Altered immunochemical reactivity of Saccharomyces cerevisiae a-cells after alpha-factor-induced morphogenesis.

Antibodies were raised against Saccharomyces cerevisiae a-cells that had been exposed to the sex pheromone, alpha-factor. After adsorption of the antiserum with diploid cells, antibodies remained that reacted specifically with the mannan from haploid cells. The characteristic determinant was observed in mannan from pheromone-treated a-cells, in mannan from untreated alpha-cells, and at a much lower concentration, in mannan from control a-cells. The antigens from these three mannans appeared to be identical. The determinant was destroyed by mild-acid hydrolysis or periodate oxidation, but not by proteolysis or digestion with exo-alpha-mannanase. Mutants with altered mannan were unable to express the antigen. Complete acid hydrolysates mannan from alpha-factor-treated a-cells contained mannose, glucose, and N-acetylglucosamine. Partial acid hydrolysis, under conditions that destroyed the antigenic determinant, released only mannose and mannobiose. The mannose fraction was labeled to high specific activity during response of a-cells to alpha-factor if radioactive glucose was the carbon source. Neither alpha- not beta-D-mannopyranosyl phosphate was a hapten. The results are consistent with the presence of a haploid-specific antigen containing an acid-labile mannose determinant and show that the amount of this antigen in a-cell mannan is increased in response to alpha-factor.     

Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.     

Isolation of an antigenic oligosaccharide fraction from Leptospira interrogans serovar canicola with a monoclonal antibody

An oligosaccharide fraction containing the antigenic determinant of lipopolysaccharide antigen (TM antigen) from Leptospira interrogans serovar canicola, recognized by a monoclonal antibody (CT3) which agglutinates serovars canicola and broomi, was isolated by formic acid and successive sulphuric acid hydrolyses. Separation of the antigenic compounds was done by Bio-Gel P-2 and Sephadex G-25 gel filtration, and high-performance liquid chromatography with two different columns. The fraction finally obtained was a mixture of two oligosaccharides, both of which migrated as a single spot having a slightly higher mobility than an authentic tetrasaccharide (stachyose) on thin layer chromatography. The fraction contained rhamnose, arabinose and two major and two minor unknown sugars which were shown to be N- or O-acetylated and/or O-methylated sugars by nuclear magnetic resonance. The fraction inhibited the binding of CT3 antibody with TM antigen in enzyme-linked immunosorbent assay and microscopic agglutination of serovar canicola with the antibody. The inhibitory activity was destroyed by periodate oxidation or mild alkaline treatment, but was resistant to sodium borohydride reduction.     

Structural studies on carcinoembryonic antigen. Periodate oxidation.

Periodate oxidation has been applied to examine the carbohydrate structure of carcinoembryonic antigen (CEA) and the possible role of the carbohydrate residues in its antigenic activity. Sialic acid (N-acetylneuraminic acid) and fucose were completely destroyed, and galactose and mannose were partially destroyed by a single periodate treatment. Serial periodate treatment (Smith degradation) destroyed additional amounts of galactose and mannose as well as significant amounts of N-acetylglucosamine. Prior removal of sialic acid by neuraminidase treatment led to increased destruction of galactose by periodate. Antigenic activity persisted indicating that the residues destroyed played little, if any, part in the antigenicity of CEA. These results yield an initial view of the structural arrangement of the carbohydrate residues in the CEA molecule.     

Immunochemical Characteristics and Localization on Cells of Protective Antigen (PAg) Prepared from Leptospira interrogans Serovar lai

Immuno-electron microscopic methods revealed that the protective antigen (PAg) of Leptospira interrogans serovar lai exists on the outer envelope sheathing the leptospiral cell body. PAg lost its protective activity after treatment by hydrolysis with 2 M formic acid at 100 C for 2 hr, or oxidation with periodate at 4 C for 40 hr. The antigenic oligosaccharide fraction was further purified from the hydrolyzed PAg by immunoaffinity column coupled with protective monoclonal antibody, LW2, and by gel filtration of HPLC. The antigenic oligosaccharide fraction contained two unknown sugars and 4-O-methylmannose (molar ratio 3:5:1). These findings suggested that these sugars are components of an antigenic determinant contributing to the protective immunity against serovar lai infection.     

Properties of an Antigenic Polysaccharide from Corynebacterium parvum

Corynebacterium parvum strain 10390 is an antitumor agent and stimulant of the reticuloendothelial system and produces a soluble antigen towards the end of its growth cycle. This material, which is a cell wall component and can also be released from the organism by acid or alkaline hydrolysis, has been purified. It is an acidic polysaccharide of molecular weight 100,000 to 150,000 and contains galactose, glucose, fucose, N-acetylgalactosamine, N-acetylglucosamine, uronic acids, sialic acids, and a small proportion of amino acids. The antigen gives a precipitin reaction with antisera raised against the whole organism and also binds to animal cells. The antigenic determinants are extremely resistant to oxidation, reduction, and enzymatic and chemical hydrolysis, but the single cell-binding site is destroyed by alkali and also by Helix pomatia digestive juice, alginase, and neuraminidase without substantially affecting the molecular weight. This site is inaccessible until the molecule is released from the cell surface. The possibility that the soluble antigen is the biologically active fraction of C. parvum is discussed.     

Isolation of Antigenically Active Components from Leptospiral Serovar-Specific Lipopoly-saccharide Antigen by Alkaline Treatment

The serovar-specific main antigen (TM antigen) of Leptospira interrogans serovar canicola, which has lipopolysaccharide properties, was treated with 0.1 n sodium hydroxide. This treatment degraded the antigen into two major antigenic components, one of high and one of low molecular weight. The component with the lower molecular weight (approximately 4,000 daltons) consisted mainly of carbohydrates, having lost almost all of the fatty acid and protein components of the original antigen. Although the substance lacked immunoprecipitable activity, it continued to show serovar-specific inhibitory potency in a radioimmunoassay system as well as in a microscopic immunoagglutination reaction of the organisms. The antigenic activity of the compound was also reduced by periodate oxidation as was that of the TM antigen. A component with the same chemical and physicochemical properties was also produced by alkaline treatment from a different serotype TM antigen (serovar kremastos Kyoto), but it showed no antigenic activity.     

Activity of the glycoprotein antigenic fractions of Mycoplasma pneumoniae in immunological reactions

Glycoprotein fractions exhibiting pronounced antigenic properties in immunological in vitro reactions have been isolated from sonicated M. pneumoniae cells by extraction with glacial acetic acid and ethanol. Glycoprotein fraction 3 of M. pneumoniae antigen shows the highest blood-sensitive and precipitating activity and may be recommended as a diagnosticum for serological tests.     

Immunochemical study of beta-glucuronidase inhibitor from porcine sublingual gland. Relationship between the antigenic determinant and the active site of the inhibitor.

Following removal of sialic acid by neuraminidase treatment the activity of the beta-glucuronidase inhibitor was remarkably decreased, but the antigenic determinant was not affected. A partial common antigen to the inhibitor was isolated from porcine small intestine, by successive fractionation of trypsin extraction of the latter on Sephadex G-150, DEAE-cellulose and Sepharose 4B column chromatography. The immunologic and characteristic properties of the common antigen were compared with those of the beta-glucuronidase inhibitor is not identical with its antigenic determinant.     

Preparation and description of high-molecular-weight soluble surface antigens from a group A Streptococcus

High-molecular-weight proteins having M protein reactivity were isolated without acid or alkaline digestion. Treatment of a heat-killed group A Streptococcus with sonic vibration released antigens which reacted strongly and specifically with absorbed type-specific antiserum. This antigen preparation was released without diminishing the total yield of acid-extractable M protein of the original heat-killed cells. Fractionation of the sonic preparation on a sucrose gradient yielded four peaks of M reactivity. When these fractions were placed on Sephadex G-200 columns, the M reactive material of three fractions appeared in the void volumes, suggesting that the active material in each had a molecular weight greater than 300,000. The reactivity of the fourth fraction followed closely the void volume of Sephadex G-100. Chemical analysis revealed heterogeneity of the fractions. Spectral analysis showed virtual absence of nucleic acid in three of the fractions and a moderate amount in the fourth. Bactericidal inhibition tests showed activity of three of the four fractions. Analysis of the fractions by Ouchterlony double-diffusion technique revealed that each of the four fractions had several antigenic constituents. All four contained M antigen. T antigen and a third unnamed antigen were present in some of the fractions. Group reactivity was present in all fractions, but did not reside on the M molecule. The enhanced potential of sonically released antigens to induce high-titer specific precipitating antibodies to M protein is discussed.     

Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly-N-acetyllactosamine backbones. An immunochemical and chromatographic study of keratan sulphate oligosaccharides after desulphation or nitrosation

Conditions were established for desulphation of hexa-, octa-, deca- and larger oligosaccharides derived from corneal keratan sulphate after treatment with endo-beta-galactosidase. The antigenicities of the desulphated oligosaccharides were compared with those of the native oligosaccharides in chromatogram binding, plastic-plate binding or inhibition of binding assays using a novel microimmunochemical approach with oligosaccharide-lipid conjugates (neoglycolipids). The results clearly show that sulphate residues are essential components of the antigenic determinant(s) recognised by three monoclonal antibodies to keratan sulphate, 5-D-4, 1-B-4 and MZ15, but they mask the i antigen activity of the linear poly-(N-acetyllactosamine) backbones of this glycosaminoglycan. Immunochemical assays, before and after beta-N-acetylglucosaminidase treatment of desulphated linear hexa-, octa- and decasaccharides derived from keratan sulphate, indicate that for reaction with one anti-i antibody, Den, there is an absolute requirement for the non-reducing beta-galactosyl residue of the i antigen structure to be in the terminal position, but with a second anti-i antibody, Tho, there is in addition some reactivity with the i antigen structure having an N-acetylglucosamine residue at the non-reducing end. The chromatographic properties after desulphation or nitrosation of a minor keratan sulphate oligosaccharide (a dodecasaccharide), which reacts especially well with antibody 5-D-4, have provided the first evidence for the presence of glucosamine residues that may be N-sulphated in corneal keratan sulphate.     

Immunochemical study of β-glucuronidase inhibitor from porcine sublingual gland Relationship between the antigenic determinant and the active site of the inhibitor

Following removal of sialic acid by neuraminidase treatment the activity of the β-glucuronidase inhibitor was remarkably decreased, but the antigenic determinant was not affected.A partial common antigen to the inhibitor was isolated from porcine small intestine, by successive fractionation of trypsin extraction of the latter on Sephadex G-150, DEAE-cellulose and Sepharose 4B column chromatography. The immunologic and characteristic properties of the common antigen were compared with those of the inhibitor, and it was concluded that the active site of the β-glucuronidase inhibitor is not identical with its antigenic determinant.     

Functional activation of immune lymphocytes by antigenic stimulation in cell-mediated immunity. III. A soluble factor released from LPS-stimulated peritoneal adherent cells effective in antigenic activation of sensitized lymphocytes.

Antigen-induced production of migration inhibitory factor (MIF) by sensitized lymphocytes requires macrophages to effectively stimulate lymphocytes with soluble antigen in vitro. The present study showed that macrophage-depleted lymphocytes of sensitized guinea pigs could be activated with antigens when the culture supernatant of peritoneal adherent cells pulse-stimulated with a macromolecular fraction of bacterial lipopolysaccharide (LPS) was added to the lymphocyte culture. The apparent macrophage-replacing activity was found in the fraction which emerged slightly ahead of serum albumin upon gel filtration of the culture supernatant, and the activity was shown to be destroyed by heating at 65 °C for 30 min or by trypsin digestion. These results appeared to show that the activity was due to a protein component, most probably released from macrophages. Two-step culture experiments revealed that the soluble factor should be present in the early stage of the culture to activate the macrophage-depleted immune lymphocytes with antigen, as well as in the later stage when the presence of antigen in the medium is no longer required. Furthermore, the factor was shown to act in the activation of a T-cell-enriched fraction of immune lymphocytes. The factor appeared to be playing some essential role in making an antigenic stimulus effective for the activation of immune lymphocytes.     

Metabolism of glucosamine in the liver of Scyliorhinus canicula (selachian)]

(1) The metabolism of the glucosamine was studied in the liver of S. canicula, after injection of D-(1-14C) glucosamine into the animal. (2) The labelled acid-soluble derivatives were separated by ion exchange columns and characterized by chromatography and electrophoresis, and were identified as glucosamine, glucosamine 6-P, N-acetylglucosamine, N-acetylmannosamine, N-acetylneuraminic acid, N-acetylglucosamine 6-P, N-acetylglucosamine 1-P, N-acetylmannosamine 6-P, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine. (3) The variation with time after glucosamine injection of the radioactivity of the fractions separated on Dowex 1-X4 was investigated. This study showed a decrease of the radioactivity in the fraction 1 (glucosamine and glucosamine 6-P), an increase in the fraction II (N-acetylneuraminic acid) and the fraction IV (UDP-N-acetylhexosamines), and a stability in the fraction III (phosphorylated N-acetylhexosamines). (4) The absence of label in neutral hexoses and their phosphorylated derivatives was interpretated as due to the weak activity of the glucosamine 6-P isomerase, which is positively modulated by the N-acetylglucosamine 6-P.     

Biochemical and immunochemical properties of the cell surface of Renibacterium salmoninarum.

The biochemical composition of the cell envelope of Renibacterium salmoninarum was investigated in a total of 13 strains isolated from different salmonid fish species at various geographical locations of the United States, Canada, and Europe. A marked similarity with the type strain R. salmoninarum ATCC 33209 was found both in the peptidoglycan and the cell wall polysaccharide. The primary structure of the peptidoglycan was found to be consistent with lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and D-alanine of adjacent peptide subunits, and a D-alanine amide substituent at the alpha-carboxyl group of D-glutamic acid in position 2 of the peptide subunit. The cell wall polysaccharide contained galactose as the major sugar component which was accompanied by rhamnose, N-acetylglucosamine, and N-acetylfucosamine. The polysaccharide amounted to more than 60% of the dry weight of the cell walls. It was found to be covalently linked to the peptidoglycan and was released by hot formamide treatment. On gel filtration chromatography the extracted polysaccharide behaved like a homogeneous polymeric compound. The purified cell wall polysaccharide showed antigenic activity with antiserum obtained by immunization of rabbits with heat-inactivated trypsinized cells of R. salmoninarum. Immunoblotting experiments with nontrypsinized cell walls and antisera raised against R. salmoninarum cells revealed that antigenic proteins were attached to the cell walls.     

Diagnostic utility of fractionated urinary filarial antigen

Urinary filarial antigen isolated from urine samples of microfilaraemic patients was analysed for its antigenic activity by immunoblotting and enzyme linked immunosorbent assay techniques. SDS-PAGE fractionation of urinary filarial antigen showed 11 protein bands, of which two showed reactivity with immunoglobulin-G fraction of filarial serum immunoglobulin in immunoblotting. Antigenic analysis of SDS-PAGE fractions of urinary filarial antigen by inhibition enzyme linked immunosorbent assay using filarial serum immunoglobulin-G andWuchereria bancrofti microfilarial excretory-secretory antigen revealed 3 fractions, numbers 5, 6 and 9 with significant activity. In indirect enzyme linked immunosorbent assay using fractions 5 and 6, filarial immunoglobulin-G antibody was detected in about 90% of microfilaraemics, 80% clinical filariasis and 20% of endemic normal individuals. Further, there was no phosphorylcholine epitope in these fractions. Fractions 5 and 6 can be a candidate antigens for the immunodiagnosis of filariasis.     

Characterization of the antigen recognized by monoclonal antibody 1D3

Human ovarian mucinous cystadenocarcinoma-associated antigen recognized by murine monoclonal antibody 1D3 (Bhattacharya et al., 1982) was characterized. Gel filtration and sodium dodecylsulfate polyacrylamide gel electrophoresis, followed by Western-blot analysis showed that 1D3 is a high molecular weight glycoprotein. Isoelectric focusing of 1D3 antigen showed 2 overlapping antigenic components with PI 2.5 and 2.6. 1D3 antigen was extremely stable (10 min at 100 degrees C) to heating. The antigenic activity was slightly stimulated by treatment with galactosidases, but neuraminidase treatment enhanced the antigenic activity about 3-fold. Antigen activity was completely stable to periodate oxidation. Pronase and trypsin treatment completely destroyed the antigenic activity. Properties of 1D3 antigen suggest that this is a high molecular weight (approximately 5-20 x 10(6) Dalton), sialomucin. Monoclonal antibody 1D3 recognizes only the protein part of this molecule.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas

The murine monoclonal antibody (MAb) designated DF3 has defined a high m.w. antigen detectable in human breast carcinomas and in human milk. DF3 antigen is detectable on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. DF3 antigen expression has been shown to correlate with the degree of human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that DF3 antigen might be useful as a biochemical marker of differentiated mammary epithelial cells. To further characterize DF3 antigen, we have developed an approach to purify the cross-reactive species by using gel filtration and antibody affinity chromatography. The affinity column-purified DF3 antigen was absorbed by wheat germ agglutinin and peanut agglutinin, but not by concanavalin A or lentil lectin. In contrast, wheat germ agglutinin inhibited MAb DF3 reactivity with the purified antigen, whereas there was little, if any, inhibition when using peanut agglutinin. These findings are thus consistent with the involvement of terminal N-acetyl-D-neuraminic acid and/or N-acetylglucosamine residues in the antigenic site. DF3 antigenicity was also sensitive to neuraminidase, but not chondroitinase ABC, chondroitinase AC, chondroitin-4-sulfatase, or hyaluronidase. Furthermore, DF3 antigen was sensitive to Pronase, subtilisin BPN', and alpha-chymotrypsin. The presence of O-glycosidic linkages between carbohydrate and protein in the DF3 antigenic site was further supported by the presence of NaBH4-sensitive sites. Together, these results suggest that sialyl oligosaccharides present on a peptide backbone are required for maintaining DF3 antigenicity. Similar findings have been demonstrated for DF3 antigen purified from both human milk and breast cancer effusions. However, the DF3 antigen in human milk consisted of a single high m.w. species, whereas the tumor-associated antigen consisted of two distinct glycoproteins with m.w. of 330,000 and 450,000. These findings may be relevant to the recent demonstration that distinct high m.w. DF3 antigens are elevated in the circulation of patients with breast carcinoma.     

Presence of an immunologically-defined class of peri-Golgi vesicles in cultured mammalian cells

Immunsera of mice injected with clathrin-depleted coated vesicle membranes, purified from rat liver, revealed a preferential labeling of some perinuclear structures by immunofluorescence microscopy on NRK cells. Subsequent production of 4 monoclonal antibodies was achieved. The antigen was strictly located in the Golgi area of the cell but was not an intrinsic element of the Golgi complex. The restricted location of the structures excluded these were lysosomes which appeared more dispersed in these cells. After nocodazole treatment the material was found dispersed in the cytoplasm. This provided a means of distinguishing the antigen from clathrin-coated structures and Golgi intrinsic elements. Immunolocalization at the electron microscope level confirmed the data obtained at the light level. Some peroxidase reaction product was rarely associated with Golgi elements, but predominantly stained small neighboring Golgi vesicles (50 nm diameter), as well as tubulo-elongated structures and some large (500 nm) irregular-shaped vesicles. A 32 kDa molecular weight antigen was characterized by immunopurification from NRK cells metabolically labeled with 35S-Met. This 32 kDa antigen appeared as part of a higher multimolecular membrane component of 300 kDa. A 170 kDa and a 70 kDa components were immunodetected in a semi-purified membrane fraction from rat liver, demonstrating that the antigen was a minor but very antigenic contaminant of the coated vesicle preparation used as immunogen. In conclusion, the labeled peri-Golgi structures may be part of the newly characterized trans-Golgi network and/or of the reticular/vesicular endosomal, prelysosomal structure recently described.