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Biomedical subjects

W C Johnson

Publications and source records attributed to W C Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Babesia bovis immunity. In vitro and in vivo evidence for IL-10 regulation of IFN-gamma and iNOS.

IL-10 has been shown to have profound immunoregulatory attributes and in the bovine appears to downregulate both Th1- and Th2-like responses. Using RT-PCR, we demonstrate IL-10 in vitro down-regulation of mRNA expression of iNOS, the cytokines involved in nitric oxide signal transduction initiation (IFN-gamma and TNF-alpha), and other mononuclear phagocyte associate cytokines. In addition, using RT-PCR with peripheral blood leukocytes and spleen leukocytes, the Griess reaction, and a killing assay, we provide evidence for the importance of iNOS in a successful immune response to B. bovis infection and for high and persistent IL-10 mRNA expression when the immune response is unsuccessful. We also provide evidence that antibody developed early after an initial infection appears to lack protective attributes (neutralizing and opsonic). Together, the data suggests that IL-10 and IFN-gamma are critical molecules involved in the response to this intraerythrocytic protozoan infection.

Animals↗
PubMed1998

Foot TcPO2 response to lumbar sympathectomy in patients with focal ischemic necrosis.

We prospectively evaluated all patients with superficial foot necrosis of 1-3 cm and transcutaneous oxygen tension (TcPO2) values of <30 mmHg who received a sympathectomy as the primary treatment of their vascular occlusive disease. Preoperatively, and every 2-3 days in the postoperative period, measurement of TcPO2 of the forefoot was performed. Clinical success was defined as healing of the necrosis or healing of a toe amputation and avoidance of a major below-knee/above-knee amputation for 1 year. Ten patients were available for long-term evaluation. During the first 4-5 days, all patients increased their foot TcPO2 and the mean increase (23 mmHg) was significant (p = 0.04). Clinical improvement was marked by an average increase of 29 mmHg by postoperative day 10. In contrast, patients with clinical failure had only an average increase of 5 mmHg in TcPO2 by the same postoperative interval. Preoperative increase in TcPO2 by at least 20 mmHg in response to dependency predicted a favorable response to sympathectomy. In addition, sustained postoperative increases in tissue oxygen levels by postoperative day 10 also favored wound healing.

Adult↗
PubMed1998

The role of IL-10 in iNOS and cytokine mRNA expression during in vitro differentiation of bovine mononuclear phagocytes.

In the study reported here, we used RT-PCR with primers specific for interleukin-1 (IL-1), IL-6, IL-10, tumor necrosis factor-alpha (TNF-alpha), and nitric oxide synthase (iNOS) to assess the cytokine mRNA expression associated with bovine blood monocytes during their differentiation to macrophages cultured on plastic (1 week). In addition, we used RT-PCR to assess the contribution of gammadelta T cells as a source of interferon-gamma (IFN-gamma), the induction signal for iNOS. Further, we evaluated cytocentrifuge preparations from the cultures for the production of IL-10 using specific antibody. We previously demonstrated that iNOS can be induced in cultured bovine monocytes in response to IFN-gamma and TNF-alpha but lose this capability in a short period of time. However, we demonstrate here that iNOS induction from monocytes cultured with IFN-gamma secreting gammadelta T cells is prolonged, suggesting that this source of IFN-gamma primes the monocytes before exogenous stimulation. Based on mRNA expression, placement of monocytes in culture resulted in activation, followed by quiescence. By 6 days in culture, the iNOS message was reduced below the basal level. In addition, the TNF-alpha message was substantially reduced, and IL-1 and IL-6 messages were reduced below detectable levels. This correlated with an increase in IL-10 message. Downregulation of these same cytokine messages as well as IFN-gamma message occurred within a 20-h period when IL-10 was added exogenously to cultures of total leukocytes. At the same time, there was an increase in the number of IL-10-positive cells and an increase in the intensity of anti-IL-10 staining within adherent cells. These results provide evidence for IL-10 regulation of some bovine mononuclear phagocyte effector functions.

Animals↗
PubMed1998

Desmoplastic fibroblastoma.

Previous reports of a distinctive, fibrous, soft-tissue tumor include eight patients with subcutaneous lesions and six patients with intramuscular lesions. We report a 48-year-old woman with a 2-cm cutaneous and subcutaneous nodule on the left arm with the same histologic features. An excisional biopsy showed a large, well circumscribed tumor replacing the reticular dermis and subcutaneous tissue. The tumor was relatively hypocellular and composed primarily of large, spindled, plump or stellate fibroblasts haphazardly dissecting between thickened fibrotic collagen bundles. The stroma contained a large amount of mucin which was positive with alcian blue at pH 2.5, and relatively numerous mast cells were present. The fibroblastic-like cells were positive with Vimentin and Factor XIIIA and negative with S-100, desmin, actin and keratin.

Collagen↗
PubMed1997

Correcting the circular dichroism spectra of peptides for contributions of absorbing side chains.

The aromatic and sulfur-containing side chains Trp, Tyr, Phe, Cys, and Met contribute to the CD spectra of peptides and proteins in the amide region, interfering with the analysis for secondary structure. We propose a method to correct the CD spectra of peptides undergoing the helix-coil transition for contributions due to absorbing side chains using singular value decomposition. The method uses the common basis vectors obtained from an analysis of the CD spectra of related peptides without the aromatic and sulfur-containing amino acids. The common basis vectors are fitted to a portion of the CD spectrum of the peptide being corrected, in the range that is unaffected by its sidechain contributions. Then the resulting coefficients from the fitting are used along with the common basis vectors to regenerate the entire corrected spectrum. The method is illustrated for the CD spectra of the peptide sequence acetyl-Y-VAXAK-VAXAK-VAXAK-amide, where X is substituted with the 20 naturally occurring amino acids. This peptide model adopts a random-coil conformation in 2 mm sodium phosphate buffer, pH 5.5, and becomes an alpha helix in methanol/buffer solutions. The difference between the original and corrected spectra shows the contribution from the aromatic and sulfur-containing side chains.

Amino Acid Sequence↗
PubMed1997

Calcium binding to recoverin: implications for secondary structure and membrane association.

Recoverin is an EF-hand calcium-binding protein reportedly involved in the transduction of light by vertebrate photoreceptor cells. It also is an autoantigen in a cancer-associated degenerative disease of the retina. Measurements by circular dichroism presented here demonstrate that the binding of calcium to recoverin causes large structural changes. increasing the alpha-helical content of the protein and decreasing its beta-turn, beta-sheet and 'other' structures. The maximum helical content (67%) was observed at 100 microM free calcium and, unlike calmodulin, decreased as the calcium concentration was modulated in either direction from this value. Fluorescence measurements indicated that recoverin may aggregate or undergo structural changes independent of calcium binding as the calcium concentration is increased above 100 microM. EGTA also appeared to affect the structure of recoverin independent of its chelation of calcium. While calcium-induced conformational changes have been proposed to alter the membrane binding of recoverin through association of its myristoylated amino terminus, in the experiments presented here the partitioning of recoverin between the cytoplasmic and membrane compartments of the rod photoreceptor outer segment was unaffected by the concentration of calcium, therefore it appears unlikely that a calcium-myristoyl switch acts alone to anchor recoverin directly to the membrane. These experiments were conducted with native recoverin which is heterogeneously acylated, but mass spectrometry confirmed that simple chromatographic methods could be devised to isolate the different forms of recoverin for further studies.

Animals↗
PubMed1997

Transcutaneous partial oxygen pressure changes following skew flap and Burgess-type below-knee amputations.

OBJECTIVE: To evaluate the degree of flap hypoxia following different types of below-knee amputations. DESIGN: Prospective preoperative and postoperative measurements of transcutaneous partial oxygen pressure (TcPo2) at the site of amputation in 10 consecutive patients who underwent a Burgess-type below-knee amputation (group 1) and in 10 consecutive patients who underwent a skew flap amputation (group 2). SETTING: An academic, tertiary care Veterans Affairs medical center. PATIENTS: Individuals with severe arterial occlusive disease of the lower extremity, in many of whom vascular reconstruction has failed. INTERVENTION: Measurements of TcPo2 (in millimeters of mercury). MAIN OUTCOME MEASUREMENT: The decrease in TcPo2 associated with the different "flaps" of a Burgess-type below-knee amputation. RESULTS: In all skin flaps, regardless of the type of amputation, an early postoperative reduction of the TcPo2 was noted. The greatest reduction (11 mm Hg) and persistence at 20 postoperative days were noted in posterior flaps. CONCLUSIONS: Strict conformity to the Burgess-type of below-knee flap design may not provide an optimal incisional blood supply. Consideration should be given to the skew flap technique in patients who require amputation for severe lower limb arterial insufficiency.

Aged↗
PubMed1997

Babesia bovis: identification of immunodominant merozoite surface proteins in soluble culture-derived exoantigen.

Babesia bovis merozoite proteins presenting as exoantigens in in vitro culture supernatants have been characterized. Bovine antisera to B. bovis exoantigens were used to immunoprecipitate [35S]-methionine metabolically labeled or lactoperoxidase-catalyzed radioiodinated B. bovis merozoite proteins. A total of 24 metabolically labeled proteins ranging in molecular weight from 24,000 to 225,000 Da and 9 radioiodinated proteins with molecular weights varying between 24,000 and 225,000 Da were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies to B. bovis merozoite surface proteins were also used to immunoprecipitate metabolically labeled exoantigens directly from in vitro culture supernatants. These results demonstrate epitopes from at least nine merozoite surface proteins present in the exoantigen fraction, among which are the recently characterized major surface antigens 1 and 2, rhoptry-associated protein 1, and spherical body protein 2.

Animals↗
PubMed1996

Reactive oxygen and nitrogen intermediates and products from polyamine degradation are Babesiacidal in vitro.

Products released from activated macrophages have been demonstrated to have microbicidal activity against a variety of microorganisms. Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) have been shown to affect the induction of degenerate (crisis) forms of Plasmodium spp. Polyamines are degraded into acrolein which has also been shown to be toxic to Plasmodium spp. We have investigated the possibility that these products act similarly with Babesia bovis. Crisis forms of B. bovis developed in erythrocyte cultures after the introduction of supernatants containing ROI, RNI, and acrolein. Xanthine degradation by xanthine oxidase leads to the formation of superoxide anion, hydrogen peroxide, and hydroxyl radicals. The degradation in the presence of B. bovis was toxic to the parasite. The toxicity was partially reversed by the addition of the ROI scavenger catalase. However, H2O2 added directly had little effect, suggesting a role for the other ROI products. Spermine degradation by polyamine oxidase and direct addition of acrolein was toxic in a dose-dependent manner. Finally, spontaneous generation of nitric oxide from sodium nitroprusside or S-nitroso-N-acetyl-penicillamine was also toxic in a dose-dependent manner. These data lead us to suggest a role for activated macrophages in the primary immune response against B. bovis.

Acrolein↗
PubMed1996

Assessment of bovine mononuclear phagocytes and neutrophils for induced L-arginine-dependent nitric oxide production.

Microbicidal activity of reactive oxygen intermediates and reactive nitrogen intermediates has been described from both murine and human cytokine activated macrophages. An L-arginine-dependent pathway of nitric oxide generation has recently been described from bovine bone marrow-derived and monocyte-derived macrophages in response to a phagocytic stimulus. We have investigated the induction and release of both reactive oxygen intermediates and reactive nitrogen intermediates from bovine neutrophils, and blood and spleen mononuclear phagocytes in response to either a phagocytic or cytokine stimulus. Mononuclear phagocytes were poor producers of hydrogen peroxide (a measure of reactive oxygen intermediate production) under conditions that readily caused release by neutrophils. In contrast, nitrite, as a measure of nitric oxide production, could not be induced from neutrophils under any stimulation conditions, while mononuclear phagocytes responded to both a phagocytic stimulus and cytokines with the induction of nitric oxide synthase message and production of nitric oxide. There appeared to be two populations of monocytes that differed both in their adherent characteristics and their level of cytokine-induced nitric oxide production. Both populations stained with a single monoclonal antibody. However, the population that had not adhered to plastic within 3 h responded to cytokine stimulation, producing up to 3 times more nitric oxide on a per cell basis than the readily adherent population. Cytokine induction required the presence of interferon-gamma and either tumor necrosis factor-alpha or lipopolysaccharide. L-arginine dependence was demonstrated by inhibition with an L-arginine analog and restoration with addition of excess L-arginine.

Animals↗
PubMed1996

Effects of alpha-hydroxy acids on photoaged skin: a pilot clinical, histologic, and ultrastructural study.

BACKGROUND: alpha-Hydroxy acids (AHAs) have been reported to improve aging skin. The mechanisms of action of AHAs on epidermal and dermal compartments need clarification. OBJECTIVE: Our purpose was to determine the effects of AHAs on photoaged human skin by clinical and microanalytic means. METHODS: Patients applied a lotion containing 25% glycolic, lactic, or citric acid to one forearm and a placebo lotion to the opposite forearm for an average of 6 months. Thickness of forearm skin was measured throughout the study. Biopsy specimens from both forearms were processed for analysis at the end of the study. RESULTS: Treatment with AHAs caused an approximate 25% increase in skin thickness. The epidermis was thicker and papillary dermal changes included increased thickness, increased acid mucopolysaccharides, improved quality of elastic fibers, and increased density of collagen. No inflammation was evident. CONCLUSION: Treatment with AHAs produced significant reversal of epidermal and dermal markers of photoaging.

Aged↗
PubMed1996

Increased factor XIIIa transglutaminase expression in dermal dendrocytes after treatment with alpha-hydroxy acids: potential physiologic significance.

BACKGROUND: Topical alpha-hydroxy acids (AHAs) have been shown to improve photoaging in human skin. OBJECTIVE: We studied factor XIIIa transglutaminase expression in dermal dendrocytes (DDs) and mast cell degranulation after treatment of the skin with AHAs. METHODS: Skin biopsy specimens obtained from patients after 4 to 8 months of treatment with lotions containing 25% AHAs were evaluated for factor XIIIa transglutaminase expression with immunoperoxidase and electron microscopy. Immunoperoxidase-stained sections were studied by means of semiquantitative methods and image analysis. Mast cell degranulation was studied by image analysis. RESULTS: Increased factor XIIIa transglutaminase expression was seen after treatment with AHAs. All treated sites had increased scores compared with control sites by semiquantitative methods. Seventy-five percent of treated sites showed an increased mean area over control sites of factor XIIIa transglutaminase positivity with image analysis. These results correlated with an increased level of mast cell degranulation in treated sites and with activation of DDs as seen by electron microscopy. CONCLUSION: Treatment of the skin with AHAs leads to mast cell degranulation and increased expression of factor XIIIa transglutaminase by activated DDs. Mast cell degranulation may lead to activation of DDs and increased factor XIIIa transglutaminase expression, via the action of tumor necrosis factor-alpha. We speculate that clinical and histologic improvement in photoaged skin after treatment with AHAs may be somehow related to this process.

Administration, Cutaneous↗
PubMed1995

Purification of recombinant porcine m2 muscarinic acetylcholine receptor from Chinese hamster ovary cells. Circular dichroism spectra and ligand binding properties.

The recombinant porcine m2 muscarinic acetylcholine receptor (rPm2R) from Chinese hamster ovary cells has been purified to homogeneity. Two mg of purified rPm2R, with a specific activity of 12 nmol of R-(-)-quinuclidinyl benzilate/mg of protein, were obtained from 30 ml of packed Chinese hamster ovary cells. The apparent molecular mass (78.5 kDa) and specific activity for the rPm2R preparation were the same as that for the Pm2R purified from atrial tissue, but the yield was 100 times greater. Purified rPm2R bound agonist and antagonist with the same affinities and coupled to the inhibitory guanine nucleotide-binding protein with the same efficiency as the purified native atrial Pm2R. Ligand binding studies were consistent with a single class of antagonist binding sites but two subclasses of agonist binding sites. The fraction of rPm2R having high affinity for agonists was increased by mM Mg2+, low detergent concentration, and low temperature. Circular dichroism spectra obtained for the purified rPm2R with and without agonists were indistinguishable, but spectra for the antagonist-occupied receptor showed reproducibly deeper characteristic negative deflections at 208 and 220 nm. Secondary structure analysis of the CD spectra predicted 53% alpha-helix for the free receptor and 49% alpha-helix for the R-(-)-quinuclidinyl benzilate-receptor complex.

Animals↗
PubMed1995

Comparison of base inclination of ribo-AU and deoxyribo-AT polymers.

The inclination angle between the base normal and the helix axis is measured for ribo-AU polymers by using flow linear dichroism (LD), and compared to measurements for deoxyribo-AT polymers under dehydrating conditions. The CD of the DNA polymers under the dehydrating conditions is not the same as the corresponding RNA polymers, which are presumed to be in the A form. However, the LD indicates that poly(dAdT)-poly(dAdT) can assume the A form in 80% 2,2,2-trifluoroethanol, although poly(dA)-poly(dT) retains B form structure in this dehydrating solvent. The inclination angles are similar for B form poly(dAdT)-poly(dAdT) and poly(dA)-poly(dT), and these parameters are also similar for A form poly(rArU)-poly(rArU) and poly(rA)-poly(rU). All inclination axes are similar.

Adenine↗
PubMed1995

Comparison of base inclination of ribo-GC and deoxyribo-GC polymers, and synthesis of poly(rGrC)-poly(rGrC).

The inclination angle between the base normal and the helix axis, and the axes around which the bases incline, are measured for ribo-GC polymers in buffer by using flow linear dichroism (LD), and compared to measurements for deoxyribo-GC polymers in buffer and under dehydrating conditions. A new method is designed to synthesize poly(rGrC)-poly(rGrC), which is not available commercially, in large quantities. The LD of this RNA reveals inclination angles that are similar to the B-form DNA in buffer, although the axes are different. The CD of poly(dGdC)-poly(dGdC) under the dehydrating conditions is similar to poly(rGrC)-poly(rGrC), indicating it is in the A form, and the LD gives larger inclination angles than either the B form or the corresponding RNA. Poly(dG)-poly(dC) is in the A form in buffer. Comparison among poly(rG)-poly(rC) in buffer, and poly(dG)-poly(dC) in buffer under dehydrating conditions, reveals similar inclination angles and axes, although the LD shows that the DNA has the largest inclination angles. Except for poly(rGrC)-poly(rGrC), which has a unique reduced dichroism, all the axes for G are similar, as are the axes for C.

Circular Dichroism↗
PubMed1995

Kinetic and biophysical analysis of the m2 muscarinic receptor.

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.

Adenylyl Cyclase Inhibitors↗
PubMed1995

Development of conventional subunit vaccines for anaplasmosis and babesiosis.

Tick-borne hemoparasitic diseases of cattle continue to impact the beef industry throughout a large portion of the world. A substantial amount of research is currently focused on development of improved vaccines. The two main approaches being followed are: (1) use of conventional inactivated or native protein subunit vaccines, and (2) development of recombinant DNA technology for expression of selected immunogens. Recombinant or synthetic peptide based vaccines hold promise owing to the exquisitely defined nature of the product. However, the development is long-term, and will require extensive testing and risk assessment before field trials can be considered. Until then, more conventional subunit immunogens may offer an attractive alternative, and can be defined immunologically better than before. This paper reviews progress in the development of improved vaccines for anaplasmosis and babesiosis with an emphasis on the characterization of culture-derived babesial exoantigens. Both in vitro and in vivo information is presented.

Anaplasmosis↗