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Biomedical subjects

A R Johnson

Publications and source records attributed to A R Johnson.

At least 37 records · Page 2Linked to original sources
PubMed1996

Mitogenic effects of mycotoxins on T4 lymphocytes.

Mycotoxins have compromising effects on varied biological systems, even when ingested at levels which do not evoke manifestations of clinical mycotoxicoses. No data have been previously found as to the therapeutic and mitogenic effects of mycotoxins. This was the objective of the present work. Human peripheral T4 lymphocytes were obtained by venipuncture, propagated in RPMI 1640 medium and challenged with varied concentrations of aflatoxins, B1, B2, G1, and G2. The cells were stained with propidium iodide and fluorescent isothiocyanate (FITC) and examined microfluorometrically. All the mycotoxins were significantly mitogenic on the basis of dose response. No adverse effects were observed when doses of 10, 50 and 100 micrograms were administered on a modest clinical basis to volunteers.

Aflatoxins↗
PubMed1995

An analysis of astrocytic cell lines with different abilities to promote axon growth.

The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin and N-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM.

Animals↗
PubMed1995

A simple method for preparing modified TES and Tris yolk buffer that is optically clear and membrane filterable.

OBJECTIVES: To determine whether modified TES and Tris (TEST) yolk buffer (TYB) made using a commercially available egg yolk extract would exhibit lab performance characteristics equal to the existing preparation made with whole egg yolk and to define the phospholipid content of the new modified TYB formulation. DESIGN: Divided ejaculates from 21 normozoospermic and 7 oligozoospermic males presenting for pre-IVF evaluation were stored at 0 to 4 degrees C for 42 hours using commercially available or modified TYB before analysis in the optimized sperm penetration assay (SPA). SETTING: A commercial tissue culture manufacturer and a clinical fertility reference laboratory. MAIN OUTCOME MEASURES: Sperm swim-up recoveries and average penetrations per ovum, determined by the SPA, were used as measures of sperm function. High-pressure liquid chromatography (HPLC) was used to profile the egg yolk extract. RESULTS: No significant differences in either sperm swim-up recovery rates or SPA results were found in normal or poor quality semen that was treated with modified or commercial TYB. The major constituent in commercial egg yolk extract is lecithin. CONCLUSIONS: Commercially available egg yolk extract passes easily through a 0.2-microns filter, is a rich source of lecithin, and can be substituted effectively for whole egg yolk in preparing TYB.

Chromatography, High Pressure Liquid↗
PubMed1995

Regulation of mesothelial cell mitogenesis by antisense oligonucleotides for the urokinase receptor.

The association of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) influences various biologic functions, including cell migration, angiogenesis, differentiation, and wound healing. Expression of uPAR at the mesothelial surface could, therefore, influence cellular responses in the pleural space. We found that a line of cultured human mesothelial cells (MeT5A) expressed specific and saturable binding sites for uPA that increased on stimulation with PMA. Ligand blotting studies showed that the mesothelial receptor is a 50 kD protein similar to that in other cell lines. Binding of active and intact, but not amino terminal or low molecular weight fragment, uPA to mesothelial cells enhanced DNA synthesis and cell proliferation, and antibodies against either the active site of uPA or uPAR abrogated this effect. We reasoned that regulation of uPAR expression could control uPA-induced mitogenesis and tested this hypothesis with antisense oligonucleotides complementary to uPAR mRNA. Phosphorothioate-modified antisense oligonucleotides inhibited uPA-mediated mesothelial cell proliferation in a concentration-dependent manner. These effects were associated with decreased binding of 125I-uPA and reduced expression of the uPAR gene product. The results indicate that uPAR is involved in signal transduction pathways that control uPA-mediated mesothelial cell proliferation, a process implicated in the pathogenesis of mesothelial inflammation and pleural neoplasia. Antisense oligonucleotides to uPAR suppress mesothelial cell mitogenesis in vitro and offer a potential means of regulating the process in vivo.

Base Sequence↗
PubMed1995

A beta-linked mannan inhibits adherence of Pseudomonas aeruginosa to human lung epithelial cells.

Adherence through carbohydrate-binding adhesins is an early step in colonization of the lung by gram-negative organisms, and because published data indicate that binding involves mannose groups, we tested the ability of a beta-linked acetyl-mannan (acemannan) to inhibit adherence of Pseudomonas aeruginosa to cultures of human lung epithelial cells. Adherence of radiolabelled P.aeruginosa to A549 cells (a type II-like pneumocyte line) increased linearly with the duration of the incubation. Acemannan inhibited adherence of bacteria, and the extent of inhibition was related to the concentration of the mannan. Inhibition required continued contact between acemannan and the target epithelial cells; cells washed free of acemannan no longer discouraged bacterial binding. Comparison of binding between seven different strains of P.aeruginosa indicated that fewer mucoid than non-mucoid bacteria adhered, but binding of either phenotype was inhibited by acemannan. Mannose, methyl alpha-D-mannopyranoside, methyl beta-D-mannopyranoside and dextran did not affect adherence of any of the non-mucoid strains. Mannose inhibited adherence by one mucoid strain, but not the other, indicating differences between strains of the same phenotype. Since prior treatment of epithelial cells with concanavalin A did not affect acemannan-induced inhibition of bacterial adherence, we concluded that the inhibitory effect of acemannan probably does not involve mannose-containing receptors.

Analysis of Variance↗
PubMed1995

Inhibition of factor Xa-mediated procoagulant activity of human lung fibroblasts and pleural mesothelial cells.

Extravascular fibrin deposition characterizes diverse forms of lung and pleural injury. Fibrin formation in these compartments is locally potentiated by the assembly and expression of the prothrombinase procoagulant complex (factors Xa, Va and II) at the surface of human lung fibroblasts and pleural mesothelial cells. We sought to identify structural domains on factor Xa that mediate expression of prothrombinase activity by these cells. In order to accomplish this objective, we used panels of monoclonal antibodies (MoAbs) to factor X to block prothrombinase assembly and function on the surface of cultured human lung fibroblasts and pleural mesothelial cells. Of 30 factor X MoAbs that recognized native factors X and Xa, 10 completely inhibited factor Xa function (prothrombin activation), and five others neutralized Xa function without affecting cell-binding, presumably by blocking the prothrombin binding site. Western blots showed that these inhibitory MoAbs reacted with the Xa heavy-chain. One MoAb that recognized the factor Xa light-chain blocked prothrombin activation at the factor Va binding site. Our results indicate that prothrombinase activity at the surface of lung parachymal or pleural cells can be blocked by MoAbs that interact with either the heavy- or light-chain of factors X. Antibodies that neutralize cell surface-expressed prothrombin activation offer a potential means to arrest pericellular fibrin formation in the lung and pleural space.

Animals↗
PubMed1995

Regulation of fibrin deposition by malignant mesothelioma.

Malignant mesothelioma (MM) is a locally aggressive tumor that spreads by poorly understood mechanisms. Because neoplastic spread has been linked to altered fibrin turnover, we used immunohistochemistry of nine MM and three fibrous tumors of the pleura to confirm in vivo fibrin deposition and expression of selected coagulation and fibrinolytic reactants in MM. Tumor-associated fibrin was readily detectable at site of tissue invasion. Little fibrin was distributed within the tumor, but tissue factor and tissue factor pathway inhibitor, urokinase, urokinase receptor, and plasminogen activator inhibitors 1 and 2 were all detected in either epithelioid or sarcomatous areas of MM. We used the MS-1 human pleural mesothelioma cell line to determine how expression of these reactants is regulated. Fibrinolytic activity of MS-1 is mainly due to urokinase and is responsive to cytokine stimulation. Functional extrinsic activation and prothrombinase complexes assemble at the cell surface. MM express procoagulants as well as fibrinolytic reactants in vivo and in vitro that promote local fibrin formation and remodeling. Fibrin deposition occurs primarily at areas of tissue invasion and could promote local extension of this neoplasm. Sparsity of fibrin within the central portions of the tumor stroma suggests that local resorption of transitional fibrin occurs at sites of established MM.

Blood Coagulation↗
PubMed1994

Growth factors for human pleural mesothelial cells in soluble products from formed clots.

Fibrin deposition within the pleural space may influence repair following pleural injury. Although the mesothelial surface can organize fibrin, the contribution of pleural mesothelial cells to pleural repair is unknown. During coagulation thrombin cleaves Fibrinopeptide A (FPA, A alpha 1-16) and fibrinopeptide B (FPB) from the A alpha and B beta chains of fibrinogen to generate fibrin monomer. Since these peptides are mitogenic for human fibroblasts, we considered that they might stimulate replication of human pleural mesothelial cells (HPMC). Application of fluid expressed from fibrin clots significantly increased cell number and stimulated uptake of 3H-thymidine by HPMC compared with untreated cells. The mitogenic response of subconfluent HPMC to dilutions of clot fluid (30-150 micrograms/ml protein) was comparable to that of 0.1 nM TGF-beta. Fibrinopeptide A (7.5-30 microM) stimulated 3H-thymidine uptake in HPMC, but FPB had only a slight effect at 30 microM. Antibody to FPA antibody significantly attenuated the mitogenic effect of clot fluid, indicating that a major component is FPA. Our study suggests that fibrinopeptides released during fibrin formation in vivo may stimulate local mesothelial regeneration following pleural injury.

Catalysis↗
PubMed1994

Effects of TGF-beta and TNF-alpha on procoagulant and fibrinolytic pathways of human tracheal epithelial cells.

The epithelial lining of the airways is subject to injury through several processes, including infections, bronchiolitis, and fume exposures. Because airway fibrin deposition influences the course of local injury, we examined how two inflammatory cytokines influenced fibrin formation and clearance in human tracheal epithelial cells (TEC). TEC were treated with transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha). TNF-alpha increased release of tissue factor (TF)-related procoagulant activity that, through generation of factor Xa, promotes assembly of the prothrombinase complex at the cell surface. Fibrinolytic activity was plasminogen dependent and due to both urokinase (uPA) and tissue plasminogen activator (tPA). The cells expressed plasminogen activator inhibitor 1 (PAI-1), but relatively little PAI-2. Depression of fibrinolysis by TGF-beta correlated with increased PAI-1. Conversely, TNF-alpha increased plasminogen activator (PA) activity due to increased uPA. Fibrinolytic activity was inhibited by actinomycin D and cyclohexamide, but changes in mRNAs for uPA, tPA, PAI-1, and TF by either cytokine were not appreciable. PAI-2 mRNA was not found. The data indicate that TGF-beta decreases the fibrinolytic capacity of TEC, suggesting that this cytokine promotes fibrin retention. TNF-alpha increases expression of both procoagulant and fibrinolytic activities; this differential regulation could favor both pericellular fibrin formation and dissolution.

Base Sequence↗
PubMed1994

Interleukin-1-mediated release of interleukin-8 by asbestos-stimulated human pleural mesothelial cells.

The pleuropulmonary response to inhaled asbestos frequently involves inflammation and release of various cytokines from lung cells. Among these, interleukin-8 (IL-8) released from the mesothelium could augment inflammation of the pleura by attracting neutrophils to the pleural space. We used cultures of human pleural mesothelial cells (HPMC) to examine the mechanism of IL-8 production by asbestos and cytokines. Suspensions of amosite, chrysotile, or crocidolite asbestos in concentrations as low as 5 micrograms/ml enhanced release of IL-8 from HPMC during 6 h of incubation at 37 degrees C. Electron microscopy of asbestos-treated HPMC showed that the cells avidly engulfed each of the different types of asbestos fibers. Two proinflammatory cytokines, interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha, enhanced IL-8 release within 2 h and had an even greater effect after 6 h. Release of IL-8 was measured by an enzyme-linked immunosorbent assay, and functional activity of the cytokine was assessed by chemotaxis of human neutrophils. Identity of IL-8 in HPMC supernatants was established by absorption with an antibody to IL-8. Preincubation of HPMC with IL-1 receptor antagonist (IL-1ra) significantly decreased release of IL-8 after stimulation with amosite or crocidolite asbestos. We conclude that HPMC release IL-8 in response to asbestos stimulation and that the response is, in part, mediated by IL-1, mainly in the form of IL-1 alpha.

Antibodies, Monoclonal↗
PubMed1994

Expression and assembly of procoagulant complexes by human pleural mesothelial cells.

Many pleural diseases involve fibrin deposition within the pleural cavity, an event that necessarily involves the mesothelium. This study of human pleural mesothelial cells (HPMC) was designed to determine how the mesothelium initiates and sustains the coagulation process. We used functional assays for activation of both factor X and prothrombin to examine expression and assembly of procoagulant activity by human pleural mesothelial cells in culture. The rates of factor Xa and thrombin formation were calcium-dependent. The rate of factor Xa formation in the presence of added factor VII increased in a concentration-dependent manner, suggesting that tissue factor is the primary procoagulant associated with HPMC. The fact that direct binding of radioiodinated factor VIIa to HPMC was specific, concentration-dependent and saturable confirms that tissue factor is expressed on the cell surface. The rate of thrombin formation increased with factor Xa concentration, and the rate was 5-, 6-fold higher in presence of added factor Va indicating that HPMC support expression of prothrombinase activity. Further, direct binding of radioiodinated factor Xa to HPMC was specific, concentration-dependent and saturable, confirming that the cells support the assembly of the prothrombinase complex.

Blood Coagulation Factors↗
PubMed1993

Site-directed mutagenesis of glutathione S-transferase YaYa. Mapping the glutathione-binding site.

Previous studies from our laboratory have shown that aspartic acid 101 plays an important role in glutathione interaction to rat glutathione S-transferase YaYa, while tyrosine 9 is directly involved in catalysis. Based on the available structural information, site-directed mutagenesis was conducted to examine the function of arginine, lysine, glutamine, and proline residues surrounding the GSH binding pocket. Arginine mutants R13K, R15K, R20K, and R20I retained partial enzymatic activities, while R13I and R15I lost most of their activities. Kinetic studies showed a marked increase in Km toward GSH for R15I suggesting that arginine 15 contributes significantly to the binding of GSH in the active site of glutathione S-transferase YaYa. A drastic decrease in enzymatic activities for R13I suggested the importance of the charged group of arginine 13 either in maintaining the structural integrity of the enzyme or in serving a vital role in enzymatic function. Replacement of glutamine 54 and 67 with glutamic acid or asparagine resulted in decreased enzymatic activities. Moreover, an 11-, 17-, and 9-fold increase in Km values toward GSH for mutant Q54E, Q54N, and Q67N was observed, respectively. These results suggested that glutamine 54 and 67 also contributed significantly to the binding of GSH. Proline at position 56 appears to be important for maintaining the structural integrity of the enzyme since mutants P56A and P56F were much less active and extremely less stable than that of the wild type enzyme. Both lysine mutants, K45R and K45I, exhibited substantially higher catalytic efficiencies toward both 1-chloro-2,4-dinitrobenzene and GSH than the wild type enzyme. Our data clearly show that lysine 45 is not an essential residue for catalysis nor for GSH binding in glutathione S-transferase YaYa.

Amino Acid Sequence↗
PubMed1993

Contact inhibition in the failure of mammalian CNS axonal regeneration.

Anamniote animals, such as fish and amphibians, are able to regenerate damaged CNS nerves following injury, but regeneration in the mammalian CNS tracts, such as the optic nerve, does not occur. However, severed adult mammalian retinal axons can regenerate into peripheral nerve segments grafted into the brain and this finding has emphasized the importance of the environment in explaining regenerative failure in the adult mammalian CNS. Following lesions, regenerating axons encounter the glial cells, oligodendrocytes and astrocytes, and their derivatives, respectively myelin and the astrocytic scar. Experiments to investigate the influence of these components on axon growth in culture have revealed cell-surface and extracellular matrix molecules that inhibit axon extension and growth cone motility. Structural and functional characterization of these ligands and their receptors is underway, and may solve the interesting neurobiological conundrum posed by the failure of mammalian CNS regeneration. Simultaneously, this might allow new possibilities for treatment of the severe clinical disabilities resulting from injury to the brain and spinal cord.

Animals↗
PubMed1993

Up-regulation of neutral endopeptidase (CALLA) in human neutrophils by granulocyte-macrophage colony-stimulating factor.

Neutral endopeptidase 24.11 (NEP/CALLA/CD10), an enzyme expressed on early lymphoid progenitors, neutrophils, and various other cell types, inactivates many biologically active peptides, including the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Inhibition of CD10/NEP on the surface of human neutrophils (PMNs) in vitro inhibits migration toward this chemotaxin, suggesting that enzymatic inactivation by NEP regulates the neutrophil response to fMLP. Because PMNs in inflammatory sites are exposed to various cytokines, we evaluated the effects of selected cytokines on CD10/NEP activity in vitro. Of five cytokines tested--interleukin-1 (IL-1), IL-6, and IL-8, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor (GM-CSF)--GM-CSF provided the most consistent increase in surface NEP activity. Low concentrations (10(-9)-10(-7) M) of GM-CSF increased NEP activity in a time- and concentration-dependent manner to more than 225% that of control (phosphate-buffered saline-treated) cells. Cytofluorometry of cells stained with a fluorescent antibody to CD10 indicated that GM-CSF increased expression of surface CD10/NEP antigen in a similar manner. The effect of GM-CSF on NEP activity was enhanced still further by simultaneous exposure to IL-1, suggesting that combinations of cytokines may direct and regulate the neutrophil response within an inflammatory site. Rapid upregulation of CD10/NEP underscores the importance of this enzyme for control of peptide mediators of inflammation.

Dose-Response Relationship, Drug↗
PubMed1993

A bacterial protease perturbs the paracellular barrier function of transporting epithelial monolayers in culture.

Tight junctions between cells and adhesion to the substratum maintain the barrier function of epithelia throughout the body. Damage to the epithelial barrier by microbial products allows penetration of bacteria and promotion of infection. We studied the effects of Pseudomonas elastase (PE) on the barrier function of epithelia by using Madin-Darby canine kidney (MDCK) epithelial cells; these cells form tight junctions (zonula occludens [ZO]) in vitro. PE decreased electrical resistance across the monolayers in a concentration- and time-dependent manner. Immunostaining of selected proteins of the ZO and zonula adherens was used to explore the effects of PE on junctional proteins. PE-treated monolayers of MDCK cells had markedly decreased immunostaining of ZO-1, a protein of the ZO, but light microscopy of PE-treated cells revealed no obvious morphologic changes. A chromium release assay indicated that, even with marked changes in transmonolayer electrical resistance, the permeability defect was not due to membrane disruption. Fluorescence staining of F-actin indicated diminution of cellular microfilaments in PE-treated cells, but E cadherin (uvomorulin), a protein of the zonula adherens, was unaffected by the enzyme. Elastases from porcine pancreas and human leukocytes with similar enzymatic activity (6 U/ml) did not decrease transmonolayer electrical resistance or degrade ZO-1. These results suggest that PE disturbs the barrier function of epithelial monolayers, in part, by changing the cell architecture and altering at least one protein of the ZO.

Animals↗
PubMed1992

Is routine endocrine testing of impotent men necessary?

Endocrine screening of impotent men is performed in an effort to identify a treatable cause of impotence. However, the prevalence of endocrinopathy in this patient population is low. We determined whether any historical or physical findings obtained during the initial office visit would identify a subgroup of patients at risk for endocrinopathy to decrease the cost of endocrine screening. The results of routine endocrine screening of 330 consecutive impotent patients formed the basis of this study. A total of 7 patients (2.1%) had endocrinopathy. Testicular atrophy was observed in 5 of these 7 patients and 6 reported decreased libido. All of the patients with endocrinopathy had either decreased libido or bilateral testicular atrophy. Our results indicate that the cost of impotence evaluation can be decreased by screening only those patients with clinical signs of hypogonadism, that is either decreased libido or bilateral testicular atrophy.

Diagnostic Tests, Routine↗