Expression of the CD15 differentiation antigen (3-fucosyl-N-acetyl-lactosamine, LeX) on putative neutrophil adhesion molecules CR3 and NCA-160.

The expression of the carbohydrate antigen 3-fucosyl-N-acetyl-lactosamine (CD15, LeX) on human neutrophil glycoproteins has been studied by immunoprecipitation and immunoblotting by using monoclonal antibody MC2. The antigen is expressed on membrane glycoproteins of approximate molecular mass 165 and 105 kDa. These glycoproteins include the complement receptor and adhesion molecule, CR3, in which the beta-chain (CD18, 105 kDa) shows much greater expression than the alpha-chain (CD11b, 165 kDa). Most of the 165 kDa CD15 antigen is accounted for by expression on the carcinoembryonic antigen (CEA)-related molecule NCA160. Other members of this family, NCA95, NCA90 and NCA55, which are also found in neutrophils, do not express the CD15 antigen. There is a marked increase in the surface expression of CD15, CR3 and the antigen recognized by anti-CEA antibodies upon activation of neutrophils by the chemotactic peptide N-formylmethionyl-leucylphenylalanine.     

Membrane-cytoskeleton interactions: Inhibition of odontoblast differentiation by a monoclonal antibody directed against a membrane protein

It is known that high-molecular-weight (HMW) membrane proteins mediate interactions with constituents of the extracellular matrix and/or with cytoskeletal elements. To study participation of HMW membrane proteins in odontoblast or ameloblast differentiation, an immunological approach has been adopted. Antibodies directed against membrane proteins (Mr, 110-190) from mouse embryos have been produced by the hybridoma technique. Supernatants of hybridoma cultures were screened for their ability to stain dental tissues and also tested for their biological activities on dental cells in primary culture or on developing tooth germs in organ culture. An IgM monoclonal antibody, MC16A16, directed against a 165-kDa antigen present in plasma membrane preparations, reacted strongly with the dental epithelium and weakly with the mesenchyme. MC16A16 also reacted with the cell surface of nonpermeabilized cultured dental cells and could detach epithelial cells cultured on glass, but not mesenchymal cells which maintained vinculin-containing focal contacts. This antibody, which affected the organization of dental-cell microfilaments in primary culture, also inhibited the polarization of odontoblasts, but not that of ameloblasts.     

Antibodies to a common outer envelope antigen of Treponema hyodysenteriae with antibacterial activity

Outer envelopes of Treponema hyodysenteriae strains P18A and VS1 were prepared and characterized by SDS-PAGE. In Western blot analysis of eleven strains of T. hyodysenteriae and two intestinal non-pathogenic spirochaetes, polyclonal antiserum raised to the outer envelopes of strain P18A contained antibodies primarily to two polypeptides. A 45 kDa polypeptide was present in only two strains of T. hyodysenteriae, P18A and MC52/80, whereas another antigen of 16 kDa was common to all eleven strains of T. hyodysenteriae but was not present in the two nonpathogens. Immunogold labelling of whole organisms suggested that the 16 kDa antigen was present on the surface of the spirochaetes. In in vitro tests the serum agglutinated and inhibited growth of only the T. hyodysenteriae strains, suggesting that antibodies to the 16 kDa antigen were responsible for these activities. Serum from a gnotobiotic pig infected with T. hyodysenteriae strain P18A had antibodies to the 16 kDa antigen alone and also possessed agglutinating and growth-inhibitory activities.     

A 165 kDa membrane antigen mediating fibronectin-vinculin interaction is involved in murine odontoblast differentiation

Membrane-mediated matrix-microfilament interactions are involved in odontoblast differentiation. In this study, we analyzed the interactions of vinculin and fibronectin with plasma membrane proteins separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene-difluoride (PVDF) paper. Vinculin was found to interact with 58, 63 and 165 kDa plasma membrane proteins. Fibronectin interacted with three high molecular weight (145, 165, and 185 kDa) membrane proteins. Attempts were made to characterize the 165 kDa protein which interacted with vinculin and with fibronectin. The interaction of the 165 kDa protein with fibronectin was not competitively inhibited by synthetic peptides such as GRGDS or GRGDSP, suggesting that the protein was not related to integrins. Antibodies directed against the 165 kDa protein allowed the identification of the precise localization and biological role of this membrane antigen. The data presented in this paper and previous observations indicate that the 165 kDa protein, involved in odontoblast elongation and polarization, mediates a fibronectin-vinculin transmembrane interaction.     

The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115,000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.     

Molecular modifications of MC31/CE9, a sperm surface molecule, during sperm capacitation and the acrosome reaction in the rat: is MC31/CE9 required for fertilization?

We examined the modification of the MC31 molecule during capacitation, the acrosome reaction, and studied its role in fertilization. These studies revealed that the molecular mass of MC31 in cauda spermatozoa was approximately 28,000-26,000 Dalton (28-26 kDa). A limited change in molecular mass was seen in capacitated spermatozoa. Treatment of sperm extracts with peptide-N-glycosidase (PN glycosidase) reduced the molecular mass of MC31 in both cauda and capacitated spermatozoa from 28-26 kDa to 23-20 kDa, suggesting that MC31 from both cauda and capacitated spermatozoa is glycosylated, and indicating that capacitation induces minor posttranslational modifications in the structure of the MC31 antigen. The MC31 antigen was redistributed from the midpiece of cauda epididymal spermatozoa to the head and equatorial segment after capacitation and acrosome reaction, respectively, when traced by indirect immunofluorescence under in vitro fertilization (IVF) conditions. Some spermatozoa did not stain for the MC31 antigen and might represent spermatozoa that have shed the antigen. IVF experiments conducted to assess the effect of an anti-MC31 monoclonal antibody (mMC31) revealed that this antibody significantly (P < 0.01) inhibited fertilization of cumulus-invested zona pellucida-intact and the zona pellucida-free oocytes in a dose-dependent manner. However, sperm-oolemma binding was not affected. These findings suggest the MC31 antigen facilitates sperm-oocyte interactions.     

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.     

Western blot antibody determination in sera from patients diagnosed with Anisakis sensitization with different antigenic fractions of Anisakis simplex purified by affinity chromatography

Using Western blot techniques, the specificities of crude and purified (PAK and PAS) Anisakis simplex antigens were compared against 24 sera from patients diagnosed with Anisakis sensitization. All patients recognized a 60 kDa protein against the A. simplex crude extract, while 37.5% and 12.5% reacted with proteins of 40 and 25 kDa, respectively, when IgG was tested. In the case of IgE determination, 41.6% of sera were negative, while 12.5% and 20.8% appeared to cross-react against Toxocara canis and Ascaris suum, respectively. When the PAK antigen (A. simplex antigen purified by means of a column of IgG anti-A. simplex) was tested, immune recognition towards the 60, 40 and 25 kDa proteins increased in 83.3%, 16.7% and 4.2%, respectively, when the Ig antibodies were tested. In the case of the PAS antigen (PAK antigen purified by means of a column of IgG anti-A. suum), the reaction against the 40 and 25 kDa proteins increased to 45.8% and 25%, respectively, when Ig antibodies were used. Finally, when the EAS antigen (eluted from the anti-A. suum column after PAK purification) was tested, 83.3% of the assayed sera reacted against the 14 kDa protein, when the Ig antibodies, IgG and IgM immunoglobulins were measured. With the IgE determination, the reactions were observed in 41.7% of patients with proteins between 60 and 35 kDa against the PAS antigen. With the EAS antigen, reactive bands of 184, 84 and 14 kDa appeared. In conclusion, in the purification process of the A. simplex larval crude extract, the proteins implicated in cross-reactions with Ascaris and Toxocara were eliminated, with an important concentration of proteins responsible for the induction of specific responses.     

Photoaffinity labelling and phosphorylation of a 165 kilodalton peptide associated with dihydropyridine and phenylalkylamine-sensitive calcium channels

Partially purified fractions of dihydropyridine and phenylalkylamine receptors associated with voltage-dependent calcium channels in rabbit skeletal muscle were found to contain two glycopeptides of similar molecular weight. A peptide of approximately 165 kDa was photoaffinity labelled with an arylazido-phenylalkylamine Ca channel inhibitor and also was phosphorylated with cAMP-dependent protein kinase. Another peptide of 170 kDa could be distinguished from the 165 kDa peptide by peptide mapping and differences in electrophoretic mobility. The results suggest that the 165 kDa peptide contains the sites responsible for regulation of calcium channel activity by calcium channel inhibitors as well as by neurotransmitters that regulate its activity in a cAMP-dependent manner.     

Identification and distribution of thioredoxin-like 2 as the antigen for the monoclonal antibody MC3 specific to colorectal cancer

MC3 is a colorectal cancer (CRC)-specific mAb previously prepared in our laboratory that can detect CRC with high sensitivity and specificity. However, the target antigen for MC3 had not been identified due to technological limitations. In the present study, immunocytochemistry and immunohistochemistry revealed the expression patterns of MC3 antigen (MC3-Ag) in colon cancer cell lines and CRC tissues. Western blotting analysis showed that the MC3 antibody reproducibly recognized two approximately 30 kDa proteins in the total cell lysates of human colon carcinoma cell lines SW480 and HT-29. Using a proteomic approach, we identified two MC3 immunoreactive spots as two isoforms of thioredoxin-like 2 (Txl-2) protein. Further paired immunostaining showed that Txl-2 had the same expression profile as probed by the MC3 antibody. Western blotting also showed that both antibodies could detect the same two bands, further verifying that Txl-2 is the antigen of MC3 antibody. Additionally, tissue arrays revealed the expression patterns of Txl-2 in various normal and cancer tissues. Further analysis showed that Txl-2 mRNA was elevated in 18 cases of CRC tissues compared to paracancerous tissues and adjacent normal tissues.     

Intra-acrosomal 155,000 dalton protein increases the antigenicity during mouse sperm maturation in the epididymis: A study using a monoclonal antibody MC101

We found an intra-acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one-dimensional SDS-PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra-acrosomal material is involved in sperm maturation in the epididymis. © 1995 wiley-Liss, Inc.     

Isolation and characterization of immunoglobulin M of Asian sea bass, <Emphasis Type="Italic">Lates calcarifer</Emphasis> and its level in serum

Asian sea bass immunoglobulin M (IgM) was purified from the sera of Lates calcarifer by affinity chromatography. Analysis of the purified IgM on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions revealed that the sea bass IgM was a tetrameric protein with a molecular weight of 896 kDa; it contained an equimolar heavy chain and light chain with molecular weight of 83 kDa and 27 kDa respectively. However, besides the covalently linked tetrameric IgM, noncovalently linked tetramer dissociated into dimeric and monomeric forms also demonstrated by non-reducing SDS-PAGE. Carbohydrate moieties were found to be linked with both heavy and light chains. A polyclonal rabbit anti-Asian sea bass IgM was prepared which showed a specific reaction of anti-fish IgM antibody with IgM of sea bass. Sea bass IgM concentration was determined in the serum by indirect ELISA. The average IgM concentration in the sera of the healthy sea bass was 5.4±1.8 mg ml⁻¹; it amounted to 16.7% of the total serum protein.     

Serum immunoglobulin M in Atlantic halibut (Hippoglossus hippoglossus): characterisation of the molecule and its immunoreactivity

Three preparations of purified immunoglobulin (IgM) were isolated from serum of Atlantic halibut (Hippoglossus hippoglossus) by means of three different methods, and each of the three IgM preparations was used to produce a polyclonal rabbit anti-halibut IgM antiserum. One of the IgM preparations was employed in the characterisation of halibut serum immunoglobulin. Halibut IgM was shown to consist of two subunits, compatible with heavy (mu) and light (L) chains. A single mu chain at approximately 76 kDa, and six possible molecular weight (MW) variants of L chain were found (range approximately 25 to approximately 28.5 kDa). IgM was glycosylated on the heavy chain and N-linked carbohydrate constituted approximately 10.3% (w/w) of the total MW of IgM. The dominant form of non-reduced IgM had a MW of approximately 780 kDa, suggesting a tetrameric structure. Non-reduced IgM also showed a number of minor protein bands. Based on estimated MW, the relative carbohydrate content and the reactivity with all three anti-halibut IgM antisera, mono-, di- and trimeric redox forms of IgM were identified. The three antisera were characterised as to specificity and reactivity by means of enzyme linked immuno-sorbent assay (ELISA), crossed immuno-electrophoresis (CIE), and immunoblotting methods. The antisera showed a considerable diversity in their specificity to the suggested MW variants of halibut Ig light chain. A method for immunohistochemical detection of IgM in tissue was established. Protein A or protein G affinity for the IgM was not detectable.     

The carboxy-terminal extension of the collagen binding domain of fibronectin mediates interaction with a 165 kDa membrane protein involved in odontoblast differentiation

Terminal differentiation of the odontoblast is characterized by an elongation and a polarization of the cell. The change in the cell shape and the reorganization of the cytoplasm involve the microfilament system. An immunological approach has previously implicated a transmembrane interaction between fibronectin and vinculin in the control of odontoblast differentiation. A 165 kDa protein localized on the cell-surface of odontoblasts mediated this interaction. In order to define the nature of the interaction of the 165 kDa protein with fibronectin, peptides were prepared by proteolytic cleavage of fibronectin with alpha-chymotrypsin. The results indicate that the 165 kDa protein interacted with a 62 kDa peptide located towards the amino-terminal extremity of fibronectin, but not with a 47 kDa related fragment. Both these 62 kDa and 47 kDa peptides included the collagen-binding domain and were retarded on a heparin-Ultrogel column. Microsequences demonstrated that the 62 kDa and 47 kDa fragments had the same amino-terminal extremity and that the larger fragment was extended in the carboxy-terminal direction. This carboxy-terminal extension of the collagen binding domain of fibronectin is implicated in the interaction of this molecule with the 165 kDa protein. On the other hand, odontoblasts differentiated normally when tooth germs were cultured in the presence of GRGDS synthetic peptide, suggesting that RGD-dependent integrins were not involved in odontoblast differentiation. Staining of dental mesenchymal cells in primary culture and of differentiated odontoblasts in situ with antibodies directed against the beta 1-subunit of integrins confirmed previous observations and showed that although beta 1 integrins are involved in the attachment of cultured dental cells, they are not implicated in the process of odontoblast differentiation.     

Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity.

Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine lambdagt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by125Iodine labeling and SDS-PAGE.     

Physical properties of methylcelluloses in relation with the conditions for cellulose modification

A high molecular weight glycoprotein antigen was isolated by size exclusion chromatography on Sepharose 4B from an extract of the yeast Saccharomyces cerevisiae. The glycoprotein antigen Sc 500 was shown to be identical to the antigen termed gp200 previously isolated (Heelan et al., 1991). The MW of Se 500 was determined to be about 500 kDa by size exclusion chromatography on Superose 6 and 460 kDa ± 20k Da by size-exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). Sc 500 contained 90% mannose and traces of N-acetylglucosamine. The amino acid composition revealed that serine and threonine were the most abundant amino acids of the protein part. By alkaline borohydride treatment some, but not all bonds between protein and carbohydrate were broken. This indicates that the main type of linkage between protein and carbohydrate is O-glycosidic and that a minor type is of N-glycosidic nature. Methylation analysis revealed that the mannose residues were connected by 1 → 2 and 1 → 3 linkages with 1 → 2, 1→ 6 linked branch points.

Purified Sc 500 was subjected to a series of chemical and enzymatic modifications followed by studies of antibody binding activity. Treatments with both periodate and alkaline sodium borohydride reduced the human serum IgA, IgG and monoclonal IgM antibody binding activity of Sc 500 whereas trypsin and pronase did not affect its ability to bind these antibodies. The mannosidase Man1 → 2,3,6Man reduced the IgM binding to Sc 500 while the other enzymes included in this experiment (Man1→2 Man, Manβ1 →4GlcNAc and PNGase F) had no effect on the antibody binding.     

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.     

Isolation of O-demethylase, an ether-cleaving enzyme system of the homoacetogenic strain MC

The O-demethylase of the methylotrophic homoacetogenic bacterium strain MC was purified to apparent homogeneity. The enzyme system consisted of four different components that were designated A, B, C, and D according to their elution sequence from the anionic-exchange chromatography column. All four components were essentially required for catalysis of the transfer of the methyl group from phenyl methyl ethers to tetrahydrofolate. According to gel filtration and SDS-PAGE, components A and B were monomers with apparent molecular masses of approximately 26 kDa (subunit 25 kDa) and 36 (subunit 41 kDa), respectively; component C appeared to be a trimeric protein (195 kDa, subunit 67 kDa); and component D was probably a dimer (64 kDa, subunit 30 kDa). Component A contained one corrinoid per monomer. In crude extracts, component D appeared to be the rate-limiting protein for the complete methyl transfer reaction. Additional requirements for the reaction were ATP and low-potential reducing equivalents supplied by either titanium(III) citrate or H₂ plus hydrogenase purified from strain MC. Received: 5 February 1997 / Accepted: 17 April 1997