Biomedical subjects
Publications and source records attributed to J Swanson.
Neisseria gonorrhoeae exhibits striking variability in several of its surface components (pili, Opa proteins and lipooligosaccharide) in vivo and in vitro. Such flagrant variation of this mucosal pathogen's surface components contrasts sharply with changes in single surface components of blood-borne trypanosomes and borreliae. Despite these differences, similar molecular events are sometimes involved.
Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.
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Colony opacity variants were detected for type III group B streptococci (GBS). Transparent colonies predominate in the parent GBS, with occasional colonies having opaque portions. Two stable opaque variants (1.1 and 1.5) were compared with three transparent clones (1.2, 1.3, and 1.4). All grew well on blood agar and on GC medium, but variant 1.1 failed to grow on Todd-Hewitt medium. Scanning and transmission electron microscopy demonstrated that colony opacity correlated with bacterial aggregation status, with opaque variants forming longer and more organized chains. Opaque-transparent switches were observed in both directions for most variants, with transparent to opaque noted most frequently, but 1.5 did not switch at all. Switching of the opacity phenotype was observed both in vitro and in neonatal mice. Relationships between colony opacity and several cell surface phenomena were explored. (i) Opaque variant 1.1 had two surface proteins (46 and 75 kDa) that were either unique or greatly overexpressed. (ii) Variant 1.1 was deficient in type III polysaccharide, while 1.5 lacked group B antigen. Diminished capsular polysaccharide of variant 1.1 was reflected in reduced negative electrophoretic mobility and in increased buoyant density. (iii) Transparent variant colonies growing closest to a penicillin disk were opaque, but colonial variants did not differ in their sensitivity to penicillin. These data indicate that GBS can exist in both opaque and transparent forms, with opaque appearance occurring by multiple routes. Opaque variants grow poorly on Todd-Hewitt medium generally used for isolation of GBS, so any possible relationships between opacity variation and pathogenesis of GBS infection are unknown.
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Variants of Neisseria gonorrhoeae MS11 show distinct colony morphologies because of the expression of a class of surface components called opacity (Opa, PII) proteins. Southern analyses combined with molecular cloning of genomic DNA from a single variant of MS11 has identified 11 opa genes contained in separate loci. These opa genes code for distinct opacity proteins which are distinguishable at their variable domains. The opa gene analyses were also extended to divergent variants of MS11. These studies have shown that, during in vitro and in vivo culture, 10 of the 11 opa genes did not undergo significant change in their primary sequence. However, in these variants, one gene (opaE) underwent non-reciprocal inter-opa recombinations to generate newer Opa variants. Phylogenic analysis of the opa gene sequences suggests that the opa gene family have evolved by a combination of gene duplication, gene replacement and partial inter-opa recombination events.
Spontaneous mutants of Neisseria gonorrheae failing to express pili or having diminished levels of piliation were studied with regard to pilin expression. All mutants displayed altered pilin processing detectable as the release of soluble, truncated pilin molecules (S-pilin). Of particular interest was the finding, in one mutant, that substitution of serine for glycine at position -1 of propilin, a highly conserved residue among N-metPhe and related pilins, abolished pilus expression but not S-pilin release. The degree of S-pilin processing and the levels of membrane-associated pilin varied among the different classes of mutants, suggesting that each was blocked at a distinct step of pilus biogenesis. The data support a model in which increased S-pilin processing is a result of a decreased rate of pilus polymerization.
A national sample of community health nurses were surveyed to assess their knowledge of, attitudes toward, and involvement with reproductive health services. Results indicated that slightly more than half of the nurses provided or administered contraceptive services to adolescents, although 73% worked in settings where contraceptive services were provided to adolescents. However, only 3.6% indicated that adolescents were their primary client population. The majority (64.3%) stated that they felt as prepared to work with adolescents as they did with adult clients. While 98% were aware that most adolescents are sexually active before seeking family planning services, a minority correctly answered questions about pregnancy and contraception among adolescents. Ninety-five percent agreed that contraceptives should be available to adolescents, and 90% agreed that parental consent should not be required for adolescents seeking contraceptive services. Yet only 21.5% were aware of the implications of the Adolescent Family Life Act.
Recombination-dependent alterations of their expressed pilin gene (pilE) enable gonococci to synthesize a myriad of structurally/antigenically different pili and to reversibly switch their pilus production on and off. These changes have been ascribed both to DNA transformation and to intragenomic recombination between pilE and silent pilin genes (pilS). We examined the pilus changes in gonococci that are incompetent for transformation because of their DNA uptake deficiency (dud) mutation, pilus- (P-) phenotype, or both. Though incompetent for DNA transformation, dud cells displayed pilus antigenic variation and underwent reversible pilus variations much like their wild-type parent. Wild-type P- with a pilE nonsense mutation were also virtually nontransformable, but they reverted to P+ at high frequencies. The pilin mRNA sequence changes that accompanied pilus transitions in these nontransformable dud and P- gonococci represent insertion of pilS stretches into their respective pilE, apparently via intragenomic recombination.
Endotoxin has been found to be a contaminant in human in vitro fertilization (IVF), leading to low pregnancy rates, mostly through embryo fragmentation. We wished to determine if standard quality control assays (one-cell and two-cell mouse embryo culture) were able to detect the presence of endotoxin and how they compared with the zona-free mouse embryo assays. Endotoxin was detected by the zona-free mouse embryo assays but not by the one- or two-cell mouse embryo assays. When excessive embryo fragmentation is noticed in the human IVF system, the use of zona-free mouse embryo assays is recommended.
Previous studies indicate that gonococcal pilin phase and antigenic variation occur by intragenomic pilin gene recombination, the outcome of which resembles that of gene conversion. During such transitions, the expressed complete pilin gene (pilE) acquires a novel sequence corresponding to that of a silent pilin gene (pilS). In the present study, we find that internal deletions of pilE can produce pilus-/pilus+ phase transitions: direct oligonucleotide repeats in the pilin-encoding portion of pilE bracket the deleted segments. A novel, orthodox pilE is formed upon repair of the internal deletions, with pilS sequence probably acting as a template for repair. Such deletion/repair of pilE is suggested as a principal mechanism underlying gonococcal pilus variation.
Unintended pregnancy is a health problem that is particularly important in the practice of public health nurses (PHNs). Data from 844 PHNs showed that they were likely to practice in settings that incorporate family planning services and were knowledgeable about specific family planning methods. They favored family planning services and an expanded supportive government role in both family planning and abortion. Since PHNs are an important resource for the community activism required to build the consensus needed to expand these services, a multiple-regression analysis was performed to determine predictors of activism. Political participation was the major predictor for activism on both family planning and abortion.
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We have examined the combined effects of transforming growth factor-beta (TGF-beta), serum and gamma-interferon (gamma-IFN) on collagen synthesis by fibroblasts and compared the response of fibroblast subpopulations to TGF-beta. Human diploid fibroblasts were treated with TGF-beta alone and with serum of gamma-IFN. Cells were labelled with radioactive amino acids, and collagen production was measured as collagenase-digestible radioactivity. Collagen mRNA was determined by a solution-hybridization assay using procollagen-alpha 1[I] cDNA clone HF 677. The results showed that either serum or TGF-beta increased incorporation, collagen production and mRNA by fibroblasts approx. 2-fold; however, collagen synthesis relative to total protein synthesis and collagen mRNA relative to total polyadenylated [poly(A)+] RNA were not affected. Only serum activated cell growth. Collagen production increased approx. 4-fold in cells exposed to both TGF-beta and serum, and this increase was equal to that expected for an additive effect by both components. Treatment with gamma-IFN decreased collagen production and collagen mRNA to 44 and 40% respectively, whereas total incorporation and poly(A)+ RNA were affected only marginally. Cells exposed simultaneously to both gamma-IFN and TGF-beta produced less collagen and contained less mRNA than did those treated with TGF-beta alone. The gamma-IFN decreased collagen synthesis in control and TGF-beta-treated cultures to a similar extent, and TGF-beta increased collagen synthesis 2-fold in cells pre-treated with gamma-IFN. Fibroblast strains obtained in medium containing plasma-derived serum synthesized approximately half as much collagen as did cells derived from the same explant in the presence of fresh human serum, and TGF-beta stimulated collagen production and mRNA in both cell strains. We conclude that TGF-beta, serum and gamma-IFN regulate collagen synthesis by independent mechanisms, and that the combined action of these components plays a significant role in regulating collagen synthesis during wound healing and tissue repair.
Because methylene blue exhibits germicidal, oxidation, and reduction properties, the authors asked whether this agent causes adverse effects on gametes, embryos, and/or secretions of the reproductive tract. Time- and dose-dependent inhibition of human sperm motility by methylene blue was observed, as was growth inhibition of 2-cell mouse embryos. Furthermore, the presence of methylene blue in uterine, fallopian tube, and peritoneal fluids altered protein mobility in polyacrylamide gels, and yielded apparent values of follicle-stimulating hormone and estradiol up to 260% of actual values (P less than 0.05). These data suggest that the presence of methylene blue in reproductive tract fluids may provide a false impression of their biochemical and biophysical compositions, and that the use of methylene blue as a chromopertubation agent be conducted with appropriate awareness.
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Expression and phase variation of Neisseria gonorrhoeae P.II genes in Escherichia coli were studied using TnphoA fusions. Fusions were created in the P.IIc gene of N. gonorrhoeae JS3 using lambda TnphoA-1 and were characterized by restriction digestion and dideoxy sequencing. Three fusions were chosen for further study; Tnp7 (fusion junction at mature amino acid 7), Tnp57 (amino acid 57), Tnp66 (amino acid 66). All fusions were in frame with the P.IIc coding sequence but were out of frame with the purported initiation codon. All fusion constructions were shown to phase vary in E. coli in an analogous fashion to that seen in N. gonorrhoeae, i.e. phase changes (in a recA background) at a frequency of c. 10(-3) accompanied by an alteration at the DNA level of the number of coding repeats (CRs). In vitro mutagenesis of the fusion constructions indicated that expression of out of frame P.II genes in E. coli was probably the result of ribosomal frameshifting within the run of 'A' residues immediately preceding the CR region and not due to low-level false initiation at codons other than the ATG initiation codon (as had previously been suggested). The mechanism for P.IIc::phoA phase variation appears to be related to the 'slipped-strand mispairing' mechanism responsible for frameshift mutations in a number of other bacterial genes containing short, direct, tandem repeats.