Home About us Contact
|
Home About us Contact | ||
|
|
||
Covalent Attachment (covalent + attachment)
Selected AbstractsCovalent Attachment of Low Molecular Weight Poly(ethylene imine) Improves Tat Peptide Mediated Gene Delivery,ADVANCED MATERIALS, Issue 16 2006F. Alexis A polymer-peptide hybrid biomaterial synthesized by coupling poly(ethylene imine) directly to resin-supported Tat peptide takes advantage of the unique features associated with the two original cationic materials and functions as a novel gene-delivery vector with good biocompatibility. The figure shows cells transfected with green fluorescent protein (GFP) using complexes of the polymer-peptide hybrid and GFP (scale bar: 100,,m). [source] Covalent Attachment of Bacteriorhodopsin Monolayer to Bromo-terminated Solid Supports: Preparation, Characterization, and Protein StabilityCHEMISTRY - AN ASIAN JOURNAL, Issue 7 2008Yongdong Jin Dr. Abstract The interfacing of functional proteins with solid supports and the study of related protein-adsorption behavior are promising and important for potential device applications. In this study, we describe the preparation of bacteriorhodopsin (bR) monolayers on Br-terminated solid supports through covalent attachment. The bonding, by chemical reaction of the exposed free amine groups of bR with the pendant Br group of the chemically modified solid surface, was confirmed both by negative AFM results obtained when acetylated bR (instead of native bR) was used as a control and by weak bands observed at around 1610,cm,1 in the FTIR spectrum. The coverage of the resultant bR monolayer was significantly increased by changing the pH of the purple-membrane suspension from 9.2 to 6.8. Although bR, which is an exceptionally stable protein, showed a pronounced loss of its photoactivity in these bR monolayers, it retained full photoactivity after covalent binding to Br-terminated alkyls in solution. Several characterization methods, including atomic force microscopy (AFM), contact potential difference (CPD) measurements, and UV/Vis and Fourier transform infrared (FTIR) spectroscopy, verified that these bR monolayers behaved significantly different from native bR. Current,voltage (I,V) measurements (and optical absorption spectroscopy) suggest that the retinal chromophore is probably still present in the protein, whereas the UV/Vis spectrum suggests that it lacks the characteristic covalent protonated Schiff base linkage. This finding sheds light on the unique interactions of biomolecules with solid surfaces and may be significant for the design of protein-containing device structures. [source] Enhancing the Methyl-Donor Activity of Methylcobalamin by Covalent Attachment of DNACHEMISTRY & BIODIVERSITY, Issue 2 2005Mario Fasching The preparation of a covalent DNA conjugate of vitamin B12 by means of heterogeneous solid-phase synthesis is reported. The cyano-corrinoid made available, dipotassium Co, -cyanocobalamin-(3,,2,),(3,,5,)-bis-2,-deoxythymidyl-3,-ate (K2 - 4), was cleanly methylated at the Co center by electrosynthetic means. Aqueous solutions of the resulting organometallic DNAB12 conjugate K2 - 5 exhibited spectroscopic properties indicative of significant weakening of the axial (CoN) bond, together with a 25-times higher basicity relative to Co, -methylcobalamin (2). Methyl-transfer equilibria of pH-neutral aqueous solutions of K2 - 5 and cob(I)alamin (K- 7) on one side, and of cob(I)alamin-(3,,2,),(3,,5,)-bis-2,-deoxythymidyl-3,-ate (K3 - 8) and methylcobalamin (2) on the other, were studied at room temperature (Scheme,3). The NMR-derived data provided an equilibrium constant of ca. 0.3. Activation of K2 - 5 for abstraction of its Co-bound Me group by a nucleophile (such as cob(I)alamin) was, thus, indicated. [source] Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved proteinPROTEIN SCIENCE, Issue 6 2002Abel Baerga-Ortiz Abstract The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen. [source] Immunoaffinity removal of xenoreactive antibodies using modified dialysis or microfiltration membranesBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Sujatha Karoor Abstract Hyperacute rejection following xenogeneic transplantation in primates is mediated by naturally occurring IgM antibodies, which are specifically directed to ,-Galactosyl residues on many nonprimate mammalian cells. Current approaches to remove these anti-,Gal IgM include plasmapheresis followed by immunoaffinity adsorption on bead columns using synthetic Gal epitopes, which requires two pieces of complex equipment. In this study, we explored the use of immunoaffinity adsorption with hollow fiber microporous or dialysis membranes to which a synthetic ,Gal trisaccharide ligand is bound. Covalent attachment of ligand directly to the surface produced negligible binding, but use of long-chain polyamines as reactive spacers yielded binding densities for anti-,Gal IgM as high as 89 mg/mL membrane volume in breakthrough curve experiments with microporous nylon membranes having an internal surface area of 4.2 m2/mL membrane volume. A crossflow microfilter fabricated from the membranes described in this study and having about 0.4 m2 luminal surface area would be able to carry out plasma separation and immunoadsorption in a single device with a large excess of binding capacity to ensure that all plasma that filters across the device and is returned to a human patient is essentially free of anti-,Gal IgM. We conclude that immunoaffinity removal of xenoreactive antibodies using microfiltration hollow fiber membranes is feasible and has potential advantages of efficiency and simplicity for clinical application. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 134,148, 2003. [source] Using the Aggregation of Latex Polymers in the Fabrication of Reproducible Enzyme ElectrodesELECTROANALYSIS, Issue 17 2003Wibowo Rahmat Abstract An enzyme electrode for glucose is described as a model system to demonstrate a fabrication method using latex aggregation and entrapment of enzyme. Electrosterically-stabilized latex particles synthesized by emulsion polymerization in batch from acrylic acid, methyl methacrylate and butyl acrylate, and glucose oxidase were coagulated together at pH,5.5 with ethanol. A platinum disk electrode dipped in the solution becomes coated with latex/enzyme. The relative thickness of the film and relative amount of enzyme may be controlled by the time the electrode is in contact with the solution. The enzyme was then immobilized by covalent attachment of amine groups to carboxylic moieties in the polymer using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride and N -hydroxysuccinimide. Five minutes contact with the latex/enzyme solution and subsequent amide coupling, gave electrodes with a reproducibility of 5.7% RSD, a wide dynamic range (0,100,mM) and good storage properties. [source] Length-dependent DNA separations using multiple end-attached peptide nucleic acid amphiphiles in micellar electrokinetic chromatographyELECTROPHORESIS, Issue 13 2008Jeffrey M. Savard Abstract End-labeled free-solution electrophoresis (ELFSE) is an alternative approach to gel-based methods for size-based electrophoretic separation of DNA. In ELFSE, an electrically neutral "drag-tag" is appended to DNA to add significant hydrodynamic drag, thereby breaking its constant charge-to-friction ratio. Current drag-tag architecture relies on covalent attachment of polymers to each DNA molecule. We have recently proposed the use of micellar drag-tags in conjunction with sequence-specific hybridization of peptide nucleic acid amphiphiles (PNAAs). This work investigates the effect of multiple PNAA attachment on DNA resolution using MEKC. Simultaneous PNAA hybridization allows for the separation of long DNA targets, up to 1012,bases, using micellar drag-tags. Each PNAA handle independently interacts with the micellar phase, reducing the overall mobility of this complex relative to individual PNAA binding. The sequence- and size-based dependence of this separation technique is maintained with multiple PNAA binding over a range of DNA sizes. Results are accurately described by ELFSE theory, yielding , = 54 for single-micelle tagging and , = 142 for dual-micelle tagging. This method is the first example of a non-covalent drag-tag used to separate DNA of 1000,bases based on both size and sequence. [source] A micropillar-integrated smart microfluidic device for specific capture and sorting of cellsELECTROPHORESIS, Issue 24 2007Yan-Jun Liu Abstract An integrated smart microfluidic device consisting of nickel micropillars, microvalves, and microchannels was developed for specific capture and sorting of cells. A regular hexagonal array of nickel micropillars was integrated on the bottom of a microchannel by standard photolithography, which can generate strong induced magnetic field gradients under an external magnetic field to efficiently trap superparamagnetic beads (SPMBs) in a flowing stream, forming a bed with sufficient magnetic beads as a capture zone. Fluids could be manipulated by programmed controlling the integrated air-pressure-actuated microvalves, based on which in situ bio-functionalization of SPMBs trapped in the capture zone was realized by covalent attachment of specific proteins directly to their surface on the integrated microfluidic device. In this case, only small volumes of protein solutions (62.5,nL in the capture zone; 375,nL in total volume needed to fill the device from inlet A to the intersection of outlet channels F and G) can meet the need for protein! The newly designed microfluidic device reduced greatly chemical and biological reagent consumption and simplified drastically tedious manual handling. Based on the specific interaction between wheat germ agglutinin (WGA) and N -acetylglucosamine on the cell membrane, A549 cancer cells were effectively captured and sorted on the microfluidic device. Capture efficiency ranged from 62 to 74%. The integrated microfluidic device provides a reliable technique for cell sorting. [source] The Assembling of Semiconductor NanocrystalsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 18 2005Alexey Shavel Abstract Recent accomplishments in arranging semiconductor nanoparticles in a desired manner are reviewed. Coupling mechanisms utilized for this purpose include electrostatic and covalent interactions, methods like layer-by-layer assembly, solvent-controlled precipitation and surface amination for covalent attachment of nanoparticles are employed. Dipole,dipole interactions are operative in nanocrystal solids and fast Förster energy transfer is observed. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Systematic Studies on Photoluminescence of Oligo(arylene-ethynylene)s: Tunability of Excited States and Derivatization as Luminescent Labeling Probes for ProteinsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 14 2006Yong-Gang Zhi Abstract Functionalized oligo(phenylene-ethynylene)s (OPEs) with different conjugation lengths, p -X(C6H4C,C)nSiMe3 (n = 1,4; X = NH2, NMe2, H) were synthesized by Sonogashira coupling of (phenylene-ethynylene)s and 1-iodo-4-(trimethylsilylethynyl)benzene, followed by desilylation of the p -substituted (trimethylsilylethynyl)benzenes with potassium hydroxide. The photoluminescent properties for the OPE series with different chain lengths and their solvatochromic responses were examined. The absorption maxima were red-shifted with increasing numbers of ,(C6H4C,C), units (n), and a linear plot of the absorption energy maxima vs. 1/n was obtained for each series. The emission spectra in dichloromethane showed a broad and structureless band, the energies of which (in wavenumbers) also fit linearly with 1/n. Both the absorption and emission wavelength maxima of the NH2 - and NMe2 -substituted OPEs exhibited significant solvent dependence, whereas the parent OPEs (X = H) showed only minor shifts of the ,max values in different solvents. Substituent effects upon the photoluminescent characteristics of the OPEs and the tunability of the excited states were examined with the p -X(C6H4C,C)nSiMe3 (n = 2, 3; X = NH2, NMe2, H, SMe, OMe, OH, and F) series. The H- and F-substituted counterparts exhibited high-energy vibronically structured emissions attributed to the 3(,,*) excited states of the (arylene-ethynylene) backbone. For compounds bearing NH2 and NMe2 groups, a broad red-shifted emission with a remarkable Stokes shift from the respective absorption maximum was observed, which can be assigned to an n , ,* transition. The n , ,* assignment was supported by MO calculations on the model compounds p -X(C6H4C,C)2SiH3 (X = NH2, H). Functionalization of the oligo(arylene-ethynylene)s with the N -hydroxysuccinimidyl (NHS) moiety enabled covalent attachment of the fluorophore to HSA protein molecules. A series of fluorescent labels, namely p -X(C6H4C,C)nC6H4NHS, (n = 1, X = NH2, NMe2, SMe, OMe, OH, F; n = 2, X = NH2, NMe2) and p -Me2NC6H4C,C(C4H2S)C,CC6H4NHS were synthesized, and their conjugates with HSA (human serum albumin) were characterized by MALDI-TOF mass spectrometry, UV/Vis absorption spectroscopy, and gel electrophoresis. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Temperature-Responsive Substrates: Adhesion and Mechanical Properties of PNIPAM Microgel Films and Their Potential Use as Switchable Cell Culture Substrates (Adv. Funct.ADVANCED FUNCTIONAL MATERIALS, Issue 19 2010Mater. Abstract Thermoresponsive poly(N -isopropylacrylamide) (PNIPAM) microgel films are shown to allow controlled detachment of adsorbed cells via temperature stimuli. Cell response occurs on the timescale of several minutes, is reversible, and allows for harvesting of cells in a mild fashion. The fact that microgels are attached non-covalently allows using them on a broad variety of (charged) surfaces and is a major advantage as compared to approaches relying on covalent attachment of active films. In the following, the microgels' physico-chemical parameters in the adsorbed state and their changes upon temperature variation are studied in order to gain a deeper understanding of the involved phenomena. By means of atomic force microscopy (AFM), the water content, mechanical properties, and adhesion forces of the microgel films are studied as a function of temperature. The analysis shows that these properties change drastically when crossing the critical temperature of the polymer film, which is the basis of the fast cell response upon temperature changes. Furthermore, nanoscale mechanical analysis shows that the films posses a nanoscopic gradient in mechanical properties. [source] Adhesion and Mechanical Properties of PNIPAM Microgel Films and Their Potential Use as Switchable Cell Culture SubstratesADVANCED FUNCTIONAL MATERIALS, Issue 19 2010Stephan Schmidt Abstract Thermoresponsive poly(N -isopropylacrylamide) (PNIPAM) microgel films are shown to allow controlled detachment of adsorbed cells via temperature stimuli. Cell response occurs on the timescale of several minutes, is reversible, and allows for harvesting of cells in a mild fashion. The fact that microgels are attached non-covalently allows using them on a broad variety of (charged) surfaces and is a major advantage as compared to approaches relying on covalent attachment of active films. In the following, the microgels' physico-chemical parameters in the adsorbed state and their changes upon temperature variation are studied in order to gain a deeper understanding of the involved phenomena. By means of atomic force microscopy (AFM), the water content, mechanical properties, and adhesion forces of the microgel films are studied as a function of temperature. The analysis shows that these properties change drastically when crossing the critical temperature of the polymer film, which is the basis of the fast cell response upon temperature changes. Furthermore, nanoscale mechanical analysis shows that the films posses a nanoscopic gradient in mechanical properties. [source] Characterization of phycoviolobilin phycoerythrocyanin-,84-cystein-lyase-(isomerizing) from Mastigocladus laminosusFEBS JOURNAL, Issue 18 2002Kai-Hong Zhao Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-,84-cystein-lyase(PVB-PEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-,-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X-100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km = 12,16 µm, depending on [PecA], kcat , 1.2 × 10,4·s,1) and the apoprotein (Km = 2.4 µm at 14 µm PCB, kcat = 0.8 × 10,4·s,1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEC-lyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore. [source] Liquid-Phase Exfoliation of Nanotubes and GrapheneADVANCED FUNCTIONAL MATERIALS, Issue 23 2009Jonathan N. Coleman Abstract Many applications of carbon nanotubes require the exfoliation of the nanotubes to give individual tubes in the liquid phase. This requires the dispersion, exfoliation, and stabilization of nanotubes in a variety of liquids. In this paper recent work in this area is reviewed, focusing on results from the author's group. It begins by reviewing stabilization mechanisms before exploring research into the exfoliation of nanotubes in solvents, by using surfactants or biomolecules and by covalent attachment of molecules. The concentration dependence of the degree of exfoliation in each case will be highlighted. In addition research into the dispersion mechanism for each dispersant type is discussed. Most importantly, dispersion quality metrics for all dispersants are compared. From this analysis, it is concluded that functionalized nanotubes can be exfoliated to the greatest degree. Finally, the extension of this work to the liquid phase exfoliation of graphite to give graphene is reviewed. [source] Synthesis and Characterization of Iron Oxide Derivatized Mutant Cowpea Mosaic Virus Hybrid Nanoparticles,ADVANCED MATERIALS, Issue 24 2008Alfredo A. Martinez-Morales A novel nanoparticle hybrid is attained by the covalent attachment of iron oxide (, -Fe2O3) nanoclusters onto the surface of a mutagenized cow pea mosaic virus (CPMV-T184C). Using a stepwise substrate-based integration, monodisperse CPMV-IO hybrids are anchored on a gold substrate. The physical and magnetic properties of individual CPMV-IO hybrids are qualitatively investigated by atomic/magnetic force microscopy (AFM/ MFM). During MFM characterization a ,boundary-effect' is observed at the CPMV/IO interface. [source] Highly Active and Recyclable Heterogeneous Iridium Pincer Catalysts for Transfer Dehydrogenation of AlkanesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 1-2 2009Zheng Huang Abstract Pincer-ligated iridium complexes have proven to be highly effective catalysts for the dehydrogenation and transfer-dehydrogenation of alkanes. Immobilization onto a solid support offers significant potential advantages in the application of such catalysts particularly with respect to catalyst separation and recycling. We describe three approaches toward such immobilization: (i) covalent attachment to a Merrifield resin, (ii) covalent bonding to silica via a pendant alkoxysilane group, and (iii) adsorption on ,-alumina (,-Al2O3), through basic functional groups on the para- position of the pincer ligand. The simplest of these approaches, adsorption on ,-Al2O3, is also found to be the most effective, yielding catalysts that are robust, recyclable, and comparable to or even more active than the corresponding species in solution. Spectroscopic evidence (NMR, IR) and studies of catalytic activity support the hypothesis that binding occurs at the para- substituent and that this has only a relatively subtle and indirect influence on catalytic behavior. [source] Covalent immobilization of ,-galactosidase on carrageenan coated with chitosanJOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2009Magdy M.M. Elnashar Abstract ,-Galactosidase was covalently immobilized to carrageenan coated with chitosan for the hydrolysis of lactose. The chitosan-carrageenan polyelectrolyte interaction was found to be dependent on the chitosan pH. At pH 4, the chitosan reached its maximum binding of 28.5% (w/w) where the chitosan surface density was 4.8 mg chitosan/cm2 g of carrageenan gel disks, using Muzzarelli method. Glutaraldehyde was used as a mediator to incorporate new functionality, aldehydic carbonyl group, to the bio-polymers for covalent attachment of ,-galactosidase. The enzyme was covalently immobilized to the biopolymer at a concentration of 2.73 mg protein per g of wet gel. FTIR proved the incorporation of the aldehydic carbonyl group to the carrageenan coated with chitosan at 1720 cm,1. The optimum time for enzyme immobilization was found to be 16 h, after which a plateau was reached. The enzyme loading increased from 2.65 U/g (control gel) to 10.92 U/g gel using the covalent technique. The gel's modification has shown to improve the carrageenan gel thermal stability as well as the immobilized enzyme. For example, the carrageenan gel treated with chitosan showed an outstanding thermal stability at 95°C compared with 35°C for the untreated carrageenan gel. Similarly, the immobilization process shifted the enzyme's optimum temperature from 50°C for the free enzyme towards a wider temperature range 45,55 °C indicating that the enzyme structure is strengthened by immobilization. In brief, the newly developed immobilization method is simple; the carrier is cheap, yet effective and can be used for the immobilization of other enzymes. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009 [source] Methods for stabilizing and activating enzymes in ionic liquids,a reviewJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2010Hua Zhao Abstract Ionic liquids (ILs) have evolved as a new type of non-aqueous solvents for biocatalysis, mainly due to their unique and tunable physical properties. A number of recent review papers have described a variety of enzymatic reactions conducted in IL solutions; on the other hand, it is important to systematically analyze methods that have been developed for stabilizing and activating enzymes in ILs. This review discusses the biocatalysis in ILs from two unique aspects (1) factors that impact the enzyme's activity and stability, (2) methods that have been adopted or developed to activate and/or stabilize enzymes in ionic media. Factors that may influence the catalytic performance of enzymes include IL polarity, hydrogen-bond basicity/anion nucleophilicity, IL network, ion kosmotropicity, viscosity, hydrophobicity, the enzyme dissolution, and surfactant effect. To improve the enzyme's activity and stability in ILs, major methods being explored include the enzyme immobilization (on solid support, sol,gel, or CLEA), physical or covalent attachment to PEG, rinsing with n -propanol methods (PREP and EPRP), water-in-IL microemulsions, IL coating, and the design of enzyme-compatible ionic solvents. It is exciting to notice that new ILs are being synthesized to be more compatible with enzymes. To utilize the full potential of ILs, it is necessary to further improve these methods for better enzyme compatibility. This is what has been accomplished in the field of biocatalysis in conventional organic solvents. Copyright © 2010 Society of Chemical Industry [source] Improvement of enantioselectivity and stability of Klebsiella oxytoca hydrolase immobilized on Eupergit C 250LJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2008Pei-Yun Wang Abstract BACKGROUND: A simple procedure was employed to covalently immobilize a Klebsiella oxytoca hydrolase (SNSM-87) onto epoxy-activated supports of Eupergit C 250L via multipoint covalent attachment. The resultant biocatalyst was explored for the hydrolytic resolution of a variety of (R,S)-2-hydroxycarboxylic acid ethyl esters. RESULTS: With the hydrolytic resolution of (R,S)-ethyl mandelate in biphasic media as the model system, optimal conditions of 55 °C, pH 6 buffer and isooctane as the organic phase were selected for improving the enzyme stability (activity retained from 10% to 50% at 96 h) and enantioselectivity (VSVR,1 value enhanced from 44 to 319) in comparison to the performance of free enzyme. Moreover, the immobilized enzyme retained its activity and enantioselectivity after eight cycles of hydrolysis at 55 °C. When applying the resolution process to other (R,S)-2-hydroxycarboxylic acid ethyl esters, 2.4- to 4.0-fold enhancements of the enantioselectivity in general were obtainable. CONCLUSIONS: The enantioselectivity enhancement, good reusability and easy recovery after reaction indicate that the immobilized SNSM-87 may have the potential as an industrial biocatalyst for the preparation of optically pure 2-hydroxycarboxylic acids. Copyright © 2008 Society of Chemical Industry [source] LIGHT REGULATION OF PHYCOBILISOME BIOSYNTHESIS AND CONTROL BY A PHYTOCHROME-LIKE PHOTORECEPTORJOURNAL OF PHYCOLOGY, Issue 2000K. Terauchi Ambient light quality changes dramatically affect the composition of light harvesting structures, the phycobilisomes, in many cyanobacterial species. In the cyanobacterium Fremyella diplosiphon, shifts in the ratio of red to green light lead to transcriptional changes and altered synthesis of several phycobilisome components. This process is called complementary chromatic adaptation (CCA). These two colors have opposite effects: red light activates an operon encoding the biliprotein phycocyanin (PC) and inactivates the operon encoding phycoerythrin (PE), whereas green light activates PE synthesis and shuts down PC synthesis. The effects of red and green light on CCA are photoreversible. Thus, CCA is similar to transcriptional processes that are controlled by phytochromes, a family of eukaryotic red/far red photoreversible photoreceptors. We are using molecular genetics to determine the mechanisms by which F. diplosiphon senses changes in the color of light of its environment. Initial mutant generation and complementation lead to the discovery of three CCA regulatory components that are part of a complex two component system. The most interesting of these is RcaE (regulator of chromatic adaptation), a histidine kinase-class protein containing a region in its amino-terminal half with similarity to the chromophore binding domains of phytochromes. Within this region, RcaE contains a cysteine residue in a similar location as that used for covalent attachment of the open-chain tetrapyrrole chromophore in phytochromes. We will present recent data characterizing RcaE, including in vivo analysis of the chromophore that is attached to RcaE, as well as results from our recent isolation of a new CCA regulatory component. [source] A versatile preparation of azobenzene-dye functionalized colored polymer nanoparticles by surface modificationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 10 2008Caroline Cannizzo Abstract A series of stable and translucent colored nanolatex, that is, colloidal aqueous suspensions of dye-tagged polymer nanoparticles (NPs) in the 15- to 20-nm diameter range, have been prepared by covalent attachment of azobenzene chromophores to the surface of reactive NPs. Primary crosslinked NPs bearing chlorobenzyl groups were produced by microemulsion copolymerization of styrene and vinylbenzylchloride. Amine-functionalized NPs were obtained after a second functionalization step with polyamines (cyclam and polypropyleneimine dendrimers of first and third generations). Dye-doped particles were obtained by reacting pyridylazo-dimethylaminobenzene (PADA) with chlorobenzyl-NPs and by reacting amine-reactive dimethylaminoazobenzene dyes (DABsyl, DAB-ITC) as well as Disperse Red 1 acrylate with polyamine-coated NPs. Regardless the dye solubility, the grafting readily proceeded in aqueous suspensions at room temperature in the presence of a cationic surfactant without added solvent. Purple, red, and orange suspensions (maximum absorption around 550, 500, 430 nm), with dye loads ranging from 0.3 to 1.2 mmol/g, corresponding to 400,1800 azobenzene residues per NP, are obtained. The reported results indicate that functional polymer NPs, with remarkably accessible multiple anchoring sites, are useful building blocks. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 3375,3386, 2008 [source] Synthesis of a [60]fullerene-functionalized poly(vinyl chloride) derivative by stereospecific chemical modification of PVCJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 23 2007Gerardo Martínez Abstract The covalent attachment of [60]fullerene (C60) to two poly(vinyl chloride) (PVC) samples with different isotactic content is achieved by direct reaction in o -dichlorobenzene (o -DCB) solution in the presence of AIBN. The extent of fullerenation is controlled by varying the C60 feed ratio. The pendant C60 -chemically modified PVC polymers are soluble in tetrahydrofuran (THF) and have been characterized by UV,vis, NMR, FTIR, DSC, TGA, cyclic voltammetry, and SEM. The quantitative microstructural analysis after covalent attachment of the bulky C60 moiety to the PVC has been followed by 13C NMR spectroscopy. From the results it can be concluded that the modification of PVC by graft reaction through free radical reaction proceeds by a stereoselective mechanism. This conclusion has been confirmed on the basis of the increase of the glass-transition temperature (Tg) and the thermal stability of the C60 -chemical modified PVC samples. The fullerenated PVCs obtained show good electron acceptor properties, as evidenced by electrochemical investigations. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 5408,5419, 2007 [source] MicroReview: Impact of the bacterial type I cytochrome c maturation system on different biological processesMOLECULAR MICROBIOLOGY, Issue 6 2005Nicholas P. Cianciotto Summary In the ,-, ,- and ,- Proteobacteria, the so-called cytochrome c maturation (Ccm) system is known to promote the covalent attachment of the haem to periplasmic apocytochrome c. However, in species of Pseudomonas, Rhizobium, Paracoccus and Legionella, mutations in ccm genes result in phenotypes that cannot be readily explained by the simple loss of a c -type cytochrome. These phenotypes include loss of siderophore production and utilization, reduced abilities to grow in low-iron conditions and in mammalian and protozoan host cells, and alterations in copper sensitivity and manganese oxidation. These various data suggest that Ccm proteins may perform one or more functions in addition to Ccm, which are critical for bacterial physiology and growth. Novel hypotheses that should be explored include the utilization of Ccm-associated haem for processes besides attachment to apocytochrome c, the export of a non-haem compound through the Ccm system, and the negative effects of protoporphyrin IX accumulation. [source] The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activityPROTEIN SCIENCE, Issue 12 2001Anne Chapman-Smith BCCP, biotin carboxyl carrier protein; IPTG, isopropyl-1-thio-,-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; S.D., standard deviation Abstract Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5,-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain. [source] Purification, crystallization and preliminary crystallographic studies of Lys48-linked polyubiquitin chainsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2010Daichi Morimoto Post-translational modification of proteins by covalent attachment of ubiquitin regulates diverse cellular events. A Lys48-linked polyubiquitin chain is formed via an isopeptide bond between Lys48 and the C-terminal Gly76 of different ubiquitin molecules. The chain is attached to a lysine residue of a substrate protein, which leads to proteolytic degradation of the protein by the 26S proteasome. In order to reveal the chain-length-dependent higher order structures of polyubiquitin chains, Lys48-linked polyubiquitin chains were synthesized enzymatically on a large scale and the chains were separated according to chain length by cation-exchange column chromatography. Subsequently, crystallization screening was performed using the hanging-drop vapour-diffusion method, from which crystals of tetraubiquitin, hexaubiquitin and octaubiquitin chains were obtained. The crystals of the tetraubiquitin and hexaubiquitin chains diffracted to 1.6 and 1.8,Å resolution, respectively. The tetraubiquitin crystals belonged to space group C2221, with unit-cell parameters a = 58.795, b = 76.966, c = 135.145,Å. The hexaubiquitin crystals belonged to space group P21, with unit-cell parameters a = 51.248, b = 102.668, c = 51.161,Å. Structural analysis by molecular replacement is in progress. [source] Surface plasmon resonance label-free monitoring of antibody antigen interactions in real timeBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2007Asta Kausaite Abstract Detection of biologically active compounds is one of the most important topics in molecular biology and biochemistry. One of the most promising detection methods is based on the application of surface plasmon resonance for label-free detection of biologically active compounds. This method allows one to monitor binding events in real time without labeling. The system can therefore be used to determine both affinity and rate constants for interactions between various types of molecules. Here, we describe the application of a surface plasmon resonance biosensor for label-free investigation of the interaction between an immobilized antigen bovine serum albumin (BSA) and antibody rabbit anti-cow albumin IgG1 (anti-BSA). The formation of a self-assembled monolayer (SAM) over a gold surface is introduced into this laboratory training protocol as an effective immobilization method, which is very promising in biosensing systems based on detection of affinity interactions. In the next step, covalent attachment via artificially formed amide bonds is applied for the immobilization of proteins on the formed SAM surface. These experiments provide suitable experience for postgraduate students to help them understand immobilization of biologically active materials via SAMs, fundamentals of surface plasmon resonance biosensor applications, and determination of non-covalent biomolecular interactions. The experiment is designed for master and/or Ph.D. students. In some particular cases, this protocol might be adoptable for bachelor students that already have completed an extended biochemistry program that included a background in immunology. [source] Crystallization and X-ray analysis of human cytoplasmic phenylalanyl-tRNA synthetaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2009Igal Finarov Human cytosolic phenylalanyl-tRNA synthetase (hcPheRS) is responsible for the covalent attachment of phenylalanine to its cognate tRNAPhe. Significant differences between the amino-acid sequences of eukaryotic and prokaryotic PheRSs indicate that the domain composition of hcPheRS differs from that of the Thermus thermophilus analogue. As a consequence of the absence of the anticodon-recognizing B8 domain, the binding mode of tRNAPhe to hcPheRS is expected to differ from that in prokaryotes. Recombinant hcPheRS protein was purified to homogeneity and crystallized. The crystals used for structure determination diffracted to 3.3,Å resolution and belonged to space group C2, with unit-cell parameters a = 362.9, b = 213.6, c = 212.7,Å, , = 125.2°. The structure of hcPheRS was determined by the molecular-replacement method in combination with phase information from multiwavelength anomalous dispersion. [source] Bone repair with a form of BMP-2 engineered for incorporation into fibrin cell ingrowth matrices,BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2005Hugo G. Schmoekel Abstract Most growth factors naturally involved in development and regeneration demonstrate strong binding to the extracellular matrix and are retained there until being locally mobilized by cells. In spite of this feedback between cell activity and growth factor mobilization in the extracellular matrix, this approach has not been extensively explored in therapeutic situations. We present an engineered bone morphogenetic protein-2 (BMP-2) fusion protein that mimics such function in a surgically relevant matrix, fibrin, incorporated into the matrix until it is locally liberated by cell surface-associated proteases. A tripartite fusion protein, denoted TG-pl-BMP-2, was designed and produced recombinantly. An N-terminal transglutaminase substrate (TG) domain provides covalent attachment to fibrin during coagulation under the influence of the blood transglutaminase factor XIIIa. A central plasmin substrate (pl) domain provides a cleavage site for local release of the attached growth factor from the fibrin matrix under the influence of cell-activated plasmin. A C-terminal human BMP-2 domain provides osteogenic activity. TG-pl-BMP-2 in fibrin was evaluated in vivo in critical-size craniotomy defects in rats, where it induced 76% more defect healing with bone at 3 weeks with a dose of 1 ,g/defect than wildtype BMP-2 in fibrin. After a dosing study in rabbits, the engineered growth factor in fibrin was evaluated in a prospective clinical study for pancarpal fusion in dogs, where it induced statistically faster and more extensive bone bridging than equivalent treatment with cancellous bone autograft. The strong healing response shown by fibrin including a bound BMP-2 variant suggests that with the combination of bound growth factor and ingrowth matrix, it may be possible to improve upon the natural growth factor and even upon tissue autograft. © 2004 Wiley Periodicals, Inc. [source] One-Step, Painting-Like Coating Procedures To Make Surfaces Highly and Permanently BactericidalBIOTECHNOLOGY PROGRESS, Issue 2 2006Daewon Park Previously we found that covalent attachment of long-chained, moderately hydrophobic polycations to surfaces of solid objects renders the latter permanently bactericidal. Herein we replaced such surface-specific, multistep immobilization techniques with a single-step, general procedure akin to common painting. Glass or polyethylene slides were briefly dipped into organic solutions of certain optimally hydrophobic N -alkyl-PEI (where PEI stands for branched 750-kDa polyethylenimine) polycations, followed by solvent evaporation. The resultant polycation-coated slides were able to kill on contact all of the encountered bacterial cells, whether the Gram-positive human pathogen Staphylococcus aureus or its Gram-negative brethren Escherichia coli. This biocidal effect was found not to be caused by N -alkyl-PEI molecules leached from the surface. Further examination of the mechanism of this bactericidal action suggested that the surface-deposited N -alkyl-PEI kills bacteria by rupturing their cellular membranes. This conclusion was further supported by studies in which the molecular weight of PEI and the hydrophobicity of the alkyl moiety were varied. [source] How microbes utilize host ubiquitinationCELLULAR MICROBIOLOGY, Issue 10 2009Thomas Spallek Summary Activity, abundance and localization of eukaryotic proteins can be regulated through covalent attachment of ubiquitin and ubiquitin-like moieties. Ubiquitination is important in various aspects of immunity. Pathogens utilize host ubiquitination for the suppression of immune signalling and reprogramming host processes to promote microbial life. They deliver so-called effector molecules into host cells, which functionally or structurally resemble components of the host ubiquitination machinery utilizing this enzymatic process or they secrete molecules to inhibit ubiquitin-mediated degradation. Since prokaryotic pathogens lack a classical ubiquitination system, effector mimicry of components of the ubiquitin machinery could be achieved through gene flow. Horizontal gene transfer allows pathogenic bacteria to access ubiquitination enzymes from a potential host, while lateral gene transfer recruits components from another pathogen providing spread within the microbial community. Additionally, convergent evolution can shape bacterial proteins to acquire ubiquitination functions. [source] | ||||||||||||||||||||||||||||