Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.     

Effects of zinc ion on the conformation of antigenic determinants on insulin.

Comparison of c.d. spectra of Zn-insulin with Zn2+-free insulin demonstrated significant differences. It has been proposed that these differences are due to either changes in the structure of insulin monomers within aggregated insulins or the results of insulin aggregation. The effect of Zn2+ on the immunological activity of insulin indicated that the antigenic determinants of insulin were also altered. The apparent loss of immunological activity of monoiodotyrosylinsulin was demonstrated to be due to the loss of Zn2+ rather than the substitution of iodine. The immunological activity of Zn-insulin and Zn2+-free insulin was compared in both the radioimmune and immune haemolysis-inhibition assays by using an identical population of antibodies and concentrations of inhibitor. Relative to Zn-insulin, Zn2+-free insulin had a markedly attenuated immunological activity in the immune haemolysis-inhibition assay, whereas in the radioimmune assay slightly greater immunological activity was observed with the Zn2+-free insulin. These observations are submitted as evidence that the removal of Zn2+ perturbs the conformation of determinants that react with antibodies operative in the immune haemolysis-inhibition assay (immune haemolysis determinants) and has a minimal effect on the conformation of determinants that react with antibodies operative in the radioimmune assay (radioimmune determinants).     

Antigenic determinants of the HLA-B7 molecule; Bw6- and B7-specific determinants are spatially separate

Monoclonal antibodies that bind HLA-B7 were used to show that the B7-specific determinant is at a topologically different site from that of the broad polymorphic, Bw6 determinant. The relationship to other antigenic determinants defined by monoclonal antibodies was also assessed. These results were independently obtained in four ways: (1) by cellular blocking assays, in which there was no inhibition of 125I-B7 antibody binding in the presence of Bw6 antibody and no inhibition of 125I-Bw6 antibody binding in the presence of B7 antibody; (2) cellular binding assays under conditions of antibody saturation showed the binding of B7-specific and Bw6 antibodies were additive; (3) solid-phase radioimmune assays demonstrated enhancement between B7-specific and Bw6 antibodies; (4) analysis of antigen antibody complexes by size-exclusion high pressure liquid chromatography showed Bw6 and B7 antibodies could form tetramolecular complexes with papain-solubilized HLA-B7. Limitations were encountered in using cellular blocking assays to map antigenic determinants of HLA-B7. These assays can produce blocking in cases where two antibodies are not competing for an antigenic determinant. Mapping antigenic determinants with assays using purified HLA-B7 as the antigenic target, in addition to cell-based assays, provided a more accurate picture.     

Antigenic structure of the cyanogen bromide peptide F-CB3 from fibrinogen alpha-chain.

The antigenic properties of the cyanogen bromide peptide F-CB3 from bovine fibrinogen alpha-chain were studied in radioimmune assays with rabbit antibodies to fibrinogen or to peptide F-CB3. Both fibrinogen and peptide F-CB3 were indistinguishable in inhibition and dissociation tests. Modification of the single disulfide bridge in peptide F-CB3 either by reduction or by cleavage with cyanide was not accompanied by loss of serologic activity. Inhibition studies with three individual fragments obtained after cyanide cleavage (molecular weight range 7000 to 23000) indicated the presence of at least three distinct antigenic determinants in peptide F-CB3. After trypsin digestion of peptide F-CB3 still 75% of its maximal inhibiting capacity was retained. Lack of change in antigenic activity of peptide F-CB3 after release from the fibrinogen molecule by cyanogen bromide and upon further fragmentation is presumably due to the presence of several sequential antigenic determinants but the presence of conformational determinants could not be entirely excluded. Since no cross-reaction was observed between bovine and human peptides F-B3 one may expect considerable variation in their amino acid sequence.     

Chondroitin Sulfate Accelerates Trans‐Golgi‐to‐Surface Transport of Proteoglycan Amyloid Precursor Protein

The amyloid precursor protein (APP) is a membrane protein implicated in the pathogenesis of Alzheimer's disease. APP is a part‐time proteoglycan, as splice variants lacking exon 15 are modified by a chondroitin sulfate glycosaminoglycan (GAG) chain. Investigating the effect of the GAG chain on the trafficking of APP in non‐polarized cells, we found it to increase the steady‐state surface‐to‐intracellular distribution, to reduce the rate of endocytosis and to accelerate transport kinetics from the trans‐Golgi network (TGN) to the plasma membrane. Deletion of the cytosolic domain resulted in delayed surface arrival of GAG‐free APP, but did not affect the rapid export kinetics of the proteoglycan form. Protein‐free GAG chains showed the same TGN‐to‐cell surface transport kinetics as proteoglycan APP. Endosome ablation experiments were performed to distinguish between indirect endosomal and direct pathways to the cell surface. Surprisingly, TGN‐to‐cell surface transport of both GAG‐free and proteoglycan APP was found to be indirect via transferrin‐positive endosomes. Our results show that GAGs act as alternative sorting determinants in cellular APP transport that are dominant over cytoplasmic signals and involve distinct sorting mechanisms.      

Cell-free Pseudomonas vaccine. III. Immunochemical analysis of purified Pseudomonas aeruginosa protein antigens and protective properties of antisera against these antigens

The immunochemical analysis of isolated and purified antigens A and B obtained from P. aeruginosa, strains 868 (serogroup O3 according to Lányi or immunotype 3/7 according to Fisher) and 170015 (serogroup O7 or immunotype 2), was carried out. Rabbit antisera to proteins A and B, as well as to the initial aqueous extracts and partially purified aqueous extracts, were obtained. Cross activity between the protein antigens of different strains was established by the methods of immunodiffusion and two-dimensional immunoelectrophoresis. Isolated proteins A and B contained both common and specific antigenic determinants detected by the method of two-dimensional immunoelectrophoresis. The immunization of rabbits with proteins A and B was found to stimulate the synthesis of protective, probably species-specific, antibodies.     

Antigenic profiles of human,bovine and canine articular chondrocytes

Summary Human, bovine and canine articular chondrocytes have been shown to bear cartilage matrix, chondrocyte-specific and histocompatibility antigens. These cell-surface antigens of chondrocytes were demonstrated both simultaneously and separately either by complement-mediated cytotoxicity or by immunohistochemical reactions. The chondrocyte-specific antigens involve subsets of species-common and species-specific determinants, which are also present on the surfaces of rib and laryngeal chondrocytes. In addition to these antigens, human and calf articular chondrocytes also express unique cell-surface components that are capable of producing a blastogenic stimulation of autologous T-lymphocytes in vitro. These putative autoantigens segregated from lymphocytes in vivo could be released in trauma and in inflammatory joint diseases triggering the immune system of the host.     

Immunological studies of bovine nasal cartilage proteoglycan "link proteins".

Bovine nasal cartilage proteoglycan aggregates are dissociated and separated by density gradient centrifugation in 4 M guanidine into proteoglycan subunit (PGS) and glycoprotein link (GPL) fractions, the latter containing hyaluronic acid and "link proteins" responsible for aggregate formation. It was previously concluded on the basis of immunodiffusion studies that GPL has two antigenic components, one in common with PGS and one specific for the link proteins. However, in the present study it was found that antisera to PGS, which should lack link proteins, reacted with both "subunit" and "link" components of GPL, and antisera to fragments of PGS derived from the hyaluronic acid-binding portion of the molecule reacted preferentially with the link component. Reduction and alkylation of GPL led to modification of the reactions of both anti-GPL and anti-PGS sera with its link component. These immunodiffusion results indicate that the proteoglycan subunit and the link proteins are immunologically related and suggest that the link proteins may be identical with and derived from the hyaluronic acid binding portion of the proteoglycan subunit.     

Species-common antigen of connective tissues.

Collagen-free extracts were prepared from bovine, porcine and canine hyaline, elastic and fibrous cartilages, articular capsule, tendon, aorta, cortical bone and regenerating articular surfaces. The extracts were investigated with antisera to bovine nasal septal cartilage, dog articular cartilage and non-collagenous protein fraction of bovine cortical bone. Immunodiffusion, immunoelectrophoresis, and immunohistochemical methods were used. In the different supporting tissues of the three animal species a common antigen, probably of proteoglycan origin, was demonstrated. The finer differences in antigenicity between the different tissues are probably due to the variations in proteoglycan composition of the given supporting tissues. Owing to the wide-spread occurrence of the antigen, the authors suggest the term "species-common connective tissue antigen" instead of the "species-common cartilage antigen" used so far.     

Detection of structural differences between nuclear and mitochondrial glutamate dehydrogenases by the use of immunoadsorbents.

Structural differences between crystalline mitochondrial and nuclear glutamate dehydrogenases from ox liver have been detected by immunological techniques. Antisera prepared against each enzyme precipitate both glutamate dehydrogenases; upon immunodiffusion, the antiserum against the nuclear enzyme gives a line of incomplete identity with the two antigens, whereas the antiserum against the mitochondrial enzyme gives a line of complete identity. Fractionation of the antibodies contained in each antiserum by means of an immunoadsorbent, to which the nuclear or the mitochondrial enzyme has been covalently linked, shows that nuclear glutamate dehydrogenase (GDH) contains specific antigenic determinants as well as determinants common to the mitochondrial enzyme, whereas the latter appears to have no antigenic portions which are not present in the nuclear antigen, in accord with the results of immunodiffusion. The antibodies against determinants common to both enzymes precipitate and inhibit them, whereas the specific anti-nuclear GDH antibodies precipitate but do not inhibit the nuclear antigen.     

A reappraisal of antigenic determinants for nitrogenase from Azotobacter and nitrate reductase from Escherichia coli

Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii. Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase.     

Small nuclear ribonucleoprotein antigens are absent from 10S translation inhibitory ribonucleoprotein but present in cytoplasmic messenger ribonucleoprotein and polysomes

A cytoplasmic 10S ribonucleoprotein particle (iRNP), which is isolated from chick embryonic muscle, is a potent inhibitor of mRNA translation in vitro and contains a 4S translation inhibitory RNA species (iRNA). The iRNP particle shows similarity in size to the small nuclear ribonucleoprotein (snRNP) particles. Certain autoimmune disease patients contain antibodies directed against snRNP antigenic determinants. The possibility that iRNP may be related to the small nuclear particles was tested by immunoreactivity with monospecific autoimmune antibodies to six antigenic determinants (Sm, RNP, PM-1, SS-A (Ro), SS-B (La), and Scl-70). By Ouchterlony immunodiffusion assays, the cytoplasmic 10S iRNP did not show any immunoreactivity. Also, a more sensitive hemagglutination inhibition assay for detecting Sm and RNP antigens failed to show reactivity with the 10S iRNP. Thus, the 10S iRNP particles are distinct from the similarly sized snRNP. However, free and polysomal messenger ribonucleoprotein (mRNP) particles and polysomes also isolated from chick embryonic muscle and analyzed by Ouchterlony immunodiffusion and hemagglutination inhibition for the presence of the antigenic determinants showed reactivity to Sm and RNP autoantibodies, but were not antigenic for the other four antibodies. Some of the Sm antigenic peptides of mRNP particles and polysomes were identical to those purified from calf thymus nuclear extract, as judged by Western blot analysis. The association of Sm with free and polysomal mRNP and polysomes suggests that Sm may be involved in some cytoplasmic aspects of mRNA metabolism, in addition to a nuclear function in mRNA processing.     

鹿茸硫酸软骨素蛋白聚糖的分离纯化研究

报道了用DEAE-纤维素(DE-23)离子交换柱层析从鹿茸二杠中分离、纯化及鉴定硫酸软骨素的方法.首先用适量蒸馏水浸泡鹿茸二杠并将其捣碎,离心取沉淀用盐酸胍浸提,浸提液对尿素液透析后经DEAE-纤维素(DE-23)离子交换柱层析,吸附大量的硫酸软骨素;再用含盐尿素溶液梯度洗脱、分离后,经软骨素酶消化及琼脂糖凝胶电泳, 与硫酸软骨素标准品比较,证实得到的物质为纯的硫酸软骨素蛋白聚糖,其得率约为48.77%.该方法使硫酸软骨素分离纯化一步完成,大大简化了纯化步骤.     

Expression of group b light chain allotypes in cottontail rabbits.

Group b allotypic determinants (b4, b5, b6, and b9) were detected on cottontail IgG by several assays including immunodiffusion, radioimmune binding, and inhibition of radiobinding. The results indicate that cottontail IgG possess some but not all of the subspecificities present on domestic rabbit IgG. Several cottontail rabbits exhibited three of the four possible allotypic markers. In these instances, however, only two populations of IgG molecules (i.e., phenogroups) could be detected, each bearing one, two, or three of the allotypes. Five separate and distinctive phenogroups were identified. The results suggest that the phenogroups represent products of multiple allelic genes for the constant region of cottontail rabbit kappa light chain.     

Studies on the antigenic structure of Japanese encephalitis virus using monoclonal antibodies

The antigenic relationships among 11 strains of Japanese encephalitis (JE) virus were analyzed by using monoclonal antibodies (NARMA) against the Nakayama-RFVL strain in hemagglutination-inhibition (HI) and neutralization (Nt) tests. Of the 14 JE virus-specific HI antibodies, all except NARMA 5 showed Nt reactivity with the homologous strain. The HI and Nt titers of these antibodies were not parallel. The 14 antibodies included the following characteristic antibodies: NARMA 3 is a species-specific antibody with HI and Nt reactivities against JE virus, NARMA 13 is a species-specific HI antibody, NARMA 6 is a Nakayama strain-specific antibody with HI and Nt reactivities, and NARMA 5 is a Nakayama strain-specific HI antibody. The 11 strains of JE virus were divided into four major antigenic groups. However, slight antigenic differences were found among some strains of the same group. Furthermore, competitive binding assays were performed to determine the distribution of antigenic determinants by enzyme-linked immunosorbent assay. The results suggest the existence of at least five HI sites on the JE virus virion, and indicate that the JE species-specific HI site and the flavivirus genus-specific HI site are topologically distinct.     

Immunochemical distinction between two different chains of type IV collagen.

Antisera against mouse and human basement-membrane type IV collagen showed in radioimmunoassays distinct binding with large pepsin fragments obtained from the C-terminal portions of alpha 1 (IV)- and alpha 2 (IV)-chains. These reactions were specific for each constituent polypeptide chain. The data were confirmed by immunoadsorption, allowing the separation of antibodies with restricted chain specificity. Inhibition assays with CNBr peptides demonstrated the different localizations of antigenic determinants, which were either species-specific or shared by the human and mouse antigens.     

Sulfated proteoglycans synthesized by Neuro 2a neuroblastoma cells: Comparison between cells with and without ganglioside-induced neurites

Mouse neuroblastoma Neuro 2a cells are known to extend neurite-like processes in response to gangliosides added to the culture medium. We compared the structural features of proteoglycans (PG) synthesized by conventional Neuro 2a cells with those of neurite-bearing cells. Two different proteoglycans labeled with [³⁵S]sulfate, namely, chondroitin sulfate proteoglycan (CS-PG) and heparan sulfate proteoglycan (HS-PG), were found both in the cell layer and in the culture medium of the conventional cells. CS-PG isolated from the cell layer had a K_av value of 0.38 on Sepharose CL-6B, and had CS side chains with Mr of 27,000. HS-PG in the cell layer was slightly larger (K_av of 0.33) in terms of hydrodynamic size than CS-PG, and the apparent Mr of the heparan sulfate side chains was 10,000. The structural parameters of CS-PG and HS-PG isolated from the medium were almost identical to those of the PGs in the cell layer. In addition to these PGs, single-chain HS, with an average Mr of 2,500, was observed only in the cell layer and this component was the major sulfated component in the cell layers of both control and ganglioside treated cells. The neurite-bearing cells also synthesized both CS-PG and HS-PG which were very similar in hydrodynamic size to those synthesized by the conventional cells, but the size of HS side chains was greater. Radioactivity, as³⁵S, of each sulfated component from the gangliosideteated culture seemed to be slightly less than that of the corresponding component from the control culture. These findings indicate that the marked morphological change in Neuro 2a cells, induced by gangliosides is not accompanied by major changes in the synthesis of PGs.     

Antigenic determinants of two components of proteoglycan complex from bovine cartilage

The immunological properties of a glycoprotein fraction and of proteoglycan subunits obtained from bovine nasal cartilage by nondisruptive methods of isolation have been studied. Using the techniques of hemagglutination and hemagglutination inhibition, we found that the glycoprotein contains most of the species-specific determinants, whereas the proteoglycan subunits contain most of the cross-reacting ones.     

Divergent regulation of proteoglycan and glycosaminoglycan free chain expression in human keratinocytes and melanocytes

Summary Keratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [³⁵S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.     

Some properties of ferredoxin-nitrite reductase from green shoots of bean and an immunological comparison with nitrite reductase from roots and etiolated shoots

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from green shoots of bean (Phaseolus angularis). The isolated enzyme (GR-NiR), having a molecular mass of 68 000, showed 1.4 times higher ferredoxin-dependent activity than methyl viologen-linked activity. The enzyme was homogeneous by polyacrylamide gel electrophoresis (PAGE). In the oxidized form, the enzyme had absorption maxima at 275, 393 (Soret band), 535 and 571 (α band) nm, indicating that siroheme is involved in the catalysis of nitrite reduction. The absorbance ratios, A₃₉₃ : A₂₇₅ and A₅₇₁ : A₃₉₃ were 0.26 and 0.32, respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion and immunoelectrophoresis suggested that it was a specific antiserum against GR-NiR. Using the antiserum, immunodiffusion and immunoprecipitation procedures were employed to compare the immunological similarity of NiR from green shoots, etiolated shoots and roots of bean. These tests revealed that the three forms of assimilatory NiR have antigenic determinants in common.