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Biomedical subjects

L Huang

Publications and source records attributed to L Huang.

At least 739 records · Page 41Linked to original sources
PubMed1985

Incorporation of acylated antibody into planar lipid multilayers: characterization and cell binding.

Multiple (up to 14) layers of lipid were deposited onto an alkylated glass surface by dialysis of egg phosphatidylcholine (PC) and deoxycholate mixed micelles in the presence of alkylated glass coverslips. The amount of lipid associated with the coverslips was measured by using radioactive PC. It was found that the number of PC molecules in the multilayer increased with increasing initial lipid concentration in the dialysis mixture. Inclusion of cholesterol resulted in a significant increase in the amount of total lipid deposited in the multilayer. However, the PC/cholesterol ratio was up to 2-fold higher in the multilayers than in the liposomes present in the same dialysis bag. In addition, mouse monoclonal anti-H2Kk antibody which had previously been derivatized with palmitic acid could be readily incorporated into the lipid multilayer during dialysis. Measurements of lateral mobility with the fluorescence recovery after photobleaching technique on fluorescently labeled lipid or antibody in the multilayer showed that the lipid molecules diffused rapidly while the antibodies were essentially immobile. Lymphoma cells such as RDM4 cells expressing surface H2Kk glycoproteins could rapidly bind to the antibody-containing multilayers. The binding was blocked by free antibody or by goat anti-mouse immunoglobulin G, indicating the immunospecificity of the binding. Cell binding to the multilayer also exhibited a threshold dependence on the antibody density of the multilayer. A lower threshold was found for cells expressing a higher surface density of H2Kk. This system may be useful for model studies of cellular recognition.

Animals↗
PubMed1985

Efficient cytoplasmic delivery of a fluorescent dye by pH-sensitive immunoliposomes.

We previously showed that liposomes composed of dioleoylphosphatidyl-ethanolamine and palmitoyl-homocysteine (8:2) are highly fusion competent when exposed to an acidic environment of pH less than 6.5. (Connor, J., M. B. Yatvin, and L. Huang, 1984, Proc. Natl. Acad. Sci. USA. 81:1715-1718). Palmitoyl anti-H2Kk was incorporated into these pH-sensitive liposomes by a modified reserve-phase evaporation method. Mouse L929 cells (k haplotype) treated with immunoliposomes composed of dioleoylphosphatidylethanolamine/palmitoyl-homocysteine (8:2) with an entrapped fluorescent dye, calcein, showed diffused fluorescence throughout the cytoplasm. Measurements by use of a microscope-associated photometer gave an approximate value of 50 microM for the cytoplasmic calcein concentration. This concentration represents an efficient delivery of the aqueous content of the immunoliposome. Cells treated with immunoliposomes composed of dioleoylphosphatidylcholine (pH-insensitive liposomes) showed only punctate fluorescence. The cytoplasmic delivery of calcein by the pH-sensitive immunoliposomes could be inhibited by chloroquine or by incubation at 20 degrees C. These results suggest that the efficient cytoplasmic delivery involves the endocytic pathway, particularly the acidic organelles such as the endosomes and/or lysosomes. One possibility is that the immunoliposomes fuse with the endosome membranes from within the endosomes, thus releasing the contents into the cytoplasm. This nontoxic method should be widely applicable to the intracellular delivery of biomolecules into living cells.

Animals↗
PubMed1985

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions.

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.

Benzoates↗
PubMed1985

Interactions of antigen-sensitized liposomes with immobilized antibody: a homogeneous solid-phase immunoliposome assay.

Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level.

Animals↗
PubMed1984

Polyhistidine mediates an acid-dependent fusion of negatively charged liposomes.

Polyhistidine facilities the fusion of negatively charged liposomes prepared by sonication. Liposome fusion was demonstrated by (a) negative-stain electron microscopy, (b) gel filtration, and (c) resonance energy transfer of the fluorescent phospholipids. Liposome fusion required the presence of polyhistidine, whereas histidine at equivalent concentrations had no effect. Little or no liposome fusion was detectable at pH 7.4, but it was greatly enhanced when the pH of the medium was reduced below 6.5. Although acidic phospholipid is necessary for fusion, liposomes made of acidic lipids alone showed only low levels of fusion activity. Liposomes composed of mixtures (1:1) of a negatively charged phospholipid and a neutral phospholipid such as phosphatidylcholine and phosphatidylethanolamine (PE), but not sphingomyelin, showed high levels (about 80%) of fusion competency. For liposomes made of PE/phosphatidylserine (PS) (1:1), fusion at pH 5.2 and 2.5 micrograms/mL polyhistidine resulted in an increase in the average liposome diameter from 296 to 2400 A, indicating multiple rounds of fusion had occurred. Liposome fusion was not very leaky as revealed by the lack of release of encapsulated calcein. For PE/PS (1:1) liposomes, about 10% of dye leakage was observed for up to about 30% liposome fusion and about 45% leakage at 80% liposome fusion. Since polyhistidine becomes a strong polycation at acidic pH, liposome fusion may be a direct result of the bilayer phase separation induced by the binding of polyhistidine with the negatively charged phospholipids. Therefore, this phenomenon is similar to the liposome fusion induced by other polycations such as polylysine at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Energy Transfer↗
PubMed1984

Thermotropic and dynamic characterization of interactions of acylated alpha-bungarotoxin with phospholipid bilayer membranes.

The interactions of palmitoyl-alpha-bungarotoxin (PBGT) with dipalmitoylphosphatidylcholine (DPPC) bilayers have been studied by using high-sensitivity differential scanning calorimetry together with steady-state and time-resolved phosphorescence and fluorescence spectroscopy. The incorporation of PBGT into large single lamellar vesicles causes a decrease in the phospholipid phase transition temperature (Tm), a broadening of the heat capacity function, and a decrease in the enthalpy change associated with the phospholipid gel to liquid-crystalline transition. Analysis of the dependence of this decreased enthalpy change on the protein/lipid molar ratio indicates that each PBGT molecule exhibits a localized effect upon the bilayer, preventing approximately six lipid molecules from participating in the lipid phase transition. Additional calorimetric experiments indicate that binding to acetylcholine receptor enriched membranes causes a small increase in the Tm of the PBGT/DPPC vesicles. Steady-state fluorescence depolarization measurements employing 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the association of PBGT with the phospholipid bilayer decreases the apparent order of the bulk lipid below Tm while increasing the order above Tm. These results have been further supported by rotational mobility measurements of erythrosin-labeled PBGT associated with giant (about 2-micron) unilamellar vesicles composed of dielaidoylphosphatidylcholine or dioleoylphosphatidylcholine using the time-dependent decay of delayed fluorescence/phosphorescence emission anisotropy. Rotational correlation times in the submillisecond time scale (about 30 microseconds) indicate that the protein is highly mobile in the fluid phase and that below Tm the rotational mobility is only slightly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Bungarotoxins↗
PubMed1984

pH-sensitive liposomes: acid-induced liposome fusion.

Sonicated unilamellar liposomes containing phosphatidylethanolamine and palmitoylhomocysteine fuse rapidly when the medium pH is lowered from 7 to 5. Liposome fusion was demonstrated by (i) mixing of the liposomal lipids as shown by resonance energy transfer, (ii) gel filtration, and (iii) electron microscopy. The pH-sensitive fusion of liposomes was observed only when palmitoylhomocysteine (greater than or equal to 20 mol%) was present in the liposomes. The presence of phosphatidyl-ethanolamine in the liposomes greatly enhanced fusion whereas the presence of phosphatidylcholine inhibited fusion. During fusion of liposomes containing phosphatidylethanolamine and palmitoylhomocysteine (8:2, mol/mol), almost all of the encapsulated calcein was released. Inclusion of cholesterol (40 mol%) in the liposomes substantially decreased leakage without impairing fusion.

Homocysteine↗
PubMed1984

Discovery, purification and characterization of the angiotensin converting enzyme inhibitor, L-681,176, produced by Streptomyces sp. MA 5143a.

L-681,176, an inhibitor of angiotensin converting enzyme was found in the culture filtrate of Streptomyces sp. MA 5143. The I50 of the crystalline inhibitor is about 1.3 micrograms/ml and the inhibition is reversed by zinc sulfate. In rats, L-681,176 exhibits a dose-related inhibition of the pressor response to angiotensin I with an ID50 of 142 mg/kg when administered intravenously. The structure of L-681,176 is similar to that of marasmine but lacking one carboxyl group. The maximum yield of L-681,176 occurs after three to four days growth at 28 degrees C.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1984

Discovery, production and purification of the Na+, K+ activated ATPase inhibitor, L-681,110 from the fermentation broth of Streptomyces sp. MA-5038.

The maximum yield for the production of L-681,110 by Streptomyces sp. MA-5038 (ATCC 31587) was observed after 5 days' incubation at 28 degrees C and pH about 8.3. L-681,110 was isolated from the fermentation broth by acetone extraction of the mycelia, absorption to Amberlite XAD-2 resin and two separations by thin-layer chromatography. The structure of L-681,110 was found to consist of a sixteen-membered lactone with a new type of substitution. The inhibition of ATPase, activity against Caenorhabditis elegans and stimulation of gamma-aminobutyric acid release indicate that L-681,110 possesses some characteristics of both oligomycin and avermectin. L-681,110 was also active against tapeworm and ticks in an in vivo assay.

Animals↗