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Biomedical subjects

R C Johnson

Publications and source records attributed to R C Johnson.

At least 199 records · Page 11Linked to original sources
PubMed1992

Rapid isolation of flanking genomic DNA using biotin-RAGE, a variation of single-sided polymerase chain reaction.

A method is described for quickly and reproducibly isolating genomic DNA contiguous with known DNA sequence by means of the polymerase chain reaction (PCR). Flanking genomic DNA is isolated using a biotinylated sequence-specific primer in combination with a generic hybrid primer that binds to a deoxyoligonucleotide sequence artificially added to the ends of the genomic DNA. Amplified sequences that include the biotinylated primer are purified from nonbiotinylated amplification products by binding to a solid-phase streptavidin matrix. The biotinylated amplification product(s) are subjected to a further round of amplification, after which they can be subcloned and analyzed. This technique was applied to the isolation of three intron-exon junctions. Verification of the identify of these junction sequences was accomplished by designing primers based on the intron sequences isolated by Biotin-RAGE, amplifying across the exon using these intron primers, and sequencing the PCR-generated product.

Animals↗
PubMed1992

The Fis protein: it's not just for DNA inversion anymore.

Higher-order nucleoprotein complexes are associated with many biological processes. In bacteria the formation of these macromolecular structures for DNA recombination, replication, and transcription often requires not only the participation of specific enzymes and co-factors, but also a class of DNA-binding proteins collectively known as 'nucleoid-associated' or 'histone-like' proteins. Examples of this class of proteins are HU, Integration Host Factor, H-NS, and Fis. Fis was originally identified as the factor for inversion stimulation of the homologous Hin and Gin site-specific DNA recombinases of Salmonella and phage Mu, respectively. This small, basic, DNA-bending protein has recently been shown to function in many other reactions including phage lambda site-specific recombination, transcriptional activation of rRNA and tRNA operons, repression of its own synthesis, and oriC-directed DNA replication. Cellular concentrations of Fis vary tremendously under different growth conditions which may have important regulatory implications for the physiological role of Fis in these different reactions. The X-ray crystal structure of Fis has been determined and insights into its mode of DNA binding and mechanisms of action in these disparate systems are being made.

Bacterial Proteins↗
PubMed1992

In vitro and in vivo induction of tumor necrosis factor alpha by Borrelia burgdorferi.

Tumor necrosis factor alpha (TNF-alpha) is an immunoregulatory cytokine with many biological activities including the mediation of inflammation. We examined sera and synovial fluids from patients seropositive for infection with Borrelia burgdorferi using a radioimmunoassay specific for TNF-alpha. Significant elevation of TNF-alpha was found in the sera and synovial fluids of patients examined, while controls showed no elevation. Sera of mice infected with B. burgdorferi contained elevated levels of TNF-alpha which varied during the course of a 24-day infection. To determine whether B. burgdorferi is capable of inducing TNF-alpha production, spirochetes were added to adherent human peripheral blood mononuclear cells or mouse peritoneal exudate cells and 24 h later supernatants were assayed. TNF-alpha induction occurred in a dose-dependent manner. The maximum stimulation occurred when a ratio of 1 to 10 spirochetes per mononuclear cell was used. At optimal concentrations, induction was not diminished by inactivation of spirochetes or pretreatment with polymyxin B. These results suggest that an increase in TNF-alpha production may occur as a result of infection with B. burgdorferi.

Animals↗
PubMed1992

Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli.

Fis is a small basic DNA-binding protein from Escherichia coli that was identified because of its role in site-specific DNA recombination reactions. Recent evidence indicates that Fis also participates in essential cell processes such as rRNA and tRNA transcription and chromosomal DNA replication. In this report, we show that Fis levels vary dramatically during the course of cell growth and in response to changing environmental conditions. When stationary-phase cells are subcultured into a rich medium, Fis levels increase from less than 100 to over 50,000 copies per cell prior to the first cell division. As cells enter exponential growth, nascent synthesis is largely shut off, and intracellular Fis levels decrease as a function of cell division. Fis synthesis also transiently increases when exponentially growing cells are shifted to a richer medium. The magnitude of the peak of Fis synthesis appears to reflect the extent of the nutritional upshift. fis mRNA levels closely resemble the protein expression pattern, suggesting that regulation occurs largely at the transcriptional level. Two RNA polymerase-binding sites and at least six high-affinity Fis-binding sites are present in the fis promoter region. We show that expression of the fis operon is negatively regulated by Fis in vivo and that purified Fis can prevent stable complex formation by RNA polymerase at the fis promoter in vitro. However, autoregulation only partially accounts for the expression pattern of Fis. We suggest that the fluctuations in Fis levels may serve as an early signal of a nutritional upshift and may be important in the physiological roles Fis plays in the cell.

Amino Acid Sequence↗
PubMed1992

Experimental Borrelia burgdorferi infection of outbred mice.

Infectivity of Borrelia burgdorferi strain 297 in normal outbred ddY mice was examined. Strain 297 was inoculated intraperitoneally in 3-week-old outbred ddY mice. B. burgdorferi was routinely cultured from the heart and urinary bladder 5 to 84 days postinoculation. The combined isolation rate from both heart and urinary bladder was significantly higher than the rate from spleen, kidney, liver, urine, and blood samples. Three- and 10-week-old mice were infected with inocula of 10(4) and 10(5) or more, respectively. Passive transfer of undiluted and 10-fold-diluted anti-297 rabbit serum and active immunization of 50 to 100 micrograms of lyophilized whole cells completely protected mice from infection with B. burgdorferi. These results suggest that the outbred mouse is a convenient model for experimental infection with B. burgdorferi.

Animals↗
PubMed1992

Cultivation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin.

Skin biopsy specimens from the peripheral aspect of erythema migrans lesions (site 1) and from clinically normal perilesional areas (site 2) were compared as sources of Borrelia burgdorferi. This spirochete was isolated from the skin of 18 of 21 (86%) patients with untreated early Lyme disease at one or both biopsy sites. Site 1 specimens were superior to site 2 specimens for the isolation of B. burgdorferi. Site 1 specimens from 18 (86%) patients were culture positive, and site 2 specimens from 12 (57%) patients were culture positive. For patients whose site 2 specimens were culture positive, site 1 specimens were also found to be culture positive. B. burgdorferi was isolated from two patients with atypical lesions and from two patients with erythema migrans lesions that were less than 5 cm in diameter. This study demonstrates that the cultivation of B. burgdorferi from skin biopsy specimens from cutaneous lesions thought to be erythema migrans can be an efficacious procedure for confirming the diagnosis of Lyme disease and that the spirochete is present in clinically normal appearing perilesional skin.

Adult↗
PubMed1992

DNA analysis of Borrelia burgdorferi NCH-1, the first northcentral U.S. human Lyme disease isolate.

The DNA of the first northcentral United States human Lyme disease isolate, Borrellia burgdorferi NCH-1, was characterized and compared with the DNAs of nine other B. burgdorferi isolates. Strain NCH-1 was isolated in August 1989 from a human skin biopsy specimen. DNA was analyzed by pulsed-field gel electrophoresis and restriction endonuclease analysis. Contour-clamped homogeneous electric field pulsed-field gel electrophoresis of in situ-lysed cells was performed to compare the plasmid profiles of the various isolates. The plasmid profile of isolate NCH-1, which included five plasmids of approximately 69, 42, 38, 32, and 23 kb, could be distinguished from those of the other isolates examined. The DNA profile of NCH-1 was most similar to those of strain 297 (human cerebrospinal fluid isolate, Connecticut) and strain PAL (human erythema migrans isolate, New York) and most dissimilar from those of strain P/Gau (human erythema migrans isolate, Germany) and strain IPF (Ixodes persulcatus tick isolate, Japan). These results indicate that genetic diversity exists among B. burgdorferi strains isolated from different geographical areas.

Base Composition↗
PubMed1992

The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along with the entire 3'-untranslated region. Analysis of hybrid cell panels indicates that the human PAM gene is situated on the long arm of chromosome 5.

Animals↗
PubMed1992

The NIH-3 immunodeficient mouse is a model for Lyme borreliosis myositis and carditis.

Experimental infection of immunodeficient NIH-3 (N:NIH-bg-nu-xid) mice with Borrelia burgdorferi was found to result in multisystem histopathologic lesions. In addition to T-cell deficiency due to the nude mutation, these mice have an x-linked defect affecting the B-cell maturation and the beige mutation resulting in the absence of NK cells. NIH-3 mice were susceptible to progressive infection with B. burgdorferi resulting in pancarditis, synovitis, and skeletal interstitial myositis whereas controls remained normal. Cardiomyopathy was characterized by inflammatory mononuclear infiltration and fibrillar necrosis. Synovial hyperplasia and inflammation were seen in the tibiotarsal and ulna-carpal joints. Advanced myositis was observed in peripheral skeletal muscle. Gastrointestinal submucosa, heart, and skeletal muscle were heavily colonized with B. burgdorferi. This mouse is proposed as a model for Lyme borreliosis carditis, synovitis, and myositis.

Acquired Immunodeficiency Syndrome↗
PubMed1991

The molecular structure of wild-type and a mutant Fis protein: relationship between mutational changes and recombinational enhancer function or DNA binding.

The 98-amino acid Fis protein from Escherichia coli functions in a variety of reactions, including promotion of Hin-mediated site-specific DNA inversion when bound to an enhancer sequence. It is unique among site-specific DNA-binding proteins in that it binds to a large number of different DNA sequences, for which a consensus sequence is difficult to establish. X-ray crystal structure analyses have been carried out at 2.3 A resolution for wild-type Fis and for an Arg-89----Cys mutant that does not stimulate DNA inversion. Each monomer of the Fis dimer has four alpha-helices, A-D; the first 19 residues are disordered in the crystal. The end of each C helix is hydrogen bonded to the beginning of helix B' from the opposite subunit in what effectively is one long continuous, although bent, helix. The four helices, C, B', C', and B, together define a platform through the center of the Fis molecule: helices A and A' are believed to be involved with Hin recombinase on one side, and helices D and D' interact with DNA lying on the other side of the platform. Helices C and D of each subunit comprise a helix-turn-helix (HTH) DNA-binding element. The spacing of these two HTH elements in the dimer, 25 A, is too short to allow insertion into adjacent major grooves of a straight B-DNA helix. However, bending the DNA at discrete points, to an overall radius of curvature of 62 A, allows efficient docking of a B-DNA helix with the Fis molecule. The proposed complex explains the experimentally observed patterns of methylation protection and DNase I cleavage hypersensitivity. The x-ray structure accounts for the effects of mutations in the Fis sequence. Those that affect DNA inversion but not DNA binding are located within the N-terminal disordered region and helix A. This inversion activation domain is physically separated in the Fis molecule from the HTH elements and may specify a region of contact with the Hin recombinase. In contrast, mutations that affect HTH helices C and D, or interactions of these with helix B, have the additional effect of decreasing or eliminating binding to DNA.

Amino Acid Sequence↗
PubMed1991

Complement-mediated killing of the Lyme disease spirochete Borrelia burgdorferi. Role of antibody in formation of an effective membrane attack complex.

Lyme disease is a multisystemic illness caused by the spirochete Borrelia burgdorferi. In the absence of specific antibody, the spirochete is resistant to the bactericidal activity of C, despite the capacity of B. burgdorferi to activate both C pathways. We examined the mechanism of serum resistance by measuring the deposition of C3 and terminal C components on B. burgdorferi in the presence and absence of immune IgG. In normal human serum antibody-sensitized borreliae bound similar amounts of C3, and similar or increased amounts of C8 and C9, in comparison to unsensitized bacteria. However, at comparable levels of C3, C8, or C9 uptake, only sensitized bacteria were killed. The requirement of antibody for killing could not be explained by differences in the rate of C deposition or by differences in the C9 to C8 ratio in the membrane attack complex (MAC). We found that bacteria incubated in C5-depleted human serum, but not in C6-depleted serum, were killed when this treatment was followed by antibody and the missing C components. Bacteria were also killed by reactive lysis (C5b-9) provided that antibody was present. Therefore, the effect of bactericidal IgG occurred at the stage of C5b binding to the bacterial surface. Elution studies of bound C9 indicated that the MAC was stably bound to the outer membrane of B. burgdorferi, whether or not the bacteria were treated with antibody. However, treatment with 0.1% trypsin released 48% of 125I-C9 from the surface of unsensitized borreliae and 24% from IgG-sensitized cells, demonstrating that the presence of the antibody changed the accessibility to trypsin of C9 in the MAC. These results indicate that the effect of antibody in the killing process is not to enhance the rate or extent of initial or terminal component binding, but rather to alter the bacterial outer membrane to allow effective MAC formation.

Antibodies, Bacterial↗
PubMed1991

Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression.

The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail. Antisera to a peptide within the PAL domain were used to identify a 50-kDa protein as the major form of PAL in bovine neurointermediate pituitary granules. This 50-kDa PAL protein was purified and found to begin at Asp434 of bPAM, indicating that it could arise through endoproteolytic cleavage of the bPAM precursor at Lys432-Lys433. With alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine as the substrate, PAL exhibits a pH optimum of 5.0; enzymatic activity is inhibited by high concentrations of salt but is relatively resistant to thiol reagents and urea. PAL activity is inhibited by EDTA and restored by a number of divalent metals, including Cd2+, Cu2+, Zn2+, and Ca2+. Kinetic studies using alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine indicate that PAL has a Km of 38 microM and a turnover number of 220/s. Expression vectors encoding only the soluble PHM domain or the PAM precursor from which the PHM domain had been deleted were constructed. hEK293 cells transfected with the PHM vector exhibited a 10-fold increase in secretion of PHM activity with no PHM activity detectable in control or transfected cells. hEK293 cells transfected with the PAL vector exhibited a 2-fold increase in secretion of PAL activity and a 15-fold increase in cellular PAL activity. Most of the PAL activity produced by the transfected cells remained membrane-associated.

Amidine-Lyases↗
PubMed1991

Endothelial cell membrane vesicles in the study of organ preference of metastasis.

Many malignancies exhibit distinct patterns of metastasis that appear to be mediated by receptor/ligand-like interactions between tumor cells and organ-specific vascular endothelium. In order to study endothelial cell surface molecules involved in the binding of metastatic cells, we developed a perfusion method to isolate outside-out membrane vesicles from the lumenal surface of rat lung microvascular endothelium. Lungs were perfused in situ for 4 h at 37 degrees C with a solution of 100 mM formaldehyde, 2 mM dithiothreitol in phosphate-buffered saline to induce endothelial cell vesiculation. Radioiodinated rat lung endothelial cell membrane vesicles bound lung-metastatic tumor cells (B16F10, R323OAC-MET) in significantly higher numbers than their low or nonmetastatic counterparts (B16F0, R323OAC-LR). In contrast, leg endothelial membrane vesicle showed no binding preference for either cell line. Neuraminidase treatment of vesicles abolished specificity of adhesion of lung-derived vesicles to lung metastatic tumor cells. These results demonstrate that in situ perfusion is an appropriate technique to obtain pure endothelial cell membrane vesicles containing functionally active adhesion molecules. The preferential binding of lung-derived endothelial cell membrane vesicles by lung metastatic tumor cells is evidence of the importance of endothelial cell adhesion molecules in the formation of metastases.

Animals↗
PubMed1991

Identification of two functional regions in Fis: the N-terminus is required to promote Hin-mediated DNA inversion but not lambda excision.

The Fis protein of E. coli binds to a recombinational enhancer sequence that is required to stimulate Hin-mediated DNA inversion. Fis is also required for efficient lambda prophase excision in vivo. The properties of mutant Fis proteins were examined in vivo and in vitro with respect to their stimulatory effects on these two different site-specific DNA recombination reactions. Both recombination reactions are dramatically affected by mutations altering a helix-turn-helix DNA binding motif located near the Fis C-terminus (residues 74-93). These mutations invariably decrease DNA binding affinity and some cause reduced DNA bending. Mutations in the Fis N-terminal region reduce or abolish the stimulation of Hin-mediated DNA recombination by Fis, but have little or no effect on DNA binding or lambda excision. We conclude that there are at least two functionally distinct domains in Fis: a C-terminal DNA binding region that is required for promoting both DNA recombination reactions and an N-terminal region that is uniquely required for Hin-mediated inversion.

Amino Acid Sequence↗
PubMed1991

Modulation of endothelial cell surface glycoconjugate expression by organ-derived biomatrices.

Cell surface molecules play an important role in cellular communication, migration, and adherence. Here, we show the effect of organ-derived biomatrices on endothelial cell surface glycosylation. Five different lectins (with and without neuraminidase treatment) have been used as probes in an enzyme-linked lectin assay to quantitatively detect glycoconjugates on endothelial cells (BAEC) grown on tissue culture plastic or biomatrices isolated from bovine lung, liver, and kidney. BAEC generally exhibit strong binding of concanavalin A (Con A), Ricinus communis agglutinin I (RCA-I), wheat germ agglutinin (WGA), and soybean agglutinin, and peanut agglutinin after neuraminidase pretreatment of cells (Neu-SBA and Neu-PNA), while SBA and PNA consistently bind weakly to BAEC. BAEC grown on organ-derived biomatrices exhibit significantly altered binding intensities of Con A, RCA-I, WGA, and Neu-PNA: BAEC cultured on lung- or kidney-derived biomatrices express significantly stronger binding affinities for Con A and RCA-I than BAEC grown on liver-derived biomatrix or tissue culture plastic. In contrast, BAEC binding of WGA and PNA (after treatment of cells with neuraminidase) is significantly reduced when BAEC are grown on liver- or kidney-derived biomatrix. Quantitative lectin immunogold electron microscopy reveals consistently stronger lectin binding over nuclear regions compared to junctional regions between neighboring cells. These results indicate that extracellular matrix components regulate endothelial cell surface glycoconjugate expression, which determines cellular functions, e.g., preferential adhesion of lymphocytes or metastatic tumor cells.

Animals↗
PubMed1991

Mechanism of site-specific DNA inversion in bacteria.

A wealth of new information regarding the structure of the synaptic complex, the mechanism of DNA strand exchange, and the role of the recombinational enhancer in promoting DNA inversion has been obtained from a combination of approaches. These include: electron microscopy of reaction intermediates, topological analysis of recombination products, and X-ray crystallography coupled with genetic analysis.

Bacteria↗
PubMed1991

Configuration of DNA strands and mechanism of strand exchange in the Hin invertasome as revealed by analysis of recombinant knots.

The Hin recombinase of Salmonella normally catalyzes a site-specific DNA inversion reaction that is very efficient when the Fis protein and a recombinational enhancer sequence are present. The mechanism of this recombination reaction has been investigated by analyzing the formation and structure of knots generated in different plasmid substrates in vitro. Hin seldom knots the wild-type substrate under standard recombination conditions. However, we show that increasing the length of DNA between the recombination sites and the enhancer and changing the sequence of the core nucleotides where strand exchange occurs increases the efficiency of the knotting reaction. The structure of the knots generated by different mutant substrates strongly supports a model involving a unique configuration of DNA strands at synapsis and DNA strand exchange mediated by rotation of one set of Hin subunits after DNA cleavage. Analysis of the stereostructure of the knots by electron microscopy of RecA-coated DNA molecules demonstrates that the direction of subunit rotation is exclusively clockwise. Because multiple subunit rotations generating knotted molecules do not occur efficiently when the enhancer is located in its native position, we suggest that the enhancer normally remains associated with the two recombination sites in the invertasome structure during strand exchange to limit strand rotation once it has been initiated. Under certain conditions, however, complex knots are formed that are probably the result of the premature release of the enhancer and multiple, unrestrained subunit exchanges.

Base Sequence↗