection of cancer cells based on the hybrid nanoelectrocatalysts and enzyme for signal amplification.

PubMed

Sun, Duanping; Lu, Jing; Zhong, Yuwen; Yu, Yanyan; Wang, Yu; Zhang, Beibei; Chen, Zuanguang

2016-01-15

Human cancer is becoming a leading cause of death in the world and the development of a straightforward strategy for early detection of cancer is urgently required. Herein, a sandwich-type electrochemical aptamer cytosensor was developed for detection of human liver hepatocellular carcinoma cells (HepG2) based on the hybrid nanoelectrocatalysts and enzyme for signal amplification. The thiolated TLS11a aptamers were used as a selective bio-recognition element, attached to the gold nanoparticles (AuNPs) modified the glassy carbon electrode (GCE) surface. Meanwhile, the electrochemical nanoprobes were fabricated through the G-quadruplex/hemin/aptamer complexes and horseradish peroxidase (HRP) immobilized on the surfaces of Au@Pd core-shell nanoparticle-modified magnetic Fe3O4/MnO2 beads (Fe3O4/MnO2/Au@Pd). After the target cells were captured, the hybrid nanoprobes were further assembled to form an aptamer-cell-nanoprobes sandwich-like system on the electrode surface. Then, hybrid Fe3O4/MnO2/Au@Pd nanoelectrocatalysts, G-quadruplex/hemin HRP-mimicking DNAzymes and the natural HRP enzyme efficiently catalyzed the oxidation of hydroquinone (HQ) with H2O2, amplifying the electrochemical signals and improving the detection sensitivity. This electrochemical cytosensor delivered a wide detection range of 1×10(2)-1×10(7)cellsmL(-1), high sensitivity with a low detection limit of 15cellsmL(-1), good selectivity and repeatability. Finally, an electrochemical reductive desorption method was performed to break gold-thiol bond and desorb the components on the AuNPs/GCE for regenerating the cytosensor. These results have demonstrated that the electrochemical cytosensor has the potential to be a feasible tool for cost-effective cancer cell detection in early cancer diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Biosynthesis and processing of a human T lymphocyte antigen.

PubMed

Bergman, Y; Levy, R

1982-03-01

The biosynthesis and processing of Leu-1, a human T lymphocyte antigen, has been studied with the use of a monoclonal antibody. This molecule exists on the cell surface as a 67,000 m.w. glycoprotein. Through a series of pulse-labeling studies, in conjunction with the use of the antibiotic tunicamycin and the enzyme Endo-H, the details of glycosylation, processing, and deposition at the cell membrane were examined. The protein backbone of the molecule is 58,000 m.w. High-mannose sugars are added to asparagine residues during synthesis. Within 20 min, these high mannose sugars are converted to complex type carbohydrates, including fucose. The fully processed glycoprotein appears at the cell surface within 30 min after synthesis. This sequence of events is similar to that for other cell surface glycoproteins, including HLA and vesicular stomatitus virus glycoprotein.

Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and β-glucosidase.

PubMed

Apiwatanapiwat, Waraporn; Murata, Yoshinori; Kosugi, Akihiko; Yamada, Ryosuke; Kondo, Akihiko; Arai, Takamitsu; Rugthaworn, Prapassorn; Mori, Yutaka

2011-04-01

In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and β-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley β-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.

Surface cell immobilization within perfluoroalkoxy microchannels

NASA Astrophysics Data System (ADS)

Stojkovič, Gorazd; Krivec, Matic; Vesel, Alenka; Marinšek, Marjan; Žnidaršič-Plazl, Polona

2014-11-01

Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor® and Topas®.

Identification of a novel 82 kDa proMMP-9 species associated with the surface of leukaemic cells: (auto-)catalytic activation and resistance to inhibition by TIMP-1

PubMed Central

Ries, Christian; Pitsch, Thomas; Mentele, Reinhard; Zahler, Stefan; Egea, Virginia; Nagase, Hideaki; Jochum, Marianne

2007-01-01

MMP-9 (matrix metalloproteinase 9) plays a critical role in tumour progression. Although the biochemical properties of the secreted form of proMMP-9 are well characterized, little is known about the function and activity of cell surface-associated proMMP-9. We purified a novel 82 kDa species of proMMP-9 from the plasma membrane of THP-1 leukaemic cells, which has substantial differences from the secreted 94 kDa proMMP-9. The 82 kDa form was not detected in the medium even upon stimulation with a phorbol ester. It is truncated by nine amino acid residues at its N-terminus, lacks O-linked oligosaccharides present in the 94 kDa proMMP-9, but retains N-linked carbohydrates. Incubation of 94 kDa proMMP-9 with MMP-3 generated the well-known 82 kDa active form, but the 82 kDa proMMP-9 was converted into an active species of 35 kDa, which was also produced by autocatalytic processing in the absence of activating enzymes. The activated 35 kDa MMP-9 efficiently degraded gelatins, native collagen type IV and fibronectin. The enzyme was less sensitive to TIMP-1 (tissue inhibitor of metalloproteinase 1) inhibition with IC50 values of 82 nM compared with 1 nM for the 82 kDa active MMP-9. The synthetic MMP inhibitor GM6001 blocked the activity of both enzymes, with similar IC50 values below 1 nM. The 82 kDa proMMP-9 is also produced in HL-60 and NB4 leukaemic cell lines as well as ex vivo leukaemic blast cells. It is, however, absent from neutrophils and mononuclear cells isolated from peripheral blood of healthy individuals. Thus, the 82 kDa proMMP-9 expressed on the surface of malignant cells may escape inhibition by natural TIMP-1, thereby facilitating cellular invasion in vivo. PMID:17489740

Tumor Associated Mesenchymal Stromal Cells Show Higher Immunosuppressive and Angiogenic Properties Compared to Adipose Derived MSCs.

PubMed

Langroudi, Ladan; Hassan, Zuhair Muhammad; Soleimani, Masoud; Hashemi, Seyed Mahmoud

2015-12-01

Differentiation, migratory properties and availability of Mesenchymal Stromal Cells (MSC) have become an important part of biomedical research. However, the functional heterogeneity of cells derived from different tissues has hampered providing definitive phenotypic markers for these cells. To characterize and compare the phenotype and cytokines of adipose derived MSCs (AD-MSCs) and tumoral-MSCs (T-MSCs) isolated from mammary tumors of BALB/c mice. Immunophenotyping and in vitro differentiation tests were used for MSC characterization. Cytokine and enzyme profiles were assessed using ELISA and Real-time PCR, respectively. T-MSCs expressed significantly higher levels of HLA-DR (p=0.04). Higher levels of PGE2 and COX-2 enzyme were also observed in T-MSCs (p=0.07 and p=0.00, respectively). Additionally, T-MSCs expressed higher levels of iNOS and MMP9 (p=0.01 and p=0.01, respectively). T-MSCs were also able to induce higher levels of proliferation and migration of HUVEC endothelial cells in wound scratch assay compared to AD-MSCs (p=0.015). Functional differences showed by the surface markers of MSCs, cytokine and enzyme production indicate the effect of different microenvironments on MSCs phenotype and function.

Simultaneous noncontact topography and electrochemical imaging by SECM/SICM featuring ion current feedback regulation.

PubMed

Takahashi, Yasufumi; Shevchuk, Andrew I; Novak, Pavel; Murakami, Yumi; Shiku, Hitoshi; Korchev, Yuri E; Matsue, Tomokazu

2010-07-28

We described a hybrid system of scanning electrochemical microscopy (SECM) and scanning ion conductance microscopy (SICM) with ion current feedback nanopositioning control for simultaneous imaging of noncontact topography and spatial distribution of electrochemical species. A nanopipette/nanoring electrode probe provided submicrometer resolution of the electrochemical measurement on surfaces with complex topology. The SECM/SICM probe had an aperture radius of 220 nm. The inner and outer radii of the SECM Au nanoring electrode were 330 and 550 nm, respectively. Characterization of the probe was performed with scanning electron microscopy (SEM), cyclic voltammetry (CV), and approach curve measurements. SECM/SICM was applied to simultaneous imaging of topography and electrochemical responses of enzymes (horse radish peroxidase (HRP) and glucose oxidase (GOD)) and single live cells (A6 cells, superior cervical ganglion (SCG) cells, and cardiac myocytes). The measurements revealed the distribution of activity of the enzyme spots on uneven surfaces with submicrometer resolution. SECM/SICM acquired high resolution topographic images of cells together with the map of electrochemical signals. This combined technique was also applied to the evaluation of the permeation property of electroactive species through cellular membranes.

Arabidopsis ketoacyl-CoA synthase 16 (KCS16) forms C36 /C38 acyl precursors for leaf trichome and pavement surface wax.

PubMed

Hegebarth, Daniela; Buschhaus, Christopher; Joubès, Jérôme; Thoraval, Didier; Bird, David; Jetter, Reinhard

2017-09-01

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a β-ketoacyl-CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C 35 and C 37 alkanes derived from C 36 and C 38 acyl-CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss-of-function mutants were depleted of C 35 and C 37 alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C 36 and C 38 fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C 34 to C 38 acyl-CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra-long compounds in adjacent pavement cells. © 2017 John Wiley & Sons Ltd.

HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi

PubMed Central

Palmeira, Vanila F.; Alviano, Daniela S.; Braga-Silva, Lys A.; Goulart, Fátima R. V.; Granato, Marcela Q.; Rozental, Sonia; Alviano, Celuta S.; Santos, André L. S.; Kneipp, Lucimar F.

2017-01-01

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics. PMID:28579986

HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi.

PubMed

Palmeira, Vanila F; Alviano, Daniela S; Braga-Silva, Lys A; Goulart, Fátima R V; Granato, Marcela Q; Rozental, Sonia; Alviano, Celuta S; Santos, André L S; Kneipp, Lucimar F

2017-01-01

Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs) currently used in the treatment of human immunodeficiency virus (HIV) have shown anti- F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM) for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi . In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i) reduction on the conidial granularity; (ii) irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii) a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv) inhibition of ergosterol and lanosterol production; (v) reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi) significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics.

The plant cell wall in the feeding sites of cyst nematodes.

PubMed

Bohlmann, Holger; Sobczak, Miroslaw

2014-01-01

Plant parasitic cyst nematodes (genera Heterodera and Globodera) are serious pests for many crops. They enter the host roots as migratory second stage juveniles (J2) and migrate intracellularly toward the vascular cylinder using their stylet and a set of cell wall degrading enzymes produced in the pharyngeal glands. They select an initial syncytial cell (ISC) within the vascular cylinder or inner cortex layers to induce the formation of a multicellular feeding site called a syncytium, which is the only source of nutrients for the parasite during its entire life. A syncytium can consist of more than hundred cells whose protoplasts are fused together through local cell wall dissolutions. While the nematode produces a cocktail of cell wall degrading and modifying enzymes during migration through the root, the cell wall degradations occurring during syncytium development are due to the plants own cell wall modifying and degrading proteins. The outer syncytial cell wall thickens to withstand the increasing osmotic pressure inside the syncytium. Furthermore, pronounced cell wall ingrowths can be formed on the outer syncytial wall at the interface with xylem vessels. They increase the surface of the symplast-apoplast interface, thus enhancing nutrient uptake into the syncytium. Processes of cell wall degradation, synthesis and modification in the syncytium are facilitated by a variety of plant proteins and enzymes including expansins, glucanases, pectate lyases and cellulose synthases, which are produced inside the syncytium or in cells surrounding the syncytium.

Binary actin-ADP-ribosylating toxins and their use as molecular Trojan horses for drug delivery into eukaryotic cells.

PubMed

Barth, Holger; Stiles, Bradley G

2008-01-01

Binary bacterial toxins are unique AB-type toxins, composed of two non-linked proteins that act as a binding/translocation component and an enzyme component. All known actin-ADP-ribosylating toxins from clostridia possess this binary structure. This toxin family is comprised of the prototypical Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, Clostridium difficile CDT, and Clostridium spiroforme toxin. Once in the cytosol of host cells, these toxins transfer an ADP-ribose moiety from nicotinamide-adenosine-dinucleotide onto G-actin that then leads to depolymerization of actin filaments. In recent years much progress has been made towards understanding the cellular uptake mechanism of binary actin-ADP-ribosylating toxins, and in particular that of C2 toxin. Both components act in a precisely concerted manner to intoxicate eukaryotic cells. The binding/translocation (B-) component forms a complex with the enzyme (A-) component and mediates toxin binding to a cell-surface receptor. Following receptor-mediated endocytosis, the enzyme component escapes from acidic endosomes into the cytosol. Acidification of endosomes triggers pore formation by the binding/translocation component in endosomal membranes and the enzyme component subsequently translocates through the pore. This step requires a host cell chaperone, Hsp90. Due to their unique structure, binary toxins are naturally "tailor made" for transporting foreign proteins into the cytosol of host cells. Several highly specific and cell-permeable recombinant fusion proteins have been designed and successfully used in experimental cell research. This review will focus on the recent progress in studying binary actin ADP-ribosylating toxins as highly effective virulence factors and innovative tools for cell physiology as well as pharmacology.

The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

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

Watanabe, H.; Grubb, J.H.; Sly, W.S.

1990-10-01

The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functionalmore » receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.« less

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process.

PubMed

Latka, Agnieszka; Maciejewska, Barbara; Majkowska-Skrobek, Grazyna; Briers, Yves; Drulis-Kawa, Zuzanna

2017-04-01

Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools.

Growth characteristics and enzyme production optimization of lipase Producing Strain

NASA Astrophysics Data System (ADS)

Zheng, Chaocheng

2018-01-01

55 samples from different regions were selected and screened by Rhodamine B flat transparent circle method to observe lipase producing effect, among which, LHY-1, identified as Serratia sp. has the characteristics of fast growth, high enzyme production and stable ability. The colony of this strain is white, the edge is smooth and tidy, the surface is moist, the cell is straight, rod-shaped, gram negative, 0.1-0.2 μm in diameter and, length 0.3-0.5 μm in length.

Dynamic nanoplatforms in biosensor and membrane constitutional systems.

PubMed

Mahon, Eugene; Aastrup, Teodor; Barboiu, Mihail

2012-01-01

Molecular recognition in biological systems occurs mainly at interfacial environments such as membrane surfaces, enzyme active sites, or the interior of the DNA double helix. At the cell membrane surface, carbohydrate-protein recognition principles apply to a range of specific non-covalent interactions including immune response, cell proliferation, adhesion and death, cell-cell interaction and communication. Protein-protein recognition meanwhile accounts for signalling processes and ion channel structure. In this chapter we aim to describe such constitutional dynamic interfaces for biosensing and membrane transport applications. Constitutionally adaptive interfaces may mimic the recognition capabilities intrinsic to natural recognition processes. We present some recent examples of 2D and 3D constructed sensors and membranes of this type and describe their sensing and transport capabilities.

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system.

PubMed

Liang, Xing-xiang; Wang, Bei-bei; Sun, Yu-fei; Lin, Ying; Han, Shuang-yan; Zheng, Sui-ping; Cui, Tang-bing

2013-03-01

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.

Chemiluminescent detection of cell apoptosis enzyme by gold nanoparticle-based resonance energy transfer assay.

PubMed

Huang, Xiangyi; Liang, Yiran; Ruan, Lingao; Ren, Jicun

2014-09-01

We report a new chemiluminescence resonance energy transfer (CRET) technique, using gold nanoparticles (AuNPs) as efficient energy acceptor, for homogeneous measurement of cell apoptosis enzyme with high sensitivity. In the design of the CRET system, we chose the highly sensitive chemiluminescence (CL) reaction between luminol and hydrogen peroxide catalysed by horseradish peroxidase (HRP) because the CL spectrum of luminol (λ max 425 nm) partially overlaps the visible absorption bands of AuNPs. In this system, the peptide substrate (DEVD) of caspase 3 was linked to the AuNP surface by Au-S linkage. HRP was attached to the AuNP surface by means of a bridge formed by the streptavidin-biotin reaction. CRET occurred as a result of formation of AuNP-peptide-biotin-streptavidin-HRP complexes. The CL of luminol was significantly reduced, because of the quenching effect of AuNPs. The quenched CL was recovered after cleavage of DEVD by caspase 3, an enzyme involved in the apoptotic process. Experimental conditions were systematically investigated. Under the optimum conditions the increase of the CL signal was linearly dependent on caspase 3 concentration within the concentration range 25 pmol L(-1) to 800 pmol L(-1) and the detection limit of caspase 3 was as low as 20 pmol L(-1), one order of magnitude lower than for FRET sensors based on graphene oxides. Our method was successfully used to detect drug-induced apoptosis of cells. This approach is expected to be extended to other assays, i.e., using other enzymes, analytes, CL substances, and even other nanoparticles (e.g., quantum dots and graphene).

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

PubMed Central

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

2016-01-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

PubMed

Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Azarnia Tehran, Domenico; Montecucco, Cesare; Barth, Holger

2016-04-01

The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins.

Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix.

PubMed

Gründel, Anne; Jacobs, Enno; Dumke, Roger

2016-12-01

Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae. Copyright © 2016 Elsevier GmbH. All rights reserved.

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.

Cell surface expression of beta 2-microglobulin (beta 2m) correlates with stages of differentiation in B cell tumours.

PubMed Central

Jones, R A; Scott, C S; Norfolk, D R; Stark, A N; Child, J A

1987-01-01

Cell surface beta 2-microglobulin (beta 2m) densities of malignant B cells were determined by enzyme immunoassay in 97 cases of immunologically defined lymphoproliferative disease. Absolute beta 2m densities were found to depend on disease category with the lowest levels found on cells from chronic lymphocytic leukaemia (mean = 5.6 ng/10(6) cells, n = 27); atypical chronic lymphocytic leukaemia (mean = 5.9 ng/10(6) cells, n = 8); and prolymphocytoid chronic lymphocytic leukaemia variant (mean = 6.0 ng/10(6) cells, n = 16). beta 2m densities for B non-Hodgkin's lymphoma (n = 14) and B prolymphocytic leukaemia (n = 17) cases were 8.1 and 10.0 ng/10(6) cells, respectively, and the highest densities were found on cells from "late-B cell" tumours (mean = 14.3 ng/10(6) cells). Plasma cells from cases of Ig secreting tumours expressed unexpectedly low beta 2m densities (mean = 9.3 ng/10(6) cells; n = 6). PMID:3108331

Scientific evidence and rationale for the development of curcumin and resveratrol as nutraceutricals for joint health.

PubMed

Mobasheri, Ali; Henrotin, Yves; Biesalski, Hans-Konrad; Shakibaei, Mehdi

2012-01-01

Interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) are key cytokines that drive the production of inflammatory mediators and matrix-degrading enzymes in osteoarthritis (OA). These proinflammatory cytokines bind to their respective cell surface receptors and activate inflammatory signaling pathways culminating with the activation of nuclear factor κB (NF-κB), a transcription factor that can be triggered by a host of stress-related stimuli including, excessive mechanical stress and ECM degradation products. Once activated, NF-κB regulates the expression of many cytokines, chemokines, adhesion molecules, inflammatory mediators, and several matrix-degrading enzymes. Therefore, proinflammatory cytokines, their cell surface receptors, NF-κB and downstream signaling pathways are therapeutic targets in OA. This paper critically reviews the recent literature and outlines the potential prophylactic properties of plant-derived phytochemicals such as curcumin and resveratrol for targeting NF-κB signaling and inflammation in OA to determine whether these phytochemicals can be used as functional foods.

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.

An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors.

PubMed

Nguyen, Thanh; Ho, Minh; Ghosh, Ambarnil; Kim, Truc; Yun, Sun Il; Lee, Seung Seo; Kim, Kyeong Kyu

2016-10-07

The ubiquitin pathway plays a critical role in regulating diverse biological processes, and its dysregulation is associated with various diseases. Therefore, it is important to have a tool that can control the ubiquitin pathway in order to improve understanding of this pathway and to develop therapeutics against relevant diseases. We found that Chicago Sky Blue 6B binds directly to the β-groove, a major interacting surface of ubiquitin. Hence, it could successfully inhibit the enzymatic activity of ubiquitin processing enzymes and the binding of ubiquitin to the CXCR4, a cell surface ubiquitin receptor. Furthermore, we demonstrated that this ubiquitin binding chemical could effectively suppress the ubiquitin induced cancer cell migration by blocking ubiquitin-CXCR4 interaction. Current results suggest that ubiquitin binding molecules can be developed as inhibitors of ubiquitin-protein interactions, which will have the value not only in unveiling the biological role of ubiquitin but also in treating related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

PubMed

Limsakul, Praopim; Peng, Qin; Wu, Yiqian; Allen, Molly E; Liang, Jing; Remacle, Albert G; Lopez, Tyler; Ge, Xin; Kay, Brian K; Zhao, Huimin; Strongin, Alex Y; Yang, Xiang-Lei; Lu, Shaoying; Wang, Yingxiao

2018-04-19

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Thermoresponsive PNIPAM Coatings on Nanostructured Gratings for Cell Alignment and Release

DOE PAGES

Zhernenkov, Mikhail; Ashkar, Rana; Feng, Hao; ...

2015-05-20

Thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) has been widely used as a surface coating to thermally control the detachment of adsorbed cells without the need for extreme stimuli such as enzyme treatment. Recently, the use of 2D and 3D scaffolds in controlling cell positioning, growth, spreading, and migration has been of a great interest in tissue engineering and cell biology. We use a PNIPAM polymer surface coating atop a nanostructured linear diffraction grating to controllably change the surface topography of 2D linear structures using temperature stimuli. Neutron reflectometry and surface diffraction are utilized to examine the conformity of the polymer coating to themore » grating surface, its hydration profile, and its evolution in response to temperature variations. Our results show that, in the collapsed state, the PNIPAM coating conforms to the grating structures and retains a uniform hydration of 63%. In the swollen state, the polymer expands beyond the grating channels and absorbs up to 87% water. Such properties are particularly desirable for 2D cell growth scaffolds with a built-in nonextreme tissue-release mechanism. Indeed, the current system demonstrates advanced performance in the effective alignment of cultured fibroblast cells and the easy release of the cells upon temperature change.« less

Thermoresponsive PNIPAM Coatings on Nanostructured Gratings for Cell Alignment and Release

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

Zhernenkov, Mikhail; Ashkar, Rana; Feng, Hao

Thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) has been widely used as a surface coating to thermally control the detachment of adsorbed cells without the need for extreme stimuli such as enzyme treatment. Recently, the use of 2D and 3D scaffolds in controlling cell positioning, growth, spreading, and migration has been of a great interest in tissue engineering and cell biology. We use a PNIPAM polymer surface coating atop a nanostructured linear diffraction grating to controllably change the surface topography of 2D linear structures using temperature stimuli. Neutron reflectometry and surface diffraction are utilized to examine the conformity of the polymer coating to themore » grating surface, its hydration profile, and its evolution in response to temperature variations. Our results show that, in the collapsed state, the PNIPAM coating conforms to the grating structures and retains a uniform hydration of 63%. In the swollen state, the polymer expands beyond the grating channels and absorbs up to 87% water. Such properties are particularly desirable for 2D cell growth scaffolds with a built-in nonextreme tissue-release mechanism. Indeed, the current system demonstrates advanced performance in the effective alignment of cultured fibroblast cells and the easy release of the cells upon temperature change.« less

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

CD86 expression as a surrogate cellular biomarker for pharmacological inhibition of the histone demethylase lysine-specific demethylase 1.

PubMed

Lynch, James T; Cockerill, Mark J; Hitchin, James R; Wiseman, Daniel H; Somervaille, Tim C P

2013-11-01

There is a lack of rapid cell-based assays that read out enzymatic inhibition of the histone demethylase LSD1 (lysine-specific demethylase 1). Through transcriptome analysis of human acute myeloid leukemia THP1 cells treated with a tranylcypromine-derivative inhibitor of LSD1 active in the low nanomolar range, we identified the cell surface marker CD86 as a sensitive surrogate biomarker of LSD1 inhibition. Within 24h of enzyme inhibition, there was substantial and dose-dependent up-regulation of CD86 expression, as detected by quantitative polymerase chain reaction, flow cytometry, and enzyme-linked immunosorbent assay. Thus, the use of CD86 expression may facilitate screening of compounds with putative LSD1 inhibitory activities in cellular assays. Copyright © 2013 Elsevier Inc. All rights reserved.

Differential actions of proteinases and neuraminidase on mammalian erythrocyte surface and its impact on erythrocyte agglutination by concanavalin A.

PubMed

Sharma, Savita; Gokhale, Sadashiv M

2012-12-01

Action of proteinases viz. trypsin and chymotrypsin, and neuraminidase on intact erythrocyte membrane proteins and glycophorins (sialoglycoproteins) exposed to cell surface and its impact on lectin (concanavalin A)-mediated agglutination were studied in Homo sapiens (human), Capra aegagrus hircus (goat) and Bubalus bubalis (buffalo). Membrane proteins and glycophorins analysis by SDS-PAGE as visualized by coomassie brilliant blue and periodic acid-schiff stains, respectively, and agglutination behaviour revealed marked differences: 1) there were prominent dissimilarities in the number and molecular weights of glycophorins in human, goat and buffalo erythrocyte membranes; 2) proteinase action(s) on human and buffalo erythrocyte surface membrane proteins and glycophorins showed similarity but was found different in goat; 3) significant differences in erythrocyte agglutinability with concanavalin A can be attributed to differences in membrane composition and alterations in the surface proteins after enzyme treatment; 4) a direct correlation was found between degradation of glycophorins and concanavalin A agglutinability; 5) action of neuraminidase specifically indicated the negative role of cell surface sialic acids in determining concanavalin A agglutinability of goat and buffalo erythrocytes, similar to human. Present studies clearly indicate that there are some basic differences in human, goat and buffalo erythrocyte membrane proteins, especially with respect to glycophorins, which determine the concanavalin A-mediated agglutination in enzyme treated erythrocytes.

Enzymic cross-linkage of monomeric extensin precursors in vitro. [Lycopersicon esculentum

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

Everdeen, D.S.; Kiefer, S.; Willard, J.J.

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, the authors purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers inmore » the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. They therefore identified the cross-linking activity as extensin peroxidase.« less

The external face of Candida albicans: A proteomic view of the cell surface and the extracellular environment.

PubMed

Gil-Bona, Ana; Amador-García, Ahinara; Gil, Concha; Monteoliva, Lucia

2018-05-30

The cell surface and secreted proteins are the initial points of contact between Candida albicans and the host. Improvements in protein extraction approaches and mass spectrometers have allowed researchers to obtain a comprehensive knowledge of these external subproteomes. In this paper, we review the published proteomic studies that have examined C. albicans extracellular proteins, including the cell surface proteins or surfome and the secreted proteins or secretome. The use of different approaches to isolate cell wall and cell surface proteins, such as fractionation approaches or cell shaving, have resulted in different outcomes. Proteins with N-terminal signal peptide, known as classically secreted proteins, and those that lack the signal peptide, known as unconventionally secreted proteins, have been consistently identified. Existing studies on C. albicans extracellular vesicles reveal that they are relevant as an unconventional pathway of protein secretion and can help explain the presence of proteins without a signal peptide, including some moonlighting proteins, in the cell wall and the extracellular environment. According to the global view presented in this review, cell wall proteins, virulence factors such as adhesins or hydrolytic enzymes, metabolic enzymes and stress related-proteins are important groups of proteins in C. albicans surfome and secretome. Candida albicans extracellular proteins are involved in biofilm formation, cell nutrient acquisition and cell wall integrity maintenance. Furthermore, these proteins include virulence factors and immunogenic proteins. This review is of outstanding interest, not only because it extends knowledge of the C. albicans surface and extracellular proteins that could be related with pathogenesis, but also because it presents insights that may facilitate the future development of new antifungal drugs and vaccines and contributes to efforts to identify new biomarkers that can be employed to diagnose candidiasis. Here, we list more than 570 C. albicans proteins that have been identified in extracellular locations to deliver the most extensive catalogue of this type of proteins to date. Moreover, we describe 16 proteins detected at all locations analysed in the works revised. These proteins include the glycophosphatidylinositol (GPI)-anchored proteins Ecm33, Pga4 and Phr2 and unconventional secretory proteins such as Eft2, Eno1, Hsp70, Pdc11, Pgk1 and Tdh3. Furthermore, 13 of these 16 proteins are immunogenic and could represent a set of interesting candidates for biomarker discovery. Copyright © 2017 Elsevier B.V. All rights reserved.

Neuroglian on hemocyte surfaces is involved in homophilic and heterophilic interactions of the innate immune system of Manduca sexta.

PubMed

Zhuang, Shufei; Kelo, Lisha; Nardi, James B; Kanost, Michael R

2007-01-01

Neuroglian, a member of the L1 family of cell adhesion molecules (L1-CAMs), is expressed on surfaces of granular cells and a subset of large plasmatocytes of Manduca sexta that act as foci for hemocyte aggregation during the innate immune response. Neuroglian expressed on surfaces of transfected Sf9 cells induced their homophilic aggregation, with the aggregation being abolished in the presence of recombinant immunoglobulin (Ig) domains of neuroglian. Neuroglian and its Ig domains also can interact with hemocyte-specific integrin (HS integrin) as demonstrated with an enzyme-linked immunoassay and a surface plasmon resonance (SPR) assay. Neuroglian double-stranded (ds) RNA not only depresses expression of neuroglian in hemocytes but also depresses the cell-mediated encapsulation response of these hemocytes to foreign surfaces. After injection of a monoclonal antibody (MAb 3B11) into M. sexta larvae that recognizes the Ig domains of neuroglian, the cell-mediated encapsulation response of hemocytes was likewise inhibited. The Ig domains of neuroglian are involved in both homophilic and heterophilic interactions, and subsets of these six different Ig domains may affect different functions of neuroglian.

Improving genetic immobilization of a cellulase on yeast cell surface for bioethanol production using cellulose.

PubMed

Yang, Jinying; Dang, Hongyue; Lu, Jian Ren

2013-04-01

In this study, Saccharomyces cerevisiae was genetically engineered to harbor the capability of utilizing celluloses for bioethanol production by displaying active cellulolytic enzymes on the cell surface. An endo-1,4-β-glucanase gene egX was cloned from Bacillus pumilus C-9 and its expression products, the EGX cellulases, were displayed on the cell surface of S. cerevisiae by fusing egX with aga2 that encodes the binding subunit of the S. cerevisiae cell wall protein α-agglutinin. To achieve high gene copies and stability, multicopy integration was obtained by integrating the fusion aga2-egX gene into the rDNA region of the S. cerevisiae chromosome. To achieve high expression and surface display efficiency, the aga2-egX gene was expressed under the control of a strong promoter. The presence of the enzymatically active cellulase fusion proteins on the S. cerevisiae cell surface was verified by carboxymethyl cellulase activity assay and immunofluorescence microscopy. This work presented a promising strategy to genetically engineer yeasts to perform efficient fermentation of cellulosic materials for bioethanol production. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evidence for a link between sphingolipid metabolism and expression of CD1d and MHC-class II: monocytes from Gaucher disease patients as a model.

PubMed

Balreira, Andrea; Lacerda, Lúcia; Miranda, Clara Sá; Arosa, Fernando A

2005-06-01

Gaucher disease (GD) is an autosomal recessive inherited defect of the lysosomal enzyme glucocerebrosidase (GluCerase) that leads to glucosylceramide (GluCer) accumulation. We previously demonstrated the existence of imbalances in certain lymphocyte populations in GD patients. We now show that GluCerase-deficient monocytes from GD patients or monocytes from healthy subjects treated with conduritol-B-epoxide (CBE), an irreversible inhibitor of GluCerase activity, display high levels of surface expression of the lipid-binding molecule CD1d. GluCerase-deficient monocytes from GD patients also showed increased surface expression of major histocompatibility complex (MHC)-class II, but not of other lysosomal trafficking molecules, such as CD63 and MHC-class I. However, CD1d and MHC-class II mRNA levels were not increased. GluCerase-deficient monocytes from GD patients undergoing enzyme replacement therapy also exhibited increased levels of CD1d and MHC-class II and imbalances in the percentage of CD4+, CD8+, and Valpha24+ T cells. Interestingly, follow-up studies revealed that enzyme replacement therapy induced a decrease in MHC-class II expression and partial correction of the CD4+ T cell imbalances. These results reveal a new link between sphingolipid accumulation in monocytes and the expression of certain MHC molecules that may result in imbalances of regulatory T cell subsets. These immunological anomalies may contribute to the clinical heterogeneity in GD patients.

Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury.

PubMed

Hasan, Djo; Blankman, Paul; Nieman, Gary F

2017-09-01

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-β, α-SMA, etc.) followed by the establishment of lung fibrosis.

Analysis of telomerase activity based on a spired DNA tetrahedron TS primer.

PubMed

Li, Yan; Wen, Yanli; Wang, Lele; Liang, Wen; Xu, Li; Ren, Shuzhen; Zou, Ziying; Zuo, Xiaolei; Fan, Chunhai; Huang, Qing; Liu, Gang; Jia, Nengqin

2015-05-15

The development of sensitive telomerase biosensors is hindered by the restricted accessibility of telomere strand (TS) primer and the limited enzyme reaction space, which is mainly confined by the vertical distance. In this work, we designed an electrochemical telomerase biosensor based on a spired DNA tetrahedron TS primer (STTS). By adding a rigid dsDNA spire onto the top of the DNA tetrahedron, we successfully regulated the distance between the TS primer and the surface, and thus greatly facilitated the telomerase elongation on surface. The signal-to-noise ratio was 2 times higher than TSP without the spire structure. The limit of detection was calculated to be lower than 10 HeLa cells, which is at least 2 magnitudes lower than other surface extension-based electrochemical telomerase sensors without amplification. The practicability of STTS sensor was also demonstrated by analysing various other cell lines including cancer cells, stem cells of high telomerase activity and somatic cells of low telomerase activity. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro response to alkaline phosphatase coatings immobilized onto titanium implants using electrospray deposition or polydopamine-assisted deposition.

PubMed

Nijhuis, Arnold W G; van den Beucken, Jeroen J J P; Jansen, John A; Leeuwenburgh, Sander C G

2014-04-01

Immobilization of biomolecules onto implant surfaces is one of the most straightforward strategies to control the interaction between an implant and its biological environment. Recently, it was shown that the enzyme alkaline phosphatase (ALP) could be efficiently immobilized onto titanium implants in a single step using polydopamine. We hypothesized that such polydopamine-ALP coatings can enhance the early attachment of cells and increase mineralization. Therefore, the current study aimed at immobilization of ALP onto titanium by means of either one- or two-step polydopamine-assisted immobilization or electrospray deposition, the comparative characterization of these experimental substrates and subsequent cell behavioral analysis using primary osteoblast-like cells. Uncoated titanium and ALP-free polydopamine coatings served as controls. Despite significant ALP surface activity and lower water contact for angles ALP-containing surface modifications, only marginal effects on early cell behavior (i.e., cell spreading) and osteogenic differentiation (i.e., proliferation, differentiation and mineralization) were observed in comparison to uncoated titanium. Copyright © 2013 Wiley Periodicals, Inc.

Phosphorus Availability, Phytoplankton Community Dynamics, and Taxon-Specific Phosphorus Status in the Gulf of Aqaba, Red Sea

NASA Astrophysics Data System (ADS)

Mackey, K. R.; Labiosa, R. G.; Calhoun, M.; Street, J. H.; Post, A. F.; Paytan, A.

2006-12-01

The relationships among phytoplankton taxon-specific phosphorus-status, phytoplankton community composition, and nutrient levels were assessed over three seasons in the Gulf of Aqaba, Red Sea. During summer and fall, stratified surface waters were depleted of nutrients and picophytoplankton populations comprised the majority of cells (80% and 88% respectively). In winter, surface nutrient concentrations were higher and larger phytoplankton were more abundant (63%). Cell specific alkaline phosphatase activity (APA) derived from enzyme labeled fluorescence was consistently low (less than 5%) in the picophytoplankton throughout the year, whereas larger cells expressed elevated APA during the summer and fall but less in the winter. A nutrient addition bioassay during the fall showed that, relative to control, APA was reduced by half in larger cells following addition of orthophosphate, whereas the APA of picophytoplankton remained low (less than 1%) across all treatments and the control. These results indicate that the most abundant phytoplankton are not limited by orthophosphate and only some subpopulations (particularly of larger cells) exhibit orthophosphate-limitation throughout the year. Our results indicate that orthophosphate availability influences phytoplankton ecology, correlating with shifts in phytoplankton community structure and the nutrient status of individual cells. The role of dissolved organic phosphorus as an important phosphorus source for marine phytoplankton in oligotrophic settings and the need for evaluating nutrient limitation at the taxa and/or single cell level (rather than inferring it from nutrient concentrations and ratios or bulk enzyme activity measurements) are highlighted.

Autodisplay of active sorbitol dehydrogenase (SDH) yields a whole cell biocatalyst for the synthesis of rare sugars.

PubMed

Jose, Joachim; von Schwichow, Steffen

2004-04-02

Whole cell biocatalysts are attractive technological tools for the regio- and enantioselective synthesis of products, especially from substrates with several identical reactive groups. In the present study, a whole cell biocatalyst for the synthesis of rare sugars from polyalcohols was constructed. For this purpose, sorbitol dehydrogenase (SDH) from Rhodobacter sphaeroides, a member of the short-chain dehydrogenase/reductase (SDR) family, was expressed on the surface of Escherichia coli using Autodisplay. Autodisplay is an efficient surface display system for Gram-negative bacteria and is based on the autotransporter secretion pathway. Transport of SDH to the outer membrane was monitored by SDS-PAGE and Western blotting of different cell fractions. The surface exposure of the enzyme could be verified by immunofluorescence microscopy and fluorescence activated cell sorting (FACS). The activity of whole cells displaying SDH at the surface was determined in an optical test. Specific activities were found to be 12 mU per 3.3 x 10(8) cells for the conversion of D-glucitol (sorbitol) to D-fructose, 7 mU for the conversion D-galactitol to D-tagatose, and 17 mU for the conversion of L-arabitol to L-ribulose. The whole cell biocatalyst obtained by surface display of SDH could also produce D-glucitol from D-fructose (29 mU per 3.3 x 10(8) cells).

Blood Type Biochemistry and Human Disease

PubMed Central

Ewald, D Rose; Sumner, Susan CJ

2016-01-01

Associations between blood type and disease have been studied since the early 1900s when researchers determined that antibodies and antigens are inherited. In the 1950s, the chemical identification of the carbohydrate structure of surface antigens led to the understanding of biosynthetic pathways. The blood type is defined by oligosaccharide structures, which are specific to the antigens, thus, blood group antigens are secondary gene products, while the primary gene products are various glycosyltransferase enzymes that attach the sugar molecules to the oligosaccharide chain. Blood group antigens are found on red blood cells, platelets, leukocytes, plasma proteins, certain tissues, and various cell surface enzymes, and also exist in soluble form in body secretions such as breast milk, seminal fluid, saliva, sweat, gastric secretions, urine, and amniotic fluid. Recent advances in technology, biochemistry, and genetics have clarified the functional classifications of human blood group antigens, the structure of the A, B, H, and Lewis determinants and the enzymes that produce them, and the association of blood group antigens with disease risks. Further research to identify differences in the biochemical composition of blood group antigens, and the relationship to risks for disease, can be important for the identification of targets for the development of nutritional intervention strategies, or the identification of druggable targets. PMID:27599872

Enhanced extraction of astragalosides from Radix Astragali by negative pressure cavitation-accelerated enzyme pretreatment.

PubMed

Yan, Ming-Ming; Chen, Cai-Yun; Zhao, Bao-Shan; Zu, Yuan-Gang; Fu, Yu-Jie; Liu, Wei; Efferth, Thomas

2010-10-01

The optimal conditions for extraction of astragalosides III and IV (AGs III and IV) in Radix Astragali by negative pressure cavitation-accelerated enzyme pretreatment were studied on the basis of a Box-Behnken design and response surface methodology. Experimental results showed that negative pressure, amount of enzyme and incubation temperature were the main factors governing the enzyme pretreatment of Radix Astragali. The optimum parameters were obtained as follows: negative pressure -0.08 Mpa, amount of enzyme 1.48% (w/w of materials) and incubation temperature 45 degrees C. Under the optimal conditions, the maximal extraction yields of AGs III and IV were 0.103 and 0.325 mg/g, which were 41.67% and 65.31% increased as compared to those without enzyme pretreatment, respectively. The effect of negative pressure cavitation and enzyme pretreatment on the structural changes of plant cells was observed by scanning electron microscopy. In conclusion, negative pressure cavitation-accelerated enzyme pretreatment was proved to be environment-friendly and economical, and could be used in secondary metabolites production. Copyright 2010 Elsevier Ltd. All rights reserved.

Surface phosphatase in Rhinocladiella aquaspersa: biochemical properties and its involvement with adhesion.

PubMed

Kneipp, Lucimar F; Magalhães, Andressa S; Abi-Chacra, Erika A; Souza, Lucieri O P; Alviano, Celuta S; Santos, André L S; Meyer-Fernandes, José R

2012-08-01

Rhinocladiella aquaspersa is an etiologic agent of chromoblastomycosis, a subcutaneous chronic infectious disease. In the present work, we found that the three morphological forms of this fungus (conidia, mycelia and sclerotic bodies) expressed different levels of ecto-phosphatase activity. Our results demonstrated that surface conidial enzyme is an acid phosphatase, inhibited by sodium salts of molybdate, orthovanadate and fluoride and that the inhibition caused by orthovanadate and molybdate was irreversible. The conidial ecto-phosphatase efficiently released phosphate groups from different phosphorylated substrates, causing a higher rate of phosphate removal when p-nitrophenylphosphate was used as substrate. This ecto-enzyme of R. aquaspersa is modulated by Co(2 +) ions and inorganic phosphate (Pi). Accordingly, removal of Pi from the culture medium resulted in a marked (121-fold) increase of ecto-phosphatase activity. Surface phosphatase activity is apparently involved in fungal adhesive properties, since the attachment of R. aquaspersa to epithelial cells was reversed by the pre-treatment of the conidia with orthovanadate, molybdate and anti-phosphatase antibody. Corroborating this finding, conidia with greater ecto-phosphatase activity (grown in Pi-depleted medium) showed higher adherence to epithelial cells than fungi cultivated in the presence of Pi.

Strong Effects of a Shelfbreak Jet on Microbial Enzyme Activities

NASA Astrophysics Data System (ADS)

Hoarfrost, A.; Balmonte, J. P.; Ziervogel, K.; Ghobrial, S.; Gawarkiewicz, G.; Arnosti, C.

2016-02-01

The activities of extracellular enzymes are critical in initiating microbial cycling of organic carbon, yet the dynamics of heterotrophic enzyme activities in marine environments are still poorly understood. Variations at a given site in rates of activity and the spectrum of organic substrates hydrolyzed may depend upon environmental context. We measured the extracellular enzymatic hydrolysis of 13 high- and low-molecular-weight organic substrates in surface and bottom waters along a closely spaced 4-station transect at 71 W on the North Atlantic continental shelf, in the vicinity of the shelfbreak front. This transect intersects a robust upwelling cell that typically shows high biologic productivity, and is locatable by changes in T/S profiles and chl a concentrations along sharp spatial gradients. At the time of sampling, cold pool waters over the continental shelf were relatively cold, 3.5 Deg. C, compared to 12 Deg. C over the upper continental slope. Satellite thermal imagery indicated that shelf water extended offshore and interacted with a large crest of the Gulf Stream. The surface and bottom waters associated with the upwelling jet were characterized by enzyme activities a factor of 20 more rapid than closer inshore waters, and surface water chl a concentrations that were two to three times higher than the inshore waters. The spectrum of enzyme activities also differed markedly between surface and bottom waters both within the jet and at near-shore stations. Microbial extracellular enzymatic activities were strongly influenced by differences in their environmental context along the continental slope and shelfbreak front. Constraining the factors controlling heterotrophic activity across the diverse marine environment is an important step in understanding microbial controls on carbon cycling.

Selective in vivo metabolic cell-labeling-mediated cancer targeting

PubMed Central

Wang, Hua; Wang, Ruibo; Cai, Kaimin; He, Hua; Liu, Yang; Yen, Jonathan; Wang, Zhiyu; Xu, Ming; Sun, Yiwen; Zhou, Xin; Yin, Qian; Tang, Li; Dobrucki, Iwona T; Dobrucki, Lawrence W; Chaney, Eric J; Boppart, Stephen A; Fan, Timothy M; Lezmi, Stéphane; Chen, Xuesi; Yin, Lichen; Cheng, Jianjun

2017-01-01

Distinguishing cancer cells from normal cells through surface receptors is vital for cancer diagnosis and targeted therapy. Metabolic glycoengineering of unnatural sugars provides a powerful tool to manually introduce chemical receptors onto the cell surface; however, cancer-selective labeling still remains a great challenge. Herein we report the design of sugars that can selectively label cancer cells both in vitro and in vivo. Specifically, we inhibit the cell-labeling activity of tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) by converting its anomeric acetyl group to a caged ether bond that can be selectively cleaved by cancer-overexpressed enzymes and thus enables the overexpression of azido groups on the surface of cancer cells. Histone deacetylase and cathepsin L-responsive acetylated azidomannosamine, one such enzymatically activatable Ac4ManAz analog developed, mediated cancer-selective labeling in vivo, which enhanced tumor accumulation of a dibenzocyclooctyne–doxorubicin conjugate via click chemistry and enabled targeted therapy against LS174T colon cancer, MDA-MB-231 triple-negative breast cancer and 4T1 metastatic breast cancer in mice. PMID:28192414

An investigation into the use of SDS-PAGE of cell surface extracts and proteolytic activity to differentiate Prevotella nigrescens and Prevotella intermedia.

PubMed

Cookson, A L; Wray, A; Handley, P S; Jacob, A E

1996-02-15

By comparison of the cell surface proteins derived from the outer membrane and fibrils from 14 Prevotella intermedia and 19 Prevotella nigrescens strains using SDS and analysed by SDS-PAGE, it was possible to distinguish the two species. A polypeptide of approx. 21 kDa distinguished P. intermedia strains, whereas two polypeptides of approx. 18 and 22 kDa could be used to identify P. nigrescens strains. Four other human oral black pigmented bacterial species (Porphyromonas gingivalis, Prevotella denticola, Prevotella loescheii and Prevotella melaninogenica) did not have the 18-, 21- or 22-kDa polypeptides shown by P. intermedia or P. nigrescens. The cell-associated proteolytic activity of eight strains of P. intermedia, 14 strains of P. nigrescens and one strain of P. gingivalis (W50) was assessed using four chromogenic substrates. The hydrolysis of the substrate GPPNA (indicative of dipeptidyl peptidase IV-like activity) and SAAPPNA (elastase-like activity) by P. intermedia strains varied from 32 to 114 units and 0.5 to 12.6 units of activity respectively, where one unit was defined as the amount of protease enzyme catalysing the formation of 1 nmol of p-nitroaniline under experimental conditions. 37.5% (3 of 8) of P. intermedia strains hydrolysed SAAPPNA (chymotrypsin-like enzyme activity) with activities of between 7 and 12 units. The hydrolysis of GPPNA and SAAAPNA by P. nigrescens strains was 32-149 and 3-16 units, respectively. 57% (8 of 14) of P. nigrescens strains hydrolysed SAAPPPNA with activities ranging from 3 to 8 units. None of the P. intermedia or P. nigrescens strains examined were found to have trypsin-like enzyme activity (BAPNA hydrolysis). The GPPNA and SAAAPNA hydrolytic activity associated with the proteases from Porphyromonas gingivalis W50 was at least twice that of P. intermedia and P. nigrescens strains. The similar peptidase activities of P. intermedia and P. nigrescens against chromogenic substrates cannot be used to differentiate the species, but SDS-PAGE of cell surface protein extracts allowed unambiguous speciation between P. intermedia and P. nigrescens. This simple technique of cell surface protein analysis can be performed in most laboratories and offers a convenient way by which to differentiate the two species.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

PubMed Central

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Microbial Biofilm Voltammetry: Direct Electrochemical Characterization of Catalytic Electrode-Attached Biofilms▿ †

PubMed Central

Marsili, Enrico; Rollefson, Janet B.; Baron, Daniel B.; Hozalski, Raymond M.; Bond, Daniel R.

2008-01-01

While electrochemical characterization of enzymes immobilized on electrodes has become common, there is still a need for reliable quantitative methods for study of electron transfer between living cells and conductive surfaces. This work describes growth of thin (<20 μm) Geobacter sulfurreducens biofilms on polished glassy carbon electrodes, using stirred three-electrode anaerobic bioreactors controlled by potentiostats and nondestructive voltammetry techniques for characterization of viable biofilms. Routine in vivo analysis of electron transfer between bacterial cells and electrodes was performed, providing insight into the main redox-active species participating in electron transfer to electrodes. At low scan rates, cyclic voltammetry revealed catalytic electron transfer between cells and the electrode, similar to what has been observed for pure enzymes attached to electrodes under continuous turnover conditions. Differential pulse voltammetry and electrochemical impedance spectroscopy also revealed features that were consistent with electron transfer being mediated by an adsorbed catalyst. Multiple redox-active species were detected, revealing complexity at the outer surfaces of this bacterium. These techniques provide the basis for cataloging quantifiable, defined electron transfer phenotypes as a function of potential, electrode material, growth phase, and culture conditions and provide a framework for comparisons with other species or communities. PMID:18849456

Enzyme-Linked Immunofiltration Assay To Estimate Attachment of Thiobacilli to Pyrite

PubMed Central

Dziurla, Marie-Antoinette; Achouak, Wafa; Lam, Bach-Tuyet; Heulin, Thierry; Berthelin, Jacques

1998-01-01

An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates. The polyclonal antiserum used in this study was raised against T. ferrooxidans DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus Thiobacillus. This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (104 bacteria were detected per well of the microtiter plate). The mean value of bacterial attachment has been estimated to be about 105 bacteria mg−1 of pyrite at a particle size of 56 to 65 μm. The geometric coverage ratio of pyrite by T. ferrooxidans ranged from 0.25 to 2.25%. This suggests an attachment of T. ferrooxidans on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties. ELIFA was shown to be compatible with the measurement of variable levels of adhesion. Therefore, this method may be used to establish adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces. PMID:9687454

CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.

PubMed Central

Shipp, M A; Tarr, G E; Chen, C Y; Switzer, S N; Hersh, L B; Stein, H; Sunday, M E; Reinherz, E L

1991-01-01

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung. Images PMID:1660144

CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.

PubMed

Shipp, M A; Tarr, G E; Chen, C Y; Switzer, S N; Hersh, L B; Stein, H; Sunday, M E; Reinherz, E L

1991-12-01

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung.

Functional roles of cell surface peptidases in reproductive organs

PubMed Central

2004-01-01

A number of biologically active peptides have been proposed to regulate function and differentiation of reproductive organs in an autocrine and/or paracrine fashion. Regulation of the local concentrations of these peptides is one of the important factors influencing their physiological effects on target cells. Membrane‐bound cell surface peptidases can activate or inactivate biologically active peptides before peptide factors access their receptors on the cell surface. Aminopeptidase A (EC 3.4.11.7), placental leucine aminopeptidase (EC 3.4.11.3), aminopeptidase‐N/CD13 (EC 3.4.11.2), dipeptidyl peptidases IV/CD26 (EC.3.4.14.5), carboxypeptidase‐M (EC 3.4.17.12), neutral endopeptidase/CD10 (EC 3.4.24.11) and endothelin converting enzyme‐1 (EC 3.4.23) are differentially expressed on the ovary, endometrium and placenta. The inhibition of enzyme activity affects steroid hormone production by granulosa and thecal cells, decidualization of endometrium and migration of extravillous trophoblasts. These findings suggest that membrane‐bound cell surface peptidases are local regulators for cellular growth and differentiation in reproductive organs by controlling extracellular concentration of peptide factors. (Reprod Med Biol 2004; 3: 165 –176) PMID:29662383

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.

Contribution of extracellular ATP on the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation.

PubMed

Kita, Toshiyuki; Arakaki, Naokatu

2015-01-01

Cell-surface F1F0-ATP synthase was involved in the cell signaling mediating various biological functions. Recently, we found that cell-surface F1F0-ATP synthase plays a role on intracellular triacylglycerol accumulation in adipocytes, and yet, the underlying mechanisms remained largely unknown. In this study, we investigated the role of extracellular ATP on the intracellular triacylglycerol accumulation. We demonstrated that significant amounts of ATP were produced extracellularly by cultured 3T3-L1 adipocytes and that the antibodies against α and β subunits of F1F0-ATP synthase inhibited the extracellular ATP production. Piceatannol, a F1F0-ATP synthase inhibitor, and apyrase, an enzyme which degrades extracellular ATP, suppressed triacylglycerol accumulation. The selective P2Y1 receptor antagonist MRS2500 significantly inhibited triacylglycerol accumulation, whereas the selective P2X receptor antagonist NF279 has less effect. The present results indicate that cell-surface F1F0-ATP synthase on adipocytes is functional in extracellular ATP production and that the extracellular ATP produced contributes, at least in part, to the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation in adipocytes through P2Y1 receptor.

Yeast cell surface display for lipase whole cell catalyst and its applications

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

Liu, Yun; Zhang, Rui; Lian, Zhongshuai

The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chainmore » length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.« less

Titania-polymeric powder coatings with nano-topography support enhanced human mesenchymal cell responses.

PubMed

Mozumder, Mohammad Sayem; Zhu, Jesse; Perinpanayagam, Hiran

2012-10-01

Titanium implant osseointegration is dependent on the cellular response to surface modifications and coatings. Titania-enriched nanocomposite polymeric resin coatings were prepared through the application of advanced ultrafine powder coating technology. Their surfaces were readily modified to create nano-rough (<100 nm) surface nano-topographies that supported human embryonic palatal mesenchymal cell responses. Energy dispersive x-ray spectroscopy confirmed continuous and homogenous coatings with a similar composition and even distribution of titanium. Scanning electron microscopy (SEM) showed complex micro-topographies, and atomic force microscopy revealed intricate nanofeatures and surface roughness. Cell counts, mitochondrial enzyme activity reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to dark purple, SEM, and inverted fluorescence microscopy showed a marked increase in cell attachment, spreading, proliferation, and metabolic activity on the nanostructured surfaces. Reverse Transcription- Polymerase Chain Reaction (RT-PCR) analysis showed that type I collagen and Runx2 expression were induced, and Alizarin red staining showed that mineral deposits were abundant in the cell cultures grown on nanosurfaces. This enhancement in human mesenchymal cell attachment, growth, and osteogenesis were attributed to the nanosized surface topographies, roughness, and moderate wetting characteristics of the coatings. Their dimensional similarity to naturally occurring matrix proteins and crystals, coupled with their increased surface area for protein adsorption, may have facilitated the response. Therefore, this application of ultrafine powder coating technology affords highly biocompatible surfaces that can be readily modified to accentuate the cellular response. Copyright © 2012 Wiley Periodicals, Inc.

Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

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

Zhang, Yun

The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with themore » firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification mainly used a fluorescence method; CL detection is limited because of the difficulty to introduce enough D-luciferin molecules. Since dehydration could easily cause proper size holes in bacterial cell membranes and facilitate D-luciferin diffusion, we used this method and recorded CL from individual cells each hour after induction. The CL light intensity from each individual cell was integrated and gene expression levels of two strain types were compared. Based on our calculation, the overall sensitivity of our system is already approaching the single enzyme level. The median enzyme number inside a single bacterium from the higher expression strain after 2 hours induction was quantified to be about 550 molecules. Finally we imaged ATP release from astrocyte cells. Upon mechanical stimulation, astrocyte cells respond by increasing intracellular Ca 2+ level and releasing ATP to extracellular spaces as signaling molecules. The ATP release imaged by direct CL imaging using free firefly luciferase and D-luciferin outside cells reflects the transient release as well as rapid ATP diffusion. Therefore ATP release detection at the cell surface is critical to study the ATP release mechanism and signaling propagation pathway. We realized this cell surface localized ATP release imaging detection by immobilizing firefly luciferase to streptavidin beads that attached to the cell surface via streptavidin-biotin interactions. Both intracellular Ca 2+ propagation wave and extracellular ATP propagation wave at the cell surface were recorded with fluorescence and CL respectively. The results imply that at close distances from the stimulation center (<120 μm) extracellular ATP pathway is faster, while at long distances (>120 μm) intracellular Ca 2+ signaling through gap junctions seems more effective.« less

New Technologies for Studying Biofilms

PubMed Central

FRANKLIN, MICHAEL J.; CHANG, CONNIE; AKIYAMA, TATSUYA; BOTHNER, BRIAN

2016-01-01

Bacteria have traditionally been studied as single-cell organisms. In laboratory settings, aerobic bacteria are usually cultured in aerated flasks, where the cells are considered essentially homogenous. However, in many natural environments, bacteria and other microorganisms grow in mixed communities, often associated with surfaces. Biofilms are comprised of surface-associated microorganisms, their extracellular matrix material, and environmental chemicals that have adsorbed to the bacteria or their matrix material. While this definition of a biofilm is fairly simple, biofilms are complex and dynamic. Our understanding of the activities of individual biofilm cells and whole biofilm systems has developed rapidly, due in part to advances in molecular, analytical, and imaging tools and the miniaturization of tools designed to characterize biofilms at the enzyme level, cellular level, and systems level. PMID:26350329

Temperature and UV light affect the activity of marine cell-free enzymes

NASA Astrophysics Data System (ADS)

Thomson, Blair; Hepburn, Christopher David; Lamare, Miles; Baltar, Federico

2017-09-01

Microbial extracellular enzymatic activity (EEA) is the rate-limiting step in the degradation of organic matter in the oceans. These extracellular enzymes exist in two forms: cell-bound, which are attached to the microbial cell wall, and cell-free, which are completely free of the cell. Contrary to previous understanding, cell-free extracellular enzymes make up a substantial proportion of the total marine EEA. Little is known about these abundant cell-free enzymes, including what factors control their activity once they are away from their sites (cells). Experiments were run to assess how cell-free enzymes (excluding microbes) respond to ultraviolet radiation (UVR) and temperature manipulations, previously suggested as potential control factors for these enzymes. The experiments were done with New Zealand coastal waters and the enzymes studied were alkaline phosphatase (APase), β-glucosidase, (BGase), and leucine aminopeptidase (LAPase). Environmentally relevant UVR (i.e. in situ UVR levels measured at our site) reduced cell-free enzyme activities by up to 87 % when compared to controls, likely a consequence of photodegradation. This effect of UVR on cell-free enzymes differed depending on the UVR fraction. Ambient levels of UV radiation were shown to reduce the activity of cell-free enzymes for the first time. Elevated temperatures (15 °C) increased the activity of cell-free enzymes by up to 53 % when compared to controls (10 °C), likely by enhancing the catalytic activity of the enzymes. Our results suggest the importance of both UVR and temperature as control mechanisms for cell-free enzymes. Given the projected warming ocean environment and the variable UVR light regime, it is possible that there could be major changes in the cell-free EEA and in the enzymes contribution to organic matter remineralization in the future.

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

PubMed

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Biochemical surface modification of Co-Cr-Mo.

PubMed

Puleo, D A

1996-01-01

Because of the limited mechanical properties of tissue substitutes formed by culturing cells on polymeric scaffolds, other approaches to tissue engineering must be explored for applications that require complete and immediate ability to bear weight, e.g. total joint replacements. Biochemical surface modification offers a way to partially regulate events at the bone-implant interface to obtain preferred tissue responses. Tresyl chloride, gamma-aminopropyltriethoxysilane (APS) and p-nitrophenyl chloroformate (p-NPC) immobilization schemes were used to couple a model enzyme, trypsin, on bulk samples of Co-Cr-Mo. For comparison, samples were simply adsorbed with protein. The three derivatization schemes resulted in different patterns and levels of activity. Tresyl chloride was not effective in immobilizing active enzyme on Co-Cr-Mo. Aqueous silanization with 12.5% APS resulted in optimal immobilized activity. Activity on samples derivatized with 0.65 mg p-NPC cm-2 was four to five times greater than that on samples simple adsorbed with enzyme or optimally derivatized with APS and was about eight times that on tresylated samples. This work demonstrates that, although different methods have different effectiveness, chemical derivatization can be used to alter the amount and/or stability of biomolecules immobilized on the surface of Co-Cr-Mo.

Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

PubMed

Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H; Boilard, Eric

2015-01-01

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

The Mechanosensitive Ca2+ Channel as a Central Regular of Prostate Tumor Cell Migration and Invasiveness

DTIC Science & Technology

2011-04-01

activation still needs to be determined (Strotmann et al. 2000). 7.2.4 The Use of MS Enzyme Inhibitors A further strategy for implicating potential MS...invasiveness and metastatic potential . 1.1 Use patch-clamp/pressure clamp techniques, confocal immunofluorescence, Westerns and surface biotinylation...9. Maroto, R. Kurosky, A. Hamill, O.P. Expression and function of canonical transient recptor potential channels in human prostate tumor cells

Expression profile of senescence-associated beta-galactosidase and activation of telomerase in human ovarian surface epithelial cells undergoing immortalization.

PubMed

Litaker, J R; Pan, J; Cheung, Y; Zhang, D K; Liu, Y; Wong, S C; Wan, T S; Tsao, S W

1998-11-01

Senescence is a specific physiological stage of cells characterized by long population doubling time. It accounts for the inability of normal somatic cells to undergo indefinite cell division. As the number of population doublings increase, cell cycle regulatory mechanisms come into play and signal cells to exit the cell cycle and become senescent. Senescence has been implicated in the aging process and may function as a tumor suppressor mechanism in human cells. The ability to measure the degree of cellular senescence is important in understanding the biological processes regulating cell aging and immortalization. Senescent cells exhibit an enzyme termed senescence-associated histochemical staining. Cells immortalized by viral oncogenes often enter a stage of crisis at the early phase of immortalization. The cells at crisis have a long population doubling time. Cells at the crisis stage resemble senescent cells and the expression of SA- beta-Gal may be used to monitor the process of immortalization. In this study the expression profile of SA-beta-Gal was examined in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalization by the human papilloma viral oncogene E6 and E7 (HPV E6 and E7). Our results showed a low percentage (12.0%) of HOSE 6-3 cells expressing SA-beta-Gal activity at the pre-crisis stage. The percentage of HOSE 6-3 cells expressing SA-beta-Gal activity was highest (39.2%) at the crisis stage. When HOSE 6-3 cells achieved immortalized status there was a sharp decrease in cells (1. 3%) expressing SA-beta-Gal activity. In addition, an inverse relationship between the expression of SA-beta-Gal activity and telomerase activity was noted in cells undergoing immortalization. The results confirm that the SA-beta-Gal enzyme is a good marker for monitoring the population of cells undergoing senescence at different stages of immortalization and that telomerase activation is a characteristic feature of post-crisis cells.

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.

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.

Rebamipide suppresses PolyI:C-stimulated cytokine production in human conjunctival epithelial cells.

PubMed

Ueta, Mayumi; Sotozono, Chie; Yokoi, Norihiko; Kinoshita, Shigeru

2013-09-01

We previously documented that ocular surface epithelial cells could regulate ocular surface inflammation and suggested that, while Toll-like receptor 3 upregulates, EP3, one of the prostaglandin E2 receptors, downregulates ocular surface inflammation. Others reported that rebamipide, a gastroprotective drug, could not only increase the gastric mucus production, but also suppressed gastric mucosal inflammation and that it was dominantly distributed in mucosal tissues. The eyedrop form of rebamipide, approved in Japan for use in the treatment of dry eye diseases, upregulates mucin secretion and production, thereby suppressing superficial punctate keratopathy on the ocular surface of patients with this disease. In the current study, we investigated whether rebamipide has anti- inflammatory effects on the ocular surface. To examine the effects of rebamipide on polyI:C-induced cytokine expression by primary human conjunctival epithelial cells, we used enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction assay. We studied the effects of rebamipide on ocular surface inflammation in our murine experimental allergic conjunctivitis (EAC) model. Rebamipide could suppress polyI:C-induced cytokine production and the expression of mRNAs for CXCL10, CXCL11, RANTES, MCP-1, and IL-6 in human conjunctival epithelial cells. In our EAC model, the topical administration of rebamipide suppressed conjunctival allergic eosinophil infiltration. The topical application of rebamipide on the ocular surface might suppress ocular surface inflammation by suppressing the production of cytokines by ocular surface epithelial cells.

Glycans and cancer: role of N-glycans in cancer biomarker, progression and metastasis, and therapeutics.

PubMed

Taniguchi, Naoyuki; Kizuka, Yasuhiko

2015-01-01

Glycosylation is catalyzed by various glycosyltransferase enzymes which are mostly located in the Golgi apparatus in cells. These enzymes glycosylate various complex carbohydrates such as glycoproteins, glycolipids, and proteoglycans. The enzyme activity of glycosyltransferases and their gene expression are altered in various pathophysiological situations including cancer. Furthermore, the activity of glycosyltransferases is controlled by various factors such as the levels of nucleotide sugars, acceptor substrates, nucleotide sugar transporters, chaperons, and endogenous lectin in cancer cells. The glycosylation results in various functional changes of glycoproteins including cell surface receptors and adhesion molecules such as E-cadherin and integrins. These changes confer the unique characteristic phenotypes associated with cancer cells. Therefore, glycans play key roles in cancer progression and treatment. This review focuses on glycan structures, their biosynthetic glycosyltransferases, and their genes in relation to their biological significance and involvement in cancer, especially cancer biomarkers, epithelial-mesenchymal transition, cancer progression and metastasis, and therapeutics. Major N-glycan branching structures which are directly related to cancer are β1,6-GlcNAc branching, bisecting GlcNAc, and core fucose. These structures are enzymatic products of glycosyltransferases, GnT-V, GnT-III, and Fut8, respectively. The genes encoding these enzymes are designated as MGAT5 (Mgat5), MGAT3 (Mgat3), and FUT8 (Fut8) in humans (mice in parenthesis), respectively. GnT-V is highly associated with cancer metastasis, whereas GnT-III is associated with cancer suppression. Fut8 is involved in expression of cancer biomarker as well as in the treatment of cancer. In addition to these enzymes, GnT-IV and GnT-IX (GnT-Vb) will be also discussed in relation to cancer. © 2015 Elsevier Inc. All rights reserved.

Chemiluminescence assay for the detection of biological warfare agents

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

Langry, K; Horn, J

A chemiluminescent homogeneous immunoassay and a hand-size multiassay reader are described that could be used for detecting biological materials. The special feature of the assay is that it employs two different antibodies that each bind to a unique epitope on the same antigen. Each group of epitope-specific antibodies has linked to it an enzyme of a proximal-enzyme pair. One enzyme of the pair utilizes a substrate in high concentration to produce a second substrate required by the second enzyme. This new substrate enables the second enzyme to function. The reaction of the second enzyme is configured to produce light. Thismore » chemiluminescence is detected with a charge-coupled device (CCD) camera. The proximal pair enzymes must be in close proximity to one another to allow the second enzyme to react with the product of the first enzyme. This only occurs when the enzyme-linked antibodies are attached to the antigen, whether antigen is a single protein with multiple epitopes or the surface of a cell with a variety of different antigens. As a result of their juxtaposition, the enzymes produce light only in the presence of the biological material. A brief description is given as to how this assay could be utilized in a personal bio-agent detector system.« less

Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis

PubMed Central

McIntosh, Avery L.; Senthivinayagam, Subramanian; Moon, Kenneth C.; Gupta, Shipra; Lwande, Joel S.; Murphy, Cameron C.; Storey, Stephen M.

2012-01-01

Despite increasing awareness of the health risks associated with excess lipid storage in cells and tissues, knowledge of events governing lipid exchange at the surface of lipid droplets remains unclear. To address this issue, fluorescence resonance energy transfer (FRET) was performed to examine live cell interactions of Plin2 with lipids involved in maintaining lipid droplet structure and function. FRET efficiencies (E) between CFP-labeled Plin2 and fluorescently labeled phosphatidylcholine, sphingomyelin, stearic acid, and cholesterol were quantitated on a pixel-by-pixel basis to generate FRET image maps that specified areas with high E (>60%) in lipid droplets. The mean E and the distance R between the probes indicated a high yield of energy transfer and demonstrated molecular distances on the order of 44–57 Å, in keeping with direct molecular contact. In contrast, FRET between CFP-Plin2 and Nile red was not detected, indicating that the CFP-Plin2/Nile red interaction was beyond FRET proximity (>100 Å). An examination of the effect of Plin2 on cellular metabolism revealed that triacylglycerol, fatty acid, and cholesteryl ester content increased while diacylglycerol remained constant in CFP-Plin2-overexpressing cells. Total phospholipids also increased, reflecting increased phosphatidylcholine and sphingomyelin. Consistent with these results, expression levels of enzymes involved in triacylglycerol, cholesteryl ester, and phospholipid synthesis were significantly upregulated in CFP-Plin2-expressing cells while those associated with lipolysis either decreased or were unaffected. Taken together, these data show for the first time that Plin2 interacts directly with lipids on the surface of lipid droplets and influences levels of key enzymes and lipids involved in maintaining lipid droplet structure and function. PMID:22744009

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars

PubMed Central

Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

2017-01-01

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2–1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus. PMID:28298443

A four-gene operon in Bacillus cereus produces two rare spore-decorating sugars.

PubMed

Li, Zi; Mukherjee, Thiya; Bowler, Kyle; Namdari, Sholeh; Snow, Zachary; Prestridge, Sarah; Carlton, Alexandra; Bar-Peled, Maor

2017-05-05

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3- C -methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM: C -methyltransferase, and NADPH-dependent CDP-3- C -methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3- C -methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3- C -methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3- C -methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C -methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3- C -methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions.

PubMed

Quan, Yong; Jin, Yisheng; Faria, Teresa N; Tilford, Charles A; He, Aiqing; Wall, Doris A; Smith, Ronald L; Vig, Balvinder S

2012-06-18

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5-7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.

Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions

PubMed Central

Quan, Yong; Jin, Yisheng; Faria, Teresa N.; Tilford, Charles A.; He, Aiqing; Wall, Doris A.; Smith, Ronald L.; Vig, Balvinder S.

2012-01-01

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5–7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells. PMID:24300234

Synthesis of the signal molecule acetylcholine during the developmental cycle of Paramecium primaurelia (Protista, Ciliophora) and its possible function in conjugation.

PubMed

Delmonte Corrado, M U; Politi, H; Ognibene, M; Angelini, C; Trielli, F; Ballarini, P; Falugi, C

2001-06-01

We recently discovered, in mating-competent Paramecium primaurelia, the presence of functionally related molecules of the cholinergic system: the neurotransmitter acetylcholine (ACh), both its nicotinic and muscarinic receptors and its lytic enzyme acetylcholinesterase (AChE). Our results on the inhibition of mating-cell pairing in vivo in mating-competent cells treated with cholinomimetic drugs support the hypothesis that the cholinergic system plays a role in cell-to-cell adhesion. To investigate the possible function of the signal molecule ACh in conjugation in P. primaurelia, we attempted to detect the intracellular sites of ACh synthesis by localizing the ACh biosynthetic enzyme choline acetyltransferase (ChAT). Using immunocytochemical and histochemical methods, we have demonstrated the presence and activity of ChAT principally on the surface membrane of mating-competent cells and of mature but non-mating-competent cells. No evidence for ChAT activity was found in immature cells. Immunoblot analysis revealed the presence of immunoreactive bands, ranging in molecular mass from 42 to 133 kDa, as reported for ChAT isolated from higher organisms. In vivo experiments showed that inhibition of ChAT activity by Congo Red, known to be a potent competitive inhibitor of acetyl coenzyme A, did not affect mating-cell pairing. Conversely, inhibition of AChE with BW 284c51 or eserine, which block enzyme activity by reacting with a specific serine within the catalytic centre, significantly inhibited mating-cell pairing. Our results suggest that ACh has a negative modulating effect on conjugation in P. primaurelia.

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

Glucose Fuel Cells with a MicroChannel Fabricated on Flexible Polyimide Film

NASA Astrophysics Data System (ADS)

Sano, Ryohei; Fukushi, Yudai; Sasaki, Tsubasa; Mogi, Hiroshi; Koide, Syohei; Ikoma, Ryuta; Akatsuka, Wataru; Tsujimura, Seiya; Nishioka, Yasushiro

2013-12-01

In this work, a glucose fuel cell was fabricated using microfabrication processes assigned for microelectromechanical systems. The fuel cell was equipped with a microchannel to flow an aqueous solution of glucose. The cell was fabricated on a flexible polyimide substrate, and its porous carbon-coated aluminum (Al) electrodes of 2.8 mm in width and 11 mm in length were formed using photolithography and screen printing techniques. Porous carbon was deposited by screen printing of carbon black ink on the Al electrode surfaces in order to increase the effective electrode surface area and to absorb more enzymes on the electrode surfaces. The microchannel with a depth of 200 μm was fabricated using a hot embossing technique. A maximum power of 0.45 μW at 0.5 V that corresponds to a power density of 1.45 μW/cm2 was realized by introducing a 200 mM concentrated glucose solution at room temperature.

Application of biofilm bioreactors in white biotechnology.

PubMed

Muffler, K; Lakatos, M; Schlegel, C; Strieth, D; Kuhne, S; Ulber, R

2014-01-01

The production of valuable compounds in industrial biotechnology is commonly done by cultivation of suspended cells or use of (immobilized) enzymes rather than using microorganisms in an immobilized state. Within the field of wastewater as well as odor treatment the application of immobilized cells is a proven technique. The cells are entrapped in a matrix of extracellular polymeric compounds produced by themselves. The surface-associated agglomerate of encapsulated cells is termed biofilm. In comparison to common immobilization techniques, toxic effects of compounds used for cell entrapment may be neglected. Although the economic impact of biofilm processes used for the production of valuable compounds is negligible, many prospective approaches were examined in the laboratory and on a pilot scale. This review gives an overview of biofilm reactors applied to the production of valuable compounds. Moreover, the characteristics of the utilized materials are discussed with respect to support of surface-attached microbial growth.

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

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

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

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 Structure of Yeast Protoplasts

PubMed Central

Streiblová, Eva

1968-01-01

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated. Images PMID:4867751

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.

Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

PubMed Central

Owen, Caroline A.

2008-01-01

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

Convection-driven aggregation of micron sized capsules

NASA Astrophysics Data System (ADS)

Shklyaev, Oleg; Shum, Henry; Balazs, Anna

Collective dynamics of microcapsules often serve as a model for understanding behavior observed in colonies of biological cells. Using computer simulations, we explore the capability of chemically generated convection to assemble microcapsules into a colony with neighbors close enough to facilitate chemical communication. The microcapsules are assumed to carry a supply of chemical fuel. When this fuel, leaking out of the capsules, reacts at enzyme-covered sites of the chamber, the reaction generates fluid density variations driving flows. These flows carry the microcapsules, which tend to aggregate into colonies on and near the enzyme-covered sites. This aggregation continues until the reagent has been depleted and convection stops. We show that capsule colonies of predesigned shapes can be assembled by patterning the enzyme-covered surface.

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.

Inhibiting the Epidermal Growth Factor Receptor | Center for Cancer Research

Cancer.gov

The Epidermal Growth Factor Receptor (EGFR) is a widely distributed cell surface receptor that responds to several extracellular signaling molecules through an intracellular tyrosine kinase, which phosphorylates target enzymes to trigger a downstream molecular cascade. Since the discovery that EGFR mutations and amplifications are critical in a number of cancers, efforts have

Kinase cogs go forward and reverse in the Wnt signaling machine.

PubMed

Dale, Trevor

2006-01-01

An important link between Wnt binding at the cell surface and nuclear -catenin-TCF-dependent transcription has been made with the identification of kinases that promote the association of the Wnt receptor and -catenin turnover complexes. Surprisingly, the enzymes implicated had previously been suggested to inhibit rather than promote Wnt signaling.

Molecular Cloning and Sequence of Channel Catfish (Ictalurus punctatus, Rafinesque 1818) Cathepsin S gene

USDA-ARS?s Scientific Manuscript database

Cathepsin S is a lysosomal cysteine endopeptidase of the papain family. This enzyme digests the invariant chain molecules so that antigenic peptides are able to load on the class II-associated invariant chain peptide of MHC. The complexes can subsequently be presented to the CD4 cell surface. In ...

The multifunctional application of microfluidic lab-on-a-chip surface enhanced Raman spectroscopy (LOC-SERS) within the field of bioanalytics

NASA Astrophysics Data System (ADS)

März, Anne; Mönch, Bettina; Walter, Angela; Bocklitz, Thomas; Schumacher, Wilm; Rösch, Petra; Kiehntopf, Michael; Popp, Jürgen

2011-07-01

This contribution will present a variety of applications of lab-on-a-chip surface enhanced Raman spectroscopy in the field of bioanalytic. Beside the quantification and online monitoring of drugs and pharmaceuticals, determination of enzyme activity and discrimination of bacteria are successfully carried out utilizing LOC-SERS. The online-monitoring of drugs using SERS in a microfluidic device is demonstrated for nicotine. The enzyme activity of thiopurine methyltransferase (TPMT) in lysed red blood cells is determined by SERS in a lab-on-a-chip device. To analyse the activity of TPMT the metabolism of 6-mercaptopurine to 6-methylmercaptopurine is investigated. The discrimination of bacteria on strain level is carried out with different E. coli strains. For the investigations, the bacteria are busted by ultra sonic to achieve a high information output. This sample preparation provides the possibility to detect SERS spectra containing information of the bacterial cell walls as well as of the cytoplasm. This contribution demonstrates the great potential of LOC-SERS in the field of bioanalytics.

Lectin histochemistry and alkaline phosphatase activity in the pia mater vessels of spontaneously hypertensive rats (SHR).

PubMed

Szumańska, G; Gadamski, R

1992-01-01

Some lectins were used to study the localization of sugar residues on the endothelial cell surface in the pia mater blood vessels of control (WKY) and hypertensive rats (SHR). The lectins tested recognized the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA-1), alpha-L-fucosyl (Ulex europaeus agglutinin, UEA-1), N-acetylglucosaminyl and sialyl (Wheat germ agglutinin, WGA), N-glycolyl-neuraminic acid (Limax flavus agglutinin, LFA), and N-acetyl-D-galactosaminyl (Helix pomatia agglutinin, HPA). Several differences were revealed in the presence of sugar receptors on the surface of endothelial cells between the control and the hypertensive rats. Our studies showed also differences in the localization of the tested glycoconjugates between pial capillaries, small, medium-size and large pial arteries. The histochemical evaluation of alkaline phosphatase revealed an increased activity of the enzyme in the pial vessels of SHRs as compared with control rats with a similar localization of the enzyme activity. Some differences in the distribution of lectin binding sites and alkaline phosphatase activity could be associated with the different functions of particular segments of the pial vascular network.

Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice.

PubMed

Bhaumik, S; Lewis, X Z; Gambhir, S S

2004-01-01

We have recently demonstrated that Renilla luciferase (Rluc) is a promising bioluminescence reporter gene that can be used for noninvasive optical imaging of reporter gene expression in living mice, with the aid of a cooled charged couple device (CCD) camera. In the current study, we explore the expression of a novel synthetic Renilla luciferase reporter gene (hRluc) in living mice, which has previously been reported to be a more sensitive reporter than native Rluc in mammalian cells. We explore the strategies of simultaneous imaging of both Renilla luciferase enzyme (RL) and synthetic Renilla luciferase enzyme (hRL):coelenterazine (substrate for RL/hRL) in the same living mouse. We also demonstrate that hRL:coelenterazine can yield a higher signal when compared to Firefly luciferase enzyme (FL): D-Luciferin, both in cell culture studies and when imaged from cells at the surface and from lungs of living mice. These studies demonstrate that hRluc should be a useful primary reporter gene with high sensitivity when used alone or in conjunction with other bioluminescence reporter genes for imaging in living rodents. (c) 2004 Society of Photo-Optical Instrumentation Engineers.

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

PubMed

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Angiotensin I-converting enzyme Gln1069Arg mutation impairs trafficking to the cell surface resulting in selective denaturation of the C-domain.

PubMed

Danilov, Sergei M; Kalinin, Sergey; Chen, Zhenlong; Vinokour, Elena I; Nesterovitch, Andrew B; Schwartz, David E; Gribouval, Olivier; Gubler, Marie-Claire; Minshall, Richard D

2010-05-03

Angiotensin-converting enzyme (ACE; Kininase II; CD143) hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD), a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death. Patient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R) resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT) vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10-20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate) or pharmacological (ACE inhibitor) chaperones with proteasome inhibitors (MG 132 or bortezomib) significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold. Homozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as concentration of primary urine.

Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A

2015-09-23

The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations.

THE ALTERATION OF INTRACELLULAR ENZYMES

PubMed Central

Kaplan, J. Gordin

1954-01-01

1. The ability of homologous series of alcohols, ketones, and aldehydes to cause alteration of intracellular catalase increases approximately threefold for each methylene group added, thus following Traube's rule. Equiactive concentrations of alcohols (methanol to octanol) varied over a 4,000-fold range, yet the average corresponding surface tension was 42 ± 2 dynes/cm., that for ketones 43 ± 2, and for aldehydes (above C1) 41 ± 3. 2. Above C8 the altering activity of alcohols ceased to follow Traube's rule, and at C18 was nil. Yet the surface activities of alcohols from nonanol to dodecanol did follow Traube's rule. These two facts show that the interface which is being affected by these agents is not the cell surface, for if it were, altering activity should not fall off between C9 and C12 where surface activity is undiminished; they show also that micelle formation by short range association of hydrocarbon "tails," usually invoked to explain decrease in biological activity of compounds above C8, is not responsible for this effect in these experiments, in which permeability of the cell membrane probably is involved. 3. The most soluble alcohols and aldehydes (alcohols C1 to C8; aldehydes C1, C2), but not ketones, cause, above optimal concentration, an irreversible inhibition of yeast catalase. 4. The critical concentration of altering agent (i.e., that concentration just sufficient to cause doubling of the catalase activity of the yeast suspension) was independent of the concentration of the yeast cells. 5. Viability studies show that the number of yeast cells killed by the altering agents was not related to the degree of activation of the catalase produced. While all the cells were invariably killed by concentrations of altering agent which produced complete activation, all the cells had been killed by concentrations which were insufficient to cause more than 50 per cent maximal activation. Further, the evidence suggested that the catalase may be partially activated by concentrations of altering agent which cause no decrease in viability at all. Hence alteration, unlike death, may not be all-or-none per cell. 6. The fact that the biological criterion being examined was the activation of a water-soluble enzyme rules out the possibility that the reason for the logarithmic increase in altering activity with chain length was increase in concentration of the altering agent in some intracellular fat phase. It is concluded that these surface-active agents cause enzyme alteration by becoming adsorbed at some intracellular interface and thus causing, directly or indirectly, the modification of catalase properties. 7. It is considered that these data support, but do not provide critical proof for, the interfacial hypothesis, which states that catalase is present at the intracellular interface in question, but is desorbed into solution as a consequence of the alteration process. PMID:13211996

The construction, fouling and enzymatic cleaning of a textile dye surface.

PubMed

Onaizi, Sagheer A; He, Lizhong; Middelberg, Anton P J

2010-11-01

The enzymatic cleaning of a rubisco protein stain bound onto Surface Plasmon Resonance (SPR) biosensor chips having a dye-bound upper layer is investigated. This novel method allowed, for the first time, a detailed kinetic study of rubisco cleanability (defined as fraction of adsorbed protein removed from a surface) from dyed surfaces (mimicking fabrics) at different enzyme concentrations. Analysis of kinetic data using an established mathematical model able to decouple enzyme transfer and reaction processes [Onaizi, He, Middelberg, Chem. Eng. Sci. 64 (2008) 3868] revealed a striking effect of dyeing on enzymatic cleaning performance. Specifically, the absolute rate constants for enzyme transfer to and from a dye-bound rubisco stain were significantly higher than reported previously for un-dyed surfaces. These increased transfer rates resulted in higher surface cleanability. Higher enzyme mobility (i.e., higher enzyme adsorption and desorption rates) at the liquid-dye interface was observed, consistent with previous suggestions that enzyme surface mobility is likely correlated with overall enzyme cleaning performance. Our results show that reaction engineering models of enzymatic action at surfaces may provide insight able to guide the design of better stain-resistant surfaces, and may also guide efforts to improve cleaning formulations. Copyright 2010 Elsevier Inc. All rights reserved.

Mycobacterial lipolytic enzymes: a gold mine for tuberculosis research.

PubMed

Dedieu, L; Serveau-Avesque, C; Kremer, L; Canaan, S

2013-01-01

Tuberculosis (TB) is one of the deadliest infectious diseases worldwide with a strong impact in developing countries. Mycobacterium tuberculosis, the etiological agent of TB, has a high capacity to evade the host immune system and establish a chronic, asymptomatic and latent infection. In a latent TB infection, persistent bacilli are present in a non-replicating dormant state within host granulomas. During reactivation, bacilli start replicating again leading to an active TB infection that can be highly contagious. Mycobacterial lipids and lipolytic enzymes are thought to play important physiological roles during dormancy and reactivation. The role of lipolytic enzymes in the physiology of M. tuberculosis and physiopathology of the disease will be discussed in this review, with an emphasis on the secreted or cell wall-associated, surface exposed lipolytic enzymes characterized to date. Studies on the localization, enzymatic activity and immunological properties of these enzymes highlighted their possible usefulness as new diagnostic markers in the fight against TB. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

A three-dimensional nitrogen-doped graphene structure: a highly efficient carrier of enzymes for biosensors

NASA Astrophysics Data System (ADS)

Guo, Jingxing; Zhang, Tao; Hu, Chengguo; Fu, Lei

2015-01-01

In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 μA mM-1 m-2), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells.In recent years, graphene-based enzyme biosensors have received considerable attention due to their excellent performance. Enormous efforts have been made to utilize graphene oxide and its derivatives as carriers of enzymes for biosensing. However, the performance of these sensors is limited by the drawbacks of graphene oxide such as slow electron transfer rate, low catalytic area and poor conductivity. Here, we report a new graphene-based enzyme carrier, i.e. a highly conductive 3D nitrogen-doped graphene structure (3D-NG) grown by chemical vapour deposition, for highly effective enzyme-based biosensors. Owing to the high conductivity, large porosity and tunable nitrogen-doping ratio, this kind of graphene framework shows outstanding electrical properties and a large surface area for enzyme loading and biocatalytic reactions. Using glucose oxidase (GOx) as a model enzyme and chitosan (CS) as an efficient molecular binder of the enzyme, our 3D-NG based biosensors show extremely high sensitivity for the sensing of glucose (226.24 μA mM-1 m-2), which is almost an order of magnitude higher than those reported in most of the previous studies. The stable adsorption and outstanding direct electrochemical behaviour of the enzyme on the nanocomposite indicate the promising application of this 3D enzyme carrier in high-performance electrochemical biosensors or biofuel cells. Electronic supplementary information (ESI) available: Procedures for CVD growth of 3D-NG, XRD and TEM measurements, a comparison with other graphene-based biosensors, a detailed study on the universality of 3D-NG as an enzyme carrier and more CV data on selectivity and stability. See DOI: 10.1039/c4nr05325g

A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro

PubMed Central

Zhang, Xiaoqian; Lu, Juan; He, Bin; Tang, Lingling; Liu, Xiaoli; Zhu, Danhua; Cao, Hongcui; Wang, Yingjie; Li, Lanjuan

2017-01-01

Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotransformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two-dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases. PMID:27959388

A tryptophan derivative, ITE, enhances liver cell metabolic functions in vitro.

PubMed

Zhang, Xiaoqian; Lu, Juan; He, Bin; Tang, Lingling; Liu, Xiaoli; Zhu, Danhua; Cao, Hongcui; Wang, Yingjie; Li, Lanjuan

2017-01-01

Cell encapsulation provides a three-dimensional support by incorporating isolated cells into microcapsules with the goal of simultaneously maintaining cell survival and function, as well as providing active transport for a bioreactor in vitro similarly to that observed in vivo. However, the biotra-nsformation and metabolic functions of the encapsulated cells are not satisfactory for clinical applications. For this purpose, in this study, hepatoma-derived Huh7 cells/C3A cells were treated with 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous non-toxic ligand for aryl hydrocarbon receptor, in monolayer cultures and on microspheres. The mRNA and protein levels, as well as the metabolic activities of drug metabolizing enzymes, albumin secretion and urea synthesis were determined. When the Huh7 and C3A cells cultured in a monolayer on two‑dimensional surfaces, ITE enhanced the protein levels and the metabolic activities of the major cytochrome P450 (CYP450) enzymes, CYP1A1, CYP1A2, CYP3A4 and CYP1B1, and slightly increased albumin secretion and urea synthesis. Moreover, when cultured on microspheres, ITE also substantially increased the protein levels and metabolic activities of CYP1A1, CYP1A2, CYP3A4 and CYP1B1 in both liver cell lines. On the whole, our findings indicate that ITE enhances the enzymatic activities of major CYP450 enzymes and the metabolic functions of liver cells cultured in monolayer or on microspheres, indicating that it may be utilized to improve the functions of hepatocytes. Thus, it may be used in the future for the treatment of liver diseases.

Cytochemical Changes in Hepatocytes of Rats with Endotoxemia and Sepsis: Localization of Fibronectin, Calcium, and Enzymes

DTIC Science & Technology

1988-01-01

rate was deposited predominantly on the outer surfaces of in the pathogn~nesis of endotoxernia and septic shock. The the RER of hepatocytes. in additron...es; and (c) tin it- the basal (perisinusoidal) surfaces and in the cisternae G-6-Pase activity. LPS treatment also leads to reduced num- of tough...by Kupffer cells (Cook et al., 1985’ Gut et al., sue fibronectin results in widening intercellular junctions anci in- 198: ~nkaa ndIwsk,18

Sulfated Glycans and Related Digestive Enzymes in the Zika Virus Infectivity: Potential Mechanisms of Virus-Host Interaction and Perspectives in Drug Discovery.

PubMed

Pomin, Vitor H

2017-01-01

As broadly reported, there is an ongoing Zika virus (ZIKV) outbreak in countries of Latin America. Recent findings have demonstrated that ZIKV causes severe defects on the neural development in fetuses in utero and newborns. Very little is known about the molecular mechanisms involved in the ZIKV infectivity. Potential therapeutic agents are also under investigation. In this report, the possible mechanisms of action played by glycosaminoglycans (GAGs) displayed at the surface proteoglycans of host cells, and likely in charge of interactions with surface proteins of the ZIKV, are highlighted. As is common for the most viruses, these sulfated glycans serve as receptors for virus attachment onto the host cells and consequential entry during infection. The applications of (1) exogenous sulfated glycans of different origins and chemical structures capable of competing with the virus attachment receptors (supposedly GAGs) and (2) GAG-degrading enzymes able to digest the virus attachment receptors on the cells may be therapeutically beneficial as anti-ZIKV. This communication attempts, therefore, to offer some guidance for the future research programs aimed to unveil the molecular mechanisms underlying the ZIKV infectivity and to develop therapeutics capable of decreasing the devastating consequences caused by ZIKV outbreak in the Americas.

Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry.

PubMed

Greis, Kenneth D; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-08-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

PubMed

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a serious fungal pathogen that kills over 600,000 people annually. It converts most of its chitin, a cell wall polysaccharide, to chitosan, which is necessary for virulence. Chitin deacetylase enzymes have been identified in the cell wall, and our studies were undertaken to understand how the deacetylase is linked to the wall and where it has activity. Our results have implications for the current model of chitosan biosynthesis and further challenge the paradigm of covalent linkages between cell wall proteins and polysaccharides through a lipid modification of the protein.

Glycosylation controls cooperative PECAM-VEGFR2-β3 integrin functions at the endothelial surface for tumor angiogenesis.

PubMed

Imamaki, Rie; Ogawa, Kazuko; Kizuka, Yasuhiko; Komi, Yusuke; Kojima, Soichi; Kotani, Norihiro; Honke, Koichi; Honda, Takashi; Taniguchi, Naoyuki; Kitazume, Shinobu

2018-05-02

Most of the angiogenesis inhibitors clinically used in cancer treatment target the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway. However, the current strategies for treating angiogenesis have limited efficacy. The issue of how to treat angiogenesis and endothelial dysfunction in cancer remains a matter of substantial debate. Here we demonstrate a glycosylation-dependent regulatory mechanism for tumor angiogenesis. St6gal1 -/- mice, lacking the α2,6-sialylation enzyme, were shown to exhibit impaired tumor angiogenesis through enhanced endothelial apoptosis. In a previous study, St6gal1 -/- endothelial cells exhibited a reduction in the cell surface residency of platelet endothelial cell adhesion molecule (PECAM). In this study, we found that cooperative functionality of PECAM-VEGFR2-integrin β3 was disturbed in St6gal1 -/- mice. First, cell surface PECAM-VEGFR2 complexes were lost, and both VEGFR2 internalization and the VEGFR-dependent signaling pathway were enhanced. Second, enhanced anoikis was observed, suggesting that the absence of α2,6-sialic acid leads to dysregulated integrin signaling. Notably, ectopic expression of PECAM increased cell surface integrin-β3, indicating that the reduction of cell surface integrin-β3 involves loss-of-endothelial PECAM. The results suggest that the cell surface stability of these glycoproteins is significantly reduced by the lack of α2,6-sialic acid, leading to abnormal signal transduction. The present findings highlight that α2,6-sialylation is critically involved in endothelial survival by controlling the cell surface stability and signal transduction of angiogenic molecules, and could be a novel target for anti-angiogenesis therapy.

The carboxypeptidase angiotensin converting enzyme (ACE) shapes the MHC class I peptide repertoire

PubMed Central

Shen, Xiao Z.; Billet, Sandrine; Lin, Chentao; Okwan-Duodu, Derick; Chen, Xu; Lukacher, Aron E.; Bernstein, Kenneth E.

2011-01-01

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8+ T cell mediated adaptive immune responses. Aminopeptidases are implicated in the editing of peptides for MHC class I loading, but C-terminal editing is thought due to proteasome cleavage. By comparing genetically deficient, wild-type and over-expressing mice, we now identify the dipeptidase angiotensin-converting enzyme (ACE) as playing a physiologic role in peptide processing for MHC class I. ACE edits the C-termini of proteasome-produced class I peptides. The lack of ACE exposes novel antigens but also abrogates some self-antigens. ACE has major effects on surface MHC class I expression in a haplotype-dependent manner. We propose a revised model of MHC class I peptide processing by introducing carboxypeptidase activity. PMID:21964607

Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

PubMed

Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

2017-11-22

Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

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".

Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

NASA Astrophysics Data System (ADS)

Liu, Jianguo; Zhang, Xiaoli; Sun, Yanhong; Lin, Wei

2010-01-01

The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O{2/-}). The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H. pluvialis during exposure to reactive oxygen species (ROS) such as O{2/-}. Astaxanthin reacted with ROS much faster than did the protective enzymes, and had the strongest antioxidative capacity to protect against lipid peroxidation. The defensive mechanisms varied significantly between the three cell types and were related to the level of astaxanthin that had accumulated in those cells. Astaxanthin-enriched red cells had the strongest antioxidative capacity, followed by brown cells, and astaxanthin-deficient green cells. Although there was no significant increase in expression of protective enzymes, the malondialdehyde (MDA) content in red cells was sustained at a low level because of the antioxidative effect of astaxanthin, which quenched O{2/-} before the protective enzymes could act. In green cells, astaxanthin is very low or absent; therefore, scavenging of ROS is inevitably reliant on antioxidative enzymes. Accordingly, in green cells, these enzymes play the leading role in scavenging ROS, and the expression of these enzymes is rapidly increased to reduce excessive ROS. However, because ROS were constantly increased in this study, the enhance enzyme activity in the green cells was not able to repair the ROS damage, leading to elevated MDA content. Of the four defensive enzymes measured in astaxanthin-deficient green cells, SOD eliminates O{2/-}, POD eliminates H2O2, which is a by-product of SOD activity, and APX and CAT are then initiated to scavenge excessive ROS.

Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

PubMed

Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

2013-06-01

A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

PubMed

Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

2017-01-31

Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement). Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10 5 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Multiwalled carbon nanotube coated polyester fabric as textile based flexible counter electrode for dye sensitized solar cell.

PubMed

Arbab, Alvira Ayoub; Sun, Kyung Chul; Sahito, Iftikhar Ali; Qadir, Muhammad Bilal; Jeong, Sung Hoon

2015-05-21

Textile wearable electronics offers the combined advantages of both electronics and textile characteristics. The essential properties of these flexible electronics such as lightweight, stretchable, and wearable power sources are in strong demand. Here, we have developed a facile route to fabricate multi walled carbon nanotube (MWCNT) coated polyester fabric as a flexible counter electrode (CE) for dye sensitized solar cells (DSSCs). A variety of MWCNT and enzymes with different structures were used to generate individual enzyme-dispersed MWCNT (E-MWCNT) suspensions by non-covalent functionalization. A highly concentrated colloidal suspension of E-MWCNT was deposited on polyester fabric via a simple tape casting method using an air drying technique. In view of the E-MWCNT coating, the surface structure is represented by topologically randomly assembled tubular graphene units. This surface morphology has a high density of colloidal edge states and oxygen-containing surface groups which execute multiple catalytic sites for iodide reduction. A highly conductive E-MWCNT coated fabric electrode with a surface resistance of 15 Ω sq(-1) demonstrated 5.69% power conversion efficiency (PCE) when used as a flexible CE for DSSCs. High photo voltaic performance of our suggested system of E-MWCNT fabric-based DSSCs is associated with high sheet conductivity, low charge transfer resistance (RCT), and excellent electro catalytic activity (ECA). Such a conductive fabric demonstrated stable conductivity against bending cycles and strong mechanical adhesion of E-MWCNT on polyester fabric. Moreover, the polyester fabric is hydrophobic and, therefore, has good sealing capacity and retains the polymer gel electrolyte without seepage. This facile E-MWCNT fabric CE configuration provides a concrete fundamental background towards the development of textile-integrated solar cells.

Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.

PubMed

Villanueva-Cabello, Tania M; Mollicone, Rosella; Cruz-Muñoz, Mario E; López-Guerrero, Delia V; Martínez-Duncker, Iván

2015-12-01

CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigen-presenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaα2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The role of electrostatic interactions in protease surface diffusion and the consequence for interfacial biocatalysis.

PubMed

Feller, Bob E; Kellis, James T; Cascão-Pereira, Luis G; Robertson, Channing R; Frank, Curtis W

2010-12-21

This study examines the influence of electrostatic interactions on enzyme surface diffusion and the contribution of diffusion to interfacial biocatalysis. Surface diffusion, adsorption, and reaction were investigated on an immobilized bovine serum albumin (BSA) multilayer substrate over a range of solution ionic strength values. Interfacial charge of the enzyme and substrate surface was maintained by performing the measurements at a fixed pH; therefore, electrostatic interactions were manipulated by changing the ionic strength. The interfacial processes were investigated using a combination of techniques: fluorescence recovery after photobleaching, surface plasmon resonance, and surface plasmon fluorescence spectroscopy. We used an enzyme charge ladder with a net charge ranging from -2 to +4 with respect to the parent to systematically probe the contribution of electrostatics in interfacial enzyme biocatalysis on a charged substrate. The correlation between reaction rate and adsorption was determined for each charge variant within the ladder, each of which displayed a maximum rate at an intermediate surface concentration. Both the maximum reaction rate and adsorption value at which this maximum rate occurs increased in magnitude for the more positive variants. In addition, the specific enzyme activity increased as the level of adsorption decreased, and for the lowest adsorption values, the specific enzyme activity was enhanced compared to the trend at higher surface concentrations. At a fixed level of adsorption, the specific enzyme activity increased with positive enzyme charge; however, this effect offers diminishing returns as the enzyme becomes more highly charged. We examined the effect of electrostatic interactions on surface diffusion. As the binding affinity was reduced by increasing the solution ionic strength, thus weakening electrostatic interaction, the rate of surface diffusion increased considerably. The enhancement in specific activity achieved at the lowest adsorption values is explained by the substantial rise in surface diffusion at high ionic strength due to decreased interactions with the surface. Overall, knowledge of the electrostatic interactions can be used to control surface parameters such as surface concentration and surface diffusion, which intimately correlate with surface biocatalysis. We propose that the maximum reaction rate results from a balance between adsorption and surface diffusion. The above finding suggests enzyme engineering and process design strategies for improving interfacial biocatalysis in industrial, pharmaceutical, and food applications.

Basigin-2 Is a Cell Surface Receptor for Soluble Basigin Ligand*S⃞

PubMed Central

Belton, Robert J.; Chen, Li; Mesquita, Fernando S.; Nowak, Romana A.

2008-01-01

The metastatic spread of a tumor is dependent upon the ability of the tumor to stimulate surrounding stromal cells to express enzymes required for tissue remodeling. The immunoglobulin superfamily protein basigin (EMMPRIN/CD147) is a cell surface glycoprotein expressed by tumor cells that stimulates matrix metalloproteinase and vascular endothelial growth factor expression in stromal cells. The ability of basigin to stimulate expression of molecules involved in tissue remodeling and angiogenesis makes basigin a potential target for the development of strategies to block metastasis. However, the identity of the cell surface receptor for basigin remains controversial. The goal of this study was to determine the identity of the receptor for basigin. Using a novel recombinant basigin protein (rBSG) corresponding to the extracellular domain of basigin, it was demonstrated that the native, nonglycosylated rBSG protein forms dimers in solution. Furthermore, rBSG binds to the surface of uterine fibroblasts, activates the ERK1/2 signaling pathway, and induces expression of matrix metalloproteinases 1, 2, and 3. Proteins that interact with rBSG were isolated using a biotin label transfer technique and sequenced by matrix-assisted laser desorption ionization tandem mass spectrophotometry. The results demonstrate that rBSG interacts with basigin expressed on the surface of fibroblasts and is subsequently internalized. During internalization, rBSG associates with a novel form of human basigin (basigin-3). It was concluded that cell surface basigin functions as a membrane receptor for soluble basigin and this homophilic interaction is not dependent upon glycosylation of the basigin ligand. PMID:18434307

Effects of Exposure to Ozone on the Ocular Surface in an Experimental Model of Allergic Conjunctivitis

PubMed Central

Lee, Hun; Kim, Eung Kweon; Kim, Hee Young; Kim, Tae-im

2017-01-01

Based on previous findings that ozone can induce an inflammatory response in the ocular surface of an animal model and in cultured human conjunctival epithelial cells, we investigated whether exposure to ozone exacerbates symptoms of allergic conjunctivitis. We evaluated the effects of exposure to ozone on conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, production of inflammatory cytokines in tears, and aqueous tear production in a mouse model of allergic conjunctivitis. To validate our in vivo results, we used interleukin (IL)-1α-pretreated conjunctival epithelial cells as an in vitro substitute for the mouse model. We evaluated whether exposure to ozone increased the inflammatory response and altered oxidative status and mitochondrial function in IL-1α-pretreated conjunctival epithelial cells. In the in vivo study, ozone induced increases in conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, and production of inflammatory cytokines, accompanied by a decrease in tear volume. In the in vitro study, exposure to ozone led to additional increases in IL-6 and tumor necrosis factor-α mRNA levels, which were already induced by treatment with IL-1α. Ozone did not induce any changes in cell viability. Pretreatment with IL-1α increased the expression of manganese superoxide dismutase, and exposure to ozone led to additional increments in the expression of this antioxidant enzyme. Ozone did not induce any changes in mitochondrial activity or expression of mitochondrial enzymes and proteins related to mitochondrial function, with the exception of phosphor-mammalian target of rapamycin. Treatment with butylated hydroxyanisole, a free radical scavenger, attenuated the ozone-induced increases in IL-6 expression in IL-1α-pretreated conjunctival epithelial cells. Therefore, we conclude that exposure to ozone exacerbates the detrimental effects on the integrity of the ocular surface caused by conjunctival allergic reactions, and further increases the inflammatory response in IL-1α-pretreated conjunctival epithelial cells. PMID:28046113

Following the compositional changes of fresh grape skin cell walls during the fermentation process in the presence and absence of maceration enzymes.

PubMed

Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Trygg, Johan; Vivier, Melané A

2015-03-18

Cell wall profiling technologies were used to follow compositional changes that occurred in the skins of grape berries (from two different ripeness levels) during fermentation and enzyme maceration. Multivariate data analysis showed that the fermentation process yielded cell walls enriched in hemicellulose components because pectin was solubilized (and removed) with a reduction as well as exposure of cell wall proteins usually embedded within the cell wall structure. The addition of enzymes caused even more depectination, and the enzymes unravelled the cell walls enabling better access to, and extraction of, all cell wall polymers. Overripe grapes had cell walls that were extensively hydrolyzed and depolymerized, probably by natural grape-tissue-ripening enzymes, and this enhanced the impact that the maceration enzymes had on the cell wall monosaccharide profile. The combination of the techniques that were used is an effective direct measurement of the hydrolysis actions of maceration enzymes on the cell walls of grape berry skin.

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.

Sequence diversity of NanA manifests in distinct enzyme kinetics and inhibitor susceptibility

NASA Astrophysics Data System (ADS)

Xu, Zhongli; von Grafenstein, Susanne; Walther, Elisabeth; Fuchs, Julian E.; Liedl, Klaus R.; Sauerbrei, Andreas; Schmidtke, Michaela

2016-04-01

Streptococcus pneumoniae is the leading pathogen causing bacterial pneumonia and meningitis. Its surface-associated virulence factor neuraminidase A (NanA) promotes the bacterial colonization by removing the terminal sialyl residues from glycoconjugates on eukaryotic cell surface. The predominant role of NanA in the pathogenesis of pneumococci renders it an attractive target for therapeutic intervention. Despite the highly conserved activity of NanA, our alignment of the 11 NanAs revealed the evolutionary diversity of this enzyme. The amino acid substitutions we identified, particularly those in the lectin domain and in the insertion domain next to the catalytic centre triggered our special interest. We synthesised the representative NanAs and the mutagenized derivatives from E. coli for enzyme kinetics study and neuraminidase inhibitor susceptibility test. Via molecular docking we got a deeper insight into the differences between the two major variants of NanA and their influence on the ligand-target interactions. In addition, our molecular dynamics simulations revealed a prominent intrinsic flexibility of the linker between the active site and the insertion domain, which influences the inhibitor binding. Our findings for the first time associated the primary sequence diversity of NanA with the biochemical properties of the enzyme and with the inhibitory efficiency of neuraminidase inhibitors.

Immobilization of Escherichia coli cells with penicillin-amidohydrolase activity on solid polymeric carriers.

PubMed

Zurková, E; Drobník, J; Kálal, J; Svec, F; Tyrácková, V; Vojtísek, V; Zeman, R

1983-09-01

Whole cells of Escherichia coli containing the enzyme penicillinamidohydrolase EC 3.5.1.11 were immobilized on the surface of modified macroporous copolymers of glycidylmethacrylate with ethylenedimethacrylate and of copolymers of methacrylaldehyde (MA) with divinylbenzene (DVB) by means of glutaraldehyde. These polymeric carriers were modified before cell binding by using ammonia or polyamines, especially ethylenediamine and hexamethylenediamine (HMDA). The highest specific activity and the largest yield in cell immobilization were achieved with the macroporous copolymer of MA and DVB modified with HMDA. The material thus obtained was used in repeated conversions of benzylpenicillin to 6-aminopenicillanic acid in a stirred batch reactor.

Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A.

PubMed

Takami, N; Oda, K; Fujiwara, T; Ikehara, Y

1990-12-27

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.

Silica Gel for Enhanced Activity and Hypochlorite Protection of Cyanuric Acid Hydrolase in Recombinant Escherichia coli.

PubMed

Radian, Adi; Aukema, Kelly G; Aksan, Alptekin; Wackett, Lawrence P

2015-11-03

Chlorinated isocyanuric acids are widely used water disinfectants that generate hypochlorite, but with repeated application, they build up cyanuric acid (CYA) that must be removed to maintain disinfection. 3-Aminopropyltriethoxysilane (APTES)-treated Escherichia coli cells expressing cyanuric acid hydrolase (CAH) from Moorella thermoacetica exhibited significantly high CYA degradation rates and provided protection against enzyme inactivation by hypochlorite (chlorine). APTES coating or encapsulation of cells had two benefits: (i) overcoming diffusion limitations imposed by the cell wall and (ii) protecting against hypochlorite inactivation of CAH activity. Cells encapsulated in APTES gels degraded CYA three times faster than nonfunctionalized tetraethoxysilane (TEOS) gels, and cells coated with APTES degraded CYA at a rate of 29 µmol/min per mg of CAH protein, similar to the rate with purified enzyme. UV spectroscopy, fluorescence spectroscopy, and scanning electron microscopy showed that the higher rates were due to APTES increasing membrane permeability and enhancing cyanuric acid diffusion into the cytoplasm to reach the CAH enzyme. Purified CAH enzyme was shown to be rapidly inactivated by hypochlorite. APTES aggregates surrounding cells protected via the amine groups reacting with hypochlorite as shown by pH changes, zeta potential measurements, and infrared spectroscopy. APTES-encapsulated E. coli cells expressing CAH degraded cyanuric acid at high rates in the presence of 1 to 10 ppm hypochlorite, showing effectiveness under swimming pool conditions. In contrast, CAH activity in TEOS gels or free cells was completely inactivated by hypochlorite. These studies show that commercially available silica materials can selectively enhance, protect, and immobilize whole-cell biocatalysts for specialized applications. Hypochlorite is used in vast quantities for water disinfection, killing bacteria on surfaces, and washing and whitening. In pools, spas, and other waters, hypochlorite is frequently delivered as chlorinated isocyanuric acids that release hypochlorite and cyanuric acid. Over time, cyanuric acid accumulates and impairs disinfection and must be removed. The microbial enzyme cyanuric acid hydrolase can potentially remove cyanuric acid to restore disinfection and protect swimmers. Whole bacterial cells expressing cyanuric acid hydrolase were encapsulated in an inert silica matrix containing an amine group. The amine group serves to permeabilize the cell membrane and accelerate cyanuric acid degradation, and it also reacts with hypochlorite to protect against inactivation of cyanuric acid hydrolase. Methods for promoting whole-cell biocatalysis are important in biotechnology, and the present work illustrates approaches to enhance rates and protect against an inhibitory substance. Copyright © 2015 Radian et al.

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

PubMed

Rouet, Romain; Thuma, Benjamin A; Roy, Marc D; Lintner, Nathanael G; Rubitski, David M; Finley, James E; Wisniewska, Hanna M; Mendonsa, Rima; Hirsh, Ariana; de Oñate, Lorena; Compte Barrón, Joan; McLellan, Thomas J; Bellenger, Justin; Feng, Xidong; Varghese, Alison; Chrunyk, Boris A; Borzilleri, Kris; Hesp, Kevin D; Zhou, Kaihong; Ma, Nannan; Tu, Meihua; Dullea, Robert; McClure, Kim F; Wilson, Ross C; Liras, Spiros; Mascitti, Vincent; Doudna, Jennifer A

2018-05-30

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

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

Belfort, Georges; Grimaldi, Joseph J.

2015-01-27

Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), andmore » (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the presence of glycocalyx filaments coating the luminal surface of our vasculature and represent a new class of synthetic membranes. They thus meet the requirements/scope of the Bimolecular Materials program, Materials Science and Engineering Div., Office of Science, US DOE.« less

Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

PubMed Central

Scurti, Gina; Thyagarajan, Krishnamurthy; Kaur, Navtej; Husain, Shahid; Fang, Quan; Naga, Osama S.; Simms, Patricia; Beeson, Gyda; Voelkel-Johnson, Christina; Garrett-Mayer, Elizabeth; Beeson, Craig C.; Nishimura, Michael I.; Mehrotra, Shikhar

2014-01-01

Ex vivo-expanded CD8+ T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced anti-tumor efficacy, relative to CD8+ T cells with a central memory-like phenotype (TCM cells). Further, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM possess relatively greater anti-oxidative capacity than TEM cells. Here we report that TCM cells exhibit a relative increase compared to TEM cells in expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity and glycolytic enzyme levels. Notably, TCR-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, up-regulated thiol levels and antioxidant genes. A comparison of anti-tumor CD8+ T cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth, and that profiling this biomarker may have benefits to T cell adoptive immunotherapy protocols. PMID:25164014

Protein secretion and surface display in Gram-positive bacteria

PubMed Central

Schneewind, Olaf; Missiakas, Dominique M.

2012-01-01

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions. PMID:22411983

Incomplete synthesis of N-glycans in congenital dyserythropoietic anemia type II caused by a defect in the gene encoding. alpha. -mannosidase II

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

Fukuda, M.N.; Masri, K.A.; Dell, A.

1990-10-01

Congenital dyserythropoietic anemia type II, or hereditary erythroblastic multinuclearity with a positive acidified-serum-lysis test (HEMPAS), is a genetic anemia in humans inherited by an autosomally recessive mode. The enzyme defect in most HEMPAS patients has previously been proposed as a lowered activity of N-acetylglucosaminyltransferase II, resulting in a lack of polylactosamine on proteins and leading to the accumulation of polylactosaminyl lipids. A recent HEMPAS case, G.C., has now been analyzed by cell-surface labeling, fast-atom-bombardment mass spectrometry of glycopeptides, and activity assay of glycosylation enzymes. Significantly decreased glycosylation of polylactosaminoglycan proteins and incompletely processed asparagine-linked oligosaccharides were detected in the erythrocytemore » membranes of G.C. These results suggest that G.C. cells contain a mutation in {alpha}-ManII-encoding gene that results in inefficient expression of {alpha}-ManII mRNA, either through reduced transcription or message instability. This report demonstrates that HEMPAS is caused by a defective gene encoding an enzyme necessary for the synthesis of asparagine-linked oligosaccharides.« less

Graphene-coated carbon fiber cloth for flexible electrodes of glucose fuel cells

NASA Astrophysics Data System (ADS)

Hoshi, Kazuki; Muramatsu, Kazuo; Sumi, Hisato; Nishioka, Yasushiro

2016-02-01

In this work, we fabricated flexible electrodes for a miniaturized, simple structured, and flexible glucose biofuel cell (BFC) using a graphene-coated carbon fiber cloth (GCFC). The areas of the anode and cathode electrodes were 3 × 10 mm2. The anode area was coated with the enzyme glucose oxidase, and the cathode area was coated with the enzyme bilirubin oxidase. No ion-exchange film was needed because glucose oxidase selectively oxidizes glucose and bilirubin oxidase selectively reduces oxygen. The power density of the BFC with GCFC electrodes in a phosphate buffer solution of 200 mM glucose solution at room temperature was 34.3 µW/cm2 at 0.43 V. The power density of a BFC using carbon fiber cloth (CFC) without graphene modification was 18.5 µW/cm2 at 0.13 V. The BFC with the GCFC electrode continued to function longer than 24 h with a power density higher than 5 µW/cm2. These effects were attributed to the much larger effective surface areas of the GCFC electrodes that maintain more enzymes than those of the CFC electrodes.

Comparison of the Virion Polymerase of Reovirus with the Enzyme Purified from Reovirus-Infected Cells

PubMed Central

Gomatos, Peter J.

1970-01-01

Reovirus has in its protein coat an enzyme which catalyzes the net synthesis of the three size classes of virus-specific, single-stranded ribonucleic acid (RNA). For synthesis of 24, 19, and 14S single-stranded RNA, Mn++ was the preferred divalent cation, and ammonium sulfate at an optimal concentration of 4.2% of saturation was an absolute requirement. During synthesis, the parental double-stranded RNA was conserved in the viral core and the newly synthesized completed RNA chains were released as free RNA. The viral cores synthesizing RNA had properties consistent with the presence of nascent RNA on their outer surface. The enzyme-template complex from the infected cells described in an earlier paper was comprised of viral cores already active in the in vivo synthesis of single-stranded RNA. This pool of viral cores was newly made during infection, and exponential increase in the number of particles in this pool, as detected by the increase in enzymatic activity, occurred 2 hr earlier than that in mature virus. PMID:5483438

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

PubMed Central

Collier, Ivan E.; Legant, Wesley; Marmer, Barry; Lubman, Olga; Saffarian, Saveez; Wakatsuki, Tetsuro; Elson, Elliot; Goldberg, Gregory I.

2011-01-01

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions. PMID:21912660

Penicillium purpurogenum cultures under ethanol-induced stress and its correlation with fungal adhesion and biodegrading ability.

PubMed

Gomaa, Ola M; Husseiny, Sherif M; Abd El Kareem, Hussein; Talaat, Riham

2016-10-01

Fungi are known to be affected by external environmental stimuli, resulting in different stress response effects, which in turn could be used to enhance its biodegrading ability. In a previous study, ethanol was used to manipulate cell-cell and cell-surface interaction to prevent cell loss and maximize the usage of Penicillium purpurogenum cells in the media, a correlation was drawn between ethanol oxidative stress, surface-bound proteins and fungal adhesion. The present study focuses on a more detailed study of the effect of ethanol on the same fungus. The results show that the presence of Yap1p gene and the detection of an oxidized form of glutathione (GSSG) suggest that a stress response might be involved in the adhesion process. The process of adhesion could be described as a signaling process and it is affected by the germ tube formation as an initial step in adhesion. Protein profile showed polymorphism in surface-bound proteins for cultures amended with ethanol when compared to control cultures. Ethanol also affected the DNA polymorphic profile of DNA, rendering the fungus genetically variable. P. purpurogenum produced phenol oxidase enzyme and could be used to degrade total phenols in olive mill waste water without the formation of biofilm on the surface of the containers.

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

PubMed Central

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

2016-01-01

Although gut host-pathogen interactions are glycan-mediated processes, few details are known about the participating structures. Here we employ high-resolution mass spectrometric profiling to comprehensively identify and quantitatively measure the exact modifications of native intestinal epithelial cell surface N-glycans induced by S. typhimurium infection. Sixty minutes postinfection, select sialylated structures showed decreases in terms of total number and abundances. To assess the effect of cell surface mannosylation, we selectively rerouted glycan expression on the host using the alpha-mannosidase inhibitor, kifunensine, toward overexpression of high mannose. Under these conditions, internalization of S. typhimurium significantly increased, demonstrating that bacteria show preference for particular structures. Finally, we developed a novel assay to measure membrane glycoprotein turnover rates, which revealed that glycan modifications occur by bacterial enzyme activity rather than by host-derived restructuring strategies. This study is the first to provide precise structural information on how host N-glycans are altered to support S. typhimurium invasion. PMID:27754876

A hemolytic factor from Haemonchus contortus alters erythrocyte morphology.

PubMed

Fetterer, R H; Rhoads, M L

1998-12-15

A hemolytic factor from adult Haemonchus contortus caused distinct morphological changes in the surface of sheep red blood cells (RBCs). After a 15 min exposure to the hemolytic factor, hemolysis was not detected in incubation media, but RBCs were spherical in shape with numerous surface projections compared to control cells that were smooth-surfaced biconcave disks. After 30 min, a time at which significant hemolysis occurred, echinocytes were formed, and after 90 min, cells were severely disrupted with many visible holes in membranes. No RBC ghosts were observed. RBCs from four other mammalian species were lysed by the H. contortus hemolytic factor. However, the rate of hemolysis varied with a relative order of sheep approximately rabbit>goat>pig>calf. The morphology of RBCs from all four species was significantly altered after 30 min incubation with the degree of morphological changes related to the degree of hemolysis. These results support the hypothesis that the hemolytic factor acts as a pore-forming agent, although a phospholipase or other enzyme might play a role in solubilization of cell membranes.

Differential localization of lipid phosphate phosphatases 1 and 3 to cell surface subdomains in polarized MDCK cells.

PubMed

Jia, Yan-Jun; Kai, Masahiro; Wada, Ikuo; Sakane, Fumio; Kanoh, Hideo

2003-09-25

Lipid phosphate phosphatases (LPPs) are integral membrane proteins with six transmembrane domains that act as ecto-enzymes dephosphorylating a variety of extracellular lipid phosphates. Using polarized MDCK cells stably expressing human LPP1 and LPP3, we found that LPP1 was located exclusively at the apical surface whereas LPP3 was distributed mostly in the basolateral subdomain. We identified a novel apical sorting signal at the N-terminus of LPP1 composed of F(2)DKTRL(7). In the case of LPP3, a dityrosine motif present in the second cytoplasmic portion was identified as basolateral targeting signal. Our work shows that LPP1 and LPP3 are equipped with distinct sorting signals that cause them to differentially localize to the apical vs. the basolateral subdomain, respectively.

Characterization of the Porphyromonas gingivalis Type IX Secretion Trans-envelope PorKLMNP Core Complex*

PubMed Central

Vincent, Maxence S.; Canestrari, Mickaël J.; Leone, Philippe; Stathopulos, Julien; Ize, Bérengère; Zoued, Abdelrahim; Cambillau, Christian; Kellenberger, Christine; Roussel, Alain

2017-01-01

The transport of proteins at the cell surface of Bacteroidetes depends on a secretory apparatus known as type IX secretion system (T9SS). This machine is responsible for the cell surface exposition of various proteins, such as adhesins, required for gliding motility in Flavobacterium, S-layer components in Tannerella forsythia, and tooth tissue-degrading enzymes in the oral pathogen Porphyromonas gingivalis. Although a number of subunits of the T9SS have been identified, we lack details on the architecture of this secretion apparatus. Here we provide evidence that five of the genes encoding the core complex of the T9SS are co-transcribed and that the gene products are distributed in the cell envelope. Protein-protein interaction studies then revealed that these proteins oligomerize and interact through a dense network of contacts. PMID:28057754

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.

High endothelin-converting enzyme-1 expression independently predicts poor survival of patients with esophageal squamous cell carcinoma.

PubMed

Wu, Ching-Fang; Lee, Ching-Tai; Kuo, Yao-Hung; Chen, Tzu-Haw; Chang, Chi-Yang; Chang, I-Wei; Wang, Wen-Lun

2017-09-01

Patients with esophageal squamous cell carcinoma have poor survival and high recurrence rate, thus an effective prognostic biomarker is needed. Endothelin-converting enzyme-1 is responsible for biosynthesis of endothelin-1, which promotes growth and invasion of human cancers. The role of endothelin-converting enzyme-1 in esophageal squamous cell carcinoma is still unknown. Therefore, this study investigated the significance of endothelin-converting enzyme-1 expression in esophageal squamous cell carcinoma clinically. We enrolled patients with esophageal squamous cell carcinoma who provided pretreated tumor tissues. Tumor endothelin-converting enzyme-1 expression was evaluated by immunohistochemistry and was defined as either low or high expression. Then we evaluated whether tumor endothelin-converting enzyme-1 expression had any association with clinicopathological findings or predicted survival of patients with esophageal squamous cell carcinoma. Overall, 54 of 99 patients with esophageal squamous cell carcinoma had high tumor endothelin-converting enzyme-1 expression, which was significantly associated with lymph node metastasis ( p = 0.04). In addition, tumor endothelin-converting enzyme-1 expression independently predicted survival of patients with esophageal squamous cell carcinoma, and the 5-year survival was poorer in patients with high tumor endothelin-converting enzyme-1 expression ( p = 0.016). Among patients with locally advanced and potentially resectable esophageal squamous cell carcinoma (stage II and III), 5-year survival was poorer with high tumor endothelin-converting enzyme-1 expression ( p = 0.003). High tumor endothelin-converting enzyme-1 expression also significantly predicted poorer survival of patients in this population. In patients with esophageal squamous cell carcinoma, high tumor endothelin-converting enzyme-1 expression might indicate high tumor invasive property. Therefore, tumor endothelin-converting enzyme-1 expression could be a good biomarker to identify patients with worse survival and higher risks of recurrence, who might benefit from the treatment by endothelin-converting enzyme-1 inhibitor.

Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability

PubMed Central

2013-01-01

Background The cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis. Results We observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain. Conclusions The physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain. This indicates that PLG layer favors biofilm formation. This study demonstrate that the nitrogen availability not only regulates GS expression and activity in M. bovis but also affects cell surface properties by modulating synthesis of PLG. PMID:24112767

Imaging the Dynamics of Cell Wall Polymer Deposition in the Unicellular Model Plant, Penium margaritaceum.

PubMed

Domozych, David; Lietz, Anna; Patten, Molly; Singer, Emily; Tinaz, Berke; Raimundo, Sandra C

2017-01-01

The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.

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.

An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes

PubMed Central

Mohamad, Nur Royhaila; Marzuki, Nur Haziqah Che; Buang, Nor Aziah; Huyop, Fahrul; Wahab, Roswanira Abdul

2015-01-01

The current demands of sustainable green methodologies have increased the use of enzymatic technology in industrial processes. Employment of enzyme as biocatalysts offers the benefits of mild reaction conditions, biodegradability and catalytic efficiency. The harsh conditions of industrial processes, however, increase propensity of enzyme destabilization, shortening their industrial lifespan. Consequently, the technology of enzyme immobilization provides an effective means to circumvent these concerns by enhancing enzyme catalytic properties and also simplify downstream processing and improve operational stability. There are several techniques used to immobilize the enzymes onto supports which range from reversible physical adsorption and ionic linkages, to the irreversible stable covalent bonds. Such techniques produce immobilized enzymes of varying stability due to changes in the surface microenvironment and degree of multipoint attachment. Hence, it is mandatory to obtain information about the structure of the enzyme protein following interaction with the support surface as well as interactions of the enzymes with other proteins. Characterization technologies at the nanoscale level to study enzymes immobilized on surfaces are crucial to obtain valuable qualitative and quantitative information, including morphological visualization of the immobilized enzymes. These technologies are pertinent to assess efficacy of an immobilization technique and development of future enzyme immobilization strategies. PMID:26019635

Production, recovery and purification of a recombinant β-galactosidase by expanded bed anion exchange adsorption.

PubMed

Boeris, Valeria; Balce, Izabella; Vennapusa, Rami Reddy; Arévalo Rodríguez, Miguel; Picó, Guillermo; Lahore, Marcelo Fernández

2012-07-01

β-Galactosidase is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides; its major application in the food industry is to reduce the content of lactose in lactic products. The aim of this work is to recover this enzyme from a cell lysate by adsorption onto Streamline-DEAE in an expanded bed, avoiding, as much as possible, biomass deposition onto the adsorbent matrix. So as to achieve less cell debris-matrix interaction, the adsorbent surface was covered with polyvinyl pyrrolidone. The enzyme showed to bind in the same extent to naked and covered Streamline-DEAE (65 mg β-gal/g matrix) in batch mode in the absence of any biomass. The kinetics of the adsorption process was studied and no effect of the polyvinyl pyrrolidone covering was found. The optimal conditions for the recovery were achieved by using a lysate made of 40% wet weight of cells, a polyvinyl pyrrolidone-covered matrix/lysate ratio of 10% and carrying out the adsorption process in expanded bed with recirculation over 2h in 20 mM phosphate buffer pH 7.4. The fraction recovered after the elution contained 65% of the initial amount of enzyme with a 12.6-fold increased specific activity with respect to the lysate. The polyvinyl pyrrolidone content in the eluate was determined and found negligible. The remarkable point of this work is that it was possible to partially purify the enzyme using a feedstock containing an unusually high biomass concentration in the presence of polyvinyl pyrrolidone onto weak anion exchangers. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Kattke, Michele D.; Chan, Albert H.; Duong, Andrew

Here, many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution ofmore » alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses β3/β4 and β6/β7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their β3/β4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.« less

Brownian dynamics simulations of interactions between aldolase and G- or F-actin.

PubMed Central

Ouporov, I V; Knull, H R; Thomasson, K A

1999-01-01

Compartmentation of proteins in cells is important to proper cell function. Interactions of F-actin and glycolytic enzymes is one mechanism by which glycolytic enzymes can compartment. Brownian dynamics (BD) simulations of the binding of the muscle form of the glycolytic enzyme fructose-1,6-bisphosphate aldolase (aldolase) to F- or G-actin provide first-encounter snapshots of these interactions. Using x-ray structures of aldolase, G-actin, and three-dimensional models of F-actin, the electrostatic potential about each protein was predicted by solving the linearized Poisson-Boltzmann equation for use in BD simulations. The BD simulations provided solution complexes of aldolase with F- or G-actin. All complexes demonstrate the close contacts between oppositely charged regions of the protein surfaces. Positively charged surface regions of aldolase (residues Lys 13, 27, 288, 293, and 341 and Arg 257) are attracted to the negatively charged amino terminus (Asp 1 and Glu 2 and 4) and other patches (Asp 24, 25, and 363 and Glu 361, 364, 99, and 100) of actin subunits. According to BD results, the most important factor for aldolase binding to actin is the quaternary structure of aldolase and actin. Two pairs of adjacent aldolase subunits greatly add to the positive electrostatic potential of each other creating a region of attraction for the negatively charged subdomain 1 of the actin subunit that is exposed to solvent in the quaternary F-actin structure. PMID:9876119

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.

Recognition and invasion of human erythrocytes by malarial parasites: contribution of sialoglycoproteins to attachment and host specificity

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

Friedman, M.J.; Blankenberg, T.; Sensabaugh, G.

1984-05-01

The receptivity of human erythrocytes to invasion by Plasmodium falciparum merozoites can be decreased by neuraminidase or trypsin treatment, an observation that supports a role for the erythrocyte sialoglycoproteins (glycophorins) in invasion. We have found that ..cap alpha../sub 1/-acid glycoprotein (AGP), added to in vitro cultures, can restore invasion of enzyme-treated human erythrocytes. AGP is structurally different from the glycophorins although it does carry 12% sialic acid. Its ability to restore receptivity to desialylated cells is dependent on its sialic acid complement, its concentration, and its binding to the erythrocyte surface. We present evidence that AGP forms a bridge betweenmore » the merozoite and the enzyme-treated erythrocyte that allows the stronger and more complex interactions of invasion to proceed. We suggest that the glycophorins play the same role on the surface of the intact erythrocyte. 31 references, 3 figures, 6 tables.« less

Differential proteome and cellular adhesion analyses of the probiotic bacterium Lactobacillus acidophilus NCFM grown on raffinose - an emerging prebiotic.

PubMed

Celebioglu, Hasan Ufuk; Ejby, Morten; Majumder, Avishek; Købler, Carsten; Goh, Yong Jun; Thorsen, Kristian; Schmidt, Bjarne; O'Flaherty, Sarah; Abou Hachem, Maher; Lahtinen, Sampo J; Jacobsen, Susanne; Klaenhammer, Todd R; Brix, Susanne; Mølhave, Kristian; Svensson, Birte

2016-05-01

Whole cell and surface proteomes were analyzed together with adhesive properties of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) grown on the emerging prebiotic raffinose, exemplifying a synbiotic. Adhesion of NCFM to mucin and intestinal HT-29 cells increased three-fold after culture with raffinose versus glucose, as also visualized by scanning electron microscopy. Comparative proteomics using 2D-DIGE showed 43 unique proteins to change in relative abundance in whole cell lysates from NCFM grown on raffinose compared to glucose. Furthermore, 14 unique proteins in 18 spots of the surface subproteome underwent changes identified by differential 2DE, including elongation factor G, thermostable pullulanase, and phosphate starvation inducible stress-related protein increasing in a range of +2.1 - +4.7 fold. By contrast five known moonlighting proteins decreased in relative abundance by up to -2.4 fold. Enzymes involved in raffinose catabolism were elevated in the whole cell proteome; α-galactosidase (+13.9 fold); sucrose phosphorylase (+5.4 fold) together with metabolic enzymes from the Leloir pathway for galactose utilization and the glycolysis; β-galactosidase (+5.7 fold); galactose (+2.9/+3.1 fold) and fructose (+2.8 fold) kinases. The insights at the molecular and cellular levels contributed to the understanding of the interplay of a synbiotic composed of NCFM and raffinose with the host. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Orientation and cellular distribution of membrane-bound catechol-O-methyltransferase in cortical neurons: implications for drug development.

PubMed

Chen, Jingshan; Song, Jian; Yuan, Peixiong; Tian, Qingjun; Ji, Yuanyuan; Ren-Patterson, Renee; Liu, Guangping; Sei, Yoshitasu; Weinberger, Daniel R

2011-10-07

Catechol-O-methyltransferase (COMT) is a key enzyme for inactivation and metabolism of catechols, including dopamine, norepinephrine, caffeine, and estrogens. It plays an important role in cognition, arousal, pain sensitivity, and stress reactivity in humans and in animal models. The human COMT gene is associated with a diverse spectrum of human behaviors and diseases from cognition and psychiatric disorders to chronic pain and cancer. There are two major forms of COMT proteins, membrane-bound (MB) COMT and soluble (S) COMT. MB-COMT is the main form in the brain. The cellular distribution of MB-COMT in cortical neurons remains unclear and the orientation of MB-COMT on the cellular membrane is controversial. In this study, we demonstrate that MB-COMT is located in the cell body and in axons and dendrites of rat cortical neurons. Analyses of MB-COMT orientation with computer simulation, flow cytometry and a cell surface enzyme assay reveal that the C-terminal catalytic domain of MB-COMT is in the extracellular space, which suggests that MB-COMT can inactivate synaptic and extrasynaptic dopamine on the surface of presynaptic and postsynaptic neurons. Finally, we show that the COMT inhibitor tolcapone induces cell death via the mechanism of apoptosis, and its cytotoxicity is dependent on dosage and correlated with COMT Val/Met genotypes in human lymphoblastoid cells. These results suggest that MB-COMT specific inhibitors can be developed and that tolcapone may be less hazardous at low doses and in specific genetic backgrounds.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

PubMed

Moulton, Elizabeth A; Bertram, Paula; Chen, Nanhai; Buller, R Mark L; Atkinson, John P

2010-09-01

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of LaCrosse virus.

PubMed

Madoff, D H; Lenard, J

1982-04-01

The intracellular transport and certain posttranslational modifications of the large glycoprotein (G1) of LaCrosse virus (LAC) in BHK cells have been studied. G1 from released LAC virus was characterized by complex oligosaccharides (endo H-resistant) and covalently attached fatty acid. Only a small fraction of total cellular G1 was present on the baby hamster kidney cell surface. Cell-surface G1 contained complex oligosaccharides, while total G1 in infected cells contained largely unprocessed (endo H-sensitive) oligosaccharides. In addition, cell G1 contained significantly less fatty acid than virion-associated G1. Pulse-chase experiments showed that the oligosaccharides of G1 were processed to the complex from much more slowly than the oligosaccharides of the vesicular stomatitis virus (VSV) glycoprotein (G). In addition, transit of LAC G1 to the cell surface and into extracellular virions was two to three fold slower than the transit of VSV G. Thus LAC G1 accumulates intracellularly and is only slowly processed by intracellular processing enzymes. Treatment with monensin caused accumulation in the cell of a form of G1 with partial sensitivity toward endo H, suggesting that monensin may act to inhibit the glycosylation process directly.

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.

Sortase Transpeptidases: Structural Biology and Catalytic Mechanism

PubMed Central

Jacobitz, Alex W.; Kattke, Michele D.; Wereszczynski, Jeff; Clubb, Robert T.

2017-01-01

Gram-positive bacteria use sortase cysteine transpeptidase enzymes to covalently attach proteins to their cell wall and to assemble pili. In pathogenic bacteria sortases are potential drug targets, as many of the proteins that they display on the microbial surface play key roles in the infection process. Moreover, the Staphylococcus aureus Sortase A (SaSrtA) enzyme has been developed into a valuable biochemical reagent because of its ability to ligate biomolecules together in vitro via a covalent peptide bond. Here we review what is known about the structures and catalytic mechanism of sortase enzymes. Based on their primary sequences, most sortase homologs can be classified into six distinct subfamilies, called class A–F enzymes. Atomic structures reveal unique, class-specific variations that support alternate substrate specificities, while structures of sortase enzymes bound to sorting signal mimics shed light onto the molecular basis of substrate recognition. The results of computational studies are reviewed that provide insight into how key reaction intermediates are stabilized during catalysis, as well as the mechanism and dynamics of substrate recognition. Lastly, the reported in vitro activities of sortases are compared, revealing that the transpeptidation activity of SaSrtA is at least 20-fold faster than other sortases that have thus far been characterized. Together, the results of the structural, computational, and biochemical studies discussed in this review begin to reveal how sortases decorate the microbial surface with proteins and pili, and may facilitate ongoing efforts to discover therapeutically useful small molecule inhibitors. PMID:28683919

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

PubMed

Salzillo, Marzia; Vastano, Valeria; Capri, Ugo; Muscariello, Lidia; Marasco, Rosangela

2017-04-01

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Digital Assays Part II: Digital Protein and Cell Assays.

PubMed

Basu, Amar S

2017-08-01

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

Near-complete genome sequence of the cellulolytic Bacterium Bacteroides ( Pseudobacteroides) cellulosolvens ATCC 35603

DOE PAGES

Dassa, Bareket; Utturkar, Sagar M.; Hurt, Richard A.; ...

2015-09-24

We report the single-contig genome sequence of the anaerobic, mesophilic, cellulolytic bacterium, Bacteroides cellulosolvens. The bacterium produces a particularly elaborate cellulosome system, whereas the types of cohesin-dockerin interactions are opposite of other known cellulosome systems: cell-surface attachment is thus mediated via type-I interactions whereas enzymes are integrated via type-II interactions.

Biogenesis of glycerol 3-phosphate acyltransferase (GPAT): influence of transmembrane domains and protein-protein interactions on the localization of GPAT to ER subdomains

USDA-ARS?s Scientific Manuscript database

Glycerolipids are the major components of cellular membranes in all plant cells, storage oils in developing seeds, and the cuticular surface of plant organs. Using the tung (Vernicia fordii) triacylglycerol (TAG) biosynthetic enzymes as model system, we previously showed that the type 1 and 2 diacyl...

Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications.

PubMed

Hyeon, Jeong Eun; Jeon, Sang Duck; Han, Sung Ok

2013-11-01

The cellulosome is one of nature's most elegant and elaborate nanomachines and a key biological and biotechnological macromolecule that can be used as a multi-functional protein complex tool. Each protein module in the cellulosome system is potentially useful in an advanced biotechnology application. The high-affinity interactions between the cohesin and dockerin domains can be used in protein-based biosensors to improve both sensitivity and selectivity. The scaffolding protein includes a carbohydrate-binding module (CBM) that attaches strongly to cellulose substrates and facilitates the purification of proteins fused with the dockerin module through a one-step CBM purification method. Although the surface layer homology (SLH) domain of CbpA is not present in other strains, replacement of the cell surface anchoring domain allows a foreign protein to be displayed on the surface of other strains. The development of a hydrolysis enzyme complex is a useful strategy for consolidated bioprocessing (CBP), enabling microorganisms with biomass hydrolysis activity. Thus, the development of various configurations of multi-functional protein complexes for use as tools in whole-cell biocatalyst systems has drawn considerable attention as an attractive strategy for bioprocess applications. This review provides a detailed summary of the current achievements in Clostridium-derived multi-functional complex development and the impact of these complexes in various areas of biotechnology. Copyright © 2013 Elsevier Inc. All rights reserved.

Monoclonal Antibody Binding to a Surface-Exposed Epitope on Cowdria ruminantium That Is Conserved among Eight Strains

PubMed Central

Shompole, Sankale; Rurangirwa, Fred R.; Wambugu, Anderson; Sitienei, John; Mwangi, Duncan M.; Musoke, Anthony J.; Mahan, Suman; Wells, Clive W.; McGuire, Travis C.

2000-01-01

Monoclonal antibodies (MAb) binding to Cowdria ruminantium elementary bodies (EB) were identified by enzyme-linked immunosorbent assay, and surface binding of one MAb (446.15) to intact EB was determined by immunofluorescence, immunogold labeling, and transmission electron microscopy. MAb 446.15 bound an antigen of approximately 43 kDa in immunoblots of eight geographically distinct strains. The MAb did not react with Ehrlichia canis antigens or uninfected bovine endothelial cell lysate and may be useful in diagnostic assays and vaccine development. PMID:11063511

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.

Down-modulation of the G-protein-coupled Estrogen Receptor, GPER, from the Cell Surface Occurs via a trans-Golgi-Proteasome Pathway*

PubMed Central

Cheng, Shi-Bin; Quinn, Jeffrey A.; Graeber, Carl T.; Filardo, Edward J.

2011-01-01

GPER is a Gs-coupled seven-transmembrane receptor that has been linked to specific estrogen binding and signaling activities that are manifested by plasma membrane-associated enzymes. However, in many cell types, GPER is predominately localized to the endoplasmic reticulum (ER), and only minor amounts of receptor are detectable at the cell surface, an observation that has caused controversy regarding its role as a plasma membrane estrogen receptor. Here, we show that GPER constitutively buds intracellularly into EEA-1+ endosomes from clathrin-coated pits. Nonvisual arrestins-2/-3 do not co-localize with GPER, and expression of arrestin-2 dominant-negative mutants lacking clathrin- or β-adaptin interaction sites fails to block GPER internalization suggesting that arrestins are not involved in GPER endocytosis. Like β1AR, which recycles to the plasma membrane, GPER co-traffics with transferrin+, Rab11+ recycling endosomes. However, endocytosed GPER does not recycle to the cell surface, but instead returns to the trans-Golgi network (TGN) and does not re-enter the ER. GPER is ubiquitinated at the cell surface, exhibits a short half-life (t½ <1 h), and is protected from degradation by the proteasome inhibitor, MG132. Disruption of the TGN by brefeldin A induces the accumulation of endocytosed GPER in Rab11+ perinuclear endosomes and prevents GPER degradation. Our results provide an explanation as to why GPER is not readily detected on the cell surface in some cell types and further suggest that TGN serves as the checkpoint for degradation of endocytosed GPER. PMID:21540189

Comprehensive Evaluation of Streptococcus sanguinis Cell Wall-Anchored Proteins in Early Infective Endocarditis▿ †

PubMed Central

Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L.; Wu, Hui; Kitten, Todd

2009-01-01

Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified—a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (∼2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in S. sanguinis infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention. PMID:19703977

Comprehensive evaluation of Streptococcus sanguinis cell wall-anchored proteins in early infective endocarditis.

PubMed

Turner, Lauren Senty; Kanamoto, Taisei; Unoki, Takeshi; Munro, Cindy L; Wu, Hui; Kitten, Todd

2009-11-01

Streptococcus sanguinis is a member of the viridans group of streptococci and a leading cause of the life-threatening endovascular disease infective endocarditis. Initial contact with the cardiac infection site is likely mediated by S. sanguinis surface proteins. In an attempt to identify the proteins required for this crucial step in pathogenesis, we searched for surface-exposed, cell wall-anchored proteins encoded by S. sanguinis and then used a targeted signature-tagged mutagenesis (STM) approach to evaluate their contributions to virulence. Thirty-three predicted cell wall-anchored proteins were identified-a number much larger than those found in related species. The requirement of each cell wall-anchored protein for infective endocarditis was assessed in the rabbit model. It was found that no single cell wall-anchored protein was essential for the development of early infective endocarditis. STM screening was also employed for the evaluation of three predicted sortase transpeptidase enzymes, which mediate the cell surface presentation of cell wall-anchored proteins. The sortase A mutant exhibited a modest (approximately 2-fold) reduction in competitiveness, while the other two sortase mutants were indistinguishable from the parental strain. The combined results suggest that while cell wall-anchored proteins may play a role in S. sanguinis infective endocarditis, strategies designed to interfere with individual cell wall-anchored proteins or sortases would not be effective for disease prevention.

How a mycoparasite employs g-protein signaling: using the example of trichoderma.

PubMed

Omann, Markus; Zeilinger, Susanne

2010-01-01

Mycoparasitic Trichoderma spp. act as potent biocontrol agents against a number of plant pathogenic fungi, whereupon the mycoparasitic attack includes host recognition followed by infection structure formation and secretion of lytic enzymes and antifungal metabolites leading to the host's death. Host-derived signals are suggested to be recognized by receptors located on the mycoparasite's cell surface eliciting an internal signal transduction cascade which results in the transcription of mycoparasitism-relevant genes. Heterotrimeric G proteins of fungi transmit signals originating from G-protein-coupled receptors mainly to the cAMP and the MAP kinase pathways resulting in regulation of downstream effectors. Components of the G-protein signaling machinery such as Gα subunits and G-protein-coupled receptors were recently shown to play crucial roles in Trichoderma mycoparasitism as they govern processes such as the production of extracellular cell wall lytic enzymes, the secretion of antifungal metabolites, and the formation of infection structures.

How a Mycoparasite Employs G-Protein Signaling: Using the Example of Trichoderma

PubMed Central

Omann, Markus; Zeilinger, Susanne

2010-01-01

Mycoparasitic Trichoderma spp. act as potent biocontrol agents against a number of plant pathogenic fungi, whereupon the mycoparasitic attack includes host recognition followed by infection structure formation and secretion of lytic enzymes and antifungal metabolites leading to the host's death. Host-derived signals are suggested to be recognized by receptors located on the mycoparasite's cell surface eliciting an internal signal transduction cascade which results in the transcription of mycoparasitism-relevant genes. Heterotrimeric G proteins of fungi transmit signals originating from G-protein-coupled receptors mainly to the cAMP and the MAP kinase pathways resulting in regulation of downstream effectors. Components of the G-protein signaling machinery such as Gα subunits and G-protein-coupled receptors were recently shown to play crucial roles in Trichoderma mycoparasitism as they govern processes such as the production of extracellular cell wall lytic enzymes, the secretion of antifungal metabolites, and the formation of infection structures. PMID:21637351

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.

Investigation of spore coat display of Bacillus subtilis β-galactosidase for developing of whole cell biocatalyst.

PubMed

Tavassoli, Setareh; Hinc, Krzysztof; Iwanicki, Adam; Obuchowski, Michal; Ahmadian, Gholamreza

2013-03-01

The production of highly efficient, recyclable and cost-effective enzymes is one of the most important goals in industrial biotechnology. Bacterial spores are highly resistant to harsh environmental conditions, easy to produce and are suitable for manipulation of genetic materials. These features make them a very efficient tool for biotechnology. Here, we show the use bacterial spores for presentation of functional enzyme. Spore coat display was used to produce a biocatalyst, which expresses β-galactiosidase (LacA). This enzyme is commonly used to produce lactose-free milk for lactose intolerant individuals. The lacA gene from Bacillus subtilis strain 168 was expressed on the surface of B. subtilis RH101(ΔcotC) spores using CotC as protein carrier. Presence of LacA protein is verified by western blotting. Results of β-galactiosidase assay show that the expressed enzyme retained its activity in condition of freezing and drying, as well as after recovery from the reaction's mixture.

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

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion

PubMed Central

Knuchel, Sarah; Anderle, Pascale; Werfelli, Patricia; Diamantis, Eva; Rüegg, Curzio

2015-01-01

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion. PMID:25973543

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion.

PubMed

Knuchel, Sarah; Anderle, Pascale; Werfelli, Patricia; Diamantis, Eva; Rüegg, Curzio

2015-06-10

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion.

Chitin: a cell-surface component of Phytomonas françai.

PubMed

Nakamura, C V; Esteves, M J; Andrade, A F; Alviano, C S; de Souza, W; Angluster, J

1993-01-01

The occurrence of chitin as a structural component of the surface of the phytopathogenic protozoan Phytomonas françai was demonstrated by paper and gas-liquid chromatographic analysis of the products of enzymatic and chemical hydrolysis of alkali-resistant polysaccharides, lectin binding, glycosidase digestion, and infrared spectra. Chitin was characterized by its insolubility in hot alkali and chromatographic immobility as well as by the release of glucosamine on hydrolysis with strong acid and of N-acetylglucosamine (GlcNAc) on hydrolysis with chitinase. The presence of chitin was also shown directly by binding of wheat-germ agglutinin (WGA), which recognizes GlcNAc units, to the parasite surface. Fluorescein-labeled WGA binding was completely abolished by treatment with chitinase. This effect was specific since it could be prevented by incubating the enzyme with chitin before treatment of the phytomonads. These findings indicate that chitin is an exposed cell-surface polysaccharide in Phytomonas françai. The data were confirmed by the infrared spectrum of an alkali-insoluble residue, which showed a pattern typical of chitin.

Potential for Aedes albopictus and Ochlerotatus j. japonicus to Change the Field Ecology of Arboviruses of Human Health Importance in the Mid-Atlantic Region of the United States

DTIC Science & Technology

2001-10-16

attachment, the viral attachment proteins on the surface of the virion bind to receptors on the microvillar membrane of the mosquito s midgut ...Penetration refers to the process through which the virions enter the midgut cells; arboviruses enter cell through receptor -mediated endocytosis. During...inactivation of the virus by digestive enzymes in the lumen of the midgut , and the absence or reduced number of cellular receptor sites for virus attachment

Watch Out for the "Living Dead": Cell-Free Enzymes and Their Fate.

PubMed

Baltar, Federico

2017-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the "gatekeepers" of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell's fate. In contrast, cell-free enzymes belong to a kind of "living dead" realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go "beyond the living things," studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles.

Propofol inhibits invasion and proliferation of C6 glioma cells by regulating the Ca2+ permeable AMPA receptor-system xc- pathway.

PubMed

Wang, Xin-Yue; Li, Yan-Li; Wang, Hai-Yun; Zhu, Min; Guo, Di; Wang, Guo-Lin; Gao, Ying-Tang; Yang, Zhuo; Li, Tang; Yang, Chen-Yi; Chen, Yi-Meng

2017-10-01

Anesthetics are documented to affect tumors; therefore, we studied the antiglioma effect of propofol on proliferation and invasiveness of glioma cells and explored the underlying mechanism. C6 glioma cells were cultured and treated with propofol, and cell viability, invasiveness, and migration were measured. Glutamate release was measured using an enzyme-catalyzed kinetic reaction. xCT protein and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR2 subunit protein expression was assessed with Western blot analysis and immunofluorescent staining. We observed that propofol significantly inhibited C6 glioma cell viability, invasiveness, and migration and decreased glutamate release. An agonist of the cystine/glutamate antiporter system (system x c - ), N-acetylcysteine (NAC), reversed propofol's effects, and propofol could inhibit C6 glioma cell proliferation by adding excess exogenous glutamate (100μM). Finally, propofol increased the surface expression of GluR2, but decreased surface expression of xCT. The effects of propofol on surface expression of GluR2 and xCT could be rescued by (R, S)-AMPA, an agonist of Ca 2+ permeable AMPA receptor (CPAR). Thus, propofol can inhibit cell viability, invasiveness, and migration of C6 glioma cells, and the CPAR-system x c - pathway contributes to these events. Copyright © 2017 Elsevier Ltd. All rights reserved.

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE PAGES

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; ...

2014-12-31

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

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

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Surface expression of ω-transaminase in Escherichia coli.

PubMed

Gustavsson, Martin; Muraleedharan, Madhu Nair; Larsson, Gen

2014-04-01

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity.

Surface Expression of ω-Transaminase in Escherichia coli

PubMed Central

Gustavsson, Martin; Muraleedharan, Madhu Nair

2014-01-01

Chiral amines are important for the chemical and pharmaceutical industries, and there is rapidly growing interest to use transaminases for their synthesis. Since the cost of the enzyme is an important factor for process economy, the use of whole-cell biocatalysts is attractive, since expensive purification and immobilization steps can be avoided. Display of the protein on the cell surface provides a possible way to reduce the mass transfer limitations of such biocatalysts. However, transaminases need to dimerize in order to become active, and furthermore, they require the cofactor pyridoxal phosphate; consequently, successful transaminase surface expression has not been reported thus far. In this work, we produced an Arthrobacter citreus ω-transaminase in Escherichia coli using a surface display vector based on the autotransporter adhesin involved in diffuse adherence (AIDA-I), which has previously been used for display of dimeric proteins. The correct localization of the transaminase in the E. coli outer membrane and its orientation toward the cell exterior were verified. Furthermore, transaminase activity was detected exclusively in the outer membrane protein fraction, showing that successful dimerization had occurred. The transaminase was found to be present in both full-length and proteolytically degraded forms. The removal of this proteolysis is considered to be the main obstacle to achieving sufficient whole-cell transaminase activity. PMID:24487538

Novel Outer Membrane Protein Involved in Cellulose and Cellooligosaccharide Degradation by Cytophaga hutchinsonii

PubMed Central

Ji, Xiaofei; Wang, Ying; Zhang, Cong; Bai, Xinfeng; Zhang, Weican

2014-01-01

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium which was reported to use a novel contact-dependent strategy to degrade cellulose. It was speculated that cellooligosaccharides were transported into the periplasm for further digestion. In this study, we reported that most of the endoglucanase and β-glucosidase activity was distributed on the cell surface of C. hutchinsonii. Cellobiose and part of the cellulose could be hydrolyzed to glucose on the cell surface. However, the cell surface cellulolytic enzymes were not sufficient for cellulose degradation by C. hutchinsonii. An outer membrane protein, CHU_1277, was disrupted by insertional mutation. Although the mutant maintained the same endoglucanase activity and most of the β-glucosidase activity, it failed to digest cellulose, and its cellooligosaccharide utilization ability was significantly reduced, suggesting that CHU_1277 was essential for cellulose degradation and played an important role in cellooligosaccharide utilization. Further study of cellobiose hydrolytic ability of the mutant on the enzymatic level showed that the β-glucosidase activity in the outer membrane of the mutant was not changed. It revealed that CHU_1277 played an important role in assisting cell surface β-glucosidase to exhibit its activity sufficiently. Studies on the outer membrane proteins involved in cellulose and cellooligosaccharide utilization could shed light on the mechanism of cellulose degradation by C. hutchinsonii. PMID:24837387

Insolubility and redistribution of GPI-anchored proteins at the cell surface after detergent treatment.

PubMed Central

Mayor, S; Maxfield, F R

1995-01-01

A diverse set of cell surface eukaryotic proteins including receptors, enzymes, and adhesion molecules have a glycosylphosphoinositol-lipid (GPI) modification at the carboxy-terminal end that serves as their sole means of membrane anchoring. These GPI-anchored proteins are poorly solubilized in nonionic detergent such as Triton X-100. In addition these detergent-insoluble complexes from plasma membranes are significantly enriched in several cytoplasmic proteins including nonreceptor-type tyrosine kinases and caveolin/VIP-21, a component of the striated coat of caveolae. These observations have suggested that the detergent-insoluble complexes represent purified caveolar membrane preparations. However, we have recently shown by immunofluorescence and electron microscopy that GPI-anchored proteins are diffusely distributed at the cell surface but may be enriched in caveolae only after cross-linking. Although caveolae occupy only a small fraction of the cell surface (< 4%), almost all of the GPI-anchored protein at the cell surface becomes incorporated into detergent-insoluble low-density complexes. In this paper we show that upon detergent treatment the GPI-anchored proteins are redistributed into a significantly more clustered distribution in the remaining membranous structures. These results show that GPI-anchored proteins are intrinsically detergent-insoluble in the milieu of the plasma membrane, and their co-purification with caveolin is not reflective of their native distribution. These results also indicate that the association of caveolae, GPI-anchored proteins, and signalling proteins must be critically re-examined. Images PMID:7579703

Starch Flocculation by the Sweet Potato Sour Liquid Is Mediated by the Adhesion of Lactic Acid Bacteria to Starch

PubMed Central

Zhang, Lili; Yu, Yang; Li, Xinhua; Li, Xiaona; Zhang, Huajiang; Zhang, Zhen; Xu, Yunhe

2017-01-01

In the current study, we focused on the mechanism underlying starch flocculation by the sweet potato sour liquid. The traditional microbial techniques and 16S rDNA sequencing revealed that Lactobacillus was dominant flocculating microorganism in sour liquid. In total, 86 bacteria, 20 yeasts, and 10 molds were isolated from the sour liquid and only eight Lactobacillus species exhibited flocculating activity. Lactobacillus paracasei subsp. paracasei L1 strain with a high flocculating activity was isolated and identified, and the mechanism of starch flocculation was examined. L. paracasei subsp. paracasei L1 cells formed chain-like structures on starch granules. Consequently, these cells connected the starch granules to one another, leading to formation of large flocs. The results of various treatments of L1 cells indicated that bacterial surface proteins play a role in flocculation and L1 cells adhered to the surface of starch granules via specific surface proteins. These surface starch-binding proteins were extracted using the guanidine hydrochloride method; 10 proteins were identified by mass spectrometry: three of these proteins were glycolytic enzymes; two were identified as the translation elongation factor Tu; one was a cell wall hydrolase; one was a surface antigen; one was lyzozyme M1; one was a glycoside hydrolase; and one was an uncharacterized proteins. This study will paves the way for future industrial application of the L1 isolate in starch processing and food manufacturing. PMID:28791000

Mediating electrostatic binding of 1-butyl-3-methylimidazolium chloride to enzyme surfaces improves conformational stability.

PubMed

Nordwald, Erik M; Kaar, Joel L

2013-08-01

We have recently developed a general approach to improve the utility of enzymes in ionic liquids (ILs) via tuning of the ratio of enzyme-containing positive to negative surface charges. In this work, the impact of enzyme surface charge ratio on the biophysical interaction of 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) with chymotrypsin and lipase was investigated to understand this approach at the molecular level. Results of fluorescence quenching assays indicated that the extent of binding of the [BMIM] cation decreased (7- and 3.5-fold for chymotrypsin and lipase, respectively) as a function of increasing ratio of positive to negative surface charges. Conformational stability assays further showed a close correlation between thermodynamic stabilization and enzyme surface charge ratio as well as [BMIM] binding. As evidence of this correlation, succinylation and acetylation resulted in the stabilization of chymotrypsin in 10% (v/v) [BMIM][Cl] by 17.0 and 6.6 kJ/mol, respectively, while cationization destabilized chymotrypsin by 3.6 kJ/mol. Combined, these results indicate that altering the surface charge ratio mediates the organization of IL molecules, namely, [BMIM] and [Cl], around the enzymes. Preferential exclusion of [Cl], in particular, via lowering of the ratio of positive to negative surface charges, correlated with increased enzyme stability. Accordingly, these results more broadly provide insight into the mechanism of stabilization in ILs via charge modification.

Role of Mitochondria in Prostate Cancer

DTIC Science & Technology

2005-12-01

outer surface of the inner mitochondrial membrane. This enzyme acts in concert with the cytoplasmic NAD-linked glycerophosphate dehydrogenase (cGPDH...dependent ROS production (Fig. 4). This observation may be attributable to the absence of a CoQ -binding protein in mGPDH. This CoQ -binding protein...evidently has a natural protection of ubisemiquinone formed during CoQ reduction by succinate dehydrogenase [28]. Evaluation of cell proliferation

Assembling high activity phosphotriesterase composites using hybrid nanoparticle peptide-DNA scaffolded architectures

NASA Astrophysics Data System (ADS)

Breger, Joyce C.; Buckhout-White, Susan; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Ancona, Mario G.; Medintz, Igor L.

2017-06-01

Nanoparticle (NP) display potentially offers a new way to both stabilize and, in many cases, enhance enzyme activity over that seen for native protein in solution. However, the large, globular and sometimes multimeric nature of many enzymes limits their ability to attach directly to the surface of NPs, especially when the latter are colloidally stabilized with bulky PEGylated ligands. Engineering extended protein linkers into the enzymes to achieve direct attachment through the PEG surface often detrimentally alters the enzymes catalytic ability. Here, we demonstrate an alternate, hybrid biomaterials-based approach to achieving directed enzyme assembly on PEGylated NPs. We self-assemble a unique architecture consisting of a central semiconductor quantum dot (QD) scaffold displaying controlled ratios of extended peptide-DNA linkers which penetrate through the PEG surface to directly couple enzymes to the QD surface. As a test case, we utilize phosphotriesterase (PTE), an enzyme of bio-defense interest due to its ability to hydrolyze organophosphate nerve agents. Moreover, this unique approach still allows PTE to maintain enhanced activity while also suggesting the ability of DNA to enhance enzyme activity in and of itself.

A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady-state RNA levels and an altered protein. Use as a liver-cell transplantation model.

PubMed Central

Thompson, N L; Hixson, D C; Callanan, H; Panzica, M; Flanagan, D; Faris, R A; Hong, W J; Hartel-Schenk, S; Doyle, D

1991-01-01

Dipeptidyl peptidase IV (DPPIV) is a serine exoproteinase expressed at high levels in epithelial cells of kidney, liver and small intestine. Recently Watanabe, Kohima & Fujimoto [(1987) Experientia 43, 400-401] and Gossrau et al. [(1990) Histochem. J. 22, 172-173] reported that Fischer 344 rats are deficient in this enzyme. We have examined DPPIV expression in Fischer 344 rats available from U.S. and German suppliers and find that livers of the U.S. Fischer rats, in contrast with their German counterparts, express active DPPIV (D+). Northern analysis of liver RNA showed comparable levels of 3.4 kb and 5.6 kb DPPIV transcripts in both D+ rats from the U.S. and German (D-) rats. Monoclonal antibody (MAb) 236.3 to DPPIV immunoprecipitated at 150 kDa enzymically active (105 kDa, denatured) protein from surface-labelled D+ hepatocytes and reacted with canalicular and sinusoidal membranes (as shown by immunofluorescence microscopy). MAb 236.3 failed to immunoprecipitate a labelled peptide from D- cell extract or to stain D- liver sections. Polyclonal antibody (PAb) specific for DPPIV immunoprecipitated an enzymically active peptide from D+ hepatocyte extracts and a smaller, inactive peptide from D- hepatocyte extracts. Peptide maps of DPPIV immunoprecipitated from D+ extracts with MAb 236.3 and PAb were identical, but differed from that of the D- hepatocyte component recognized by PAb. The molecular basis of the DPPIV deficiency in the D- rats thus appears to be the translation of an enzymically inactive protein missing the epitope recognized by MAb 236.3. We have exploited these D- rats as hosts for syngeneic transplantation of liver cells from D+ Fischer rats. DPPIV expression is stable in the transplanted cells and allows them to be readily distinguished from the surrounding D- tissue. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1705112

An alternative mode of CD43 signal transduction activates pro-survival pathways of T lymphocytes.

PubMed

Bravo-Adame, Maria Elena; Vera-Estrella, Rosario; Barkla, Bronwyn J; Martínez-Campos, Cecilia; Flores-Alcantar, Angel; Ocelotl-Oviedo, Jose Pablo; Pedraza-Alva, Gustavo; Rosenstein, Yvonne

2017-01-01

CD43 is one of the most abundant co-stimulatory molecules on a T-cell surface; it transduces activation signals through its cytoplasmic domain, contributing to modulation of the outcome of T-cell responses. The aim of this study was to uncover new signalling pathways regulated by this sialomucin. Analysis of changes in protein abundance allowed us to identify pyruvate kinase isozyme M2 (PKM2), an enzyme of the glycolytic pathway, as an element potentially participating in the signalling cascade resulting from the engagement of CD43 and the T-cell receptor (TCR). We found that the glycolytic activity of this enzyme was not significantly increased in response to TCR+CD43 co-stimulation, but that PKM2 was tyrosine phosphorylated, suggesting that it was performing moonlight functions. We report that phosphorylation of both Y 105 of PKM2 and of Y 705 of signal transducer and activator of transcription 3 was induced in response to TCR+CD43 co-stimulation, resulting in activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway. ERK5 and the cAMP response element binding protein (CREB) were activated, and c-Myc and nuclear factor-κB (p65) nuclear localization, as well as Bad phosphorylation, were augmented. Consistent with this, expression of human CD43 in a murine T-cell hybridoma favoured cell survival. Altogether, our data highlight novel signalling pathways for the CD43 molecule in T lymphocytes, and underscore a role for CD43 in promoting cell survival through non-glycolytic functions of metabolic enzymes. © 2016 John Wiley & Sons Ltd.

Adenosine Deaminase Enhances the Immunogenicity of Human Dendritic Cells from Healthy and HIV-Infected Individuals

PubMed Central

Massanella, Marta; Rodríguez-García, Marta; Blanco, Julià; Gatell, José M.; García, Felipe; Gallart, Teresa; Lluis, Carme; Mallol, Josefa

2012-01-01

ADA is an enzyme implicated in purine metabolism, and is critical to ensure normal immune function. Its congenital deficit leads to severe combined immunodeficiency (SCID). ADA binding to adenosine receptors on dendritic cell surface enables T-cell costimulation through CD26 crosslinking, which enhances T-cell activation and proliferation. Despite a large body of work on the actions of the ecto-enzyme ADA on T-cell activation, questions arise on whether ADA can also modulate dendritic cell maturation. To this end we investigated the effects of ADA on human monocyte derived dendritic cell biology. Our results show that both the enzymatic and non-enzymatic activities of ADA are implicated in the enhancement of CD80, CD83, CD86, CD40 and CCR7 expression on immature dendritic cells from healthy and HIV-infected individuals. These ADA-mediated increases in CD83 and costimulatory molecule expression is concomitant to an enhanced IL-12, IL-6, TNF-α, CXCL8(IL-8), CCL3(MIP1-α), CCL4(MIP-1β) and CCL5(RANTES) cytokine/chemokine secretion both in healthy and HIV-infected individuals and to an altered apoptotic death in cells from HIV-infected individuals. Consistently, ADA-mediated actions on iDCs are able to enhance allogeneic CD4 and CD8-T-cell proliferation, globally yielding increased iDC immunogenicity. Taken together, these findings suggest that ADA would promote enhanced and correctly polarized T-cell responses in strategies targeting asymptomatic HIV-infected individuals. PMID:23240012

Surface Display of the Receptor-Binding Region of the Lactobacillus brevis S-Layer Protein in Lactococcus lactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

PubMed Central

Åvall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

2003-01-01

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium. PMID:12676705

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells.

PubMed

Avall-Jääskeläinen, Silja; Lindholm, Agneta; Palva, Airi

2003-04-01

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.

Release of Membrane-associated Mucins from Ocular Surface Epithelia

PubMed Central

Blalock, Timothy D.; Spurr-Michaud, Sandra J.; Tisdale, Ann S.; Gipson, Ilene K.

2008-01-01

Purpose Three membrane-associated mucins (MAMs)—MUC1, MUC4 and MUC16—are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. Methods An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to MUC16’s cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in release/shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor (TNF), TNF-α-converting enzyme, and matrix metalloproteinases-7 and –9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. Results MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced release of MUC16 but not MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16—78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. Conclusions Results suggest that extracellular domains of MUC1, 4, and 16 can be released from the ocular surface by agents present in tears. Neutrophil elastase and TNF present in higher amounts in dry eye patients’ tears may cause MAM release—allowing rose bengal staining. PMID:18436821

Erythrocyte enzymes in sheep: comparison of activity in fetal, newborn, maternal and nonpregnant ewe erythrocytes.

PubMed

Noble, N A; Cabalum, T C; Nathanielsz, P W; Tanaka, K R

1982-01-01

Hematological data and the activities of 21 red cell enzymes were measured in 8 nonpregnant ewes, 13 chronically catheterized fetuses at 125-135 days of gestation, and 8 of their mothers. In addition, 7 lambs were followed from birth to 17 days of age. Fetal sheep red cells have dramatically increased activities for 17 of 21 enzymes measured compared with adult nonpregnant ewes. The pattern of decline of enzyme activities with development varies considerably among enzymes. The activity of seven enzymes showed an orderly decline from fetal to adult life. For seven enzymes very little or no decline in activity was observed between 125 and 135 days of gestation and birth. Pyruvate kinase activity declined to adult levels by birth. Phosphoglucose isomerase and nucleoside phosphorylase activity increased, and glutathione peroxidase activity decreased in newborn lamb red cells compared to fetal cells. Differences in blood cell variables were also found among these groups.

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.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

PubMed Central

Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

2011-01-01

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

Development and Validation of a Whole-Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by Surface-Enhanced Laser Desorption Ionization-Time of Flight Mass Spectrometry

PubMed Central

Greis, Kenneth D.; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-01-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP. PMID:16048957

Stabilization of surface-immobilized enzymes using grafted polymers

NASA Astrophysics Data System (ADS)

Moskovitz, Yevgeny; Srebnik, Simcha

2004-03-01

Vast research efforts focus on improving the biocompatibility and biofunctionality of surfaces for artificial implants and organs. A relatively successful approach involves grafting of polymer (usually PEG) on the artificial surface, which significantly improves its biocompatibility. In addition, positioning enzymes on or in the vicinity of the surface can significantly enhance bioseparation processes. However, the catalytic activity of the anchored enzyme is often drastically impaired by the nonnatural environment, leading to loss of activity and denaturation. We study protein adsorption and stabilization by grafted polymers using a mean-field lattice model. The model protein is designed as a compact HP with a specific bulk conformation reproducing a catalytic cleft of natural enzymes. Using hydrophilic grafted polymers of tailored length and density, we show that the conformation as well as hydrophobic and active centers of the model enzyme can be restored. This research is inspired by the problem of biocompatibility and biofunctionality of surfaces for artificial implants and organs.

Synchrotron Time-Lapse Imaging of Lignocellulosic Biomass Hydrolysis: Tracking Enzyme Localization by Protein Autofluorescence and Biochemical Modification of Cell Walls by Microfluidic Infrared Microspectroscopy

PubMed Central

Devaux, Marie-Françoise; Jamme, Frédéric; André, William; Bouchet, Brigitte; Alvarado, Camille; Durand, Sylvie; Robert, Paul; Saulnier, Luc; Bonnin, Estelle; Guillon, Fabienne

2018-01-01

Tracking enzyme localization and following the local biochemical modification of the substrate should help explain the recalcitrance of lignocellulosic plant cell walls to enzymatic degradation. Time-lapse studies using conventional imaging require enzyme labeling and following the biochemical modifications of biopolymers found in plant cell walls, which cannot be easily achieved. In the present work, synchrotron facilities have been used to image the enzymatic degradation of lignocellulosic biomass without labeling the enzyme or the cell walls. Multichannel autofluorescence imaging of the protein and phenolic compounds after excitation at 275 nm highlighted the presence or absence of enzymes on cell walls and made it possible to track them during the reaction. Image analysis was used to quantify the fluorescence intensity variations. Consistent variations in the enzyme concentration were found locally for cell cavities and their surrounding cell walls. Microfluidic FT-IR microspectroscopy allowed for time-lapse tracking of local changes in the polysaccharides in cell walls during degradation. Hemicellulose degradation was found to occur prior to cellulose degradation using a Celluclast® preparation. Combining the fluorescence and FT-IR information yielded the conclusion that enzymes did not bind to lignified cell walls, which were consequently not degraded. Fluorescence multiscale imaging and FT-IR microspectroscopy showed an unexpected variability both in the initial biochemical composition and the degradation pattern, highlighting micro-domains in the cell wall of a given cell. Fluorescence intensity quantification showed that the enzymes were not evenly distributed, and their amount increased progressively on degradable cell walls. During degradation, adjacent cells were separated and the cell wall fragmented until complete degradation. PMID:29515611

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.

Inactivation of glutamate racemase (MurI) eliminates virulence in Streptococcus mutans.

PubMed

Zhang, Jianying; Liu, Jia; Ling, Junqi; Tong, Zhongchun; Fu, Yun; Liang, Min

2016-01-01

Inhibition of enzymes required for bacterial cell wall synthesis is often lethal or leads to virulence defects. Glutamate racemase (MurI), an essential enzyme in peptidoglycan biosynthesis, has been an attractive target for therapeutic interventions. Streptococcus mutans, one of the many etiological factors of dental caries, possesses a series of virulence factors associated with cariogenicity. However, little is known regarding the mechanism by which MurI influences pathogenesis of S. mutans. In this work, a stable mutant of S. mutans deficient in glutamate racemase (S. mutans FW1718) was constructed to investigate the impact of murI inactivation on cariogenic virulence in S. mutans UA159. Microscopy revealed that the murI mutant exhibited an enlarged cell size, longer cell chains, diminished cell⬜cell aggregation, and altered cell surface ultrastructure compared with the wild-type. Characterization of this mutant revealed that murI deficiency weakened acidogenicity, aciduricity, and biofilm formation ability of S. mutans (P<0.05). Real-time quantitative polymerase chain reaction (qRT-PCR) analysis demonstrated that the deletion of murI reduced the expression of the acidogenesis-related gene ldh by 44-fold (P<0.0001). The expression levels of the gene coding for surface protein antigen P (spaP) and the acid-tolerance related gene (atpD) were down-regulated by 99% (P<0.0001). Expression of comE, comD, gtfB and gtfC, genes related to biofilm formation, were down-regulated 8-, 43-, 85- and 298-fold in the murI mutant compared with the wild-type (P<0.0001), respectively. Taken together, the current study provides the first evidence that MurI deficiency adversely affects S. mutans virulence properties, making MurI a potential target for controlling dental caries. Copyright © 2016 Elsevier GmbH. All rights reserved.

Optimization of culture medium for novel cell-associated tannase production from Bacillus massiliensis using response surface methodology.

PubMed

Belur, Prasanna D; Goud, Rakesh; Goudar, Dinesh C

2012-02-01

Naturally immobilized tannase (tannin acyl hydrolase, E.C. 3.1.1.20) has many advantages, as it avoids the expensive and laborious operation of isolation, purification, and immobilization, plus it is highly stable in adverse pH and temperature. However, in the case of cell-associated enzymes, since the enzyme is associated with the biomass, separation of the pure biomass is necessary. However, tannic acid, a known inducer of tannase, forms insoluble complexes with media proteins, making it difficult to separate pure biomass. Therefore, this study optimizes the production of cell-associated tannase using a "protein-tannin complex" free media. An exploratory study was first conducted in shake-flasks to select the inducer, carbon source, and nitrogen sources. As a result it was found that gallic acid induces tannase synthesis, a tryptose broth gives higher biomass, and lactose supplementation is beneficial. The medium was then optimized using response surface methodology based on the full factorial central composite design in a 3 l bioreactor. A 2(3) factorial design augmented by 7 axial points (alpha = 1.682) and 2 replicates at the center point was implemented in 17 experiments. A mathematical model was also developed to show the effect of each medium component and their interactions on the production of cell-associated tannase. The validity of the proposed model was verified, and the optimized medium was shown to produce maximum cell-associated tannase activity of 9.65 U/l, which is 93.8% higher than the activity in the basal medium, after 12 h at pH 5.0, 30 degrees C. The optimum medium consists of 38 g/l lactose, 50 g/l tryptose, and 2.8 g/l gallic acid.

Protein disulfide isomerase and Nox: new partners in redox signaling.

PubMed

Trevelin, Silvia Cellone; Lopes, Lucia Rossetti

2015-01-01

Reactive oxygen species (ROS) contribute to the pathogenesis of cardiovascular disease, including hypertension, atherosclerosis, cardiac hypertrophy, heart failure and restenosis. Thiol proteins and thiol oxidoreductases are key players in cell signaling, and their altered expression and/or activity has been associated with a disrupture in cardiac and vascular homeostasis. Protein disulfide isomerase (PDI) is a thiol oxidoreductase member of the thioredoxin family that has multiple roles in cellular function. Originally discovered in the endoplasmic reticulum (ER), PDI is essential for protein folding. However, it can also be found in the cytosol and closely associated with the surface of platelets, smooth muscle cells, neutrophils and endothelial cells. On the cell surface, PDI is imperative for platelet aggregation and transnitrosation, which are related to thrombosis and control of vascular tone by nitric oxide, respectively. Furthermore, PDI signaling contributes to redox-dependent events such as smooth muscle cell migration induced by PDGF and TNFα-dependent angiogenesis. Studies from our group have shown that intracellular PDI regulates the expression and activity of the NADPH oxidase family of proteins (Nox), which are enzymes dedicated to ROS generation. PDI acts as a new organizer of leukocyte Nox2 by redox dependently associating with p47phox and controlling its recruitment to the plasma membrane, an essential step for assembly of the active enzyme. Such multiple effects of PDI suggest that specific targeting of this oxidoreductase could represent a new approach in the treatment of vascular disease. In this review, we present a novel role for PDI as an adaptor protein involved in redox processes and Nox signaling and propose PDI as a potential therapeutic target in the treatment of atherosclerosis, thrombosis and hypertension.

Redefining the effect of salt on thermophilic starter cell viability, culturability and metabolic activity in cheese.

PubMed

Hickey, C D; Fallico, V; Wilkinson, M G; Sheehan, J J

2018-02-01

This study investigated the differential effect of salt concentration in the outside and inside layers of brine salted cheeses on viability, culturability and enzyme activity of starter bacteria. The high-salt environment of the outside layer caused a sharp decrease in L. helveticus viability as measured by traditional plate counts. Remarkably, this was associated with lower release of intracellular enzymes (LDH), reduced levels of proteolysis and larger membrane integrity as measured by flow cytometry (FC) following classical Live/Dead staining. FC analysis of light scattering properties highlighted a significant reduction in size and granularity of the microbiota located in the cheese surface, suggestive of cell shrinkage and condensation of internal macromolecules probably due to hyperosmotic stress. The microbiota of the cheese surface were found to experience greater oxidative stress, as measured by FC analysis of the total levels of reactive oxygen species, compared to that of the interior layer. These results lead us to postulate that the physiology and health status of the microbiota were significantly different in the outer and inner layers of the cheese. The hyperosmotic environment of the outer layer resulted in reduced cell lysis, as measurable by assays based upon membrane integrity, but rather triggered cell death via mechanisms involving cell shrinkage and ROS-mediated damage of vital intracellular components. This study challenges the current thinking on how salt controls microbial activity in ripening cheese, especially in cheeses which are brine salted as local variations in biochemical ripening indices can differ significantly from the outside to the inside of a ripening cheese. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enzyme Prodrug Therapy Achieves Site-Specific, Personalized Physiological Responses to the Locally Produced Nitric Oxide

PubMed Central

2018-01-01

Nitric oxide (NO) is a highly potent but short-lived endogenous radical with a wide spectrum of physiological activities. In this work, we developed an enzymatic approach to the site-specific synthesis of NO mediated by biocatalytic surface coatings. Multilayered polyelectrolyte films were optimized as host compartments for the immobilized β-galactosidase (β-Gal) enzyme through a screen of eight polycations and eight polyanions. The lead composition was used to achieve localized production of NO through the addition of β-Gal–NONOate, a prodrug that releases NO following enzymatic bioconversion. The resulting coatings afforded physiologically relevant flux of NO matching that of the healthy human endothelium. The antiproliferative effect due to the synthesized NO in cell culture was site-specific: within a multiwell dish with freely shared media and nutrients, a 10-fold inhibition of cell growth was achieved on top of the biocatalytic coatings compared to the immediately adjacent enzyme-free microwells. The physiological effect of NO produced via the enzyme prodrug therapy was validated ex vivo in isolated arteries through the measurement of vasodilation. Biocatalytic coatings were deposited on wires produced using alloys used in clinical practice and successfully mediated a NONOate concentration-dependent vasodilation in the small arteries of rats. The results of this study present an exciting opportunity to manufacture implantable biomaterials with physiological responses controlled to the desired level for personalized treatment. PMID:29570264

Cloning, expression, and characterization of a peculiar choline-binding beta-galactosidase from Streptococcus mitis.

PubMed

Campuzano, Susana; Serra, Beatriz; Llull, Daniel; García, José L; García, Pedro

2009-09-01

A Streptococcus mitis genomic DNA fragment carrying the SMT1224 gene encoding a putative beta-galactosidase was identified, cloned, and expressed in Escherichia coli. This gene encodes a protein 2,411 amino acids long with a predicted molecular mass of 268 kDa. The deduced protein contains an N-terminal signal peptide and a C-terminal choline-binding domain consisting of five consensus repeats, which facilitates the anchoring of the secreted enzyme to the cell wall. The choline-binding capacity of the protein facilitates its purification using DEAE-cellulose affinity chromatography, although its complete purification was achieved by constructing a His-tagged fusion protein. The recombinant protein was characterized as a monomeric beta-galactosidase showing a specific activity of around 2,500 U/mg of protein, with optimum temperature and pH ranges of 30 to 40 degrees C and 6.0 to 6.5, respectively. Enzyme activity is not inhibited by glucose, even at 200 mM, and remains highly stable in solution or immobilized at room temperature in the absence of protein stabilizers. In S. mitis, the enzyme was located attached to the cell surface, but a significant activity was also detected in the culture medium. This novel enzyme represents the first beta-galactosidase having a modular structure with a choline-binding domain, a peculiar property that can also be useful for some biotechnological applications.

Effects of the propeptide of group X secreted phospholipase A(2) on substrate specificity and interfacial activity on phospholipid monolayers.

PubMed

Point, Vanessa; Bénarouche, Anaïs; Jemel, Ikram; Parsiegla, Goetz; Lambeau, Gérard; Carrière, Frédéric; Cavalier, Jean-François

2013-01-01

Group X secreted phospholipase A(2) (GX sPLA(2)) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA(2) activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA(2) activity of recombinant mouse GX sPLA(2) (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA(2) activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30-35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA(2) activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA(2). This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Evidence of an Unidentified Extracellular Heat-Stable Factor Produced by Lysobacter enzymogenes (OH11) that Degrade Fusarium graminearum PH1 Hyphae.

PubMed

Odhiambo, Benard Omondi; Xu, Gaoge; Qian, Guoliang; Liu, Fengquan

2017-04-01

Lysobacter enzymogenes OH11 produces heat-stable antifungal factor (HSAF) and lytic enzymes possessing antifungal activity. This study bio-prospected for other potential antifungal factors besides those above. The cells and extracellular metabolites of L. enzymogenes OH11 and the mutants ΔchiA, ΔchiB, ΔchiC, Δclp, Δpks, and ΔpilA were examined for antifungal activity against Fusarium graminearum PH1, the causal agent of Fusarium head blight (FHB). Results evidenced that OH11 produces an unidentified extracellular heat-stable degrading metabolite (HSDM) that exhibit degrading activity on F. graminearum PH1 chitinous hyphae. Interestingly, both heat-treated and non-heat-treated extracellular metabolites of OH11 mutants exhibited hyphae-degrading activity against F. graminearum PH1. Enzyme activity detection of heat-treated metabolites ruled out the possibility of enzyme degradation activity. Remarkably, the PKS-NRPS-deficient mutant Δpks cannot produce HSAF or analogues, yet its metabolites exhibited hyphae-degrading activity. HPLC analysis confirmed no HSAF production by Δpks. Δclp lacks hyphae-degrading ability. Therefore, clp regulates HSDM and extracellular lytic enzymes production in L. enzymogenes OH11. ΔpilA had impaired surface cell motility and significantly reduced antagonistic properties. ΔchiA, ΔchiB, and ΔchiC retained hyphae-degrading ability, despite having reduced abilities to produce chitinase enzymes. Ultimately, L. enzymogenes OH11 can produce other unidentified HSDM independent of the PKS-NRPS genes. This suggests HSAF and lytic enzymes production are a fraction of the antifungal mechanisms in OH11. Characterization of HSDM, determination of its biosynthetic gene cluster and understanding its mode of action will provide new leads in the search for effective drugs for FHB management.

Autodigestion: Proteolytic Degradation and Multiple Organ Failure in Shock

PubMed Central

Altshuler, Angelina E.; Kistler, Erik B.; Schmid-Schönbein, Geert W.

2015-01-01

There is currently no effective treatment for multiorgan failure following shock other than alleviation supportive care. A better understanding of the pathogenesis of these sequelae to shock is required. The intestine plays a central role in multiorgan failure. It was previously suggested that bacteria and their toxins are responsible for the organ failure seen in circulatory shock, but clinical trials in septic patients have not confirmed this hypothesis. Instead, we review here evidence that the digestive enzymes, synthesized in the pancreas and discharged into the small intestine as requirement for normal digestion, may play a role in multi-organ failure. These powerful enzymes are non-specific, highly concentrated and fully activated in the lumen of the intestine. During normal digestion they are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. However, if this barrier becomes permeable, e.g. in an ischemic state, the digestive enzymes escape into the wall of the intestine. They digest tissues in the mucosa and generate small molecular weight cytotoxic fragments such as unbound free fatty acids. Digestive enzymes may also escape into the systemic circulation and activate other degrading proteases. These proteases have the ability to clip the ectodomain of surface receptors and compromise their function; for example cleaving the insulin receptor causing insulin resistance. The combination of digestive enzymes and cytotoxic fragments leaking into the central circulation causes cell and organ dysfunction, and ultimately may lead to complete organ failure and death. We summarize current evidence suggesting that enteral blockade of digestive enzymes inside the lumen of the intestine may serve to reduce acute cell and organ damage and improve survival in experimental shock. PMID:26717111

Studies of the kallikrein-kinin system and prostaglandins in epithelial ion transport.

PubMed

Margolius, H S; Halushka, P V; Chao, J; Miller, D H; Cuthbert, A W; Spayne, J A

1985-01-01

Tissue kallikrein of colon mucosa is synthesized rapidly, and this synthetic process can now be examined in relation to hormonal or dietary manipulations or pathological circumstances that affect intestinal ion transport. Although the identical renal tissue enzyme is known to be enriched in membranes of distal convoluted tubular epithelial cells, the precise localization of the intestinal enzyme is uncertain. An understanding of the intestinal cellular locale of kallikrein will help in defining its local role. That tissue kallikreins can be inhibited by monovalent cations and some drugs (e.g., amiloride) and that kallikrein inhibitors affect cation transport across epithelial surfaces containing such enzymes must be reconciled with the new observations of kinin-induced chloride secretion. Extracellular calcium, eicosanoid synthesis, and cyclic nucleotide production are involved in the secretory response to kinins, although an absolute requirement for intact eicosanoid synthesis may not exist.

Rapid enzyme regeneration results in the striking catalytic longevity of an engineered, single species, biocatalytic biofilm.

PubMed

Tong, Xiaoxue; Barberi, Tania Triscari; Botting, Catherine H; Sharma, Sunil V; Simmons, Mark J H; Overton, Tim W; Goss, Rebecca J M

2016-10-21

Engineering of single-species biofilms for enzymatic generation of fine chemicals is attractive. We have recently demonstrated the utility of an engineered Escherichia coli biofilm as a platform for synthesis of 5-halotryptophan. E. coli PHL644, expressing a recombinant tryptophan synthase, was employed to generate a biofilm. Its rapid deposition, and instigation of biofilm formation, was enforced by employing a spin-down method. The biofilm presents a large three-dimensional surface area, excellent for biocatalysis. The catalytic longevity of the engineered biofilm is striking, and we had postulated that this was likely to largely result from protection conferred to recombinant enzymes by biofilm's extracellular matrix. SILAC (stable isotopic labelled amino acids in cell cultures), and in particular dynamic SILAC, in which pulses of different isotopically labelled amino acids are administered to cells over a time course, has been used to follow the fate of proteins. To explore within our spin coated biofilm, whether the recombinant enzyme's longevity might be in part due to its regeneration, we introduced pulses of isotopically labelled lysine and phenylalanine into medium overlaying the biofilm and followed their incorporation over the course of biofilm development. Through SILAC analysis, we reveal that constant and complete regeneration of recombinant enzymes occurs within spin coated biofilms. The striking catalytic longevity within the biofilm results from more than just simple protection of active enzyme by the biofilm and its associated extracellular matrix. The replenishment of recombinant enzyme is likely to contribute significantly to the catalytic longevity observed for the engineered biofilm system. Here we provide the first evidence of a recombinant enzyme's regeneration in an engineered biofilm. The recombinant enzyme was constantly replenished over time as evidenced by dynamic SILAC, which suggests that the engineered E. coli biofilms are highly metabolically active, having a not inconsiderable energetic demand. The constant renewal of recombinant enzyme highlights the attractive possibility of utilising this biofilm system as a dynamic platform into which enzymes of interest can be introduced in a "plug-and-play" fashion and potentially be controlled through promoter switching for production of a series of desired fine chemicals.

Shifted Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65 results in formation of high mannose N-glycans in aggressive prostate cancer cells.

PubMed

Bhat, Ganapati; Hothpet, Vishwanath-Reddy; Lin, Ming-Fong; Cheng, Pi-Wan

2017-11-01

There is a pressing need for biomarkers that can distinguish indolent from aggressive prostate cancer to prevent over-treatment of patients with indolent tumor. Golgi targeting of glycosyltransferases was characterized by confocal microscopy after knockdown of GM130, giantin, or both. N-glycans on a trans-Golgi enzyme β4galactosyltransferase-1 isolated by immunoprecipitation from androgen-sensitive and independent prostate cancer cells were determined by matrix-assisted laser desorption-time of flight-mass spectrometry. In situ proximity ligation assay was employed to determine co-localization of (a) α-mannosidase IA, an enzyme required for processing Man 8 GlcNAc 2 down to Man 5 GlcNAc 2 to enable synthesis of complex-type N-glycans, with giantin, GM130, and GRASP65, and (b) trans-Golgi glycosyltransferases with high mannose N-glycans terminated with α3-mannose. Defective giantin in androgen-independent prostate cancer cells results in a shift of Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65. Consequently, trans-Golgi enzymes and cell surface glycoproteins acquire high mannose N-glycans, which are absent in cells with functional giantin. In situ proximity ligation assays of co-localization of α-mannosidase IA with GM130 and GRASP65, and trans-Golgi glycosyltransferases with high mannose N-glycans are negative in androgen-sensitive LNCaP C-33 cells but positive in androgen-independent LNCaP C-81 and DU145 cells, and LNCaP C-33 cells devoid of giantin. In situ proximity ligation assays of Golgi localization of α-mannosidase IA at giantin versus GM130-GRASP65 site, and absence or presence of N-glycans terminated with α3-mannose on trans-Golgi glycosyltransferases may be useful for distinguishing indolent from aggressive prostate cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Binding of heparin to plasma proteins and endothelial surfaces is inhibited by covalent linkage to antithrombin.

PubMed

Chan, Anthony K C; Paredes, Nethnapha; Thong, Bruce; Chindemi, Paul; Paes, Bosco; Berry, Leslie R; Monagle, Paul

2004-05-01

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are used for prophylaxis and treatment of thrombosis. However, UFH has a short plasma half-life and variable anticoagulant response in vivo due to plasma or vessel wall protein binding and LMWH has a decreased ability to inactivate thrombin, the pivotal enzyme in the coagulation cascade. Covalent linkage of antithrombin to heparin gave a complex (ATH) with superior anticoagulant activity compared to UFH and LMWH, and longer intravenous half-life compared to UFH. We found that plasma proteins bound more to UFH than ATH, and least to LMWH. Also, UFH bound significantly more to endothelial cells than ATH, with 100% of UFH and 94% of ATH binding being on the cell surface and the remainder was endocytosed. Competition studies with UFH confirmed that ATH binding was likely through its heparin moiety. These findings suggest that differences in plasma protein and endothelial cell binding may be due to available heparin chain length. Although ATH is polydisperse, the covalently-linked antithrombin may shield a portion of the heparin chain from association with plasma or endothelial cell surface proteins. This model is consistent with ATH's better bioavailability and more predictable dose response.

Beyond the hydrophobic effect: Critical function of water at biological phase boundaries--A hypothesis.

PubMed

Damodaran, Srinivasan

2015-07-01

Many life-sustaining processes in living cells occur at the membrane-water interface. The pertinent questions that need to be asked are what is the evolutionary reason for biology to choose the membrane-water interface as the site for performing and/or controlling crucial biological reactions and what is the key physical principle that is singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this review, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes and receptor activated processes via manipulating the thermodynamic activity of water at the membrane-water interfacial region. In support of this hypothesis, first we establish that the surface pressure of a lipid monolayer is a direct measure of a reduction in the thermodynamic activity of interfacial water. Second, we show that the surface pressure-dependent activation/inactivation of interfacial enzymes is fundamentally related to their dependence on interfacial water activity. We extend this argument to infer that cells might manipulate activities of membrane-associated biological processes via manipulating the activity of interfacial water via localized compression or expansion of the interface. In this paper, we critically analyze literature data on mechano-activation of large pore ion channels in Escherichia coli spheroplasts and G-proteins in reconstituted lipid vesicles, and show that these pressure-induced activation processes are fundamentally and quantitatively related to changes in the thermodynamic state of interfacial water, caused by mechanical stretching of the bilayer. Copyright © 2015 Elsevier B.V. All rights reserved.

Feedback control of AHR signalling regulates intestinal immunity.

PubMed

Schiering, Chris; Wincent, Emma; Metidji, Amina; Iseppon, Andrea; Li, Ying; Potocnik, Alexandre J; Omenetti, Sara; Henderson, Colin J; Wolf, C Roland; Nebert, Daniel W; Stockinger, Brigitta

2017-02-09

The aryl hydrocarbon receptor (AHR) recognizes xenobiotics as well as natural compounds such as tryptophan metabolites, dietary components and microbiota-derived factors, and it is important for maintenance of homeostasis at mucosal surfaces. AHR activation induces cytochrome P4501 (CYP1) enzymes, which oxygenate AHR ligands, leading to their metabolic clearance and detoxification. Thus, CYP1 enzymes have an important feedback role that curtails the duration of AHR signalling, but it remains unclear whether they also regulate AHR ligand availability in vivo. Here we show that dysregulated expression of Cyp1a1 in mice depletes the reservoir of natural AHR ligands, generating a quasi AHR-deficient state. Constitutive expression of Cyp1a1 throughout the body or restricted specifically to intestinal epithelial cells resulted in loss of AHR-dependent type 3 innate lymphoid cells and T helper 17 cells and increased susceptibility to enteric infection. The deleterious effects of excessive AHR ligand degradation on intestinal immune functions could be counter-balanced by increasing the intake of AHR ligands in the diet. Thus, our data indicate that intestinal epithelial cells serve as gatekeepers for the supply of AHR ligands to the host and emphasize the importance of feedback control in modulating AHR pathway activation.

Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

PubMed Central

Baltar, Federico

2018-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

Immobilization of glucoamylase on ceramic membrane surfaces modified with a new method of treatment utilizing SPCP-CVD.

PubMed

Ida; Matsuyama; Yamamoto

2000-07-01

Glucoamylase, as a model enzyme, was immobilized on a ceramic membrane modified by surface corona discharge induced plasma chemical process-chemical vapor deposition (SPCP-CVD). Characterizations of the immobilized enzyme were then discussed. Three kinds of ceramic membranes with different amounts of amino groups on the surface were prepared utilizing the SPCP-CVD method. Each with 1-time, 3-times and 5-times surface modification treatments and used for supports in glucoamylase immobilization. The amount of immobilized glucoamylase increased with the increase in the number of surface modification treatments and saturated to a certain maximum value estimated by a two-dimensional random packing. The operational stability of the immobilized glucoamylase also increased with the increase in the number of the surface treatment. It was almost the same as the conventional method, while the activity of immobilized enzyme was higher. The results indicated the possibility of designing the performance of the immobilized enzyme by controlling the amount of amino groups. The above results showed that the completely new surface modification method using SPCP was effective in modifying ceramic membranes for enzyme immobilization.

A synthetic urinary probe–coated nanoparticles sensitive to fibroblast activation protein α for solid tumor diagnosis

PubMed Central

Feng, Xinwei; Wang, Qifan; Liao, Yuehua; Zhou, Xie; Wang, Yidan; Liu, Wanli; Zhang, Ge

2017-01-01

We developed fibroblast activation protein α (FAPα)-sensitive magnetic iron oxide nanoparticles (MNPs) by conjugating a substrate-reporter tandem peptide as a synthetic biomarker to the surface of MNPs (marker-MNPs). In vitro, the marker-MNPs showed stability when treated with serum or urine and exhibited high susceptibility and specificity for FAPα enzyme and 3T3/FAPα cell line. Furthermore, the marker-MNPs were administered to esophageal squamous cell carcinoma xenograft tumor mice; they reached the tumor tissues in the mice, where they were cleaved effectively by the local overexpressed FAPα to release the reporter peptide and filter it into the urine. The tumor targeting and biodistribution of marker-MNPs were verified by in vivo imaging. The cleaved reporter peptides in urine detected by enzyme-linked immunosorbent assay have high diagnostic accuracy for esophageal squamous cell carcinoma (area under the receiver-operating characteristic curve =1.0). Our study implies a promising strategy of utilizing the low-cost and noninvasive synthetic urinary probe–coated nanoparticles for the diagnosis of FAPα-positive solid tumors, except for in renal cancer. PMID:28794628

Contact activation: a revision.

PubMed

Schmaier, A H

1997-07-01

In conclusion, a revised view of the contact system has been presented. This system has little to do with the initiation of hemostasis. Like lupus anticoagulants, deficiencies of contact proteins give prolonged APTTs but may be risk factors for thrombosis. BK from kininogens is a potent modulator of vascular biology inducing vasodilation, tissue plasminogen activator release, and prostacyclin liberation. Kininogens, themselves, are selective inhibitors of alpha-thrombin-induced platelet activation preventing alpha-thrombin from cleaving the cloned thrombin receptor after arginine41. Kininogens' alpha-thrombin inhibitory activity exists in intact kininogens, BK, and all of BK's breakdown products. HK also is the pivotal protein for contact protein assembly on endothelium. It is the receptor for prekallikrein which when bound to HK becomes activated to kallikrein by an endothelial cell enzyme system independent of activated forms of plasma factor XII. Prekallikrein activation on endothelial cells results in kinetically favorable single chain urokinase and plasminogen activation. Thus the "physiologic, negatively charged surface" for contact system activation is really the assembly of these proteins on cell membranes and activation by membrane-associated enzymes.

Carbonic anhydrase enzymes regulate mast cell–mediated inflammation

PubMed Central

Soteropoulos, Patricia

2016-01-01

Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine–mediated inflammation through their enhanced ability to develop into mast cells. In this study, we show that carbonic anhydrase (Car) enzymes are up-regulated in type 2–associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent mouse mast cell responses and inflammation after Trichinella spiralis infection or the induction of food allergy–like disease. Further, we used CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively, these experiments identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be used to treat mast cell–mediated inflammation. PMID:27526715

Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1*

PubMed Central

Awad, Wael; Adamczyk, Barbara; Örnros, Jessica; Karlsson, Niclas G.; Mani, Katrin; Logan, Derek T.

2015-01-01

Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation. PMID:26203194

Fiber Optic Surface Plasmon Resonance-Based Biosensor Technique: Fabrication, Advancement, and Application.

PubMed

Liang, Gaoling; Luo, Zewei; Liu, Kunping; Wang, Yimin; Dai, Jianxiong; Duan, Yixiang

2016-05-03

Fiber optic-based biosensors with surface plasmon resonance (SPR) technology are advanced label-free optical biosensing methods. They have brought tremendous progress in the sensing of various chemical and biological species. This review summarizes four sensing configurations (prism, grating, waveguide, and fiber optic) with two ways, attenuated total reflection (ATR) and diffraction, to excite the surface plasmons. Meanwhile, the designs of different probes (U-bent, tapered, and other probes) are also described. Finally, four major types of biosensors, immunosensor, DNA biosensor, enzyme biosensor, and living cell biosensor, are discussed in detail for their sensing principles and applications. Future prospects of fiber optic-based SPR sensor technology are discussed.

Intravenous anesthetic propofol suppresses prostaglandin E2 production in murine dendritic cells.

PubMed

Inada, Takefumi; Kubo, Kozue; Ueshima, Hironobu; Shingu, Koh

2011-01-01

Propofol is an intravenous anesthetic that is widely used for anesthesia and sedation. Dendritic cells (DC) are one of the crucial immune cells that bridge innate and adaptive immunity, in which DC process antigens during innate immune responses to present them to naïve T-cells, leading to an establishment of adaptive immunity. Prostaglandin (PG)-E(2) may be secreted by DC into the microenvironment, considerably influencing DC phenotype and function, and thus determining the fate of adaptive immunity. Since propofol suppresses PGE(2) production in murine macrophages, the primary purpose of the present study was to determine whether propofol also suppresses PGE(2) production in DC. Assuming a positive finding of such suppression, we tested whether this also leads to alterations of interleukin (IL)-12 and IL-10 production and DC surface marker expression, both of which can be modulated by PGE(2). In bone marrow-derived DC, propofol significantly suppressed the PGE(2) production after lipopolysaccharide stimulation. Cyclo-oxygenase (COX) protein expression and arachidonic acid release were unaffected, while COX enzyme activity was significantly inhibited by propofol. The propofol-induced COX inhibition did not lead to the increased production of cysteinyl leukotrienes and leukotriene-B(4). Endogenous COX inhibition with propofol, as well as with the selective COX-2 inhibitor, NS-398, did not affect IL-12 and IL-10 production from DC. The surface expression of I-A(b) and CD40 on DC was not changed, while that of CD86 slightly increased, with both propofol and NS-398; expression of CD80 was not affected with propofol, but increased slightly with NS-398. Finally, endogenous COX inhibition with either propofol or NS-398 did not significantly affect the ability of DC to induce allogeneic T-cell proliferation. It is concluded that the intravenous anesthetic propofol suppresses COX enzyme activity in DC, with no consequences with respect to IL-12/IL-10 production and allogeneic T-cell proliferation, while minimal consequences were observed in surface molecule expression.

Proteomic analyses for profiling regulated proteins/enzymes by Fucus vesiculosus fucoidan in B16 melanoma cells: A combination of enzyme kinetics functional study.

PubMed

Wang, Zhi-Jiang; Zheng, Li; Yang, Jun-Mo; Kang, Yani; Park, Yong-Doo

2018-06-01

Fucoidans are complex sulfated polysaccharides that have a wide range of biological activities. Previously, we reported the various effects of Fucus vesiculosus fucoidan on tyrosinase and B16 melanoma cells. In this study, to identify fucoidan-targeted proteins in B16 melanoma cells, we performed a proteomics study and integrated enzyme kinetics. We detected 19 candidate proteins dysregulated by fucoidan treatment. Among the probed proteins, the enzyme kinetics of two candidate enzymes, namely lactate dehydrogenase (LDH) as an upregulated protein and superoxide dismutase (SOD) as a downregulated enzyme, were determined. The enzyme kinetics results showed that Fucus vesiculosus fucoidan significantly inhibited LDH catalytic function while it did not affect SOD activity even at a high dose, while only slightly decreased activity (up to 10%) at a low dose. Based on our previous and present observations, fucoidan could inhibit B16 melanoma cells growth via regulating proteins/enzymes expression levels such as LDH and SOD known as cell survival biomarkers. Interestingly, both expression level and enzyme catalytic activity of LDH were regulated by fucoidan, which could directly induce the apoptotic effect on B16 melanoma cells along with SOD downregulation. This study highlights how combining proteomics with enzyme kinetics can yield valuable insights into fucoidan targets. Copyright © 2018 Elsevier B.V. All rights reserved.

Glucose-Driven Fuel Cell Constructed from Enzymes and Filter Paper

ERIC Educational Resources Information Center

Ge, Jun; Schirhagl, Romana; Zare, Richard N.

2011-01-01

A glucose-driven enzymatic filter-paper fuel cell is described. A strip of filter paper coated with carbon nanotubes and the glucose oxidase enzyme functions as the anode of the enzyme fuel cell. Another strip of filter paper coated with carbon nanotubes and the laccase enzyme functions as the cathode. Between the anode and the cathode, a third…

Kinase Activity Studied in Living Cells Using an Immunoassay

ERIC Educational Resources Information Center

Bavec, Aljos?a

2014-01-01

This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function

PubMed Central

Smith, Gina A.; Fearnley, Gareth W.; Abdul-Zani, Izma; Wheatcroft, Stephen B.; Tomlinson, Darren C.; Harrison, Michael A.

2017-01-01

ABSTRACT Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response. PMID:28798148

Ligation of CD8α on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity

PubMed Central

Addison, Elena G; North, Janet; Bakhsh, Ismail; Marden, Chloe; Haq, Sumaira; Al-Sarraj, Samia; Malayeri, Reza; Wickremasinghe, R Gitendra; Davies, Jeffrey K; Lowdell, Mark W

2005-01-01

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric α/α form are more cytotoxic than their CD8– counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8– NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8α+ cells after lysis of the same targets. We report that cross-linking of the CD8α chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK-cell apoptosis from which CD8α+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK-cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti-MHC Class I antibody. Thus, in contrast to the CD8– subset, CD8α+ NK cells are capable of sequential lysis of multiple target cells. PMID:16236125

A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

USGS Publications Warehouse

Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

2005-01-01

Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Ku??tzing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L-1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method. ?? Springer 2005.

A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

USGS Publications Warehouse

Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

2005-01-01

Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Kützing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L−1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method.

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

Future of biosensors: a personal view.

PubMed

Scheller, Frieder W; Yarman, Aysu; Bachmann, Till; Hirsch, Thomas; Kubick, Stefan; Renneberg, Reinhard; Schumacher, Soeren; Wollenberger, Ulla; Teller, Carsten; Bier, Frank F

2014-01-01

Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the "100-dollar" personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday "consumables" such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of "autonomous" biosensors will emerge.

Iron-sulfur-based single molecular wires for enhancing charge transport in enzyme-based bioelectronic systems.

PubMed

Mahadevan, Aishwarya; Fernando, Teshan; Fernando, Sandun

2016-04-15

When redox enzymes are wired to electrodes outside a living cell (ex vivo), their ability to produce a sufficiently powerful electrical current diminishes significantly due to the thermodynamic and kinetic limitations associated with the wiring systems. Therefore, we are yet to harness the full potential of redox enzymes for the development of self-powering bioelectronics devices (such as sensors and fuel cells). Interestingly, nature uses iron-sulfur complexes ([Fe-S]), to circumvent these issues in vivo. Yet, we have not been able to utilize [Fe-S]-based chains ex vivo, primarily due to their instability in aqueous media. Here, a simple technique to attach iron (II) sulfide (FeS) to a gold surface in ethanol media and then complete the attachment of the enzyme in aqueous media is reported. Cyclic voltammetry and spectroscopy techniques confirmed the concatenation of FeS and glycerol-dehydrogenase/nicotinamide-adenine-dinucleotide (GlDH-NAD(+)) apoenzyme-coenzyme molecular wiring system on the base gold electrode. The resultant FeS-based enzyme electrode reached an open circuit voltage closer to its standard potential under a wide range of glycerol concentrations (0.001-1M). When probed under constant potential conditions, the FeS-based electrode was able to amplify current by over 10 fold as compared to electrodes fabricated with the conventional pyrroloquinoline quinone-based composite molecular wiring system. These improvements in current/voltage responses open up a wide range of possibilities for fabricating self-powering, bio-electronic devices. Copyright © 2015 Elsevier B.V. All rights reserved.

Albumin/asparaginase capsules prepared by ultrasound to retain ammonia.

PubMed

Tinoco, Ana; Ribeiro, Artur; Oliveira, César; Parpot, Pier; Gomes, Andreia; Cavaco-Paulo, Artur

2016-11-01

Asparaginase reduces the levels of asparagine in blood, which is an essential amino acid for the proliferation of lymphoblastic malign cells. Asparaginase converts asparagine into aspartic acid and ammonia. The accumulation of ammonia in the bloodstream leads to hyperammonemia, described as one of the most significant side effects of asparaginase therapy. Therefore, there is a need for asparaginase formulations with the potential to reduce hyperammonemia. We incorporated 2 % of therapeutic enzyme in albumin-based capsules. The presence of asparaginase in the interface of bovine serum albumin (BSA) capsules showed the ability to hydrolyze the asparagine and retain the forming ammonia at the surface of the capsules. The incorporation of Poloxamer 407 in the capsule formulation further increased the ratio aspartic acid/ammonia from 1.92 to 2.46 (and 1.10 from the free enzyme), decreasing the levels of free ammonia. This capacity to retain ammonia can be due to electrostatic interactions and retention of ammonia at the surface of the capsules. The developed BSA/asparaginase capsules did not cause significant cytotoxic effect on mouse leukemic macrophage cell line RAW 264.7. The new BSA/asparaginase capsules could potentially be used in the treatment of acute lymphoblastic leukemia preventing hyperammonemia associated with acute lymphoblastic leukemia (ALL) treatment with asparaginase.

Intestinal epithelial cell surface glycosylation in mice. I. Effect of high-protein diet.

PubMed

Gupta, R; Jaswal, V M; Meenu Mahmood, A

1992-01-01

The effects of variation in dietary protein content have been investigated on brush border glycosylation and enzyme activities in mice small intestine. The comparison of different parameters was made between the mice fed 30% (high protein, HP) and 18% protein (pair-fed, PF, and ad libitum-fed) for 21 days. The activities of brush border sucrase, lactase, p-nitrophenyl (PNP)-beta-D-glucosidase and PNP-beta-D-galactosidase were reduced in the HP diet-fed mice compared to PF and ad libitum-fed controls. Alkaline phosphatase and leucine amino-peptidase activities were significantly enhanced while gamma-glutamyl transpeptidase activity was unaltered under these conditions. Total hexoses and sialic acid content in the brush borders were reduced significantly in the test group compared to the controls while hexosamine and fucose contents remained essentially similar in different groups. The results on the binding of wheat germ agglutinin and Ulex europaeus agglutininI to microvillus membranes corroborated the chemical analysis data on sialic acid and fucose contents of the membranes. Peanut agglutinin binding was enhanced in mice from the HP group. Incorporation of (14C)-mannose into membranes was significantly less in HP diet-fed mice. These results indicate that the feeding of HP diet to mice brings about marked alterations in small intestinal epithelial cell surface glycosylation and enzyme functions.

Study of solid/liquid and solid/gas interfaces in Cu-isoleucine complex by surface X-ray diffraction

NASA Astrophysics Data System (ADS)

Ferrer, Pilar; Rubio-Zuazo, Juan; Castro, German R.

2013-02-01

The enzymes could be understood like structures formed by amino acids bonded with metals, which act as active sites. The research on the coordination of metal-amino acid complexes will bring light on the behavior of metal enzymes, due to the close relation existing between the atomic structure and the functionality. The Cu-isoleucine bond is considered as a good model system to attain a better insight into the characteristics of naturally occurring copper metalloproteins. The surface structure of metal-amino acid complex could be considered as a more realistic model for real systems under biologic working conditions, since the molecular packing is decreased. In the surface, the structural constrains are reduced, keeping the structural capability of surface complex to change as a function of the surrounding environment. In this work, we present a surface X-ray diffraction study on Cu-isoleucine complex under different ambient conditions. Cu(Ile)2 crystals of about 5 mm × 5 mm × 1 mm have been growth, by seeding method in a supersaturated solution, presenting a surface of high quality. The sample for the surface diffraction study was mounted on a cell specially designed for solid/liquid or solid/gas interface analysis. The Cu-isoleucine crystal was measured under a protective dry N2 gas flow and in contact with a saturated metal amino acid solution. The bulk and the surface signals were compared, showing different atomic structures. In both cases, from surface diffraction data, it is observed that the atomic structure of the top layer undergoes a clear structural deformation. A non-uniform surface relaxation is observed producing an inhomogeneous displacement of the surface atoms towards the surface normal.

Optimization of thermophilic trans-isoprenyl diphosphate synthase expression in Escherichia coli by response surface methodology.

PubMed

Piccolomini, Angelica A; Fiabon, Alex; Borrotti, Matteo; De Lucrezia, Davide

2017-01-01

We optimized the heterologous expression of trans-isoprenyl diphosphate synthase (IDS), the key enzyme involved in the biosynthesis of trans-polyisoprene. trans-Polyisoprene is a particularly valuable compound due to its superior stiffness, excellent insulation, and low thermal expansion coefficient. Currently, trans-polyisoprene is mainly produced through chemical synthesis and no biotechnological processes have been established so far for its large-scale production. In this work, we employed D-optimal design and response surface methodology to optimize the expression of thermophilic enzymes IDS from Thermococcus kodakaraensis. The design of experiment took into account of six factors (preinduction cell density, inducer concentration, postinduction temperature, salt concentration, alternative carbon source, and protein inhibitor) and seven culture media (LB, NZCYM, TB, M9, Ec, Ac, and EDAVIS) at five different pH points. By screening only 109 experimental points, we were able to improve IDS production by 48% in close-batch fermentation. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

A facile and efficient method of enzyme immobilization on silica particles via Michael acceptor film coatings: immobilized catalase in a plug flow reactor.

PubMed

Bayramoglu, Gulay; Arica, M Yakup; Genc, Aysenur; Ozalp, V Cengiz; Ince, Ahmet; Bicak, Niyazi

2016-06-01

A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120 h at 30 °C with only a loss of about 36 % of its initial activity.

Quantifying the importance of galactofuranose in Aspergillus nidulans hyphal wall surface organization by atomic force microscopy.

PubMed

Paul, Biplab C; El-Ganiny, Amira M; Abbas, Mariam; Kaminskyj, Susan G W; Dahms, Tanya E S

2011-05-01

The fungal wall mediates cell-environment interactions. Galactofuranose (Galf), the five-member ring form of galactose, has a relatively low abundance in Aspergillus walls yet is important for fungal growth and fitness. Aspergillus nidulans strains deleted for Galf biosynthesis enzymes UgeA (UDP-glucose-4-epimerase) and UgmA (UDP-galactopyranose mutase) lacked immunolocalizable Galf, had growth and sporulation defects, and had abnormal wall architecture. We used atomic force microscopy and force spectroscopy to image and quantify cell wall viscoelasticity and surface adhesion of ugeAΔ and ugmAΔ strains. We compared the results for ugeAΔ and ugmAΔ strains with the results for a wild-type strain (AAE1) and the ugeB deletion strain, which has wild-type growth and sporulation. Our results suggest that UgeA and UgmA are important for cell wall surface subunit organization and wall viscoelasticity. The ugeAΔ and ugmAΔ strains had significantly larger surface subunits and lower cell wall viscoelastic moduli than those of AAE1 or ugeBΔ hyphae. Double deletion strains (ugeAΔ ugeBΔ and ugeAΔ ugmAΔ) had more-disorganized surface subunits than single deletion strains. Changes in wall surface structure correlated with changes in its viscoelastic modulus for both fixed and living hyphae. Wild-type walls had the largest viscoelastic modulus, while the walls of the double deletion strains had the smallest. The ugmAΔ strain and particularly the ugeAΔ ugmAΔ double deletion strain were more adhesive to hydrophilic surfaces than the wild type, consistent with changes in wall viscoelasticity and surface organization. We propose that Galf is necessary for full maturation of A. nidulans walls during hyphal extension.

Some lessons from the tissue transglutaminase knockout mouse.

PubMed

Sarang, Z; Tóth, B; Balajthy, Z; Köröskényi, K; Garabuczi, E; Fésüs, L; Szondy, Z

2009-04-01

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca(2+)-dependent protein modifications. It acts as a G protein in transmembrane signaling and as a cell surface adhesion mediator, this distinguishes it from other members of the transglutaminase family. The sequence motifs and domains revealed in the TG2 structure, can each be assigned distinct cellular functions, including the regulation of cytoskeleton, cell adhesion, and cell death. Though many biological functions of the enzyme have already been described or proposed previously, studies of TG2 null mice by our laboratory during the past years revealed several novel in vivo roles of the protein. In this review we will discuss these novel roles in their biological context.

Host cell capable of producing enzymes useful for degradation of lignocellulosic material

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

Los, Alrik Pieter; Sagt, Cornelis Maria Jacobus; Schoonneveld-Bergmans, Margot Elisabeth Francoise

The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

Osteoblast Differentiation on Collagen Scaffold with Immobilized Alkaline Phosphatase.

PubMed

Jafary, F; Hanachi, P; Gorjipour, K

2017-01-01

In tissue engineering, scaffold characteristics play an important role in the biological interactions between cells and the scaffold. Cell adhesion, proliferation, and activation depend on material properties used for the fabrication of scaffolds. In the present investigation, we used collagen with proper characteristics including mechanically stability, biodegradability and low antigenicity. Optimization of the scaffold was done by immobilization of alkaline phosphatase on the collagen surface via cross-linking method, because this enzyme is one of the most important markers of osteoblast, which increases inorganic phosphate concentration and promote mineralization of bone formation. Alkaline phosphatase was immobilized on a collagen surface by 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride, as a reagent. Then, rat mesenchymal stem cells were cultured in osteogenic medium in control and treated groups. The osteogenesis-related genes were compared between treatments (differentiated cells with immobilized alkaline phosphatase/collagen scaffold) and control groups (differentiated cells on collagen surface without alkaline phosphatase) on days 3 and 7 by quantitative real-time PCR (QIAGEN software). Several genes, including alkaline phosphatase, collagen type I and osteocalcine associated with calcium binding and mineralization, showed upregulation in expression during the first 3 days, whereas tumor necrosis factor-α, acting as an inhibitor of differentiation, was down-regulated during osteogenesis. Collagen scaffold with immobilized alkaline phosphatase can be utilized as a good candidate for enhancing the differentiation of osteoblasts from mesenchymal stem cells.

Fructose 1,6-Bisphosphate Aldolase, a Novel Immunogenic Surface Protein on Listeria Species

PubMed Central

Conceição, Fabricio Rochedo; Hust, Michael; Mendonça, Karla Sequeira; Moreira, Ângela Nunes; França, Rodrigo Correa; da Silva, Wladimir Padilha; Aleixo, José Antonio G.

2016-01-01

Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on Listeria would provide potential diagnostic and therapeutic targets. In the present work, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested Listeria species from close-related bacteria. Localization studies indicated that FBA is present in every fraction of Listeria cells, including supernatant and the cell wall, setting Listeria spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope in FBA allowed distinguishing Listeria spp. from closely-related bacteria, and was identified as part of the active site in the dimeric enzyme. However, its function in cell surface seems not to be host cell adhesion-related. Western and dot blot assays further demonstrated that mAb-3F8 together with anti-InlA mAb-2D12 could differentiate pathogenic from non-pathogenic Listeria isolated from artificially contaminated cheese. In summary, we report FBA as a novel immunogenic surface target useful for the detection of Listeria genus. PMID:27489951

Fructose 1,6-Bisphosphate Aldolase, a Novel Immunogenic Surface Protein on Listeria Species.

PubMed

Mendonça, Marcelo; Moreira, Gustavo Marçal Schmidt Garcia; Conceição, Fabricio Rochedo; Hust, Michael; Mendonça, Karla Sequeira; Moreira, Ângela Nunes; França, Rodrigo Correa; da Silva, Wladimir Padilha; Bhunia, Arun K; Aleixo, José Antonio G

2016-01-01

Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on Listeria would provide potential diagnostic and therapeutic targets. In the present work, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested Listeria species from close-related bacteria. Localization studies indicated that FBA is present in every fraction of Listeria cells, including supernatant and the cell wall, setting Listeria spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope in FBA allowed distinguishing Listeria spp. from closely-related bacteria, and was identified as part of the active site in the dimeric enzyme. However, its function in cell surface seems not to be host cell adhesion-related. Western and dot blot assays further demonstrated that mAb-3F8 together with anti-InlA mAb-2D12 could differentiate pathogenic from non-pathogenic Listeria isolated from artificially contaminated cheese. In summary, we report FBA as a novel immunogenic surface target useful for the detection of Listeria genus.

Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases.

PubMed

Wheeler, Richard; Turner, Robert D; Bailey, Richard G; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A S; Hayhurst, Emma J; Horsburgh, Malcolm; Hobbs, Jamie K; Foster, Simon J

2015-07-28

Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. Understanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. Using Staphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We show that these enzymes are required for normal growth and define the mechanism through which cellular enlargement is accomplished, i.e., by breaking bonds in the peptidoglycan, which reduces the stiffness of the cell wall, enabling it to stretch and expand, a process that is likely to be fundamental to many bacteria. Copyright © 2015 Wheeler et al.

Mucopolysaccharidosis enzyme production by bone marrow and dental pulp derived human mesenchymal stem cells.

PubMed

Jackson, Matilda; Derrick Roberts, Ainslie; Martin, Ellenore; Rout-Pitt, Nathan; Gronthos, Stan; Byers, Sharon

2015-04-01

Mucopolysaccharidoses (MPS) are inherited metabolic disorders that arise from a complete loss or a reduction in one of eleven specific lysosomal enzymes. MPS children display pathology in multiple cell types leading to tissue and organ failure and early death. Mesenchymal stem cells (MSCs) give rise to many of the cell types affected in MPS, including those that are refractory to current treatment protocols such as hematopoietic stem cell (HSC) based therapy. In this study we compared multiple MPS enzyme production by bone marrow derived (hBM) and dental pulp derived (hDP) MSCs to enzyme production by HSCs. hBM MSCs produce significantly higher levels of MPS I, II, IIIA, IVA, VI and VII enzyme than HSCs, while hDP MSCs produce significantly higher levels of MPS I, IIIA, IVA, VI and VII enzymes. Higher transfection efficiency was observed in MSCs (89%) compared to HSCs (23%) using a lentiviral vector. Over-expression of four different lysosomal enzymes resulted in up to 9303-fold and up to 5559-fold greater levels in MSC cell layer and media respectively. Stable, persistent transduction of MSCs and sustained over-expression of MPS VII enzyme was observed in vitro. Transduction of MSCs did not affect the ability of the cells to differentiate down osteogenic, adipogenic or chondrogenic lineages, but did partially delay differentiation down the non-mesodermal neurogenic lineage. Copyright © 2015 Elsevier Inc. All rights reserved.

[Effects of fibronectin on cytodifferentiation (T56) of human squamous cell carcinoma of tongue].

PubMed

Huang, Y; Yang, F

1994-12-01

Fibronectin (FN) are large glycoproteins that have been implicated in a wide variety of cellular properties, including cell adhesion, morphology, cytoskeletal organization, migration, differentiation, and oncogenic transformation. T56 cells were treated with 20, 50, and 100 micrograms/ml of FN for 48 h, and the changes in cells were analysed qualitatively and quantitatively with the methods of transmission electron microscopy, enzyme cytochemistry, and cell electrophoresis, etc. The studies were made to test the effects of FN on T56 cells in biological behaviour in terms of cell growth, multiplication, cell metabolism, cell electrophoresis and surface morphology. The results indicated that FN could partially restore T56 cell normal epidermic cell's phenotype, inhibit cell mitosis, increase contact of cells, decrease the number of microvilli and ruffles, and promote cell oxybiotic metabolism. These results suggest that FN might relate in many aspects to the biological behaviour of T56 cell and could affect changes in the behaviour.

Crystal structure of the Streptomyces coelicolor sortase E1 transpeptidase provides insight into the binding mode of the novel class E sorting signal

DOE PAGES

Kattke, Michele D.; Chan, Albert H.; Duong, Andrew; ...

2016-12-09

Here, many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution ofmore » alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses β3/β4 and β6/β7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their β3/β4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.« less

Enzymes involved in L-carnitine biosynthesis are expressed by small intestinal enterocytes in mice: implications for gut health.

PubMed

Shekhawat, Prem S; Sonne, Srinivas; Carter, A Lee; Matern, Dietrich; Ganapathy, Vadivel

2013-07-01

Carnitine is essential for mitochondrial β-oxidation of long-chain fatty acids. Deficiency of carnitine leads to severe gut atrophy, ulceration and inflammation in animal models of carnitine deficiency. Genetic studies in large populations have linked mutations in the carnitine transporters OCTN1 and OCTN2 with Crohn's disease (CD), while other studies at the same time have failed to show a similar association and report normal serum carnitine levels in CD patients. In this report, we have studied the expression of carnitine-synthesizing enzymes in intestinal epithelial cells to determine the capability of these cells to synthesize carnitine de novo. We studied expression of five enzymes involved in carnitine biosynthesis, namely 6-N-trimethyllysine dioxygenase (TMLD), 4-trimethylaminobutyraldehyde dehydrogenase (TMABADH), serine hydroxymethyltransferase 1 and 2 (SHMT1 and 2) and γ-butyrobetaine hydroxylase (BBH) by real-time PCR in mice (C3H strain). We also measured activity of γ-BBH in the intestine using an ex vivo assay and localized its expression by in situ hybridization. Our investigations show that mouse intestinal epithelium expresses all five enzymes required for de novo carnitine biosynthesis; the expression is localized mainly in villous surface epithelial cells throughout the intestine. The final rate-limiting enzyme γ-BBH is highly active in the small intestine; its activity was 9.7 ± 3.5 pmol/mg/min, compared to 22.7 ± 7.3 pmol/mg/min in the liver. We conclude that mouse gut epithelium is able to synthesize carnitine de novo. This capacity to synthesize carnitine in the intestine may play an important role in gut health and can help explain lack of clinical carnitine deficiency signs in subjects with mutations with OCTN transporters. Copyright © 2012 European Crohn's and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

Effects of cryopreservation and hypothermic storage on cell viability and enzyme activity in recombinant encapsulated cells overexpressing alpha-L-iduronidase.

PubMed

Mayer, Fabiana Quoos; Baldo, Guilherme; de Carvalho, Talita Giacomet; Lagranha, Valeska Lizzi; Giugliani, Roberto; Matte, Ursula

2010-05-01

Here, we show the effects of cryopreservation and hypothermic storage upon cell viability and enzyme release in alginate beads containing baby hamster kidney cells overexpressing alpha-L-iduronidase (IDUA), the enzyme deficient in mucopolysaccharidosis type I. In addition, we compared two different concentrations of alginate gel (1% and 1.5%) in respect to enzyme release from the beads and their shape and integrity. Our results indicate that in both alginate concentrations, the enzyme is released in lower amounts compared with nonencapsulated cells. Alginate 1% beads presented increased levels of IDUA release, although this group presented more deformities when compared with alginate 1.5% beads. Importantly, both encapsulated groups presented higher cell viability after long cryopreservation period and hypothermic storage. In addition, alginate 1.5% beads presented higher enzyme release after freezing protocols. Taken together, our findings suggest a benefic effect of alginate upon cell viability and functionality. These results may have important application for treatment of both genetic and nongenetic diseases using microencapsulation-based artificial organs.

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.

A microfabricated low cost enzyme-free glucose fuel cell for powering low-power implantable devices

NASA Astrophysics Data System (ADS)

Oncescu, Vlad; Erickson, David

In the past decade the scientific community has showed considerable interest in the development of implantable medical devices such as muscle stimulators, neuroprosthetic devices, and biosensors. Those devices have low power requirements and can potentially be operated through fuel cells using reactants present in the body such as glucose and oxygen instead of non-rechargeable lithium batteries. In this paper, we present a thin, enzyme-free fuel cell with high current density and good stability at a current density of 10 μA cm -2. A non-enzymatic approach is preferred because of higher long term stability. The fuel cell uses a stacked electrode design in order to achieve glucose and oxygen separation. An important characteristic of the fuel cell is that it has no membrane separating the electrodes, which results in low ohmic losses and small fuel cell volume. In addition, it uses a porous carbon paper support for the anodic catalyst layer which reduces the amount of platinum or other noble metal catalysts required for fabricating high surface area electrodes with good reactivity. The peak power output of the fuel cell is approximately 2 μW cm -2 and has a sustainable power density of 1.5 μW cm -2 at 10 μA cm -2. An analysis on the effects of electrode thickness and inter electrode gap on the maximum power output of the fuel cell is also performed.

MTA-enriched nanocomposite TiO(2)-polymeric powder coatings support human mesenchymal cell attachment and growth.

PubMed

Shi, Wen; Mozumder, Mohammad Sayem; Zhang, Hui; Zhu, Jesse; Perinpanayagam, Hiran

2012-10-01

The objective of the study described in this paper was the development of novel polymer/ceramic nanocomposite coatings for implants through the application of ultrafine powder coating technology. Polyester resins were combined with µm-sized TiO(2) (25%) as the biocompatibility agent, nTiO(2) (0.5%) as the flow additive and mineral trioxide aggregates (ProRoot® MTA, 5%) as bioactive ceramics. Ultrafine powders were prepared and applied to titanium to create continuous polymeric powder coatings (PPCs) through the application of electrostatic ultrafine powder coating technology. Energy dispersive x-ray analysis confirmed that MTA had been incorporated into the PPCs, and elemental mapping showed that it had formed small clusters that were evenly distributed across the surface. Scanning electron microscopy (SEM) revealed continuous and smooth, but highly textured surface coatings that contrasted with the scalloped appearance of commercially pure titanium (cpTi) controls. Atomic force microscopy revealed intricate nano-topographies with an abundance of submicron-sized pits and nano-projections, evenly dispersed across their surfaces. Inverted fluorescence microscopy, SEM and cell counts showed that human embryonic palatal mesenchymal cells attached and spread out onto PPC and MTA-enriched PPCs within 24 h. Mitochondrial enzyme activity measured viable and metabolically active cells on all of the surfaces. After 72 h of growth, cell counts and metabolic activity were significantly higher (P < 0.05) on the grey-MTA enriched PPC surfaces, than on unmodified PPC and cpTi. The novel polymer/ceramic nanocomposites that were created with ultrafine powder coating technology were continuous, homogenous and nano-rough coatings that enhanced human mesenchymal cell attachment and growth.

A Redundant Role of Human Thyroid Peroxidase Propeptide for Cellular, Enzymatic, and Immunological Activity

PubMed Central

Góra, Monika; Buckle, Ashley M.; Porebski, Benjamin T.; Kemp, E. Helen; Sutton, Brian J.; Czarnocka, Barbara; Banga, J. Paul

2014-01-01

Background: Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. Methods: An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPOΔpro; amino acid residues 21–108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. Results: The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro was enzymatically active and recognized by patients' autoantibodies and a panel of epitope-specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPOΔpro was also maintained. Molecular modeling and dynamics simulations were consistent with these observations. Conclusions: Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of TPOΔpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and importantly is recognized by patients' autoantibodies, is a key advance for purification of substantial quantities of homogeneous preparation of TPO for crystallization, structural, and immunological studies. PMID:23668778

A redundant role of human thyroid peroxidase propeptide for cellular, enzymatic, and immunological activity.

PubMed

Godlewska, Marlena; Góra, Monika; Buckle, Ashley M; Porebski, Benjamin T; Kemp, E Helen; Sutton, Brian J; Czarnocka, Barbara; Banga, J Paul

2014-02-01

Thyroid peroxidase (TPO) is a dimeric membrane-bound enzyme of thyroid follicular cells, responsible for thyroid hormone biosynthesis. TPO is also a common target antigen in autoimmune thyroid disease (AITD). With two active sites, TPO is an unusual enzyme, and thus there is much interest in understanding its structure and role in AITD. Homology modeling has shown TPO to be composed of different structural modules, as well as a propeptide sequence. During the course of studies to obtain homogeneous preparations of recombinant TPO for structural studies, we investigated the role of the large propeptide sequence in TPO. An engineered recombinant human TPO preparation expressed in Chinese hamster ovary (CHO) cells lacking the propeptide (TPOΔpro; amino acid residues 21-108) was characterized and its properties compared to wild-type TPO. Plasma membrane localization was determined by cell surface protein biotinylation, and biochemical studies were performed to evaluate enzymatic activity and the effect of deglycosylation. Immunological investigations using autoantibodies from AITD patients and other epitope-specific antibodies that recognize conformational determinants on TPO were evaluated for binding to TPOΔpro by flow cytometry, immunocytochemistry, and capture enzyme-linked immunosorbent assay. Molecular modeling and dynamics simulation of TPOΔpro comprising a dimer of myeloperoxidase-like domains was performed in order to investigate the impact of propeptide removal and the role of glycosylation. The TPOΔpro was expressed on the cell surface at comparable levels to wild-type TPO. The TPOΔpro was enzymatically active and recognized by patients' autoantibodies and a panel of epitope-specific antibodies, confirming structural integrity of the two major conformational determinants recognized by autoantibodies. Faithful intracellular trafficking and N-glycosylation of TPOΔpro was also maintained. Molecular modeling and dynamics simulations were consistent with these observations. Our results point to a redundant role for the propeptide sequence in TPO. The successful expression of TPOΔpro in a membrane-anchored, enzymatically active form that is insensitive to intramolecular proteolysis, and importantly is recognized by patients' autoantibodies, is a key advance for purification of substantial quantities of homogeneous preparation of TPO for crystallization, structural, and immunological studies.

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

PubMed Central

Owen, Peter; Salton, Milton R. J.

1977-01-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions. Images PMID:144722

Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed immunoelectrophoresis.

PubMed

Owen, P; Salton, M R

1977-12-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

A novel film-pore-surface diffusion model to explain the enhanced enzyme adsorption of corn stover pretreated by ultrafine grinding.

PubMed

Zhang, Haiyan; Chen, Longjian; Lu, Minsheng; Li, Junbao; Han, Lujia

2016-01-01

Ultrafine grinding is an environmentally friendly pretreatment that can alter the degree of polymerization, the porosity and the specific surface area of lignocellulosic biomass and can, thus, enhance cellulose hydrolysis. Enzyme adsorption onto the substrate is a prerequisite for the enzymatic hydrolysis process. Therefore, it is necessary to investigate the enzyme adsorption properties of corn stover pretreated by ultrafine grinding. The ultrafine grinding pretreatment was executed on corn stover. The results showed that ultrafine grinding pretreatment can significantly decrease particle size [from 218.50 μm of sieve-based grinding corn stover (SGCS) to 17.45 μm of ultrafine grinding corn stover (UGCS)] and increase the specific surface area (SSA), pore volume (PV) and surface composition (SSA: from 1.71 m(2)/g of SGCS to 2.63 m(2)/g of UGCS, PV: from 0.009 cm(3)/g of SGCS to 0.024 m(3)/g of UGCS, cellulose surface area: from 168.69 m(2)/g of SGCS to 290.76 m(2)/g of UGCS, lignin surface area: from 91.46 m(2)/g of SGCS to 106.70 m(2)/g of UGCS). The structure and surface composition changes induced by ultrafine grinding increase the enzyme adsorption capacity from 2.83 mg/g substrate of SGCS to 5.61 mg/g substrate of UGCS. A film-pore-surface diffusion model was developed to simultaneously predict the enzyme adsorption kinetics of both the SGCS and UGCS. Satisfactory predictions could be made with the model based on high R (2) and low RMSE values (R (2) = 0.95 and RMSE = 0.16 mg/g for the UGCS, R (2) = 0.93 and RMSE = 0.09 mg/g for the SGCS). The model was further employed to analyze the rate-limiting steps in the enzyme adsorption process. Although both the external-film and internal-pore mass transfer are important for enzyme adsorption on the SGCS and UGCS, the UGCS has a lower internal-pore resistance compared to the SGCS. Ultrafine grinding pretreatment can enhance the enzyme adsorption onto corn stover by altering structure and surface composition. The film-pore-surface diffusion model successfully captures features on enzyme adsorption on ultrafine grinding pretreated corn stover. These findings identify wherein the probable rate-limiting factors for the enzyme adsorption reside and could, therefore, provide a basis for enhanced cellulose hydrolysis processes.

LipC (Rv0220) Is an Immunogenic Cell Surface Esterase of Mycobacterium tuberculosis

PubMed Central

Shen, Guomiao; Singh, Krishna; Chandra, Dinesh; Serveau-Avesque, Carole; Maurin, Damien; Canaan, Stéphane; Singla, Rupak; Behera, Digambar

2012-01-01

We have reported previously the identification of novel proteins of Mycobacterium tuberculosis by the immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained from M. tuberculosis-infected rabbits at 5 weeks postinfection. In this study, we report the further characterization of one of these antigens, LipC (Rv0220). LipC is annotated as a member of the Lip family based on the presence of the consensus motif “GXSXG” characteristic of esterases. Although predicted to be a cytoplasmic enzyme, we provide evidence that LipC is a cell surface protein that is present in both the cell wall and the capsule of M. tuberculosis. Consistent with this localization, LipC elicits strong humoral immune responses in both HIV-negative (HIV−) and HIV-positive (HIV+) tuberculosis (TB) patients. The absence of anti-LipC antibodies in sera from purified protein derivative-positive (PPD+) healthy subjects confirms its expression only during active M. tuberculosis infection. Epitope mapping of LipC identified 6 immunodominant epitopes, 5 of which map to the exposed surface of the modeled LipC protein. The recombinant LipC (rLipC) protein also elicits proinflammatory cytokine and chemokine responses from macrophages and pulmonary epithelial cells. rLipC can hydrolyze short-chain esters with the carbon chain containing 2 to 10 carbon atoms. Together, these studies demonstrate that LipC is a novel cell surface-associated esterase of M. tuberculosis that is highly immunogenic and elicits both antibodies and cytokines/chemokines. PMID:22038913

Enzymatic Production of Biodiesel: Strategies to Overcome Methanol Inactivation.

PubMed

Lotti, Marina; Pleiss, Jürgen; Valero, Francisco; Ferrer, Pau

2018-05-01

Lipase-catalyzed transesterification of triglycerides and alcohols to obtain biodiesel is an environmentally friendly and sustainable route for fuels production since, besides proceeding in mild reaction conditions, it allows for the use of low-cost feedstocks that contain water and free fatty acids, for example non-edible oils and waste oils. This review article reports recent advances in the field and focus in particular on a major issue in the enzymatic process, the inactivation of most lipases caused by methanol, the preferred acyl acceptor used for alcoholysis. The recent results about immobilization of enzymes on nano-materials and the use of whole-cell biocatalysts, as well as the use of cell-surface display technologies and metabolic engineering strategies for microbial production of biodiesel are described. It is discussed also insight into the effects of methanol on lipases obtained by modeling approaches and report on studies aimed at mining novel alcohol stable enzymes or at improving robustness in existing ones by protein engineering. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vivo sensing of proteolytic activity with an NSET-based NIR fluorogenic nanosensor.

PubMed

Ku, Minhee; Hong, Yoochan; Heo, Dan; Lee, Eugene; Hwang, Seungyeon; Suh, Jin-Suck; Yang, Jaemoon

2016-03-15

Biomedical in vivo sensing methods in the near-infrared (NIR) range, which that provide relatively high photon transparency, separation from auto-fluorescence background, and extended sensitivity, are being used increasingly for non-invasive mapping and monitoring of molecular events in cancer cells. In this study, we fabricated an NIR fluorogenic nanosensor based on the nanoparticle surface energy transfer effect, by conjugation of fluorescent proteolytic enzyme-specific cleavable peptides with gold nanorods (GNRs). Membrane-anchored membrane type 1-matrix metalloproteinases (MT1-MMPs), a family of zinc-dependent proteolytic enzymes, can induce the metastatic potential of cancer cells by promoting degradation of the extracellular matrix. Therefore, sensitive detection of MT1-MMP activity can provide essential information in the clinical setting. We have applied in vivo NIR sensing to evaluate MT1-MMP activity, as an NIR imaging target, in an MT1-MMP-expressing metastatic tumor mouse model. Copyright © 2015 Elsevier B.V. All rights reserved.

Harnessing the natural inhibitory domain to control TNFα Converting Enzyme (TACE) activity in vivo.

PubMed

Wong, Eitan; Cohen, Tal; Romi, Erez; Levin, Maxim; Peleg, Yoav; Arad, Uri; Yaron, Avraham; Milla, Marcos E; Sagi, Irit

2016-12-16

Dysregulated activity of A Disintegrin And Metalloproteinase 17 (ADAM17)/TNFα Converting Enzyme (TACE) is associated with inflammatory disorders and cancer progression by releasing regulatory membrane-tethered proteins like TNFα, IL6R and EGFR ligands. Although specific inhibition of TACE is thought to be a viable strategy for inflammatory disorders and for malignancies treatment, the generation of effective inhibitors in vivo has been proven to be challenging. Here we report on the development of a protein inhibitor that leverages the endogenous modulator of TACE. We have generated a stable form of the auto-inhibitory TACE prodomain (TPD), which specifically inhibits in vitro and cell-surface TACE, but not the related ADAM10, and effectively modulated TNFα secretion in cells. TPD significantly attenuated TACE-mediated disease models of sepsis, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and reduced TNFα in synovial fluids from RA patients. Our results demonstrate that intervening with endogenous ADAM sheddase modulatory mechanisms holds potential as a general strategy for the design of ADAM inhibitors.

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.

Subcellular localization of pituitary enzymes

NASA Technical Reports Server (NTRS)

Smith, R. E.

1970-01-01

A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

Environmental and genetic factors that contribute to Escherichia coli K-12 biofilm formation

PubMed Central

Prüß, Birgit M.; Verma, Karan; Samanta, Priyankar; Sule, Preeti; Kumar, Sunil; Wu, Jianfei; Christianson, David; Horne, Shelley M.; Stafslien, Shane J.; Wolfe, Alan J.; Denton, Anne

2010-01-01

Biofilms are communities of bacteria whose formation on surfaces requires a large portion of the bacteria’s transcriptional network. To identify environmental conditions and transcriptional regulators that contribute to sensing these conditions, we used a high-throughput approach to monitor biofilm biomass produced by an isogenic set of Escherichia coli K-12 strains grown under combinations of environmental conditions. Of the environmental combinationsd, growth in tryptic soy broth at 37°C supported the most biofilm production. To analyze the complex relationships between the diverse cell surface organelles, transcriptional regulators, and metabolic enzymes represented by the tested mutant set, we used a novel vector-item pattern-mining algorithm. The algorithm related biofilm amounts to the functional annotations of each mutated protein. The pattern with the best statistical significance was the gene ontology ‘pyruvate catabolic process,’ which is associated with enzymes of acetate metabolism. Phenotype microarray experiments illustrated that carbon sources that are metabolized to acetyl-coenzyme A, acetyl phosphate, and acetate are particularly supportive of biofilm formation. Scanning electron microscopy revealed structural differences between mutants that lack acetate metabolism enzymes and their parent and confirmed the quantitative differences. We conclude that acetate metabolism functions as a metabolic sensor, transmitting changes in environmental conditions to biofilm biomass and structure. PMID:20559621

ATP-dependent chromatin remodeling in T cells.

PubMed

Wurster, Andrea L; Pazin, Michael J

2012-02-01

One of the best studied systems for mammalian chromatin remodeling is transcriptional regulation during T cell development. The variety of these studies have led to important findings in T cell gene regulation and cell fate determination. Importantly, these findings have also advanced our knowledge of the function of remodeling enzymes in mammalian gene regulation. First we briefly present biochemical and cell-free analysis of 3 types of ATP dependent remodeling enzymes (SWI/SNF, Mi2, and ISWI) to construct an intellectual framework to understand how these enzymes might be working. Second, we compare and contrast the function of these enzymes during early (thymic) and late (peripheral) T cell development. Finally, we examine some of the gaps in our present understanding.

Beta-glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells.

PubMed

Izagirre, U; Angulo, E; Wade, S C; ap Gwynn, I; Marigómez, I

2009-02-01

In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system

PubMed Central

Atilano, Magda Luciana; Pereira, Pedro Matos; Vaz, Filipa; Catalão, Maria João; Reed, Patricia; Grilo, Inês Ramos; Sobral, Rita Gonçalves; Ligoxygakis, Petros; Pinho, Mariana Gomes; Filipe, Sérgio Raposo

2014-01-01

Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host. DOI: http://dx.doi.org/10.7554/eLife.02277.001 PMID:24692449

Transepithelial and endothelial transport of poly (amidoamine) dendrimers.

PubMed

Kitchens, Kelly M; El-Sayed, Mohamed E H; Ghandehari, Hamidreza

2005-12-14

This article summarizes our efforts to evaluate the potential of poly (amidoamine) (PAMAM) dendrimers as carriers for oral drug delivery. Specifically, the permeability of a series of cationic PAMAM-NH2 (G0-G4) dendrimers across Caco-2 cell monolayers was evaluated as a function of dendrimer generation, concentration, and incubation time. The influence of dendrimer surface charge on the integrity, paracellular permeability, and viability of Caco-2 cell monolayers was monitored by measuring the transepithelial electrical resistance (TEER), 14C-mannitol permeability, and leakage of lactate dehydrogenase (LDH) enzyme, respectively. Microvascular extravasation of PAMAM-NH2 dendrimers in relation to their size, molecular weight, and molecular geometry is also discussed. Results of these studies show that transepithelial transport and microvascular extravasation of PAMAM dendrimers are dependent on their structural features including molecular size, molecular geometry, and surface chemistry. These results suggest that by optimizing the size and surface charge of PAMAM dendrimers, it is possible to develop oral delivery systems based on these carriers for targeted drug delivery.

Fucosylation is a common glycosylation type in pancreatic cancer stem cell-like phenotypes.

PubMed

Terao, Naoko; Takamatsu, Shinji; Minehira, Tomomi; Sobajima, Tomoaki; Nakayama, Kotarosumitomo; Kamada, Yoshihiro; Miyoshi, Eiji

2015-04-07

To evaluate/isolate cancer stem cells (CSCs) from tissue or cell lines according to various definitions and cell surface markers. Lectin microarray analysis was conducted on CSC-like fractions of the human pancreatic cancer cell line Panc1 by establishing anti-cancer drug-resistant cells. Changes in glycan structure of CSC-like cells were also investigated in sphere-forming cells as well as in CSC fractions obtained from overexpression of CD24 and CD44. Several types of fucosylation were increased under these conditions, and the expression of fucosylation regulatory genes such as fucosyltransferases, GDP-fucose synthetic enzymes, and GDP-fucose transporters were dramatically enhanced in CSC-like cells. These changes were significant in gemcitabine-resistant cells and sphere cells of a human pancreatic cancer cell line, Panc1. However, downregulation of cellular fucosylation by knockdown of the GDP-fucose transporter did not alter gemcitabine resistance, indicating that increased cellular fucosylation is a result of CSC-like transformation. Fucosylation might be a biomarker of CSC-like cells in pancreatic cancer.

EVIDENCE FOR AN EXOCELLULAR SITE FOR THE ACID PHOSPHATASE OF SACCHAROMYCES MELLIS1

PubMed Central

Weimberg, Ralph; Orton, William L.

1964-01-01

Weimberg, Ralph (Northern Regional Research Laboratory, Peoria, Ill.), and William L. Orton. Evidence for an exocellular site for the acid phosphatase of Saccharomyces mellis. J. Bacteriol. 88:1743–1754. 1964.—Evidence is presented which demonstrates an exocellular location for acid phosphatase in Saccharomyces mellis. Derepressed intact cells exhibit acid phosphatase activity. The properties of the system are similar to those shown by the enzyme in cell-free extracts. There is no increase in total activity when cell-free extracts are prepared. Enzymatically active cell walls were prepared by leaching acetone-dried cells of this yeast in dilute acetate buffer (pH 6.5) plus β-mercaptoethanol. The insoluble residue, consisting mainly of cell-wall material and containing the phosphatase, was treated with a variety of hydrolytic enzymes and other chemicals. Only papain and crude snail gut extracts dissociated the enzyme from the particulate fraction in nearly quantitative amounts. The mechanism of release by these two enzymes probably differs. Of all enzymes tested, only the snail gut extract digested the cell walls. By dividing the procedure for making protoplasts of S. mellis into two steps, acid phosphatase may be dissociated from resting cells and recovered as an active soluble enzyme. The first step is to pretreat the cells with a thiol reagent. The second step is to digest the cell wall by enzymes present in crude snail gut extracts. Arsenite must be included in the second step to protect the phosphatase from inactivation. The phosphatase is quantitatively released before the cell becomes osmotically fragile. Images PMID:14240965

How Ebola and Marburg Viruses Battle the Immune System

DTIC Science & Technology

2007-07-01

macrophages, neutrophils) Asialoglycoprotein receptor ( hepatocytes ) TLR Other? NP VP35 VP40 GP VP30 VP24 L 3′ 5′ Cell-surface GP Filovirus Matrix... hepatocytes are particularly susceptible, elevated liver enzymes are among the first telling signs of disease and liver damage seems to account for much...of monocytic origin (such as immature DCs) also promotes filoviral entry10. Another C-type lectin, the asialoglycoprotein *US Army Medical Research

Functional Study of the Vitamin K Cycle Enzymes in Live Cells

PubMed Central

Tie, J.-K.; Stafford, D.W.

2018-01-01

Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle. PMID:28065270

Screening And Optimizing Antimicrobial Peptides By Using SPOT-Synthesis

NASA Astrophysics Data System (ADS)

López-Pérez, Paula M.; Grimsey, Elizabeth; Bourne, Luc; Mikut, Ralf; Hilpert, Kai

2017-04-01

Peptide arrays on cellulose are a powerful tool to investigate peptide interactions with a number of different molecules, for examples antibodies, receptors or enzymes. Such peptide arrays can also be used to study interactions with whole cells. In this review, we focus on the interaction of small antimicrobial peptides with bacteria. Antimicrobial peptides (AMPs) can kill multidrug-resistant (MDR) human pathogenic bacteria and therefore could be next generation antibiotics targeting MDR bacteria. We describe the screen and the result of different optimization strategies of peptides cleaved from the membrane. In addition, screening of antibacterial activity of peptides that are tethered to the surface is discussed. Surface-active peptides can be used to protect surfaces from bacterial infections, for example implants.

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.

A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts.

PubMed

Naveilhan, P; Baudet, C; Jabbour, W; Wion, D

1994-09-01

A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do--namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occurred since the cells involved differentiated during development.

The trans-sialidase from Trypanosoma cruzi induces thrombocytopenia during acute Chagas' disease by reducing the platelet sialic acid contents.

PubMed

Tribulatti, María Virginia; Mucci, Juan; Van Rooijen, Nico; Leguizamón, María Susana; Campetella, Oscar

2005-01-01

Strong thrombocytopenia is observed during acute infection with Trypanosoma cruzi, the parasitic protozoan agent of American trypanosomiasis or Chagas' disease. The parasite sheds trans-sialidase, an enzyme able to mobilize the sialyl residues on cell surfaces, which is distributed in blood and is a virulence factor. Since the sialic acid content on the platelet surface is crucial for determining the half-life of platelets in blood, we examined the possible involvement of the parasite-derived enzyme in thrombocytopenia induction. We found that a single intravenous injection of trans-sialidase into naive mice reduced the platelet count by 50%, a transient effect that lasted as long as the enzyme remained in the blood. CD43(-/-) mice were affected to a similar extent. When green fluorescent protein-expressing platelets were treated in vitro with trans-sialidase, their sialic acid content was reduced together with their life span, as determined after transfusion into naive animals. No apparent deleterious effect on the bone marrow was observed. A central role for Kupffer cells in the clearance of trans-sialidase-altered platelets was revealed after phagocyte depletion by administration of clodronate-containing liposomes and splenectomy. Consistent with this, parasite strains known to exhibit more trans-sialidase activity induced heavier thrombocytopenia. Finally, the passive transfer of a trans-sialidase-neutralizing monoclonal antibody to infected animals prevented the clearance of transfused platelets. Results reported here strongly support the hypothesis that the trans-sialidase is the virulence factor that, after depleting the sialic acid content of platelets, induces the accelerated clearance of the platelets that leads to the thrombocytopenia observed during acute Chagas' disease.

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

PubMed

Sand, Sverre L; Nissen-Meyer, Jon; Sand, Olav; Haug, Trude M

2013-02-01

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts. Copyright © 2012 Elsevier B.V. All rights reserved.

Ecto-F1-ATPase: A moonlighting protein complex and an unexpected apoA-I receptor

PubMed Central

Vantourout, Pierre; Radojkovic, Claudia; Lichtenstein, Laeticia; Pons, Véronique; Champagne, Eric; Martinez, Laurent O

2010-01-01

Mitochondrial ATP synthase has been recently detected at the surface of different cell types, where it is a high affinity receptor for apoA-I, the major protein component in high density lipoproteins (HDL). Cell surface ATP synthase (namely ecto-F1-ATPase) expression is related to different biological effects, such as regulation of HDL uptake by hepatocytes, endothelial cell proliferation or antitumor activity of Vγ9/Vδ2 T lymphocytes. This paper reviews the recently discovered functions and regulations of ecto-F1-ATPase. Particularly, the role of the F1-ATPase pathway(s) in HDL-cholesterol uptake and apoA-I-mediated endothelial protection suggests its potential importance in reverse cholesterol transport and its regulation might represent a potential therapeutic target for HDL-related therapy for cardiovascular diseases. Therefore, it is timely for us to better understand how this ecto-enzyme and downstream pathways are regulated and to develop pharmacologic interventions. PMID:21157968

Soil Minerals Affect Extracellular Enzyme Activities in Cold and Warm Environments

NASA Astrophysics Data System (ADS)

Yang, Z.; Morin, M. M.; Graham, D. E.; Wullschleger, S. D.; Gu, B.

2017-12-01

Extracellular enzymes are mainly responsible for degrading and cycling soil organic matter (SOM) in both cold and warm terrestrial ecosystems. Minerals can play important roles in affecting soil enzyme activities, however, the interactions between enzyme and soil minerals remain poorly understood. In this study, we developed a model soil-enzyme system to examine the mineral effects on a hydrolytic enzyme (i.e., β-glucosidase) under both cold (4°C) and relatively warm (20 and 30°C) conditions. Minerals including iron oxides and clays (e.g., kaolinite and montmorillonite) were used to mimic different types of soils, and enzyme adsorption experiments were conducted to determine the enzyme interactions with different mineral surfaces. Time-series experiments were also carried out to measure enzymatic degradation of the organic substrates, such as cellobiose and indican. We observed that fractions of adsorbed enzyme and the hydrolytic activity were higher on iron oxides (e.g., hematite) compared to kaolinite and montmorillonite at given experimental conditions. The degradation of cellobiose was significantly faster than that of indican in the presence of minerals. We also found that the adsorption of enzyme was not dependent on the mineral surface areas, but was controlled by the mineral surface charge. In addition, temperature increase from 4 to 30°C enhanced mineral-assisted glucosidase hydrolysis by 2 to 4 fold, suggesting greater degradation under warmer environments. The present work demonstrates that the enzyme activity is influenced not only by the soil temperature but also by the surface chemistry of soil minerals. Our results highlight the need to consider the physical and chemical properties of minerals in biogeochemical models, which could provide a better prediction for enzyme-facilitated SOM transformations in terrestrial ecosystems.

Non-tumor cell IDO1 predominantly contributes to enzyme activity and response to CTLA-4/PD-L1 inhibition in mouse glioblastoma.

PubMed

Zhai, Lijie; Ladomersky, Erik; Dostal, Carlos R; Lauing, Kristen L; Swoap, Kathleen; Billingham, Leah K; Gritsina, Galina; Wu, Meijing; McCusker, Robert H; Binder, David C; Wainwright, Derek A

2017-05-01

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a median survival of 14.6months. A contributing factor to GBM aggressiveness is the intratumoral expression of the potently immunosuppressive enzyme, indoleamine 2,3 dioxygenase 1 (IDO1). The enzymatic activity of IDO1 is associated with the conversion of tryptophan into downstream kynurenine (Kyn), which has previously been hypothesized to contribute toward the suppression of tumor immunity. Utilizing the syngeneic, immunocompetent, intracranial GL261 cell GBM model, we previously demonstrated that tumor cell, but not non-tumor cell IDO1, suppresses T cell-mediated brain tumor regression in mice. Paradoxically, we also showed that the survival advantage mediated by immune checkpoint blockade is abrogated by non-tumor cell IDO1 deficiency. Here, we have built on our past observations and confirm the maladaptive role of tumor cell IDO1 in a novel mouse GBM model. We also demonstrate that, non-tumor cells, rather than mouse GBM cells, are the dominant contributor to IDO1-mediated enzyme activity. Finally, we show the novel associations between maximally-effective immune-checkpoint blockade-mediated survival, non-tumor cell IDO1 and intra-GBM Kyn levels. These data suggest for the first time that, GBM cell-mediated immunosuppression is IDO1 enzyme independent, while the survival benefits of immune checkpoint blockade require non-tumor cell IDO1 enzyme activity. Given that current clinical inhibitors vary in their mechanism of action, in terms of targeting IDO1 enzyme activity versus enzyme-independent effects, this work suggests that choosing an appropriate IDO1 pharmacologic will maximize the effectiveness of future immune checkpoint blockade approaches. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Ultrarapid sonochemical synthesis of enzyme-incorporated copper nanoflowers and their application to mediatorless glucose biofuel cell

NASA Astrophysics Data System (ADS)

Chung, Minsoo; Nguyen, Tuan Loi; Tran, Thao Quynh Ngan; Yoon, Hyon Hee; Kim, Il Tae; Kim, Moon Il

2018-01-01

We have developed a mediatorless glucose biofuel cell based on hybrid nanoflowers incorporating enzymes including glucose oxidase (GOx), laccase, or catalase with copper phosphate, which were further mixed and compressed with conductive multi-walled carbon nanotube (CNT). The nanoflowers were simply synthesized within 5 min at room temperature using sonication method but yielded greatly improved stability as well as highly retained activity by the proper incorporation of enzyme molecules inside the flower-like structure. With glucose as biofuel, GOx and laccase nanoflowers were applied to form enzyme anode and cathode, respectively, and catalase nanoflowers were additionally employed to catalyze the decomposition of hydrogen peroxide, which may be deleterious for GOx, into oxygen and water. Using the enzyme nanoflowers-based biofuel cell system without any involved mediator, a high power density up to 200 μW cm-2 were obtained, which was approximately 80% to that from the biofuel cell system prepared with the corresponding free enzymes. Importantly, the enzyme nanoflowers-based biofuel cell maintained their initial power density over 90% during storage for two months at 4 °C, while most of the glucose biofuel cells in the literature present meaningful stability only in the range of one or two weeks. Based on this result, we expect that this simple but efficient strategy to prepare highly stable glucose biofuel cell using the rapidly-synthesized enzyme-inorganic hybrid nanoflowers can be readily extended to diverse applications in medical and environmental chemistry.

Biological indicators for steam sterilization: characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase enzyme.

PubMed

Albert, H; Davies, D J; Woodson, L P; Soper, C J

1998-11-01

The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953. Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5. The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured. The optimal pH for stability of the enzyme was approximately 7.2. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700. The vegetative cell and spore-associated enzymes were cross-reactive. The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.

Surface hydrophobicity and roughness influences the morphology and biochemistry of streptomycetes during attached growth and differentiation.

PubMed

Petráčková, Denisa; Buriánková, Karolína; Tesařová, Eva; Bobková, Šárka; Bezoušková, Silvia; Benada, Oldřich; Kofroňová, Olga; Janeček, Jiří; Halada, Petr; Weiser, Jaroslav

2013-05-01

Streptomycetes, soil-dwelling mycelial bacteria, can colonise surface of organic soil debris and soil particles. We analysed the effects of two different inert surfaces, glass and zirconia/silica, on the growth and antibiotic production in Streptomyces granaticolor. The surfaces used were in the form of microbeads and were surrounded by liquid growth media. Following the production of the antibiotic granaticin, more biomass was formed as well as a greater amount of antibiotic per milligram of protein on the glass beads than on the zirconia/silica beads. Comparison of young mycelium (6 h) proteomes, obtained from the cultures attached to the glass and zirconia/silica beads, revealed three proteins with altered expression levels (dihydrolipoamide dehydrogenase, amidophosphoribosyltransferase and cystathionine beta-synthase) and one unique protein (glyceraldehyde-3-phosphate dehydrogenase) that was present only in cells grown on glass beads. All of the identified proteins function primarily as cytoplasmic enzymes involved in different parts of metabolism; however, in several microorganisms, they are exposed on the cell surface and have been shown to be involved in adhesion or biofilm formation. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

Pak, J.; Sharon, C; Satkunarajah, M

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the firstmore » example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.« less

Enzyme Armoring by an Organosilica Layer: Synthesis and Characterization of Hybrid Organic/Inorganic Nanobiocatalysts.

PubMed

Correro, M Rita; Sykora, Sabine; Corvini, Philippe F-X; Shahgaldian, Patrick

2017-01-01

The availability of highly stable and reusable enzymes is one of the main challenges in bio-based industrial processes. Enzyme immobilization and encapsulation represent promising strategies to reach this goal. In this chapter, the synthetic strategy to produce hybrid organic/inorganic nanobiocatalysts (NBC) is reported. This strategy is based on the sequential immobilization of an enzyme on the surface of silica nanoparticles followed by the growth, at the surface of the nanoparticles, of a shielding layer which serves as an armor to protect the enzyme against denaturation/degradation. This armor is produced through a thickness-controlled organosilane poly-condensation onto the nanoparticle surface around the enzyme to form a protective organosilica layer. The armored nanobiocatalysts present enhanced catalytic activity and improved stability against heat, pH, chaotropic agents, proteases, and ultrasound. The method is versatile in that it can be successfully adapted to a number of different enzymes. © 2017 Elsevier Inc. All rights reserved.

Lecithin/chitosan controlled release nanopreparations of tamoxifen citrate: loading, enzyme-trigger release and cell uptake.

PubMed

Barbieri, Stefano; Sonvico, Fabio; Como, Caterina; Colombo, Gaia; Zani, Franca; Buttini, Francesca; Bettini, Ruggero; Rossi, Alessandra; Colombo, Paolo

2013-05-10

Tamoxifen citrate (TAM), an anticancer drug with amphiphilic properties, was loaded in lecithin/chitosan nanoparticles (LCN) with a view to oral administration. The influence of tamoxifen loading on the physico-chemical properties of nanoparticles was studied. Size, surface charge and morphological properties of tamoxifen-loaded nanoparticles (LCN-TAM) were assessed. The increase in the tamoxifen amount in the LCN-TAM preparation up to 60 mg/100 ml maintained the positive zeta potential value of about +45 mV. A statistically significant decrease in particle size was observed for TAM amounts between 5 and 20mg. A strong influence of loaded tamoxifen on the structure of lecithin/chitosan nanoparticles was observed, supported by the quantification of free chitosan and morphological analysis. A loading of tamoxifen in nanoparticles of around 19% was obtained. The release of the drug from the LCN-TAM colloidal dispersion was measured, showing that tamoxifen citrate was released very slowly in simulated gastro-intestinal fluids without enzymes. When enzymes able to dismantle the nanoparticle structure were added to the dissolution medium, drug release was triggered and continued in a prolonged manner. Tamoxifen-loaded nanoparticles showed cytotoxicity towards MCF-7 cells comparable to that obtained with tamoxifen citrate solution, but the rate of this toxic effect was dependent on drug release. Caco-2 cells, used as a model of the intestinal epithelium, were shown to take up the TAM loaded nanoparticles extensively. Copyright © 2013 Elsevier B.V. All rights reserved.

Nanoporous Gold for Enzyme Immobilization.

PubMed

Stine, Keith J; Jefferson, Kenise; Shulga, Olga V

2017-01-01

Nanoporous gold (NPG) is a material of emerging interest for immobilization of biomolecules, especially enzymes. The material provides a high surface area form of gold that is suitable for physisorption or for covalent modification by self-assembled monolayers. The material can be used as a high surface area electrode and with immobilized enzymes can be used for amperometric detection schemes. NPG can be prepared in a variety of formats from alloys containing between 20 and 50 % atomic composition of gold and less noble element(s) by dealloying procedures. Materials resembling NPG can be prepared by hydrothermal and electrodeposition methods. Related high surface area gold structures have been prepared using templating approaches. Covalent enzyme immobilization can be achieved by first forming a self-assembled monolayer on NPG bearing a terminal reactive functional group followed by conjugation to the enzyme through amide linkages to lysine residues. Enzymes can also be entrapped by physisorption or immobilized by electrostatic interactions.

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

Cytochrome P450 in the central nervous system as a therapeutic target in neurodegenerative diseases.

PubMed

Navarro-Mabarak, Cynthia; Camacho-Carranza, Rafael; Espinosa-Aguirre, Jesús Javier

2018-05-01

Cytochromes P450 (CYPs) constitute a family of enzymes that can be found in the endoplasmic reticulum (ER), mitochondria or the cell surface of the cells. CYPs are characterized by carrying out the oxidation of organic compounds and they are mainly recognized as mediators of the biotransformation of xenobiotics to polar hydrophilic metabolites that can be eliminated from the organism. However, these enzymes play a key role in many other physiological processes, being involved in diverse indispensable metabolic pathways since they metabolize many endogenous substrates. Various CYP isoforms are expressed in the brain, and it is believed that this could be in part due to the particular function of brain CYPs. In the brain, CYPs are involved in the cholesterol turnover, the biosynthesis of dopamine, serotonin, morphine, hormones, and protective lipid mediators (epoxyeicosatrienoic acids), in addition to their already recognized role in xenobiotics detoxification and psychotropic drug metabolism. Increasing evidence suggests that this group of enzymes is fundamental for the normal functioning and maintenance of brain homeostasis. This review is focused on highlighting the importance of CYP-mediated endogenous metabolism in the central nervous system (CNS) and its relationship with recent findings regarding CYP involvement in neurodegenerative diseases. Some therapeutic approaches focused on CYP regulation are also discussed.

Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

PubMed Central

Federici, L.; Caprari, C.; Mattei, B.; Savino, C.; Di Matteo, A.; De Lorenzo, G.; Cervone, F.; Tsernoglou, D.

2001-01-01

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 Å resolution of PG from the phytopathogenic fungus Fusarium moniliforme (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding. PMID:11687632

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.

Cellular Uptake of the Clostridium perfringens Binary Iota-Toxin

PubMed Central

Blöcker, Dagmar; Behlke, Joachim; Aktories, Klaus; Barth, Holger

2001-01-01

The binary iota-toxin is produced by Clostridium perfringens type E strains and consists of two separate proteins, the binding component iota b (98 kDa) and an actin-ADP-ribosylating enzyme component iota a (47 kDa). Iota b binds to the cell surface receptor and mediates the translocation of iota a into the cytosol. Here we studied the cellular uptake of iota-toxin into Vero cells. Bafilomycin A1, but not brefeldin A or nocodazole, inhibited the cytotoxic effects of iota-toxin, indicating that toxin is translocated from an endosomal compartment into the cytoplasm. Acidification (pH ≤ 5.0) of the extracellular medium enabled iota a to directly enter the cytosol in the presence of iota b. Activation by chymotrypsin induced oligomerization of iota b in solution. An average mass of 530 ± 28 kDa for oligomers was determined by analytical ultracentrifugation, indicating heptamer formation. The entry of iota-toxin into polarized CaCo-2 cells was studied by measuring the decrease in transepithelial resistance after toxin treatment. Iota-toxin led to a significant decrease in resistance when it was applied to the basolateral surface of the cells but not following application to the apical surface, indicating a polarized localization of the iota-toxin receptor. PMID:11292715

Extraction of immune and inflammatory cells from human lung parenchyma: evaluation of an enzymatic digestion procedure.

PubMed Central

Holt, P G; Robinson, B W; Reid, M; Kees, U R; Warton, A; Dawson, V H; Rose, A; Schon-Hegrad, M; Papadimitriou, J M

1986-01-01

The inflammatory and immune cell populations of the human lung parenchyma have not been characterized in detail. This report describes a novel and efficient procedure for their extraction. Histologically normal human lung tissue samples from pneumonectomy specimens were sliced to 0.5 mm, and digested in collagenase/DNAse. Viable mononuclear cell yields ranged from 15-48 X 10(6)/g, and were markedly in excess of reported methods employing mechanical tissue disruption, which normally yield populations containing almost exclusively macrophages. The lung digest population was examined by flow cytometry using monoclonal antibodies against cell surface receptors, and found to comprise up to 40% T lymphocytes, 10% B lymphocytes and 30% macrophages, contaminated by less than 1% peripheral blood cells. Based upon these figures, the recoverable lung parenchymal lymphoid cell pool appears considerably larger than previously recognized, being of the same order as the peripheral blood pool. Initial functional studies suggest that such cellular activities as antigen-specific T cell proliferation, antigen-presentation, interleukin 1 production and natural killer cell activity survive the extraction process, and controlled enzymatic digestion experiments with peripheral blood cells indicate that the degree of enzyme-mediated damage to these functions and to cell-surface structures, was minimal. The extraction method thus appears suitable for studying the types and functions of human parenchymal lung cells in health and disease. Images Fig. 2 p195-a PMID:3026698

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.

ATP-dependent chromatin remodeling in T cells

PubMed Central

Wurster, Andrea L.; Pazin, Michael J.

2012-01-01

One of the best studied systems for mammalian chromatin remodeling is transcriptional regulation during T cell development. The variety of these studies have led to important findings in T cell gene regulation and cell fate determination. Importantly, these findings have also advanced our knowledge of the function of remodeling enzymes in mammalian gene regulation. In this review, first we briefly present biochemical/cell-free analysis of 3 types of ATP dependent remodeling enzymes (SWI/SNF, Mi2, and ISWI), to construct an intellectual framework to understand how these enzymes might be working. Second, we compare and contrast the function of these enzymes, during early (thymic) and late (peripheral) T cell development. Finally, we examine some of the gaps in our present understanding. PMID:21999456

Defining the Pathway for Tat-mediated Delivery of β-Glucuronidase in Cultured Cells and MPS VII Mice

PubMed Central

Orii, Koji O.; Grubb, Jeffrey H.; Vogler, Carole; Levy, Beth; Tan, Yun; Markova, Kamelia; Davidson, Beverly L.; Mao, Q.; Orii, Tadao; Kondo, Naomi; Sly, William S.

2008-01-01

We used recombinant forms of human β-glucuronidase (GUS) purified from secretions from stably transfected CHO cells to compare the native enzyme to a GUS-Tat C-terminal fusion protein containing the 11-amino-acid HIV Tat protein transduction domain for: (1) susceptibility to endocytosis by cultured cells, (2) rate of clearance following intravenous infusion, and (3) tissue distribution and effectiveness in clearing lysosomal storage following infusion in the MPS VII mouse. We found: (1) Native GUS was more efficiently taken up by cultured human fibroblasts and its endocytosis was exclusively mediated by the M6P receptor. The GUS-Tat fusion protein showed only 30-50% as much M6P-receptor-mediated uptake, but also was taken up by adsorptive endocytosis through binding of the positively charged Tat peptide to cell surface proteoglycans. (2) GUS-Tat was less rapidly cleared from the circulation in the rat (t1/2 = 13 min vs 7 min). (3) Delivery to most tissues of the MPS VII mouse was similar, but GUS-Tat was more efficiently delivered to kidney. Histology showed that GUS-Tat more efficiently reduced storage in renal tubules, retina, and bone. These studies demonstrate that Tat modification can extend the range of tissues corrected by infused enzyme. PMID:16043103

Positive in vitro wound healing effects of functional inclusion bodies of a lipoxygenase from the Mexican axolotl.

PubMed

Stamm, Anne; Strauß, Sarah; Vogt, Peter; Scheper, Thomas; Pepelanova, Iliyana

2018-04-07

AmbLOXe is a lipoxygenase, which is up-regulated during limb-redevelopment in the Mexican axolotl, Ambystoma mexicanum, an animal with remarkable regeneration capacity. Previous studies have shown that mammalian cells transformed with the gene of this epidermal lipoxygenase display faster migration and wound closure rate during in vitro wound healing experiments. In this study, the gene of AmbLOXe was codon-optimized for expression in Escherichia coli and was produced in the insoluble fraction as protein aggregates. These inclusion bodies or nanopills were shown to be reservoirs containing functional protein during in vitro wound healing assays. For this purpose, functional inclusion bodies were used to coat cell culture surfaces prior cell seeding or were added directly to the medium after cells reached confluence. In both scenarios, AmbLOXe inclusion bodies led to faster migration rate and wound closure, in comparison to controls containing either no AmbLOXe or GFP inclusion bodies. Our results demonstrate that AmbLOXe inclusion bodies are functional and may serve as stable reservoirs of this enzyme. Nevertheless, further studies with soluble enzyme are also necessary in order to start elucidating the exact molecular substrates of AmbLOXe and the biochemical pathways involved in the wound healing effect.

Bovine brain ribonuclease is the functional homolog of human ribonuclease 1.

PubMed

Eller, Chelcie H; Lomax, Jo E; Raines, Ronald T

2014-09-19

Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Enzyme nanoparticle fabrication: magnetic nanoparticle synthesis and enzyme immobilization.

PubMed

Johnson, Patrick A; Park, Hee Joon; Driscoll, Ashley J

2011-01-01

Immobilized enzymes are drawing significant attention for potential commercial applications as biocatalysts by reducing operational expenses and by increasing process utilization of the enzymes. Typically, immobilized enzymes have greater thermal and operational stability at various pH values, ionic strengths and are more resistant to denaturation that the soluble native form of the enzyme. Also, immobilized enzymes can be recycled by utilizing the physical or chemical properties of the supporting material. Magnetic nanoparticles provide advantages as the supporting material for immobilized enzymes over competing materials such as: higher surface area that allows for greater enzyme loading, lower mass transfer resistance, less fouling effect, and selective, nonchemical separation from the reaction mixture by an applied a magnetic field. Various surface modifications of magnetic nanoparticles, such as silanization, carbodiimide activation, and PEG or PVA spacing, aid in the binding of single or multienzyme systems to the particles, while cross-linking using glutaraldehyde can also stabilize the attached enzymes.

A Multifunctional Surface That Simultaneously Balances Hydrophilic Enzyme Catalysis and Hydrophobic Water Repellency.

PubMed

Lawton, Timothy J; Uzarski, Joshua R; Filocamo, Shaun F

2016-08-16

The compatibility of multiple functions at a single interface is difficult to achieve, but is even more challenging when the functions directly counteract one another. This study provides insight into the creation of a simultaneously multifunctional surface formed by balancing two orthogonal functions; water repellency and enzyme catalysis. A partially fluorinated thiol is used to impart bulk hydrophobicity on the surface, and an N-hydroxysuccinimide ester-terminated thiol provides a specific anchoring sites for the covalent enzyme attachment. Different ratios of the two thiols are mixed together to form amphiphilic self-assembled monolayers, which are characterized with polarization-modulation infrared reflection-absorption spectroscopy and contact angle goniometry. The enzyme activity is measured by a fluorescence assay. With the results collected here, specific surface compositions are identified at which the orthogonal functions of water repellency and enzyme catalysis are balanced and exist simultaneously. An understanding of how to effectively balance orthogonal functions at surfaces can be extended to a number of higher-scale applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

ENZYMES OF GLUCOSE AND PYRUVATE CATABOLISM IN CELLS, SPORES, AND GERMINATED SPORES OF CLOSTRIDIUM BOTULINUM1

PubMed Central

Simmons, Richard J.; Costilow, Ralph N.

1962-01-01

Simmons, R. J. (Michigan State University, East Lansing), and R. N. Costilow. Enzymes of glucose and pyruvate catabolism in cells, spores, and germinated spores of Clostridium botulinum. J. Bacteriol. 84:1274–1281. 1962.—An investigation was made of the enzymes of vegetative cells, spores, and germinated spores of Clostridium botulinum 62-A to elucidate a pathway of glucose metabolism. Manometric studies were conducted with intact cells, and various enzymes and enzyme systems were assayed in cell-free and spore-free extracts by use of spectrophotometric and colorimetric procedures. Glucose fermentation was found to be inducible; glucokinase was the controlling enzyme. All other enzymes of the Embden-Meyerhof-Parnas (EMP) pathway were found in both induced and non-induced cells, but they were in relatively low concentrations in the latter. This, plus the fact that no glucose-6-phosphate dehydrogenase was detected, led to the conclusion that glucose is catabolized primarily by the EMP system. A number of glycolytic enzymes were also found in extracts of spores and germinated spores of this organism, but the activities were extremely low as compared with activities in cell extracts. A phosphoroclastic-type reaction was readily demonstrated in both glucose-adapted and non-adapted cells, but not in spores and germinated spores. However, both acetokinase and phosphotransacetylase, as well as coenzyme A transphorase, were detected in spores and germinated-spore extracts, although at very low activity levels as compared with cell extracts. The specific activity of diaphorase in spore extracts was about one-half that of corresponding cell extracts, and the activity of reduced diphosphopyridine nucleotide (DPNH) oxidase was actually higher in the spore extracts. In addition, the DPNH oxidase in spore extracts was considerably more heat-stable than that in extracts of cells or germinated spores. PMID:13977433

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

PubMed Central

Salamone, Monica; Carfì Pavia, Francesco

2016-01-01

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells.

PubMed

Varma, Sandeep R; Sundaram, R; Gopumadhavan, S; Vidyashankar, Satyakumar; Patki, Pralhad S

2013-01-01

HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000  μ g/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380  μ g/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.

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

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.

Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

PubMed

Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

2006-01-01

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture*

PubMed Central

Tape, Christopher J.; Norrie, Ida C.; Worboys, Jonathan D.; Lim, Lindsay; Lauffenburger, Douglas A.; Jørgensen, Claus

2014-01-01

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDCM.tub-KDEL) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (LyrM37-KDEL) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDCM.tub-KDEL and LyrM37-KDEL facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell–cell phospho-signaling experiments, we propose DDCM.tub-KDEL and LyrM37-KDEL as excellent enzymes for cell-specific labeling with amino acid precursors. PMID:24820872

Improvement in Saccharification Yield of Mixed Rumen Enzymes by Identification of Recalcitrant Cell Wall Constituents Using Enzyme Fingerprinting.

PubMed

Badhan, Ajay; Wang, Yu-Xi; Gruninger, Robert; Patton, Donald; Powlowski, Justin; Tsang, Adrian; McAllister, Tim A

2015-01-01

Identification of recalcitrant factors that limit digestion of forages and the development of enzymatic approaches that improve hydrolysis could play a key role in improving the efficiency of meat and milk production in ruminants. Enzyme fingerprinting of barley silage fed to heifers and total tract indigestible fibre residue (TIFR) collected from feces was used to identify cell wall components resistant to total tract digestion. Enzyme fingerprinting results identified acetyl xylan esterases as key to the enhanced ruminal digestion. FTIR analysis also suggested cross-link cell wall polymers as principal components of indigested fiber residues in feces. Based on structural information from enzymatic fingerprinting and FTIR, enzyme pretreatment to enhance glucose yield from barley straw and alfalfa hay upon exposure to mixed rumen-enzymes was developed. Prehydrolysis effects of recombinant fungal fibrolytic hydrolases were analyzed using microassay in combination with statistical experimental design. Recombinant hemicellulases and auxiliary enzymes initiated degradation of plant structural polysaccharides upon application and improved the in vitro saccharification of alfalfa and barley straw by mixed rumen enzymes. The validation results showed that microassay in combination with statistical experimental design can be successfully used to predict effective enzyme pretreatments that can enhance plant cell wall digestion by mixed rumen enzymes.

Clinical and biochemical studies support smokeless tobacco’s carcinogenic potential in the human oral cavity

PubMed Central

Mallery, Susan R.; Tong, Meng; Michaels, Gregory C.; Kiyani, Amber R.; Hecht, Stephen S.

2014-01-01

In 2007, International Agency for Cancer Research presented compelling evidence that linked smokeless tobacco use to the development of human oral cancer. While these findings imply vigorous local carcinogen metabolism, little is known regarding levels and distribution of Phase I, II and drug egress enzymes in human oral mucosa. In the study presented here, we integrated clinical data, imaging and histopathologic analyses of an oral squamous cell carcinoma that arose at the site of smokeless tobacco quid placement in a patient. Immunoblot and immunohistochemical (IHC) analyses were employed to identify tumor and normal human oral mucosal smokeless tobacco-associated metabolic activation and detoxification enzymes. Human oral epithelium contains every known Phase I enzyme associated with nitrosamine oxidative bioactivation with ~2 fold inter-donor differences in protein levels. Previous studies have confirmed ~3.5 fold inter-donor variations in intraepithelial Phase II enzymes. Unlike the superficially located enzymes in non-replicating esophageal surface epithelium, IHC studies confirmed oral mucosal nitrosamine metabolizing enzymes reside in the basilar and suprabasilar region which notably is the site of ongoing keratinocyte DNA replication. Clearly, variations in product composition, nitrosamine metabolism and exposure duration will modulate clinical outcomes. The data presented here form a coherent picture consistent with the abundant experimental data that links tobacco-specific nitrosamines to human oral cancer. PMID:24265177

Enhancing enzymatic efficiency by attachment to semiconductor nanoparticles for biosensor applications

NASA Astrophysics Data System (ADS)

Breger, Joyce C.; Walper, Scott A.; Oh, Eunkeu; Susumu, Kimihiro; Stewart, Michael H.; Deschamps, Jeffrey R.; Ancona, Mario G.; Medintz, Igor L.

2015-05-01

Nanosensors employing quantum dots (QDs) with appended biofunctional moieties offer tremendous promise for disease surveillance/diagnostics and chemical/biological threat activity. Their small size permits cell penetration and their inherent photochemical properties are well-suited for rapid, optical measurement. The effectiveness of enzymes immobilized on QDs, however, are not completely understood, hindering development of chemical/biological sensors and remediation materials. Here, we analyze enzyme effectiveness for the neutralization of a simulant nerve agent when attached to two distinctly-sized QDs. Two sizes of QDs, 525 or 625 nm, were appended with DHLA ligands to improve aqueous stability and prevent aggregation. Various molar ratios of de novo phosphotriesterase trimer (PTE3) were rapidly self-assembled via spontaneous metal coordination of the PTE oligohistidine tag onto the Zn2+-rich QD surface. PTE catalyzes the detoxification of organophosphate pesticides (e.g, paraoxon, an analog of sarin) to p-nitrophenol whose absorbance can be measured at 405 nm. The optimal ratio of PTE3 to 525 nm and 625 nm QD's was determined to be 12 and 24, respectively. The enhanced enzyme performance in both cases is most likely due to increased enzyme-substrate interactions from improvements in enzyme orientation, enzyme density, and substrate diffusion on or near the QD. Development of these nansosensors as optical-based biosensors (e.g., within compact microfluidic devices) may greatly improve the sensitivity of conventional biological/chemical detection schemes.

Effects of Mg2+ and adenine nucleotides on thymidylate synthetase from different mouse tumors.

PubMed

Rode, W; Jastreboff, M M

1984-01-01

Magnesium ions variably influenced activity of highly purified thymidylate synthetase preparations from different mouse tumors, activating the enzyme from Ehrlich ascites carcinoma (EAC) cells and inhibiting the enzyme from L1210 and L5178Y cells and from 5-fluorodeoxyuridine (FdUrd)-resistant EAC cells. In the presence of Mg2+ in a concentration resulting in either maximum activation or inhibition (25-30 mM) the enzymes from both the sensitive and FdUrd-resistant EAC lines and L5178Y cells were activated by ATP. Under the same conditions of Mg2+ concentration ADP and AMP inhibited the enzyme from the parental but not from the FdUrd-resistant EAC cells.

Evaluation of Staphylococcus aureus DNA aptamer by enzyme-linked aptamer assay and isothermal titration calorimetry.

PubMed

Bayraç, Ceren; Öktem, Hüseyin Avni

2017-02-01

To monitor the specificity of Staphylococcus aureus aptamer (SA-31) against its target cell, we used enzyme-linked aptamer assay. In the presence of target cell, horseradish peroxidase-conjugated streptavidin bound to biotin-labeled SA-31 showed specific binding to S  aureus among 3 different bacteria with limit of detection of 10 3 colony-forming unit per milliliter. The apparent K a was 1.39 μM -1  ± 0.3 μM -1 . The binding of SA-31 to membrane proteins extracted from cell surface was characterized using isothermal titration calorimetry, and the effect of changes in binding temperature and salt concentrations of binding buffer was evaluated based on thermodynamic parameters (K a , ΔH, and ΔG). Since binding of aptamer to its targets solely depends on its 3-dimensional structure under experimental conditions used in selection process, the change in temperature and ion concentration changed the affinity of SA-31 to its target on surface of bacteria. At 4°C, SA-31 did not show an affinity to its target with poor heat change upon injection of membrane fraction to aptamer solution. However, the apparent association constants of SA-31 slightly varied from K a  = 1.56 μM -1  ± 0.69 μM -1 at 25°C to K a  = 1.03 μM -1  ± 0.9 μM -1 at 37°C. At spontaneously occurring exothermic binding reactions, affinities of S aureus aptamer to its target were also 9.44 μM -1  ± 0.38 μM -1 at 50mM, 1.60 μM -1  ± 0.11 μM -1 at 137mM, and 3.28 μM -1  ± 0.46 μM -1 at 200 mM of salt concentration. In this study, it was demonstrated that enzyme-linked aptamer assay and isothermal titration calorimetry were useful tools for studying the fundamental binding mechanism between a DNA aptamer and its target on the outer surface of S aureus. Copyright © 2016 John Wiley & Sons, Ltd.

Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects.

PubMed

Ward, Keeran; Xi, Jingshu; Stuckey, David C

2015-12-01

The use of non-ionic colloidal liquid aphrons (CLAs) as a support for enzyme immobilisation was investigated. Formulation required the mixing of an aqueous-surfactant solution with a relatively non-polar solvent-surfactant solution, forming a solvent droplet surrounded by a thin stabilised aqueous film (soapy shell). Studies utilising anionic surfactants have showed increased retention, however, very little have been understood about the forces governing immobilisation. This study seeks to determine the effects of enzyme properties on CLA immobilisation by examining a non-ionic/non-polar solvent system comprised of two non-ionic surfactants, Tween 20 and 80, mineral oil and the enzymes lipase, aprotinin and α-chymotrypsin. From these results it was deduced that hydrophobic interactions strongly governed immobilisation. Confocal Scanning Laser Microscopy (CSLM) revealed that immobilisation was predominantly achieved by surface adsorption attributed to hydrophobic interactions between the enzyme and the CLA surface. Enzyme surface affinity was found to increase when added directly to the formulation (pre-manufacture addition), as opposed to the bulk continuous phase (post-manufacture addition), with α-chymotrypsin and aprotinin being the most perturbed, while lipase was relatively unaffected. The effect of zeta potential on immobilisation showed that enzymes adsorbed better closer to their pI, indicating that charge minimisation was necessary for immobilisation. Finally, the effect of increasing enzyme concentration in the aqueous phase resulted in an increase in adsorption for all enzymes due to cooperativity between protein molecules, with saturation occurring faster at higher adsorption rates. Copyright © 2015 Elsevier B.V. All rights reserved.

Strategies for "wiring" redox-active proteins to electrodes and applications in biosensors, biofuel cells, and nanotechnology.

PubMed

Nöll, Tanja; Nöll, Gilbert

2011-07-01

In this tutorial review the basic approaches to establish electrochemical communication between redox-active proteins and electrodes are elucidated and examples for applications in electrochemical biosensors, biofuel cells and nanotechnology are presented. The early stage of protein electrochemistry is described giving a short overview over electron transfer (ET) between electrodes and proteins, followed by a brief introduction into experimental procedures for studying proteins at electrodes and possible applications arising thereof. The article starts with discussing the electrochemistry of cytochrome c, the first redox-active protein, for which direct reversible ET was obtained, under diffusion controlled conditions and after adsorption to electrodes. Next, examples for the electrochemical study of redox enzymes adsorbed on electrodes and modes of immobilization are discussed. Shortly the experimental approach for investigating redox-active proteins adsorbed on electrodes is outlined. Possible applications of redox enzymes in electrochemical biosensors and biofuel cells working by direct ET (DET) and mediated ET (MET) are presented. Furthermore, the reconstitution of redox active proteins at electrodes using molecular wire-like units in order to "wire" the proteins to the electrode surface and possible applications in nanotechnology are discussed.

Release of complement regulatory proteins from ocular surface cells in infections.

PubMed

Cocuzzi, E; Guidubaldi, J; Bardenstein, D S; Chen, R; Jacobs, M R; Medof, E M

2000-11-01

The decay accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59), two complement regulatory proteins that protect self cells from autologous complement-mediated injury, are attached to corneal and cqonjunctival epithelial cells by glycosylphos-phatidylinositol (GPI) anchors. We sought to 1) determine the frequency with which bacteria recovered from patients with infections of the eye elaborate factors that can remove these surface proteins from ocular cells, 2) determine the spectrum of bacteria from other sites that have similar effects, and 3) establish the time interval required for reconstitution of the two regulators. Culture supernatants of 18 ocular isolates [P. aeruginosa (n = 3), S. marcescens (n = 1), S. epidermidis (n = 9), and S. aureus (n = 5)], and > 100 other clinical specimens isolated in the hospital's microbiology laboratory [P. mirabilis (n = 1), S. aureus (n = 65), S. epidermidis (n = 24), B. cereus (n = 12), H. influenzae (n = 15), and Enterobacter sp. (n = 21)] were incubated at 37 degrees C for various times with conjunctival epithelial cells, conjunctival fibroblasts or HeLa cells and the release of DAF and CD59 proteins from the surfaces of the cells analyzed by 2-site immunoradiometric assays and by Western blotting. The kinetics of recovery of DAF and CD59 expression on the cells was measured by flow cytometry. DAF and/or CD59 release from the cell monolayers varied from < 5% to > 99% at as much as a 1:81 dilution of the supernatant from some bacteria. On conjunctival epithelial cells, more than 8 hr was required for 44% recovery of DAF expression and for 50% recovery of CD59 expression. Bacteria produce phospholipases and/or other enzymes which can efficiently remove DAF and CD59 from ocular cell surfaces. This phenomenon may correlate with their in vivo pathogenicity.

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.

Real-time Analysis of the Enzymatic Biodegradation of Polyhydroxyalkanoate Thin Films Using Microfabricated Polymer Microstructures

NASA Astrophysics Data System (ADS)

Delfaus, Stephen; Latuga, Brian M.; Morse, Clinton; McCarney, Evan R.; Rossini, Connie J.; Augustine, Brian H.; Flythe, Michael D.; Rowe, Sean; Baron, Stephen F.; Dennis, Douglas E.

2003-11-01

In-situ atomic force microscopy (AFM) allows for the real-time acquisition and analysis of materials undergoing biological and chemical alterations. A co-polymer blend of poly 3-hydroxybutyrate / poly 3-hydroxyvalerate P(3HB-3HV) were spun-cast onto glass slides to create thin films with film thickness of 40 nm. This polymer is naturally biodegradable by a variety of bacterially produced enzymes. In this study, these materials were degraded by an untyped and concentrated Strptomyces sp. enzyme produced from soil. Using liquid-cell AFM in contact mode, we were able to observe biodegradation uniformly across the surface of the P(3HB-3HV) films beginning within 2 min of introduction of the enzyme. Height standards have been developed using microcontact printing of self assembled monolayers and selective dewetting to produce P(3HB-3HV) structures with dimensions as small as 10 mm. We will discuss the use of microfabricated height standards to measure biodegradation kinetics in these polymers.

Senescense

PubMed Central

De Leo, Pietro; Sacher, Joseph A.

1970-01-01

During ripening of banana (Musa sapientum L., var. Gros Michel or Valery) acid phosphatase activity increases 13-to 26-fold in the precipitate and 2- to 4-fold in the supernatant fraction of tissue homogenates. These increases are closely correlated with the onset and peak of the climacteric. The precipitate enzyme may be extracted with Triton X-100, CaCl2 or NaCl; about 80% of it is in a 500g precipitate. Studies on effect of tonicity of the grinding medium indicate that the precipitate enzyme is desorbed from membrane or cell wall surfaces, and is not released as a result of lysis of membranes. The development of acid phosphatase during aging of tissue slices is the same as in intact fruit. Short term studies of tissue slices with cycloheximide and actinomycin D indicate that the increase in activity is owed to new enzyme synthesis, which is dependent upon synthesis of RNA. The possible effects of the increase in acid phosphatase on ripening are discussed. PMID:16657436

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.

Differential requirement for cathepsin D for processing of the full length and C-terminal fragment of the malaria antigen MSP1.

PubMed

Tulone, Calogero; Sponaas, Anne-Marit; Raiber, Eun-Ang; Tabor, Alethea B; Langhorne, Jean; Chain, Benny M

2011-01-01

Merozoite Surface Protein 1 is expressed on the surface of malaria merozoites and is important for invasion of the malaria parasite into erythrocytes. MSP1-specific CD4 T cell responses and antibody can confer protective immunity in experimental models of malaria. In this study we explore the contributions of cathepsins D and E, two aspartic proteinases previously implicated in antigen processing, to generating MSP1 CD4 T-cell epitopes for presentation. The absence of cathepsin D, a late endosome/lysosomal enzyme, is associated with a reduced presentation of MSP1 both following in vitro processing of the epitope MSP1 from infected erythrocytes by bone marrow-derived dendritic cells, and following in vivo processing by splenic CD11c+ dendritic cells. By contrast, processing and presentation of the soluble recombinant protein fragment of MSP1 is unaffected by the absence of cathepsin D, but is inhibited when both cathepsin D and E are absent. The role of different proteinases in generating the CD4 T cell repertoire, therefore, depends on the context in which an antigen is introduced to the immune system.

Differential Requirement for Cathepsin D for Processing of the Full Length and C-Terminal Fragment of the Malaria Antigen MSP1

PubMed Central

Raiber, Eun-Ang; Tabor, Alethea B.; Langhorne, Jean; Chain, Benny M.

2011-01-01

Merozoite Surface Protein 1 is expressed on the surface of malaria merozoites and is important for invasion of the malaria parasite into erythrocytes. MSP1-specific CD4 T cell responses and antibody can confer protective immunity in experimental models of malaria. In this study we explore the contributions of cathepsins D and E, two aspartic proteinases previously implicated in antigen processing, to generating MSP1 CD4 T-cell epitopes for presentation. The absence of cathepsin D, a late endosome/lysosomal enzyme, is associated with a reduced presentation of MSP1 both following in vitro processing of the epitope MSP1 from infected erythrocytes by bone marrow-derived dendritic cells, and following in vivo processing by splenic CD11c+ dendritic cells. By contrast, processing and presentation of the soluble recombinant protein fragment of MSP1 is unaffected by the absence of cathepsin D, but is inhibited when both cathepsin D and E are absent. The role of different proteinases in generating the CD4 T cell repertoire, therefore, depends on the context in which an antigen is introduced to the immune system. PMID:22053177

Extracellular Electron Uptake: Among Autotrophs and Mediated by Surfaces.

PubMed

Tremblay, Pier-Luc; Angenent, Largus T; Zhang, Tian

2017-04-01

Autotrophic microbes can acquire electrons from solid donors such as steel, other microbial cells, or electrodes. Based on this feature, bioprocesses are being developed for the microbial electrosynthesis (MES) of useful products from the greenhouse gas CO 2 . Extracellular electron-transfer mechanisms involved in the acquisition of electrons from metals by electrical microbially influenced corrosion (EMIC), from other living cells by interspecies electron transfer (IET), or from an electrode during MES rely on: (i) mediators such as H 2 ; (ii) physical contact through electron-transfer proteins; or (iii) mediator-generating enzymes detached from cells. This review explores the interactions of autotrophs with solid electron donors and their importance in nature and for biosustainable technologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multiscale simulations give insight into the hydrogen in and out pathways of [NiFe]-hydrogenases from Aquifex aeolicus and Desulfovibrio fructosovorans.

PubMed

Oteri, Francesco; Baaden, Marc; Lojou, Elisabeth; Sacquin-Mora, Sophie

2014-12-04

[NiFe]-hydrogenases catalyze the cleavage of molecular hydrogen into protons and electrons and represent promising tools for H2-based technologies such as biofuel cells. However, many aspects of these enzymes remain to be understood, in particular how the catalytic center can be protected from irreversible inactivation by O2. In this work, we combined homology modeling, all-atom molecular dynamics, and coarse-grain Brownian dynamics simulations to investigate and compare the dynamic and mechanical properties of two [NiFe]-hydrogenases: the soluble O2-sensitive enzyme from Desulfovibrio fructosovorans, and the O2-tolerant membrane-bound hydrogenase from Aquifex aeolicus. We investigated the diffusion pathways of H2 from the enzyme surface to the central [NiFe] active site, and the possible proton pathways that are used to evacuate hydrogen after the oxidation reaction. Our results highlight common features of the two enzymes, such as a Val/Leu/Arg triad of key residues that controls ligand migration and substrate access in the vicinity of the active site, or the key role played by a Glu residue for proton transfer after hydrogen oxidation. We show specificities of each hydrogenase regarding the enzymes internal tunnel network or the proton transport pathways.

Helicobacter pylori Resists the Antimicrobial Activity of Calprotectin via Lipid A Modification and Associated Biofilm Formation

PubMed Central

Gaddy, Jennifer A.; Radin, Jana N.; Cullen, Thomas W.; Chazin, Walter J.; Skaar, Eric P.; Trent, M. Stephen

2015-01-01

ABSTRACT Helicobacter pylori is one of several pathogens that persist within the host despite a robust immune response. H. pylori elicits a proinflammatory response from host epithelia, resulting in the recruitment of immune cells which manifests as gastritis. Relatively little is known about how H. pylori survives antimicrobials, including calprotectin (CP), which is present during the inflammatory response. The data presented here suggest that one way H. pylori survives the nutrient sequestration by CP is through alteration of its outer membrane. CP-treated H. pylori demonstrates increased bacterial fitness in response to further coculture with CP. Moreover, CP-treated H. pylori cultures form biofilms and demonstrate decreased cell surface hydrophobicity. In response to CP, the H. pylori Lpx lipid A biosynthetic enzymes are not fully functional. The lipid A molecules observed in H. pylori cultures treated with CP indicate that the LpxF, LpxL, and LpxR enzyme functions are perturbed. Transcriptional analysis of lpxF, lpxL, and lpxR indicates that metal restriction by CP does not control this pathway through transcriptional regulation. Analyses of H. pylori lpx mutants reveal that loss of LpxF and LpxL results in increased fitness, similar to what is observed in the presence of CP; moreover, these mutants have significantly increased biofilm formation and reduced cell surface hydrophobicity. Taken together, these results demonstrate a novel mechanism of H. pylori resistance to the antimicrobial activity of CP via lipid A modification strategies and resulting biofilm formation. PMID:26646009

Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

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

Rudzok, S., E-mail: susanne.rudzok@ufz.d; Schlink, U., E-mail: uwe.schlink@ufz.d; Herbarth, O., E-mail: olf.herbarth@medizin.uni-leipzig.d

2010-05-01

The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-likemore » enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue (registered) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.« less

Xeroderma pigmentosum cells contain low levels of photoreactivating enzyme.

PubMed Central

Sutherland, B M; Rice, M; Wagner, E K

1975-01-01

Fibroblasts from patients with xeroderma pigmentosum contain low levels of photoreactivating enzyme in comparison to normal cells. Levels vary from 0 (line 1199) to 50 (line 1259) percent of normal. The depressed enzyme levels are not an artifact of low growth rate, age of cell donor, cell culture conditions, assay conditions, the presence of inhibitors, or mycoplasma contamination. We show that human fibroblasts can monomerize pyrimidine dimers in vivo. PMID:1054487

Variable ligand- and receptor-binding hot spots in key strains of influenza neuraminidase

PubMed Central

Votapka, Lane; Demir, Özlem; Swift, Robert V; Walker, Ross C; Amaro, Rommie E

2012-01-01

Influenza A continues to be a major public health concern due to its ability to cause epidemic and pandemic disease outbreaks in humans. Computational investigations of structural dynamics of the major influenza glycoproteins, especially the neuraminidase (NA) enzyme, are able to provide key insights beyond what is currently accessible with standard experimental techniques. In particular, all-atom molecular dynamics simulations reveal the varying degrees of flexibility for such enzymes. Here we present an analysis of the relative flexibility of the ligand- and receptor-binding area of three key strains of influenza A: highly pathogenic H5N1, the 2009 pandemic H1N1, and a human N2 strain. Through computational solvent mapping, we investigate the various ligand- and receptor-binding “hot spots” that exist on the surface of NA which interacts with both sialic acid receptors on the host cells and antiviral drugs. This analysis suggests that the variable cavities found in the different strains and their corresponding capacities to bind ligand functional groups may play an important role in the ability of NA to form competent reaction encounter complexes with other species of interest, including antiviral drugs, sialic acid receptors on the host cell surface, and the hemagglutinin protein. Such considerations may be especially useful for the prediction of how such complexes form and with what binding capacity. PMID:22872804

Absence of cell surface expression of human ACE leads to perinatal death

PubMed Central

Michaud, Annie; Acharya, K. Ravi; Masuyer, Geoffrey; Quenech'du, Nicole; Gribouval, Olivier; Morinière, Vincent; Gubler, Marie-Claire; Corvol, Pierre

2014-01-01

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized most often by perinatal death. It is due to the inactivation of any of the major genes of the renin-angiotensin system (RAS), one of which is the angiotensin I-converting enzyme (ACE). ACE is present as a tissue-bound enzyme and circulates in plasma after its solubilization. In this report, we present the effect of different ACE mutations associated with RTD on ACE intracellular trafficking, secretion and enzymatic activity. One truncated mutant, R762X, responsible for neonatal death was found to be an enzymatically active, secreted form, not inserted in the plasma membrane. In contrast, another mutant, R1180P, was compatible with life after transient neonatal renal insufficiency. This mutant was located at the plasma membrane and rapidly secreted. These results highlight the importance of tissue-bound ACE versus circulating ACE and show that the total absence of cell surface expression of ACE is incompatible with life. In addition, two missense mutants (W594R and R828H) and two truncated mutants (Q1136X and G1145AX) were also studied. These mutants were neither inserted in the plasma membrane nor secreted. Finally, the structural implications of these ACE mutations were examined by molecular modelling, which suggested some important structural alterations such as disruption of intra-molecular non-covalent interactions (e.g. salt bridges). PMID:24163131

Heterogeneous distribution of glutamine synthetase among rat liver parenchymal cells in situ and in primary culture.

PubMed Central

Gebhardt, R; Mecke, D

1983-01-01

The distribution of glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.1)] among rat liver parenchymal cells in situ and in primary culture was investigated by indirect immunofluorescence using a specific antiserum. In intact liver, the enzyme was found to be localized exclusively within a very small population of the parenchymal cells surrounding the terminal hepatic venules. Other parts of the parenchyma including non-parenchymal cell types did not stain for this enzyme. Heterogeneity was preserved during isolation of liver parenchymal cells and persisted in cultured cells for at least 3 days. Despite alterations in enzyme activity due to the adaptation of the cells to the culture conditions or due to the hormonal stimulation of the enzyme activity, no change in the relative number of cells expressing this enzyme could be detected. This rather peculiar localization of glutamine synthetase demonstrates an interesting aspect of liver zonation and might have important implications for liver glutamine and, more generally, nitrogen metabolism. Furthermore, it raises the question of whether there might be a phenotypic difference among liver parenchymal cells. Images Fig. 1. PMID:6138251

The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

PubMed

Berthouze, Magali; Venkataramanan, Vidya; Li, Yi; Shenoy, Sudha K

2009-06-17

Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

Surface vimentin is critical for the cell entry of SARS-CoV.

PubMed

Yu, Yvonne Ting-Chun; Chien, Ssu-Chia; Chen, I-Yin; Lai, Chia-Tsen; Tsay, Yeou-Guang; Chang, Shin C; Chang, Ming-Fu

2016-01-22

Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex. A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein. A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.

Enzyme microheterogeneous hydration and stabilization in supercritical carbon dioxide.

PubMed

Silveira, Rodrigo L; Martínez, Julian; Skaf, Munir S; Martínez, Leandro

2012-05-17

Supercritical carbon dioxide is a promising green-chemistry solvent for many enzyme-catalyzed chemical reactions, yet the striking stability of some enzymes in such unconventional environments is not well understood. Here, we investigate the stabilization of the Candida antarctica Lipase B (CALB) in supercritical carbon dioxide-water biphasic systems using molecular dynamics simulations. The preservation of the enzyme structure and optimal activity depend on the presence of small amounts of water in the supercritical dispersing medium. When the protein is at least partially hydrated, water molecules bind to specific sites on the enzyme surface and prevent carbon dioxide from penetrating its catalytic core. Strikingly, water and supercritical carbon dioxide cover the protein surface quite heterogeneously. In the first solvation layer, the hydrophilic residues at the surface of the protein are able to pin down patches of water, whereas carbon dioxide solvates preferentially hydrophobic surface residues. In the outer solvation shells, water molecules tend to cluster predominantly on top of the larger water patches of the first solvation layer instead of spreading evenly around the remainder of the protein surface. For CALB, this exposes the substrate-binding region of the enzyme to carbon dioxide, possibly facilitating diffusion of nonpolar substrates into the catalytic funnel. Therefore, by means of microheterogeneous solvation, enhanced accessibility of hydrophobic substrates to the active site can be achieved, while preserving the functional structure of the enzyme. Our results provide a molecular picture on the nature of the stability of proteins in nonaqueous media.

Cloning of gene-encoded stem bromelain on system coming from Pichia pastoris as therapeutic protein candidate

NASA Astrophysics Data System (ADS)

Yusuf, Y.; Hidayati, W.

2018-01-01

The process of identifying bacterial recombination using PCR, and restriction, and then sequencing process was done after identifying the bacteria. This research aimed to get a yeast cell of Pichia pastoris which has an encoder gene of stem bromelain enzyme. The production of recombinant stem bromelain enzymes using yeast cells of P. pastoris can produce pure bromelain rod enzymes and have the same conformation with the enzyme’s conformation in pineapple plants. This recombinant stem bromelain enzyme can be used as a therapeutic protein in inflammatory, cancer and degenerative diseases. This study was an early stage of a step series to obtain bromelain rod protein derived from pineapple made with genetic engineering techniques. This research was started by isolating the RNA of pineapple stem which was continued with constructing cDNA using reserve transcriptase-PCR technique (RT-PCR), doing the amplification of bromelain enzyme encoder gene with PCR technique using a specific premiere couple which was designed. The process was continued by cloning into bacterium cells of Escherichia coli. A vector which brought the encoder gene of stem bromelain enzyme was inserted into the yeast cell of P. pastoris and was continued by identifying the yeast cell of P. pastoris which brought the encoder gene of stem bromelain enzyme. The research has not found enzyme gene of stem bromelain in yeast cell of P. pastoris yet. The next step is repeating the process by buying new reagent; RNase inhibitor, and buying liquid nitrogen.

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.

Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

PubMed Central

Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna

2013-01-01

Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. PMID:24038690

Effect of functionalized and non-functionalized nanodiamond on the morphology and activities of antioxidant enzymes of lung epithelial cells (A549).

PubMed

Solarska-Ściuk, Katarzyna; Gajewska, Agnieszka; Glińska, Sława; Michlewska, Sylwia; Balcerzak, Łucja; Jamrozik, Agnieszka; Skolimowski, Janusz; Burda, Květoslava; Bartosz, Grzegorz

2014-10-05

The development of nanotechnology opens up new ways for biomedical applications of unmodified and modified diamond nanoparticles which are one of the most popular nanomaterials used in biology, biotechnology, medicine, cosmetics and engineering. They have been applied as diagnostic and therapeutic agents because they can be targeted to and localized in cells causing apoptosis and necrosis. The problem of biocompatibility of nanodiamonds at higher concentrations is thus of primary importance. The first step in the modification of DNPs is usually the introduction of hydrogen groups, which can bind other functional groups. The basic method to introduce -OH groups onto nanoparticles is the Fenton reaction. The aim of this study was to compare the effect of unmodified nanodiamond particles and nanoparticles modified by introduction of -OH groups and etoposide onto their surface reaction on human non-small lung cancer cells. A549 cells were incubated with 2-100μg/ml nanopowders and at 0.6-24μg/ml etoposide in the DMEM medium. We observed a decrease of cells viability and generation of reactive oxygen/ nitrogen species in the cells after incubation, estimated by oxidation of H2DCF-DA and DAF-FM-DA. Modified detonation nanoparticles affected also the cellular content of glutathione and activities of main antioxidant enzymes (glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase and catalase). The results of TEM microscopy show changes in cell morphology. These data demonstrate that modified nanoparticles induce oxidative stress in the target cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Biomolecular hybrid material and process for preparing same and uses for same

DOEpatents

Kim, Jungbae [Richland, WA

2010-11-23

Disclosed is a composition and method for fabricating novel hybrid materials comprised of, e.g., carbon nanotubes (CNTs) and crosslinked enzyme clusters (CECs). In one method, enzyme-CNT hybrids are prepared by precipitation of enzymes which are subsequently crosslinked, yielding crosslinked enzyme clusters (CECs) on the surface of the CNTs. The CEC-enzyme-CNT hybrids exhibit high activity per unit area or mass as well as improved enzyme stability and longevity over hybrid materials known in the art. The CECs in the disclosed materials permit multilayer biocatalytic coatings to be applied to surfaces providing hybrid materials suitable for use in, e.g., biocatalytic applications and devices as described herein.

Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus.

PubMed

Nan, Li; Yang, Ke; Ren, Guogang

2015-06-01

Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu(2+) ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1ppm (2days) to 4.5ppm (7days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu(2+) ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface. Copyright © 2015. Published by Elsevier B.V.

Surface Plasmon Resonance Imaging of the Enzymatic Degradation of Cellulose Microfibrils

NASA Astrophysics Data System (ADS)

Reiter, Kyle; Raegen, Adam; Clarke, Anthony; Lipkowski, Jacek; Dutcher, John

2012-02-01

As the largest component of biomass on Earth, cellulose represents a significant potential energy reservoir. Enzymatic hydrolysis of cellulose into fermentable sugars, an integral step in the production of biofuel, is a challenging problem on an industrial scale. More efficient conversion processes may be developed by an increased understanding of the action of the cellulolytic enzymes involved in cellulose degradation. We have used our recently developed quantitative, angle-scanning surface plasmon resonance imaging (SPRi) device to study the degradation of cellulose microfibrils upon exposure to cellulosic enzymes. In particular, we have studied the action of individual enzymes, and combinations of enzymes, from the Hypocrea Jecorina cellulase system on heterogeneous, industrially-relevant cellulose substrates. This has allowed us to define a characteristic time of action for the enzymes for different degrees of surface coverage of the cellulose microfibrils.

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

PubMed Central

Itoh, Takafumi; Hibi, Takao; Fujii, Yutaka; Sugimoto, Ikumi; Fujiwara, Akihiro; Suzuki, Fumiko; Iwasaki, Yukimoto; Kim, Jin-Kyung; Taketo, Akira

2013-01-01

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine. PMID:24077704

Role of mesenchymal stem cells versus angiotensin converting enzyme inhibitor in kidney repair.

PubMed

Ahmed, Hanaa H; Toson, Elshahat A; El-Mezayen, Hatem A; Rashed, Laila A; Elsherbiny, Eslam S

2017-07-01

The current study sought to clarify the role of bone marrow derived mesenchymal stem cells (BM-MSCs) and adipose tissue derived mesenchymal stem cells (AD-MSCs) in repressing nephropathy in the experimental model. Moreover, the aim of this work was extended to compare between stem cells role and angiotensin converting enzyme inhibitor in kidney repair. Isolation and preparation of MSCs culture, flow cytometry using CD34, CD44 and CD105 cell surface markers, biochemical analyses for determination of serum creatinine, urea, transforming growth factor β (TGF-β), cystatin C (CYS-C) and urinary N-Acetyl-ß-D-Glucosaminidase (UNAG), and histopathological investigation of kidney tissue sections were performed. The results of the present study revealed that single intravenous infusion of MSCs either derived from bone marrow or adipose tissue was able to enhance renal reparative processes through significantly decreased serum creatinine, urea, TGF-β and CYS-C levels as well as UNAG level and significantly increase glomerular filtration rate. Additionally, the histopathological investigations of kidney tissues showed that MSCs have significant regenerative effects as evidenced by the decrease in focal inflammatory cells infiltration, focal interstitial nephritis and congested glomeruli as well as degenerated tubules. The current data provided distinct evidence about the favourable impact of AD-MSCs and BM-MSCs in attenuation of cyclosporine-induced nephropathy in rats through their ability to promote functional and structural kidney repair via transdifferentiation. © 2016 Asian Pacific Society of Nephrology.

Annexin-directed β-glucuronidase for the targeted treatment of solid tumors.

PubMed

Guillen, Katrin P; Ruben, Eliza A; Virani, Needa; Harrison, Roger G

2017-02-01

Enzyme prodrug therapy has the potential to remedy the lack of selectivity associated with the systemic administration of chemotherapy. However, most current systems are immunogenic and constrained to a monotherapeutic approach. We developed a new class of fusion proteins centered about the human enzyme β-glucuronidase (βG), capable of converting several innocuous prodrugs into chemotherapeutics. We targeted βG to phosphatidylserine on tumor cells, tumor vasculature and metastases via annexin A1/A5. Phosphatidylserine shows promise as a universal marker for solid tumors and allows for tumor type-independent targeting. To create fusion proteins, human annexin A1/A5 was genetically fused to the activity-enhancing 16a3 mutant of human βG, expressed in chemically defined, fed-batch suspension culture, and chromatographically purified. All fusion constructs achieved >95% purity with yields up to 740 μg/l. Fusion proteins displayed cancer selective cell-surface binding with cell line-dependent binding stability. One fusion protein in combination with the prodrug SN-38 glucuronide was as effective as the drug SN-38 on Panc-1 pancreatic cancer cells and HAAE-1 endothelial cells, and demonstrated efficacy against MCF-7 breast cancer cells. βG fusion proteins effectively enable localized combination therapy that can be tailored to each patient via prodrug selection, with promising clinical potential based on their near fully human design. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

Nano and Microparticle-Enhanced Immunosensor Approaches for the Detection of Cancer Biomarker Proteins

NASA Astrophysics Data System (ADS)

Mani, Vigneshwaran

Accurate, sensitive, point-of-care multiplexed protein measurements are critical for early disease detection and monitoring, impacting biomarker and drug discovery, and personalized medicine. Significant application involves monitoring panels of proteins in the blood that are biomarkers for diagnosing cancer. However, measurements of biomarker panels in blood or other bodily fluids have been slow to integrate into current practice of cancer diagnostics partly due to the lack of technically simple, low-cost, sensitive, point-of-care multiplexed measurement devices, as well as the lack of rigorously validated protein panels. The present thesis in part addresses these limitations by the development of electrochemical and surface plasmon resonance (SPR) immunosensors utilizing 1mum superparamagnetic labels for accurate detection of prostate cancer biomarker proteins in patient serum samples. Electrochemical discrete immunosensors featuring nanostructured surface with densely packed 5 nm glutathione-coated gold nanoparticles coupled with multi-enzyme magnetic particle (MP) labels enabled measurement of prostate specific antigen (PSA) with a detection limit (DL) of 0.5 pg mL-1 in undiluted serum. Such low DLs are attributed to high surface area, conductivity of nanostructured surface, and multi-enzyme signal amplification. DLs are further improved by utilizing MP bioconjugated with more than 100,000 antibody labels to offline capture proteins from the serum sample matrix, minimizing nonspecific binding of interfering proteins on sensor surface before detection. This approach provided an unprecedented 10 fg DL mL-1 for PSA in undiluted serum using a flow SPR biosensor. Finally electrochemical microfluidic immunoarrays featuring nanostructured surface and offline protein capture by multi-label MPs enabled multiplexed detection of prostate cancer biomarkers PSA and interleukin-6 (IL-6). These approaches provided up to 1000-fold lower DLs compared to commercial bead based assays. The high sensitivity of these approaches will allow monitoring of biomarker levels in diseases states where proteins are in sub pg mL -1 concentrations that are normally challenging to detect using traditional methods such as enzyme linked immunosorbent assays (ELISA). Further emphases will be on SPR-based fundamental studies on binding affinity enhancement of MP conjugates to protein surfaces. In addition, this thesis describes the assembly of glucose/O2 enzymatic biofuel cells for power generation utilizing layer-by-layer films of osmium redox polymers and enzymes. Towards the end, the present thesis describes a simple, low-cost and accurate paper-based electrochemical device fabrication methods and its applications towards monitoring genotoxic activities in the environmental samples.