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Biomedical subjects

K Hong

Publications and source records attributed to K Hong.

At least 127 records · Page 7Linked to original sources
PubMed1988

Fluorometric detection of the bilayer-to-hexagonal phase transition in liposomes.

We have used the fluorescent probe N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) to detect the bilayer-to-hexagonal phase transition. The fluorescence intensity of the probe was found to increase during the bilayer-to-hexagonal transition. The bilayer-to-hexagonal transitions of various types of phosphatidylethanolamine or cardiolipin measured by this method are consistent with results obtained by differential scanning calorimetry. To establish this method for wider use, agents known to alter the bilayer-to-hexagonal transition were examined, and the results are comparable with the published data. The added advantage of this fluorometric method over other currently available techniques is that it is applicable not only for aggregated lipid samples but also for dilute liposome suspensions. This is especially important when one of the components of the system under study can partition between lipid and aqueous phase. Since NBD is located near the headgroup region of the bilayer, it most likely detects the change of the environment surrounding that region. On the basis of our present study, it appears that NBD-PE is sufficiently sensitive to detect bilayer-to-hexagonal phase transition.

Fluorescent Dyes↗
PubMed1988

Monoclonal antibodies to mouse complement receptor type 1 (CR1). Their use in a distribution study showing that mouse erythrocytes and platelets are CR1-negative.

mAb to murine C receptor type 1 (CR1) were produced and three of them were characterized. One antibody, designated as 8C12, immunoprecipitated a protein of 190,000 Mr from a detergent extract of surface-labeled spleen cells and stained spleen B but not T lymphocytes in fluorescent flow cytometry. It inhibited both CR1-mediated rosette formation and the cofactor activity of CR1 for factor I-mediated cleavage of C3b, suggesting that it recognizes the ligand-binding site of CR1. The two other antibodies, designated as 7G6 and 7E9, recognized different epitopes from that recognized by 8C12, and they cross-reacted with a protein of 150,000 Mr that is present in a spleen extract. The distribution of CR1 in murine hemopoietic cells was studied by binding experiments with radiolabeled 8C12 and fluorescent flow cytometry. When CR1 was not detected by 8C12 alone, the two other antibodies were used in combination with 8C12 to confirm the negative results. Almost all B lymphocytes from the spleen, lymph nodes, and peripheral blood were CR1 positive. Most of the Thy-1-positive lymphocytes from these tissues were CR1 negative. Thymus lymphocytes were also CR1 negative. Peritoneal macrophages and chemotactic factor stimulated but not unstimulated peripheral blood granulocytes were CR1 positive. In contrast to human E, mouse E were CR1 negative. This pattern of distribution was consistent with previous results obtained by rosette assays. Although mouse platelets cause immune adherence hemagglutination with C3b-bearing SRBC, they are CR1 negative. Three other lines of evidence also indicated that platelets are CR1 negative. First, no band of CR1 was demonstrated by immunoprecipitation with 8C12 of an extract of surface-labeled platelets. Second, 8C12, which inhibited rosette formation by lymphocytes, alone or in combination with 7G6 and 7E9, did not inhibit immune adherence between platelets and C3b-bearing SRBC. Third, polyclonal rabbit IgG prepared from anti-mouse CR1 antiserum did not inhibit immune adherence by platelets. These results strongly suggest that the C3b-binding factor(s) on mouse platelets is different from CR1 and that processing of C3b-bearing immune complexes in mouse blood may be mediated by a new and as yet unidentified C3b-binding factor(s).

Animals↗
PubMed1988

Semisolid state fermentation of baker's yeast in an air-fluidized bed fermentor.

In an attempt to grow microorganisms other than fungi using a solid-state fermentation process, a model system of Baker's yeast (Saccharomyces cerevisiae) was cultured in an air-fluidized bed fermentor. A semisolid potato mixture (pretreated with alpha-amylase) was used for the substrate in this highly aerated system. The growth of Baker's yeast in this air-fluidized bed process was easily controllable and very reproducible. Once feasible moisture levels and air flow rates were determined, the independent variables studied were the amount of the enzyme used for digesting the potato starch, the size of the yeast inoculum, and the concentration of the added defined medium.

Culture Media↗
PubMed1988

Relation of monophasic action potential recorded with contact electrode to underlying transmembrane action potential properties in isolated cardiac tissues: a systematic microelectrode validation study.

Monophasic action potentials, recorded with contact non-suction electrodes, have been used both clinically and experimentally. However, to date no systematic microelectrode validation studies have been done to underlying myocardial cell populations from different myocardial regions with different transmembrane potential profiles. In the present study transmembrane action potential properties, recorded with standard microelectrodes, were compared with monophasic action potentials recorded with contact electrodes in three different (endocardium, epicardium, and free running Purkinje fibre) isolated canine preparations during pacing and during spontaneous automatic activity. The mean transmembrane durations at 50% and 90% repolarisations (APD50 and APD90) of 19-30 cells at a monophasic action potential recording site was not statistically significant from monophasic action potential duration in all three tissue preparations studied. However, in endocardial preparations, composed of superficial (1-2 cell layers) Purkinje fibres with deeper ventricular muscle cells, the APD50 (139(17) ms) and APD90 (181(26) ms) of monophasic action potentials more closely reflected (but not significantly different) the underlying deeper ventricular muscle cells (APD50 134(14) ms and APD90 167(15) ms) rather than the mean transmembrane action potential durations of the underlying most superficial Purkinje fibres (166(22) ms for APD50 and 210(30) ms for APD90) (p less than 0.025). Tetrodotoxin (TTX) at 1 x 10(-6) mol.litre-1 shortened Purkinje fibre action potential duration and slightly lengthened that of ventricular muscle. Simultaneously recorded monophasic action potential showed an intermediate change in action potential duration. Incremental pacing and applied single premature stimuli resulted in similar degrees of shortening of action potential duration for both monophasic action potential and transmembrane potential in all three preparations. In endocardial preparations, barium chloride (4 mmol.litre-1) superfusion induced early afterdepolarisations, and spontaneous phase 4 depolarisations (n = 6) in both Purkinje and ventricular muscle cells giving rise to spontaneous automatic activity. These abnormal automatic activities were accurately detected by simultaneous monophasic action potential recordings. Suppression of automaticity by verapamil (0.2-0.5 micrograms.ml-1) as confirmed by transmembrane action potential recordings were similarly detected by monophasic action potential recordings (ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1987

Divalent cation and hydrogen ion effects on the structure and surface activity of pulmonary surfactant.

The structure and surface activity of the extracellular fraction of pulmonary surfactant known as tubular myelin are Ca2+ dependent. Previous studies have demonstrated surfactant-specific proteins with monomeric molecular weights of 28,000-36,000 (SP28-36) are associated with this fraction. In reassembled lipoprotein mixtures, SP28-36 promotes the Ca2+-induced aggregation and surface activity of surfactant lipids, but the detailed interactions between Ca2+, SP28-36, and surfactant lipids have not been established. In this study, we investigated the effect of various cations on the aggregation of surfactant lipid liposomes in the presence of SP28-36. SP28-36 reduced the threshold ion concentration for liposome aggregation from greater than 10 to 0.5 mM for Ca2+, Ba2+, and Sr2+ but not Mg2+ or Mn2+. The liposome aggregation was reversed by ethylenediaminetetraacetic acid and not associated with leakage of carboxyfluorescein. SP28-36 promoted similar liposome aggregation at pH less than 5 in the absence of divalent cations. Surfactant lipids adsorbed slowly to an air-fluid interface in all ionic conditions unless SP28-36 was present. Both Ca2+ and H+ induced rapid lipid adsorption in the presence of SP28-36. The surface activity of native surfactant had a similar ion dependence. Electron micrographs of native surfactant showed typical tubular myelin structures at pH 7.4 only in the presence of Ca2+. At pH 4.4 in the absence of Ca2+, similar but not identical structures were seen. In the reconstituted system, SP28-36 in the presence of Ca2+ induced the formation of larger multilayered structures including parallel bilayers and small areas of squares and triangles with dimensions similar to structures found in the native material.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Rapid and simple measurement of human C5a-des-Arg level in plasma or serum using monoclonal antibodies.

A new sandwich immunoassay method for measuring human C5a-des-Arg was developed using monoclonal antibody specifically reactive with C5a-des-Arg. Monoclonal antibodies were obtained from a panel of hybridomas produced by fusion of mouse myeloma cells, P3 X 63-AG8,653, with spleen cells from a CBF1(C57BL/6 X BALB/c) mouse immunized with purified C5a. The reactivities of these monoclonal antibodies against C5a, C5a-des-Arg and C5 were tested by solid-phase radioimmunoassay. One of the antibodies reacted with C5a-des-Arg, but not with C5a and C5. By use of this antibody for capturing antibody in sandwich immunoassay, a rapid and simple method was developed for measuring C5a-des-Arg without previous removal of C5. The sensitivity of this assay system was approximately 1 ng/ml for C5a-des-Arg.

Animals↗
PubMed1987

Covalent association of C3b with C4b within C5 convertase of the classical complement pathway.

The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

Animals↗
PubMed1987

Comparison of regional pulmonary perfusion in lobar pneumonia during high frequency and conventional mechanical ventilation in sheep.

We compared the effects of high frequency jet ventilation (HFV), conventional ventilation (CMV), and spontaneous breathing (SB) on regional pulmonary blood flows (QLLL), standard cardiopulmonary measurements and the serum levels of the first generation metabolites of prostacyclin (6-keto-PGF1 alpha) and thromboxane A2 (TxB2) in established left lower lobe pseudomonas aeruginosa pneumonia in 11 sheep. Gram negative pneumonia resulted in significant increases in alveolar-arterial oxygen gradients [(A-a)DO2] and pulmonary shunt fractions (Qs/Qt), as well as a significant decrease in QLLL during SB. Significant differences in standard haemodynamics, (A-a)DO2, Qs/Qt, and QLLL were not observed when HFV was compared to CMV. However, serum levels of 6-keto-PGF1 alpha were elevated when the animals underwent HFV. We conclude that HFV is a safe and efficient method of oxygenation and ventilation in unilobar gram negative pneumonia and also results in a significant increase in the serum levels of 6-keto-PGF1 alpha when compared to CMV in sheep. The exact significance of the latter finding is the subject of current investigation.

6-Ketoprostaglandin F1 alpha↗
PubMed1987

Exposure of hydrophobic domains of clathrin in its membrane fusion-inducible pH region.

Clathrin induces fusion of phospholipid membranes containing phosphatidylserine when the pH of the medium is reduced below about 6. The hydrophobicity of clathrin in the membrane fusion-inducible pH region was examined. In the presence of clathrin, the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate was shifted to shorter wavelengths and its fluorescence intensity increased at pH values below about 6. Steep increase of the fluorescence intensity of the fluorescent probe, N-(1-anilinonaphthyl-4)maleimide covatently bound to clathrin was observed in a similar pH region. The efficiency of resonance energy transfer from tryptophan to anilinonaphthyl residues in the clathrin molecule showed similar pH dependency. When Triton X-114 solutions containing clathrin were subjected to phase separation by raising the temperature from 0 degree to 30 degrees C at different pH values, clathrin remained in the aqueous phase above pH 6, whereas the protein was partitioned between the aqueous and detergent phases at pH 5-6, and was present only in the detergent phase below pH 5. The effective hydrophobicity of clathrin determined by the fluorescence method using cis-parinaric acid also increased at pH values below 6. Moreover, clathrin was aggregated by lowering the pH below 6. These results show that exposure of hydrophobic domains of clathrin through conformational change occurs in its membrane fusion-inducible pH region, suggesting the crucial role of protein hydrophobicity in the initiation of membrane fusion.

Clathrin↗
PubMed1986

Membrane-bound C4b interacts endogenously with complement receptor CR1 of human red cells.

Activation of the classical complement pathway on the membrane of autologous cells results in the deposition of C4b on their surface and in the assembly of the C3 convertase C4b2a, one of the amplifying enzymes of the cascade. Here we study the sequence of events leading to irreversible inactivation of the potentially harmful C4b bound to human red cells. We show that deposited C4b interacts endogenously with complement receptor type 1 (CR1) present on the membrane of the same red cell. Complexes containing CR1 and C4b are found in extracts of membranes of C4b-bearing red cells after treatment of the intact cells with a bifunctional crosslinking reagent. The amount of complexed CR1 increases with the number of deposited C4b molecules. Only small amounts of free CR1 are observed on red cells bearing as few as 1,900 molecules of C4b, suggesting that the binding avidity between C4b and endogenous CR1 is high. In agreement with this observation, we find that the deposited C4b inhibits the exogenous cofactor activity of the red cell CR1 for the factor I-mediated cleavage of target-bound clustered C3b. The C4b bound to the human red cells is cleaved by the serum enzyme C3b/C4b inactivator (factor I) and a large fragment (C4c) is released in the incubation medium. The cleavage is totally inhibited by mAbs against CR1, showing that the complement receptor is an essential cofactor for the activity of I. When the number of bound C4b per red cell is relatively small (less than 1,000 molecules) the substrate for the enzymatic activity of factor I is mostly or exclusively the C4b bound endogenously to CR1. Indeed, the kinetics or the extent of cleavage of C4b are not affected by greatly augmenting the concentration of exogenous CR1 or of C4b-bearing red cells in the incubation mixture, thereby increasing the frequency of collisions between CR1 on the surface of one cell with C4b deposited on the membrane of a different cell. On the basis of the present and prior observations, we speculate that both DAF and CR1 act endogenously to inactivate the function of autologous red cell-bound C4b and prevent the progression of the cascade. DAF binding prevents the formation of the C3 convertase, C4b2a. The cleavage and irreversible inactivation of C4b only occurs after the concerted activities of endogenous CR1 and serum factor I.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Proteins↗
PubMed1986

Spermine as a modulator of membrane fusion: interactions with acidic phospholipids.

The interaction of spermine with acidic phospholipids was investigated for its possible relevance to membrane fusion. Equilibrium dialysis was used to measure the binding of spermine and calcium to large unilamellar vesicles (liposomes) of phosphatidate (PA) or phosphatidylserine (PS). Spermine bound to isolated PA and PS liposomes with intrinsic association constants of approximately 2 and 0.2 M-1, respectively. Above the aggregation threshold of the liposomes, the binding of spermine increased dramatically, especially for PA. The increased binding upon aggregation of PA liposomes was interpreted as evidence for the formation of a new binding complex after aggregation. Spermine enhanced calcium binding to PA, while it inhibited calcium binding to PS, under the same conditions. This difference explained the small effect of spermine on the overall rate of calcium-induced fusion of PS liposomes as opposed to the large effect on PA liposomes. The rate increase could be modeled by a spermine-induced increase in the liposome aggregation rate. The preference for binding of spermine to PA over PS suggested a preference for accessible monoesterified phosphate groups by spermine. This preference was confirmed by the large effects of spermine on aggregation and overall fusion rates of liposomes containing phosphatidylinositol 4,5-diphosphate. The large spermine effects on these liposomes compared with phosphatidate- or phosphatidylinositol-containing liposomes suggested that spermine has a strong specific interaction with phosphatidylinositol 4,5-diphosphate. Clearly, phosphorylation of phosphatidylinositol can lead to a large change in the spermine sensitivity of membrane fusion.

Calcium↗
PubMed1986

Fusion of liposomes induced by a cationic protein from the acrosome granule of abalone spermatozoa.

Lysin, a protein of Mr 16 000 from the acrosome granule of the abalone, is responsible for the dissolution of the egg vitelline layer. The primary structure of this cationic protein projects some hydrophobic domains in the secondary structure. Lysin was found to associate nonselectively with phospholipid bilayers and cause a spontaneous release of encapsulated carboxyfluorescein in liposomes. The association of lysin with phosphatidylcholine liposomes suggests that there is a hydrophobic interaction between lysin and lipid bilayers. Binding of lysin to phospholipid resulted in the aggregation of phosphatidylserine-containing liposomes, but aggregation was not observed in neutral phosphatidylcholine liposomes. Resonance energy transfer and dequenching of fluorescent 1-palmitoyl-2-cis-parinaroylphosphatidylcholine were both used to determine the fusogenic activity of lysin in aggregated liposomes. Results from both assays are consistent. Lysin-induced fusion was observed in all the phosphatidylserine-containing liposomes, and the general trend of fusion susceptibility was phosphatidylserine/phosphatidylcholine (1:2) approximately equal to phosphatidylserine/phosphatidylcholine/phosphatidylethanolamine (1:1:1) greater than phosphatidylserine/phosphatidylethanolamine (1:2). Cholesterol up to 30% did not affect the intrinsic fusion susceptibility. A hydrophobic penetration by protein molecules and the packing of phospholipid bilayers are used to interpret the fusion susceptibility. Lysin-induced liposome aggregation was highly independent of the state of self-association of lysin in ionic medium. However, the fusogenic activity of self-associated lysin was found to be much less than the monodispersed one. Liposomes preincubated with Ca2+ did not fuse initially as readily as those without Ca2+ treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗
PubMed1986

Number of hits necessary for complement-mediated hemolysis.

The number of hits necessary for the C8 and C9 steps of immune hemolysis was reexamined with a previously unemployed experimental design, in which various numbers of EAC1-7, excess of the supplementary component and a constant amount of the component tested were incubated in a constant volume (Inoue et al. 1976. Infect. Immun. 13: 337). Our results were consistent with previous findings; the steps of guinea pig C8 and C9, the human C8 each followed a one-hit mechanism, while that of human C9 showed ka multi-hit response. When lysis of sensitized erythrocytes (EA) by normal human serum was analysed in a similar way, one-hit curves were obtained. This result, taken together with the above results, suggests that immune hemolysis occurs by a single lesion including a single C8 and multiple C9 in the case of human complement and that normal human serum contains sufficient excess of C9. On the other hand, when C9-deficient human serum was used for lysis of EA, multiple-hit curves were obtained. The mechanism of lysis by C5b-8 may differ from that by C5b-9.

Animals↗
PubMed1986

Effect on cardiopulmonary changes of gram-negative endotoxemia in sheep after type-specific, cross-reactive, and nonspecific immune stimulation.

The purpose of this study was to examine the effects of prior nonspecific immune stimulation (BCG), cross-reactive immunization (E coli J5 0111 whole cells [J5 WC], and core glycolipid [J5 CGL]), and type-specific immunization (Serratia marcescens core glycolipid [SM CGL]) on the cardiopulmonary variables and white blood cell counts of awake, monitored sheep following IV Serratia marcescens endotoxin. Comparison of cardiac output, pulmonary artery pressure, pulmonary capillary wedge pressure, pulmonary vascular resistance, alveolar-arterial oxygen gradients, and total white blood cells lead us to conclude that type-specific immunization (SM CGL) most effectively ameliorates the changes of gram-negative endotoxemia contrasted to nonimmunization. Core glycolipid cross-reactive (J5 CGL) immunization was somewhat more effective than whole-cell cross-reactive (J5 WC) immunization in this regard. Nonspecific immune stimulation (BCG) was able only to significantly decrease the changes in pulmonary vascular resistance compared to nonimmunization.

Animals↗
PubMed1985

Interaction of clathrin with liposomes: pH-dependent fusion of phospholipid membranes induced by clathrin.

Clathrin, dissociated from coated vesicles of bovine brain and purified by gel chromatography, was found to interact with the lipid bilayer as shown by the spontaneous release of encapsulated fluorescent dye in liposome. Clathrin-induced dye release was enhanced at acidic pH in phosphatidylserine-containing vesicles. A strong correlation between dye release and fusion of liposomes was observed. In general, when there was a fast release of encapsulated dye induced by clathrin, a pH-dependent, clathrin-induced fusion was observed. Clathrin did not induce either dye release or fusion of egg phosphatidylcholine liposomes. The self-association of clathrin at low pH diminished the fusogenic activity. Fusion induced by clathrin at low pH could be stopped at pH above 5.0 and resumed by lowering the pH below 5.0. This suggests that the interaction of clathrin with phospholipid membranes can be regulated by pH.

Animals↗
PubMed1984

Lectins facilitate calcium-induced fusion of phospholipid vesicles containing glycosphingolipids.

Ca2+-induced fusion of phospholipid vesicles containing globoside (GL-4) or disialoganglioside (GDla) is several-fold slower than the fusion of the pure phospholipid vesicles. Lectins specific for these glycosphingolipids, soybean agglutinin and wheat germ agglutinin, respectively, enhance the rate of fusion when added to the vesicle suspension before the introduction of Ca2+. The enhancement depends on the lectin concentration and the time of preincubation with the lectin. We propose that lectins facilitate membrane fusion by inducing intermembrane contact, which is the first step in the overall process of membrane fusion, or by laterally phase separating the inhibitory glycolipids.

Calcium↗