Search PubMed⌕ Search

Biomedical subjects

S J Norris

Publications and source records attributed to S J Norris.

53 records · Page 3Linked to original sources
PubMed1988

Isolation and characterization of recombinant Escherichia coli clones secreting a 24-kilodalton antigen of Treponema pallidum.

Escherichia coli clones containing Treponema pallidum DNA in the pUC8 vector and secreting a 24-kilodalton antigen of T. pallidum have been isolated. Both syphilitic human and syphilis-immune rabbit sera reacted with the recombinant p24 antigen, indicating that an equivalent protein in T. pallidum is capable of eliciting antibody responses during natural infections. The p24 antigen of T. pallidum was identified by using two-dimensional gel electrophoresis and immunoblotting with monospecific anti-p24 serum. We tentatively concluded that this cloned antigen is a secreted protein or a labile or minor component of T. pallidum because (i) p24 was secreted by the recombinant E. coli cells; (ii) recombinant p24 in E. coli cells was processed into several smaller species with molecular masses ranging from 12 to 20 kilodaltons, which correlate well with the masses of secreted antigens described by others; and (iii) p24 protein appeared to be highly antigenic during natural infections, but only a very small amount of this antigen was associated with or retained by the purified organisms. The possible role of the p24 protein in determining the growth characteristics of T. pallidum is suggested by the ability of recombinant p24 to induce growth changes in E. coli cells. All E. coli colonies expressing the p24 polypeptide exhibited a flat and rough colony morphology and a filamentous growth pattern that were different from those of other E. coli cells. The DNA sequence coding for the p24 polypeptide is located on a 1.7-kilobase-pair BamHI fragment of the T. pallidum genomic DNA and is absent in the nonpathogenic Treponema phagedenis DNA. However, any possible relationship between the p24 antigen and the virulence of T. pallidum remains to be determined. In preliminary studies, rabbits immunized with the purified p24 were not protected from the infection with live T. pallidum organisms.

Antibodies, Bacterial↗
PubMed1988

Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis.

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.

Amino Acid Sequence↗
PubMed1986

Factors affecting the multiplication and subculture of Treponema pallidum subsp. pallidum in a tissue culture system.

Limited multiplication of Treponema pallidum subsp. pallidum (Nichols strain) can be obtained in the presence of Sf1Ep rabbit epithelial cell cultures, but continuous culture has not yet been achieved. In the system currently employed, growth is exponential for the first 10 to 15 days of culturing, after which multiplication and the percentage of motile organisms decrease. In an effort to identify culture conditions which may adversely affect treponemal viability and growth, eight culture parameters were monitored over a 12-day period of incubation. Several of these parameters, including pH, redox potential, dissolved oxygen concentration, and glucose levels were found to change dramatically during the course of incubation, indicating that they may be responsible for the cessation of treponemal multiplication. The feasibility of extending the period of growth by subculturing was also investigated. In preparation for planned serial subcultivation experiments, several subculture procedures were tested and found to be effective in allowing the transfer of T. pallidum from 3-day-old primary cultures to secondary cultures without loss of motility or growth potential. Increases of up to 55-fold were observed in secondary cultures, but increased growth due to subculturing was not a consistent finding. Use of subculture intervals of greater than or equal to 6 days resulted in a progressive decrease in treponemal multiplication in secondary cultures, although retention of motility was extended in the subcultures compared with motility in the primary cultures. These results indicate that the lack of continued multiplication of T. pallidum in subcultures is not due to damage to the treponemes during subculture. Prolonged multiplication of T. pallidum may be obtained through the stabilization of culture conditions by either performing subcultures at regular intervals or by medium replacement techniques. It was also found that primary T. pallidum cultures could be established by using as the inoculum treponemes that had been stored at -70 degrees C in a medium containing 15% glycerol.

Animals↗
PubMed1985

Reinfection of chancre-immune rabbits with Treponema pallidum. I. Light and immunofluorescence studies.

Inoculation of infectious Treponema pallidum into the skin of chancre-immune rabbits results in a limited inflammatory response. Intact organisms are identifiable by immunofluorescence in the dermis of the infection site for 1-2 days. By Day 3 structurally intact T pallidum are seen localized in hair follicles, erector pili muscles, and cutaneous nerves, while inflammatory cells containing fluorescent (T pallidum) fragments are seen in the dermis. After Day 6 intact organisms are no longer found. It is proposed that hair follicles, erector pili muscles, and particularly nerves may provide relatively protected sites for T pallidum, and that T pallidum may migrate within nerves. Clearance of organisms from the infected site appears to be mediated by phagocytosis and digestion by macrophages as a result of an accelerated delayed hypersensitivity response, but antibody-mediated destruction and T pallidum migration may also be involved.

Animals↗
PubMed1984

Antigenic complexity of Treponema pallidum: antigenicity and surface localization of major polypeptides.

The protein structure of Treponema pallidum was characterized by two-dimensional electrophoresis (2DE), consisting of isoelectric focusing (IEF, pH 5 to 7) in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Up to 85 major polypeptide species could be detected in the organisms in 2DE gels by Coomassie Blue staining. The antigenicity of the individual polypeptides was determined by transferring the 2DE pattern to nitrocellulose paper and utilizing a sensitive immunoperoxidase procedure to demonstrate the reactivity of immunoglobulins in sera obtained from rabbits infected intratesticularly at least 6 mo previously. The infected rabbit serum reacted with virtually every major polypeptide detectable by protein staining techniques, indicating that infected rabbits produce antibodies against nearly all major T. pallidum proteins at the time when the animals exhibit systemic resistance to reinfection. Surface radioiodination of freshly purified T. pallidum by an Iodogen procedure yielded preferential labeling of a major polypeptide with an apparent m.w. of 39,000. The results of this study indicate that the antigenic complexity of T. pallidum is much greater than described previously. The 39-kd polypeptide appears to be a major surface constituent of T. pallidum and as such may play an important role in the induction of immunity to syphilis.

Animals↗
PubMed1983

Demonstration of Treponema pallidum in a cutaneous gumma by indirect immunofluorescence.

Treponema pallidum was demonstrated in a cutaneous, tertiary syphilitic lesion by indirect immunofluorescence microscopy, but not by darkfield microscopy illumination or silver stain. The numerous organisms observed by this method may help explain the histologically vigorous tissue reaction in tertiary syphilis, despite the scarcity of organisms demonstrable by other methods.

Aged↗
PubMed1982

In vitro cultivation of Treponema pallidum: independent confirmation.

In vitro cultivation of the virulent Nichols strain of Treponema pallidum was achieved in a tissue culture system as described by A. H. Fieldsteel, D. L. Cox, and R. A. Moeckli (Infect. Immun. 32:908-915, 1981). In 7 of 8 experiments, 8.9- to 26.2-fold increases in the number of T. pallidum were observed over a 12- to 12-day period of incubation.

Bacteriological Techniques↗
PubMed1980

Long-term incorporation of tritiated adenine into deoxyribonucleic acid and ribonucleic acid by Treponema pallidum (Nichols strain).

Treponema pallidum (Nichols strain), extracted in medium containing Eagle minimal essential medium 50% fresh, heat-inactivated normal rabbit serum, and 1.0 mM dithiothreitol, was incubated under 3% oxygen in the presence of tritiated nucleic acid precursors. [8-3H]adenine was incorporated with high efficiency into trichloroacetic acid-insoluble material; 2'-deoxyadenosine and uridine were incorporated in lower quantities, and thymine and thymidine were not incorporated. Incorporation of [3H]adenine was inhibited by penicillin G, mitomycin C, actinomycin D, and erythromycin, but was not affected by cycloheximide. Partial purification of nucleic acids from T. pallidum incubated with [8-3H]adenine for 36 to 72 h and subsequent treatment with ribonuclease and deoxyribonuclease revealed that 15 to 20% of the trichloroacetic acid-precipitable counts were resistant to ribonuclease but susceptible to deoxyribonuclease. A simple assay was developed in which NaOH treatment was used to distinguish incorporation into ribonucleic acid and deoxyribonucleic acid. Both ribonucleic acid and deoxyribonucleic acid synthesis continued for 6 days of incubation under 3% O2, whereas incorporation was limited to the first day of incubation in samples incubated under aerobic or anaerobic conditions. T. pallidum thus appears to be capable of significant de novo deoxyribonucleic acid and ribonucleic acid synthesis under microaerobic conditions.

Adenine↗
PubMed1978

Influence of oxygen tension, sulfhydryl compounds, and serum on the motility and virulence of Treponema pallidum (Nichols strain) in a cell-free system.

The motility and virulence of Treponema pallidum (Nichols strain) were monitored during incubation in a modified tissue culture medium to study the effects of oxygen tension and medium composition on survival of the organism. A basal medium of Eagle minimal essential medium with 50% fresh, heat-inactivated normal rabbit serum was used inasmuch as better survival occurred with 50% normal rabbit serum than with lower concentrations. Addition of 0.5 to 2.0 mM dithiothreitol or 2.0 mM dithioerythritol to the basal medium led to significantly longer retention of T. pallidum viability in the presence of 3% oxygen than under aerobic or anaerobic conditions. The results of this investigation lend support to the classification of T. pallidum as a microaerophilic organism and provide direction for the design of potentially successful culture systems, with or without tissue culture cells.

Blood↗
PubMed1977

Vaccination of cattle and sheep with a combined Clostridium perfringens types C and D toxoid.

Cattle and sheep (30 each) were vaccinated with a combined Clostridium perfringens type C and C perfringens type D toxoid. Vaccination and blood sample collections were made every 2 weeks over a period of 8 weeks. Increases in antitoxin titers occurred after the 2nd administration of the 2.0-ml combined product. Highest titers were recorded when the 2nd vaccinal dose was given 2 weeks after the first. This confirms reports that response to clostridial antigens is greater when the 2nd immunizing dose is delayed 2 to 6 weeks after the initial dose.

Animals↗
PubMedDate not supplied

Genome structure of spirochetes.

The genome structures of several pathogenic spirochetes have recently been determined. The genomes of Borrelia species consist of a linear chromosome of approximately one million base pairs (Mb) and various linear and circular plasmids. Analysis of restriction fragment length polymorphisms and 16S ribosomal RNA sequence data indicate the division of Borrelia burgdorferi into at least three distinct genetic groups. Leptospira interrogans has a circular chromosome 5 Mb in size and a 0.35 Mb extrachromosomal element. Repetitive sequence elements similar to insertion sequences have been identified in the Leptospira interrogans genome. The chromosome of Treponema pallidum subsp. pallidum is circular and has a size of approximately one Mb. Genetic studies conducted to date indicate that B. burgdorferi and L. interrogans have a high degree of genetic diversity, whereas remarkably few genetic differences have been observed among the pathogenic Treponema. Knowledge of the genomic structure of these organisms will serve as a basis for future genetic studies.

Borrelia burgdorferi Group↗
PubMedDate not supplied

Purification of Treponema pallidum, Nichols strain, by Percoll density gradient centrifugation.

The purification of motile and virulent Treponema pallidum, Nichols strain, from rabbit testicular tissue is reported. Suspensions of T. pallidum were overlayed onto 20-ml cushions of 43% Percoll and in-situ density gradients were formed by centrifugation at 34,800 g for 30 min. Gradient fractionation indicated that T. pallidum banded at a density of 1.051 g/cc3 and that soluble proteineous testicular components remained in the upper portion of the gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the removal of host testicular and serum components. Purified suspensions of T. pallidum were greater than 95% actively motile and fully virulent, and greater than 50% motility could be maintained in vitro for up to five days. As determined by electron microscopy, Percoll-purified T. pallidum was structurally unaltered and contained much less tissue debris than did crude extracts or T. pallidum prepared by differential centrifugation. The Percoll purification method has been applied successfully to physiology, recombinant DNA, and antigenic structure studies, and to the preparation of antigen for the fluorescent treponemal antibody-absorbed (FTA-Abs) test for syphilis.

Animals↗
PubMedDate not supplied

Serum requirement for the multiplication of Treponema pallidum in a tissue-culture system: association of growth-promoting activity with the protein fraction.

The nature of the serum requirement of Treponema pallidum subspecies pallidum (Nichols strain) was examined in a culture system utilizing Sf1Ep cottontail rabbit cells. In this system, significant multiplication of treponemes occurs in the presence of select lots of fetal bovine serum (FBS) or calf serum (CS) at concentrations of greater than or equal to 5% (vol/vol). Heat-inactivation of the serum greatly enhances treponemal multiplication, and normal human serum was found to be as effective as FBS in supporting the growth of T. pallidum. The protein fraction of FBS obtained by membrane ultrafiltration was capable of supporting the multiplication of T. pallidum when added to the basal tissue culture medium; an average increase of 23-fold was observed in these cultures, as compared with a mean increase of 25-fold in the 20% FBS controls. In contrast, the ultrafiltrate fraction of FBS (consisting of compounds with molecular weights of less than 10,000 daltons) did not support either growth or the retention of motility. Proteins precipitable with 25% (wt/vol) polyethylene glycol (i.e., albumin, transferrin, ceruloplasmin, and other proteins) also promoted the growth of T. pallidum. This observation provides further evidence that the required serum components are associated with the protein fraction.

Animals↗
PubMedDate not supplied

The microaerophilic nature of Treponema pallidum: enhanced survival and incorporation of tritiated adenine under microaerobic conditions in the presence or absence of reducing compounds.

Treponema pallidum, although sensitive to atmospheric concentrations of O2, requires low levels of O2 for optimal survival and metabolic activity. The addition of 0.0125-0.2 mg/ml of sodium metabisulfite to a basal medium consisting of Eagle's minimal essential medium and 50% fresh, heat-inactivated normal rabbit serum was found to have an effect similar to that of dithiothreitol in extending the survival of T. pallidum (Nichols strain) under 3% O2. Detailed analysis of the effect of O2 tension revealed that 50% motility was retained longest at atmospheric O2 concentrations of 1-5%, whether or not dithiothreitol or sodium metabisulfite were present. Concentrations of O2 of 3-10% were optimal for nucleic acid synthesis, as determined by [3H]adenine incorporation during the first 24 hr of incubation. Sodium metabisulfite was less effective than dithiothreitol in stimulating nucleic acid synthesis. Neither sodium metabisulfite nor dithiothreitol at their effective concentrations had any effect on levels of dissolved O2. During incubation under 3% O2, motility was maintained at > 50% for 15 days and virulence for at least 13 days by dilution of the treponemal suspensions every three days with fresh medium containing sodium metabisulfite. The optimal retention of motility and nucleic acid synthesis under microaerobic conditions in the absence of reducing compounds provides further evidence that T. pallidum is a microaerophilic organism.

Adenine↗