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Biomedical subjects

A R Johnson

Publications and source records attributed to A R Johnson.

At least 91 records · Page 5Linked to original sources
PubMed1985

Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide.

Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acids. While the enzyme is known to be in organs such as kidney and brain, we found it in human neutrophils. These cells cleaved the NEP substrate glutaryl (Glut)-Ala-Ala-Phe-(4-methoxynaphthylamine) (Glut-Ala-Ala-Phe-MNA) at a rate of 9.5 nmol X hr-1 per 10(6) cells, and phosphoramidon (1 microM) inhibited the hydrolysis by 90%. Intact neutrophils from donors who smoked had NEP activities about twice that of nonsmokers. Subcellular fractionation and sucrose density gradient centrifugation of lysed neutrophils showed that most of the NEP activity was membrane bound. A washed membrane fraction from human neutrophils rapidly cleaved 0.5 mM Glut-Ala-Ala-Phe-MNA (96 nmol X min-1 X mg-1) and the hydrolysis was inhibited by phosphoramidon and by specific antiserum to human renal NEP. The washed membrane fraction also rapidly cleaved 0.1 mM bradykinin (34 nmol X min-1 mg-1) and 0.1 mM fMet-Leu-Phe (49 nmol X min-1 X mg-1). The membrane-bound enzyme cleaved the peptide substrates at the same site as the homogeneous human renal NEP, and phosphoramidon and thiorphan inhibited the hydrolysis. Kinetic studies with pure human renal NEP showed that the chemotactic peptide fMet-Leu-Phe was one of the best biologically active substrates (Km, 59 X 10(-6) M; kcat, 3654 min-1). Immunocytochemistry at the light microscopic level revealed a high concentration of NEP on the cell membrane of neutrophils. This was confirmed with electron microscopy using the immunogold technique on ultrathin cryosections. These studies indicate that NEP in neutrophils may have important functions in inflammation and chemotaxis.

Bradykinin↗
PubMed1985

Enhancement of the antiaggregatory activity of prostacyclin by propranolol in human platelets.

The beta-adrenergic antagonist propranolol has been found to inhibit platelet aggregation. We investigated the possibility that propranolol exerts this action by stimulating the synthesis or enhancing the antiaggregatory activity of prostaglandin (PG) I2. The media from cultures of human endothelial cells inhibited thrombin-induced platelet aggregation, an effect attributed to PGI2 production by the cells. When endothelial cells were incubated with dl- or d-propranolol, the media had two to three times the inhibitory activity of control media. However, this increased activity was not due to increased synthesis of PGI2 because control and propranolol-treated cultures synthesized similar amounts of the PGI2 metabolite, 6-keto-PGF1 alpha. Instead, propranolol enhanced the antiaggregatory activity of PGI2. Propranolol (1 microM) and PGI2 (0.05 nM), when tested separately, inhibited aggregation by 19% and 13%, respectively, whereas the combination inhibited aggregation by 51%. PGI2 inhibited platelet aggregation and thromboxane (Tx) B2 production but stimulated cyclic AMP formation. The adenyl cyclase inhibitor 2',5'-dideoxyadenosine (DDA) had no effect of its own on these parameters, but blocked the actions of PGI2. Propranolol inhibited aggregation and TxB2 synthesis without changing cyclic AMP levels. Unlike PGI2, propranolol's effects were not altered by DDA. While the combination of propranolol and PGI2 inhibited aggregation to a greater extent than either agent alone, this enhanced effect with the combination did not extend to TxB2 or cyclic AMP production. Propranolol, PGI2, and the combination inhibited TxB2 synthesis to a similar extent, and PGI2 produced a similar increase in cyclic AMP in the presence and absence of propranolol. These findings indicate that propranolol and PGI2 inhibit platelet aggregation through cyclic AMP-independent and dependent mechanisms, respectively. While propranolol does not alter the synthesis of PGI2, it enhances the inhibition of aggregation by PGI2, and this may contribute to its antiplatelet effect.

Cells, Cultured↗
PubMed1985

Neutral metalloendopeptidase in human lung tissue and cultured cells.

The distribution of a neutral metalloendopeptidase (NEP), or "enkephalinase," in human lung tissue and cultured cells was compared with that of angiotensin I converting enzyme (ACE). The specific activities of NEP and ACE were measured in homogenates of fetal lung tissue and in isolated airways and pulmonary vessels. NEP activity was highest in airway tissue, and ACE activity was highest in isolated vessels. Human endothelial cells from either umbilical veins or pulmonary arteries had high ACE activity (80 to 90 nmol/h/10(6) cells) but only a trace of NEP activity (0.5 to 0.6 nmol/h/10(6) cells). Fibroblasts cultured from human lungs were low in ACE but richer in NEP than cultured endothelial cells. Fibroblasts from human foreskins or caesarean section skin were the richest source of NEP activity (60 to 80 nmol/h/10(6) cells). Immunohistochemical studies confirmed the biochemical assays. As expected, ACE was localized on the luminal surface of blood vessels, with a distribution similar to that of factor VIII antigen, an endothelial marker. In contrast, NEP was localized within the alveolar septa. Cultured endothelial cells stained only weakly for NEP in contrast to cultured fibroblasts. The location of these 2 enzymes in different cells and the differences in peptide substrate specificity suggests that they act sequentially on circulating peptides or those released within microvascular beds.

Cells, Cultured↗
PubMed1985

Neutral endopeptidase in serum samples from patients with adult respiratory distress syndrome. Comparison with angiotensin-converting enzyme.

The activities of 2 peptidases, angiotensin-I converting enzyme (ACE) and neutral metalloendopeptidase (NEP), were measured in serum from patients with adult respiratory distress syndrome (ARDS). As noted by others, we found that the specific activity of serum ACE was reduced in patients with severe alveolocapillary damage caused by ARDS. In addition, patients who were severely ill with chronic obstructive lung disease had lower serum ACE than did normal ambulatory control subjects. Patients with cardiogenic pulmonary edema, however, had no consistent loss of the enzyme. The most striking changes occurred in the serum levels of NEP. Whereas the specific activity of this enzyme was very low in the normal subjects, it was elevated as much as 50- to 60-fold in serum samples from patients with ARDS. Serum from patients with cardiogenic pulmonary edema had high levels of NEP, and it was elevated also in a subset of patients with chronic obstructive lung disease. On the assumption that patients with ARDS sustain significant damage at the alveolar-capillary level, these changes in ACE and NEP could signal endothelial damage; ACE loss could result from direct injury to the vascular lumen, and NEP from subendothelial tissues could enter the bloodstream through damaged endothelium. Alternatively, NEP might be released from leukocytes sequestered in the lung and leak into the bloodstream.

Adult↗
PubMed1985

Metabolism of androsterone and 5 alpha-androstane-3 alpha,17 beta-diol in human lung tissue and in pulmonary endothelial cells in culture.

The metabolism of [3H]androsterone and [3H] 5 alpha-androstane-3 alpha,17 beta-diol ( [3H] 3 alpha-diol) was studied in slices of human lung tissue and cultures of human pulmonary artery endothelial cells. Lung tissue metabolized [3H]androsterone (0.25 microM) to 5 alpha-androstane-3,17-dione (30.3 pmol 100 mg-1 tissue h-1), isoandrosterone (0.7 pmol 100 mg-1 tissue h-1), 5 alpha-dihydrotestosterone (5 alpha-DHT; 0.1 pmol 100 mg-1 tissue h-1), 3 alpha-diol (0.1 pmol 100 mg-1 tissue h-1), and two polar metabolites. Pulmonary arterial endothelial cells produced the same metabolites of [3H]androsterone (0.083 microM), with the exception of the polar compounds [5 alpha-androstane-3,17-dione (1.3 pmol mg-1 protein h-1), isoandrosterone (0.1 pmol mg-1 protein h-1), 5 alpha-DHT (0.2 pmol mg-1 protein h-1), and 3 alpha-diol (0.2 pmol mg-1 protein h-1)]. Thus, the principal metabolite of [3H]androsterone in both lung tissue and endothelial cells was 5 alpha-androstane-3,17-dione. Human lung tissue metabolized [3H]3 alpha-diol (0.28 microM) to 5 alpha-DHT (8.8 pmol 100 mg-1 tissue h-1), androsterone (2.2 pmol 100 mg-1 tissue h-1), 5 alpha-androstane-3,17-dione (0.8 pmol 100 mg-1 tissue h-1), isoandrosterone (0.1 pmol 100 mg-1 tissue h-1), and four polar metabolites (0.2 pmol 100 mg-1 tissue h-1). 5 alpha-DHT was the principal metabolite of [3H]3 alpha-diol within the first hour of incubation, but the concentration of this androgen declined thereafter to 3.6 pmol 100 mg-1 tissue after 4 h of incubation. This decline was correlated with increased 5 alpha-androstane-3,17-dione synthesis (6.7 pmol 100 mg-1 tissue 4 h-1). Androsterone formation from [3H]3 alpha-diol, however, was linear with time of incubation for 4 h (8.9 pmol 100 mg-1 tissue 4 h-1). The formation of these products demonstrates that the principal 5 alpha-reduced-C19-steroid-metabolizing enzymes in human lung are 3 alpha-hydroxysteroid oxidoreductase.

Adolescent↗
PubMed1985

Human gamma interferon increases the binding of T lymphocytes to endothelial cells.

Binding of lymphocytes to human umbilical vein endothelial cells (EC) was quantitated by measuring adhesion of 51Cr labelled lymphocytes to endothelial cell monolayers and rosette formation between lymphocytes and EC in suspension. Mitogen stimulated human peripheral blood mononuclear cell culture supernatants and mixed lymphocyte reaction supernatants enhanced the binding of T lymphocytes to EC monolayers or suspensions preincubated with such supernatants. The active component of these supernatants appeared to be gamma interferon (IFN-gamma) since culture supernatants lost activity after heating at 56 degrees C for 60 min, exposure to pH 2.0 or treatment with anti-IFN-gamma. In addition, purified IFN-gamma increased the binding of T lymphocytes to EC (T-EC). This occurred in a concentration dependent manner when IFN-gamma was preincubated with EC but not with lymphocytes. While the optimum concentration of IFN-gamma was 250 u/ml, a significant enhancement was seen with as little as 10 u/ml. These findings suggest that IFN-gamma may play a part in the emigration of lymphocytes to perivascular chronic inflammatory sites by augmenting the adhesion of lymphocytes to the endothelium of small blood vessels.

Cell Adhesion↗
PubMed1985

Arachidonic acid metabolites and endothelial injury: studies with cultures of human endothelial cells.

Human endothelial cells in culture can metabolize arachidonic acid to prostaglandin (PG) I2 (prostacyclin), PGE2, and PGF2 alpha, and to various nonpolar products. Similar metabolites are formed by cultured pulmonary arterial and venous cells and umbilical venous cells. In studies with umbilical venous endothelial cells, PG formation was stimulated by mechanical agitation or by treatment with histamine or melittin. Histamine stimulated prostacyclin release into cell culture media without damaging the cells, but melittin injured the cell membrane as indicated by the loss of 51Cr from labeled cells. Media collected from mechanically agitated cells inhibited the release of chromium by melittin. Heating to 56 C or acidification of the media did not reverse this protective effect. Prostacyclin added to labeled cells before challenge with melittin partially inhibited 51Cr release, but PGE2 or leukotrienes B4, C4, and D4 did not. In experiments with pulmonary endothelial cells, histamine augmented the release of hydroxyeicosatetraenoic acids (HETEs) and other nonpolar metabolites. The major nonpolar product comigrated on high-pressure liquid chromatography with di-HETEs. 11-HETE and 15-HETE were identified in media from histamine-stimulated cells by comigration with standards. Neither of these compounds nor 12-HETE protected endothelial cells from melittin-induced injury. It is not yet known if any arachidonic acid metabolites can modulate endothelial cell injury.

Arachidonic Acids↗
PubMed1984

Hydrolysis of opioid hexapeptides by carboxypeptidase N. Presence of carboxypeptidase in cell membranes.

Carboxypeptidase N, purified to homogeneity from human plasma, rapidly hydrolyzed Lys6- or Arg6-enkephalins when measured by high pressure liquid chromatography. Comparison of the kinetics of hydrolysis of the enkephalin hexapeptides and bradykinin by carboxypeptidase N revealed the following values for the Km and kcat: Arg6-Met5-enkephalin, 49 microM, 1024 min-1; Arg6-Leu5-enkephalin, 57 microM, 375 min-1; Lys6-Met5-enkephalin, 216 microM, 6204 min-1; bradykinin, 19 microM, 58 min-1. Thus, while bradykinin had the lowest Km, the specificity constants (kcat/Km) for all the enkephalin hexapeptides were higher than that of bradykinin due to their high turnover numbers. Preincubation of the enzyme with 0.1 mM CoCl2 increased both the kcat and Km of bradykinin and Arg6-Met5-enkephalin. Similar results were obtained when the above experiments were conducted with the active 48,000 dalton subunit of carboxypeptidase N. Basic carboxypeptidase activity was found in the amniotic fluid, in membrane fractions of various human and bovine tissues, and in cultured cells in the following order of decreasing specific activity: human placental microvilli, human kidney, human amniotic fluid, human lung, bovine lung, bovine pulmonary artery, human foreskin fibroblasts, human pulmonary arterial endothelial cells, and human lung fibroblasts. The membrane-bound carboxypeptidase activity had a neutral pH optimum and behaved similarly to plasma carboxypeptidase N in the presence of various inhibitors and activators. It was different from the carboxypeptidase activity in bovine adrenal chromaffin granules which had an acid pH optimum and was inhibited by sulfhydryl reagents. These studies show that human carboxypeptidase N, an enzyme found in high concentration in blood, readily hydrolyzes Arg6- or Lys6-enkephalins. It could thus control the levels of these peptides if they are released into the circulation from the adrenal gland. In addition, a membrane-bound carboxypeptidase N-like enzyme in various tissues may regulate the local levels of biologically active peptides containing C-terminal basic amino acids such as hexapeptide enkephalins, kinins, anaphylatoxins or fibrinopeptides.

Adrenal Glands↗
PubMed1984

Synthesis of lactosaminoglycan-containing glycoproteins by vascular endothelial cells.

Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [3H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (Mr greater than 5000) 3H-labeled glycopeptide. Digestion of these 3H-labeled glycopeptides with endo-beta-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid----Gal----GlcNAc----Gal. Treatment of [3H]glucosamine-labeled cells with melittin caused 3H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total 3H-labeled glycoproteins from the cell and 3% of the 3H-labeled proteoglycans. The 3H-labeled glycoproteins were digested with Pronase and the resulting 3H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (Mr greater than 5000) now comprised about 30% of these released 3H-labeled glycopeptides. These high molecular weight 3H-labeled glycopeptides were degraded with endo-beta-galactosidase but not with testicular hyaluronidase. Analysis of the released 3H-labeled glycoproteins indicated a preferential release of glycoproteins of 70-90 kDa enriched in lactosaminoglycan-type oligosaccharides.

Amino Sugars↗
PubMed1984

Elimination of oral food challenge reaction by injection of food extracts. A double-blind evaluation.

Twenty subjects underwent a double-blind evaluation by analyzing six variables to determine if subcutaneous injection of the food extract neutralizing dose would protect subjects from reactions. Twelve subjects had four of the six variables neutralized 60% of the time following the food antigen neutralizing dose. The placebo trials neutralized four of six variables 15% of the time. The sign/symptom results show statistical significance favoring food extract neutralization over placebo. The remaining eight subjects had at least two of the six variables neutralized by the food extract up to 85% of the time. It appears that the phenomenon of subcutaneous food neutralization can be scientifically endorsed for clinical use in the treatment of food reactions.

Administration, Oral↗
PubMed1984

Some effects of trichloroethylene on mouse lungs and livers.

Repeated administration of trichloroethylene (TCE) to mice by either i.p. injection or by inhalation increased the activity of hepatic microsomal NADPH cytochrome-c reductase. The NADPH cytochrome c reductase activity in microsomes isolated from lungs of animals treated with TCE by inhalation was decreased relative to controls (untreated animals). TCE inhalation was associated with pathologic changes in lungs, but not in livers of the treated animals. The duration of exposure is probably an important factor however, since animals exposed for only 1 hr per day exhibited neither pathologic changes in the lungs nor an alteration of enzyme activity. These findings indicate that inhalation of TCE, without prior treatment with inducers, can enhance activity of the hepatic mixed function oxidase system. The reduced activity of the pulmonary mixed function oxidase system in animals that inhaled TCE may reflect injury to the lungs.

Animals↗
PubMed1984

Conditions influencing human sperm capacitation and penetration of zona-free hamster ova.

The ability of human spermatozoa to penetrate zona-free hamster ova was examined following low-temperature capacitation (4 degrees C) in TES-Tris (TEST)-yolk buffer for periods of up to 66 hours. Results obtained from 66 individuals demonstrated that the number of penetrations per ova were increased by an average of 2.5-fold when spermatozoa were capacitated for 42 hours, as compared with 18 hours. Furthermore sperm with extremely poor penetration rates after 18-hour capacitation was often improved by longer capacitation periods. Patients from our infertility clinic with less than 20 X 10(6) spermatozoa/ml of ejaculate had significantly lower penetration rates when compared with patients and donors with greater than 20 X 10(6) spermatozoa/ml (P less than or equal to 0.001). The observed effects of TEST-yolk buffer appear to be related to its ability to preserve sperm motility over prolonged periods, during which increased capacitation of the total sperm population is achieved.

Animals↗
PubMed1984

Genetic Adaptation to Elevated Carbon Dioxide Atmospheres by Pseudomonas-Like Bacteria Isolated from Rock Cod (Sebastes spp.).

The microorganisms on rock cod fillets stored in a modified atmosphere (MA; 80% CO(2)-20% air) at 4 degrees C for 21 days were isolated. Only Lactobacillus sp. (71 to 87%) and tan-colored Pseudomonas sp.-like isolates (TAN isolates) were found. The TAN isolates grew more slowly in MA than in air at 8 degrees C. When TAN isolates were grown in air at 8 degrees C and then transferred to MA at 8 degrees C, there was an initial decline in viable counts for 10 to 30 h followed by exponential growth. During this exponential growth phase in MA, the growth rates of the TAN isolates from MA-stored fish were significantly greater than those of the TAN isolates from fresh fish. When a TAN isolate from fresh fish was grown under MA for 21 days, it then grew as rapidly under MA as isolates from MA-stored fish. These results suggest that the TAN isolates genetically adapt to high levels of CO(2).

Journal Article↗
PubMed1984

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells.

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Studies on human spermatozoa with round head syndrome.

The ability of spermatozoa with round head syndrome to penetrate zona pellucida-free hamster ova and to undergo nuclear decondensation was studied in three infertile patients. Whereas spermatozoa from pregnancy-proven donors bound to hamster ova and achieved a penetration rate of 100%, those with round heads did not bind to or penetrate any ova. While spermatozoa nuclear decondensation occurs after ovum penetration and indicates a positive result, it has now been demonstrated that this phenomenon can also be induced by incubating spermatozoa with crushed hamster ova. In addition, nuclei from normal and round-headed spermatozoa decondense in the presence of these ova to a similar degree. These observations suggest that infertility related to round head sperm morphology is associated with an inability to interact with and penetrate the oolemma, rathern than dysfunctional spermatozoan changes following penetration.

Animals↗
PubMed1983

Cytotoxicity of sera from patients with scleroderma. Effects on human endothelial cells and fibroblasts in culture.

Sera from patients with progressive systemic sclerosis were compared with sera from normal individuals and from patients with other connective tissue diseases for cytotoxic effects on cultured human cells. More than 40% of the sera from patients with active progressive systemic sclerosis were cytotoxic by several criteria for pulmonary arterial or umbilical venous endothelial cells, foreskin fibroblasts, and neuroblastoma cells. Cytotoxic sera caused morphologic changes, uptake of trypan blue dye, and a decrease in the incorporation of 3H-thymidine into DNA. In contrast, only 4 sera from normal individuals or patients with other rheumatic diseases affected cell morphology, staining, or uptake of 3H-thymidine. Partial characterization of the cytotoxic factor indicated that it is sensitive to proteolysis by trypsin. The molecular weight of the factor was estimated to be similar to that of albumin.

Cells, Cultured↗