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Biomedical subjects

D T Brandau

Publications and source records attributed to D T Brandau.

9 recordsLinked to original sources

The interaction of cryoimmunoglobulins with a model surface.

Cryoimmunoglobulins are associated with numerous clinical problems ranging from collagen vascular disorders (rheumatoid arthritis and systemic lupus erythematosus) to infectious processes including HIV infection. The precise role of cryoglobulins in the pathophysiology of these disorders remains unresolved. Although cold insolubility may account for some of the observed processes, it cannot explain the entire array of findings in cryoglobulinemia. An alternative hypothesis suggests that the subtle differences responsible for cold precipitation of these proteins renders them intrinsically more sticky, resulting in deposition of cryoimmunoglobulins on vascular surfaces. We have explored this hypothesis by characterizing the binding of monoclonal cold soluble and cryoimmunoglobulins to silica beads as a model biological surface. It is found that monoclonal, type I, IgM and IgG cryoglobulins have only a slight tendency to bind to a greater extent to this surface than cold soluble immunoglobulins. Physical studies utilizing front surface fluorescence measurements and differential scanning calorimetry show surface interaction leads to partial thermal destabilization of the proteins. To a limited extent, this destabilization is more pronounced with the cryoglobulins compared to cold-soluble control homologues. Surface bound IgM cryoimmunoglobulin was also found to fix complement less efficiently than their cold soluble surface bound counterparts. These studies do not strongly support the hypothesis that pathological mechanisms of cryoimmunoglobulins primarily involve abnormal surface interactions, although surface effects could play a limited role in some situations.

Calorimetry, Differential Scanning

Lack of binding between cryoimmunoglobulins, immunoglobulins and fibronectin: implications for immune complex vasculitis.

Immune-complex-mediated vasculitis is a frequent complication of rheumatoid arthritis and systemic lupus erythematosus. The mechanism of deposition of immune complexes within the vessel wall in these diseases remains unknown, but probably involves other proteins. Fibronectin is a likely candidate since it possesses the ability to bind to collagen, endothelial cells, and possibly immunoglobulins and immune complexes. In this study, the binding of fibronectin to IgG and IgM cryoglobulins, cold soluble IgM, IgG, IgG subclasses and IgG fragments was investigated in the solution phase. Static light scattering, fluorescence anisotropy, fluorescence intensity, and PEG precipitation studies were used to investigate binding under different conditions of temperature and ionic strength. These studies failed to demonstrate significant binding between fibronectin and IgM, IgG, IgG subclasses and IgG fragments under the conditions studied. These findings argue against solution phase binding of fibronectin and immunoglobulins contributing to immune complex vasculitis. The possibility of important surface interactions between these proteins has not been ruled out.

Cryoglobulins

Electrostatic properties of cryoimmunoglobulins.

Inhibition of the cryoprecipitation of cryoimmunoglobulins by neutral salts suggests that intermolecular electrostatic (charge-charge) interactions are responsible for their abnormal solution properties. To test this hypothesis, H+ titration curves and isoelectric points were measured for two monoclonal IgG cryoglobulins (Ger and Muk) and compared with four normal (cold soluble) monoclonal IgG. The cryoglobulin Ger manifested values outside the range encountered for the other proteins. The partitioning of the IgG proteins was also examined in aqueous polyethylene glycol-dextran two-phase systems in the presence of both positive and negative salt-induced electrostatic potentials across the phase interface. Both cryoglobulins were found to behave as if they were more negatively charged than the noncryoglobulins. The experiments support the hypothesis that the differences in solubility behavior of monoclonal cryoglobulin and noncryoglobulin proteins are caused by differences in the electrostatic properties of the proteins.

Antibodies, Monoclonal

Kinetics of the precipitation of cryoimmunoglobulins.

The kinetics of the cryoprecipitation of two monoclonal IgG and two monoclonal IgM cryoimmunoglobulins, two IgM/IgG mixed cryoglobulins and a series of cold soluble monoclonal IgG and IgM immunoglobulins in the presence of polyethylene glycol have been compared by time dependent turbidity measurements. The effects of temp and ionic strength on kinetic processes are described in detail. The monoclonal cryoimmunoglobulins display lag times which are not seen with the other proteins, suggesting a critical nucleation event. The protein concn dependence of the lag times indicate that these nucleation centers contain only a few immunoglobulin molecules. Direct evidence for the existence of precipitation nuclei was obtained from dynamic light scattering studies of two of the monoclonal proteins during their lag periods. Both proteins manifested an approx. 20% decrease in their mean diffusion coefficients (corresponding to a 25% increase in Stokes' radius) prior to detectable precipitation. This suggests the formation of nuclei between 2 and 8 times the size of the monomeric proteins. It is postulated that the increasing size of mixed cryoglobulin complexes with decreasing temp provides analogous nucleation sites. The latter stages of precipitation appear to be kinetically similar for all proteins examined, although the size and shape of the aggregates are quite variable.

Antibodies, Monoclonal

Thermodynamics of monoclonal and mixed cryoimmunoglobin solubilization.

The direct calorimetric determination of heats of solution for four monoclonal and three mixed (IgM/IgG) cryoglobulins is described. Values obtained by differential scanning calorimetry (DSC) are compared to values of the apparent delta Hsol obtained by a polyethylene glycol (PEG) precipitation method. The four monoclonal cryoglobulins manifest heats of solution determined by DSC to be of the same order of magnitude as heats obtained by PEG precipitation, although DSC values were 25 to 125% lower than the corresponding van't Hoff enthalpies. Values of delta Hsol for mixed cryoglobulins were significantly greater than monoclonal cryoglobulins on a molar basis. These higher values are primarily attributed to the greater surface area of these complexes which results in more extensive contact between molecules in the solid phase. No evidence was found that conformational changes contributed to the calorimetric delta Hsol values employing a variety of spectroscopic methods.

Antibodies, Monoclonal

The temperature-dependent stoichiometry of mixed cryoimmunoglobulins.

The interaction of three monoclonal rheumatoid factor IgM molecules with IgG antigens has been studied utilizing immunoglobulins isolated from three mixed cryoglobulins. Static light scattering measurements show that the stoichiometry of these immune complexes changes in a temperature-dependent manner from IgM(IgG)0-2 at temperatures greater than 37 degrees C to IgM(IgG)5 complexes at temperatures below 15 degrees C. These results were confirmed by the analysis of the composition of polyethyleneglycol-precipitated complexes. For one mixed cryoglobulin (Glo), temperature-dependent changes in stoichiometry were also verified by chemical cross-linking studies. Binding constants were determined by Scatchard analysis of light scattering data and by fluorescence polarization measurements. Values on the order of 10(5) M-1 were obtained for three monoclonal rheumatoid factor IgM molecules. Glo was further investigated by dynamic light scattering and partial specific volume measurements. Both dynamic light scattering and partial specific volume measurements provided evidence for surprising shape changes of the IgM X IgG complex as a function of temperature and IgG stoichiometry. Collectively, the data support the simple hypothesis that cryoprecipitation of mixed cryoglobulins occurs as a consequence of increases in the size (stoichiometry) of the complexes that are formed at low temperatures.

Cryoglobulinemia

The effect of interchain disulfide bond cleavage on the cold induced precipitation of cryoimmunoglobulins.

Selective cleavage of the interchain disulfide bonds present in the two IgG1-kappa monoclonal cryoglobulins Ger and Muk results in a partial loss of cryoprecipitability of the parent proteins at 0 degree C. The progressive loss of cryoprecipitability which occurs as a function of increasing reductant concentration parallels the successive cleavage of interheavy-light and interheavy-heavy chain disulfides. Circular dichroism shows that reduction and alkylation of hinge region disulfides induces small conformational changes in the IgG molecules that could alter cryoprecipitability. The N-terminal amino acid sequence of the Fc component derived by restricted proteolysis with trypsin of protein Muk was found to be completely homologous with N-terminal Fc sequences of noncryoglobulin IgG reference proteins, indicating identical hinge regions. Reduction and alkylation of two monoclonal IgM cryoglobulins also reduces cryoprecipitability. After reduction and alkylation of either the monoclonal IgM rheumatoid factor or the polyclonal IgG component of two mixed cryoglobulins recombination results in decreased cryoprecipitation of the intact cryoglobulin complex. In all cases inhibition of cryoprecipitation is greater when iodoacetic acid rather than iodoacetamide is employed as the S-alkylating group. These results do not support a direct role for the hinge region in the precipitation of cryoimmunoglobulins.

Alkylation

Entropic DNA.

The presence of mobile genetic elements without apparent biological function within genomes requires explanation. It is argued that consideration of DNA as an open thermodynamic system leads to the simple hypothesis that mobile genetic material increases the internal entropy thereby lowering the free energy of the DNA relative to DNA of the same size. This phenomenon enhances the survival of such sequences by simple structural stabilization. The consequences of this idea are discussed in terms of the proposed Bekenstein limit to the entropy to energy ratio of thermodynamic systems and biological entropy/information flow.

Animals

New bovine adrenal medullary peptide and its precursor.

We have isolated a previously unknown peptide and its precursor from bovine adrenal medullary chromaffin granules. The peptide sequence is Leu-Pro-Val-Asn-Ser-Pro-Met-Asn-Lys-Gly-Asn-Glu-Val-Met-Lys. The peptide is cleaved from the precursor at a Lys site. The sequence shows no homology to any known protein in the largest sequence data bank available.

Adrenal Medulla