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The extracellular antigens of Actinobacillus actinomycetemcomitans Y4 (serotype b) contain a 37-kDa protein which is a major target for IgGs from patients suffering from severe alveolar bone loss. Since the 37-kDa protein has not been studied sufficiently, our investigation focused on its characteristics, e.g., its localization, specificity, and whether it directly stimulates macrophages to produce cytokines. The 37-kDa protein was purified from the culture supernatant of the Y4 strain by means of chromatofocusing and gel filtration. The 37-kDa protein is a unique glycoprotein which forms immune complexes with monoclonal antibodies against rhamnose-fucose polysaccharide. Patients with A. actinomycetemcomitans-associated periodontitis had higher antibody titers to the purified 37-kDa protein than healthy subjects (p < 0.001). Anti-37-kDa protein antibodies recognized a 37-kDa band in the cytosolic, ribosomal, and total membrane fractions from Y4 cells. Extracellular substances from other strains of A. actinomycetemcomitans (serotypes a and c) also reacted in the Western blots, but Haemophilus spp. or several periodontopathic bacteria did not. These results suggested that the 37-kDa protein is a cytosolic protein that is passed through the cell membrane, and its protein portion is specific for A. actinomycetemcomitans but common to serotypes. This protein induced Il-1 beta, Il-6, and TNF-alpha release from murine macrophages. The Il-6-inducing activity of the 37-kDa protein was higher than that of LPS. These findings suggested that the 37-kDa protein which is released from live cells plays a role in A. actinomycetemcomitans-associated periodontitis, as antigen inducing the release of inflammatory cytokines which are associated with alveolar bone loss.

Fucosylated antigen expression, fucosyltransferase activities and expression of Fuc-TIV and Fuc-TVII genes have been measured in the human leukemic cell lines KG1a, arrested at the undifferentiated myeloblast stage of maturation and KG1, arrested at the myeloblast and early promyelocytic stage. The results are compared with those we earlier found for the later promyelocytic cell line HL-60 and the myelocyte form into which HL-60 cells can be induced to differentiate. These leukemic cell lines, and the differentiated HL-60 cells, are believed to correspond to four successive stages of myeloid maturation in the bone marrow. Fuc-TVII mRNA was strongly expressed in the myeloblastic KG1a cells but expression was less in KG1 and HL-60 cells. In contrast to the sharp fall in Fuc-TIV expression observed on differentiation of HL-60 cells, the expression of Fuc-TIV mRNA showed a progressive increase from KG1a to HL-60 cells; thus the peak of expression of this gene was at the HL-60 promyelocyte stage. This peak correlated with an increase in fucosyltransferase activity with nonsialylated acceptors and the transitory downregulation of cell surface sialyl-Le(x) expression and upregulation of Le(x), VIM-2 and Le(y) expression. The variations in levels of expression of the fucosylated antigens on the surface of the developing myeloid cells therefore correlate with variations in mRNA expression arising from the independent regulation of Fuc-TIV and Fuc-TVII genes.

IgA and IgG antibodies eluted from the surface of spermatozoa obtained from 11 infertile men were used to analyse the antigens defined by each class of sperm-associated antibodies. An enhanced chemiluminescent Western blot technique was developed to detect the low concentrations of immunoglobulins present in the eluted samples. The same analysis was performed with the sperm membrane-specific antibodies isolated from the sera of 8 of the patients included in the study. Sperm-eluted antibodies reacted with a total of 18 protein bands migrating with molecular masses of between 110 and 18 kDa. Individual antibody-binding patterns differed. Furthermore, IgA and IgG antibodies from any one patient recognised different sets of antigens. In spite of the apparent heterogeneity of the antigens defined by sperm-associated antibodies, the majority of these antibodies reacted with three protein zones of 68/64, 37/36 and 20/18 kDa. The antigens defined by the sperm surface-specific antibodies obtained from the sera of eight infertile patients differed from one patient to another and, in the majority of the patients, differed from those defined by the corresponding sperm-associated antibodies. Nevertheless, two protein zones of 68/64 and 20/18 kDa were recognised by both local and systemic antibodies in 6 and 4 patients, respectively.

Dendritic cells (DC) are a subset of leukocytes whose major function is antigen presentation. We investigated the phenotype and function of enriched (95-98.5%) rat DC. We show that both spleen and thymus DC express the natural killer cell receptor protein 1 (NKR-P1) as a disulfide linked homodimer of 60 kD. Freshly isolated DC express a low level of NKR-P1, which is strongly upregulated after overnight culture. Spleen, but not thymus DC, were able to kill the NK-sensitive YAC-1 cell line in vitro, and since this killing was Ca2+ dependent, a Fas ligand-Fas interaction was probably not involved. Besides their potent antigen-presenting function, DC can thus be cytotoxic for some tumor targets.

Lactadherin, a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and expressed in human breast carcinomas. Previously, we have shown that the mature protein, formerly known as BA46, has three domains: an epidermal growth factor (EGF)-like domain containing an Arg-Gly-Asp (RGD) cell adhesion sequence and C1 and C2 domains similar to those found in coagulation factors V and VIII. An alignment of lactadherin with its bovine (MGP57/53) and murine (MFG-E8) homologs shows that the RGD sequence has been conserved during evolution, suggesting that the RGD sequence is not fortuitous. We demonstrate that lactadherin purified using Triton X-114 phase partitioning promotes RGD-dependent cell attachment of green monkey kidney cells (MA104), mouse fibroblast cells (3T3-L1), and breast carcinoma cells (ELL-G). A lactadherin-specific monoclonal antibody, Mc3, inhibits attachment to purified lactadherin, suggesting that contaminants in the purification are not responsible for binding. In addition, the anti-integrin alpha(v)beta3 monoclonal antibody LM609 inhibits cell attachment of MA104 cells to lactadherin. These results demonstrate that lactadherin promotes RGD-dependent cell adhesion via integrins. Denaturation of lactadherin with heat and reducing conditions diminished cell attachment, suggesting that optimal cell attachment to RGD is dependent on the structural presentation of the sequence.

RP105 is a 105-kDa type I membrane protein of the leucine-rich repeat (LRR) family. Anti-RP105 sensitizes B cells to antigen-receptor-mediated apoptosis, but protects B cells from radiation-induced apoptosis and stimulates B cell proliferation. The sequence of the mouse RP105 has been reported. Here, we report the characterization of the human RP105. The 2.6-kb cDNA encodes a protein of 661 amino acids which displays 78% homology with mouse RP105. The 22 LRR and the 9 potential N-linked glycosylation sites within the extracellular region are conserved. While previous studies have shown that RP105 is expressed on surface IgM+IgD+2 B cells in mice, human RP105 was shown to be expressed on all subsets of mature B cells and dendritic cells. Human RP105 gene was mapped to the long arm of chromosome 5, where numerous cytokines and receptors have been localized.

We previously identified two surface-exposed Mycoplasma arthritidis protein antigens, designated MAA1 and MAA2, that may be involved in cytadherence. Since adherence to host tissues is an important first step in most bacterial infections, we suggest that MAA1 and MAA2 may be virulence factors for M. arthritidis. In order to provide evidence for such a role, we conducted a series of experiments in which rats were actively immunized with each of these proteins purified from sodium dodecyl sulfate-polyacrylamide gels or passively immunized with poly- or monoclonal antibodies against MAA1 and MAA2. In each case, immunity against MAA1 and MAA2 conferred at least partial protection against M. arthritidis-induced disease. The greatest protection was achieved by passive immunization with monoclonal antibody A9a, directed against a surface-exposed epitope of putative adhesin MAA1. Because protective immunity in most bacterial infections is directed against major virulence factors, these results suggest that MAA1 and MAA2 may play a role in the pathogenesis of M. arthritidis-induced arthritis of rats, possibly by mediating initial colonization of joint tissues.

We describe a method for the production of baculovirus-based cDNA libraries. By staining with monoclonal antibodies, single positive cells can be sorted and the virus encoding for the surface epitope can be isolated by limiting dilution. We have used this method to isolate cDNAs encoding several cell-surface antigens.

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123(lo), Thy-1+, and CD38(-/lo) CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.

To establish a screening procedure for tumor cell-surface reactive Fabs, we used a model antigen/antibody system including the epidermal growth factor receptor (EGF-R) and the anti-EGF-R monoclonal antibody 425. The 425 Fab was displayed on the surface of M13 filamentous phage. In a screening assay for 425 phage binding to tumor cell surfaces, biotinylated 425-phage bound specifically to EGF-R-positive A431 epidermoid carcinoma cells and not to K562 non-expressor erythroleukemia cells. With a model library, the sensitivity of phage enrichment by phage binding to cell surfaces was one 425-phage in 20,000 unrelated phages after 4 rounds of panning on A431 cells. In a phage tissue screening assay, 425-phage, but not unrelated phage, bound specifically to melanoma cells expressing EGF-R. Epitope and idiotope specificity of 425-phage was demonstrated in phage competition assays, using as targets A431 cells and anti-idiotypic antibodies to monoclonal antibody 425, respectively. Finally, the EGF-R protein was directly isolated from A431 cell extracts, using biotinylated 425-phage. The data obtained with the 425 model library system demonstrate the usefulness of antibody phage display for the rapid identification and isolation of tumor or other disease-related cell surface antigens.

The high-frequency blood group antigen Ok(a) is carried on a red cell membrane glycoprotein (gp) of 35-69 kDa that is widely distributed on malignant cells of different origins. Immunostaining of hemopoietic cells and a range of normal human tissues demonstrated a wide distribution of the Ok(a) gp that appears to be nonlineage-restricted, although certain tissues show differentiation-related expression. Ok(a) gp was purified from red cell membranes by immunoaffinity chromatography using mAb A103 and amino acid sequence analysis was performed. The N-terminal 30 amino acids are identical to the predicted sequence of M6 leukocyte activation antigen (M6), a member of the Ig superfamily (IgSF) with two IgSF domains. There are homologs in rat (MRC OX-47 or CE9), in mouse (basigin or gp42), and in chicken (HT7 or neurothelin). The molecular basis of the Ok(a) mutation was established by sequencing M6 cDNA derived from normal and Ok(a-) EBV-transformed B cell lines. A point mutation in the translated portion of M6 cDNA, G331AG-->AAG gives rise to a predicted E92-->K amino acid change in the first Ig-like domain of the Ok(a-) form of the protein. Transfection of mouse NS-0 cells with normal or Ok(a-) cDNA confirmed the identity of the protein and only the Ok(a-) transfectants failed to react with monoclonal anti-Ok(a) Ab.

Fourteen protein antigens were identified on SDS-PAGE of Afipia felis culture supernatant. Immunoblotting against 10 monoclonal antibodies obtained from mice infected with live A. felis showed that 4 antibodies reacted with a 56 kDa band and 3 with both 56 kDa and 62 kDa bands. Compared with A. felis sonicate, the reacting proteins in culture supernatant showed an increase in molecular mass of 2-3 kDa, suggesting that they were more glycosylated. Purified antigen obtained by affinity chromatography of culture supernatant on the seven immobilized antibodies was tested against antibodies reacting with the 56 kDa and 62 kDa bands. All eluates contained both components, suggesting that the antibodies were directed against different epitopes of a double antigen held together during the affinity chromatography but cleaved by reduction and SDS-PAGE. The molecular size of the uncleaved protein in culture supernatant was determined by size-exclusion chromatography as > 1000 kDa. Testing of pre- and post-infection rabbit sera in immunoblotting against culture supernatant demonstrated that the 56 kDa and 62 kDa components gave the most prominent specific reactions with post-infection sera. One of fifty human sera submitted for testing for cat-scratch disease and 1 of 50 sera from healthy blood donors reacted with several bands in A. felis culture supernatant, including the 56 kDa and 62 kDa bands.

An 8kb gene coding for a putative serine/threonine protein kinase from Plasmodium falciparum has been cloned and sequenced. It is arranged in two exons: exon I is 2 kb and exon II is 5.6 kb. The gene codes for a large protein of 2510 amino acids. Antibodies raised against a fusion protein were used to localize the putative kinase. By immunofluorescence microscopy, it was found in the cytoplasm of infected red cells. By immunoelectron microscopy it was associated with membranous structures in the red cell and with the red cell membrane, particularly at parasite-induced knobs. This is the first putative protein kinase of P. falciparum to be exported from the parasite into its host cell.

Female BALB/c mice were infested four times with pathogen-free Ixodes scapularis nymphs prior to infestation with nymphs infected with Borrelia burgdorferi B31. Each infestation was separated by a 14-day tick-free period. Mean weights of fed ticks and percentage reaching repletion did not indicate development of acquired resistance. Only 16.7% of mice repeatedly infested with pathogen-free ticks prior to infected I. scapularis nymph challenge became positive for B. burgdorferi. One hundred percent of control mice infested only with infected ticks were culture positive for B. burgdorferi.

The method for the extraction of native surface protein antigen with a mol. wt. of 92 kD from vegetative cells of B.anthracis STI-1 and its purification was developed. The antigen was extracted with 3% sodium lauryl sarcosylate at 4 degrees C from bacterial mass previously treated with 0.1 M tris-HCl buffer solution, pH 8.0 Purification was carried out by adsorption chromatography on hydroxylapatite. The isolated protein antigen with a mol. wt. of 92 kD was electrophoretically and immunochemically homogeneous. The study of the protective properties of this protein in combination with Freund's adjuvant, made on white mice, revealed that it had good prospects for use in the creation of chemical vaccines against the causative agent of anthrax.

A unique physical feature of Treponema pallidum, the venereally transmitted agent of human syphilis, is that its outer membrane contains 100-fold less membrane-spanning protein than the outer membranes of typical gram-negative bacteria, a property that has been related to the chronicity of syphilitic infection. These membrane-spanning T. pallidum rare outer membrane proteins, termed TROMPs, represent potential surface-exposed virulence determinants and targets of host immunity. Only recently has the outer membrane of T. pallidum been isolated and its constituent proteins identified. Five proteins of molecular mass 17-, 28-, 31-, 45-, and 65-kDa were outer membrane associated. The 17- and 45-kDa proteins, which are also present in greater amounts with the T. pallidum inner membrane protoplasmic cylinder complex, had been previously characterized lipoproteins and are, therefore, not membrane-spanning but rather membrane-anchored by their lipid moiety. In contrast, the 28-, 31-, and 65-kDa proteins are exclusively associated with the outer membrane. Both the purified native and an Escherichia coli recombinant outer membrane form of the 31-kDa protein, designated Tromp1, exhibit porin activity, thereby confirming the membrane-spanning outer membrane topology of Tromp1. The 28-kDa protein, designated Tromp2, has sequence characteristics in common with membrane-spanning outer membrane proteins and has also been recombinantly expressed in E. coli, where it targets exclusively to the E. coli outer membrane. The 65-kDa protein, designated Tromp3, is present in the least amount relative to Tromps1 and 2. Tromps 1, 2, and 3 were antigenic when tested with serum from infection and immune syphilitic rabbits and humans. These newly identified TROMPs provide a molecular foundation for the future study of syphilis pathogenesis and immunity.

The mucosal addressin cell adhesion molecule-1 (MAdCAM-1), expressed selectively on high endothelial venules (HEV) and lamina propria venules, directs lymphocyte traffic by binding the lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1, alpha 4 beta 7). Full-length DNA encoding human MAdCAM-1 was obtained by combining sequences from an expressed sequence tag (EST) identified in an early stage human brain cDNA library, a polymerase chain reaction-derived clone, and a MAdCAM-1 genomic clone. The deduced amino acid sequence revealed an 18 amino acid signal peptide, two N-terminal immunoglobulin (Ig)-like domains conserved (59-65%) in sequence with those of the mouse homologue, an 86 amino acid mucin-like region rich in serine-threonine residues, a 20 amino acid transmembrane domain and a 43 amino acid charged cytoplasmic domain. No counterpart to the third IgA-like domain of mouse MAdCAM-1 was present; however, the serine-threonine-rich mucin domain was extended as two distinguishable major and minor mucin regions unrelated to the mouse domain. The major domain is formed from six tandem repeats of an eight amino acid sequence having the MUC-2-related consensus DTTSPEP/SP. Human MAdCAM-1 mRNA transcripts were restricted to small intestine, colon, spleen, pancreas and brain. Alternatively spliced MAdCAM-1 variants were identified that lack parts of the second Ig domain and all or part of the major mucin domain, indicating that the function of this vascular addressin is regulated by extensive modifications to its multi-domain structure.

Monoclonal antibody BB1 was previously shown to recognize the CD80 protein as well as a putative B7.3 molecule, both ligands of the important T-cell receptors CD28 and CTLA-4. We report that BB1 coprecipitated from detergent lysates of human B- and pre-B-cell lines a protein kinase activity, as detected by solid phase immunoprecipitation followed by in vitro kinase assay. As other monoclonal antibodies to CD80 did not co-precipitate any kinase activity, it seems likely that this protein kinase is associated with the so far poorly characterized putative B7.3 molecule.

Laminins are a family of multifunctional basement membrane glycoproteins. Over the last years, many laminin isoforms have been characterized, which were shown to be composed of distinct combinations of variant alpha, beta and gamma chains. Some of these isoforms show remarkable tissue specificity, which suggests functional involvement in local processes. In this study the previously described mAb 4C7, which recognize epithelial basement membranes as well as endothelial basement membranes in lymphoid follicles, was identified as an anti-laminin-5 antibody. Using a set of mAbs against various variant laminin chains we established that specifically the gamma 2 chain of laminin-5 was confined to the endothelial basement membranes of vessels in lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid follicles, whereas other variant laminin chains were also expressed elsewhere in the lymphoid tissue. Additionally, the expression of the known integrin receptors of laminin-5 was also examined. The alpha 6 beta 4 integrin-receptor for laminin was found to be colocalized with the laminin-5 gamma 2 chain on the abluminal surface of endothelial cells, whereas the alpha 3 integrin chain could not be detected in lymphoid follicles. This finding suggests that the alpha 6 beta 4 integrin (and not the alpha 3 beta 1 integrin) serves as a laminin-5 receptor on endothelial cells in the follicular compartment of lymphoid tissue. Furthermore, alpha 6 beta 4 was also found in the same punctuated pattern on FDCs as laminin-5. The function of the laminin-alpha 6 beta 4 complex in this particular localisation is still obscure, but a role in the maintainance of the follicular compartment via hemidesmosome-like attachment sites is postulated.

A carbohydrate enriched soluble fraction (CHP) was prepared by mild treatment of viable Echinococcus granulosus protoscoleces (PSC) with the enzyme endoglycosidase-F (endo-F) and characterized by SDS-PAGE, glycoside- inhibition ELISA, and immunoblotting. Three groups of four BALB/c mice were immunized with the CHP, with the remaining deglycosylated PSC (DGP) and with dead PSC (DPSC) to analyse the relative immunogenicity of carbohydrates on the surface of PSC. A fourth sentinel group was not immunized. The antibody response was analyzed during primary and hyperimmune responses. Specific antibody titres (IgG and IgM) against somatic PSC antigens (PSA) were evaluated by ELISA and their antigen recognition pattern by immunoblotting, discriminating carbohydrate and peptide specific antibody responses by periodate treatment of PSA. The avidity index of those antibodies and the titer of non-specific immunoglobulins during the whole protocol were evaluated by ELISA in vitro mitogenic activity of CHP was also evaluated. The results indicated: 1) immunodominance of surface carbohydrate epitopes from PSC, 2) predominant IgM and low avidity response against these epitopes, 3) a dramatic increase of non-specific antibody titres and 4) in vitro mitogenic activity of CHP.

A cell surface antigen that is expressed by normal and 95% of transformed colonic epithelium and is recognized by the monoclonal antibody A33 (Welt, S., Divgi, C. R., Real, F. X., Yeh, S. D., Garin-Chesa, P., Finstad, C. L., Sakamoto, J., Cohen, A., Sigurdson, E. R., Kemeny, N., Carswell, E. A., Oettgen, H. F., and Old, L. J. (1990) J. Clin. Oncol. 8, 1894-1906) has been purified to homogeneity from the human colonic carcinoma cell line LIM1215. The A33 protein was purified from Triton X-114 extracts of LIM1215 cells under nondenaturing conditions. These extracts were applied sequentially to Green-Sepharose HE-4BD, Mono-Q HR 10/10, Superose 12 HR 10/30, and micropreparative Brownlee Aquapore RP 300. The purification was monitored by biosensor analysis using surface plasmon resonance detection with a F(ab')2 fragment of the humanized A33 monoclonal antibody immobilized on the sensor surface and Western blot analysis following SDS-polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions using humanized A33 monoclonal antibody. The purified A33 antigen has a Mr on SDS-PAGE of 43,000 under nonreducing conditions. By contrast, the purified protein displayed a Mr of approximately 180,000 under native conditions on both size exclusion chromatography and native PAGE, possibly due to the formation of a homotetramer. N-terminal amino acid sequence analysis of the purified protein identified 34 amino acid residues of a unique sequence: ISVETPQDVLRASQGKSVTLPXTYHTSXXXREGLIQWD. A polyclonal antibody was raised against a synthetic peptide corresponding to residues 2-20 of this sequence. The antipeptide serum recognized the purified protein using Western blot analysis under both nonreducing (Mr 43,000) and reducing (Mr 49,000) conditions.

The fatty acid synthase (FAS) from Brevibacterium ammoniagenes is a homohexameric multienzyme complex that catalyzes the synthesis of both saturated and unsaturated fatty acids. By immunological screening of a B. ammoniagenes expression library, an fas DNA fragment was isolated and subsequently used to clone the entire gene together with its flanking sequences. Within 10,525 bp of sequenced DNA, the 9,189-bp FAS coding region was identified, corresponding to a protein of 3,063 amino acids with a molecular mass of 324,910 Da. This gene (fasA) encodes, at its 5' end, the same amino acid sequence as is observed with purified B. ammoniagenes FAS. A second reading frame encoding another B. ammoniagenes FAS variant (FasB) had been identified previously. Both sequences are colinear and exhibit 61 and 47% identity at the DNA and protein levels, respectively. By using specific antibodies raised against a unique peptide sequence of FasB, this enzyme was shown to represent only 5 to 10% of the cellular FAS protein. Insertional inactivation of the FasB coding sequence causes no defective phenotype, while fasA disruptants require oleic acid for growth. Correspondingly, oleate-dependent B. ammoniagenes cells obtained by ethyl methanesulfonate mutagenesis were complemented by transformation with fasA DNA but not with fasB DNA. The data indicate that B. ammoniagenes contains two related though differently expressed type I FASs. FasA represents the bulk of cellular FAS protein and catalyzes the synthesis of both saturated and unsaturated fatty acids, while the minor variant, FasB, cannot catalyze the synthesis of oleic acid.

Surface antigen 8, one of pathogenicity factors of P.pseudomallei and having an antiphagocytic action, contains glycoprotein with a mol. wt. of 200 kD and proteins with mol wt. of 25, 30 and 34 kD. According to its chemical structure, the carbohydrate part of antigen 8 is the homogeneous polymer of 6-d-D-mannoheptose, and its protein component is a collection of monomer proteins with mol. wt. of 12-120 kD. The consecutive fractionation of antigen 8 by gel and ion exchange chromatography has made it possible to isolate its individual fragments, differing by the collection of antigenic components, as well as by their antiphagocytic and protective activity. Experiments have shown that glycoprotein with a mol. wt. of 200 kD is an active immunosuppressive agent, while protein with a mol. wt. of 34 kD produces a pronounced immunogenic effect.

Transformation of cercariae of Schistosoma mansoni into schistosomula is accompanied by release of a soluble 28-kDa serine protease (s28) from the acetabular glands. The postulated activities of s28 include cleavage of skin connective tissue proteins (elastin, etc.), release of the cercarial glycocalyx, and cleavage of complement proteins. Our previous results demonstrated the presence of an antigenically cross-reactive protein on the surface of mechanically transformed schistosomula. As shown here, schistosomula express on their surface a 28-kDa serine protease (m28) which can be immunoprecipitated with anti-s28 antibodies. m28 eluted from the schistosomular tegumental membrane with NP-40 was purified to homogeneity in one step by adsorption on a chymotrypsin inhibitor column: 6-aminocaproyl-D-tryptophan methyl ester-Sepharose. Proteolytic activity of m28 was completely inhibited by the chymotrypsin inhibitor N-succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone. Efficient removal of m28 from schistosomula was achieved with NP-40, deoxycholate, cholate, Tween 20, and phospholipases A2 and C, but not with papain, trypsin, pronase, or proteinase K. Furthermore, treatment with phosphatidyl inositol-specific phospholipase C (PI-PLC) followed by hydroxylamine also released m28. Anti-cross-reactive determinant antibodies which recognize a neo epitope exposed in glycosyl phosphatidyl inositol-containing molecules cleaved by PI-PLC bind to purified m28. The latter results suggest that m28 is anchored to the tegumental membrane of schistosomula by a lipid anchor and that perhaps some of the m28 molecules are bound via glycosylphosphatidyl inositol. Based on inhibitor sensitivity and antigenic cross-reactivity, it is conceivable that s28 and m28 are related, if not identical, proteins. Finally, m28 was detected antigenically also on lung-stage and adult worms of S. mansoni.

An enzyme-linked immunosorbent assay (ELISA) to determine IgG antibody levels against K88 (F4) fimbrial antigen from porcine enterotoxigenic Escherichia coli (ETEC) has been developed. The ELISA method was checked with serum samples obtained from rabbits and pigs, and the parameters affecting the method were also analysed. ELISA plates were optimally coated with K88 antigen 0.5 microgram/ml for testing rabbit antiserum or with 1.25 microgram/ml for testing pig serum. Optimal concentrations of H202 (0.5%) and orthophenylene-diamine (OPD) (0.125%) were chosen when a 10-min incubation period was used. The expression of antibody levels as enzyme-immunosorbent units (EIU) significantly decreased the variability of results between duplicate plates, when compared with the expression of results as direct OD values. ELISA-K88 applied to a field study with serum samples from 141 vaccinated and 52 unvaccinated sows was shown to be useful in differentiating between samples from vaccinated and unvaccinated animals.

Intercellular adhesion molecules play an important role in the generation of T lymphocyte-mediated immune responses. Here, we describe a novel accessory molecule, DNAX accessory molecule-1 (DNAM-1), that is constitutively expressed on the majority of peripheral blood T lymphocytes. DNAM-1 is a 65 kDa transmembrane glycoprotein consisting of 318 aa including two immunoglobulin-like domains. Anti-DNAM-1 monoclonal antibody (MAb) inhibits T and NK cell-mediated cytotoxicity against a variety of tumor cell targets and blocks cytokine production by alloantigen-specific T cells. In addition, DNAM-1 is a tyrosine-phosphorylated signal-transducing molecule that participates in primary adhesion during cytotoxic T lymphocyte (CTL)-mediated cytotoxicity.

IgG hybridomas were produced with preferentially reacted with cell-surface antigens of either yeast cells or hyphae of Candida albicans. Four mAbs were used in an immunostaining procedure to follow the expression dynamics of these antigens in media supplemented with glucose or galactose. Yeast cell growth was analysed during the lag phase, the early- and late-exponential phases and the stationary phase, and mycelium formation was analysed between 0.5 and 24 h induction at 37 degrees C. It appears that yeast cell-surface antigens 5C11 and 2E11 are expressed throughout all phases of yeast cell growth as well as on young hyphae after up to 1 h induction. Longer hyphae only faintly react with these two mAbs as they switch to hyphal cell-surface antigens 2G8 and 4E1 after 3 h induction. The reactivity to mAbs 2G8 and 4E1 was induced after a 3 h temperature shift and was confined to the terminal third of growing mycelia. Growth and hyphae induction in galactose prolonged the reactivity of young hyphae with the two anti-yeast-cell mAbs, whereas the expression of surface antigens 2G8 and 2E11 appeared delayed and desynchronized on hyphae. Whereas a similar reactivity was found with ten ATCC strains of C. albicans, four clinical isolates had a unique pattern of reactivity. Immunoblot analyses of DTT extracts of cell-surface constituents indicated that the antigens were proteinaceous in nature and showed that yeast-cell antigens 5C11 and 2E11 are detected in four bands between 68 and 104 kDa, whereas mycelial antigens 4E1 and 2G8 are detected in 117 kDa and 104 kDa bands found in mycelial but not in yeast-cell extracts. Present data support the concept of a dynamic balance in the expression of phase-specific antigens in C. albicans.

A new and rapid method for co-purification of haemagglutinin (HA) and neuraminidase (NA) proteins from influenza A/H3N2 viruses is described. Surface glycoproteins were first solubilized using a non-ionic detergent under high ionic strength conditions, then they were separated by chromatography on sepharose previously bound to monoclonal antibodies (MAbs) directed either against HA (IaH-chromatography) or against NA (IaN-chromatography). Depending on the protein specificity of the MAb immobilized on the column, HA or NA was bound to sepharose and the counterpart protein was free in the flow-through volume. IaH-chromatography and IaN-chromatography proved equally efficient in term of recoveries (> 75%) and purity (> or = 99%) of both HA and NA but differences appeared when considering functional and antigenic properties of pure proteins. Those properties were highly retained in IaH- and IaN-derived HA as well as in IaH-derived NA while IaN-NA was partially degraded. IaH-chromatography allowed the co-purification of HA and NA proteins in heterologous antigen-antibody system with a 50% rate of cross reactivity. IaH-HA and IaH-NA may be suitable for immunity studies, standardization of influenza vaccine and for diagnostic purposes.

A purification process was developed for the manufacture of NS1-OspA as a candidate vaccine for Lyme disease. NS1-OspA was expressed as a soluble recombinant protein in E. coli from 50 and 200 liter fermentations. A multistep bench scale procedure, including detergent treatment, sonication, Q-Sepharose chromatography, dialysis, and SP-Sepharose chromatography, was used to effectively purify NS1-OspA from E. coli to > 90% purity with a 35% yield. This procedure was subsequently modified and specific steps eliminated to create a manufacturing scale process for the purification of NS1-OspA. Continuous flow centrifugation, mechanical lysis, QA-52 anion exchange chromatography, acid induced precipitation, and cross flow filtration gave a final purity of > 75%, a recovery of > 70%, and measured endotoxin levels of < 5 micrograms/mg of protein. Unique features of this process include using unclarified lysates and a single use (disposable) resin for the batch chromatography step. This simple, cost effective production process provides a recombinant protein with the yield and purity suitable for use as a subunit veterinary vaccine.

We have examined autoantigens for IgA anti-keratinocyte cell surface antibodies in 17 intercellular IgA vesiculopustular dermatosis (IAVPD) cases showing only IgA antibodies and 5 cases showing both IgG and IgA antibodies (G A cases). IAVPD cases were divided into two subtypes: (1) intraepidermal neutrophilic IgA dermatosis type showing pustule formation throughout the epidermis and IgA antibodies reactive with the entire epidermis and (2) subcorneal pustular dermatosis type containing IgA antibodies reactive with the uppermost portion of the epidermis. Most G A cases showed atypical clinical features. With immunoblot analysis of normal human epidermal extracts, IgA antibodies in these cases showed no specific reactivity with either pemphigus foliaceus antigen (desmoglein 1) or pemphigus vulgaris antigen (desmoglein 3), except that IgC antibodies in one G/A case each recognized one of the two antigens. With immunoblotting of desmosome enriched fraction obtained from bovine snout epidermis, IgA antibodies in 10 IAVPD and 3 G/A cases and IgG antibodies in 4 G/A cases showed reactivity with either desmoglein 1 or desmocollin another desmosomal cadherin. These results indicate that IAVPD and G/A cases are heterogeneous in terms of both clinical features and antigens and that the IgA autoantibodies in these cases may react with different antigens from those for IgG autoantibodies.

Five cDNA clones were selected from the positive clones detected by screening a germ tube expression library constructed in lambda gt11 with rabbit antisera raised against cell wall extracts of Candida albicans. The selected clones were amplified and used to obtain affinity purified antibodies by eluting from the expressed proteins that had been previously transferred onto nitrocellulose discs. The antibodies obtained were used as probes in immunoblots of the cell wall extracts separated by denaturing polyacrylamide electrophoresis. A single protein band was detected for each clone. Detection of products of the cloned sequences varied according to the extraction procedure and/or cell morphology. These products included bands exhibiting apparent molecular weights of 40, 58, 68 and 70 kDa present in beta-mercaptoethanol (beta ME) extracts from both yeast and germ tubes, and a 30 kDa beta ME extracted protein specific for germ tubes. The expression of these products at the cell surface was confirmed by indirect immunofluorescence. Expression of the mRNAs of the different cDNA clones varied according to growth- and morphology-related factors and showed no direct correlation between expression and presence in the cell wall. These observations suggest that complex mechanisms are involved in the regulation and expression of cell surface components of C. albicans.

The plasma membrane of the avian pathogen Mycoplasma gallisepticum contains about 200 polypeptides including the major lipoprotein pMGA. We have developed a simple and efficient procedure for the purification of three membrane proteins of this wall-less bacterium. Proteins were selectively extracted from isolated plasma membranes with the mild zwitterionic detergent (N-dodecyl-N,N-dimethylammonio) undecanoate (DDMAU) and subjected to size-exclusion chromatography (FPLC) in the presence of the same detergent. Two of the thus separated protein fractions were subjected to a third step involving an anion-exchange chromatography (HPLC), also in the presence of DDMAU, which led to the purification to homogeneity of p67, the major acyl protein of M. gallisepticum plasma membrane (yield, 40%; purification factor, 11), p52 (yield, 38%; purification factor, 20), and p77 (yield, approximately 45%; purification factor, 500). Analyses performed by Western blotting and crossed immunoelectrophoresis showed that the three purified proteins are distinct antigens. Furthermore, N-terminal sequencing confirmed that p67 is pMGA. The method described in this paper is simple, efficient, and nondenaturing; it provides pure proteins, at the milligram level for p52 and p67, and should prove easy to being scaled-up if necessary.

Fimbriae extracts obtained using the thermal shock method, from bovine and porcine enterotoxigenic Escherichia coli strains with K99 and/ or F41 antigens, were analyzed by SDS-PAGE, immunoblotting and haemagglutinating activity. Three major protein bands with molecular weights 17 kDa, 29.3 kDa and 30.9 kDa were detected depending on the strain assayed. A 17 kDa band was identified as the fimbrial subunit for K99 fimbriae and was detected in strains of bovine and porcine origin. The 30.9 kDa band was identified as the fimbriae subunit for F41 fimbriae and was detected in all porcine strains with F41 antigen and only in the bovine strains of serogroups O9 and 0101 that proved positive for F41 antigen. The 29.3 kDa band was shown to be antigenically related to F41 and K88, and was only detected in bovine strains of serogroups O8 (5 strains) and O20 (a single strain). We speculate that the 29.3 kDa band may be related to the CS31A antigen.

A 'new', inherited, high-frequency blood group antigen has been named PEL and is numbered 901014. Two PEL-propositi with anti-PEL have been found, both French-Canadians. Two other French-Canadian propositi with very weak expression of PEL on their red cells have an antibody, provisionally named anti-MTP, which does not react with PEL-cells.

The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate.

CD6 is a 105/130 kDa monomeric T cell surface glycoprotein that has been shown to play a role in human T cell activation. Recently a partial mouse CD6 cDNA sequence was described. We have isolated full-length cDNA clones including the initiation codon and sequence encoding the full signal peptide, as well as an additional 39 amino acids within the cytoplasmic domain as compared to the previously reported clone. The predicted full-length mouse CD6 protein contains 665 amino acids and has the features of a type I integral membrane protein. The extracellular domain of mouse CD6 is composed of three repeated cysteine-rich domains similar to those in human CD6, mouse and human CD5, and other members of a family of proteins whose prototype is the type I macrophage scavenger receptor. In marked contrast to the previously published human CD6 sequence, the mouse sequence predicts a long cytoplasmic tail that is not closely related to other proteins and possesses two proline-rich motifs containing the SH3-domain binding consensus sequence, three protein kinase C phosphorylation site motifs, nine casein kinase-2 phosphorylation site motifs, and a serine-threonine-rich motif repeated three times. Northern blot analysis revealed that mouse CD6 mRNA is expressed predominantly in thymus, lymph node, and spleen. A polyclonal antiserum was raised against mouse CD6 by gene gun plasmid DNA immunization of rabbits with the mouse CD6 cDNA in an expression vector. In immunofluorescence analysis this polyclonal antiserum positively stained the surface of cells transfected with the mouse CD6 cDNA in an expression vector, as well as most normal mouse thymocytes and peripheral T cells. CD6 protein is expressed on most CD4+CD8+ double-positive and CD4+ or CD8+ single-positive thymocytes, and is expressed at highest levels on mature CD3high thymocytes. The expression of mouse CD6 in thymocytes and peripheral T cells correlates closely with the expression of the related CD5 molecule. The polyclonal rabbit anti-mouse CD6 Abs immunoprecipitated a major polypeptide of 128 kDa from resting and 130 kDa from PMA- and FCS-activated mouse thymocytes and lymph node cells; it is likely that this increase in size upon activation is due to phosphorylation of mouse CD6 as has been described for human CD6. These data demonstrate that mouse thymocytes and T cells express a 130-kDa cell surface protein homologous to human CD6.

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.

Thirty-seven Borrelia burgdorferi strains, isolated in 1992 from Ixodes ricinus in Finland, were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting and indirect immunofluorescence assay (IFA) with five to nine monoclonal antibodies (MAbs). By SDS-PAGE results and reactivities to MAbs H3TS, J 8.3, I 17.3, and D6, the 37 isolates were assigned to the species B. burgdorferi sensu stricto (n = 7), Borrelia afzelii (n = 17), or Borrelia garinii (n = 13). Twenty more isolates examined only by IFA and with part of the MAbs were distributed as follows: 9 B. burgdorferi sensu stricto and 11 other species. Among 16 of 37 isolates displaying a SDS-PAGE patterns considered typical of that of B. garinii, 3 were negative by the test with MAb D6; the rest were positive. The three MAb D6-negative isolates reacted with MAb J 8.3 but not with MAb I 17.3. It is suggested that these isolates of a previously undescribed type represent atypical B. afzelii strains deficient in the expression of OspB proteins. The misleading species designation by the SDS-PAGE result is described. The IFA results were generally consistent with those obtained by immunoblotting. The exception was for 3 of 29 isolates that were positive with MAb H5332 by immunoblotting but that were IFA negative. In the present material of 57 strains, all 16 B. burgdorferi sensu stricto isolates originated from the Aland Islands. B. afzelii and B. garinii were isolated from all three regions where ticks were collected. The distributive difference seems to offer a basis for comparative clinico-epidemiological studies of Lyme borreliosis.

The 987P fimbria of enterotoxigenic Escherichia coli is a heteropolymeric structure which consists essentially of a major FasA subunit and a minor subunit, the FasG adhesin. The latter harbors the binding moiety for receptor molecules on piglet intestinal epithelial cells. In this study, anti-FasF antibody probes were developed and used to demonstrate that the FasF protein represents a new minor fimbrial component. FasF was identified in highly purified fimbriae, and its sequence demonstrated significant levels of similarity with that of FasA. Immune electron microscopy localized both the FasG and FasF proteins at the fimbrial tip as well as at broken ends and at various intervals along the fimbrial length. The presence of these minor proteins in purified 987P fimbriae was corroborated by enzyme-linked immunosorbent assay inhibitions. Finally, the use of nonfimbriated fasG, fasF, and fasA mutants indicated that subunit translocation through the outer membrane follows a specific order, FasG being the first, FasF being the second, and FasA being the third type of exported subunit. Since fimbriae are thought to grow from the base, FasG is proposed to be a tip adhesin and FasF is proposed to be a linker molecule between the adhesin and the fimbrial shaft. Moreover, export of FasG (or FasF) in the absence of FasF (or FasA) indicates that during the process of fimbrial biogenesis in the outer membrane, translocating events precede the initiation of subunit heteropolymerization.

Cryptosporidium parvum is a protozoal pathogen which infects the gastrointestinal epithelium of mammals causing diarrhoea, the duration and severity of which is determined by the immunocompetency of the host. Currently, there is no effective treatment or prevention. We evaluated the ability of surface antigen-1 (SA-1), defined as those antigens recognized by neutralizing mAb 17.41, to elicit a protective antibody response when used as an immunogen. A SA-1 enriched fraction was obtained by immunoaffinity chromatography and was used to immunize a naive Holstein calf. SA-1 immune serum from this calf detected C. parvum epitopes to a 1:10,000 dilution in a dot blot assay, and sporozoite surface epitopes at a 1:10,000 dilution in a live immunofluorescence assay. Western blot analysis showed that SA-1 immune bovine serum recognized a similar pattern of C. parvum antigens as the defining mAb 17.41. Oral passive transfer of SA-1 immune bovine serum did not protect severe combined immunodeficient (scid) mice or suckling BALB/c mice from initial infection with C. parvum, or terminate a persistent infection in scid mice.

During a 3-day period, glucose starvation of wild-type Escherichia coli produced thermotolerant, H2O2-resistant, small cells with a round morphology. These cells contained elevated levels of the DnaK protein, adjusted either for total protein or on a per-cell basis. Immunoprecipitation of [35S]methionine-labeled protein produced by such starving cells demonstrated that DnaK underwent continuous synthesis but at decreasing rates throughout this time. Glucose resupplementation of starving cells resulted in rapid loss of thermotolerance, H2O2 resistance, and the elevated DnaK levels. A dnaK deletion mutant, but not an otherwise isogenic wild-type strain, failed to develop starvation-induced thermotolerance or H2O2 resistance. The filamentous phenotype associated with DnaK deficiency was suppressed by cultivation in a defined glucose medium. When starved for glucose, the nonfilamentous and rod-shaped dnaK mutant strain failed to convert into the small spherical form typical of starving wild-type cells. The dnaK mutant retained the ability to develop adaptive H2O2 resistance during growth but not adaptive resistance to heat. Complementation of DnaK deficiency by using Ptac-regulated dnaK+ and dnaK+J+ expression plasmids confirmed a specific role for the DnaK molecular chaperone in these starvation-induced phenotypes.

The two monoclonal antibodies MAX.1 and MAX.11 recognize cell surface antigens that are almost undetectable on monocytes but highly expressed on differentiated macrophages. Biochemical characterization revealed that both antibodies detect the same 58-64-kDa glycoprotein anchored to the plasma membrane by glycosyl-phosphatidylinositol linkage. We purified the MAX.1/11 antigen by immunoaffinity chromatography using monoclonal antibody MAX.11. The NH2-terminal amino acid sequence was determined and turned out to be identical to the NH2-terminal sequence of the membrane-bound carboxypeptidase M. By precipitation with antibodies MAX.1 and MAX.11, membrane preparations of macrophages and placental microvilli were almost completely depleted of enzyme activity, indicating that the two antibodies indeed recognize carboxypeptidase M. Immunoreactivity of both antibodies correlates with the reported tissue distribution of enzyme activity. Expression of carboxypeptidase M on mRNA level and enzymatic activity markedly increase during in vitro differentiation of monocytes, according to the described increase in MAX.1 and MAX.11 antigen expression. Moreover, in vitro differentiated macrophages show the highest specific activity yet described in any tissue. In addition, carboxypeptidase M expression could be detected in HL-60, U937, and THP-1 myeloid cell lines. Vitamin D3-induced monocytic differentiation resulted in an increased carboxypeptidase M expression in all three cell lines. Further studies are needed to elucidate the functional role of carboxypeptidase M during monocytic differentiation and activation.

Giardia lamblia, an intestinal parasite of humans and other vertebrates, undergoes surface antigenic variation by modulating the expression of different variant-specific surface proteins (VSP). VSPs are cysteine-rich surface proteins that bind zinc and other heavy metals in vitro. We developed an immunoaffinity chromatographic method to purify a VSP in order to determine its biochemical properties. The sequences of two different proteolytic fragments agreed with the sequence deduced from the cloned gene, and amino-terminal sequence indicated the removal of a 14-residue signal peptide, consistent with the transport of VSP to the cell surface. The protein is not glycosylated and has an isoelectric point of 5.3. X-ray microanalyses indicated that the major metals in Giardia trophozoites, as well as purified VSP, are zinc and iron. The zinc concentration in Giardia cells was found to be 0.43 mM and the iron concentration 0.80 mM when compared with standard samples (zinc) or calculated from a known physical constants (iron). We propose that metal coordination stabilizes VSPs, rendering them resistant to proteolytic attack in the upper small intestine. Moreover, the ability to bind ions by Giardia may play a role in nutritional deficiency and/or malabsorption in heavily infected persons.

The subcellular localization of the K88 usher FaeD was studied in Escherichia coli whole cells by using isopycnic sucrose density gradient centrifugation of isolated membranes, the detergents Triton X-100 and sodium lauryl sarcosinate and immunoblotting with a specific FaeD antiserum. Cells containing the complete K88 operon, as well as cells containing the subcloned faeD gene in various expression vectors, were used. Most of the FaeD was present in the outer membranes in a detergent-resistant form. Agglutination experiments with E. coli cells expressing FaeD confirmed an outer membrane localization and indicated the presence of FaeD at the cell surface. Automated Edman degradation indicated that the mature FaeD contained 777 amino acid residues and confirmed that FaeD is synthesized with a rather long signal sequence of 35 amino acid residues. Twelve different FaeD-PhoA fusion proteins were prepared and characterized by nucleotide sequencing and immunoblotting. Most of these fusion sites were located in the amino-terminal and carboxyl-terminal regions of FaeD. Six amino-terminal fusion proteins were soluble proteins in the periplasm, whereas the other fusion proteins were associated with the outer membrane. The protease accessibility of FaeD and of the six outer membrane-bound FaeD-PhoA fusion proteins was studied using whole cells, cells with permeabilized outer membranes, and isolated membranes. Collagenase H, kallikrein, trypsin and proteinase K were used. Based on the results of these experiments and computer predictions, a model for the membrane topology of FaeD was developed in which FaeD contains a large central domain containing 24 membrane-spanning segments and two relatively large periplasmic regions, at the amino-terminal and carboxyl-terminal end of the protein, respectively.

The purpose of this study was to investigate the frequency of antibodies in the serum of patients with endometriosis reacting with endometrial, ovarian, thyroid and skeletal muscle antigens. A total of 55 fertile patients with pelvic pain who had endometriosis diagnosed by laparoscopy and confirmed by histology formed the study group, while 43 fertile patients without pelvic pain and undergoing tubal sterilization formed the control group. Eutopic endometrial membrane antigen was prepared from biopsies taken from patients or controls by an ultracentrifugation technique and used in an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig) G, IgA and IgM endometrial antibodies. Ovarian, thyroid and skeletal muscle antigens were prepared similarly and used in an ELISA. There was a significant difference in the prevalence of IgG antibodies to eutopic endometrial antigens derived from either control or endometriosis patients (P < 0.05). IgA or IgM antibodies against endometrial antigens were not detected. Endometrial antibodies were detectable in both the luteal and the follicular phases of the menstrual cycle. Immunoblot analysis demonstrated the presence of endometrial antigens with molecular weights of 60 and 66 kDa in the eutopic endometrium of patients with endometriosis. Antibodies to thyroid, ovary or skeletal muscle were not detected in these patients. These findings indicate that endometrial antibodies of the IgG class can be detected in approximately 50% of patients with endometriosis but it remains unclear whether these represent a pathologically distinct subgroup of such patients.

The primary structure of the acidic capsular polysaccharide of Escherichia coli 08:K43:H11 was shown by monosaccharide analysis, methylation analysis, beta-elimination, and by 1D and 2D 1H and 13C NMR spectroscopy to be composed of branched pentasaccharide repeating units with the structure.

Galactosyl receptor, a cell surface Ca(2+)-dependent lectin with binding affinity for galactose, was evaluated by immunoblotting, immunoprecipitation, Northern blotting, and immunocytochemistry in human liver, testis, and sperm. Polyclonal antisera raised against the minor asialoglycoprotein receptor variant of rat hepatocytes (designated rat hepatic lectin-2/3, RHL-2/3), and its human liver-equivalent (designated H2), recognize native galactosyl receptor in the testis and sperm in immunoblotting, immunoprecipitation, and immunocytochemical experiments. An equivalent to the major hepatocyte asialoglycoprotein receptor variant (rat RHL-1 and human H1) was not detected. Human testis and sperm galactosyl receptor was resolved, after immunoprecipitation and immunoblotting, as a single protein component of molecular mass 50 kD. The single protein component in human testis and sperm contrasted with the doublet nature of rat testis and sperm galactosyl receptor, consisting of two components of molecular masses of 54 and 49 kD. Northern blotting experiments using radiolabeled H1 and H2 cDNA probes confirmed the presence of H2 mRNA and the lack of H1 mRNA in the human testis. Immunocytochemical studies detected specific antigenic sites on the entire surfaces of spermatogenic cells. However, immunoreactivity in epididymal and ejaculated sperm was confined to head surfaces overlying the acrosome. Results from these studies, and from previous studies in the rat, suggest that the testis/sperm galactosyl receptor is a C-type Ca(2+)-dependent lectin with possible roles in cell-cell interaction during spermatogenesis and sperm-zona pellucida binding at fertilization.

Monoclonal antibody 175 recognizes a cell-surface antigen on more than 80% of nucleated ovine bone marrow cells (BMC). The distribution is unusual, as the majority of differentiated myeloid and erythroid cells express the antigen (175 antigen), whereas mast cells, basophils, and the majority of lymphocytes do not. The level of 175 antigen expression has been shown to increase as BMC differentiate during hematopoiesis. Previous attempts to identify the 175 antigen have been unsuccessful. In this study, the 175 antigen was affinity-purified and shown to contain serine protease activity. Immunoblot analysis following sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of bone marrow cell lysates run under reducing or nonreducing conditions showed two closely adjacent protein bands (a doublet) of 28 to 30 kD molecular weight. N-linked deglycosylation showed that the 30-kD band was a glycosylated form of the 28-kD protein. Both protein bands shared the same N-terminal amino acid sequence over 20 residues, with high homology with serine proteases. Affinity-purified 175 antigen was proteolytic in substrate gels, the activity being inhibited by the 175 monoclonal antibody (Mab) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), but not by metallo, thiol, or acid protease-specific inhibitors. The 175 antigen is therefore part of a growing family of cell-surface proteases associated with hematopoietic cell differentiation.