Biomedical subjects
Publications and source records attributed to Alan J Russell.
Nicatinamide adenine dinucleotide (NAD) with an attached perfluoropolyether tail acts as an affinity surfactant in the extraction of the enzyme horse liver alcohol dehydrogenase (HLADH) from an aqueous medium into a fluorous solvent.
We report herein an efficient method to control pH in reactions catalyzed by hydrolytic enzymes, such as the degradation of paraoxon by phosphotriesterase (E.G. 3.1.8.1; OPH), using urease-catalyzed (E.G. 3.5.1.5) urea hydrolysis as a buffering agent. Given the distinct pH profiles of urease and OPH activities, urease produces base on demand in response to pH drop during paraoxon detoxification. As pH changes, the enzyme activities fluctuate to finally stabilize at a pH "set-point," where the rates of acid and base generation are equal. By varying the urease to OPH ratio, various pH "set-points" ranging between 6.5 and 8.5 were achieved within minutes and could be predicted theoretically. This dynamic approach for pH control was successfully applied to the development of a positive-response inhibition-based sensor.
BACKGROUND: Platelet deposition after angioplasty remains problematic and may contribute to intimal hyperplasia and restenosis. We proposed that polyethylene glycol diisocyanate (PEG-DISO), a polymer that rapidly forms covalent linkages with amine residues on proteins, could mask thrombogenic vascular wall proteins from platelets, thereby abrogating acute platelet deposition. METHODS AND RESULTS: To test this hypothesis, we isolated the femoral arteries of 10 New Zealand White rabbits and injured them with 3 passes of a 2F Fogarty catheter which was inserted through a distal arteriotomy. Immediately after balloon injury, (111)indium-labeled autologous platelets were infused peripherally and the injured femoral arteries were randomly treated for 1 minute with a PEG-DISO solution in one artery and a control solution of the phosphate buffered saline vehicle in the contralateral artery. Following treatment, reflow was initiated. The vessels were harvested after 1 hour and radioactivity was quantified in a gamma counter. Platelet counts were standardized by weight and expressed as platelets/mg (mean +/- SEM). Platelet deposition onto arteries treated with PEG-DISO was (1.2 +/- 0.5) x 10(6) platelets/mg compared to (5.6 +/- 4.2) x 10(6) platelets/mg onto the contralateral control arteries treated with vehicle (P < 0.005). Scanning electron micrographs of the injured vessel segment confirmed qualitatively less platelet deposition on the treated segments than on the control segments. CONCLUSION: Treatment with PEG-DISO significantly inhibited platelet deposition after vascular injury. These data support the hypothesis that treatment with PEG-DISO masks surface adhesive proteins from platelet receptors in vivo and that the resulting molecular barrier significantly reduces platelet deposition onto the damaged vessel wall for at least one hour. The formation of a molecularly thin barrier to platelet deposition may thus be a novel and effective treatment to abrogate acute intravascular thrombosis and may have value in the treatment of restenosis.
BACKGROUND: The identification of somatic mutations in tissues is often difficult when the number of normal alleles in the tissue far exceeds the number of mutant ones. We found that the identification of gsp mutation was not possible by direct sequencing and present a new approach that improves the identification of gsp somatic mutations. MATERIAL/METHODS: Genomic DNA was extracted from frozen tissue of a human ovarian stromal Leydig cell tumor. Exons 8 and 9 of the Gsa gene were amplified by PCR and despite the abnormal migration pattern at this first DGGE, direct sequencing of the PCR product did not reveal mutations, probably due to the small amount of mutant alleles. To improve this amount, the PCR products were re-amplified using as template the excised products of the mutant homoduplex and heteroduplex bands obtained at the first DGGE. RESULTS: This approach resulted in the enhancing of the mutant homoduplex bands whereas the heteroduplex bands remained unchanged at the second DGGE. Direct sequencing of the second round PCR clearly identified the mutation R201C in the ovarian Leydig cell tumor. CONCLUSIONS: We have demonstrated a relatively rapid, convenient and reliable method to improve gsp somatic mutation detection combining a second DGGE of the PCR products obtained from the heteroduplexes and mutant homoduplex bands disclosed in a first DGGE followed by direct sequencing.
The development of smart coatings for a variety of uses depends on the ability of the coating material to perform specific functions. We have used water dispersible polyurethane preparations for the immobilization of binding proteins under mild conditions. In these experiments, antibodies against the enzyme beta-galactosidase or the bacterium Escherichia coli were immobilized in polyurethane coatings and then used to effectively capture their cognate antigen. Further, a second, more general, capture protocol was developed which involves the incorporation of the protein avidin in the plastics. This system efficiently captures biotinylated beta-galactosidase. Biotinylated anti-E. coli antibody captured by avidin bioplastics resulted in a nearly 5-fold increase in the number of bound bacteria when compared to blank polyurethane. The use of avidin in a bioplastic allows any biotinylated antibody to be applied to all or part of the surface resulting in a patterning of capture agents on a preformed surface.
A two-step method was developed to homogeneously insert carbonic anhydrase (CA, E.C. 4.2.1.1) into Michael-adduct-based coatings. CA was first covalently coupled to an N-vinylformamide-based water-soluble polymer. Unlike native CA, the resulting polymer/CA system could be dispersed within a film matrix. The enzyme-containing coating (ECC) hydrolyzes p-nitrophenyl propionate in buffered media at high rates retaining approximately 7% apparent activity. In comparison, other two-step techniques for the chemical coupling of CA to the coating surface were less efficient and led to coatings with significantly less activity. A three-step immobilization process coupling the enzyme to the surface of a partially hydrolyzed coating also raised retention of activity after coating synthesis. CA-ECC is stable under ambient conditions retaining 45% activity after 90 days of storage at room temperature.
Amphiphilic random, gradient, and block copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and n-butyl methacrylate (BMA) were synthesized by atom transfer radical polymerization (ATRP) in water/2-propanol mixtures using a methoxy-poly(ethylene glycol) (MPEG) (M(n) = 2000) macroinitiator. Kinetic studies indicate that the copolymerization is well controlled with molecular weights increasing linearly with conversion. Copolymers with molecular weights up to M(n) = 34000 and low polydispersities (M(w)/M(n) = 1.11-1.47) were prepared. The reactivity ratios were calculated for the copolymerizations catalyzed by CuBr/bpy, (r(DMAEMA) = 1.07, r(BMA) = 1.24). The thermosensitivity and aggregation properties of the random, gradient, and block copolymers significantly depended on the architecture of the copolymers. The lower critical solution temperature of MPEG-b-PDMAEMA(84) was 38 degrees C (5 wt % in water).
We have grown an antimicrobial polymer directly on the surfaces of glass and paper using atom transfer radical polymerization (ATRP). The method described here results in potentially permanent nonleaching antibacterial surfaces without the need to chemically graft the antimicrobial material to the substratum. The tertiary amine 2-(dimethylamino)ethyl methacrylate was polymerized directly onto Whatman #1 filter paper or glass slides via atom transfer radical polymerization. Following the polymerization, the tertiary amino groups were quaternized using an alkyl halide to produce a large concentration of quaternary ammonium groups on the polymer-modified surfaces. Incubating the modified materials with either Escherichia coli or Bacillus subtilis demonstrated that the modified surfaces had substantial antimicrobial capacity. The permanence of the antimicrobial activity was demonstrated through repeated use of a modified glass without significant loss of activity. Quaternary amines are believed to cause cell death by disrupting cell membranes allowing release of the intracellular contents. Atomic force microscopic imaging of cells on modified glass surfaces supports this hypothesis.
The use of enzymes in conjunction with inorganic photocatalysts requires stability against photooxidation. In this paper, we describe enhanced stabilization of a model enzyme, chymotrypsin, to photooxidation driven by titanium dioxide exposed to ultraviolet light (TiO(2)-UV). Stabilization is achieved conjugating the enzyme with an oligomeric adduct of UV-absorbing (2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate) (HBMA) and free radical-absorbing 2-methacryloyloxyethyl-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylate (Trolox-HEMA). Juxtaposition of the antioxidant Trolox with the UV absorber HBMA within a single chain reduced the rate of deactivation of the former by TiO(2)-UV. This enables modified enzyme, which is adsorbed on TiO(2), to absorb both UV-light and free radicals and locally reduce the rate of photooxidation. Interestingly, Trolox was more readily deactivated by TiO(2)-UV when it was conjugated separately to chymotrypsin that had been pre-modified with HBMA moieties.
Photoexcited TiO2 degrades biomolecules such as nucleic acids, cell membrane proteins, and enzymes. Stabilization of enzyme activity against the deactivation caused by the combination of TiO2-UV is essential if we are to develop novel hybrid materials exhibiting photocatalytic and biocatalytic activities useful for decontamination applications. In this paper we describe the stabilization of a model enzyme, chymotrypsin, against TiO2-UV-induced deactivation by conjugating the enzyme with UV-absorbing, carboxyl-terminated oligo[2-[3-(2H-benzotriazol-2-yl)-4-hydroxyphenyl]ethyl methacrylate] [oligo(HBMA)-COOH]. Chymotrypsin was completely deactivated within 3 h, whereas the chymotrypsin-oligo(HBMA) conjugate retained > 50% activity even after 5 h of exposure to TiO2-UV (lambdamax 365 nm). The degree of enzyme stabilization induced by the conjugated UV absorber was 2-fold higher than that from the equivalent number of conjugated PEG chains. Spectroscopic characterizations revealed that chymotrypsin-oligo(HBMA) absorbs UV light and initially resists photoexcitation of TiO2. Modified chymotrypsin also exhibited resistance to changes in the secondary structure during the deactivation. This method of stabilizing enzymes against photodegradation could be also useful in photolithographic enzyme immobilizations for sensors and arrays or for stabilization of any UV-sensitive protein.
We have developed a novel technique to synthesize near-uniform protein-polymer conjugates by initiating atom transfer radical polymerization of monomethoxy poly(ethylene glycol)-methacrylate from 2-bromoisobutyramide derivatives of chymotrypsin (a protein-initiator). Polymerization initiated from the monosubstituted protein-initiator resulted in the conjugate containing a single, near-monodisperse polymer chain per protein molecule with polydispersity index 1.05. Increasing the number of conjugated 2-bromoisobutyramide initiators per molecule of protein increased the molecular weights and polydispersity indices of the final protein-polymer conjugates. The generic nature of this technique was demonstrated by initiating polymerization of nonionic, cationic, and anionic monomers from the protein-initiator. Protein-polymer conjugates synthesized by this novel technique retained 50-86% of the original enzyme activity. The technique described herein should be useful in synthesizing well-defined protein-polymer conjugates exhibiting a wide range of physical and chemical properties.
Salt hydrate pairs were used to control water activity in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate. It was shown that salt hydrate pairs behave essentially the same in ionic liquids as they do in organic solvents as long as they do not dissolve. Initial rate-water activity profiles were prepared for the immobilized Candida antarctica lipase catalyzed synthesis of 2-ethylhexyl methacrylate. The ability to use salt hydrate pairs for the control of water activity in ionic liquids should allow for improved comparison of enzyme activity and specificity in ionic liquids and conventional solvents.
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