Functional activities and epitope specificity of human and murine antibodies against the class 4 outer membrane protein (Rmp) of Neisseria meningitidis.
Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane. (+info)
Autoantibodies to RNA polymerases recognize multiple subunits and demonstrate cross-reactivity with RNA polymerase complexes.
OBJECTIVE: To determine the subunit specificity of autoantibody directed to RNA polymerases (RNAP) I, II, and III, which is one of the major autoantibody responses in patients with systemic sclerosis (SSc). METHODS: Thirty-two SSc sera with anti-RNAP antibodies (23 with anti-RNAP I/III, 5 with anti-RNAP I/III and II, and 4 with anti-RNAP II alone) were analyzed by immunoblotting using affinity-purified RNAP and by immunoprecipitation using 35S-labeled cell extracts in which RNAP complexes were dissociated. Antibodies bound to individual RNAP subunits were eluted from preparative immunoblots and were further analyzed by immunoblotting and immunoprecipitation. RESULTS: At least 15 different proteins were bound by antibodies in anti-RNAP-positive SSc sera in various combinations. All 9 sera immunoprecipitating RNAP II and all 28 sera immunoprecipitating RNAP I/III recognized the large subunit proteins of RNAP II and III, respectively. Reactivity to RNAP I large subunits was strongly associated with bright nucleolar staining by indirect immunofluorescence. Affinity-purified antibodies that recognized a 62-kd subunit protein cross-reacted with a 43-kd subunit protein and immunoprecipitated both RNAP I and RNAP III. Antibodies that recognized a 21-kd subunit protein obtained from sera that were positive for anti-RNAP I/III and II antibodies immunoprecipitated both RNAP II and RNAP III. CONCLUSION: Anti-RNAP antibodies recognize multiple subunits of RNAP I, II, and III. Moreover, the results of this study provide the first direct evidence that antibodies that recognize shared subunits of human RNAPs or epitopes present on different human RNAP subunits are responsible for the recognition of multiple RNAPs by SSc sera. (+info)
Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis.
BACKGROUND: Decreased synthesis of the predominant secretory human colonic mucin (MUC2) occurs during active ulcerative colitis. AIMS: To study possible alterations in mucin sulphation and mucin secretion, which could be the cause of decreased mucosal protection in ulcerative colitis. METHODS: Colonic biopsy specimens from patients with active ulcerative colitis, ulcerative colitis in remission, and controls were metabolically labelled with [35S]-amino acids or [35S]-sulphate, chase incubated and analysed by SDS-PAGE, followed by quantitation of mature [35S]-labelled MUC2. For quantitation of total MUC2, which includes non-radiolabelled and radiolabelled MUC2, dot blotting was performed, using a MUC2 monoclonal antibody. RESULTS: Between patient groups, no significant differences were found in [35S]-sulphate content of secreted MUC2 or in the secreted percentage of either [35S]-amino acid labelled MUC2 or total MUC2. During active ulcerative colitis, secretion of [35S]-sulphate labelled MUC2 was significantly increased twofold, whereas [35S]-sulphate incorporation into MUC2 was significantly reduced to half. CONCLUSIONS: During active ulcerative colitis, less MUC2 is secreted, because MUC2 synthesis is decreased while the secreted percentage of MUC2 is unaltered. Furthermore, sulphate content of secreted MUC2 is unaltered by a specific compensatory mechanism, because sulphated MUC2 is preferentially secreted while sulphate incorporation into MUC2 is reduced. (+info)
Induction of hepatic cytochromes P450 in dogs exposed to a chronic low dose of polychlorinated biphenyls.
Induction of cytochrome P450 isoforms, specifically CYP1A1, and their catalytic activities are potential biomarkers of environmental contamination by polychlorinated biphenyls (PCBs). In this study, dogs were exposed to 25 ppm or 5 ppm Aroclor 1248 (PCB mixture) daily in their diet for 10 or 20 weeks, respectively. Relative to controls, hepatic microsomes from dogs dosed with PCBs had higher levels of CYP1A1 detected in immunoblots and higher levels of EROD activity, but low levels of induction for CYP2B and PROD activity. Concentrations of 96 PCB congeners in serum and liver were evaluated using capillary chromatography. Results showed that all dogs exposed to PCB mixtures had higher levels of PCB in serum and liver. Dogs preferentially sequestered highly chlorinated PCB congeners in liver relative to serum. With these experiments, we demonstrated that EROD activity was a potentially sensitive marker of PCB exposure at 5 and 25 ppm. Furthermore, CYP1A1 and EROD activity were maximally induced in dogs consuming dietary concentrations only 2.5 times the maximal permissible level for human food (FDA). The value of CYP1A1 induction as a biomarker of PCB exposure was tenuous because neither CYP1A1 levels nor EROD activity correlated with total PCB body burden. However, a small subset of congeners were identified in liver that may strongly influence EROD and PROD induction. Finally, two dogs in the 25 ppm dose group were fasted for 48 h. After 24 h of fasting, several new congeners appeared in the serum and remained in the serum for the remainder of the fast. The fast caused a 293% increase in PCB concentration in serum. This increase has strong implications regarding mobilization of toxic PCBs in wildlife during fasting (e.g., migration, hibernation). (+info)
Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage.
Four expression cassettes containing strong fungal promoters, a signal sequence for protein translocation, a KEX protease cleavage site, and a synthetic gene (tha) encoding the sweet protein thaumatin II were used to overexpress this protein in Aspergillus awamori lpr66, a PepA protease-deficient strain. The best expression results were obtained with the gdhA promoter of A. awamori or with the gpdA promoter of Aspergillus nidulans. There was good correlation of tha gene dosage, transcript levels, and thaumatin secretion. The thaumatin gene was expressed as a transcript of the expected size in each construction (1.9 or 1.4 kb), and the transcript levels and thaumatin production rate decayed at the end of the growth phase, except in the double transformant TB2b1-44-GD5, in which secretion of thaumatin continued until 96 h. The recombinant thaumatin secreted by a high-production transformant was purified to homogeneity, giving one major component and two minor components. In all cases, cleavage of the fused protein occurred at the KEX recognition sequence. This work provides new expression systems in A. awamori that result in very high levels of thaumatin production. (+info)
Immunochemical detection and isolation of DNA from metabolically active bacteria.
Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community. (+info)
In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes.
Individual cyanobacterial cells are normally identified in environmental samples only on the basis of their pigmentation and morphology. However, these criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identification of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since fluorescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later, it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, TSA-based detection of HRP was more sensitive than that based on nonfluorescent substrates. Critical points of the assay, such as cell fixation and permeabilization, specificity, and sensitivity, were systematically investigated by using four oligonucleotides newly designed to target groups of cyanobacteria. (+info)
RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae.
The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A genetic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation groups. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53, which was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlike other RAD53 alleles, we demonstrate that the rad53-31 allele retains an intact checkpoint function. Thus, the checkpoint function and the DNA replication function of RAD53 can be functionally separated. The activation of DNA replication through RAD53 most likely occurs through DBF4. Two-hybrid analysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the steady-state level of DBF4 message and Dbf4p protein is reduced in several rad53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulated through the common intermediate RAD53. (+info)