Common epitopes of protein antigens in Neisseria and Moraxella

Cross reactions between N. meningitidis and M. catarrhalis proteins were studied with the use of a panel of monoclonal antibodies to M. catarrhalis protein antigens. All antigenic preparations under study were shown to give cross reactions between N. meningitidis serotype porin of 39 kD (strain B125) and M. catarrhalis proteins of 40-41 kD. These M. catarrhalis proteins belonged to main proteins of class F and had the function of porins in the cell. In addition, the epitope of 41-kD antigen, detected by monoclonal antibodies 3E10, is common for both N. meningitidis porin and N. meningitidis iron-regulated proteins of 70 and 50 kD. The epitope of M. catarrhalis protein of 67 kD, detected by monoclonal antibodies 1G6, is common for N. meningitidis porin and N. meningitidis iron-regulated proteins of 50 and 55 kD.     

Immunobiological properties of monoclonal antibodies to Mycoplasma hominis antigens

Approaches to obtaining stable mouse hybridomas synthesizing monoclonal antibodies (McAb) to M. hominis key antigens were developed. 4 clones capable of the stable synthesis of McAb of different IgG classes were obtained. Clones A3/2 and A5/D produced antibodies to the thermostable determinant to with a mol. wt. of 80-120 kD, sensitive to sodium periodate and resistant to potassium proteinase. Clone H9/B2 synthesized McAb which interacted with potassium proteinase-sensitive M. hominis thermolabile determinant with a mol. wt. of 80 kD. McAb of clone A3/2, labeled with fluorescein isothiocyanate and horse-radish peroxidase, specifically reacted with M. hominis antigens in the immunofluorescence test and the immunoenzyme assay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data may serve as prerequisites for the development of diagnostic test systems aimed at the detection of M. hominis antigens in different clinical substances.     

Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica

Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.     

Electrophoretic Studies on Seed Protein of the Genus Lathyrus

SDS-polyacrylamide gel electrophoresis of total seed extracts revealed the presence of legumin-like polypeptides ranging in molecular weight from 42 kD to 89 kD in Lathyrus sativus and L. odoratus. The polypeptides of higher mol wt were however, absent in L. aphaca. Vicilin-like polypepides of mol wt 76, 54, 36, 33, 31, 20 and 17 kD were seen in L. sativus and L. odoratus and of mol wt 72, 58, 54, 52, 36, 33 and 20 kD in L. aphaca. Analysis of various seed protein fractions of L. sativus revealed the presence of a large number of albumin polypeptides varyingin mol wt range from 12.5 to 95 kD, when as glutelin (mol wt 18 to 80 kD) and prolamin (mol wt 25.5 and 26 kD) fraction polypeptides were relatively fewer. On the basis of similarities in seed polypeptide profiles, L. sativus and L. odoratus seem to be closely related, whereas L. aphaca appears to be distantly related.     

Inhibitor analysis of protein phosphorylation/dephosphorylation in maize mitochondria in relation to redox conditions

The role of protein phosphorylation/ dephosphorylation in the redox regulation of mitochondrial functioning was investigated. Incubation of isolated mitochondria of maize (Zea mays L.) in the presence of γ-³²P-ATP revealed phosphorylation of polypeptides with mol wt of 66, 60, 55, 48/50 doublet, 45, 29, 22, and 19 kD. The presence in the incubation medium of oxidized glutathione significantly reduced the level of protein phosphorylation. The addition of reduced glutathione diminished phosphorylation of proteins with mol wt of 60 and 48/50 kD and slightly increased phosphorylation of proteins with mol wt of 66, 55, and 45 kD. The reducing agent, sodium dithionite decreased phosphorylation of proteins with mol wt of 60, 45, 29, 22, and 19 kD but increased phosphorylation of 55 kD protein. The inhibitors of protein kinases and protein phosphatases significantly modified the effects of redox agents. For example, simultaneous action of an oxidant K₃[Fe(CN)₆] and NaF enhanced phosphorylation level compared to separate treatments with these agents. The combined application of sodium dithionite and NaF elevated phosphorylation level of 55 kD protein. Phosphoprotein with mol wt of about 66 kD was identified immunochemically as a heat shock protein (HSP 60). The results indicate the presence in mitochondria of redox-sensitive protein kinases and protein phosphatases. Differential changes in the pattern of mitochondrial phosphoproteins under the action of various redox agents suggest that phosphorylation is probably involved in the transduction of redox signal in plant mitochondria.     

Antibodies to iron-regulated proteins of meningococci in blood sera of healthy persons of different age groups

One hundred and twenty individual sera obtained from healthy persons of different age groups were studied for the presence of antibodies to meningococcal iron-regulated proteins (IRP). The study revealed that occurrence of such antibodies in sera under study was IRP nature- and age-dependent. Antibodies to two IRP were found to occur most frequently: 85 kD (TbpB) and 72 kD (FrpB). Antibodies to the former IRP were detected in more than 50% and antibodies to the latter IRP, in more than 90% of sera. This was probably due to the presence of epitopes common with those in protein antigens of some other microorganisms, such as Moraxella catarrhalis and Haemophilus influenzae. The occurrence of antibodies to periplasmatic IRP with 34 kD (FbpA) in blood sera varied within the range of 5 to 30%. At the same time the occurrence of antibodies to this protein in the sera under study was age-depended: children until five years exhibited the minimal occurrence (about 5%), while in adults it reached 30%.     

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.     

The use of monoclonal antibodies for detecting the common antigenic determinants of Brucella spp. and Yersinia enterocolitica O:9

Monoclonal antibodies (McAb) 2AH10, specifically reacting with protein preparations having mol. wt. of 18 and 38 kD and not interacting with brucellar lipopolysaccharide (LPS) and protein-polysaccharide antigen, have been obtained. As shown with the use of McAb 2AH10, Brucella spp. and Yersinia enterocolitica O:9 possess common antigenic determinants, localized not only in the area of their LPS, which is generally known, but also in the area of their outer cell-wall proteins with mol. wt. 18 and 38 kD. The sensitivity of the solid-phase enzyme immunoassay and the latex agglutination test with the use of McAb 2AH10 is essentially higher in the detection of B. abortus and B. suis, than B. melitensis and B. rangiferi, as well as Y. enterocolitica O:9. Essential differences observed in the detected concentrations of different Brucella species and Y. enterocolitica O:9 are seemingly linked with different expression of specific antigenic determinants, detected with the use of McAb 2AH10 in the corpuscular antigens under study.     

Analysis of Antigens in Mycobacterium Paratuberculosis

Analysis of antigens in Mycobacterium paratuberculosis. Acta vet. scand. 1979, 20, 200–215. — Using crossed immunoelectrophoresis (GIE) and crossed line immunoelectrophoresis (GLIE), antigens from different strains and variants of Mycobacterium paratuberculosis were compared, and cross-reactions between 1 of these strains and Mycobacterium avium and BGG studied. In each of 4 bovine laboratory strains of M. paratuberculosis examined, altogether 44 different antigens were demonstrated. This is the largest number of antigens in M. paratuberculosis which has been described so far. No important difference in the antigenic structure of the strains was found. The 4 laboratory strains are being used routinely in the production of vaccine against Johne’s disease in Norway and Iceland. One of the aims of the present work was to investigate the antigenic relationship between these strains and the goat-pathogenic Norwegian and the Icelandic variant of M. paratuberculosis. Out of 44 different antigens demonstrated in the laboratory strains, 39 and 31 gave cross-reactions against the Norwegian and the Icelandic variant, respectively. This is in accordance with practical experience, as the results of vaccination against Johne’s disease, performed in Norway for many years, are very good. Twenty-seven and 24 cross-reacting antigens between M. paratuberculosis and strains of M. avium and BGG, respectively, were observed. This finding agrees with clinical observations. Another aim of the investigation was to identify species-specific antigens as regards M. paratuberculosis. One antigen showed a marked cross-reaction between the strains of M. paratuberculosis examined, but did not react with antisera against M. avium and BGG. Some other antigens showed partial specificity. The results obtained stress the complicated antigenic situation in mycobacteria which is of decisive significance as regards the diagnosis and classification of mycobacterial infections.     

The molecular weight cut-off of microcapsules is determined by the reaction between alginate and polylysine

Mammalian cells encapsulated in alginate-polylysine microcapsules are used as artificial organs in cancer research and in biotechnology. These applications require microcapsules with a reproducible mol. wt. cut-off. The high cost of the polycation, polylysine, requires an efficient preparation procedure. This article shows that the overall reported contact time of 5 minutes at ambient conditions should be increased several times in order to reach a maximal binding between the calcium alginate beads and 0.1% (w/v) polylysine solutions. An increase of the polylysine concentration from 0.0125% to 0.8% (w/v) resulted in a faster maximal binding, but the amount of polylysine bound increased also. Immersion of calcium alginate beads with a diameter of 750 mum, prepared from 1 mL alginate, in 30 mL of a 0.8% (w/v) polylysine solution, resulted in a polylysine spill of more than 89%. The time required to reach a maximal binding was related to the reaction temperature. The interaction zone between calcium alginate beads and fluorescein isothiocyanate-labeled polylysine solutions was visualized with a confocal laser scanning microscope as a function of time. Microcapsules, prepared at 40 degrees C with 0.1% (w/v) polylysine solutions with mol. wts. between 12 and 249.2 kD, were permeable for fluorescein isothiocyanate-labeled dextran, mol. wt. 4.7, but not for 40.5 kD. Higher polylysine concentrations resulted in a membrane with a mol. wt. cut-off lower than 4.7 kD. (c) 1993 John Wiley & Sons, Inc.     

Exploration of Moraxella catarrhalis outer membrane proteins, CD and UspA, as new carriers for lipooligosaccharide-based conjugates

Moraxella catarrhalis outer membrane proteins, CD and ubiquitous surface protein A (UspA), were used as carriers for M. catarrhalis detoxified lipooligosaccharide (dLOS)-based conjugates. Our study was designed to investigate the feasibility of CD and UspA as protein carriers for dLOS-based conjugates and their possible synergic effects on protection from both anti-LOS and anti-CD or anti-UspA antibody responses. Female Balb/c mice were immunized subcutaneously three times with dLOS-CD or dLOS-UspA conjugate in Ribi adjuvant. Antisera elicited by the conjugates showed high titers of specific anti-LOS antibodies with complement-dependent bactericidal activity towards M. catarrhalis strain 25238. In a mouse aerosol challenge model, mice immunized with both conjugates showed a significant enhancement of the clearance of strain 25238 from lungs as compared with the control mice. Although both conjugates elicited reduced (relative to unconjugated CD or UspA) but significant levels of anti-CD or UspA antibodies, they did not show synergetic effects with anti-LOS antibodies on the bactericidal activity or the pulmonary bacterial clearance. Nevertheless, CD and UspA are safe and effective new carriers for dLOS-based or other potential carbohydrate-based conjugate vaccines to help thymus-independent carbohydrate antigens for production of anti-carbohydrate antibodies against target pathogens.     

Z-DNA-binding proteins from bull testis.

Three Z-DNA-binding proteins of Mr 31, 33 and 58 kD were isolated from mature bull testis. They were obtained in a native state suitable for binding studies. These are the first examples of Z-DNA-binding proteins from a mammalian tissue. Purification involved tissue extraction with 0.35 M NaCl, cation exchange chromatography on CM-Trisacryl M and two consecutive anion exchange FPLC runs on Mono Q. The proteins appeared virtually homogeneous by anion exchange FPLC, SDS polyacrylamide gel electrophoresis and reverse phase HPLC (58 kD protein only). Yields from 50 g of testis tissue were: 31 kD protein, 40 micrograms; 33 kD protein, 100 micrograms; and 58 kD protein, 150 micrograms. Z-DNA binding was determined by Scatchard analysis of filter binding data using brominated poly(dG-dC).poly(dG-dC) as a conformation-specific ligand. Dissociation constants (Kz, in mol nucleotide/liter) were: 31 kD protein, 7 X 10(-7) M; 33 kD protein, 8 X 10(-7) M; 58 kD protein, 6 X 10(-8) M (primary binding site) and 6 X 10(-7) M (secondary binding site). B-DNA binding to poly(dG-dC).poly(dG-dG) was too low for reliable determination under the conditions of assay. This attested to a high degree of conformational specificity of the three proteins. The 58 kD protein bound Z-DNA at the primary site with an affinity almost equivalent to that of a polyclonal anti-Z-DNA antiserum raised in a rabbit (Kz, 4 X 10(-8) M).     

Sequence homology of group A streptococcal Pep M5 protein with other coiled-coil proteins

Group A streptococcal Pep M5 protein, an antiphagocytic determinant of the bacteria, is an alpha-helical coiled-coil molecule, and exhibits significant sequence homology with tropomyosin and myosin, but to a lesser degree with other coiled-coil proteins. Moreover, Pep M5 is more homologous to myosin than to tropomyosin, and the homologies are more numerous between the C-terminal domain of the Pep M5 protein and the S2 fragment of myosin. The C-terminal domain of the Pep M5 protein exhibits extensive sequence identity with the C-terminal region of Pep M6 molecule, another M protein serotype. Thus, regions within two M protein serotypes are homologous to the S2 region of the myosin molecule. These observations are consistent with the immunological findings of other investigators and thus may explain some of the previously reported immunological cross-reactions between antigens of the group A streptococcus and mammalian heart tissue.     

Proteins of cotyledons of mature horse chestnut seeds

This is the first characterization of proteins from storage parenchyma of cotyledons of mature dormant recalcitrant horse chestnut (Aesculus hippocastanum L.) seeds and evaluation the cell protein-synthesizing capacity. It was established that the content of protein in cotyledons did not exceed 0.5% of tissue fresh weight. Soluble proteins (the proteins of the postmitochondrial supernatant or cytosol) comprised the bulk (up to 90%) of total proteins. Protein of subcellular structures (20000 g-pellet) comprised 5–7% of total protein. Cotyledon proteins were heterogenous in their charges and molecular weights of subunits. Cotyledon protein was easily extracted with a salt (1 M NaCl); they comprised 90% of water-soluble albumin-like proteins. The proportion of globulins was insignificant; it did not exceed 5%. Most water-soluble proteins (more than 80%) were tolerant to heat denaturing. Among these heat-stable proteins, two major groups of polypeptides dominated: an electrophoretically homogeneous component with a mol wt of 24–25 kD and a complex group from three to five polypeptides with mol wts in the range between 6 and 12 kD. Native heat-stable proteins had disulfide bonds. Four fractions of heat-stable proteins were obtained by ammonium sulfate fractionation; three of them were alike in their polypeptide composition and contained major components with mol wts of 24–25 and 5–12 kD. It was established that the active translational machinery functioned in the cells of storage parenchyma in cotyledons of mature dormant horse chestnut seeds. During each stage of stratification, cotyledon fragments incorporated ³⁵S-methionine into TCA-insoluble material more actively than axial organs. We discuss cotyledon protein composition, their function as a storage organ, and a possible role of heat-stable proteins.     

Isolation of a novel rat testicular metalloprotein binding cadmium and zinc

Various testicular metal-binding proteins having apparent mol wt in the range of 10–30 kD have been demonstrated by gel filtration of¹⁰⁹Cd- or⁶⁵Zn-labeled cytosol, but in no case has a purified metalloprotein been isolated that contains stoichiometric amounts of the metal. The purpose of this work was to purify from rat testes a testes-specific 30 kD Cd-binding protein (Cd-testin) following in vitro addition of¹⁰⁹Cd to testis cytosol. Conventional purification methods similar to those used for purification of metallothionein could not be used because Cd was not retained in stoichiometric amounts by the 30 kD species when these methods were employed. However, using ammonium sulfate fractionation, hydrophobic interaction and gel filtration chromatography, a 30 kD protein containing 2.6 mol of Cd/ mol of protein was isolated. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the isolated protein contained one major polypeptide with a mol mass of 22 kD and a pI of 4.6 (22 kD/pI 4.6) and two minor polypeptides (16 kD/pI 4.6 and 10±4 kD/pI 6.3) Two-dimensional gel electrophoresis demonstrated that the 22 kD species is a major low mol mass (<60 kD) protein in rat testic cytosol. The 22 kD protein was not detectable in cytosol of rooster testis, a tissue that is insensitive to Cd-induced damage and devoid of the 30 kD Cd-binding protein. Gel filtration and hydrophobic interaction chromatography of¹⁰⁹Cd- and⁶⁵Zn-labeled cytosol demonstrated that¹⁰⁹Cd and⁶⁵Zn cochromatography with the 30 kD protein. The function of this novel 30 kD testicular metal-binding protein is not known, but our work and other studies suggest that its occurrence in testes is linked to the production of a unique 22 kD polypeptide.     

Cross-reacting antigens of pathogenic Burkholderia and some dangerous causative agents of infectious diseases

Cross-reacting antigens in B. mallei, B. pseudomallei, B. thailandensis, Francisella tularensis, Yersinia pestis and Mycobacterium tuberculosis were studied with the use of immuno- and electrophoretic techniques. The set of antigens was shown to be almost identical in the causative agents of glanders, melioidosis, as well as in B. thailandensis, though in the latter organism 200-kD glycoprotein was absent. The analysis of immuno- and proteinograms demonstrated the presence of cross-reactions in the representatives of the genus Burkholderia with the causative agents of plague, tularemia and tuberculosis, which served as the basis for making the scheme of their antigenic relationships. The use of immunosorption techniques with subsequent analysis of the preparations by means of the SDS polyacryl gel electrophoresis and immunoblotting made it possible to characterize cross-reacting antigens of the pathogenic microorganisms under study, to establish their molecular weights (81-15 kD) and to show that some detected antigens are analogous to B. pseudomallei outer membrane proteins (34 and 30 kD).     

Conjugative plasmids of Salmonella strains resistant to antibiotics

Plasmid DNA profile and conjugative R-plasmids were detected in Salmonella clinical isolates. The study revealed that drug resistance of Salmonella clinical strains of different serovars was determined by R-plasmids with a mol. wt. of 60-90 kD, carrying a certain spectrum of resistance to antibiotics. 9 types of conjugative plasmids, differing in their mol. wt. and resistance markers, were detected.     

Monoclonal antibodies for carcinoembryonic antigen (CEA) as a model system: identification of two novel CEA-related antigens in meconium and colorectal carcinoma tissue by Western blots and differential immunoaffinity chromatography

In a previous study, five monoclonal antibodies against the carcinoembryonic antigen (CEA) with different epitope specificities were delineated. One of these antibodies which exhibits a high affinity for CEA binds to different carcinoma tissues, to liver tissue, and to granulocytes. This antibody was selected for the immunoaffinity purification of CEA and related antigens from colorectal carcinoma tissue, from spleen tissues, from bile, and from meconium. After elution from the immunosorbent, the antigens were separated by SDS-PAGE, were transferred to nitrocellulose, and were incubated with the five different antibodies. Antibody T84.1 bound to the following antigens: 177 kD and 128 kD from colonic carcinoma, 81 kD from bile, 49 kD from spleen, as well as 165 kD and 100 kD from meconium. Two additional antibodies showed a similar binding pattern. The fourth antibody (CEA.11) bound to the 165 kD meconium antigen and to the two colorectal carcinoma antigens. The fifth antibody (T84.66) showed a strong reaction with the 177 kD colorectal carcinoma antigen and a faint reaction with a 183 kD antigen in meconium. As judged from m.w. and immunochemical properties, the 128 kD colorectal carcinoma antigen and the 100 kD meconium antigen are two novel CEA-related antigens. Because antibody CEA.11 did not bind to the 100 kD meconium antigen in Western blots, the 165 kD antigen could be eluted from a CEA.11 immunosorbent without contamination by the 100 kD antigen. Similarly, as predicted from the binding pattern in the Western blots, the two colorectal carcinoma antigens were separated from each other by a T84.66 immunosorbent.     

Identification of a novel glycosyltransferase involved in LOS biosynthesis of Moraxella catarrhalis

Moraxella catarrhalis is an important human mucosal pathogen that contributes to otitis media in infants and exacerbates conditions such as chronic obstructive pulmonary disease in the elderly. This study describes the identification of a novel gene, lgt5 that encodes a glycosyltransferase involved in the LOS biosynthesis of M. catarrhalis. Analysis of NMR data of LOS-derived oligosaccharide from a Serotype A lgt5 mutant strain of M. catarrhalis indicate that lgt5 encodes an alpha-(1-->4)-galactosyltransferase.     

Specific antigen of Gnathostoma spinigerum for immunodiagnosis of human gnathostomiasis

Sera from four patients with parasitologically confirmed gnathostomiasis, 15 patients with presumptive gnathostomiasis, 64 patients with various parasitic infections and 19 healthy adults were studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for their reactivities against somatic extract of Gnathostoma spinigerum third-stage larvae (L3). It was found that the L3 extract was highly complex consisting of more than 20 antigenic components, a few of which gave reactions with sera from the healthy controls. Extensive cross-reactions of the parasite's antigen with sera from patients with other parasitic infections occurred. A specific antigen of G. spinigerum with a mol. wt of 24,000 (24k) was found to react with all parasitologically proven patients, five of the presumptive patients, one of the patients with other parasitic infections and none of the healthy individuals. This 24k component of G. spinigerum is a potential diagnostic antigen for use in the immunodiagnosis of human gnathostomiasis.