Physicochemical and immunochemical properties of carcinoembryonic antigen (CEA) from different tumour sources.

The physical, chemical and immunochemical properties of carcinoembryonic antigen (CEA) purified from hepatic metastases of eight tumours, originating in the colon (6), stomach (1) and lung (1), have been examined. Differences were observed in the overall molecular charge, and also in the carbohydrate composition of the different preparations (both total % carbohydrate, and mole % of the individual sugars). Negligible differences in amino acid composition were found. Gel filtration analysis of these CEA preparations and an additional four partially purified preparations (from pancreatic, hepatic, breast and oesophageal tumour tissues) revealed a single CEA-active peak of similar molecular weight (about 200,000-300,000 daltons) in all preparations. Radioimmunoassay data for the twelve CEA preparations indicated that all preparations contain the same antigenic determinants, as detected by our antiserum, but that there are differences in the expression of these determinants in different preparations.     

Purification and immunochemical properties of a protein antigen from serotype g Streptococcus mutans

A proteinaceous antigen (PAg) was purified from the culture supernatant of Streptococcus mutans 6715 (serotype g) by ultrafiltration, ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic chromatography, and subsequent Sephacryl S-300 gel filtration. A yield of 0.1 mg of PAg was obtained from a liter of culture supernatant. The isoelectric point and molecular weight of PAg were pH 4.6 and 210,000, respectively. It contained 35% sugar, which was identified as glucose by gas-liquid chromatography. Amino acid analysis revealed that PAg contains 28% acidic and 11% basic amino acid residues. PAg retained its antigenicity after heating at 80 C for 10 min in deionized water, or after treatment with 0.1 M HC1 or 0.1 M NaOH at 37 C for 1 hr. Immunodiffusion and immunoelectrophoresis analyses revealed that PAg is serologically distinct from other cell-surface antigens such as serotype-specific polysaccharide and lipoteichoic acid. A cross-reaction between PAg and a protein antigen similarly prepared from serotype c S. mutans was observed in immunodiffusion tests.     

Purification and immunochemical properties of a wall protein antigen from Clostridium difficile ATCC 11011

A wall-surface protein antigen, designated 32K antigen, was extracted from whole cells of Clostridium difficile strain ATCC 11011 with phosphate buffered saline and purified by ion-exchange chromatography, gel filtration, and chromatofocusing. The 32K antigen preparation was determined to be highly homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the antigen was characteristic in the predominance of the acidic amino acids, the very low contents of methionine and histidine, and the lack of cysteine. A monomeric molecular weight of the 32K antigen was estimated to be 32,000 by SDS-PAGE and 30,200 by sedimentation equilibrium. The antigen exhibited two isoelectric forms (IP, 4.12 and 3.96). Neither carbohydrate nor phosphorus was detectable in the antigen. The antigen was relatively resistant to trypsin but sensitive to pepsin. Immunoblot analysis of the wall proteins isolated from other strains of C. difficile probed with monospecific antiserum against the antigen from ATCC 11011 showed that the antigenicity of 32K wall protein was common among some of the strains containing 32K wall proteins.     

Composition and immunochemical properties of the cell surface proteins of Vibrio cholerae

The composition and immunochemical properties of cell surface proteins of Vibrio cholerae belonging to both the biotypes (classical and El Tor) and the serotypes (Ogawa and Inaba) were investigated. Proteins were isolated by extraction with EDTA/NaCl. When the extract was further treated with sodium deoxycholate, a product significantly enriched with the major protein was obtained. The surface localization of these proteins was confirmed by immunoelectron microscopy using protein A-colloidal gold particles as probes. Antisera to these proteins possessed complement-mediated bactericidal activities towards V. cholerae strains belonging to both the biotypes and the serotypes, and upon crossed immunoelectrophoresis produced several immunoprecipitation reactions towards whole-cell sonicates belonging to all types of V. cholerae. These proteins were immunogenic in the rabbit intestine, as antibodies of two classes (IgG and IgA) were detected in the intestinal fluids. The intestinal immune response was greatly enhanced when cell surface proteins were administered with liposomes. These results suggest that cell surface proteins represent common antigens of V. cholerae and can be explored as vaccine candidates against cholera.     

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.     

Physicochemical properties of kidney-specific antigen

Using specific antirenal sera obtained from rabbits and absorbed with a mixture of extracts from heterologous organs, a specific antigen was detected in human and CBA mouse renal extracts. Its molecular weight was found to amount to about 100 000 dalton. It is salted out with ammonium sulfate at 50-70% saturation of renal extract and is destroyed on extract heating for 30 min at 75 degrees C. This antigen is sensitive to trypsin and papain but resistant to hyaluronidase. It is partially destroyed by DNase and RNase, provided the latter ones are used in comparatively high doses (1 mg per 0.3 ml extract) and exposure lasts one day. Based on the study of the physicochemical properties it is suggested that the kidney-specific antigen may be a ribonucleoprotein or a deoxyribonucleoprotein but cannot be attributed to glycoproteins.     

Expression in and purification of Hepatitis B surface antigen (S-protein) from methylotrophic yeast Pichia pastoris

The study describes expression and purification of recombinant hepatitis B small surface antigen (rHBsAg hereafter) in methylotrophic yeast Pichia pastoris strain GS115. For expression of the rHBsAg, a single copy of 678 bp cDNA was inserted at the unique EcoRI site downstream of the alcohol oxidase (AOX 1) promoter of the 8.2 kb pHIL-D2 vector. The cDNA-pHIL-D2 construct was used to transform the strain GS115, resulting in a Mut(S) (Methanol Utilizing Slow) phenotype in which the 226 amino acids containing active and full-length rHBsAg protein could be expressed intra-cellularly during slow growth and induction with methanol. The recombinant protein from the Mut(S) expressor was harvested by cell disruption, and purified first by adsorption-desorption on aerosil followed by two-step chromatographic separation i.e. anion exchange on DEAE resin followed by gel permeation on Superdex 75. Reversed passive hem-agglutination assay (RPHA) was used to test the antigenicity while SDS-PAGE was performed to check the purity of the 27 kDa rHBsAg and its aggregates. The results showed that disruption at 12 Kpsi (three cycles), or 30 Kpsi (1 cycle), desorption with 10mM carbonate buffer (pH 9-10), and storage at 4 degrees C without detergent did not adversely affect the antigenicity of the rHbsAg. However, the presence of detergents such as TritonX100 and deoxycholate in the disruption and desorption buffers, respectively resulted in reduced antigenicity during storage both at 4 and -20 degrees C in spite of higher initial yields.     

Physicochemical surface properties of brewing yeast influencing their immobilization onto spent grains in a continuous reactor

Immobilization of brewing yeast onto a cellulose-based carrier obtained from spent grains, a brewing byproduct, by acid/base treatment has been studied in a continuously operating bubble-column reactor. The aim of this work was to study the mechanisms of brewing yeast immobilization onto spent grain particles through the information on physicochemical surface properties of brewing yeast and spent grain particles. Three mechanisms of brewing yeast immobilization onto spent grains carrier were proposed: cell-carrier adhesion, cell-cell attachment, and cell adsorption (accumulation) inside natural shelters (carrier's surface roughness). The possibility of stable cell-carrier adhesion regarding the free energy of interaction was proved and the relative importance of long-range forces (Derjaguin-Landau-Verwey-Overbeek theory) and interfacial free energies was discussed. As for the cell-cell attachment leading to a multilayer yeast immobilization, a physicochemical interaction through localized hydrophobic regions on cell surface was hypothesized. However, neither flocculation nor chain formation mechanism can be excluded so far. The adsorption of brewing yeast inside sufficiently large crevices (pores) was documented with photomicrographs. A positive effect of higher dilution rate and increased hydrophobicity of base-treated spent grains on the yeast immobilization rate has also been found.     

Purification, chemical, and immunochemical properties of a new lectin from Mimosoideae (Parkia discolor)

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P. discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.     

Physicochemical properties of apolipoprotein(a) and lipoprotein(a-) derived from the dissociation of human plasma lipoprotein (a)

Chemical reduction of human plasma lipoprotein(a) (Lp(a)) yielded two water-soluble products which were separated by rate zonal ultracentrifugation. Apolipoprotein(a) (apo(a)) was completely recovered from the bottom of the gradient, whereas lipoprotein(a-) (Lp(a-)), which contained all of the lipids and apo-B100 of Lp(a), floated. By the techniques of circular dichroism and viscometry Lp(a-) was identical to low density lipoprotein (LDL). Lp(a-) was slightly larger in mass than autologous LDL and contained proportionally more triglyceride. The difference in mass between Lp(a) and Lp(a-) was accounted for by the loss of 2 molecules of apo(a) from the Lp(a) particle. The molecular weight of reduced and carboxymethylated apo(a) was 281,000 as determined by sedimentation equilibrium in 6 M guanidine HCl. By circular dichroism the structure of apo(a) was mostly random (71%) with the remainder representing 8% alpha-helix and 21% beta-sheet; its intrinsic viscosity, 28.3 cm3/g, was consistent with an extended flexible coil. The amino acid composition was characterized by an unusually high content of proline (11.4 mol %) as well as tryptophan, tyrosine, arginine, threonine, and a low amount of lysine, phenylalanine, and isoleucine. Apo(a) contained 28.1% carbohydrate by weight represented by mannose, galactose, galactosamine, glucosamine, and sialic acid in an approximate molar ratio of 3:7:5:4:7, respectively. Overall, the structure of Lp(a) appears to be consistent with a rigid spherical LDL-like core particle which, as a consequence of its association with a flexible glycoprotein such as apo(a), favors the entrapment of significant amounts of hydrodynamically associated solvent. Furthermore, the Lp(a-) remnant generated by the removal of apo(a) from Lp(a) was similar in structure but not identical to autologous LDL.     

Purification and properties of Myxococcus xanthus cell surface antigen 1604.

A cell surface antigen complex from Zwittergent-solubilized Myxococcus xanthus has been purified by immunoaffinity chromatography with monoclonal antibody (MAb) 1604 and by subsequent gel filtration. We propose that the cell surface antigen (CSA) 1604 complex participates in intercellular interactions. The apparent total molecular mass of the CSA 1604 complex is 200 kilodaltons (kDa), as determined by gel filtration and by electrophoresis and Western immunoblot probing with MAb 1604. The antigen epitope recognized by MAb 1604 is on a 51-kDa polypeptide. The CSA complex also contains 14% neutral carbohydrate and a 23-kDa polypeptide that lacks the 1604 epitope. The carbohydrate is most likely part of a lipopolysaccharide (LPS) associated with the CSA, because an MAb recognizing an O antigen epitope from the LPS of M. xanthus also reacted with CSA 1604 on Western immunoblots. Thus, the 200-kDa CSA complex consists of 97 +/- 6 kDa of protein and many associated LPS molecules. The LPS evidently produces the multiplicity of bands observed on Western immunoblots between 100 and 200 kDa. The association with LPS may contribute to the negative charge of the CSA 1604 complex, which has a pI of 4.3. The CSA was clustered on the surface of intact M. xanthus cells after labeling with MAb 1604 and immunogold. Furthermore, fractionation studies indicated that cells grown on a plastic surface had 50% of their total CSA 1604 in the cytosol, 39% in the membrane fraction, and 8% in the periplasm. Saturable binding studies with 125I-MAb 1604 indicated that there were 2,400 CSA 1604 sites per cell. The Kd for MAb 1604 binding to the cell was 9 nM.     

Physicochemical and immunochemical characterization of allergenic proteins from rye-grass (Lolium perenne) pollen prepared by a rapid and efficient purification method.

Three fractions of rye-grass (Lolium perenne) pollen extract have been isolated by preparative isoelectric focusing (i.e.f.) and characterized in terms of physicochemical and immunochemical properties. The purified components were designated 'R7' and 'R14' on the basis of their positions in relation to other rye-grass pollen extract components on SDS/polyacrylamide-gel electrophoresis and their apparent molecular masses were assessed as 31 and 11 kDa respectively. On i.e.f., R14 split into two components, one acidic (pI 5.0) and one basic (pI 9.0), termed 'R14a' and 'R14b' respectively, and R7 focused at pI 5.8. R7 and R14a were shown to be allergenic by skin-prick test and all three components were recognized by rye-grass-pollen-specific human IgE. On SDS/polyacrylamide-gel electrophoresis and i.e.f., R7 behaved in a manner identical with that shown by an authentic sample of Rye I and gave an amino acid analysis similar to published data [Johnson & Marsh (1966) Immunochemistry 3, 91-100] for Rye group-I isoallergens; the amino acid sequence of the first 27 N-terminal amino acids was also determined. Physicochemical analysis revealed that R14a was equivalent to Rye II and 14b to Rye III. Preparative i.e.f. followed by gel-permeation chromatography proved to be a rapid and efficient method for purifying the allergenic components of Rye I (R7), Rye II (R14a) and Rye III (R14b) from rye-grass pollen extract.     

Physicochemical and immunochemical properties of heavy chains of immunoglobulin G typical of malignant growth

Molecular weight of heavy chains of immunoglobulin G typical of cancer is studied immunoglobulin and may be responsible for manifestation of certain anomalous acid and peptide composition of this protein heavy chains as compared with immunoglobulin G in blood serum of healthy people. Immunochemical methods helped detecting an antigenic determinant (or determinants) which is arranged in the heavy chains of the studied immunoglobulin and may be responsible for manifestation of certain anomalous properties of cancer-typical immunoglobulin G molecules. A set of bromo-cyanogenic fragments differing from the spectrum of these fragments in the heavy chains of normal immunoglobulin G is formed following a specific chemical effect of bromo-cyanogen on the heavy chains of immunoglobulin G typical of cancer. Essential differences are found in dancyl-fingerprints of the heavy chains of the compared proteins. Everything mentioned is a result of changes in the primary structure of the heavy chains of immunoglobulin G typical of cancer.     

Isolation and characterization of polyphosphates from the yeast cell surface

When cells of Saccharomyces fragilis are subjected to osmotic shock, they release a limited amount of inorganic polyphosphate into the medium, which represents about 10% of the total cellular content. The osmotic shock procedure causes no substantial membrane damage, as judged from the unimpaired cell viability, limited K⁺ leakage and low percentage of stained cells. It is therefore suggested that this polyphosphate fraction is localized outside the plasma membrane. The released polyphosphate fraction differs from the remaining cellular polyphosphates in two respects: the mean chain length of the shock-sensitive fraction is significantly higher than that of the total cellular polyphosphates and its metabolic turnover rate, subsequent to pulsing with [³²P]orthophosphate is much lower compared to the rest of the cellular polyphosphate. Incubation of intact cells with the anion exchange resin Dowex AG 1-X4 results in the release of high molecular weight polyphosphates. These results suggest that the osmotic shock-sensitive polyphosphate fraction has specific characteristics in both its cellular localization and metabolism.     

Galactose-regulated expression of hepatitis B surface antigen by a recombinant yeast

Summary A yeast expressing the hepatitis B surface antigen (HBsAg) gene under the control of a regulated promoter was grown in batch and fed-batch modes. In batch fermentations, the addition of galactose caused the rapid production of HBsAg. Cells grown in fed-batch mode did not produce HBsAg unless the conditioned medium was replaced prior to induction.     

Purification and properties of a membrane-bound phospholipase B from baker''s yeast (Saccharomyces cerevisiae)

Two kinds of E. coli K-12 mutants for lysophospholipase L2 (located in the inner membrane) were isolated, using an improved version of the colony autoradiographic method developed by Raetz; these were, 1) strains carrying an elevated level of the enzyme and 2) strains defective or temperature-sensitive in the enzyme. Characterization of the crude lysates of these mutants revealed that the differences of lysophospholipase L2 activity are not due to the presence or absence of regulatory factors. Evidence was obtained, by using these mutants, that this lysophospholipase L2 transfers the acyl group of 2-acyl lysophospholipid to phosphatidylglycerol, forming acyl phosphatidylglycerol.     

E antigen (HBeAG) and surface antigen (HBsAg) in bladder schistosomiasis

The problem of the relationship between surface B antigen and schistosomiasis or other parasitic infections which are transmitted though the skin is not still resolved. Serum samples from 54 Somalian patients infected by Schistosoma haematobium were tested for the presence of the surface B antigen (HBsAg) and the e-antigen (HBeAg). The HbsAg was found in 14.8 per cent of these patients, while among controls (47 cases) the frequency was of 34.0 p]er cent; no e-antigen was found among the patients and controls, the prevalence of anti-HBs antibodies was of 57.4 per cent among the patients with urinary schistosomiasis and of 44.6 per cent among the controls; a low rate of anti-e antibodies was found in the patients (7.4%) and in the controls (10.6%). These observation seem to indicate that the problem of an increased frequency of hepatitis B virus markers among patients with urinary schistosomiasis needs for further investigation.