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Biomedical subjects

K Hong

Publications and source records attributed to K Hong.

At least 145 records · Page 8Linked to original sources
PubMed1984

Effects of immunization on cardiopulmonary alterations of gram-negative endotoxemia.

We studied the effects of an infusion of Serratia marcescens endotoxin on hemodynamic function, white blood cells, platelets, and the plasma levels of thromboxane B2 (TXB2) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) in awake monitored sheep. The animals were immunized using the core glycolipid (CGL) fraction of Escherichia coli J5 0111. An additional group of animals was studied after passive transfer of immune serum from the actively immunized CGL animals. Active immunization with CGL was effective in preventing the hematologic and hemodynamic changes as well as the elevations in TXB2 and 6-keto-PGF1 alpha seen during gram-negative endotoxemia. Passive immunization also reduced some of the deleterious effects of endotoxin infusion but was less effective than active immunization. This study demonstrates the potential of cross-specific immunization to protect against the cardiopulmonary alterations caused by gram-negative endotoxemia.

6-Ketoprostaglandin F1 alpha↗
PubMed1984

Inhibition of immune precipitation by complement.

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.

Animals↗
PubMed1983

Polyamines as modulators of membrane fusion: aggregation and fusion of liposomes.

We have studied the effect of the polyamines (spermine, spermidine, and putrescine) on the aggregation and fusion of large (approximately 100 nm in diameter) unilamellar liposomes in the presence of 100 mM NaCl, pH 7.4. Liposome fusion was monitored by the Tb/dipicolinic acid fluorescence assay for the intermixing of internal aqueous contents, and the release of contents was followed by carboxyfluorescein fluorescence. Spermine and spermidine at physiological concentrations aggregated liposomes composed of pure phosphatidylserine (PS) or phosphatidate (PA) and mixtures of PA with phosphatidylcholine (PC) but did not induce any fusion. However, liposomes composed of mixtures of acidic phospholipids, cholesterol, and a high mole fraction of phosphatidylethanolamine could be induced to fuse by spermine and spermidine in the absence of divalent cations. Putrescine alone in the physiological concentration range was ineffective for both aggregation and fusion of these liposomes. Liposomes made of pure PC did not aggregate in the presence of polyamines. Addition of aggregating concentrations of spermine caused a drastic increase in the rate of Ca(2+)-induced fusion of PA liposomes and a large decrease in the threshold Ca(2+) concentration required for fusion. This effect was less pronounced in the case of PS or PA/PC vesicles. Preincubation of PA vesicles with spermine before the addition of Ca(2+) resulted in a 30-fold increase in the initial rate of fusion. We propose that polyamines may be involved in the regulation of membrane fusion phenomena accompanying cell growth, cell division, exocytosis, and fertilization.

Cholesterol↗
PubMed1983

Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation.

Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.

Animals↗
PubMed1983

Liposomes containing colloidal gold are a useful probe of liposome-cell interactions.

A method is described for the preparation of liposomes containing colloidal gold as an electron-dense marker to trace liposome-cell interactions. Since gold sols would precipitate at the high concentrations necessary for loading a large proportion of liposomes, gold sols were formed within preformed liposomes which had encapsulated gold chloride. The optimal conditions for encapsulating the marker were ascertained for liposomes prepared by the method of reverse-phase evaporation. Gold sols formed rapidly at ambient temperature and without organic solvent, and produced homogeneous populations of gold granules inside liposomes. Most vesicles contained the marker, allowing us to determine unambiguously the intracellular fate of liposomes and their contents. The in vitro experiments showed that gold-liposomes were internalized by African green monkey kidney cells in a manner similar to receptor-mediated endocytosis of well-characterized ligands. Preliminary in vivo studies also indicated that liposomes were endocytosed by Kupffer cells via the coated vesicle pathway.

Animals↗
PubMed1983

Endocytosis of liposomes and intracellular fate of encapsulated molecules: encounter with a low pH compartment after internalization in coated vesicles.

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.

Ammonium Chloride↗
PubMed1983

Interaction of the eighth component of guinea pig complement (C8) with the membrane-bound C5b-7 complex. The binding site of C8 to C5b-7 is formed on association of the alpha-gamma subunit with the beta-chain.

The eighth component (c8) of guinea pig complement consists of three polypeptide chains, the alpha-, beta-, and gamma-chains with M.W. of 60,000, 60,000, and 24,000, respectively. The alpha- and gamma-chains are bound by a disulfide bond(s) forming an alpha-gamma subunit, which is linked noncovalently to the beta-chain. The alpha-gamma subunit and the beta-chain were separated and purified from C8 by treatment with sodium dodecyl sulfate (SDS) and gel chromatography on Sephacryl S-300 in the presence of SDS. After removal of SDS, neither alpha-gamma nor beta showed the hemolytic activity of C8 when assayed independently, but showed significant activity in combination, indicating reconstitution of active C8. The recovery of hemolytic activity was 3.48%. When alpha-gamma and beta were incubated successively with EAC-7 with intervening reconstitution of active C8 on the cells was insignificant, irrespective of the order of the reactions. alpha-gamma and beta did not bind to EAC-7 when added separately, but after recombination 7% of alpha-gamma and 9% of beta bound to EAC-7 when EAC-7 was in excess. These results indicate that the binding site of guinea pig C8 to the membrane-bound C5b-7 complex does not exist on either alpha-gamma or beta only but stretches over both or is formed on one subunit after recombination of the subunits.

Animals↗
PubMed1982

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.

Animals↗
PubMed1982

Synexin facilitates fusion of specific phospholipid membranes at divalent cation concentrations found intracellularly.

The effect of synexin (an adrenal medullary protein) on the kinetics of Ca2+- and Mg2+-mediated membrane fusion was examined. Membrane fusion was studied by monitoring intermixing of the aqueous contents of phospholipid vesicles. Synexin facilitated Ca2+-mediated, but not Mg2+-mediated, fusion of phosphatidate/phosphatidylethanolamine (1:3) and phosphatidate/phosphatidylserine/phosphatidylethanolamine/cholesterol (1:2:3:2) vesicles. The threshold concentration of Ca2+ for fusion was decreased to approximately equal to 10 microM in the presence of synexin at 6 micrograms/ml and 1.5 mM Mg2+ in vesicle suspensions containing 50 microM lipid. This effect of synexin was drastically inhibited by including 25% phosphatidylcholine (mol/mol) in the vesicle membrane. It is proposed that the Ca2+-dependent lipid-specific enhancement of membrane fusion by synexin contributes to an increase in the sensitivity of specific intracellular membranes to Ca2+ with respect to fusion.

Animals↗
PubMed1981

Role of synexin in membrane fusion. Enhancement of calcium-dependent fusion of phospholipid vesicles.

Synexin, a soluble adrenal medullary and liver protein which causes calcium-dependent aggregation of isolated chromaffin granules, was isolated and purified according to published procedures. The effects of synexin on the kinetics of membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Synexin lowers the threshold of CA2+ concentration required for fusion of large unilamellar vesicles of phosphatidylserine and a mixture of phosphatidylserine with phosphatidylethanolamine. synexin also increases drastically the initial rate of fusion. the initial rate of fusion increases with the quantity of synexin present in the reaction mixture. In the presence of 1-2 mM Ca2+ and 50 microM phospholipid, synexin at 20 to 40 micrograms/ml increases the rate of fusion by two orders of magnitude. Mg2+ does not support synexin-induced fusion. With vesicles containing a mixture of phosphatidylserine with phosphatidylcholine, synexin enhances aggregation in the presence of CA2+, without promoting fusion. Synexin may play a role in exocytosis by promoting fusion of membranes containing specific phospholipids in the presence of Ca2+.

Adrenal Medulla↗
PubMed1981

Fifth component of guinea pig complement: purification and characterization.

The 5th component of complement (C5) was purified from guinea pig serum. The 6-step procedure, involving removal of C1 by precipitation at pH 7.5, mu = 0.04, 2.0 M ammonium sulfate precipitation, acid precipitation at pH 5.6 mu = 0.1, and successive chromatographies on Sephadex G-200, DEAE-cellulose, and hydroxylapatite columns, yielded 1.6 to 4 mg of C5 from 250 ml of serum. Purified C5 gave 1 protein band on disc polyacrylamide gel electrophoresis (PAGE) and sodium dodecylsulfate-(SDS) PAGE. Guinea pig C5, like human C5, consisted of 2 polypeptide chains designated as alpha (m.w. of 108,000) and beta (m.w. of 79,000) linked together by disulfide bonds. The amino acid composition was also very similar to that of human C5. The amino-terminus of the alpha-chain was aspartic acid or asparagine, and that of the beta-chain was undetectable by the dansyl method. Limited proteolysis of C5 with trypsin caused virtually no alteration in its mobility on immunoelectrophoresis and SDS-PAGE without reduction. Cleavage with trypsin was restricted to the alpha-chain: the beta-chain was totally resistant to the digestion. The alpha-chain was split into at least 4 fragments of 58,000, 34,000, 29,000, and 27,000 daltons linked to one another and/or to the beta-chain by disulfide bonds.

Amino Acids↗