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J N Miller

Publications and source records attributed to J N Miller.

At least 55 records · Page 3Linked to original sources
PubMed1994

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.

Animals↗
PubMed1993

Novel instrumentation and biomedical applications of very near infrared fluorescence.

A high wavelength fluorescent probe, Nile Red, was added to four proteins, viz., bovine albumin, alpha 1-acid glycoprotein, beta-lactoglobulin and ovomucoid. Nile Red showed an enhancement in fluorescence and a shift in emission wavelength, suggesting it was bonding hydrophobically to these proteins. Drug displacement of Nile Red from alpha 1-acid glycoprotein was achieved with both D,L-propranolol and flufenamic acid, showing that the binding site is less electrostatic and more hydrophobic in nature. In order to monitor these interactions, a simple spectrofluorimeter was constructed from solid-state components; the sensitivity of this instrument compared well with that of standard laboratory spectrofluorimeters.

Binding Sites↗
PubMed1993

Use of the "blue halo" assay in the identification of genes encoding exported proteins with cleavable signal peptides: cloning of a Borrelia burgdorferi plasmid gene with a signal peptide.

We have recently reported a phoA expression vector, termed pMG, which, like TnphoA, is useful in identifying genes encoding membrane-spanning sequences or signal peptides. This cloning system has been modified to facilitate the distinction of outer membrane and periplasmic alkaline phosphatase (AP) fusion proteins from inner membrane AP fusion proteins by transforming pMG recombinants into Escherichia coli KS330, the strain utilized in the "blue halo" assay first described by Strauch and Beckwith (Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988). The lipoprotein mutation lpp-5508 of KS330 results in an outer membrane that is leaky to macromolecules, and its degP4 mutation greatly reduces periplasmic proteolytic degradation of AP fusion proteins. pMG AP fusions containing cleavable signal peptides, including the E. coli periplasmic protein beta-lactamase, the E. coli and Chlamydia trachomatis outer membrane proteins OmpA and MOMP, respectively, and Tp 9, a Treponema pallidum AP recombinant, diffused through the leaky outer membrane of KS330 and resulted in blue colonies with blue halos. In contrast, inner membrane AP fusions derived from E. coli proteins, including leader peptidase, SecY, and the tetracycline resistance gene product, as well as Tp 70, a T. pallidum AP recombinant which does not contain a signal peptide, resulted in blue colonies without blue halos. Lipoprotein-AP fusions, including the Borrelia burgdorferi OspA and T. pallidum Tp 75 and TmpA showed halo formation, although there was significantly less halo formation than that produced by either periplasmic or outer membrane AP fusions. In addition, we applied this approach to screen recombinants constructed from a 9.0-kb plasmid isolated from the B31 virulent strain of B. burgdorferi. One of the blue halo colonies identified produced an AP fusion protein which contained a signal peptide with a leader peptidase I cleavage recognition site. The pMG/KS330r- cloning and screening approach can identify genes encoding proteins with cleavable signal peptides and therefore can serve as a first step in the identification of genes encoding potential virulence factors.

Alkaline Phosphatase↗
PubMed1993

Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane protein for which the structural gene has been cloned and sequenced.

Amino Acid Sequence↗
PubMed1992

New fluorescent derivatives of cyclosporin for use in immunoassays.

The synthesis of new fluorescent derivatives of cyclosporin is described and their affinity with the specific Sandoz monoclonal antibody investigated. Synthesis was carried out using cyclosporin-C-hemisuccinate as the starting material with monodansylcadaverine, 4-bromomethyl-7-methoxycoumarin, and 4-bromomethyl-6,7-dimethoxycoumarin as labels. After extraction the derivatives were purified by HPLC and their binding affinity with the monoclonal antibody evaluated by the Incstar Cyclo-Trac SP radioimmunoassay. All three derivatives showed good binding and it is suggested that they may be of use in an immunoassay for measuring cyclosporin.

Chromatography, High Pressure Liquid↗
PubMed1991

The pathophysiology of acute syphilitic funisitis.

There is an increase in the number of cases of syphilis in pregnancy in the United States. Fetal death may occur in syphilis from acute or chronic infections. A case is presented in which an acute fetal infection occurred. The patient presented at 31 weeks' gestation, with a decrease in fetal movements and non-reactive cardiotocography. Ultrasound and Doppler analysis of the fetal heart, cerebral and umbilical arteries, aorta and umbilical vein led to the suspicion of acute cardiac failure. An amniocentesis yielded a white cell count of 1122 white blood cells, with 91% polymorphs, but the Gram stain was negative. The fetus developed a persistent bradycardia and was delivered. The diagnosis of acute severe syphilitic funisitis was suspected from histological sections of the cord. Diagnosis was established from maternal and fetal blood. Modern ultrasound techniques, including imaging, Doppler and cardiotocography, can lead to the analysis of the pathophysiology of disease states. An acute syphilitic infection should be suspected when this constellation of findings is found.

Journal Article↗
PubMed1991

Flow injection immunoassay using a protein A immunoreactor.

Competitive immunoassays have been developed for the immunosuppressant cyclosporin A and the anti-asthmatic drug theophylline utilizing identical controlled pore glass-protein A microcolumns and flow injection techniques. For cyclosporin A the assay was based on a monoclonal antibody with fluorescence detection whilst for theophylline sheep antiserum and electrochemical detection was used.

Cyclosporine↗
PubMed1991

An on-line immunoassay method for theophylline using a protein A immunoreactor.

A heterogeneous fluorescence immunoassay for theophylline has been automated using a flow injection analysis system containing a protein A solid phase reactor to separate antibody-bound and unbound fluorescein-theophylline. For each sample the antibody-protein A reaction takes place at near neutral pH, and the complexes are eluted at acid pH. The antibody-binding capacity of the reaction greatly exceeds the antibody level in each sample incubation mixture, and a single reactor can be repeatedly cycled between neutral and acid pHs. Experimental variations such as reactor size, flow rate, pH values, and reactant concentrations have been studied. Theophylline could readily be determined at the micrograms ml-1 level with on-line incubation with antibodies.

Automation↗
PubMed1991

Radiation hybrid map of 13 loci on the long arm of chromosome 5.

Radiation hybrid mapping was used in conjunction with a natural deletion mapping panel to predict the order of and distance between 13 loci in the distal portion of the long arm of human chromosome 5. A panel of irradiation hybrids containing fragments of 5q was generated from an HPRT+ Chinese hamster-human cell hybrid containing a derivative chromosome 5 [der(5)t(4;5)(5qter----5p15.1::4p15.1----4pter)] as its only human DNA. One hundred nine radiation hybrids containing human DNA were screened with polymerase chain reaction primer sets representing nine genes encoding growth factors, growth factor receptors, or hormone receptors (IL3, IL4, IL5, CSF1R, FGFA, ADRB2, GRL, GABRA1, and DRD1) as well as four other loci (FER, SPARC, RPS14, and CD14) to generate a radiation hybrid map of the area 5q21-q35. A physical map predicting the order of and distance between the 13 loci was constructed based on segregation of the 13 loci in hybrid clones. The radiation hybrid panel will be useful as a mapping tool for determining the location and order of other genes and polymorphic loci in this region as well as for generating new DNA probes from specific regions.

Animals↗
PubMed1991

Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector.

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain. Immunoblot analysis of T. pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa. [35S]-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein. DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites.

Alkaline Phosphatase↗
PubMed1991

Antimicrobial activity of rabbit leukocyte defensins against Treponema pallidum subsp. pallidum.

Defensins, which are peptides with broad antimicrobial activity, are major constituents of rabbit neutrophils and certain macrophages. We tested six rabbit defensins, NP-1, NP-2, NP-3a, NP-3b, NP-4, and NP-5, for activity against Treponema pallidum subsp. pallidum. Mixtures of T. pallidum and defensin in 10% normal rabbit serum (NRS) or heat-inactivated NRS (HI-NRS) were incubated anaerobically for various time periods ranging between 0 and 16 h and then examined by dark-field microscopy for treponemal motility or inoculated intradermally into rabbits to assess treponemal virulence. Immobilization of T. pallidum by NP-1 (400 micrograms/ml) occurred after 4 and 8 h of coincubation in mixtures containing NRS and HI-NRS, respectively. Similarly, neutralization of T. pallidum by NP-1 occurred more rapidly and was complete when incubations were performed in NRS as compared with that in HI-NRS. Endpoint titration confirmed the augmentation of NP-1 antitreponemal activity by heat-labile serum factors; NP-1 showed neutralizing activity at 4 micrograms/ml (about 1 microM) in NRS and at 40 micrograms/ml in HI-NRS. When NP-1 was tested in serum that was deficient in C6, the T. pallidum neutralizing activity of NP-1 was reduced to levels slightly greater than that observed in HI-NRS. NP-1 that had been reduced and alkylated was inactive against T. pallidum. When NP-2, NP-3a, NP-3b, NP-4, and NP-5 were tested at 400 micrograms/ml, all exerted potent treponemicidal activity, manifested by abrogation or delayed development of cutaneous lesions relative to that of controls. These data suggest that defensins may equip certain macrophages and neutrophils to participate in host defense against T. pallidum, that the direct activity of defensins against T. pallidum is enhanced by heat-labile serum factors (presumably complement), and that conformational factors influence the biological activity of the defensin molecule.

Animals↗
PubMed1991

Contribution of rabbit leukocyte defensins to the host response in experimental syphilis.

In the companion paper (L. A. Borenstein, M. E. Selsted, R. I. Lehrer, and J. N. Miller, Infect. Immun. 59:1359-1367, 1991), we report that rabbit alveolar macrophage and neutrophil derived defensins possess antimicrobial activity against Treponema pallidum subsp. pallidum, the etiologic agent of syphilis. In this study, antisera specific for NP-1 and NP-2 (defensins present in certain macrophages and polymorphonuclear leukocytes) and NP-5 (a defensin produced only in neutrophils) were used to detect these peptides by immunoperoxidase staining in testicular lesions from infected rabbits. Profound amounts of cell-free and cell-associated defensins were detected in the tunica albuginea and interstitial spaces during the first 24 h of infection. The presence of defensins was transient and almost undetectable by day 4. Interstitial defensins were detected again at day 10 and increased through day 16, at which time lesion healing was evident by hematoxylin and eosin staining. The appearance and increase in detectable defensins between days 10 and 16 of infection correlated with a reduction in numbers and disappearance of T. pallidum, as demonstrated by using silver staining. The extent and pattern of immunostaining for NP-1 and NP-2 corresponded with immunostaining for NP-5 and identified neutrophils as the cellular source of the defensins. These findings indicate that defensins may contribute to the control of local T. pallidum infection and suggest a role for acute inflammatory processes in the resolution of early experimental syphilis.

Animals↗
PubMed1991

Analysis of outer membrane ultrastructure of pathogenic Treponema and Borrelia species by freeze-fracture electron microscopy.

We analyzed the outer membrane (OM) ultrastructure of four pathogenic members of the family Spirochaetaceae by freeze fracture. The OM of Treponema pallidum subsp. pertenue contained a low intramembranous particle concentration, indicating that it contains few OM transmembrane proteins. The concave OM fracture faces of Treponema hyodysenteriae and Borrelia burgdorferi contained dense populations of particles, typical of gram-negative organisms. A relatively low concentration of particles which were evenly divided between a small and a large species was present in the concave OM fracture face of Borrelia hermsii; the convex OM fracture face contained only small particles. As for gram-negative bacteria, the convex OM fracture face particle concentrations of these pathogens were low. These spirochetes cleaved preferentially within the OM, in contrast to typical gram-negative bacteria, which tend to fracture within the inner membrane. The OM ultrastructure of T. pallidum subsp. pertenue provides an explanation for the lack of antigenicity of the treponemal surface and may reflect a mechanism by which this pathogen evades the host immune response.

Borrelia↗
PubMed1990

Complement activation limits the rate of in vitro treponemicidal activity and correlates with antibody-mediated aggregation of Treponema pallidum rare outer membrane protein.

A modification of the in vitro immobilization assay together with freeze-fracture analysis was used to determine the factors responsible for the prolonged time required in vitro to achieve killing of Treponema pallidum subsp. pallidum. The modified immobilization assay permitted separate determination of the time required for binding of antibody to the surface of T. pallidum and for C activation. Treponemes were preincubated in heat-inactivated immune rabbit serum (IRS) followed by washing the organisms in 2.5% BSA/PBS to remove unbound IRS antibody before the addition of C. The results showed that a comparable degree of C-dependent killing occurred when treponemes were preincubated in heat-inactivated IRS for either 30 min or 16 h, indicating that treponemicidal antibody rapidly binds to the surface of T. pallidum. Preincubation of treponemes for 17 h in heat-inactivated IRS followed by a 1-h incubation in C resulted in the loss of 80% treponemal motility, indicating that C activation results in rapid killing of T. pallidum. Treponemes preincubated in IRS for 1 h, then incubated for 8 h and 16 h in heat-inactivated normal serum also lost a significant level of motility after the addition of C; in contrast, motility was unaffected after 30 min and 4 h of incubation in heat-inactivated normal serum under similar conditions. These results demonstrate that, whereas antibody binding to and C-mediated killing of treponemes can proceed rapidly, the prolonged time to C activation limits the rate at which treponemicidal activity occurs in vitro. In addition, treponemicidal activity using the modified immobilization assay could not be demonstrated with antiserum against T. pallidum endoflagella, antiserum against proteins solubilized from T. pallidum using the detergent Triton X-114, and a mAb to the T. pallidum r190-kDa "4D" protein, suggesting that these molecules are not accessible to surface binding antibody. Freeze-fracture analysis, recently used in our laboratory to demonstrate that the outer membrane of T. pallidum has rare constituent protein, was utilized to demonstrate outer membrane target Ag of IRS antibody. T. pallidum rare outer membrane protein (TROMP) molecules were shown in freeze-fracture electron micrographs to be consistently aggregated following a 16-h incubation of treponemes in IRS. In contrast, no aggregation of TROMP was present in treponemes incubated in normal rabbit serum for 16 h or in treponemes incubated in IRS for 2 h. These findings suggest that the rate of C activation leading to in vitro treponemicidal activity is limited by the time required for aggregation of antibody-bound TROMP molecules.

Animals↗
PubMed1990

Cloning, sequencing, and expression of two class B endoflagellar genes of Treponema pallidum subsp. pallidum encoding the 34.5- and 31.0-kilodalton proteins.

Two structural endoflagellar genes of Treponema pallidum that encode the 34.5- and 31.0-kilodalton (kDa) polypeptides as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were cloned, sequenced, and expressed. We designated these genes flaB1 and flaB3. A DNA sequence analysis of flaB1 and flaB3 showed that each gene possesses a single open reading frame that encodes a polypeptide; these polypeptides have molecular masses of 31.1 and 31.0 kDa, respectively. Shine-Dalgarno ribosome-binding sequences were identified upstream from the initiation codons of each gene. In addition, a single consensus promoter sequence was identified 121 base pairs upstream from the initiation codon of flaB1, suggesting polycistronic transcription of flaB1 and flaB3. Computer-induced alignment showed that the FlaB1 amino acid sequence was identical at 206 positions (72%) to the FlaB3 sequence. Both genes were subcloned into pATH vectors and were expressed under the control of the trpE promoter. The expression products of flaB1 and flaB3 revealed fusion proteins having molecular masses of 61.0 and 59.0 kDa, respectively, which were identified on immunoblots by using specific anti-T. pallidum endoflagellar serum.

Amino Acid Sequence↗
PubMed1990

Immunization with Treponema pallidum endoflagella alters the course of experimental rabbit syphilis.

Rabbits were immunized over a 32-week period with a total of 450 micrograms of purified Treponema pallidum endoflagella. As measured by enzyme-linked immunosorbent assay, sera from immunized rabbits had antiendoflagellar antibody titers that were fivefold greater than titers of sera from infected immune rabbits and patients with secondary disease. Sera from all immunized animals possessed complement-dependent treponemicidal activity as measured by in vitro immobilization. Immunized animals challenged with virulent T. pallidum were not protected from symptomatic infection but showed an altered course of lesion development.

Animals↗
PubMed1990

Cross-reactivity of nonspecific treponemal antibody in serologic tests for Lyme disease.

Serum samples obtained from 59 persons who had acute necrotizing ulcerative gingivitis, periodontitis, syphilis, or Lyme disease were tested against Treponema phagedenis biotype Reiter, Treponema denticola, Treponema vincentii, and Treponema scoliodontum by indirect fluorescent-antibody staining methods. Although there were positive reactions for sera representing each of these study groups and for 20 (13%) of 156 samples collected from the general population (premarital screening for syphilis), titration endpoints were relatively low (less than or equal to 1:256). Serum samples from 18 persons who had gingivitis or periodontitis but no history of Lyme borreliosis were tested by enzyme-linked immunosorbent assay for antibodies to Borrelia burgdorferi. Of these, five (28%) had immunoglobulin M antibody and four (22%) contained immunoglobulin G antibodies to this spirochete. Adsorption with either sorbent commercially prepared from T. phagedenis biotype Reiter or with washed, whole cells of T. phagedenis biotype Reiter reduced cross-reactivity in the enzyme-linked immunosorbent assay for Lyme borreliosis.

Antibodies, Bacterial↗