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L Huang

Publications and source records attributed to L Huang.

At least 793 records · Page 44Linked to original sources
PubMed1978

Transport of 5-hydroxytryptamine by dense granules from porcine platelets.

A method is described for the isolation of a homogeneous preparation of dense granules from procine platelets. The purified dense granule fraction contained approximately 400 nmol of 5-hydroxytryptamine/mg of protein and appeared to be homogeneous when examined by electron microscopy. Isolated dense granules transport exogenously added 5-hydroxytryptamine via two mechanisms: 1) a carrier-mediated process predominating at low substrate concentrations and 2) a diffusion-controlled process predominating at high substrate concentrations. Temperature studies revealed an apparent energy of activation of 14.9 kcal/mol for the carrier-mediated transport. Kinetic data yielded a Km of 3.3 micron and a Vmax of 0.79 nmol/min/mg of protein for the mediated transport process. Steady state uptake was sensitive to changes in medium osmotic pressure and a decline in uptake below 300 mosM was correlated with release of endogenous 5-hydroxytryptamine. The transport was inhibited by a number of structural analogs of 5-hydroxytryptamine. These results demonstrate the existence of a carrier-mediated transport system for 5-hydroxytryptamine in the membranes of the platelet dense granules.

Animals↗
PubMed1978

Interactions of phospholipid vesicles with murine lymphocytes. I. Vesicle-cell adsorption and fusion as alternate pathways of uptake.

The interactions of mouse thymocytes with unilamellar phospholipid vesicles comprised of dimyristoyl lecithin (DML), dipalmitoyl (DPL), dioleoyl lecithin (DOL), and egg yolk lecithin (EYL) were examined in vitro. In cells treated with [3H]DML or [3H]DPL vesicles, electron croscope (EM) autoradiographic analysis showed most of the radioactive lipids to be confined to the cell surface. Transmission EM studies showed the presence of intact vesicles (DPL) and collapsed or ruptured vesicle fragments (DML) adsorbed to the surfaces of treated cells. In cells treated with DPL vesicles containing a water-soluble dye (6-carboxyfluorescein; 6-CF), most of the fluorescent vesicles were localized at the periphery of the treated cells. Furthermore, substantial fractions of the cell-associated DPL and DML could be released by a mild trypsinization without damaging the cells. These results suggest that the uptake of DML and DPL is primarily due to vesicle-cell adsorption. Such an adsorption process appears to be enhanced at or below the thermotropic-phase transition temperature of the vesicle lipid. Under certain conditions these adherent vesicles also formed patches or caps on the cell surface. In cells treated with DOL or EYL vesicles, transmission EM and EM autoradiography showed relatively little exogenous vesicle lipid located at the cell surface. Thymocytes incubated (37 degrees C) with [14C] EYL vesicles containing a trapped marker, [3H]inulin, incorporated both isotopes at identical rates. In separate experiments it was found that this marker was located inside the treated cells. Thymocytes treated with DOL vesicles containing 6-CF exhibited a uniform and diffuse distribution of dye in the internal volume of the cells. Little cell-associated EYL or DOL could be released by trypsinization. Evidence against endocytosis of intact vesicles as a major pathway of vesicle uptake is also presented. These observations, coupled with the demonstration of vesicle-cell lipid exchange as a minor component of vesicle uptake suggest that incorporation of EYL and DOL vesicles by thymocytes is primarily by vesicle-cell fusion.

Adsorption↗
PubMed1978

Interactions of phospholipid vesicles with murine lymphocytes. II. Correlation between altered surface properties and enhanced proliferative response.

The effect of unilamellar lipid vesicles composed of dioleoyl lecithin (DOL), egg yolk lecithin (EYL), 1:1 EYL:cholesterol (Chol), dipalmitoyl lecithin (DPL), and dimyristoyl lecithin (DML) on the mitogenic response in mouse lymphocytes was tested. Cortisone-resistant thymocytes were briefly treated with lipid vesicles and subsequently stimulated with concanavalin A (con A). All of the lipid vesicles induced an enhanced mitogenic response on day 3 as tested by [3H]TdR incorporation and by counting total cells. The order of enchanced [3H]TdR incorporation (less than or equal to 5.3 times the control) was DML greater than DPL greater than 1:1 EYL:Chol greater than EYL congruent to DOL greater than untreated control cells. These increases were paralleled by increased numbers of total cells. The response of spleen cells to a B-cell mitogen, bacterial lipopolysaccharide, was similarly enhanced by vesicle pretreatments in the same order. Vesicle treatments alone were not mitogenic Pretreatment of cells with lipid vesicles modified lectin binding: DML and DPL increased the binding of [125I]con A by three to four times the control, whereas 1:1 EYL:Chol, EYL, or DOL had little or no effect. The binding of [125I]phytohemagglutinin-P (PHA-P) to vesicle-treated cells was indistinguishable from untreated cells. The lectin (con A; PHA-P)-induced agglutination of vesicle-treated cells was also modified by different lipid vesicles in the same order as the mitogenic response. Based on the results presented in the accompanying report [6], we find that the cell surface adsorption properties of the applied lipid vesicles correlate with their ability to enhance the mitogenic response, and that they modify agglutinability and lectin binding. These results are further discussed in terms of the possible alteration of membrane properties and subsequent cellular activity.

Animals↗
PubMed1977

Accelerated calcium ion uptake in murine thymocytes induced by concanavalin A.

The mechanism of enhancement of Ca2+ uptake by the T cell mitogen concanavalin A (Con A) was studied in murine thymocytes. Native Con A enhanced the rate of Ca2+ uptake as much as 9-fold, an increase being observed within five minutes after Con A addition. The effect of Con A was reversed completely by alpha-methyl mannopyranoside (alpha-MM). Increased Ca2+ uptake was observed with increasing concentrations of Con A, between 2 and 400 microgram/ml, indicating that the stimulation of Ca2+ uptake is not restricted to mitogenic lectin concentrations (0.5-2 microgram/ml). Succinyl Con A exhibits only a slight effect in the same concentration ranges as native Con A. Ca2+ uptake, both in the absence and presence of Con A, is strongly dependent on energy metabolism and is carrier mediated. The augmentation of Ca2+ uptake by native Con A is due to an enhanced Vmax. Uptake of the anion, CrO42-, by thymocytes, found to be a non-saturable process, was also enhanced by Con A. The effect of Con A on CrO42- permeability appears to be independent of its effect on Ca2+ uptake.

Azides↗
PubMed1976

The identification of fibrous proteins in fetal rat epidermis by electrophoretic and immunologic techniques.

Two proteins have been identified in extracts of fetal rat skin which are related to the two major fibrous proteins of newborn rat stratum corneum. The relative amount of these proteins increases daily from the 16th to the 20th day (d) of gestation when judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunoelectrophoresis using antibody to the purified fibrous protein. Two-dimensional analysis by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis demonstrates that these two proteins are the only cross-reactive species in the fetal skin from 16d to 19d development. Some additional lower-molecular-weight components can be detected at 20d and 21d. In double-diffusion analysis, cross-reactive proteins in 19d fetal extracts show partial identity but have fewer antigenic sites than proteins in 20d extracts. The 20d protein shows a reaction of identity with purified newborn fibrous protein. Immunofluorescence studies on fetal skin support the prescence of cross-reacting components at 16d development related to the newborn fibrous protein. Intensity of fluorescence increases at 18d and 20d in the spinous and granular cell cytoplasm and in the keratohyaline granules. The stratum corneum, first seen at 20d, is intensely fluorescent. The cellular localization and time of appearance of the cross-reactive proteins suggest that they may be associated with tonofilaments.

Animals↗
PubMed1976

Mechanism of maleic acid-induced glucosuria in dog kidney.

The multiple indicator-dilution technique in vivo and isolated brush-border membranes in vitro have been used to explore the mechanism of maleic acid-induced glucosuria in dog kidney. The interaction of D-glucose with the antiluminal membrane from the peritubular fluid surface is unaltered. It is demonstrated that alpha-methyl-D-glucoside (alpha MG) enters and exits from the proximal tubular cell only across the brush-border membrane. Then using alphaMG as a reference indicator, it is shown that maleic acid does not cause complete inhibition of D-glucose interaction with the antiluminal membrane from the cytoplasmic surface. The binding of [3H]phlorizin both in vivo and in vitro is not affected by prior administration of maleic acid, indicating that D-glucose interaction with the outside surface of the brush border is also not affected by maleic acid. The data are therefore consistent with the concept that maleic acid-induced glucosuria is due either to i) partial inhibition of D-glucose movement from cytoplasm across the antiluminal membrane into the blood, ii) stimulated movement back across the brush-border membrane into urine, or iii) a combination of the two effects.

Adenosine Triphosphatases↗
PubMed1975

Purification and characterization of shikimate kinase enzyme activity in Bacillus subtilis.

In Bacillus subtilis shikimate kinase enzyme activity can be demonstrated when a small polypeptide forms a trifunctional complex with the bifunctional enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase-chorismate mutase. The shikimate kinase polypeptide whoch carries the catalytic site has been purified to homogeneity by a five-step procedure. The skikimate kinase was determined to have a molecular weight of 10,000 by superfine Sephadex G-75 thin layer chromatography and by calculation of the minimum chemical molecular weight from its amino acid composition. This number corresponds closely to the molecular weight determined by the mobility of the protein following electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The enzyme aggregates with itself forming larger molecular weight proteins. Thes aggregational pattersn depend on protein concentration and sulfhydryl bridges. The enzyme activity is completely inhibited by EDTA and the requirement for Mg2+ can be partially replaced by Mn2+, Ca2+, and Co2+. The inhibition of shikimate kinase activity by p-hydroxymercuribenzoate is reversed completely when the enzyme complex is treated with dithiothreitol, suggesting the sulfhydryl groups may be involved with the active site. The trifunctional complex is relatively unstable, and the nonidentical subunits dissociate readily. This dissociation results in a 99% loss in shikimate kinase activity and a 30% decrease in the chorismate mutase-DAHP synthetase activities. Shikimate kinase activity is subject to a variety of controls. It is inhibited by the allosteric effectors chorismate and prephenate, the products of the reaction, ADP, and shikimate 5-phosphate. The activity responds to changes in the energy charge of the cell. Because of the variety of controls exerted on this enzyme, this member of the regulatory complex may represent the key enzyme in the allosteric control of the synthesis of the common precursors of aromatic acid synthesis.

Adenosine Diphosphate↗
PubMed1975

Interaction of phospholipid vesicles with cultured mammalial cells. I. Characteristics of uptake.

The interaction of monolayer cultures of Chinese hamster V79 cells with artificially generated, unilamellar lipid vesicles (approximately 500 A diameter) was examined. Vesicles prepared from a variety of natural and synthetic radiolabeled phosphatidyl cholines (lecithins) were incubated with V79 cells bathed in a simple balanced salt solution. After incubation, the cells were analyzed for exogenous lipid incorporation. Large quantities (approximately 10(8) molecules/cell/h) of lecithin became cell associated without affecting cell viability. The effects of pH, charged lipids, and the influence of the vesicle lipid phase transition on the uptake process were examined. Glutaraldehyde fixation of cells before vesicle treatment, or incubation in the presence of metabolic inhibitors, failed to reduce the lecithin uptake by more than 25-50%, suggesting that the lipid uptake is largely energy independent. Cells in sparse culture took up about ten times more lipid than dense cultures. Prolonged incubation (greater than 15 h) of sparse cell cultures with lecithin vesicles resulted in significant cell death while no deleterious effect was found in dense cultures, or with 1:1 lecithin/cholesterol vesicles. When vesicle-treated cells were homogenized and fractionated, about 20-30% of the exogenous lipid was found in the plasma membrane fraction, with the remainder being distributed into intracellular fractions. Electron microscope radioautography further demonstrated that most of the internalized lipid was present in the cytoplasm, with little in the nucleus. These results are discussed in terms of possible modification of cell behavior by lipid vesicle treatment.

Azides↗
PubMed1975

Interaction of phospholipid vesicles with cultured mammalian cells. II. Studies of mechanism.

The mechanism of interaction of artificially generated lipid vesicles (approximately 500 A diameter) with Chinese hamster V79 cells bathed in a simple balanced salt solution was investigated. The major pathways of exogenous lipid incorporation in vesicle-treated cells are vesicle-cell fusion and vesicle-cell lipid exchange. At 37 degrees C, the fusion process is dominant, while at 2 degrees C or with energy depleted cells, exchange of lipids between vesicles and cells is important. The fusion mechanism was demonstrated using vesicles of [14C]lecithin containing trapped [13H]inulin. Consistent with a fusion hypothesis, both components became cell associated at 37 degrees C in nearly the same proportions as they were present in the applied vesicles. Additional arguments in favor of vesicle-cell fusion and against phagocytosis or adsorption of intact vesicles are presented. At 2 degrees C or with inhibitor-treated cells, the [3H]inulin uptake was largely suppressed, while the lipid uptake was reduced to a lesser extent. Evidence for vesicle-cell lipid exchange was obtained using V79 cells grown on 3H precursors for cellular lipids. [14C]lecithin vesicles, incubated with such cells, showed no change in their elution properties when subjected to molecular sieve chromatography on Sepharose 4B. However, radioactivity and thin-layer chromatographic analyses revealed that a variety of cell lipiids had been exchanged into the uniamellar vesicles. Further evidence for the fusion and exchange processes was obtained using vesicles prepared from mixtures of [3H]lecithin and [14C]cholesterol. A two-step fusion mechanism consistent with the present findings is proposed as a working model for other fusion studies.

Azides↗