A practically sensitive radioimmunoassay for cyclic CMP by 2'-O-acetylation

An improved method for the determination of subnanogram quantities of zinc has been devised using a tungsten filament for vaporization in a low-pressure microwave-induced helium plasma emission spectrometer. Desolvation and ashing in an air atmosphere of zinc containing samples in the presence of 1,10-phenanthroline (8 mm) and potassium chloride (3 mm) prevent fractional vaporization and yields a single, sharp emission signal. Analysis of nanogram quantities of zinc metalloenzymes contained in sample volumes of 5 μl illustrates the use of this method. The coefficient of variation for 0.14 ng of zinc in 75 ng of carboxypeptidase A is 3.5%, with a detection limit of 3 pg.     

Determination of picogram quantities of zinc in zinc metalloproteins by atomic absorption spectrometry using a graphite furnace atomizer

Optimum operating conditions have been determined for the atomization of zinc from metalloproteins in a graphite furnace. Addition of 50 mm ammonium dihydrogen phosphate to to protein and measurement of the integrated absorbance suppresses or eliminates matrix interference effects. Using a 5-μl sample both the sensitivity and the detection limit are 0.3 ng of Zn/ml, i.e., 1.5 pg of zinc on an absolute basis. For 10 ng/ml of zinc in 5-μl samples of a zinc metalloenzyme, the coefficient of variation is 1.5%. Accuracy has been established by analysis of zinc metalloenzymes of known zinc stoichiometry. The method has been applied successfully to the determination of zinc in several proteins for which zinc stoichiometry had been unknown.     

Mediator-induced macrophage activation, as shown by enhanced cytotoxicity for tumor, requires macrophage surface fucose and sialic acid

Macrophage-activating factor (MAF) activates macrophages so that their cytotoxic capacity is enhanced. This effect of MAF is inhibited by removing fucose from the macrophage cell surface by incubation with fucosidase, or by removing sialic acid by treatment with neuraminidase. After incubation with fucosidase or neuraminidase the average inhibition of cytotoxicity was 92 and 73%, respectively. β-Galactosidase had no effect. Addition of the specific products, fucose or sialic acid, to the incubation mixture of macrophages and enzyme blocked the effect of the enzymes. Taken together these observations indicate that macrophage surface fucose and sialic acid are essential for the interaction of MAF with macrophages which results in enhanced cytotoxicity for tumor cells.     

Glycolipid-dependent interaction between human migration-inhibitory factor and mononuclear phagocytes

It has been previously established in the guinea pig that the response of peritoneal macrophages to migration inhibitory factor (MIF) is enhanced by a macrophage glycolipid and that gangliosides reversibly bind MIF. This suggests that glycolipids function as cell surface receptors for MIF. In this report, it is demonstrated that the response of human peripheral blood monocytes to human MIF is augmented by preincubation of these cells with glycolipidenriched material extracted from the human macrophage-like cell line U937 or human peripheral blood monocytes and with a purified glycolipid from guinea pig peritoneal macrophages. In addition, a mixed ganglioside preparation from bovine brain shows the same effect. In contrast, the pure gangliosides, G_M1 and G_D1a, and glycolipids from the HL-60 cell line, which is a MIF-unresponsive cell line, were not able to enhance the response to human MIF. The specificity of enhancement by particular glycolipids could not be attributed to an increased uptake of only enhancing glycolipids since there was no significant difference between the association of monocytes with radioactive liposomes containing biologically active or inactive glycolipids. Pronase treatment did not affect the enhancing activity of the U937 glycolipidenriched material. Incubation of cells with glycolipids results in enhancement only if done at 37 °C and not at 4 °C. Therefore, the association of lipid with the monocyte surface appears to be dependent on temperature.Further evidence for the receptor nature of these enhancing glycolipids is provided by experiments involving affinity purification experiments. Coupling of bovine brain mixed gangliosides to agarose resulted in a matrix capable of reversibly binding MIF. G_D1a-agarose was inactive in this respect.     

Dissociation of human interferon-gamma-like activity from migration-inhibition factor

Supernatants harvested from concanavalin A-stimulated human peripheral mononuclear cells after 24 hr of incubation contain one interferon species similar to human interferon-gamma (IFN-γ) with a pI of 4.6–5.3 (first day pH 5 IFN-γ). In contrast, during the subsequent 24 hr of incubation two species with properties of IFN-γ are produced with pI of 3.6–4.0 (second day pH 4 IFN-γ) and 4.6–5.6 (second day pH 5 IFN-γ), respectively. First day pH 5 IFN-γ and second day pH 5 IFN-γ have been found to differ on the basis of trypsin sensitivity. This pattern of polymorphism is similar to the pattern previously described for human migration-inhibitory factor (MIF) which can be separated into first day pH 5 MIF, second day pH 3 MIF, and second day pH 5 MIF. However, IFN-γ-like species can be differentiated from MIF biochemically and antigenically. Fractions with second day pH 4 IFN-γ have no MIF activity and fractions with second day pH 3 MIF contain no IFN activity. In addition, first and second day pH 5 MIF, which also contain IFN-γ activity, can be separated from the latter by precipitation as well as neutralization with polyclonal and monoclonal anti-human MIF antibodies.     

Ontogeny of T-cell mitogen response in Lewis rats. III. Juvenile adherent suppressor cells block adult mitogen responses

Spleen cells from suckling female Lewis rats (4 to 20 days old) were able to suppress mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) of spleen or thymus cells from adult female Lewis rats and thymus cells from suckling Lewis rats. Thymus cells from suckling rats were unable to suppress adult spleen cell mitogenic responses to Con A. Removal of carbonyl iron (cFe)-, plastic-, or nylon-wool-adherent cells removed the suppressive action of juvenile spleen cells, but irradiation did not. Separated plastic-adherent spleen cells from suckling animals suppressed adult mitogenic responses to Con A. at optimal Con A doses 2-mercaptoethanol (2-ME, 2 X 10(-5) M) abolished the suppressive effect of juvenile cells, however, at the hyperoptimal dose of Con A (125 micrograms/ml) even higher doses of 2-ME did not relieve suppression by juvenile cells. These suppressor cells in suckling pups were affected by early weaning which decreased suppression, resulting in enhanced mitogenic responses of juvenile cells and removal of the ability to suppress adult mitogenic response.     

Further characterization of the putative glycolipid receptor for mif: Role of fucose associated with an acidic glycolipid

Water soluble glycolipids were extracted from guinea pig macrophages. These glycolipids, when incubated with macrophages, augment the cells' response to migration inhibitory factor. The glycolipids were fractionated by diethylaminoethyl-Sephadex ion exchange chromatography into neutral and acidic fractions. Only the acidic glycolipid fraction was able to enhance the responsiveness of macrophages to migration inhibitory factor. Additional studies indicate that the enhancing activity of these glycolipid preparations can be abrogated by the removal of terminal fucose residues with α-L-fucosidase. The possibility that fucose functions as an essential component of a macrophage glycolipid receptor for migration inhibitory factor is discussed.     

Characterization of an immune suppressor from transformed human trophoblastic JEG-3 cells

Cultured human choriocarcinoma JEG-3 cells secrete an immunosuppressor that inhibits lymphocyte proliferation stimulated by either an antigen or a mitogen. In this study, the immunosuppressive factor was characterized by three methods: ion-exchange and exclusion chromatography, partition in organic solvents, and thin-layer chromatography on silicic acid. This JEG-3 cell factor appeared to be a protein complex of about 150,000–200,000 Da that contained an immunologically active polar lipid. The structural and functional characteristics of JEG-3 cell immunosuppressor are similar if not identical to those of SIF, a suppressor lymphokine derived from T cells. These secretions from transformed trophoblastic cells may correspond to normal placental products or represent a function of malignant cells.     

Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca2+

Extracellular Ca2+ regulated the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear leukocytes (PMN) stimulated with N'-formyl-methionyl-leucyl-phenylalanine (FMLP) in the presence of cytochalasin B. Maximum PAF synthesis and release required the presence of 0.14 mM Ca2+ whereas 1.4 mM Ca2+ was necessary for maximum lysosomal enzyme secretion. The synthesis of PAF occurred within 2.5 min after PMN stimulation in the presence of 1.4 mM Ca2+; however, PAF release did not occur until 5 min after stimulation. Peak PAF release occurred by 7.5 min but accounted for only 30-40% of the total amount of PAF synthesized, the remainder being retained on or within the PMN. Stimulation of PMN in the presence of 0.01 M EDTA or EGTA decreased PAF synthesis and release by greater than 95%. In the absence of extracellular Ca2+, stimulated PMN synthesized PAF in amounts that were 10-30% of maximum, but there was no release of the newly synthesized PAF. At Ca2+ concentrations greater than 0.01 mM, there was a dose-dependent (up to 0.14 mM) increase in PAF synthesis that was associated with the initiation and concomitant increase in the amount of PAF released. These data suggest the presence of a PAF synthesis-release coupling mechanism in which the extracellular Ca2+-dependent release of PAF stimulates additional PAF synthesis.     

Reversal of macrophage-augmented MLR reactivity by tumor-induced splenic suppressor T cells and their soluble factor(s)

Augmenting concentrations of macrophages or their supernatants failed to reverse T-cell hyporeactivity in tumor-bearing mice (TBM). Serial passaging over nylon wool columns depleted TBM spleen cells of a mildly adherent tumor-induced suppressor cell and restored mixed lymphocyte reaction (MLR) reactivity to the purified TBM T-cell population. The tumor-induced suppressor cell was extensively plated to remove macrophages and characterized as a T cell by its anti-Thy 1 serum sensitivity. This suppressor T cell, when added to normal T cells, abrogated all enhancing effects caused by addition of macrophages. Suppressor T-cell inhibition was non-contact dependent, since suppressor T-cell supernatants inhibited MLR activity in T cells treated with enhancing concentrations of macrophage supernatants. Thus it appears that tumor-induced T-cell debilitation is a reversible phenomenon, mediated not by macrophages but by soluble factor(s) from a nonphagocytic, mildly adherent, suppressor T cell.     

Effect of atrial natriuretic factor (ANF)-related peptides on aldosterone secretion by adrenal glomerulosa cells: critical role of the intramolecular disulphide bond

We previously demonstrated that synthetic 48-73 atrial natriuretic factor (ANF) (previously called 8-33 ANF) blocked the response of rat adrenal glomerulosa cells to angiotensin II, ACTH and potassium. We have now investigated the effects of natural 43-73 ANF, oxidised synthetic 48-73 ANF and the natural 1-73 ANF on aldosterone output by rat glomerulosa cells. The natural 43-73 ANF and the natural 1-73 ANF were equipotent to 48-73 ANF in inhibiting the stimulation of aldosterone secretion produced by angiotensin II with an IC50 of 2 X 10(-9)M. Similar results were obtained with ACTH and potassium. After oxidation with performic acid, 48-73 ANF was completely devoid of activity on the response of aldosterone to angiotensin II, ACTH and potassium. We conclude that the intramolecular disulphide bond in 48-73 ANF is critical for maintaining the active conformation of ANF.     

A series of murine interleukin molecules which stimulate both murine and human lymphocytes: I. Production by Phytolacca americana (pokeweed) lectin 2 (Pa-2)-stimulated thymus and thymus-derived cells

Cell-free supernatant fluid, from cultures of Phytolacca americana (pokeweed) lectin 2 (Pa-2)-pulsed murine spleen or thymus cells, contains factors which induce cultured lymphocytes to differentiate into IgM-secreting cells (assayed by a reverse plaque technique) and to proliferate (measured by the incorporation of tritiated thymidine) without the addition of mitogen. The factors in this supernatant fluid responsible for these activities have been designated as lymphocyte stimulating factors (LSF). LSF showed no genetic restrictions related to the major histocompatability complex; LSF made in one strain of mice worked in other strains. Indeed, LSF is not restricted by species barriers; human peripheral blood mononuclear cells were also stimulated by murine LSF to proliferate and differentiate into immunoglobulin-secreting cells without further addition of antigen or mitogen. Maximum production of LSF was achieved within 12 hr of culture and was independent of cell division. In contrast to TRF, no further production of LSF was detectable after 24 hr of culture. Unlike T-cell growth factor, this material stimulated increased mitosis of thymic, T, and B lymphocytes without the addition of mitogen or antigen. LSF also stimulated polyclonal B-cell differentiation into IgM-secreting cells. Maximal numbers of immunoglobulin-secreting cells were generated when LSF was added at the initiation of the culture. Indeed, unlike TRF, LSF needed to be present only during the first 6 hr of culture to achieve maximum stimulation, and did not require the presence of antigen. The production of LSF by a T-cell population in the spleen was shown by two independent methods. Spleen cells treated with anti-Thy 1 plus complement failed to produce detectable levels of LSF. On the other hand, purified populations of surface immunoglobulin-negative spleen cells produced LSF. Furthermore, the subset of thymocytes responsible for LSF production was the small population (approximately 10%) of cells in the thymus, which are not agglutinated by peanut agglutinin.     

Influence of cortisone on immunologic tolerance in vivo: Cortisone prolongs the duration of unresponsiveness but fails to affect its induction

The influence of cortisone administration on either the induction or the duration of immunologic tolerance was examined in vivo. Tolerance induced by isologous IgG coupled to fluorescein was chosen because the hapten-bearing cell can be directly visualized and the hapten-specific immune response to either a TD antigen or a TI₂ antigen can be tested. It was found that cortisone facilitates the maintenance of tolerance, but fails to affect its induction to either class of antigen. Fluorescein-IgG-bearing cells are cortisone resistant. They are seen for a longer period of time in animals treated with cortisone and tolerogen than in animals treated with tolerogen, and fluorescent cells are either T or B cells. We propose that cortisone facilitates the maintenance of tolerance by maintaining a receptor blockade in vivo. This finding might have clinical implications for the treatment of autoimmunity.     

Nonspecific inhibitor of contact sensitivity made by T-acceptor cells: triggering of T cells armed with antigen-specific T-suppressor factor (TsF) requires both occupancy of the major histocompatibility complex recognition site by soluble I-J product and cross-linking of the antigen recognition sites of the TsF

The phenomenon of associative recognition, i.e., the recognition of antigen together with major histocompatibility complex products (MHC) was studied in a model system. T-acceptor cells armed with antigen-specific T-suppressor factor (TsF) released a nonspecific inhibitor of the transfer of contact sensitivity when exposed to antigen together with MHC. The MHC product occurred in a KCl extract of cells and behaved genetically and serologically as I-J. Cells armed with anti-picryl or anti-"oxazolone" TsF could be triggered by the corresponding "bis-picryl-L-lysine" and "bis-oxazolone-L-lysine" together with MHC. This suggested that cross-linking of antigen recognition sites on separate molecules of TsF might be required. To investigate this possibility the bifunctional "mixed" hapten "N alpha-picryl-N epsilon-oxazolone-L-lysine," which is univalent with respect to the picryl and oxazolone haptenic groups, was synthesized. This triggered cells armed with a mixture of anti-picryl and anti-oxazolone TsF but not cells armed with either TsF alone. It was concluded that both occupancy of the I-J recognition site and the cross-linking of separate molecules of TsF was required for triggering. Moreover the hapten and the KCl extract could be given sequentially and in either order. This finding suggested that the triggering of the release of nonspecific inhibitor was due to the separate recognition of I-J and antigen and not to new antigenic determinants produced by their interaction.     

Physiochemical characterization of lymphocyte inhibitory factor (LyIF) isolated from avian B and T cells

Thymic (T) or bursal (B) lymphocytes from chicks sensitized to Mycobacterium tuberculosis produce an avian lymphocyte inhibitory factor (LyIF). The physiochemical properties of both T and B LyIF were established by ultrafiltration which yielded four fractions with molecular weight ranges of greater than 100,000; 50,000-100,000; 10,000-50,000; and less than 10,000; enzymatic treatment with chymotrypsin and neuraminidase; varying pH; and heat exposure. These studies demonstrated that the maximum activity for both T and B LyIF was within a molecular weight range of 10,000-50,000. Both were sensitive to chymotrypsin and neuraminidase treatment. Both were stable at 56 degrees C for 30 min and resistant to changes in pH from 5 to 9. T-Cell migration was inhibited equally by B or T LyIF, while B-cell migration was inhibited to a lesser extent by T LyIF and B LyIF. Further experiments should establish the reasons for these observed differences in cross-reactivity.     

Conformational comparison of human pituitary growth hormone and human chorionic somatomammotropin (human placental lactogen) by second-order absorption spectroscopy

Second-order absorption spectra strongly suggest the presence of a hydrogen bond between the single Trp of human pituitary growth hormone (hGH) and a carboxylate ion. This hydrogen-bonded complex is buried within the hydrophobic interior of the hGH molecule. Although the homologous Trp in human chorionic somatomammotropin [human placental lactogen, HCS(hPL)] is also buried within the hydrophobic interior of the molecule, there is no evidence that it is hydrogen bonded in the native protein. However, during the early stages of thermolysin digestion of HCS(hPL), both difference and second-order absorption spectra do indicate the transient presence of a similar hydrogen-bonded Trp-carboxylate complex. The molar extinction coefficients of hGH and HCS(hPL) have been refined.     

Hapten-specific carrier-dependent tolerance induction in man in vitro

We sought to determine whether hapten-specific tolerance can be induced in cultured human lymphocytes in vitro. Unfractionated as well as T and B cells from peripheral blood lymphocytes of healthy human volunteers were cultured with different hapten-carrier conjugates before in vitro challenge with dinitrophenyl (DNP) linked to keyhole limpet hemocyanin. Hapten-specific antibody was detected in the supernatant by solid-phase radioimmunoassay. Both hapten specificity and carrier dependence in addition to the cellular basis of tolerance induction were examined. The results show that hapten-specific tolerance of antibody production was induced by human gamma-globulin (HGG) conjugated to DNP but not by other conjugates of DNP nonhuman gamma-globulin, as well as human serum albumin. Moreover, both T and B cells are involved in tolerance induction to DNP-HGG in vitro. The significance of tolerance in human in vitro for the specific therapy of autoimmune disease is discussed.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Inhibitory effect of a low dose of prednisone on PHA-induced Ia antigen expression by human T cells and on proliferation of T cells stimulated with autologous PHA-T cells

Administration of a small dose of prednisone markedly reduced (1) the PHA-induced expression of Ia antigens by T cells, (2) the stimulatory activity of Ia antigen-bearing T cells in autologous and allogeneic mixed lymphocyte reactions (MLRs), and (3) the proliferative response of T cells stimulated with autologous PHA-activated T cells or autologous or allogeneic non-T cells. The inhibitory effects of prednisone are reversible and are not detectable on T cells isolated from blood drawn 24 hr following prednisone administration. The kinetics of the prednisone-mediated inhibition of MLRs with autologous PHA-T cells is different from that of MLRs with autologous non-T cells. These data in conjunction with the information available in the literature suggest that the mechanisms underlying these two types of autologous MLRs are different.     

Degradation of poly(adenosine diphosphate ribose) by homogenates of various normal tissues and tumors of rats.

The extent of increase in the activity of phenylalanine ammonia-lyase upon illumination for 15 hr of cultured Petroselinum hortense cells was greatly dependent upon the age of the culture. Two distinct peaks in specific activity were observed during the growth cycle, one occurring at the beginning and the other at the end of the period of increase in cell fresh weight. High yields in both cell fresh weight and enzyme activity were obtained with the second peak shortly before the stationary phase of the culture was reached. Cells were harvested at this stage and stored at ?20 °C.The enzyme was purified from the frozen cells to apparent homogeneity by precipitation with (NH₄)₂SO₄, chromatography on DEAE-cellulose, Sephadex G-200 and hydroxyapatite columns, and preparative polyacrylamide gel electrophoresis. An over-all yield of 16% was achieved with a 440-fold increase in the specific activity by purification of the enzyme through the hydroxyapatite step.Upon analytical polyacrylamide gel electrophoresis either in the presence or in the absence of sodium dodecyl sulfate, the purified protein migrated essentially as a single band. Molecular weights of about 330,000 and 83,000, respectively, were estimated for the enzyme and for its protein subunits. Thus, the enzyme molecule seems to be composed of four probably identical protein subunits. Two Michaelis constants for l-phenylalanine (K_m^L, 3.2 × 10^?5 M, and K_m^H, 2.4 × 10^?4 M) and a Hill coefficient of h = 0.6 were obtained. This suggests that the enzyme is subject to regulation of its catalytic properties by negative cooperativity of the protein subunits.