Composition and properties of the extracellular polysaccharide produced by arthrobacter stabilis NRRL B-3225

The extracellular polysaccharide produced by Arthrobacter stabilis NRRL B-3225 contains d-glucose, d-galactose, pyruvic acid, O-succinyl, and O-acetyl in the approximate molar ratio of 6:3:1:1:1.5. Succinyl is linked as its half-ester, making it a readily removable, acidic function that imparts versatility to the polysaccharide both for fundamental research and for commercial use. The viscosity of aqueous, salt-free solutions of both native and deacylated polymer is relatively low, but atypical of anionic polysaccharides, increases rapidly in the presence of salts, acids, or alkali.     

HLA-A2-restricted T-cell epitopes specific for prostatic acid phosphatase

Prostatic acid phosphatase (PAP) has been investigated as the target of several antigen-specific anti-prostate tumor vaccines. The goal of antigen-specific active immunotherapies targeting PAP would ideally be to elicit PAP-specific CD8+ effector T cells. The identification of PAP-specific CD8+ T-cell epitopes should provide a means of monitoring the immunological efficacy of vaccines targeting PAP, and these epitopes might themselves be developed as vaccine antigens. In the current report, we hypothesized that PAP-specific epitopes might be identified by direct identification of pre-existing CD8+ T cells specific for HLA-A2-restricted peptides derived from PAP in the blood of HLA-A2-expressing individuals. 11 nonamer peptides derived from the amino acid sequence of PAP were used as stimulator antigens in functional ELISPOT assays with peripheral blood mononuclear cells from 20 HLA-A2+ patients with prostate cancer or ten healthy blood donors. Peptide-specific T cells were frequently identified in both groups for three of the peptides, p18–26, p112–120, and p135–143. CD8+ T-cell clones specific for three peptides, p18–26, p112–120, and p299–307, confirmed that these are HLA-A2-restricted T-cell epitopes. Moreover, HLA-A2 transgenic mice immunized with a DNA vaccine encoding PAP developed epitope-specific responses for one or more of these three peptide epitopes. We propose that this method to first identify epitopes for which there are pre-existing epitope-specific T cells could be used to prioritize MHC class I-specific epitopes for other antigens. In addition, we propose that the epitopes identified here could be used to monitor immune responses in HLA-A2+ patients receiving vaccines targeting PAP to identify potentially therapeutic immune responses.     

Hydrophobic surfaces of tubulin probed by time-resolved and steady-state fluorescence of nile red

Binding of Nile Red to tubulin enhances and blue-shifts fluorescence emission to about 623 nm with a "shoulder" around 665 nm. Binding is reversible and saturable with an apparent Kd of approximately 0.6 microM. Nile Red does not alter tubulin polymerization, and polymerization in 2-(N-morpholino)ethanesulfonic acid (Mes) buffer does not alter the spectrum of the Nile Red-tubulin complex. In contrast, polymerization in glutamate buffer results in a red shift, reduction of intensity, and a decrease in lifetime, suggesting an increase in "polarity" of the binding environment. Lifetimes of 4.5 and 0.6 ns fluorescence in Mes buffer are associated with the 623-nm peak and the 665-nm shoulder, respectively. Indirect excitation spectra for these components are distinct and the 4.5-ns component exhibits tryptophan to Nile Red energy transfer. Acrylamide quenching yields linear Stern-Volmer plots with unchanged lifetimes, indicating static quenching. Apparent quenching constants are wavelength-dependent; global analysis reveals a quenchable component corresponding to the 4.5 ns component and an "unquenchable" component superposing the 0.6-ns spectrum. Analysis of anisotropy decay required an "associative" model which yielded rotational correlation times of greater than 50 ns for the 4.5-ns lifetime and 0.3 ns for the 0.6-ns lifetime. Dilution of tubulin in Mes results in an apparent red shift of emission without lifetime changes, due only to loss of the 623-nm component. These data are reconciled in terms of a model with two binding sites on the tubulin dimer. The more "nonpolar" site is located in a region of subunit-subunit contact which accounts for the fluorescence changes upon dilution; this permits estimation of a subunit dissociation constant of 1 microM.     

Preparation of monoclonal antibody-ferritin conjugates of high specific activity

125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 1:1.4, containing over 90% ferritin-IgG "monomers"; 70-90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.     

CO2 and fluorinated solvent-based technologies for protein microparticle precipitation from aqueous solutions

Precipitation with a compressed or supercritical fluid antisolvent (PCA) has been used to produce microparticles of biologically active proteins, pharmaceuticals, and polymers. However, the application of PCA to a wider range of proteins is limited by the low mutual solubility of water (necessary to dissolve most proteins) and CO(2) (traditionally used as the compressed antisolvent). This investigation extends PCA to proteins in aqueous solutions by utilizing ethanol as a cosolvent to enhance the antisolvent properties of CO(2) toward aqueous systems. alpha-Chymotrypsin, a model protein, was precipitated from both compressed CO(2) and a liquid fluorinated antisolvent, a hydrofluoroether (HFE). The equilibrium phase behavior of the antisolvent/ethanol/water systems was examined to identify a one-phase region suitable for protein precipitation. Spherical protein microparticles with a primary particle size of approximately 0.2-0.6 microm were recovered using both the compressed CO(2) and fluorinated antisolvents. Although the proteins retained significant activity using both antisolvent systems, the HFE-precipitated chymotrypsin retained higher activity than the CO(2)-precipitated protein.