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Biomedical subjects

C R Middaugh

Publications and source records attributed to C R Middaugh.

At least 19 recordsLinked to original sources
PubMed2001

Complex effects of molecular chaperones on the aggregation and refolding of fibroblast growth factor-1.

Fibroblast growth factor one (FGF-1) exists in a molten globule (MG)-like state under physiological conditions (neutral pH, 37 degrees C). It has been proposed that this form of the protein may be involved in its atypical membrane transport properties. Macromolecular chaperones have been shown to bind to MG states of proteins as well as to be involved in protein membrane transport. We have therefore examined the effect of such proteins on the aggregation and refolding of FGF-1 to evaluate whether they might play a role in FGF-1 transport. The proposed chaperone alpha-crystallin was found to strongly inhibit the aggregation of the MG state of FGF-1. Curiously, two other proteins of similar size and charge (thyroglobulin and a monoclonal IgM immunoglobulin) with no previously reported chaperone properties were also found to have a related effect. In contrast, the chaperone GroEL/ES induced further aggregation of MG-like FGF-1 but had no effect on the native conformation. Both chaperones stimulated refolding to the native state (25 degrees C) but had no detectable effect when FGF-1 was refolded to the MG state (37 degrees C). This suggests that disordered intermediates are present in the folding pathways of the native and MG-like FGF conformations which differ from the MG-like state induced under physiological conditions. FGF-1 does, therefore, interact with molecular chaperones, although this may involve both the MG and the native states of the protein.

Adenosine Triphosphate↗
PubMed2001

The potential role of proteoglycans in cationic lipid-mediated gene delivery. Studies of the interaction of cationic lipid-DNA complexes with model glycosaminoglycans.

Recent evidence supports a role for proteoglycans in polycation-mediated gene delivery. Therefore, the interaction of glycosaminoglycans with cationic lipid-DNA complexes (CLDCs) has been characterized using a combination of biophysical approaches. At low ionic strength, CLDCs bind to heparin-derivatized Sepharose particles, with the ratio of cationic lipid to DNA controlling the binding. Incorporation of the helper lipids cholesterol or 1,2-dioleoyl-phosphatidylethanolamine increases the amount of bound CLDC. Heparin also induces the aggregation of CLDCs, with cholesterol reducing this effect. Isothermal titration calorimetry demonstrates an endothermic heat for the binding of heparin to CLDCs at low ionic strength, whereas circular dichroism studies suggest a heparin-stimulated release of DNA from CLDCs at a greater than 20-fold charge excess. Increasing the ionic strength to 0.11 reduces CLDC binding to heparin beads, and greatly enhances the release of DNA from CLDCs by heparin. The ability of the cell surface glycosaminoglycan heparan sulfate to release DNA from CLDCs is more sensitive than heparin to the incorporation of the cholesterol or 1,2-dioleoyl-phosphatidylethanolamine. Titration calorimetry reveals an exothermic heat for the interaction glycosaminoglycans with CLDCs at higher ionic strength. These results are consistent with the direct involvement of proteoglycans in transfection.

Calorimetry↗
PubMed2001

Isothermal titration calorimetric analysis of the interaction between cationic lipids and plasmid DNA.

The effects of buffer and ionic strength upon the enthalpy of binding between plasmid DNA and a variety of cationic lipids used to enhance cellular transfection were studied using isothermal titration calorimetry at 25.0 degrees C and pH 7.4. The cationic lipids DOTAP (1,2-dioleoyl-3-trimethyl ammonium propane), DDAB (dimethyl dioctadecyl ammonium bromide), DOTAP:cholesterol (1:1), and DDAB:cholesterol (1:1) bound endothermally to plasmid DNA with a negligible proton exchange with buffer. In contrast, DOTAP: DOPE (L-alpha-dioleoyl phosphatidyl ethanolamine) (1:1) and DDAB:DOPE (1:1) liposomes displayed a negative enthalpy and a significant uptake of protons upon binding to plasmid DNA at neutral pH. These findings are most easily explained by a change in the apparent pKa of the amino group of DOPE upon binding. Complexes formed by reverse addition methods (DNA into lipid) produced different thermograms, sizes, zeta potentials, and aggregation behavior, suggesting that structurally different complexes were formed in each titration direction. Titrations performed in both directions in the presence of increasing ionic strength revealed a progressive decrease in the heat of binding and an increase in the lipid to DNA charge ratio at which aggregation occurred. The unfavorable binding enthalpy for the cationic lipids alone and with cholesterol implies an entropy-driven interaction, while the negative enthalpies observed with DOPE-containing lipid mixtures suggest an additional contribution from changes in protonation of DOPE.

Calorimetry↗
PubMed2001

Conformational origin of the aggregation of recombinant human factor VIII.

Aggregation of proteins is a major problem in their use as drugs and is also involved in a variety of pathological diseases. In this study, biophysical techniques were employed to investigate aggregate formation in the pharmaceutically important protein, recombinant human factor VIII (rhFVIII). Recombinant human factor VIII incubated in solution at 37 degrees C formed soluble aggregates as detected by molecular sieve chromatography and dynamic light scattering. This resulted in a corresponding loss of biological activity. Fluorescence and CD spectra of the thermally stressed rhFVIII samples did not, however, suggest significant differences in protein conformation. To identify conformational changes in rhFVIII that may be involved in rhFVIII aggregation, temperature and solutes were used to perturb the native structure of rhFVIII. Far-UV CD and FTIR studies of rhFVIII as a function of temperature revealed conformational changes corresponding to an increase in intermolecular beta-sheet content beginning at approximately 45 degrees C with significant aggregation observed above 60 degrees C. Fluorescence and DSC studies of rhFVIII also indicated conformational changes initiating between 45 and 50 degrees C. An increase in the exposure of hydrophobic surfaces was observed beginning at approximately 40 degrees C, as monitored by increased binding of the fluorescent probe, bis-anilinonaphthalene sulfonic acid (bis-ANS). Perturbation by various solutes produced several transitions prior to extensive unfolding of rhFVIII. In all cases, a common transition, characterized by an increase in the wavelength of the fluorescence emission maximum of rhFVIII from approximately 330 to 335 nm, was observed during thermal and solute perturbation of factor VIII. Moreover, this transition was correlated with an increased association of factor VIII upon incubation at 37 degrees C in the presence of various solutes. These results suggest that association of rhFVIII in solution was initiated by a small transition in the tertiary structure of the protein which produced a nucleating species that led to the formation of inactive soluble aggregates.

1-Propanol↗
PubMed2001

Antigen levels and antibody titers after DNA vaccination.

DNA vaccination generates strong cellular and humoral immunity in animal models. The mechanisms by which plasmid DNA uptake and expression after intramuscular injection lead to immune responses are not well understood. In particular, the importance of antigen expression levels on subsequent antibody immune responses has not been established. We found that a chemiluminescent assay for alkaline phosphatase allows measurement of antigen levels of secreted alkaline phosphatase (SEAP) in vivo after intramuscular injection of a wide range of plasmid doses. The mice produced antibodies to the alkaline phosphatase reporter gene and both antigen levels and antibody titers were measured over time. We found that the correlation between initial antigen level and antibody response was high (r = 0.74, p < 0.001) and remained high even after accounting for the dose of plasmid injected (r = 0.61, p < 0.001). The correlation between DNA dose and antibody titer was statistically significant (r = 0.53, p < 0.001) but was reduced to almost zero after we accounted for initial antigen levels.

Alkaline Phosphatase↗
PubMed2001

Formulation and characterization of amphotericin B-polyethylenimine-dextran sulfate nanoparticles.

A new aqueous nanoparticle system has been developed using complex coacervation employing the oppositely charged polymers polyethylenimine (PEI) and dextran sulfate (DS), with zinc sulfate as a stabilizing agent. Amphotericin B (AmB) was loaded into the nanoparticles as a model drug. The nanoparticles contained PEI and DS in the weight ratio of approximately 1:2. They possessed a zeta potential of approximately +30 mV and demonstrated a narrow size distribution in the range 100-600 nm with a polydispersity index of 0.2. Electron microscopy revealed spherical nanocapsules with a smooth surface. Very favorable drug entrapment and recovery efficiencies of up to 85% were routinely observed. Processing parameters, such as the pH of the PEI solutions, ratio of the two polymers, as well as the concentrations of DS and zinc sulfate, all played a significant role in controlling particle size. Dissolution studies demonstrated a fast release that is dependent on the model drug solubility. The AmB-loaded nanoparticles displayed no toxicity in tissue culture in contrast to free drug and were almost as efficacious as free drug in killing Candida albicans. Advantages of this simple technique are (1) ease of manufacturing and mild preparation conditions, (2) employment of completely aqueous processing conditions, (3) use of biocompatible polymers that can be prepared aseptically, (4) ability to control their size, and (5) a high level of drug entrapment.

Amphotericin B↗
PubMed2000

Infrared spectroscopic characterization of the interaction of cationic lipids with plasmid DNA.

Fourier transform infrared spectroscopy was used to characterize the interaction of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dioctadecyldimethylammonium bromide with plasmid DNA. The effect of incorporating the neutral colipids cholesterol and dioleoylphosphatidylethanolamine on this interaction was also examined. Additionally, dynamic and phase analysis light scattering were used to monitor the size and zeta potential of the resulting complexes under conditions similar to the Fourier transform infrared measurements. Results suggest that upon interaction of cationic lipids with DNA, the DNA remains in the B form. Distinct changes in the frequency of several infrared bands arising from the DNA bases, however, suggest perturbation of their hydration upon interaction with cationic lipids. A direct interaction of the lipid ammonium headgroup with and dehydration of the DNA phosphate is observed when DNA is complexed with these lipids. Changes in the apolar regions of the lipid bilayer are minimal, whereas the interfacial regions of the membrane show changes in hydration or molecular packing. Incorporation of helper lipids into the cationic membranes results in increased conformational disorder of the apolar region and further dehydration of the interfacial region. Changes in the hydration of the DNA bases were also observed as the molar ratio of helper lipid in the membranes was increased.

Cholesterol↗
PubMed2000

Conformational lability of herpesvirus protein VP22.

The herpesvirus protein VP22 traffics between cells, being exported from expressing cells in a non-Golgi-dependent manner and localizing in the nuclei of surrounding cells. This transport is retained in certain VP22 fusion proteins, making VP22 a candidate for use in macromolecular drug delivery. In an effort to understand the physical basis for this activity, we have initiated structural studies of VP22.C1, the C-terminal half of VP22, which possesses the full transport activity of the native protein. CD and Fourier transform infrared analyses indicate a secondary structure consisting of approximately 30% alpha-helix, 17% beta-sheet, and 51% disordered and turn structure. Unfolding studies conducted by CD, differential scanning calorimetry, and fluorescence reveal a series of discrete structural transitions in the range of 20-60 degrees C. CD and fluorescence studies of interactions between VP22.C1 and divalent cations and model polyanions indicate that Mg(2+), Zn(2+), oligonucleotides, and heparin interact with the protein, causing changes in secondary structure and thermal stability. Additionally, the interaction of VP22.C1 with model lipids was examined. Fluorescence titrations of the protein with trans-parinaric acid at various temperatures suggest the binding of one to two molecules of parinaric acid to VP22.C1 at temperatures >40 degrees C, suggesting the possibility of conformation dependent membrane interaction under physiological conditions.

Capsid↗
PubMed2000

Potential use of non-classical pathways for the transport of macromolecular drugs.

Since an increasing number of drug delivery strategies utilising proteins and peptides exhibiting 'non-classical' transport activities have been proposed, studies have begun to establish underlying functional relationships between different vectors. These attempts to find common factors have been hampered by a lack of biophysical data for the various potential protein and peptide transporters, as well as by the structural and functional diversity of the group as a whole. We describe the various types of vectors being considered for use and the preliminary therapeutic successes that have been achieved. Additionally, the various models that have been proposed for non-classical import and export are outlined and discussed in relation to therapeutic delivery. Possible future developments are also discussed.

Animals↗
PubMed1998

Analysis of plasmid DNA from a pharmaceutical perspective.

The advent of gene therapy and polynucleotide-based vaccines has resulted in the use of plasmid DNA as a drug substance. Although biologically (cell or animal) based assays must currently be employed to establish the identity and potency of such drugs, we argue that in the future, a combination of microchip-based mutation detection devices combined with an array of chromatographic, electrophoretic, hydrodynamic, and spectroscopic methods can be employed to rigorously establish these properties. We review a variety of such methods in this context and also consider the issue of the chemical stability of plasmids. Extensive comparison is made to protein-based pharmaceuticals with the unique importance of polynucleotide sequence emphasized in comparison to protein tertiary structure.

Chemistry Techniques, Analytical↗
PubMed1997

Degradative covalent reactions important to protein stability.

Commonly observed chemical modifications that occur in proteins during their in vitro purification, storage, and handling are discussed. Covalent modifications described include deamidation and isoaspartate formation, cleavage of peptide bonds at aspartic acid residues, cystine destruction and thiol-disulfide interchange, oxidation of cysteine and methionine residues, and the glycation and carbamylation of amino groups.

Aspartic Acid↗
PubMed1997

Size exclusion HPLC method for the determination of acidic fibroblast growth factor in viscous formulations.

A size exclusion HPLC method has been developed to determine the protein concentration of pharmaceutical formulations of recombinant acidic fibroblast growth factor (aFGF). These topical aFGF formulations not only contain low levels of protein mass (50 micrograms ml-1), but also include buffer ions, polysaccharide polyanions to conformationally stabilize aFGF and 1% hydroxyethylcellulose to increase the solution's viscosity. A cesium chloride mobile phase is utilized during SEC-HPLC to dissociate aFGF from the pharmaceutical excipients and to minimize nonspecific interaction of the protein with the column matrix. The protein content of a viscous aFGF formulation is determined by comparison of aFGF peak areas to standards of known concentration. Fluorescence spectroscopy was utilized to directly demonstrate that the protein remains in its native conformation during sample preparation and analysis.

Chemistry, Pharmaceutical↗
PubMed1997

Size and conformational stability of the hepatitis A virus used to prepare VAQTA, a highly purified inactivated vaccine.

A variety of biophysical techniques have been employed to examine the size and conformational integrity of highly purified hepatitis A virus (HAV) in solution (purified HAV particles are subsequently formalin-inactivated and adsorbed to aluminum salts for use as the vaccine VAQTA). The size of HAV particles was assessed by a combination of electron microscopy, sedimentation velocity, and dynamic light scattering. The effect of ionic strength and temperature on the overall conformational stability of HAV was determined by a combination of intrinsic HAV protein fluorescence, fluorescent probes of both RNA and protein, and UV-visible spectroscopy. A major structural change in HAV occurs near 60 degrees C with the addition of 0.2 M magnesium chloride enhancing the thermal stability of HAV by approximately 10 degrees C. Salt concentrations above 0.2 M, however, decrease the solubility of HAV. The effect of pH on the physical properties of HAV particles was monitored by dynamic light scattering, analytical size exclusion HPLC, and interaction with fluorescent dyes. HAV particles undergo a substantially reversible association/aggregation at pH values below 6 with the concomitant exposure of previously buried hydrophobic surfaces below pH 4. These results are in good agreement with previous studies of HAV thermal stability under extreme conditions in which the irreversible inactivation of the viral particles was measured primarily by the loss of viral infectivity. The wide variety of biophysical measurements described in this work, however, directly monitor structural changes as they occur, thus providing a molecular basis with which to monitor HAV stability during purification and storage.

Chromatography, High Pressure Liquid↗
PubMed1996

Biochemical and biophysical comparison of native and chemically synthesized phospholamban and a monomeric phospholamban analog.

Phospholamban (PLB) was rapidly isolated from canine cardiac sarcoplasmic reticulum using immunoaffinity chromatography and prepared by solid phase peptide synthesis. The two proteins are indistinguishable when analyzed by SDS-polyacrylamide gel electrophoresis and exhibit pentameric oligomeric states. They are similarly detected on Western blots, are phosphorylation substrates, have identical amino acid compositions that directly reflect their predicted values, yield the same internal amino acid sequences upon CNBr digestion, and have molecular mass values agreeing with the expected value (approximately 6123 Da). Native and synthetic PLB reduced the calcium sensitivity of Ca2+ATPase, which is reversed by anti-PLB antibody. A Cys-to-Ser PLB analog, where the cysteines (36, 41, and 46) were substituted by serines, is monomeric on SDS-polyacrylamide gel electrophoresis, can be phosphorylated, and is recognized by polyclonal antisera. PLB migrates with a sedimentation coefficient of 4.8 S in sedimentation velocity ultracentrifugation experiments, whereas Cys-to-Ser PLB does not sediment, consistent with a monomeric state. Circular dichroism spectral analysis of PLB indicates about 70% alpha-helical structure, whereas Cys-to-Ser PLB manifests only about 30%. Because the physiochemical properties of native and synthetic PLB appear identical, the more readily available synthetic protein should be suitable for more extensive structural studies.

Adenosine Triphosphatases↗