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Biomedical subjects

L Huang

Publications and source records attributed to L Huang.

At least 685 records · Page 38Linked to original sources
PubMed1988

Kinetic and ultrastructural studies of interactions of target-sensitive immunoliposomes with herpes simplex virus.

The bilayer phase of dioleoylphosphatidylethanolamine (PE) can be stabilized with palmitoyl-IgG monoclonal antibody to the glycoprotein gD of the herpes simplex virus (HSV). Interactions of PE immunoliposomes with the target virions were characterized by analyzing the kinetics of lipid mixing, by liposomal content release, and by ultrastructural studies. As revealed by a resonance energy transfer assay, lipid mixing between PE immunoliposomes and virions was very rapid, with a second-order rate constant (kapp) of 0.173 (min)-1 (microgram/mL virus)-1. In comparison, content release from PE immunoliposomes was much slower and exhibited multiple-phase, mixed-order kinetics, indicating that liposome destabilization involved fusion of liposomes with HSV. The extent and the apparent rate of liposome destabilization were strongly dependent on liposome concentration. This was evident by the fact that only one to two liposomes were destabilized by each virus particle at low liposome concentration (0.1 microM). For higher liposome concentrations (1-10 microM), this value was 35-104. This finding implies that collision among the virus-bound liposomes is essential for the eventual collapse of PE immunoliposomes to form the hexagonal (HII) equilibrium phase which was observed using freeze-fracture electron microscopy. Studies employing soluble gD, immobilized on latex beads, indicated that a multivalent antigen source is essential for PE immunoliposome destabilization. Immediately after liposome-virus binding, fusion of liposome with the viral membrane then follows. Upon growth of the fusion complexes, which increase to 35-104 liposomes for each virus, an eventual collapse of the structure results, driving PE to its equilibrium structure of HII phase.

Antibodies, Monoclonal↗
PubMed1988

Gadolinium-labeled liposomes containing paramagnetic amphipathic agents: targeted MRI contrast agents for the liver.

Unique paramagnetic liposomal contrast agents were synthesized and utilized for selective augmentation of T1 MR imaging of the livers of normal Balb/c mice. Amphipathic gadolinium complexes, which mimic phospholipids, were incorporated into the lamella of small unilamellar liposomes (SUV) such that they became an integral part of its surface. T1 signal enhancement of normal liver approached 150% after iv administration of the paramagnetic liposomes, determined by experiments performed on a 1.9-T, experimental whole-body MRI unit. Tracer studies utilizing gadolinium-153-tagged SUV revealed that the agents exhibited excellent in vivo stability, compared to liposomal preparations in which paramagnetic agents are simply entrapped in the aqueous core of the liposome vesicle.

Animals↗
PubMed1988

Purification and Comparison of the Periplasmic and Extracellular Forms of the Cellodextrinase from Bacteroides succinogenes.

Both the periplasmic and the extracellular cellodextrinases from Bacteroides succinogenes S85 grown on Avicel microcrystalline cellulose were purified to homogeneity by column chromatography and characterized. Over 70% of the total cellobiosidase activity displayed by cells was accounted for by these enzymes. The periplasmic and extracellular cellodextrinases had identical molecular weights (50,000), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identical isoelectric points (4.9). In addition, the two enzymes were similar in catalytic properties, with K(m) and V(max) values of approximately 0.24 mM and 21 mumol/min per mg of protein, respectively. Examination of the two enzymes by using peptide mapping and immunoblotting techniques provided additional evidence indicating their identical nature. Immunoblotting of the extracellular culture fluid with affinity-purified antibody to the periplasmic cellodextrinase revealed one band with a molecular weight corresponding to that of the periplasmic cellodextrinase. The stability of the purified periplasmic cellodextrinase in aqueous solution was markedly enhanced by increased protein content. This enzyme showed a low affinity for crystalline cellulose.

Journal Article↗
PubMed1988

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85.

A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gram-negative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39 degrees C and pH 6.5, the activity of the enzyme with p-nitrophenyl-beta-D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.0 mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45 degrees C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharrides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl cellulose.

Bacteroides↗
PubMed1988

Insulin and a putative insulin metabolic mediator fraction from liver and muscle stimulate p33 messenger ribonucleic acid accumulation by apparently different mechanisms.

We have previously shown that in rat H4 hepatoma cells insulin enhances the nuclear transcription of p33 mRNA in a dose- and time-dependent manner, with no alteration in mRNA half-time (t1/2). Presumably, this effect is mediated by the cell surface receptor. In this report, we have investigated the effect of putative insulin mediator fractions which act to control metabolic events on p33 mRNA accumulation in these cells. Initial experiments originally demonstrated an insulin-like effect of an added putative metabolic fraction to enhance p33 mRNA concentrations. However, when the fetal calf serum supply was changed, the effect of insulin remained, but that of added mediator was no longer observed. After a series of experimental approaches designed to alter the permeability of the cell membrane, it was found that in the presence of increased Ca2+, the effect of mediator could again be observed. The present data demonstrate that the partially purified cAMP-dependent protein kinase/adenylate cyclase inhibitory putative mediator fractions from liver and muscle enhance p33 mRNA accumulation in intact H4 hepatoma cells by a mechanism that is differentiated from that of insulin. The action of the putative mediator is inhibited by cycloheximide, while the action of insulin itself is not. These results suggest that insulin may control nuclear transcription by multiple signaling mechanisms. Alternatively, the added putative metabolic mediator may not enter the cell in the presence of cycloheximide or is inactive as such within the cell and must first be converted to an active species by a step requiring protein synthesis.

Adenylyl Cyclase Inhibitors↗
PubMed1988

A homogeneous, liposome-based signal amplification for assays involving enzymes.

Exploiting the fact that phosphatidylethanolamine (PE) liposome can be stabilized by a membrane lipid or protein, and that destruction of this stabilizer leads to rapid lysis of the liposome, we have designed a liposome-based signal enhancement mechanism for assays that involve enzyme as the final read-out step. Stable liposomes with entrapped glucose-6-phosphate dehydrogenase (G6PDH) were prepared with unsaturated PE stabilized with 5 mol percent of ganglioside GM1. Addition of beta-galactosidase caused rapid (3-5 min) lysis of liposomes, revealing the latent G6PDH activity, owing to the enzymatic degalactosylation of GM1. We have used Microgenic's CEDIA assay for digoxin as an example. The magnitude of signal was 25 mA/min per microgram of digoxin per liter for the unamplified assay and 1000 mA/min per microgram of digoxin per liter for the liposome-enhanced assay--i.e., a 40-fold amplification. This simple, rapid, and homogeneous signal-amplification mechanism is likely to be useful in many enzyme-dependent assays, such as ELISA, CEDIA, gene-probe assays, and immunoliposome assays.

Blood↗
PubMed1987

Target-sensitive immunoliposomes as an efficient drug carrier for antiviral activity.

We have shown previously that target-sensitive immunoliposomes composed of palmitoyl antibody stabilized phosphatidylethanolamine bilayers could be destabilized by binding to the target cells (Ho, R. J. Y., Rouse, B. T., and Huang, L., Biochemistry (1986) 25, 5500-5506). Target-sensitive immunoliposome-encapsulated and free cytotoxic drugs of nucleoside analogs cytosine-beta-D-arabinoside (AraC) or acycloguanosine (acyclovir, ACV) were compared for their antiviral efficacy and cell cytotoxicity. Target-insensitive immunoliposomes and nontargeted liposomes were also investigated. When the mouse fibroblast L929 cells were infected at low multiplicity with herpes simplex virus, AraC encapsulated in target-sensitive immunoliposomes composed of transphosphatidylated egg phosphatidylethanolamine effectively inhibited virus replication and had far less cell cytotoxicity than free drug. As a measure of cytotoxicity, the drug concentration required to inhibit 50% of [3H]thymidine incorporation from 6 to 42 h (CD50) was determined. For free AraC, this value was 0.3 ng/ml, whereas for target-sensitive immunoliposome-encapsulated AraC, the CD50 exceeded 1 microgram/ml. However, target-sensitive immunoliposome-encapsulated AraC was virus inhibitory (50% effective dose = ED50) at 1.8 ng/ml. A free drug concentration of at least 1000-fold greater was required for comparable antiviral activity. A similar phenomenon was observed when ACV was administered via target-sensitive immunoliposomes. The CD50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 12.5 ng/ml and 1.4 micrograms/ml, respectively, whereas the ED50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 1.1 and 125 ng/ml, respectively. Consequently, our results indicated the superiority of target-sensitive immunoliposomes at drug delivery, especially when drugs were cytotoxic to cells. The use of liposomes of the target-insensitive variety provided some enhancement of activity, but this was several-fold less than that observed with target-sensitive immunoliposomes. In addition, the nucleoside transport inhibitors, p-nitrothiobenzylinosine and dipyridamole, were shown to inhibit the liposome-mediated antiviral activity of AraC. This finding indicated that site-specific cytosolic delivery of nucleoside analogs by target-sensitive immunoliposomes involved a cellular nucleoside transport system. A mechanism of action is proposed.

Animals↗
PubMed1987

Interactions of target-sensitive immunoliposomes with herpes simplex virus. The foundation of a sensitive immunoliposome assay for the virus.

Interactions between target-sensitive (TS) immunoliposomes and herpes simplex virus (HSV) were investigated. Target sensitivity of phosphatidylethanolamine (PE) immunoliposomes is a result of the ability of acylated monoclonal anti-HSV glycoprotein D (gD) to stabilize the bilayer phase of PE, whereas by itself, PE does not form stable liposomes (Ho, R. J. Y., Rouse, B. T., and Huang, L. (1986) Biochemistry 25, 5500-5506). Upon binding of these immunoliposomes to HSV antigen-containing gD, destabilization of PE immunoliposomes was observed. By encapsulating either a self-quenching fluorescent dye, calcein, or alkaline phosphatase inside the liposomal compartment, the HSV-induced destabilization of TS immunoliposomes was shown to be target-specific. Neither Sendai, Semliki Forest, nor Sindbis virus could significantly destabilize the TS immunoliposomes. Moreover, HSV-induced liposome destabilization could be inhibited by free anti-gD (the same antibody used in TS immunoliposomes) but not by monoclonal anti-HSV glycoprotein B, indicating that the interaction was antigen-specific. Destabilization could also be induced by binding to truncated gD (tgD), but only when in a multivalent form immobilized on latex beads. Truncated gD is a cloned, 312-amino acid fragment of HSV-gD that lacks the transmembrane segment. Preincubation of soluble tgD with the TS immunoliposomes failed to induce destabilization and, in addition, abolished the tgD-bead-induced destabilization. This finding strongly indicated that multivalent binding is essential for TS immunoliposome destabilization. Using alkaline phosphatase encapsulated in the liposomes, TS immunoliposomes could be used to detect HSV in fluid phase with 50% signal recorded at 5 microliters of 3.2 x 10(3) pfu/ml; at least 10-fold more sensitive than the standard double-antibody sandwich enzyme-linked immunosorbent assay. The interactions described here may be useful in designing a homogeneous and sensitive immunoliposome assay.

Animals↗
PubMed1987

Plasmid DNA adsorbed to pH-sensitive liposomes efficiently transforms the target cells.

We have previously reported that plasmid DNA entrapped in the pH-sensitive immunoliposomes effectively transforms the target cells (Proc. Natl. Acad. Sci. USA, in press). In the present study, we demonstrate that DNA adsorbed on the same liposome also transforms the target cells. The transformation activity is antibody dependent, as liposomes containing no targeting antibody had reduced activity. The activity could be significantly inhibited by excess non-specific DNA (salmon sperm). Since some DNA are likely adsorbed to the liposomes during the entrapment process, the activity of the entrapped DNA is partially accounted for by the adsorbed DNA. The possibility of developing a simple DNA-mediated transfection protocol using liposome adsorbed DNA is discussed.

Adsorption↗
PubMed1987

L-652,469--a dual receptor antagonist of platelet activating factor and dihydropyridines from Tussilago farfara L.

L-652,469, 14-acetoxy-7 beta-(3'-ethylcrotonoyloxy)-notonipetranone, isolated from the methylene chloride extracts of the buds of Tussilago farfara L, was found to inhibit both platelet activating factor (PAF) and Ca2+ entry blocker binding to membrane vesicles. It inhibits the [3H]PAF specific binding to rabbit platelet membranes with equilibrium inhibition constants (Ki) of 3.2 and 4.0 microM in the presence of 150 mM NaCl and 10 mM MgCl2 respectively. It is a competitive PAF receptor antagonist with an equilibrium dissociation constant (KB) of 5.16 microM. It also competitively inhibits the specific binding of Ca2+ channel blockers (e.g. [3H]nitrendipine; Ki = 1.2 microM) in cardiac sarcolemmal vesicles. At 10(-5) M, L-652,469 causes a 60% relaxation of Ca2+-induced contraction of rat thoracic aorta strips. Due to its dual antagonistic activities, L-652,469 potently inhibits the gel-filtered rabbit platelet aggregation with a pA2 of 5.79. It was also found to be orally active with a beneficial effect to inhibit the PAF-induced rat foot edema and the first phase of carrageenan-induced rat hindpaw edema.

Animals↗
PubMed1987

The role of multivalency in antibody mediated liposome targeting.

We have investigated the role of multivalency in immunoliposome binding to cells displaying different amounts of surface antigen using liposomes with increasing numbers of palmitoyl anti-H2Kk antibodies incorporated into the bilayer. RDM-4 lymphoma cells were treated with proteinase k to generate a series of cells with various amounts of H2Kk antigen. Percent binding of immunoliposomes was related to the number of antigens displayed by the RDM-4 cell. Increasing liposome binding was observed with increasing number of antibody molecules per liposome. However, the ratio of binding of the high-antigen-density cells to that of the low-antigen-density cells was higher with immunoliposomes of lower antibody density than the ones with higher antibody density. This result suggests that for better discrimination between cells differing in antigen density, liposomes with lower numbers of antibody molecules per liposome may be more useful as a discriminatory tool for cells with a low level antigen expression than liposomes with greater antibody densities.

Animals↗
PubMed1987

Thermodynamic and phase characterization of phosphatidylethanolamine and ganglioside GD1a mixtures.

By employing diphenylhexatriene steady-state fluorescence anisotropy, pyrenedecanoic acid excimer formation, and high sensitivity scanning calorimetry we have demonstrated that the liposomes containing phosphatidylethanolamine (PE) and various mole fractions of ganglioside GD1a had a gel-liquid crystalline phase transition between 15 and 25 degrees C. Calorimetric measurements indicated that these phase transitions were broad and centered between 17 and 21 degrees C. The enthalpy change of the transition was linearly dependent on the ganglioside concentration up to 10.0 mol% and plateaued between 11.4-16.2 mol%. The high enthalpy change (37 kcal/mol of GD1a added into the PE bilayer) indicates the existence of PE-GD1a complex structure in the liposomal membrane. It is proposed that semi-fluid domains containing six PE and one ganglioside molecule are present in the PE-GD1a membranes at temperatures above gel-liquid crystalline phase transition. The Sendai virus induced leakage of PE-GD1a liposomes has been investigated by using an entrapped, self-quenching fluorescent dye, calcein. The leakage rate was dependent on the mole fraction of ganglioside GD1a and was maximal at 6.3 mol%. Arrhenius plots of the leakage rates showed breaks in the 20-25 degrees C temperature range, which correspond to the gel-liquid crystalline phase transition of the target liposomes. These data suggest that the rate of Sendai virus-induced leakage can be regulated via fluidity modulation by changing the PE to GD1a ratio at constant temperatures.

Calorimetry, Differential Scanning↗
PubMed1987

Solid core liposomes with encapsulated colloidal gold particles.

Solid core liposomes with encapsulated colloidal gold particles were prepared through four major steps: Preparation of prevesicles with encapsulated solid cores of agarose-gelatin by emulsification of agarose-gelatin sol in organic solvent containing emulsifiers followed by cooling. Extraction of lipophilic components from prevesicles to obtain microspherules of agarose-gelatin. Introducing colloidal gold particles into microspherules and coating with protein molecules. Encapsulation of colloidal gold-bearing microspherules with the modified organic solvent spherule evaporation method for preparation of liposomes (Kim et al. (1983) Biochim. Biophys. Acta 728, 339-348 and Kim et al. (1984) Biochim. Biophys. Acta 812, 793-801). Electron micrographs showed that if liposomes were prepared by using a lipid mixture containing dioleoylphosphatidylcholine/cholesterol/dioleoylphosphatidylglycerol/tri olein (molar ratio 4.5:4.5:1:1), there was only a single continuous bilayer membrane for each solid core liposome. However, if no triolein was added to the lipid mixture, it would cause the formation of multilamellar liposomes. In both cases, there were hundreds to thousands of colloidal gold particles within each solid core liposome.

Cholesterol↗
PubMed1987

ATP-Mn2+ stimulates the generation of a putative mediator of insulin action.

Substantial evidence suggests that insulin receptor-associated protein kinase may play a pivotal role in the expression of the intracellular effects of insulin. This study was undertaken to determine whether insulin receptor kinase contributes to the generation of putative insulin mediators. The effect of ATP and divalent cation addition on the production of insulin mediators from liver plasma membranes was investigated. ATP (1 mM) added to liver plasma membranes in the absence of divalent cations enhanced insulin-stimulated release/generation of mediator slightly (approximately 3-fold). ATP in the presence of Mn2+ further increased release/generation of mediator markedly (approximately 100-fold). In contrast, ATP in the presence of Mg2+ had no stimulatory effect. Mn2+ and Mg2+ alone were ineffective. Addition of EDTA completely diminished the stimulatory effects of insulin, ATP, and Mn2+. The stimulation was ATP-specific since other nucleotides and nonhydrolyzable analogues of ATP had no or very weak activity. ATP-Mn2+ stimulated insulin-dependent mediator release/generation in a dose-dependent manner. These results suggest that insulin mediator release/generation is markedly stimulated by an ATP-Mn2+-dependent phosphorylation reaction, similar to insulin-stimulated receptor tyrosine kinase phosphorylation.

Adenosine Triphosphate↗
PubMed1987

Cytotoxicity of diphtheria toxin A fragment to toxin-resistant murine cells delivered by pH-sensitive immunoliposomes.

pH-sensitive immunoliposomes composed of dioleoylphosphatidylethanolamine and oleic acid (8:2 molar ratio) mediated the delivery of the cytotoxic fragment A of diphtheria toxin to the cytoplasm of target L-929 cells. Free fragment A, fragment A encapsulated in antibody-free liposomes, or fragment A encapsulated in pH-insensitive immunoliposomes was not effective in the inhibition of the cellular protein synthesis. pH-sensitive immunoliposomes containing diphtheria fragment A were not toxic to nontarget diphtheria-resistant A31 cells or to nontarget diphtheria-sensitive Vero cells. Pretreatment of target L-929 cells with the weak bases NH4Cl or chloroquine, agents which raise the endosome/lysosome pH, blocked the cytotoxic effect of the pH-sensitive immunoliposomes containing fragment A. Excess free antibody or excess empty pH-sensitive immunoliposomes also blocked the cytotoxic effect. Since it is known that fragment A alone cannot cross lipid membranes, the results of this study indicate that pH-sensitive immunoliposomes are able to release the toxin into the cytoplasm, probably by fusing with the endosome membrane following a receptor-mediated endocytosis of the immunoliposome.

Animals↗
PubMed1987

Resolution of racemic gossypol.

Treating racemic gossypol with chiral amine (S)-1-methylphenethyl amine gave the expected pair of diastereomeric amino condensates in which the binaphthyl nuclei racemized during further treatment. Acetylation was applied to avoid the change. On acetylation, 7 products were separated. Their structures were assigned based upon their spectroscopic data. After hydrolysis they gave optically active (+)- and (-)-gossypol correspondingly. A simple and practical method of quick separation of the diastereomers was also discussed.

Chemical Phenomena↗