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Biomedical subjects

R C Johnson

Publications and source records attributed to R C Johnson.

At least 181 records · Page 10Linked to original sources
PubMed1993

Lung endothelial dipeptidyl peptidase IV is an adhesion molecule for lung-metastatic rat breast and prostate carcinoma cells.

Attachment of circulating tumor cells to endothelial cell adhesion molecules restricted to select vascular compartments is thought to be responsible for site-specific metastasis. Lung-metastatic rat R3230AC-MET breast and RPC-2 prostate carcinoma cells bound outside-out endothelial cell membrane vesicles, prepared by perfusion of the rat lung vasculature with a low-strength formaldehyde solution, in significantly higher numbers than their nonmetastatic counterparts R3230AC-LR and RPC-LR. In contrast, vesicles derived from the vasculature of a nonmetastasized organ (e.g., hind leg muscle) showed no binding preference for either of the four tumor cell lines. Lung-derived endothelial vesicles were used here to generate mAbs against lung endothelial cell adhesion molecules. The first group of mice were actively immunized against lung endothelial vesicles, whereas the second group was injected with syngeneic mouse antiserum against leg endothelial vesicles before active immunization with lung endothelial vesicles. 17 hybridoma supernatants obtained from the two fusions bound lung vesicles with at least a 10-fold higher affinity than leg vesicles. Seven (four obtained by a passive/active immunization protocol) stained rat capillary endothelia. One mAb, mAb 8.6A3, inhibited specific adhesion of lung-derived vesicles to lung-metastatic breast and prostate carcinoma cells. Purification of the antigen (endothelial cell adhesion molecule) from rat lung extracts revealed a protein with a 110-kD mol wt. NH2-terminal sequencing established identity with dipeptidyl peptidase IV which had been reported to serve as a fibronectin-binding protein. These results indicate that vesicles obtained from in situ perfused organs are a convenient immunogen for the production of antibodies to compartment-specific endothelial cell surface molecules, and reinforce the concept that endothelial cell surface components are selectively recognized by circulating cancer cells during metastasis formation.

Animals↗
PubMed1993

Adhesion to and invasion of cultured tick (Acarina: Ixodidae) cells by Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) and maintenance of infectivity.

Lyme disease spirochetes, Borrelia burgdorferi, interact with cultured tick cells in ways similar to those reported to occur in the vector Ixodes dammini Spielman, Clifford, Piesman & Corwin. Spirochete adhesion and penetration were examined using a cell line from embryos of Rhipicephalus appendiculatus Neumann that morphologically resembles tick gut cells, RAE25. Cocultivation of B. burgdorferi with these cells permitted prolonged maintenance of infectivity for hamsters. Borrelial adherence to RAE25 cells was time- and density-dependent and increased by 10-15% per h during the first 5.5 h of cocultivation when we used a concentration of 4 x 10(7) spirochetes/ml. After 6 h, > 90% of the cells bound an average of 3-5 spirochetes per cell. Low passage, hamster-infective strains of B. burgdorferi (JMNT and CD16) showed a 2-3-fold higher rate of adhesion to RAE25 cells than the highly passaged, noninfectious strain B31. Inactivation of CD16 or JMNT by heat, starvation, or treatment with puromycin reduced adherence by 40-60%, whereas pretreatment with monoclonal antibodies to the outer surface proteins had no effect. Spirochetes adhered to young I. dammini cell lines to a similar degree as they did to RAE25, whereas lines from the ticks Dermacentor variabilis (Say) (RML15) and Boophilus microplus (Canestrini) (BME26) bound 30-60% fewer spirochetes. Electron microscopy revealed epicellular borreliae associated with coated pits and vesicles before endocytosis, and intracellular spirochetes were surrounded by a host cell-derived membrane.

Animals↗
PubMed1993

The nonspecific DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of complex nucleoprotein structures.

The mammalian high mobility group proteins HMG1 and HMG2 are abundant, chromatin-associated proteins whose cellular function is not known. In this study we show that these proteins can substitute for the prokaryotic DNA-bending protein HU in promoting the assembly of the Hin invertasome, an intermediate structure in Hin-mediated site-specific DNA inversion. Formation of this complex requires the assembly of the Hin recombinase, the Fis protein, and three cis-acting DNA sites, necessitating the looping of intervening DNA segments. Invertasome assembly is strongly stimulated by HU or HMG proteins when one of these segments is shorter than 104 bp. By use of ligase-mediated circularization assays, we demonstrate that HMG1 and HMG2 can bend DNA extremely efficiently, forming circles as small as 66 bp, and even 59-bp circles at high HMG protein concentrations. In both invertasome assembly and circularization assays, substrates active in the presence of HMG1 contain one less helical turn of DNA compared with substrates active in the presence of HU protein. Analysis of different domains of HMG1 generated by partial proteolytic digestion indicate that DNA-binding domain B is sufficient for both bending and invertasome assembly. We suggest that an important biological function of HMG1 and HMG2 is to facilitate cooperative interactions between cis-acting proteins by promoting DNA flexibility. A general role for HMG1 and HMG2 in chromatin structure is also suggested by their ability to wrap DNA duplexes into highly compact forms.

Animals↗
PubMed1993

A guinea pig model for Lyme disease.

We report that outbred Hartley guinea pigs are susceptible to Borrelia burgdorferi. We recovered spirochetes from 57 of 60 (95%) guinea pigs inoculated when < or = 3 months of age. In contrast, animals inoculated when > or = 6 months of age were resistant to infection as defined by recovery of organisms at > or = 4 weeks postinoculation. Infection was widely disseminated: B. burgdorferi was recovered from 83% of bladders, 64% of knee joints, 57% of hearts, 48% of spleens, and 38% of spinal cords examined within 4 weeks of inoculation. Histopathologic changes were common in the heart (88%) (preferential involvement of perineural tissues near the annulus fibrosus) and bladder (76%) and were also noted in a minority of spinal cords (13%) and knee joints (9%). Western immunoblots demonstrated an immunoglobulin G response to B. burgdorferi, particularly to the 24-, 31- (OspA), 39-, and 41-kDa (flagellin) antigens. Infection was cleared from most tissues with the passage of time; spirochetes were recovered from 63% of tissues removed from guinea pigs at < or = 4 weeks after inoculation but from only 32% at > or = 8 weeks postinoculation (P < 0.001). An exception was the failure to clear spirochetes from infected knees, 90% of which were culture positive even when evaluated at > or = 8 weeks postinoculation. The guinea pig provides a new model useful for studying host-spirochete interactions in Lyme disease.

Animals↗
PubMed1993

Protective immunity is induced by a Borrelia burgdorferi mutant that lacks OspA and OspB.

A mutant of virulent Borrelia burgdorferi 297 was apparently selected for by long-term storage at 5 degrees C. This mutant was found to lack the plasmid which encodes outer surface protein A (OspA) and OspB. In addition to the loss of the OspA and OspB proteins, the mutant lacked two lipoproteins, of 20 and 7.5 kDa, that were observed in the wild type. Since the mutant was not recovered from the tissues or blood of hamsters injected with the mutant, the mutant was determined to be noninfectious. Hamsters vaccinated with noninfectious mutant 297 were protected completely from challenge with virulent wild-type 297 spirochetes. Prechallenge sera from hamsters vaccinated with mutant 297 lacked antibodies to OspA and OspB, while those from hamsters vaccinated with virulent wild-type 297 or avirulent 297 exhibited antibodies to these proteins. Hamsters vaccinated with virulent wild-type 297 or mutant 297 elicited antibodies to OspC and a 39-kDa protein (P39), whereas hamsters vaccinated with avirulent 297 lacked these antibodies. These results suggest that OspC and/or P39 are important for the development of a protective immune response. Study of this mutant may elucidate factors important to the development of a Lyme disease vaccine.

Animals↗
PubMed1993

Facilitation of complement-dependent killing of the Lyme disease spirochete, Borrelia burgdorferi, by specific immunoglobulin G Fab antibody fragments.

In the absence of specific antibody, Borrelia burgdorferi is resistant to the bactericidal action of complement, despite the capacity of the spirochete to activate complement. Complement-mediated killing of B. burgdorferi requires the presence of antiborrelial immunoglobulin G (IgG). The effect of bactericidal IgG takes place after formation of the C5 convertase. Therefore, we examined the ability of Fab fragments from bactericidal IgG to mediate killing of B. burgdorferi by complement. The complement-activating domain of IgG, the Fc fragment, was not required for killing of borreliae, as monovalent Fab fragments prepared from immune IgG were also able to mediate killing. However, the killing efficiency of the Fab fragments was less than that of intact IgG, suggesting that the bactericidal activity of IgG is enhanced by divalency. IgG Fab-mediated killing occurred without increased complement activation or C3 fluid-phase consumption. Cell killing proceeded via the classical complement pathway, as no killing of Fab fragment-sensitized cells was observed in human serum deficient in C2. These results demonstrate directly that the bactericidal effect of anti-B. burgdorferi IgG is independent of the complement-activating properties of the antibody.

Animals↗
PubMed1993

DNA binding and bending are necessary but not sufficient for Fis-dependent activation of rrnB P1.

The Escherichia coli Fis protein binds to three sites in the upstream activation region of the rrnB P1 promoter and enhances transcription 5- to 10-fold in vivo. In this report, we investigate the mechanism of Fis-dependent activation of transcription. We show that stimulation of rrnB P1 transcription by Fis can occur on linear DNA templates and does not require DNA upstream of the promoter-proximal Fis site I. Mutants of Fis defective for Hin-mediated recombination have been isolated previously and have defined an N-terminal domain required for DNA inversion by Hin in addition to the C-terminal domain which is required for DNA binding. Several of these mutants were found to be defective in stimulation of rrnB P1 transcription in vivo and in vitro. Activation-defective mutants fall into three classes: those that fail to bind to the upstream activation region, those that bind but fail to bend the DNA normally, and those that bind and bend but still fail to activate transcription. We conclude that it is unlikely that Fis functions by simply bringing upstream sequences or bound factors into the proximity of RNA polymerase to activate transcription. Rather, the data are most easily interpreted in terms of transcription activation by direct interactions between Fis and RNA polymerase, requiring precise positioning of the two proteins facilitated by bending of the DNA binding site.

Carrier Proteins↗
PubMed1993

Comparative analysis of genetic variability among Borrelia burgdorferi isolates from Europe and the United States by restriction enzyme analysis, gene restriction fragment length polymorphism, and pulsed-field gel electrophoresis.

The genomes of 62 North American and European Borrelia burgdorferi isolates were examined by restriction endonuclease analysis (REA), gene probe restriction fragment length polymorphism, and pulsed-field gel electrophoresis (PFGE). Hybridization of restriction fragments with the immunologically relevant 83-kDa antigen gene revealed polymorphisms and divided the isolates into three major groups. Group I included all but two of the American isolates and some of the European isolates. One of two Californian isolates (DN 127) and one Ixodes dammini isolate from New York (strain 25015), previously described as atypical, were distinct from the isolates in the three groups. Plasmid profile analysis and REA, the method with the highest level of discrimination, revealed extensive heterogeneity among isolates of the same major group. Our study demonstrates the usefulness of the 83-kDa antigen gene probe for dividing the isolates into major genogroups, whereas REA and plasmid profile analysis allow for a distinction of individual strains within these groups.

Animals↗
PubMed1993

Serologic analysis of white-tailed deer sera for antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay and western immunoblotting.

White-tailed deer serum samples were collected in the Minneapolis-St. Paul, Minn., metropolitan area during the fall and winter months from 1989 to 1992 and analyzed for antibodies to Borrelia burgdorferi, the etiologic agent of Lyme borreliosis. Ninety-eight percent of the serum samples were collected from regions where currently the vector tick, Ixodes dammini, is nonexistent. Antibodies to B. burgdorferi were detected in 2.2% of 508 samples by enzyme-linked immunosorbent assay, and their presence was confirmed by Western immunoblot analysis. Western immunoblotting yielded mean numbers of reactive bands of 0.1 and 6.0 for samples that were negative and positive for antibodies by enzyme-linked immunosorbent assay, respectively. The molecular weights of the antigens in many of the reactive bands from positive samples were similar to the molecular weights of antigens reactive with samples from humans with Lyme borreliosis. An antibody response to the major outer surface proteins A and B was not detected. Serologic analysis of deer sera may provide a valuable method for surveillance programs designed to monitor the spread of B. burgdorferi in nature.

Animals↗
PubMed1993

Isolation and characterization of Borrelia burgdorferi from blood of a bird captured in the Saint Croix River Valley.

Field investigations were conducted to further evaluate the role of birds in the maintenance and dissemination of Borrelia burgdorferi. Blood specimens were taken from 39 passerine birds of 17 species captured during June 1991 at the Saint Croix National Riverway in Wisconsin, and one isolate, WI91-23, was cultured from an adult song sparrow (Melospiza melodia). This isolate was shown to be infectious for Peromyscus leucopus and Mesocricetus auratus (golden hamster). Isolate WI91-23 was confirmed as B. burgdorferi by immunofluorescence assay by using species-specific anti-OspA monoclonal antibodies H3TS and H5332 and anti-OspB antibody H5TS. Isolate WI91-23 was compared with Borrelia anserina Es, Borrelia hermsii MAN-1, and other B. burgdorferi strains (ATCC 53210, CT-1, and Catharus fuscescens [veery] liver 10293). Pulsed-field gel electrophoresis of in situ-lysed spirochetes revealed that the DNA plasmid profile of WI91-23 was most similar to those of plasmids from B. burgdorferi and most different from those of plasmids from B. anserina and B. hermsii. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the protein profile of WI91-23 was like that of other B. burgdorferi strains studied, with dominant proteins corresponding to OspA and OspB, and that it differed from the protein profiles of B. anserina and B. hermsii. These findings indicate that passerine birds may serve as reservoirs for B. burgdorferi.

Animals↗
PubMed1993

Prevalence of the Lyme disease spirochete, Borrelia burgdorferi, in deer ticks (Ixodes dammini) collected from white-tailed deer (Odocoileus virginianus) in Saint Croix State Park, Minnesota.

During a special two-day hunt (11, 12 November 1989) in Saint Croix State Park, Minnesota (USA), one side of the neck for each of 146 white-tailed deer (Odocoileus virginianus) was examined for ticks. Of the 5,442 ticks collected, 90% (4,893) were the winter tick, Dermacentor albipictus, and 10% (549) were the deer tick, Ixodes dammini, the primary vector of the causative agent of Lyme disease, Borrelia burgdorferi. Adult males had the greatest frequency of infestation of either D. albipictus (100%) or I. dammini (88%) and had on average more ticks, compared to other deer. Based on an examination of midgut material from 435 I. dammini by polyclonal antibody analysis, spirochetes were observed in 22% of the ticks. Species-specific monoclonal antibody analysis of the spirochetes confirmed that the bacteria were B. burgdorferi.

Animals↗
PubMed1993

Calcinosis cutis following electromyographic examination.

We report the first case of calcinosis cutis following electromyographic examination. All previously reported cases of iatrogenic calcinosis cutis occurred following electroencephalographic examination using calcium-containing electrode compounds or following the extravasation of calcium-containing solutions along infiltrated intravenous lines. We also review the literature on calcinosis cutis due to iatrogenic and occupational exposure to calcium salts. The clinical presentation of calcinosis cutis is variable, but the histologic appearance is diagnostic. This entity may be underreported because of its benign, self-limited course and lack of recognition by physicians. Calcinosis cutis should be recognized by family practitioners, dermatologists, and subspecialists who encounter patients who have undergone prolonged monitoring studies that involve the use of electrodes.

Calcinosis↗
PubMed1992

[The characterization of a Spanish strain of Borrelia burgdorferi].

BACKGROUND: The characteristics of a Spanish strain of Borrelia burgdorferi, the spirochete which causes Lyme's disease, and which, up to the present, has not been isolated in Spain, are described. METHODS: The organism was obtained from ticks (Ixodes ricinus) from the northern part of Spain. It was studied in culture by dark field microscopy and the internal structure observed by electron transmission. The antigenic composition was analyzed under polyacrylamide electrophoresis, immunoblot and reactivity against monoclonal antibodies. Plasmid analysis was carried out by Southern blot. RESULTS: In culture the length of the organism is somewhat shorter than normal. It grows slowly and tends to autoagglutinate. It has 6-13 periplasmic flagella. The antigenic analysis of this microorganism through immunoblot and reactivity against different monoclonal antibodies showed differences with regards to other North American strains, with the most significant being the composition of certain proteins of the surface of the organism. These differences may have clinical repercussion. DNA analysis by Southern blot demonstrated slight differences in regard to the composition of plasmids compared to other strains analyzed. CONCLUSIONS: Borrelia burgdorferi exists in Spain. The isolated strain shows peculiar characteristics with respect to others analyzed. The availability of an autochthonous strain may allow more reliable serological diagnosis in Spain.

Animals↗
PubMed1992

Treatment of early Lyme disease.

PURPOSE: To compare the safety and efficacy of azithromycin, amoxicillin/probenecid, and doxycycline for the treatment of early Lyme disease, to identify risk factors for treatment failure, and to describe the serologic response in treated patients. PATIENTS AND METHODS: Fifty-five patients with erythema migrans and two patients with flu-like symptoms alone and fourfold changes in antibody titers to Borrelia burgdorferi were randomized to receive (1) oral azithromycin, 500 mg on the first day followed by 250 mg once a day for 4 days; (2) oral amoxicillin 500 mg and probenecid 500 mg, three times a day for each for 10 days; or (3) doxcycline, 100 mg twice a day for 10 days. If symptoms were still present at 10 days, treatment was extended with amoxicillin/probenecid or doxycycline for 10 more days. Evaluations were done at study entry and 10, 30, and 180 days later. RESULTS: Three of the patients who initially had symptoms suggestive of spread of the spirochete to the nervous system, one from each antibiotic treatment group, subsequently developed neurologic abnormalities, but symptoms in the other 54 patients resolved within 3 to 30 days after study entry. Six of the 19 patients (32%) (95% confidence interval, 13% to 57%) given amoxicillin/probenecid developed a drug eruption, whereas none of the patients given azithromycin or doxycycline had this complication. The presence of dysesthesias at study entry was the only risk factor significantly associated with treatment failure (p less than 0.001). By convalescence, 72% of the patients were seropositive, and 56% still had detectable IgM responses to the spirochete 6 months later. CONCLUSIONS: The three antibiotic regimens tested in this study were generally effective for the treatment of early Lyme disease, but the regimens differ in the frequency of side effects and in ease of administration.

Adult↗
PubMed1992

Failure of Borrelia burgdorferi to survive in the skin of patients with antibiotic-treated Lyme disease.

BACKGROUND: Borrelia burgdorferi has been cultivated from clinically normal skin (previous erythema migrans sites) after antibiotic therapy for Lyme disease. OBJECTIVE: We investigated the possibility of similar findings in 13 of our patients with antibiotic-treated Lyme disease from whom B. burgdorferi was cultivated from their erythema migrans lesions before antibiotic therapy. METHODS: After treatment with doxycycline or a combination of amoxicillin and probenecid, skin biopsy specimens were obtained from clinically normal skin adjacent to the previous biopsy sites and cultured. RESULTS: B. burgdorferi was not cultivated from these posttreatment biopsy sites. CONCLUSION: The failure of B. burgdorferi to survive in the former erythema migrans sites of our antibiotic-treated patients, as well as their favorable clinical response, supports the use of doxycycline or combined amoxicillin and probenecid in the treatment of early Lyme disease but does not preclude the survival of the organism in other tissues.

Adult↗
PubMed1992

Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules.

Peptidylglycine alpha-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membrane-associated were all packaged into regulated secretory granules in AtT-20 cells.

Base Sequence↗