Characterization of the sperm receptor on the surface of eggs of Strongylocentrotus purpuratus

Eggs of the sea urchins Strongylocentrotus purpuratus and Arbacia punctulata bind sperm with a high degree of species specificity. By use of an in vitro assay that utilizes bindin (the protein from sperm that mediates sperm-egg binding) egg surface-derived glycoconjugates that function as receptors in this adhesion process have been identified and purified. These glycoconjugates are of extraordinarily high molecular weight and exhibit some properties expected for a proteoglycan. The isolated receptors from both species bind to sperm and inhibit fertilization species specifically. Both receptors contain active carbohydrate-rich fragments that can be liberated by proteolytic digestion. The carbohydrate-rich receptor fragment from S. purpuratus is a very high-molecular-weight (>10⁶), negatively charged glycosaminoglycan-like polymer containing fucose, galactosamine, iduronic acid, and sulfate esters. By contrast, the carbohydrate-rich fragment derived from the A. punctulata receptor is of defined molecular weight (6000) and has no net charge. Incubation of acrosome-reacted sperm with nanomolar amounts of the carbohydrate-rich fragments from either species results in inhibition of fertilization, indicating that these receptor fragments retain sperm binding activity. However, studies utilizing heterologous gametes show that the carbohydrate-rich receptor fragments are not species specific in binding. Thus, it appears that although the carbohydrate chains of the receptor are an adhesive element of the receptor, the intact glycoconjugate is required for species-specific binding.     

A rapid enzymatic procedure for production of 1-beta-D-(3H)arabinofuranosyl cytosine 5'-triphosphate.

A rapid method of sequentially phosphorylating picomole quantities of [³H]-araC to [³H]araCTP is described (ara = 1-β-d-arabinofuranosyl). The procedure utilizes a system of phosphorylating enzymes isolated from rat spleen and requires a single incubation step. The [³H]araCTP product is isolated by ion-exchange chromatography and analyzed by PEI-cellulose thin-layer chromatography. At low concentrations of [³H]araC as much as 80% can be phosphorylated to the triphosphate, and the produet may be obtained in radiochemical purity greater than 97%.     

Exposure of dog thyroid slices to acetylcholine induces refractoriness to its subsequent stimulation of glucose oxidation

An initial incubation of dog thyroid slices with 0.1 or 1 microM acetylcholine (ACH) for at least 2 h decreases its subsequent stimulation of [1-14C]glucose oxidation. Refractoriness persists for as long as 6 h in the absence of ACH. While new protein synthesis is essential for recovery, it is not necessary for its induction. Refractoriness is prevented when 25 microM tropicamide, an atropine-like drug, is present from the beginning of the initial incubation, but not when it is added after 2 h of incubation of slices with ACH, indicating that at this time ACH is no longer necessary for refractoriness. During refractoriness induced by ACH, stimulation of glucose oxidation by thyroid-stimulating hormone, prostaglandin E1, dibutyryl cyclic AMP, and cholera toxin, but not menadiol, is also significantly diminished. Incubation of thyroid slices with ACH does not modify its stimulation of iodide organification or 32Pi incorporation into phospholipids. These results suggest that the desensitization is not due to changes in the ACH receptor but rather to intracellular metabolic effects. This phenomenon may be important in the regulation of cholinergic effects on the thyroid.     

Induction of delayed-type hypersensitivity to measles virus in mice

Intracutaneous injection of inactivated measles virus (MV) into hind footpads of BALB/c mice infected 5 to 11 days previously with MV produces a strong delayed-type hypersensitivity (DTH) response. Pretreatment of mice with cyclophosphamide (CP) results in a significantly stronger response. In CP-pretreated mice, the optimal infecting dose of live MV and the restimulating amount of inactivated MV are approximately 10(7) plaque-forming units and 2 micrograms/mouse, respectively. The optimal time after infection for measuring DTH to MV is 7 days, while the optimal CP-pretreatment concentration is 200 mg/kg. The DTH response generated by MV is specific and not caused by fetal calf serum or Vero cell antigens. MV DTH is transferable to uninfected mice with lymph node cells. Transfer of DTH is sensitive to treatment with anti-Thy 1.2 serum plus complement, indicating the response is T cell dependent. With this sensitive assay for measuring cell-mediated immunity to MV, it will now be possible to analyze T cell cross-reactivity among paramyxoviruses and assess viral cell-mediated immunity in mice infected with neuroadapted MV.     

Secondary delayed type hypersensitivity to sheep red blood cells in mice: Dependence on long-lived memory cells

Secondary delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is a long-lived memory phenomenon which is characterized by the accelerated reappearance of the state of DTH after a booster injection of the antigen. In this paper the nature of the DTH-related T memory cells accounting for secondary DTH was investigated. Parabiosis of primed and nonprimed mice for a period of 4 weeks resulted in an equally large secondary DTH responsiveness in both partners. This ability was maintained in both members for at least 6 months after termination of the parabiosis. These results indicate that (a) DTH-related T memory cells are potentially circulating cells, and (b) the persistence of these memory cells is not dependent on the presence of the antigen which induced their generation. Subcutaneous (sc) injection of intravenously (iv) primed mice with a small dose of antithymocyte serum before boosting did prevent the development of secondary DTH responsiveness in sc boosted mice, but not in iv boosted mice. Treatment of primed mice with vinblastine or azathioprine did not decrease the capacity of adoptive transfer of secondary DTH by means of spleen cells, but passive transfer of secondary DTH was completely abolished by this treatment. These results suggest that (a) SRBC-induced DTH-related T memory cells are nonproliferating, partially sessile, partially recirculating cells, and (b) these memory cells proliferate before they become DTH-related effector cells.     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

Antibody to immunoselected L-cell antigens mimics stimulating activity of antibody to whole L cells

A heterogeneous IgG antibody raised in rabbits in response to injections of whole L cells was used to identify and select relevant antigens in a nonionic detergent extract of L cells prelabeled with [35S]methionine by means of immunoprecipitation and immunoaffinity chromatography. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the immunoprecipitate and immunoeluate contained far fewer protein bands than the whole cell extract but selectively retained a 42,000-MW protein species. In response to injections of the immunoprecipitate, rabbits produced a new antiserum which reacted predominantly with the 42,000-MW protein when reacted with L-cell proteins separated by sodium dodecyl sulfate-gel electrophoresis and transferred to nitrocellulose paper by the Western blot technique. The new antiserum (raised to the immunoprecipitate) and the original antiserum (raised to whole cells) were equipotent in stimulating calcium transport, phospholipid metabolism, and DNA synthesis in L cells. Binding of the IgG fractions of the two antisera displayed identical high affinity binding to L-cell surface antigens, with the same average association constant of 1.5 X 10(6) M-1. These studies have shown that an antiserum raised to whole L cells has a much narrower reactive spectrum with L-cell membrane antigens than might be imagined and has identified a 42,000-MW membrane protein as an important immunogen which itself elicits a potent immune response resulting in an antibody capable of mimicking the cell stimulatory properties of the original antiserum.     

Methods of selective staining of polyacrylamide gels for -SH groups and cadmium

A new and very sensitive procedure for analysis of polyunsaturated phospholipids is described. The method involves resolution of lipids by high-performance thin-layer chromatography, conversion of lipids to a fluorescent derivative, and quantitation by densitometric scanning. The formation of the fluorophore is dependent on the presence of at least two double bonds in an acyl chain. As little as 20 pmol of phosphatidylinositol can be determined with a precision of better than 5%.     

Acute experimental autoimmune encephalomyelitis in mice. I. Adjuvant action of Bordetella pertussis is due to vasoactive amine sensitization and increased vascular permeability of the central nervous system

Experimental autoimmune encephalomyelitis (EAE) in mice is dependent upon the use of Bordetella pertussis suspensions as an adjuvant. Intravenous administration of B. pertussis causes an increased vascular permeability in brain tissue and an increased vascular sensitivity to vasoactive amines which promotes the development of EAE. The efficacy of different batches and strains of B. pertussis in the expression of EAE closely correlates with the vasoactive amine sensitization activity of each material tested. Pertussigen, the histamine sensitizing factor (HSF), is responsible for these adjuvant properties whereas purified endotoxin is inactive. The effect of cimetidine, diphenhydramine, methysergide, reserpine, and cyproheptadine on B. pertussis induced histamine sensitivity and the expression of EAE are examined. Cyproheptadine, an agent with mixed histamine and serotonin blocking properties, blocks both B. pertussis-induced vasoactive amine sensitization and the expression of EAE.     

Protein phosphorylation in anti-actin IgG and [(IgG)2protein A]2 complex-stimulated L cells

Phosphorylation of cellular proteins was stimulated in a dose-dependent manner by the surface binding of IgG antibodies to antigens on L cells. Most prominent among the phosphorylated cellular proteins were Mr = 115,000, 93,000, 58,000, 38,000, and 33,000 proteins. Stimulation of protein phosphorylation was maximal at 48 hr of incubation and was preceeded by maximal stimulated uridine incorporation into RNA (0-24 hr) and thymidine incorporation into DNA (24-48 hr), and followed by maximal stimulated cell proliferation occurring at 72 hr (P less than 0.001 for all differences). Modification of the ligand IgG molecule by formation of complexes with protein A (PA) altered the stimulation patterns of protein phosphorylation: [(IgG)2(PA)]2, Mr = 716,000, enhanced and (IgG)(PA), Mr = 200,000, inhibited phosphorylation. The nature of the cell surface antigen(s) was partially clarified by the demonstration that affinity-purified antibodies to cytoskeletal proteins (principally a surface actin molecule) accounted for a significant part of the stimulation effect. Thus, perturbation of the L-cell membrane by certain molecular forms of anti-actin IgG antibody produces a transmembrane signal resulting in an orderly series of metabolic events including enhanced protein phosphorylation at 48 hr occurring just prior to enhanced cell growth.     

Gap junction amplification in rat ovarian granulosa cells: I. A direct response to follicle-stimulating hormone

The ability of follicle-stimulating hormone (FSH), the estrogens, estradiol-17β and diethylstilbestrol, and the estrogen antagonists, clomiphene and enclomiphene citrate to affect the growth and internalization of hypophysectomized rat granulosa cell gap junction membranes was compared in ovarian follicles assigned to one of four follicle size classes (60–149, 150–249, 250–319, and 320–450 μm diameter). In the absence of exogenous hormone stimulation, atresia prevents follicle growth beyond 320 μm in diameter but surface gap junction membrane increases throughout this early follicle growth. Internalization of gap junction membrane is first detected at the 150- to 249-μm follicle stage and also increases with follicle size. Therefore, growth and turnover of gap junction membrane occur at a basal rate in the absence of gonadotropin or steroid hormone stimulation. Estrogen and estrogen antagonist injections result in no significant differences in the amount of surface or internalized junction membrane in the three smallest follicle size classes when compared to the untreated hypophysectomized animals. However, estrogen but not estrogen antagonists rescues growing follicles from atresia and permits their further growth into the 320- to 450-μm follicle size class. As a result of the additional follicle growth, both surface and internalized junction membrane increase beyond that seen in the largest follicles from hypophysectomized animals. In contrast to other treatments, FSH stimulation promotes amplification of gap junction membrane in all size classes and, like estrogen, rescues follicles from atresia and promotes their entry into the 320- to 450-μm follicle size class. Surface gap junction membrane is amplified two- to fourfold over other treatments in the first three follicle size classes, but reaches maximal levels in the 250- to 319-μm follicles. The internalized junction membrane which first appears in the 150- to 249-μm size class is dramatically increased over other treatments in the 250- to 319- and 320- to 450-μm size classes. These studies indicate that exogenous estrogen stimulation promotes gap junction growth indirectly by sustaining the basal rate of junction synthesis in follicles rescued from atresia. In contrast, exogenous FSH stimulation directly amplifies the developmental sequence of gap junction growth and turnover. During early follicle growth, FSH stimulation preferentially promotes increases in surface gap junctions while internalization of surface junctions is increased during later follicle growth.     

Prolyl hydroxylase activity during newt forelimb regeneration

Prolyl hydroxylase activity was measured to obtain insight into changes in collagen metabolism during forelimb regeneration in the adult newt, Notophthalmus viridescens. Activity increased markedly during the redifferentiative stages, remained elevated during digit formation, and decreased as regeneration neared completion. The greatest activity, seen during early digit formation, represents a 20-fold increase in activity over the level seen in nonregenerating limbs. The profile of enzyme activity correlates with the rate of collagen synthesis and the soluble collagen content of the regenerating tissue. Enzyme extracted from limbs in the early stages of regeneration can be activated in vitro upon preincubation with cofactors; however, preincubation has little, if any, effect on enzyme extracted from limbs at the later digit-forming stages.     

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.     

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.     

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.     

Nucleoside-specific suppression in MRL/MP +/+ mice

The induction of nucleoside-specific nonresponsiveness was further studied in the autoimmune strain MRL/MP +/+ (MRL/n). Experiments were undertaken to determine (i) whether nucleoside-conjugated spleen cells are able to induce specific nonresponsiveness to T-dependent nucleoside antigens in MRL/n mice, and (ii) whether periodic treatment with nucleoside-conjugated spleen cells would retard the development of spontaneous anti-DNA antibodies and associated indicators of autoimmunity. The results show that nonresponsiveness to nucleoside antigens is inducable in male, but not in female, MRL/n mice. Nonresponsiveness in male MRL/n was transferable and mediated by T cells. Treatment of male MRL/n mice with nucleoside-conjugated spleen cells (NSC) appeared to attenuate the progress of autoimmune symptoms in experimental animals. These results are discussed in the context of recent studies exploring the etiology of autoantibody production and the loss of self-tolerance in murine models of autoimmunity.     

Vitamin K-dependent carboxylase: Evidence for a semiquinone radical intermediate

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, PheLeuGluGluIle. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.     

Cytolytic T lymphocyte responses to metabolically inactivated stimulator cell. I. Metabolic inactivation impairs both CD and LD antigen signals

The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T_c) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T_c responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T_c precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T_c from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T_c. It is concluded that, in addition to the traditional view that these treatments damage the “LD” signal to helper T lymphocytes, metabolic inactivation also impairs recognition of “CD” determinants by precursor T_c.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Synthesis of a naphthylvinylpyridine derivative and its use for affinity chromatography of choline acetyltransferase

N-(10-carboxy)decamethylene-4(1-naphthylvinyl)pyridinium chloride, a derivative of the choline acetyltransferase (CAT) inhibitor naphthylvinylpyridine (NVP) was synthesized and used as a ligand for affinity chromatography of choline acetyltransferase. The preparation of this inhibitor included the quaternization of naphthylvinylpyridine with 11-Br-undecanoic acid methyl ester to obtain N-(10-carbomethoxy)decamethylene-4-(1-naphthylvinyl)pyridinium bromide, followed by hydrolysis to free the carboxylic group. This inhibitor (C11-NVP+) had a potency comparable to that of N-methyl-4(1-naphthylvinyl) pyridinium iodide (C1-NVP+) which is the most potent derivative of NVP but which lacks a functional group for conjugation to Sepharose. The C11-NVP+ was then bound through the carboxylic group to aminoalkyl Sepharose by a carbodiimide promoted condensation reaction. Interaction of CAT with the inhibitor retarded its elution from a column of Sepharose-C11-NVP+ and permitted the purification of the enzyme to electrophoretic homogeneity starting from a preparation in which CAT represented about 20% of the total proteins. Conventional procedures of protein purification had previously been unsuccessful in isolating the enzyme in pure form. Inhibition studies showed that CAT could exhibit either a "high" or a "low" sensitivity to inhibition by naphthylvinylpyridine and its derivatives (I50 with C1-NVP+ = 0.57 microM or 5.2 microM). A direct relationship existed between the sensitivity of CAT to these inhibitors and the retention of the enzyme by the affinity column.