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Biomedical subjects

L Huang

Publications and source records attributed to L Huang.

At least 775 records · Page 43Linked to original sources
PubMed1981

Immunoliposome labeling: a sensitive and specific method for cell surface labeling.

A simple, one-step procedure for fluorescence labeling of cultured cells with high sensitivity and specificity is described. We term this method immunoliposome labeling. Monoclonal IgG antibody was first covalently coupled with palmitic acid. The palmitoyl IgG was then incorporated into unilamellar liposomes (about 100 nm diameter) containing either N-(4-nitrobenzo-2-oxa-1,3-diazole)- or N-(fluorescein isothiocyanyl)-phosphatidylethanolamine by a detergent-dialysis procedure. A monoclonal antibody to the mouse major histocompatibility antigen, H-2k, was tested as a model system. Fluorescent liposomes with covalently coupled anti-H-2k specifically labeled the mouse L-929 cells (H-2k type), but not the A-31 cells (H-2d type). The degree of labeling was quantitated by a microscope photometer. Cells labeled with fluorescent liposomes showed 4--6-fold stronger fluorescence than cells labeled with either fluorescein-conjugated antibody or with primary antibody followed by fluorescein-conjugated secondary antibody (indirect immunofluorescence). Since different types of label (fluorescent, radioactive, etc.) can be incorporated into liposomes, this specific and sensitive method is potentially very versatile.

4-Chloro-7-nitrobenzofurazan↗
PubMed1981

Cell-mediated immunity to herpes simplex virus: induction of cytotoxic T lymphocyte responses by viral antigens incorporated into liposomes.

The immunogenicity of inactivated herpes simplex virus type 1 (HSV-1) antigens incorporated into liposomes was measured by their ability to induce secondary anti-HSV-1 specific cytotoxic T lymphocyte (CTL) responses in splenocyte cultures from virus-primed mice. Such virus-specific CTL could be induced provided the liposomes contained virus along with plasma membrane antigen of the same H-2 type as that of the virus-primed responser cells. Responses did not occur in cultures stimulated with liposomes containing only viral antigens or with a mixture of liposomes composed respectively of lipid and virus with those composed of lipid and plasma membrane proteins. Moreover F1 responder cells stimulated with liposomes composed of virus and plasma membrane protein of one of the parental haplotypes produced CTL restricted in their cytotoxicity to infected targets of the same haplotype as was used in the liposome. These results show that liposomes can be used to induce anti-HSV-1 CTL with inactivated viral antigens but recognition of both viral and H-2 antigen is required for this process to occur in vitro. The implications of our findings to the preparation of subunit vaccines against HSV-1 are briefly discussed.

Animals↗
PubMed1980

Transient permeabilization induced osmotically in membrane vesicles from Torpedo electroplax: a mild procedure for trapping small molecules.

During hypoosmotic stress, membrane vesicles enriched in acetylcholine receptors become more permeable to external tracer molecules. When vesicles are immersed in 3 volumes of water containing 22Na+, 50-70% of 22Na+ equilibration is attained within 90 s. On the other hand, the uptake of 22 Na+ is greatly diminished only 6-10 s after an osmotic shock, and vesicle resealing is completed within 15 s. Furthermore, 90 s after osmotic shock, efflux rates are comparable to those of native vesicles, which also indicates that the vesicles have resealed. During osmotic shock, the entry of molecules into the vesicles increases with the strength of the osmotic shock and also depends on the size of the permeant. With a given strength of osmotic shock, the large molecule [3H]inulin (Mr 5000) is taken up less than the smaller molecules 22Na+ and [3H]sucrose. In addition, alpha-bungarotoxin binding latency of the vesicles is not affected by osmotic shock, indicating that the sideness of the vesicles remains unchanged. The acetylcholine receptors in the vesicles remain functional after osmotic shock. For example, 90 s after 22Na+ and [3H]sucrose are loaded into vesicles by osmotic shock, only 22Na+ is released by dilution in a buffer containing carbamoylcholine (carbamylcholine). Also, the influx of 22 Na+ into previously shocked vesicles can be specifically stimulated by carbamoylcholine. Such stimulations in the shocked vesicles can be blocked by d-tubocurarine or alpha-bungarotoxin, and they can be desensitized by preincubation with carbamoylcholine. These results suggest the possibility of using osmotic shock to load molecular probes into these membrane vesicles, which could provide a powerful tool for studying inner surfaces of the intact vesicles.

Acetylcholine↗
PubMed1980

Monoclonal antibody covalently coupled with fatty acid. A reagent for in vitro liposome targeting.

Monoclonal antibody to the mouse histocompatibility antigen, H-2k, was derivatized with palmitic acid using an activated ester of N-hydroxysuccinimide. About 70% of the resulting amphipathic antibody could be incorporated into liposomes by a detergent-dialysis method. The antibody-bound liposomes were about 900 A in diameter and were heterogeneous in terms of the number of antibody molecules per liposome. These liposomes showed specific binding affinity to mouse L-929 cells (H-2k), but not to A-31 cells (H-2d), whereas native liposomes showed no detectable binding to either cell type. The specific binding of anti-H-2k-bound liposomes to L-929 cells could be blocked by a preincubation of cells with an excess of free, underivatized anti-H-2k antibody but not by normal mouse IgG. Using a fluorescent phospholipid, liposomes containing anti-H-2k specifically labeled L-929 cells but not A-31 cells in a mixed culture. Liposomes containing normal mouse IgG did not significantly label either cell type. These results clearly demonstrated the effectiveness of the monoclonal antibody for liposome targeting.

Animals↗
PubMed1980

Studies on the insulin mediator. II. Separation of two antagonistic biologically active materials from fraction II.

Insulin treatment significantly altered the elution profile of deproteinized muscle extracts chromatographed on Sephadex G-25 columns, particularly in fraction II, which contains the insulin mediator. Further purification of fraction II by high-voltage paper electrophoresis at pH 1.9 and 3.5 resulted in two active fractions. Fraction 1 leads to 4 stimulated the cyclic AMP-dependent protein kinase and inhibited glycogen synthase phosphoprotein phosphatase, and may be a novel substance. Fractions 1 leads to 6 and 3 leads to 6 inhibited the cyclic AMP-dependent protein kinase and stimulated glycogen synthase phosphatase. It is proposed that the insulin mediator is present in fractions 1 leads to 6 and 3 leads to 6.

Animals↗
PubMed1979

Generation by insulin of a chemical mediator that controls protein phosphorylation and dephosphorylation.

Deproteinized skeletal muscle extracts free of major nucleotides from control and insulin-treated rats were fractionated and assayed for inhibition of protein phosphorylation by cyclic adenosine monophosphate (AMP)-dependent and -independent protein kinases. A differential effect of insulin on a particular fraction was observed on cyclic AMP-dependent protein kinase but not on cyclic AMP-independent protein kinases. This fraction that inhibited cyclic AMP-dependent protein kinase also stimulated glycogen synthase phosphoprotein phosphatase. It is proposed that this fraction may contain a mediator substance generateed in the presence of insulin.

Animals↗
PubMed1979

Entrapment of proteins in phosphatidylcholine vesicles.

The trapping efficiency of globular proteins in four different types of phosphatidylcholine vesicles was systematically studied. Vesicles were generated in a mixture of 125I-labeled proteins of various molecular weights. The trapped proteins were separated from untrapped proteins by gel filtration and ultrafiltration and subsequently analyzed by gel electrophoresis and autoradiography. Entrapment of proteins was demonstrated by their resistance to trypsin digestion. The relative amount of each entrapped protein species was then compared to that of the original protein solution. In multilamellar vesicles and large unilamellar vesicles, proteins of molecular weight up to 97 000 had the same trapping efficiency as sucrose. In small unilamellar vesicles generated by either sonication or ethanol injection, however, the relative trapping efficiency of protein decreased progressively as the molecular weight of the protein became greater. For example, the trapping efficiency of alpha-amylase (Mr 97 000) was only half of that for sucrose. The apparent decrease in trapping efficiency with the protein's molecular weight in small unilamellar vesicles canbe accounted for by the combination of the bound water layer at the vesicle's internal surface and the steric hindrance when protein is captured during vesicle formation.

Chymotrypsinogen↗
PubMed1979

Synthesis and characterization of a new fluorescent phospholipid.

A novel fluorescent phospholipid, whose structure was tentatively assigned as 1-(2'-thio-1'-hydroxyethyl)-2-(ethylphosphatidyl)isoindole), was synthesized by reacting O-phthalaldehyde and beta-mercaptoethanol with phosphatidylethanolamine. The fluorescent lipid product was purified by silicic acid chromatography. The purity was demonstrated by thin-layer chromatography. This fluorescent phospholipid could not form stable lipid vesicles. However, a mixture of phosphatidylcholine and this fluorescent phospholipid did form stable vesicles after sonication, as demonstrated by Sepharose 4B column chromatography and electron microscopy. The absorption and fluorescence properties of this lipid, both as aqueous micelles or incorporated into vesicles, have been determined. The potential usage of this new fluorescent phospholipid in membrane studies is discussed.

Fluorescent Dyes↗