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Biomedical subjects

R C Willson

Publications and source records attributed to R C Willson.

At least 19 recordsLinked to original sources
PubMed2001

Development of an effective gene delivery system: a study of complexes composed of a peptide-based amphiphilic DNA compaction agent and phospholipid.

We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/microg DNA produced the highest luciferase gene expression, exceeding 1 x 10(9) relative light units/s/mg protein (>3 microg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems.

Amino Acid Sequence↗
PubMed2001

RNA isolation and fractionation with compaction agents.

A new approach to the isolation of RNA from bacterial lysates employs selective precipitation by compaction agents, such as hexammine cobalt and spermidine. Using 3.5 mM hexammine cobalt, total RNA can be selectively precipitated from a cell lysate. At a concentration of 2 mM hexammine cobalt, rRNA can be fractionated from low molecular weight RNA. The resulting RNA mixture is readily resolved to pure 5S and mixed 16S/23S rRNA by nondenaturing anion-exchange chromatography. Using a second stage of precipitation at 8 mM hexammine cobalt, the low molecular weight RNA fraction can be isolated by precipitation. Compaction precipitation was also applied to the purification of an artificial stable RNA derived from Escherichia coli 5S rRNA and to the isolation of an Escherichia coli-expressed ribozyme.

Chemical Fractionation↗
PubMed2001

Optimization of a peptide/non-cationic lipid gene delivery system for effective microinjection into chicken embryo in vivo.

Here we report the characterization and optimization of a peptide/non-cationic lipid gene delivery system that successfully produces high levels of gene expression when delivered by microinjection into chicken embryos in vivo. In addition to plasmid DNA, the delivery complex consisted of four components: 1) a "condensing" peptide with both hydrophobic and cationic amino acid segments; 2) a "fusogenic" peptide with both membrane insertion and amphipathic helical segments; 3) a relatively short-chain phosphatidylcholine (14:1 cis-9); and 4) polyethyleneglycol conjugated to dioleoylphosphatidylethanolamine through a disulfide linkage. Optimum amounts of each component were determined by measuring expression of a luciferase reporter gene following a 24-hour incubation with chick embryo fibroblast (CEF) cells in culture. When relatively low amounts of condensing peptide, fusogenic peptide, or lipid were assembled into the complexes, relatively large concentrations of complex were required to reach maximum gene expression. When the amounts of peptide or lipid were increased, less complex was required to achieve maximum expression, but expression fell substantially with higher amounts of added complex. The polyethyleneglycol component significantly increased gene expression. With some preparations, luciferase activities in the CEF cells reached 1x10(10) relative light units per second per mg protein within 24 hours. Following the optimization experiments with the CEF cells, formulations containing low levels, intermediate levels, and high levels of the delivery system components were assembled with green fluorescent protein plasmid DNA, then microinjected into somite regions of chicken embryos in vivo. It was found that intermediate levels of the components gave the most reliable formulations for inducing localized gene expression in the somitic cells.

Animals↗
PubMed2001

Microbial detection with low molecular weight RNA.

The need to monitor microorganisms in the environment has increased interest in assays based on hybridization probes that target nucleic acids (e.g., rRNA). We report the development of liquid-phase assays for specific bacterial 5S rRNA sequences or similarly sized artificial RNAs (aRNAs) using molecular beacon technology. These beacons fluoresce only in the presence of specific target sequences, rendering as much as a 27-fold fluorescence enhancement. The assays can be used with both crude cell lysates and purified total RNA preparations. Minimal sample preparation (e.g., heating to promote leakage from cells) is sufficient to detect many Gram-negative bacteria. Using this approach it was possible to detect an aRNA-labeled Escherichia coli strain in the presence of a large background of an otherwise identical E. coli strain. Finally, by using a longer wavelength carboxytetramethylrhodamine beacon it was possible to reduce the fraction of the signal due to cellular autofluorescence to below 0.5%.

Biomarkers↗
PubMed2000

Expression of marker RNAs in Pseudomonas putida.

A broad-host-range vector that expresses a unique artificial RNA in Pseudomonas putida has been developed. This vector was derived from the plasmid pBBR1MCS and incorporates regulatory regions from the Escherichia coli ribosomal operon, rrnB. These include the promoters P1 and P2, and the terminators T1 and T2. The gene for the artificial RNA was derived from Vibrio proteolyticus 5S rRNA. The artificial RNA product accumulates to a level that is 10-20% of the total 5S rRNA in P. putida. The RNA product is not incorporated into ribosomes and has a minimal effect on cell growth rate. In contrast, when wild-type V. proteolyticus 5S rRNA was expressed from the vector, it was incorporated into ribosomes. It is expected that this new vector system will allow artificial RNA expression systems to be readily developed for a large variety of species.

Escherichia coli↗
PubMed1999

Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.

The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu(50), and Arg(45) and Arg(68) of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg(68) --> Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (DeltaDeltaG degrees estimated at 4.0 and 6.4 kcal mol(-1), respectively). The same mutations reduce affinity for BQL by only 7- and 55-fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol(-1).

Animals↗
PubMed1999

Boronate affinity adsorption of RNA: possible role of conformational changes.

Batch equilibrium adsorption isotherm determination is used to characterize the adsorption of mixed yeast RNA on agarose-immobilized m-aminophenylboronic acid. It is shown that the affinity-enhancing influence of divalent cations depends strongly on the precise nature of the cation used, with barium being far more effective than the conventionally-used magnesium. This adsorption-promoting influence of barium is suggested to arise primarily from ionic influences on the structure and rigidity of the RNA molecule, as the adsorption of ribose-based small molecules is not similarly affected. The substitution of barium for the standard magnesium counterion does not greatly promote the adsorption of DNA, implying that the effect is specific to RNA and may be useful in boronate-based RNA separations. RNA adsorption isotherms exhibit sharp transitions as functions of temperature, and these transitions occur at different temperatures with Mg2+ and with Ba2+. Adsorption affinity and capacity were found to increase markedly at lower temperatures, suggestive of an enthalpically favored interaction process. The stoichiometric displacement parameter, Z, in Ba2+ buffer is three times the value in Mg2+ buffer, and is close to unity.

Adsorption↗
PubMed1999

Effects of mutations of the alpha His45 residue of Vibrio harveyi luciferase on the yield and reactivity of the flavin peroxide intermediate.

This work was undertaken to investigate the functional consequences of mutations of the essential alpha His45 residue of Vibrio harveyi luciferase, especially with respect to the yield and reactivity of the flavin 4a-hydroperoxide intermediate II. A total of 14 luciferase variants, each with a different single-residue replacement for the alpha His45, were examined. These variants showed changes, mostly slight, in their light decay rates of the nonturnover luminescence reaction and in their Km values for decanal and reduced riboflavin 5'-phosphate (FMNH2). All alpha His45 mutants, however, showed markedly reduced bioluminescence activities, the magnitude of the reduction ranging from about 300-fold to 6 orders of magnitude. Remarkably, a good correlation was obtained for the wild-type luciferase, 12 alpha His45-mutated luciferases, and six additional variants with mutations of other alpha-subunit histidine residues between the degrees of luminescence activity reduction and the dark decay rates of intermediate II. Such a correlation further indicates that the activation of the O-O bond fission is an important function of the flavin 4a-hydroperoxide intermediate II. Both alpha H45G and alpha H45W were found to bind near-stoichiometric amounts of FMNH2. Moreover, each variant catalyzed the oxidation of bound FMNH2 by two mechanisms, with a minor pathway leading to the formation of a luminescence-active intermediate II and a major dark pathway not involving any detectable flavin 4a-hydroperoxide species. This latter pathway mimics that in the normal catalysis by flavooxidases, and its elicitation in luciferase was demonstrated for the first time by single-residue mutations.

Aldehydes↗
PubMed1999

Cloning, expression, and characterization of the Fab fragment of the anti-lysozyme antibody HyHEL-5.

Hybridoma cDNAs encoding the individual chains of the Fab fragment of the well characterized murine monoclonal antibody HyHEL-5 were cloned and sequenced. The recombinant Fab fragment was produced by expressing each chain in a separate Escherichia coli pET vector, denaturing inclusion bodies and co-refolding. Characterization of the purified Fab by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing demonstrated proper processing of the individual chains. The association of the recombinant Fab fragment with hen egg lysozyme and the avian epitope variant bobwhite quail lysozyme was found by isothermal titration calorimetry to have energetics very similar to that of the HyHEL-5 IgG. Heterologous expression of the HyHEL-5 Fab fragment opens the way to structure/function studies in this well-known system.

Amino Acid Sequence↗
PubMed1999

Association and dissociation kinetics of bobwhite quail lysozyme with monoclonal antibody HyHEL-5.

The anti-hen egg lysozyme monoclonal antibody HyHEL-5 and its complexes with various species-variant and mutant lysozymes have been the subject of considerable experimental and theoretical investigation. The affinity of HyHEL-5 for bobwhite quail lysozyme (BWQL) is over 1000-fold lower than its affinity for the original antigen, hen egg lysozyme (HEL). This difference is believed to arise almost entirely from the replacement in BWQL of the structural and energetic epitope residue Arg68 by lysine. In this study, the association and dissociation kinetics of BWQL with HyHEL-5 were investigated under a variety of conditions and compared with previous results for HEL. HyHEL-5-BWQL association follows a bimolecular mechanism and the dissociation of the antibody-antigen complex is a first-order process. Changes in ionic strength (from 27 to 500 mM) and pH (from 6.0 to 10.0) produced about a 2-fold change in the association and dissociation rates. The effect of viscosity modifiers on the association reaction was also studied. The large difference in the HEL and BWQL affinities for HyHEL-5 is essentially due to differences in the dissociation rate constant.

Animals↗
PubMed1999

Carbon dioxide transport by proteic and facilitated transport membranes.

Membrane separation of gases is governed by the permeability of each species across the membrane. The ratio of permeabilities yields the selectivity. Use of certain organic carriers in facilitated transport membranes and the CO2 converting enzyme carbonic anhydrase (CA) in proteic and facilitated transport membranes allows a dramatic increase in CO2 selectivity over other gases. CA has a low Km (9 mM), which we predicted would allow it to scavenge CO2 to very low partial pressures. Our goal was to determine if CA could remove CO2 from an environment at levels of 0.1% or less. Prior measurements of CO2 transport across thin supported liquid membranes showed that addition of CA enhanced CO2 flux by 3- to 100-fold. Proteic films use bifunctional reagents (e.g., glutaraldehyde) to cross-link the enzyme forming a gel. Bovine serum albumin (BSA) is often added for structural stability. Using such a preparation we examined the ability of proteic films to improve CO2 selectivity and to scavenge CO2 from a mixed gas stream. Proof-of-concept results, measured by mass spectrometry, showed a fivefold improvement in CO2 capture rate with maximal improvement at CO2 values of 1% partial pressure difference in the presence of 0 atm absolute difference. At 0.1% CO2 the membrane exhibited a 76% improvement over controls. At 0.3% CO2 the improvement is about threefold. CA proteic membranes exhibit selectivity for CO2 over oxygen and nitrogen in excess of three orders of magnitude. A CA-based proteic or facilitated transport membrane should readily achieve CO2 partial pressures of 0.05% under CELSS conditions. In addition to proteic membranes we are exploring direct immobilization of engineered CA to ultra-high-permeability teflon membranes. Site-directed mutagenesis was used to add functional groups while retaining full enzymatic activity. These results provide a basis for development of far more efficient CO2 capture proteic and facilitated transport membranes with increased selectivity to values closer to 100-fold at 1% CO2. The result will be CO2 selectivity at 0.1% on the order of 400-fold. These results exceed those obtained with other technologies.

Biological Transport↗
PubMed1998

Association and dissociation kinetics of anti-hen egg lysozyme monoclonal antibodies HyHEL-5 and HyHEL-10.

The immunoglobulin G1 (IgG1) kappa antibodies HyHEL-5 and HyHEL-10 interact with nonoverlapping epitopes on hen egg lysozyme (HEL); the HyHEL-5/HEL interface has two energetically and structurally important salt links, whereas the HyHEL-10/HEL interface involves predominantly hydrogen bonds and van der Waals interactions. The kinetics of association and dissociation of antibodies HyHEL-5 and HyHEL-10 with HEL under a variety of conditions were investigated in this study. The association of each antibody with HEL follows second-order kinetics. The association process is significantly diffusion-limited, as indicated by the viscosity dependence of the interaction of both antibodies with HEL, although detailed energetics suggest that the association process may be more complex. The association rate constant for the HyHEL-5/HEL system is within a factor of 2 of the modified Smoluchowski estimate for proteins of this size, whereas HyHEL-10 interacts with HEL with an association rate an order of magnitude lower. The association reactions are insensitive to ionic strength, showing only a twofold decrease in the association rate constant when the ionic strength was increased from 27 mM to 500 mM. Interestingly, the association rate constant for the interaction of HyHEL-5 with HEL varies with pH in the range 6.0-10.0, whereas HyHEL-10/HEL association is not affected by pH in the same range. The dissociation of the HyHEL-5/HEL and HyHEL-10/HEL complexes follow first-order kinetics with half-lives at 25 degrees C of approximately 3,150 s and approximately 21,660 s, respectively.

Animals↗
PubMed1998

Microbial denitrogenation of fossil fuels.

The microbial degradation of nitrogen compounds from fossil fuels is important because of the contribution these contaminants make to the formation of nitrogen oxides (NOx) and hence to air pollution and acid rain. They also contribute to catalyst poisoning during the refining of crude oil, thus reducing process yields. We review the current status of microbial degradation of aromatic nitrogen compounds and discuss the potential of microbial processes to alleviate these problems.

Acid Rain↗
PubMed1997

Association of the anti-hen egg lysozyme antibody HyHEL-5 with avian species variant and mutant lysozymes.

The energetics of association of the murine anti-hen egg lysozyme antibody HyHEL-5 with bobwhite quail lysozyme, California quail lysozyme, and the Arg45-->Lys mutant of hen egg lysozyme was characterized by isothermal titration calorimetry. The association of each lysozyme with HyHEL-5 is enthalpically driven in the temperature range 10 degrees C to 37 degrees C. The calorimetric results indicate that the salt-links between Arg45 and Arg68 of hen egg lysozyme and GluH50 on the HyHEL-5 paratope are energetically important in HyHEL-5/HEL association. In contrast to previous studies, the results suggest that the three characteristic 'quail' mutations affect the energetics of antibody/antigen association, even though they are buried and not in direct contact with the antibody.

Animals↗
PubMed1997

Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme.

Fluorescence polarization spectroscopy and isothermal titration calorimetry were used to study the influence of osmolytes on the association of the anti-hen egg lysozyme (HEL) monoclonal antibody HyHEL-5 with bobwhite quail lysozyme (BWQL). BWQL is an avian species variant with an Arg-->Lys mutation in the HyHEL-5 epitope, as well as three other mutations outside the HyHEL-5 structural epitope. This mutation decreases the equilibrium association constant of HyHEL-5 for BWQL by over 1000-fold as compared to HEL. The three-dimensional structure of this complex has been obtained recently. Fluorescein-labeled BWQL, obtained by labeling at pH 7.5 and purified by hydrophobic interaction chromatograpy, bound HyHEL-5 with an equilibrium association constant close to that determined for unlabeled BWQL by isothermal titration calorimetry. Fluorescence titration, stopped-flow kinetics, and isothermal titration calorimetry experiments using various concentrations of the osmolytes glycerol, ethylene glycol, and betaine to perturb binding gave a lower limit of the uptake of approximately 6-12 water molecules upon formation of the HyHEL-5/BWQL complex.

Animals↗
PubMed1996

Refined structures of bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment.

The HyHEL-5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg-white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL-5, an antihen egg-white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side-chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL-5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side-chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL-5 is similar to that in previously determined lysozyme-HyHEL-5 complexes.

Animals↗
PubMed1995

Microcalorimetric characterization of the anion-exchange adsorption of recombinant cytochrome b5 and its surface-charge mutants.

The adsorption of recombinant soluble tryptic fragment of rat cytochrome b5 on the strong anion exchanger Mono Q was studied using isothermal titration calorimetry and differential scanning calorimetry (DSC). Titration calorimetry results obtained at low levels of adsorbed protein show increasingly endothermic (unfavorable) enthalpies of binding with increasing surface coverage, confirming the heterogeneous nature of binding. The enthalpy of adsorption declines toward zero at higher loadings. At low surface coverage, enthalpies increase linearly with temperature, giving rise to a positive value of delta Cp. Enthalpies of adsorption depend strongly on the history of the adsorbent. DSC is used to show that cytochrome b5 is stable in both free and adsorbed states at all temperatures used in the titration calorimetric experiments. Site-directed mutants of recombinant cytochrome b5 carrying single charge-neutralizing substitutions are used to test the contributions of particular residues to the thermodynamics of adsorption. Like those derived from van't Hoff analysis of equilibrium adsorption isotherms and HPLC retention data, calorimetric enthalpies of adsorption are positive, confirming the dominant role of entropic effects in ion-exchange adsorption in this system.

Adsorption↗