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R C Johnson

Publications and source records attributed to R C Johnson.

At least 145 records · Page 8Linked to original sources
PubMed1995

Analysis and comparison of plasmid profiles of Borrelia burgdorferi sensu lato strains.

The relationship between plasmid profiles and genospecies of the Lyme disease borreliae was investigated by using 40 strains from diverse biological and geographical sources. The genospecies of the strains were determined by examination of rRNA gene restriction patterns with cDNA probes complementary to the 16S and 23S rRNAs of Escherichia coli. Plasmid profiles were obtained by pulsed-field gel electrophoresis. The number of plasmids per strain and the size of these plasmids ranged from 4 to 10 and from 13.3 to 57.7 kb, respectively. The strains all contained a single large plasmid of 50 to 57.7 kb, with the exception of two Borrelia garinii strains that contained two or three of the large plasmids. The large plasmids of Borrelia burgdorferi sensu stricto strains ranged in size from 51.4 to 52.7 kb and were consistently smaller than the 54.0- to 57.7-kb plasmids present in B. garinii and Borrelia afzelii. The exceptions of this observation were the two B. garinii strains with multiple large plasmids; in this case the large plasmids were 50.6 to 53 kb. Although a large degree of heterogeneity in the sizes and frequencies of occurrence of smaller plasmids was observed, there were some differences among the three genospecies. The differences in plasmids were further studied by using two BamHI DNA fragments from a 28.7-kb plasmid of B. burgdorferi sensu stricto 297 as probes. Both probes hybridized with the 27- to 29-kb plasmids of B. burgdorferi sensu stricto strains. In contrast, two patterns of hybridization were observed with B. garinii and B. afzelii. One pattern was the hybridization of both probes, with each probe hybridizing with a different plasmid. The other pattern was the hybridization of a plasmid of a strain with only one of the two probes. some strains of the three genospecies did not hybridize with either probe. Our results suggest that the plasmid profiles of B. burgdorferi sensu lato have genospecies characteristics and that the hybridization patterns of similar-sized plasmids are different for the three genospecies.

Blotting, Southern↗
PubMed1995

Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera.

Serum specimens from persons with or without Lyme borreliosis were analyzed by indirect fluorescent antibody staining methods for total immunoglobulins to Babesia microti, Ehrlichia chaffeensis (Arkansas strain), and Ehrlichia equi (MRK strain). There was serologic evidence of human exposure to multiple tick-borne agents in 15 (6.6%) of 227 serum samples obtained in Connecticut and Minnesota. Of these, 10 serum samples were from Connecticut patients who had erythema migrans and antibodies to Borrelia burgdorferi (range, 1:160 to 1:40, 960). A maximal antibody titer of 1:640 was noted for a B. microti infection, whereas titration end points of 1:640 and 1:1,280 were recorded for E. chaffeensis and E. equi seropositives, respectively. In specificity tests, there was no cross-reactivity among the antisera and antigens tested for the four tick-borne pathogens. On the basis of serologic testing, a small group of persons who had Lyme borreliosis had been exposed to one or more other tick-borne agents, but there was no clinical diagnosis of babesiosis or ehrlichiosis. Therefore, if the clinical picture is unclear or multiple tick-associated illnesses are suspected, more extensive laboratory testing is suggested.

Animals↗
PubMed1995

Immunoblot interpretation criteria for serodiagnosis of early Lyme disease.

We monitored the antibody responses of 55 treated patients with early Lyme disease and physician-documented erythema migrans. Six sequential serum samples were obtained from patients before, during, and until one year after antibiotic therapy and analyzed by in-house enzyme-linked immunosorbent (ELISA) and immunoblot assays. An immunoblot procedure utilizing a gradient gel and an image analysis system was developed. A relational database management system was used to analyze the results and provide criteria for early disease immunoblot interpretation. Recommended criteria for the immunoglobulin M (IgM) immunoblot are the recognition of two of three proteins (24, 39, and 41 kDa). The recommended criteria for a positive IgG immunoblot are the recognition of two of five proteins (20, 24 [> 19 intensity units], 35, 39, and 88 kDa). Alternatively, if band intensity cannot be measured, the 22-kDa protein can be substituted for the 24-kDa protein with only a small decrease in sensitivity. Monoclonal antibodies were used to identify all these proteins except the 35-kDa protein. With the proposed immunoblot interpretations, the sequential serum samples were examined. At visit 1, the day of diagnosis and initiation of treatment, 54.5% of the serum samples were either IgM or IgG positive. The peak antibody response, with 80% of the serum samples positive, occurred at visit 2, 8 to 12 days into treatment. The sensitivities of the IgM and IgG immunoblot for detecting patients that were seropositive into the study period were 58.5 and 54.6%, respectively, at visit 1 and 100% at visit 2. Twenty percent of the patients remained seronegative throughout the study. The specificities of the IgM and IgG immunoblots were 92 to 94% and 93 to 96%, respectively. The IgM immunoblot and ELISA were similar in sensitivities, whereas the IgG immunoblot had greater sensitivity than the IgG ELISA (P = 0.006).

Antibodies, Bacterial↗
PubMed1995

Effects of ground flaxseed in swine diets on pig performance and on physical and sensory characteristics and omega-3 fatty acid content of pork: I. Dietary level of flaxseed.

Forty-eight barrows and gilts were fed diets containing 0 (CO), 5, 10, or 15% ground flaxseed (FS) for the final 25 d before slaughter. Flaxseed treatments did not affect any production or carcass traits (P > .10). No pork processing problems due to lack of firmness were encountered. Amounts (milligrams/gram of tissue) of alpha-linolenic acid (ALA [18:3n-3]) and eicosapentaenoic acid (EPA [20:5n-3]) increased (P < .01) in both backfat layers and ALA increased (P < .01) in kidney (leaf) fat after FS. Alpha-linolenic acid and EPA increased (P < .001) in the raw belly in response to FS; the effect was maintained throughout processing (P < .01) to microwaved bacon. Alpha-linolenic acid and EPA increased (P < .01, P < .05, respectively) with amount of FS in longissimus thoracis and liver. In the brain, DHA decreased (P < .05) with amount of FS. Trained panelists in triangle tests were able to identify bacon from pigs fed 10 and 15% flaxseed. Panelists could not identify various treatments in the loin tests.

Animals↗
PubMed1995

Effects of ground flaxseed in swine diets on pig performance and on physical and sensory characteristics and omega-3 fatty acid content of pork: II. Duration of 15% dietary flaxseed.

Four barrows and four gilts were assigned to each of five dietary treatments: a control (CO) diet or a 15% flaxseed (FS) diet fed for 7, 14, 21, or 28 d prior to slaughter. Flaxseed treatments did not affect any production or carcass traits (P > .10). No pork processing problems due to lack of firmness were encountered. Amounts (milligrams/gram of tissue) and percentages of alpha-linolenic acid (ALA [18: 3n-3]), arachidonic acid (AA [20:4n-6]), and eicosapentaenoic acid (EPA [20:5n-3]) increased (P < .001) in both backfat layers and in kidney (leaf) fat and docosahexaenoic acid (DHA [22:6n-3]) increased (P < .006) in the middle/inner backfat layer after FS. The effects of dietary FS on fatty acid profiles were maintained in lard and pastry. Alpha-linolenic acid, AA, and EPA increased (P < .001) and DHA increased (P < .021) in the raw belly in response to FS; the effect was maintained throughout processing to microwaved bacon. Percentage of oleic acid (OA [18:1n-9]) decreased (P < .016) in raw bellies after FS. Alpha-linolenic acid and EPA increased (P < .001) with duration of FS in longissimus thoracis and liver neutral lipids, polar lipids, and combined lipid fractions, but DHA increased (P < .004) only in longissimus thoracis polar and combined lipid fractions. Arachidonic acid decreased in the longissimus thoracis polar lipid fractions (P < .037) and in liver neutral (P < .01) and combined lipid fractions (P < .006) with FS. All ALA:AA and EPA:AA ratios increased (P < .001 to P < .048) with increased duration of FS (except ALA:AA in pastry [P = .56]). Processing of bellies into bacon and storing at -18 degrees C for 6 wk did not cause thiobarbituric acid (TBA) values to exceed a value of 1.0 after either the CO or FS diets. After microwaving bacon when TBA values approached 2.0, there was no FS dietary effect on TBA values. Trained panelists rated FS bacon more flavor-intense than CO bacon and recorded more flavor defects for FS bacon. A 105-member consumer group showed a higher frequency of "dislikes" for FS bacon than for CO bacon.

Adipose Tissue↗
PubMed1995

Analyses of mammalian sera in enzyme-linked immunosorbent assays with different strains of Borrelia burgdorferi sensu lato.

Blood samples were collected from cottontail rabbits (Sylvilagus floridanus), raccoons (Procyon lotor), white-footed mice (Peromyscus leucopus), and white-tailed deer (Odocoileus virginianus) between 1977 and 1991 in southern Connecticut and New York State (USA) and were tested for antibodies against eight strains of Borrelia burgdorferi sensu lato in enzyme-linked immunosorbent assays. Among these spirochetes were six strains of B. burgdorferi sensu stricto, one strain of B. garinii (=IP90) and a strain (IPF) in group VS461. Sera from each study group reacted positively to all strains having origins in North America and Eurasia. Assay sensitivities normally ranged between 85% and 100% for all study groups. The lowest sensitivity (66%) was noted when mouse sera were tested with B. garinii, an isolate from Ixodes persulcatus in the former Soviet Union. Differences in serum reactivity to various strains were noted for all study groups, but because of multiple shared antigens among the closely related spirochetes tested, the selection of a particular North American strain of B. burgdorferi sensu stricto did not appear to be a critical factor for optimal assay performance. Locally obtained strains of this bacterium are preferred as coating antigens for serologic testing because of their availability.

Animals↗
PubMed1995

An audit of stab wounds in Cardiff.

The incidence of stabbings in the United Kingdom has not been determined. We audited all cases of stabbings presenting to the Accident and Emergency Department of the Cardiff Royal Infirmary over an 18-month period from 1 January 1991. Over 122 000 new cases presented, 92 (0.075) with stab wounds, 48% of which required hospital admission. Stabbings accounted for 62 060 pounds of the surgical budget. There was a high incidence of self-inflicted stab wounds accounting for a significant proportion of the expenditure. This has not been reported previously.

Adolescent↗
PubMed1994

The structure of Fis mutant Pro61Ala illustrates that the kink within the long alpha-helix is not due to the presence of the proline residue.

The influence of proline on bending of the alpha-helix was investigated by replacement of the proline residue located in the middle of the long alpha-helix of the Fis protein with alanine, serine, or leucine. Each of the three substitutions folded into a stable protein with the same or higher melting points than the wild-type, but only Pro61Ala was functionally active in stimulating Hin-mediated DNA inversion. Pro61Ala formed crystals that were isomorphous with the wild-type protein allowing the structure to be determined at 1.9-A resolution by x-ray diffraction methods. The structure of the Pro61Ala mutant is almost identical to the wild-type protein, consistent with its near wild-type activity. One of the alpha-helices, the B-helix, is kinked in the wild-type Fis protein by 20 degrees which was previously assumed to be caused solely by the presence of proline 61 in the center of the helix. However, the B-helix is still kinked by 16 degrees when proline 61 is replaced by alanine. Local peptide backbone movement around residue 57 adjusts the geometry of the helix to accommodate the new main chain hydrogen bond between the -CO group in Glu57 and the -NH group in Ala61. Thus, the kink of the alpha-helix in Pro61Ala does not require the presence of proline.

Alanine↗
PubMed1994

The persistence of spirochetal nucleic acids in active Lyme arthritis.

Six of seven patients with Lyme arthritis were positive by PCR. In contrast, all 18 synovial fluid samples from patients with other disorders, including rheumatoid arthritis, spondyloarthropathy, gout, pseudogout, hemarthrosis, degenerative joint disease, lupus, papillary synovitis, and trauma, were negative by PCR (P < 0.001, Lyme arthritis compared with controls, Fisher exact test). All 38 laboratory controls were negative by PCR. The assay reproducibly detected 20 or fewer B. burgdorferi cells directly or when added to extracted synovial fluid that was previously negative by PCR. Polymerase chain reaction was done four times with identical results, including analyses with both outer surface protein A primer sets.

Adult↗
PubMed1994

Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex.

The Escherichia coli Fis (factor for inversion stimulation) protein functions in many diverse biological systems including recombination, transcription, and DNA replication. Although Fis is a site-specific DNA-binding protein, it lacks a well-defined consensus recognition sequence. The electrophoretic mobility of Fis-DNA complexes, along with considerations of the Fis crystal structure, indicates that significant deformation of DNA occurs upon Fis binding. To investigate the structure of Fis-DNA complexes, the chemical nuclease 1,10-phenanthroline-copper complex (OP-Cu) has been linked to four specific sites within the Fis DNA-binding domain. Two of these Fis-OP derivatives were active in cleaving DNA. The scission patterns obtained on four different Fis binding sites indicate that Fis positions itself on these highly divergent DNA sequences in a very similar fashion. The patterns of cleavage of a derivative at Asn-98 generally support a model of a Fis-DNA complex that contains specific bends within the core-recognition sequence. Data from a second Fis-OP derivative at Asn-73 provides evidence for greater wrapping of flanking DNA around the sides of the Fis protein than was previously postulated. The cleavage efficiency of flanking segments varies, suggesting that the extent of DNA wrapping is sequence dependent. Specific amino acids on Fis are implicated in promoting this DNA wrapping.

Base Sequence↗
PubMed1994

Structure, distribution, and functional expression of the phosphofructokinase C isozyme.

To elucidate the structure, tissue-specific expression, and allosteric properties of phosphofructokinase-C (PFK-C), we cloned the cDNA for PFK-C from a rat hypothalamic cDNA library. The cDNA is 2643 base pairs long and encodes a protein of 765 amino acids. The deduced amino acid sequence is highly homologous to PFK-M (muscle) and PFK-L (liver), 69 and 65% amino acid identity, respectively, especially at substrate binding and catalytic sites, while the allosteric binding sites are less conserved. Tissue-specific expression of PFK-C was investigated by Northern blot analysis. PFK-C mRNA was detected in several brain regions and the anterior pituitary but not in liver, skeletal muscle, or several other tissues. In situ hybridization showed that PFK-C is expressed at a higher level in higher brain regions such as the cortex, compared with the midbrain and basal ganglia, while PFK-L is expressed at approximately equal levels throughout the brain. Expression plasmids containing PFK-C and PFK-L coding sequences were constructed and expressed by transient transfection into CMT cells. Expression of transfected PFKs was demonstrated by PFK enzymatic activity and by Western blotting with anti-rat brain and liver PFK antisera. Allosteric regulatory properties of PFK-C and PFK-L expressed in CMT cells were compared. Fructose 2,6-bisphosphate, a potent activator of PFK, decreased the Km of PFK-C for fructose 6-phosphate from 200 to 60 microM while decreasing that of PFK-L from 300 to 55 microM. The properties of PFK-C and PFK-L expressed in CMT cells clearly demonstrate the allosteric differences between the different PFK isozymes.

Allosteric Regulation↗
PubMed1994

Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions.

The structure of the 52-amino acid DNA-binding domain of the prokaryotic Hin recombinase, complexed with a DNA recombination half-site, has been solved by x-ray crystallography at 2.3 angstrom resolution. The Hin domain consists of a three-alpha-helix bundle, with the carboxyl-terminal helix inserted into the major groove of DNA, and two flanking extended polypeptide chains that contact bases in the minor groove. The overall structure displays features resembling both a prototypical bacterial helix-turn-helix and the eukaryotic homeodomain, and in many respects is an intermediate between these two DNA-binding motifs. In addition, a new structural motif is seen: the six-amino acid carboxyl-terminal peptide of the Hin domain runs along the minor groove at the edge of the recombination site, with the peptide backbone facing the floor of the groove and side chains extending away toward the exterior. The x-ray structure provides an almost complete explanation for DNA mutant binding studies in the Hin system and for DNA specificity observed in the Hin-related family of DNA invertases.

Amino Acid Sequence↗
PubMed1994

Differentiation of endothelial cells: analysis of the constitutive and activated endothelial cell phenotypes.

Endothelial cells line the inside of all blood vessels, forming a structurally and functionally heterogeneous population of cells. Their complexity and diversity has long been recognized, yet very little is known about the molecules and regulatory mechanisms that mediate the heterogeneity of different endothelial cell populations. The constitutive organ- and microenvironment-specific phenotype of endothelial cells controls internal body compartmentation, regulating the trafficking of circulating cells to distinct vascular beds. In contrast, surface molecules associated with the activated cytokine-inducible endothelial phenotype play a critical role in pathological conditions including inflammation, tumor angiogenesis, and wound healing. Differentiation of the endothelial cell phenotypes appears to follow similar mechanisms to the differentiation of hematopoietic cells, with the exception that endothelial cells maintain transdifferentiating competence. The present review offers a scheme of endothelial cell differentiation and discusses the possible applications of differentially expressed endothelial cell molecules as targets for directed therapeutic intervention.

Animals↗
PubMed1994

HU and functional analogs in eukaryotes promote Hin invertasome assembly.

The prokaryotic protein HU functions as an accessory factor in many different biochemical reactions. We have characterized the role of HU in assembling the invertasome, an intermediate nucleoprotein complex involved in Hin-mediated site-specific recombination. Formation of this complex requires the looping of intervening DNA segments between sites bound by the Hin recombinase and the Fis protein. HU stimulates this process on substrates containing intervening segments of length < 100 bp. Characterization of the activity of HU in Hin-mediated recombination in vitro and in vivo yields evidence that its role in this reaction is primarily to facilitate the looping of the intervening DNA segment. By using this reaction as an assay, we identify proteins from mammals, yeast, trypanosomes, and wheat which can fulfill the same function in vitro. Using ligase-mediated circularization of short DNA fragments we also show that HU, the high mobility group (HMG) 1 and 2 proteins from mammals, and a protein from yeast can bend DNA extremely efficiently. These results support the view that this ubiquitous class of proteins enhance the assembly of nucleoprotein complexes under conditions of limited DNA flexibility.

Animals↗
PubMed1994

Cultivation of Borrelia burgdorferi from the blood of two patients with erythema migrans lesions lacking extracutaneous signs and symptoms of Lyme disease.

BACKGROUND: We recently demonstrated that cultivation of Borrelia burgdorferi from skin biopsy specimens obtained from erythema migrans lesions was an efficacious procedure to confirm the diagnosis of Lyme disease. OBJECTIVE: Our purpose was to investigate the efficacy of our microbiologic technique on blood samples obtained from patients with Lyme disease and erythema migrans. METHODS: Whole blood samples were obtained from 52 patients with erythema migrans and early localized or early disseminated Lyme disease and placed into polystyrene tubes containing 6 ml of modified Barbour-Stoenner-Kelly medium, processed, and examined for B. burgdorferi by dark-field microscopy. RESULTS: B. burgdorferi was cultured from the blood of two of our 52 patients (4%). Clinically, both of these patients were considered to have early localized Lyme disease. CONCLUSION: The culture of B. burgdorferi from the blood of patients with early Lyme disease does not appear to be an efficacious procedure to confirm the diagnosis of Lyme disease. However, the demonstration of spirochetemia in patients with erythema migrans without any extracutaneous evidence of disseminated illness does have therapeutic significance.

Adult↗
PubMed1994

Characterization of monoclonal antibodies for identification of Borrelia japonica, isolates from Ixodes ovatus.

Monoclonal antibodies for identification of Borrelia japonica isolated from tick, Ixodes ovatus and long-tailed shrew, Sorex unguiculatus in Japan and Borrelia related to Lyme disease (Borrelia burgdorferi sensu lato) were prepared and characterized. All isolates belonging to B. japonica and isolates from I. dentatus and cottontail rabbit in North America reacted with MAb O1441b against flagellin which was prepared from immunized mice with strain HO14, type strain of B. japonica, but isolates from I. persulcatus, patient, and wood mouse, Apodemus speciosus ainu, in Japan, and isolates belonging to B. burgdorferi, B. garinii and B. afzelii from North America and Europe did not. Strains used in this study reacted with MAb P62 against common antigen which was prepared from immunized mice with strain NT24 isolated from I. persulcatus in Japan, but B. japonica did not. These MAbs are useful for identification and differentiation of B. japonica and B. burgdorferi sensu lato in Japan.

Animals↗
PubMed1994

Sustained cellular immune responses to Borrelia burgdorferi: lack of correlation with clinical presentation and serology.

Fifty-one patients with erythema migrans were followed up prospectively with serial clinical evaluations, serologic determinations for antiborrelial antibodies, and lymphocyte stimulation responses to Borrelia burgdorferi antigens to determine (i) the factors associated with sustained cellular immune responses and (ii) whether lymphocyte stimulation is a good indicator of prior exposure to B. burgdorferi in patients treated early after erythema migrans. Positive lymphocyte stimulation responses ( > 2 standard deviations above normal control values) were found in 15 (29%) of 51 patients 3 months after treatment for erythema migrans and in 8 (18%) of 44 patients 1 year posttreatment. Heightened lymphocyte responses were not associated with the number or duration of erythema migrans lesions prior to treatment, the mean size of the largest erythema migrans lesion, or the number of symptoms at the time of presentation. The development of Jarisch-Herxheimer reaction, choice of antibiotic, and clinical outcome also were not associated with a positive lymphoproliferation assay result. Changes in the lymphocyte stimulation indices between the two time points assessed (3 months and 1 year posttreatment) also did not correlate with the above variables. When serologic results and lymphoproliferative responses were evaluated as categorical or continuous variables, there were no correlations between values. One year after treatment for early Lyme disease, lymphocyte reactivity is not a good indicator of prior infection with B. burgdorferi.

Adult↗