Chemical processing as a tool to generate ovalbumin variants with changed stability.

Processing of ovalbumin may result in proteins that differ more than 23 degrees C in denaturation temperature while the structural fold is not significantly affected. This is achieved by 1) conversion of positive residues into negative ones (succinylation); 2) elimination of negative charges (methylation); 3) reducing the proteins hydrophobic exposure (glycosylation); 4) increasing the hydrophobic exposure (lipophilization); or by 5) processing under alkaline conditions and elevated temperature (S-ovalbumin). The effect on the structural fold was investigated using a variety of biochemical and spectroscopic tools. The consequences of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry and by monitoring the tryptophan fluorescence or ellipticity at 222 nm of protein samples dissolved in different concentrations of guanidine-HCl. The impact of the modification on the denaturation temperature scales for all types of modifications with a free energy change of about 1 kJ per mol ovalbumin per Kelvin (or 0.0026 kJ per mol residue per K). The nature of the covalently coupled moiety determines the impact of the modification on the protein thermodynamics. It is suggested that especially for lipophilized protein the water-binding properties are substantially lowered. Processing of globular proteins in a controlled manner offers great opportunities to control a desired functionality, for example, as texturizer in food or medical applications.

Immunohistology of antigen-presenting cells in vivo: a novel method for serial observation of fluorescently labeled cells.

PURPOSE: Dendritic cells and macrophages are phagocytic antigen-presenting cells that bridge the innate and acquired immune systems. The coexistence of subtypes of dendritic cells and macrophages with overlapping properties complicates resolution of their precise roles in an immune response within a given tissue. This report documents a method to identify and observe these cells over time in a living animal and thereby to visualize them during a dynamic immune response. METHODS: To label potential antigen-presenting cells, fluorescently tagged ovalbumin was injected into the anterior chambers of mouse eyes. Fluorescently tagged antibodies to cell surface proteins were injected to label specific cell types. Intravital fluorescence microscopy with digital image recording was used to visualize the labeled cells in the irises at various times after the injection. RESULTS: The pattern and density (116-148 cells/mm(2)) of cells labeled in vivo by fluorescent ovalbumin or F4/80 antibodies were similar to that identified by conventional wholemount immunostaining for macrophages and dendritic cells. Fluorescent antibodies specific for CD11b, CD11c, CD80, CD86, or major histocompatibility complex (MHC) class II protein each labeled selective populations of cells in vivo. In contrast to conventional histology, in vivo immunohistology permitted serial observations. The phenotype of cells labeled by fluorescent ovalbumin was not the same at 6 (95% CD11c(+)) and 24 hours (24% CD11c(+)) after injection. CONCLUSIONS: This method of in vivo immunohistology provides a tool for studying cell kinetics and dynamic interactions that cannot be assessed by conventional immunohistology. Furthermore, it avoids potential artifacts from tissue fixation and may work with antibodies that label cells poorly in vitro.

Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid.

The non-beta-oxidisable tetradecylthioacetic acid (TTA) is incorporated into cellular membranes when C3H/10T1/2 cells are cultured in TTA-containing medium. We here demonstrate that this alteration in cellular membranes affect the nuclear translocation of proteins involved in signal transduction. Analysis of cellular fatty acid composition shows that TTA and TTA:1n-8 constitute approximately 40 mol% of total fatty acids in cellular/nuclear membranes. Activation of c-fos expression is significantly inhibited in TTA-treated cells but the enzymatic activation of mitogen activated protein kinase (ERK) is not affected. Immunofluorescence and confocal microscopy studies demonstrate that in mitogene-stimulated TTA-treated cells, the translocation of phosphorylated ERK1/2, protein kinase C alpha (PKC alpha), and PKC beta(1) from the cytoplasm into the nucleus is considerably decreased and delayed. Concomitant with a decreased nuclear import, ERK1/2 dephosphorylation is decreased in TTA-treated cells. There is no TTA-induced inhibition of nuclear import of proteins with a classical nuclear localization signal (NLS), as seen by in vitro nuclear import experiments of BSA fused to the NLS from SV40 large T, or in vivo studies of hnRNP A1 nuclear import. The expression levels of Importin alpha, Importin beta, Importin 7, and NTF2 are not altered in the TTA-treated cells. Taken together, our data indicate that TTA treatment causes changes in cellular fatty acid composition that negatively affect NLS-independent mechanisms of protein translocation through the nuclear pore complex.

Interferon-gamma can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase.

Most antigenic peptides presented on major histocompatibility complex class I molecules are generated during protein breakdown by proteasomes, whose specificity is altered by interferon-gamma (IFN-gamma). When extended versions of the ovalbumin-derived epitope SIINFEKL are expressed in vivo, the correct C terminus is generated by proteasomal cleavage, but distinct cytosolic protease(s) generate its N terminus. To identify the other protease(s) involved in antigen processing, we incubated soluble extracts of HeLa cells with the 11-mer QLESIINFEKL, which in vivo is processed to the antigenic 8-mer (SIINFEKL) by a proteasome-independent pathway. This 11-mer was converted to the 9-mer by sequential removal of the N-terminal residues, but surprisingly the extract showed little or no endopeptidase or carboxypeptidase activity against this precursor. After treatment of cells with IFN-gamma, this N-terminal trimming was severalfold faster and proceeded to the antigenic 8-mer. The IFN-treated cells also showed greater aminopeptidase activity against many model fluorogenic substrates. Upon extract fractionation, three bestatin-sensitive aminopeptidase peaks were detected. One was induced by IFN-gamma and was identified immunologically as leucine aminopeptidase (LAP). Purified LAP, like the extracts of IFN-gamma-treated cells, processed the 11-mer peptide to SIINFEKL. Thus, IFN-gamma not only promotes proteasomal cleavages that determine the C termini of antigenic peptides, but also can stimulate formation of their N termini by inducing LAP. This enzyme appears to catalyze the trimming of the N terminus of this and presumably other proteasome-derived precursors. Thus, susceptibility to LAP may be an important influence on the generation on immunodominant epitopes.

Differential production of chemokines and their role in neutrophil infiltration in rat allergic inflammation.

BACKGROUND: Recently we demonstrated that activated rat macrophages produced neutrophil chemotactic factors (chemokines) including cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-2alpha, CINC-2beta, CINC-3/rat macrophage inflammatory protein (MIP)-2 and rat MIP-1alpha (rMIP-1alpha). METHODS: In the present study, by using an enzyme-linked immunosorbent assay specific for each chemokine, we determined the levels of the chemokines in the pouch fluid (inflammatory site) of the fluorescein isothiocyanate-labeled ovalbumin (FITC-OVA)-induced allergic inflammation in rats. Effects of anti-chemokine antibodies on neutrophil chemotaxis were determined in vivo and in vitro. RESULTS: CINC-1 was the major chemokine which rapidly increased after challenge with FITC-OVA, whereas CINC-3 was a minor one, and CINC-2, CINC-3 and rMIP-1alpha increased slowly with a lag time of about 2 h. Anti-CINC-1/CINC-2 antibodies, which inhibited all the CINCs, suppressed both neutrophil infiltration in vivo and neutrophil chemotactic activity of the 8-hour pouch fluid in vitro, whereas anti-rMIP-1alpha antibody slightly suppressed the chemotaxis in vivo and in vitro. CONCLUSION: Our results suggest that CINCs, especially CINC-1 and CINC-2, play an important role in the infiltration of neutrophils into the inflammatory site of FITC-OVA-induced allergic inflammation in rats.

Effect of cationization on anti-hapten antibody responses in sheep and mice.

The effect on anti-hapten immune responses of altering the isoelectric point of hapten-carrier conjugates by cationization was examined in sheep and mice. No adjuvants were employed in these studies. In sheep the cationization of ovalbumin-dinitrophenyl (OVA-DNP) resulted in an enhanced primary anti-dinitrophenyl (DNP) response. The secondary anti-DNP responses were not consistently greater for animals primed with cationized (CAT) OVA-DNP than in animals primed with OVA-DNP. Similar results were obtained with mice immunized with bovine serum albumin (BSA)-DNP or CAT BSA-DNP. Mice and sheep primed with OVA-DNP failed to mount an anamnestic response following secondary immunization with CAT OVA-DNP. Failure of a cationized hapten-carrier to stimulate a secondary anti-DNP response following priming with uncationized hapten-carrier was also observed in mice using BSA-DNP as the model antigen.

Histamine and prostacyclin. Primary and secondary release in allergic conjunctivitis.

The relationship between the release of histamine, a major mast cell mediator of conjunctival type I reactions, and the production of a prostanoid, prostacyclin (prostaglandin I2, PGI2), was examined in a guinea pig model of allergic conjunctivitis. Guinea pigs were sensitized topically and challenged by repeated conjunctival instillation of fluoresceinyl ovalbumin. Histamine and 6-keto-PGF1 alpha, the stable product of the spontaneous degradation of PGI2, were measured in tears by radioimmunoassays. Clinical type I reactions and tear histamine appeared by 8 days and increased up to 22 days during the initial sensitization, with notable variations between animals. The kinetics of histamine and 6-keto-PGF1 alpha release in tears were examined over a 24-hr period after the antigen challenge. Histamine release was maximal during the first 10 min and returned to baseline values by 1 hr in all instances. The 6-keto-PGF1 alpha release also peaked during the first 10 min but continued for an extended period. The ratio of tear 6-keto-PGF1 alpha to histamine increased more than 16-fold over the 2 hr after antigen challenge. Late-phase reactions with second peaks of histamine or 6-keto-PGF1 alpha in the tears were observed in two different guinea pigs 4-8 hr after antigen challenge. Histamine applied to the eyes of naive guinea pigs also induced the release of 6-keto-PGF1 alpha in tears. Histamine appeared to act as a primary mediator, stimulating the secondary production and release of PGI2 by constitutive (eg, vascular) and possibly infiltrating inflammatory cells during an allergic conjunctival reaction.

Effect of conformational propensity of peptide antigens in their interaction with MHC class II molecules. Failure to document the importance of regular secondary structures.

In an attempt to define some of the conformational requirements for binding of the antigenic peptide OVA 323-336 to purified IAd molecules, three distinct experimental approaches were applied. First, the effect of introducing proline or glycine residues within the region of OVA 323-336 crucial for its IAd binding capacity was analyzed. In most instances these substitutions had little or no effect, suggesting that neither alpha-helical nor beta-sheet regular structures may be strictly required for productive interaction with MHC molecules. Some of the same substitutions were also found to have no effect on the capacity of the peptide to stimulate OVA 323-336 specific T cell hybridomas, suggesting that regular structures such as alpha-helices or beta-sheets may not be strictly required for T cell stimulation, either. Second, we introduced, within the OVA 323-336 molecule, structural modifications predicted to alter its dipole characteristics and stabilize helical structures. No improvement of the IAd binding capacity was detected following these structural alterations. Surprisingly, some but not others of these analogs displayed increased antigenicity for OVA 323-336 specific T cell hybridomas. Third, a panel of analogs of OVA 323-336 were synthesized in which the crucial IAd binding core region was linked to non-native sequences of differing conformational propensities. When 22 such analogs were tested for IAd binding, it was found that these non-native sequences could drastically influence the binding capacity, but no correlation was found between their effect and their alpha-helical, beta-sheet, or beta-turn conformational propensity as calculated by the Chou and Fasman algorithm. In summary, all the data presented herein suggest that, at least in the case of OVA 323-336 and IAd, the propensity of the antigen molecule to form secondary structures such as alpha-helices, beta-sheets, or beta-turns does not correlate with its capacity to bind MHC molecules.

Massive follicular lymphoid hyperplasia in experimental allergic conjunctivitis. Local antibody production.

Acute and recurrent allergic conjunctival reactions were induced in guinea pigs by repeated conjunctival applications of fluoresceinyl ovalbumin (FL-OA) for up to 30 months. Early type I conjunctival reactions developed 11 to 25 days after the initial conjunctival exposure to FL-OA. Continuous topical challenges during a six- to 30-month period caused a variety of reactions, including papillary changes and massive hyperplasia of the conjunctival-associated lymphoid tissues. Hyperplasia of lymphoid tissues was induced during a shorter period (two to five months) with a mixture of FL-OA and phorbol ester. Culture fluid from hyperplastic conjunctival lymphoid tissue showed a ratio of IgG1/IgG2 antibody production of up to 15. A low level of recurrence of type I reactivity, after an initial desensitization phenomenon due to a loss of reactive mast cells, correlated with prominent follicular hyperplasia of the conjunctival-associated lymphoid tissue.

Experimental allergic conjunctivitis: production of different isotypes of antibody by conjunctival-associated lymphoid tissue in culture.

Long-term (greater than 2 years) topical, conjunctival application of fluoresceinyl ovalbumin (FL-OA) induced allergic conjunctivitis-like lesions and hyperplasia of conjunctival-associated lymphoid tissue (CALT) in guinea pigs. Single-cell suspensions of CALT and spleen were prepared by collagenase digestion and cultured with or without FL-OA or lipopolysaccharide; the culture supernatants were assayed for IgG, IgA, IgM, and IgE antibody. Absolute values (ng Ab protein/ml) of anti-FL-OA IgG subclasses (IgG1 and IgG2) were measured using purified preparations of IgG1 and IgG2 anti-FL-OA antibody standards in an enzyme-linked immunosorbent assay. Immunohistochemical studies were performed using frozen sectioned CALT tissues as well as cultured single cells. IgG1, IgG2, IgA, IgM, but not IgE, anti-FL-OA antibodies were detected in the culture supernatants of both CALT and spleen. IgG- and IgA-secreting plasma cells were demonstrated in immunoperoxidase-stained CALT and single-cell cultures. The ratio of IgG1 to IgG2 isotypes produced by CALT in vitro was significantly higher than that produced by spleen and also that found in serum. These findings indicated that a site-specific regulation of antibody isotypes may exist within the hyperplastic CALT induced by the long-term topical exposure to FL-OA.

The observation of immune complex formation and deposition in the eyes of living rabbits.

Fluorescence microscopy of the conjunctival and episcleral circulations of the rabbit's eye has revealed for the first time the formation and deposition of intravascular and perivascular immune complexes in undisturbed capillaries in vivo. During intravascular immune complex formation, immune aggregates appear abruptly, at a critical antigen/antibody ratio, and embolize to the arterial side of the capillary bed. This is accompanied by a fall in the circulating platelet count. The emboli disperse over the ensuing 120 min. In frozen sections immune aggregates are coarsely granular and intraluminal at the moment they embolize. However, after they disperse, subendothelial granules of antigen can be found. IgG and C3 are associated with both intraluminal and subendothelial antigen. Perivascular immune complex formation is accompanied by an intense inflammatory response, which is absent after the deposition of an intravascular immune precipitate. The mechanism by which immune complexes may accomplish their transition across the capillary endothelium is discussed.

Estrogen regulation of the biological activity of the avian oviduct progesterone receptor and its ability to induce avidin.

During primary estrogen stimulation of chick oviduct development, estrogen withdrawal, or secondary estrogen treatment, changes in the oviduct progesterone receptor (PR) occur. The presence of estrogen appears to regulate not only PR concentration but also its biochemical activity, i.e. its capacity to bind to nuclear acceptor sites and alter RNA synthesis. This study reports that estrogen regulates the nuclear binding capacity of the PR even more rapidly than previously reported in fully developed oviducts of chicks that have been injected daily for 4 weeks with diethylstilbestrol (DES). Further, the nuclear binding capacity of the PR correlates with the ability of progesterone (P) to induce avidin protein concentrations in the oviducts in vivo. The PR concentration in the oviducts increases 2-fold within 8 h of the last injection and the decreases to a minimal value by 24 h. Injection of [3H]P into the chicks shows that the in vivo nuclear localization of the steroid increases almost 4-fold at 8 h, followed by a similar decrease to minimal values by 24 h. Cell-free nuclear binding assays, using PR isolated at various times after the last DES injection and oviduct nucleoprotein complexes, indicate that the capacity of the receptors to bind to nuclear acceptor sites is regulated by the estrogen. The enhanced nuclear binding capacity of the isolated PR increases to maximal values by 12-14 h after the last estrogen treatment and then begins to decrease to minimal values by 24 h. Similarly, the ability of P to induce in vivo avidin protein concentrations and to alter general RNA synthesis in the oviducts is reduced by 70% (of the estrogen non-withdrawn chick levels) by 24 h after the last estrogen injection. These changes over the 24-h period after the last DES treatment are not due to changes in the serum DES concentrations. The following 10-day period of estrogen withdrawal reveals a cyclic decaying pattern in the capacity of the PR for nuclear binding. The P induction of avidin and alteration of RNA polymerase II activity, using nuclear run-off experiments, also show a similar cyclic decaying pattern. By 6 days of estrogen withdrawal, the PR is incapable of any nuclear binding, and P cannot induce avidin protein concentrations in the oviducts. Serum DES concentrations over this 10-day period display only a gradual decay.(ABSTRACT TRUNCATED AT 400 WORDS)

Effective avidin coating of erythrocytes by disulfide bond formation. Production of a stable rosetting agent for multiple applications.

A method is described for covalently coupling avidin to the membrane of glutaraldehyde-treated erythrocytes. The method is based on the use of the heterobifunctional coupling reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and appears to be more effective than other protein coupling techniques. The resulting avidin-coated red cells are very stable and can be stored for more than 1 year without aggregation or loss of biotin-binding activity. They can be used in various rosetting assays and were tested for T cell depletion or negative cell selection with five biotinylated monoclonal antibodies.

Synthesis of an amplifiable reporter RNA for bioassays.

The replacement of reporter groups, such as fluorescent molecules or enzymes, by an amplifiable reporter should lead to bioassays of greatly increased sensitivity, since a very large number of copies of the reporter can be accumulated in a short time. Midivariant RNA is an appropriate reporter, since it is autocatalytically replicated by Q beta RNA polymerase in vitro. This RNA can be amplified exponentially, with a population doubling time of 36 seconds, resulting in the synthesis of 10(6) copies of each molecule in 12 minutes. We have used chemical methods to attach biotin to the 5' terminus of midivariant RNA via a disulfide linker. This biotinylated RNA combines with avidin to give a product that is readily purified by gel electrophoresis. The RNA-biotin-avidin adduct, and the RNA released from it by reductive cleavage of the linker arm, replicate normally. The RNA-biotin-avidin adduct should be a suitable reporter for a variety of replication-assisted bioassays involving biotinylated antibodies or biotinylated nucleic acid probes.

Avidin as a probe of the conformational changes induced in pyruvate carboxylase by acetyl-CoA and pyruvate.

Sheep liver pyruvate carboxylase was mixed with avidin at a molar ratio of 1:1 in the presence of various combinations of the components of the assay systems required for either the acetyl-CoA-dependent or the acetyl-CoA-independent activity and negatively stained samples were examined by electron microscopy. Significant numbers of chain-like polymers of enzyme-avidin complexes were evident only when acetyl-CoA or high levels of pyruvate were present in the media. Similar results were also obtained for chicken liver pyruvate carboxylase despite this enzyme's almost complete lack of acetyl-CoA-independent activity. Thus, although acetyl-CoA and high concentrations of pyruvate may induce pyruvate carboxylase to adopt a 'tight' tetrahedron-like conformation which can interact with avidin to form chains, this structural change alone does not result in an enzymic form that is maximally active. This suggests that the allosteric activation of pyruvate carboxylase by acetyl-CoA is attributable, at least in part to more subtle conformational changes; especially in the case of the chicken enzyme.

Hybridoma production by simplified avidin-mediated electrofusion.

A simplified method for producing cell hybrids by avidin-mediated electrofusion has been developed. First, biotin was attached both to immunogen and to the surface of murine myeloma cells using N-hydroxysuccinimidobiotin. Biotinylated immunogen was incubated with splenocytes derived from immunized mice and allowed to bind to surface immunoglobulins of B cells. Using streptavidin, biotinylated myeloma cells then were bridged to those B cells bearing biotinylated immunogen. Selective fusion of bridged cells was accomplished by their limited exposure to high-voltage potentials. A high frequency of hybridomas was obtained all of which secreted high titers of antibodies to a selected model immunogen, keyhole limpet hemocyanin.

Avidin induction by theophylline in vivo and in vitro.

Avidin induction in chick tissues in vivo and in vitro was studied by a phosphodiesterase inhibitor, theophylline, and compared to progesterone-dependent induction. Theophylline (100 mg/kg, ip) caused a significant increase in avidin content only in the oviduct of diethylstilbestrol-treated chicks, but not in the lung, muscle, intestine, plasma, or in the bursa of Fabricius. Diethylstilbestrol priming was necessary for oviductal avidin induction in vivo by theophylline. In the oviduct culture, theophylline at a concentration between 100 and 500 micrograms/ml caused a dose-dependent increase in avidin production. Effects of theophylline and progesterone on avidin synthesis in oviduct culture were synergistic. Avidin production was dependent on protein and RNA synthesis, since induction was inhibited by cycloheximide and actinomycin D. Avidin induction by theophylline resembled progesterone-dependent induction, beginning 9 h after the injection in vivo and 12 h after administration of these drugs in vitro. Avidin induced by theophylline showed heat-induced biotin exchange identical to that of progesterone-induced avidin, indicating close similarity of these proteins. The results suggest that theophylline can mimic the action of progesterone on avidin production, and that cyclic nucleotides may have a role in the regulation of avidin synthesis.

Use of the biotin-avidin system for detecting a broad range of serologically related plant viruses by ELISA.

The biotin-avidin detection system was used in direct and indirect ELISA for detecting a broad range of serologically related tobamoviruses. When compared to standard ELISA procedures that use antibodies labelled with alkaline phosphatase, the biotin-avidin system increased the assay sensitivity and allowed a wider range of related viral serotypes to be detected.