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Biomedical subjects

Y K Yip

Publications and source records attributed to Y K Yip.

At least 37 records · Page 2Linked to original sources
PubMed1983

Human interferon-gamma is internalized and degraded by cultured fibroblasts.

Human interferon-gamma (IFN-gamma) binds specifically and with high affinity to receptors on the surface of cultured fibroblasts (GM-258). At 37 degrees C about 50% of the receptor-bound IFN-gamma was rapidly internalized (t 1/2 = 4-5 min) by these cells. Following an initial lag of 15-30 min, internalized IFN-gamma was continuously degraded over a period of at least 8 h. The total uptake of IFN-gamma over this time period was found to exceed by 5 times the number of occupied IFN receptors present on the surface of these cells, suggesting that either there is a large intracellular pool of IFN-gamma receptors, or that receptors are recycled during the course of incubation. Cycloheximide (100 micrograms/ml) inhibited uptake only after the first 2 h of incubation and then only moderately. It is therefore unlikely that de novo receptor synthesis plays a major role in the observed uptake process. Both sodium azide (15 mM) and methylamine (20 mM) inhibited both the uptake and degradation of IFN-gamma at all times up to 6 h. While uptake was only slightly reduced in the presence of chloroquine (25 microM), degradation was markedly inhibited, suggesting that degradation occurs intracellularly, probably within lysosomes.

Azides↗
PubMed1983

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon.

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.

Antigens, Surface↗
PubMed1982

Specific binding of 125I-human interferon-gamma to high affinity receptors on human fibroblasts.

Highly purified human interferon-gamma (IFN-gamma) was iodinated with 125I-Bolton-Hunter reagent to a specific activity of 34 microCi/micrograms of protein. After iodination, molecular sieve chromatography was used to isolate fractions containing IFN-gamma at approximately 80-90% radioactive purity. This preparation of 125I-IFN-gamma bound specifically to high affinity binding sites on human GM-258 fibroblasts. Scatchard analysis of binding data revealed the presence of 2400 binding sites/cell, each binding with an apparent Kd of 1.5 X 10(-10) M. Binding was competitively inhibited in the presence of unlabeled IFN-gamma and, to a lesser degree, by IFN-beta, but not by IFN-alpha. Neutralizing antibodies specific for IFN-gamma efficiently inhibited the specific binding of 125I-IFN-gamma to cells. We conclude that the specific high affinity binding site is the receptor for IFN-gamma.

Cell Line↗
PubMed1982

Molecular weight of human gamma interferon is similar to that of other human interferons.

The molecular weight (as determined by molecular sieve chromatography) of human gamma interferon, formerly referred to as immune or type II interferon, is between 40,000 and 70,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gamma interferon activity was recovered mainly from two regions of the gels corresponding to molecular weights of 20,000 and 25,000. The results suggest that in native form human gamma interferon may be aggregated.

Glycoproteins↗
PubMed1982

Purification of two subspecies of human gamma (immune) interferon.

Interferon (IFN)-gamma was produced in cultures of human leukocytes by combined stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) and phytohemagglutinin (PHA). IFN-gamma was purified by sequential adsorption and elution from controlled-pore glass and concanavalin A-Sepharose and by subsequent adsorptive removal of contaminating proteins on DEAE-Sephacel at pH 8.0. Treatment of such partially purified IFN-gamma preparations with the anionic detergent NaDodSO4 (0.1% at 20-25 degrees C) decreased biological activity to approximately 5-20%. When analyzed by NaDodSO4/polyacrylamide gel electrophoresis the bulk of IFN activity not destroyed by NaDodSO4 treatment was recovered from two peaks with apparent molecular weights of 20,000 and 25,000. The two activity peaks showed close correspondence with Coomassie blue-stained bands regularly demonstrable in purified supernatants from induced cultures but absent from culture supernatants from uninduced cells. Available evidence suggests that the two bands, isolated in pure form, represent subspecies of IFN-gamma. Native IFN-gamma was found to have a lower affinity for alkyl agarose columns than human IFN-alpha or IFN-beta did, suggesting that IFN-gamma is a relatively hydrophilic protein. Sulfhydryl-specific binding of native IFN-gamma to an Affi-Gel 501 column suggested that this IFN contains free sulfhydryl.

Humans↗
PubMed1981

Partial purification and characterization of human gamma (immune) interferon.

Human gamma (immune) interferon (IFN-gamma) was produced in lymphocyte cultures stimulated with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) and purified phytohemagglutinin. Physicochemical analysis showed that human IFN-gamma is a glycoprotein with an isoelectric point around 8.6 and an apparent molecular weight of 58,000 +/- 3000. A purification process for IFN-gamma was developed consisting of sequential chromatographic separations on controlled-pore glass, concanavalin A-Sepharose, and Bio-Gel P-200. This purification process resulted in an increase in specific activity from about 10(4) (crude culture fluid) to an estimated 10(7) units per mg of protein with a cumulative recovery of about 40% of the IFN activity.

Chromatography, Affinity↗
PubMed1981

Partial characterization of gamma (immune) interferon mRNA extracted from human lymphocytes.

gamma (immune) interferon (IFN-gamma) was induced in cultures of fresh human lymphocytes by combined treatment with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA). Compared to the IFN-gamma yields obtained with PHA induction alone, the inclusion of TPA caused a significant enhancement of IFN-gamma production. Poly(A)-containing mRNA was isolated from mononuclear cells induced with TPA and PHA. Injected into Xenopus laevis oocytes, this mRNA preparation gave rise to IFN activity with characteristic properties of human IFN-gamma. Sucrose density gradient centrifugation analysis showed that IFN-gamma mRNA sedimented at 15 S, suggesting that it contains a total of about 1400 nucleotides.

Animals↗
PubMed1981

Stimulation of human gamma interferon production by diterpene esters.

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.

Cell Separation↗
PubMed1980

Pharmacokinetic properties of human fibroblast and leukocyte interferon in rabbits.

When rabbits were given intramuscular injections of the same quantities of human leukocyte or fibroblast interferons, the former produced moderately higher levels of circulating interferon. Fibroblast interferon was not cleared faster from circulation, nor was direct inactivation by rabbit blood responsible for this difference.

Animals↗
PubMed1978

Characteristics of human lymphoblastoid (Namalva) interferon.

Interferon derived from the human lymphoblastoid cell line, Namalva, was fractionated by antibody affinity chromatography into two antigenically distinct interferon subspecies. At least 13% of the total Namalva interferon activity possessed the F antigenic determinant found on human interferon derived from fibroblast cultures, while the bulk of the Namalva interferon activity had the Le antigenic determinant characteristic for human leukocyte interferon. The separated Le and F subspecies of Namalva interferon differed in the degree of their heterospecific activities on bovine cells. The Le moiety resembled crude leukocyte interferon in that it was highly active in bovine cells. The F component of Namalva interferon showed a lower degree of activity in bovine cells, thus resembling crude fibroblast interferon. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by isoelectric focusing, crude Namalva interferon qualitatively resembled crude leukocyte interferon.

Burkitt Lymphoma↗
PubMed1977

Reconstitution of native human hemoglobin from separated globin chains and alloplex intermediates.

A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free alpha- and beta-globin chains (alpha degrees, beta degrees) and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the allosteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at high dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For alpha-globin, the yield is about 50% because of incomplete combination with the heme-containing beta chain. For beta-globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.

Allosteric Site↗
PubMed1975

Studies on catalytic properties of purified high molecular weight pancreatic lipase.

The properties of the 500-fold purified high-molecular-weight lipase have been studied. The rate of hydrolysis of the triglycerides decreases with increasing fatty acid chain length. The lipolytic activity also increases with increase in unsaturation in the fatty acyl moiety. Diglycerides are hydrolyzed at more than twice the rate for triglycerides while monoglycerides are not hydrolyzed. Methyl esters are generally hydrolyzed at a higher rate which increases with increasing chain length of the fatty acid but the enzyme does not act on phospholipids. Emulsifying agents such as Tween 20, gum arabic, and albumin increase the rate of hydrolysis. Metal ions such as Hg2+, Zn2+, Cu2+, and Fe2+ strongly inhibit the lipolytic activity of the high-molecular-weight lipase while Ca2+ or Mg2+ by themselves show no stimulating effect. Treatment of the high-molecular-weight lipase with P-chloromercurybenzoate inhibits hydrolytic activity by 70% while iodoacetic acid has no effect.

Albumins↗