Cytotoxic activity of lymphocytes. VIII. Lymphotoxin activity in cell-free extracts of activated lymphocytes.

Cell-free extracts of human lymphocytes activated by PHA contain a cytotoxin that kills mouse L cells. Such cells elaborate two different kinds of toxins into the culture supernatant fluids, called α- and β-lymphotoxin (-LT), which differ in size, stability, and antigenicity. The amount of intracellular toxin is 1 to 24% of that found in supernatants of different tonsil donors. Equal amounts of intracellular toxin appear in both microsomal fraction (100,000g pellet) and soluble supernatant fractions of the cell-free extracts (CFE). The toxin can be solubilized from the membrane by digestion with papain or extraction with a nonionic detergent, but not by repeated sonication. The molecular weight of both the microsomal and soluble cellular cytotoxin is 45,000 ± 5000. The intracellular toxin differs from the extracellular toxins secreted by the same cells in two major characteristics: one, although its size approximates that of supernatant β-LT (and is smaller than the 76,000 M_r α-LT), antibody-inhibiting α-LT but not β-LT inhibits both the microsomal and soluble CFE-LT. Two, the intracellular LT does not display the charge heterogeneity so characteristic of supernatant α-LT. Supernatant α-LT and CFE-LT are similar in their patterns of inactivation by heating to 80 °C and treatments with sodium dodecylsulfate (SDS), guanidine, proteases, and heavy metal ions, and are similarly unaffected by treatment with 8 M urea, N-ethylmaleimide, and sodium periodate. These results suggest that the single polypeptide intracellular LT is the precursor of the more complex secreted α-LT molecule.     

Characterization of β-Lactotensin,a Bioactive Peptide Derived from Bovine β-Lactoglobulin,as a Neurotensin Agonist

β-Lactotensin (β-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine β-lactoglobulin. The ileum-contracting activity of β-LT was blocked by the NT₁ antagonist SR48692. β-LT was selective for the neurotensin NT₂ receptor while neurotensin was selective for the NT₁ receptor. β-LT is the first natural ligand showing selectivity for the NT₂ receptor. β-LT showed hypertensive activity after intravenous administration at a dose of 30 mg/kg in conscious rats, while neurotensin showed hypotensive activity. The hypertensive activity of β-LT was blocked by levocabastine (1 mg/kg, i.v.), an NT₂ antagonist. SR48692, which blocked the hypotensive activity of neurotensin, had no effect on the hypertensive activity of β-LT. These results suggest that the hypertensive activity of β-LT is mediated by the NT₂ receptor. It was concluded that the NT₁ and NT₂ receptors mediate the opposite effect on blood pressure.     

Elaboration of lymphotoxin by freshly isolated human T lymphocytes and continuous lymphoid-cell lines.

Freshly isolated human T lymphocytes were demonstrated to produce lymphotoxin (LT) after mitogenic stimulation with phytohemagglutinin (PHA). In contrast, freshly isolated B lymphocytes, stimulated with two B-cell mitogens [pokeweed mitogen (PWM) and Staphylococcus protein A (Staph A)] did not produce the lymphokine, although thymidine incorporation was increased in these cells. We also examined a series of nine continuous human lymphoid-cell lines with B-cell markers and observed the spontaneous release of either large or small amounts of cytotoxin, or none at all. Cytotoxin from one of the productive cell-lines (H4218) was compared in detail with that obtained from PHA-stimulated, freshly isolated human lymphocytes. The behavior of the two cytotoxins was found to be identical in respect to migration on polyacrylamide gel, neutralization with rabbit anti-human α-LT serum, ultracentrifugation on 5–30% sucrose gradients, and stability for 15 min at 75 °C. Observation of these identical parameters strongly suggests that the α-LT elaborated by PHA-stimulated, freshly isolated human lymphoid cells is the same as the cytotoxin obtained from the continuous human lymphoid-cell line H4218. Thus α-LT may also be produced in quantity from continuous lymphoid-cell lines by mass tissue-culture techniques, which are more readily applicable to large-scale production than is purification from freshly cultured human lymphoid tissues. Notably, in cultures of freshly isolated human lymphoid cells, only T cells, and not B cells, generated lymphotoxin. However, continuous human lymphoid-cell lines with B-cell markers can also secrete this lymphokine.     

Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase.     

Identification of membrane-associated lymphotoxin (LT) on mitogen-activated human lymphocytes using heterologous anti-LT antisera in vitro.

Surface-associated lymphotoxin (LT) molecules have been identified on mitogen-activated human lymphocytes employing heterologous anti-α-LT serum in vitro. These membrane-associated LT molecules are present on PHA- or Con A-activated lymphocytes but do not appear to be expressed on unstimulated cells. Furthermore, these molecules were detected primarily on activated T lymphocytes, with little detectable on activated B- or null-cell populations. The removal of surface LT-bearing lymphocytes, using anti-α-LT serum + C′, does not dramatically affect the capacity of the remaining cells to release LT after mitogen restimulation. In addition, the presence of toxic molecules on the surface of activated lymphocytes suggests that these materials may be expressed in an inactive, noncytotoxic form.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. III. Interferon and cytotoxin induced by the specific antigen as compared with those induced by bacterial lipopolysaccharide

The time course of development and decline of the ability of BCG-infected mice to produce interferon in the serum in response to the intravenous infection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG-infected mice after stimulation with PPD. The duration of the period in which cytotoxin-production responsiveness to PPD was definitely detectable was much shorter than that for interferon-production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG-infected mice after stimulation with PPD did not parallel that of release of cytotoxin. The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG-infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti-L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG-infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.     

Snake toxin secondary structure predictions. Structure activity relationships

Modified Chou &; Fasman (1974a,b) secondary structure prediction rules have been successfully applied to the 57 snake venom toxins described as being neurotoxic or cytotoxic. Despite the different toxicities involved, a common distribution of secondary structure was detected throughout these toxins. The results also highlight the contrasts between short and long neurotoxins, and neurotoxins and cytotoxins. From comparisons of the typical structure of each toxin group with the known X-ray data an erabutoxin b and Philippines sea-snake toxin b, regions that dictate neurotoxicity or cytotoxicity can be tentatively identified. These deductions are discussed with regard to known chemical properties of these molecules. Similarly, the relevance of the differences between short and long neurotoxins to the superior binding of the latter to the cholinergic receptor is considered.It appears that the cytotoxins and neurotoxins are variations on a central toxic theme, but have differing specificities, whose origin can be traced to certain regions of the toxin in question.     

Feedback repression of polyamine uptake into mammalian cells requires active protein synthesis.

Two mammalian cell lines, rat hepatoma (HTC) and Chinese hamster ovary (CHO), were fed 10 to 50 microM spermidine while changes were monitored in intracellular polyamine levels and spermidine uptake activity. Normal feedback control preventing excessive polyamine uptake was found to be completely blocked by the addition of inhibitors of protein synthesis at the time of polyamine exposure. Under these conditions the cells accumulated abnormally high, toxic concentrations of spermidine. Further, continuous protein synthesis was needed to maintain repression of polyamine transporter proteins that had been inhibited previously by normal or elevated intracellular polyamines. These results suggest that a major factor in the regulation of polyamine uptake is the rapid, reversible inactivation of existing polyamine carrier molecules by an unstable protein whose synthesis is stimulated by intracellular polyamines.     

Decay of messenger ribonucleic acid from the lactose operon of Escherichia coli as a function of growth temperature

The inactivation rates of the first, β-galactosidase, and last, transacetylase, messages of the lactose operon of Escherichia coli were measured at different growth temperatures. The inactivation rate of each message appears to increase exponentially with temperature. The rate constant for this increase is almost twice as high for transacetylase message as it is for β-galactosidase message. The inactivation rate is more a direct function of growth temperature than of growth rate. At 15 °C transacetylase message is inactivated about 2.5 times more slowly than is β-galactosidase message. This difference is not paralleled by a different rate of chemical loss of the β-galactosidase message compared to the distal lac mRNA; all parts of the molecule appear to be lost at the same rate. This same pattern is observed in decay of the total mRNA; loss of capacity to direct peptide synthesis (functional inactivation) occurs at variable rates whereas loss of mRNA mass (chemical degradation) seems to occur at a uniform rate.We conclude that each message has a unique target for inactivation with a specifie temperature coefficient of sensitivity, and the inactivation of a message need not be associated with chemical destruction of the molecule.     

Steroid hormone inactivation is required during the juvenile-adult transition in Drosophila

Steroid hormones are systemic signaling molecules that regulate juvenile-adult transitions in both insects and mammals. In insects, pulses of the steroid hormone 20-hydroxyecdysone (20E) are generated by increased biosynthesis followed by inactivation/clearance. Although mechanisms that control 20E synthesis have received considerable recent attention, the physiological significance of 20E inactivation remains largely unknown. We show that the cytochrome P450 Cyp18a1 lowers 20E titer during the Drosophila prepupal to pupal transition. Furthermore, this reduction of 20E levels is a prerequisite to induce βFTZ-F1, a key factor in the genetic hierarchy that controls early metamorphosis. Resupplying βFTZ-F1 rescues Cyp18a1-deficient prepupae. Because Cyp18a1 is 20E-inducible, it appears that the increased production of steroid is responsible for its eventual decline, thereby generating the regulatory pulse required for proper temporal progression of metamorphosis. The coupling of hormone clearance to βFTZ-F1 expression suggests a general mechanism by which transient signaling drives unidirectional progression through a multistep process.     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).     

Primer-mediated inhibition of the hydrolysis of template DNA by T5-induced DNA polymerase.

Bacteriophage T5-induced DNA polymerase has an associated 3′→5′ exonuclease activity for which both single-stranded and duplex DNA serve as substrate (1). In this report, we demonstrate that hydrolysis of single-stranded DNA homopolymers (template) is inhibited in the presence of complementary (Watson-Crick sense) oligonucleotides (primer). Almost complete inhibition is observed at a primer/template ratio of ? 0.1. Formation of “H-bonded” primer-template complex seems to be necessary for the inhibition of template hydrolysis because (a) similar amounts of noncomplementary oligonucleotides have no detectable effect on the rate of template hydrolysis, and (b) complementary oligonucleotides lose their inhibitory potential at temperatures where the H-bonded primer-template complex is expected to be unstable. From our data, it appears that the inhibition of template hydrolysis in the presence of primer molecules is due to the preferential binding of the enzyme at the 3′-OH terminus of the primer in the primer-template complex.     

Aminoguanidine-mediated inactivation and alteration of neuronal nitric-oxide synthase

It is established that aminoguanidine (AG) is a metabolism-based inactivator of the three major isoforms of nitric-oxide synthase. AG is thought to be of potential use in diseases, such as diabetes, where pathological overproduction of NO is implicated. We show here that during the inactivation of neuronal nitric-oxide synthase (nNOS) by AG that the prosthetic heme is altered, in part, to dissociable and protein-bound adducts. The protein-bound heme adduct is the result of cross-linking of the heme to residues in the oxygenase domain of nNOS. The dissociable heme product is unstable and reverts back to heme upon isolation. The alteration of the heme is concomitant with the loss in the ability to form the ferrous-CO complex of nNOS and accounts for at least two-thirds of the activity loss. Studies with [(14)C]AG indicate that alteration of the protein, in part on the reductase domain of nNOS, also occurs but at low levels. Thus, heme alteration appears to be the major cause of nNOS inactivation. The elucidation of the mechanism of inactivation of nNOS will likely lead to a better understanding of the in vivo effects of NOS inhibitors such as AG.     

Maltosylamine, a specific inhibitor of beta-amylase.

β-Maltosylamine has been synthesized for the first time. It is an effective specific inhibitor of sweet potato β-amylase. This result extends the observation that 1-aminoglycosides are specific inhibitors of exoglycosidases which hydrolyze the corresponding glycose and also demonstrates that an enzyme acting with inversion, as well as those acting with retention of anomeric configuration, can be inhibited by glycosylamines. Maltosylamine, which acts as a reversible inhibitor of β-amylase, appears to be directed to the active site since it protects the essential sulfhydryl group of the enzyme from inactivation by N-ethylmaleimide.     

Occurrence of unstable PEP carboxylase in C4 grasses in the genus Spartina Schreb.

Abstract The aims of this work were to discover the distribution within the C₄ grass Spartina anglica of a PEP carboxylase which is very unstable during and after extraction, and to determine whether this unstable form occurs in other members of the genus. In S. anglica, only the leaf contains an unstable PEP carboxylase. Within the leaf only the major one of two isoenzymes is unstable, and this is located in the mesophyll cells. The unstable isoenzyme is inactivated during extraction and storage unless protected by bovine serum albumin or Triton X-100, and is inactivated in assay mixtures at optimum pH in the absence of PEP. Evidence is presented that inactivation is not due to degradation or inhibition during extraction and storage. The enzyme from leaves of Spartina species taxonomically closely related to S. anglica is also very unstable during and after extraction, but that from less closely related species is much more stable.     

Effects of temperature on the activity of cloacin DF13 and cloacin DF13-immunity protein.

During the interaction of cloacin DF13 and sensitive cells the cloacin molecules display different functions which can be distinguished on the basis of their heat-sensitivity. Binding to cell envelope receptors, binding of immunity protein and in vitro inactivation of ribosomes are heat-stable functions in contrast with the entire killing action in vivo. Cloacin DF13-immunity protein appears to be a heat-stable inhibitor of the fibosome inactivation caused by cloacin DF13.     

Effect of beta-lactotensin on acute stress and fear memory

β-Lactotensin (β-LT) is a bioactive peptide derived from bovine milk β-lactoglobulin and is a natural ligand for neurotensin receptors. We examined the effect of β-LT on restraint stress and fear memory in mice. Mice subjected to acute restraint stress exhibited a decreased number of head-dips and increased head-dip latency compared to non-stressed controls in the hole-board test, reflecting increased stress-induced behaviors. However, prior administration of β-LT improved the behaviors caused by stress. The anti-stress effect of β-LT was blocked by levocabastine, a neurotensin receptor subtype 2 (NTR2) antagonist. In the fear-conditioning test, the duration of freezing responses by cued fear conditioning was significantly reduced in mice administered β-LT compared with control mice. These results suggest that β-LT has an anti-stress effect and promotes the extinction of fear memory, which may be mediated by NTR2.     

The human LT system. II. Immunological relationships of LT molecules released by mitogen activated human lymphocytes in vitro.

Materials with LT activity present in supernatants from PHA stimulated human lymphocytes in vitro are very heterogeneous and can be separated into multiple molecular weight classes, termed complex, α, β, and γ. Several of these classes can be further resolved into subclasses by other physical and chemical methods. The immunologic relationships of these materials one to another were examined employing various rabbit anti-humn LT sera which will neutralize LT activity on L-929 cells in vitro. These studies reveal: (a) LT activities are due to a distinct group of substances which are immunologically related one to another and can exist in several molecular weight forms; (b) a high MW class of molecules, termed complex, appears to contain all currently known LT classes and subclasses; (c) LT classes and subclasses both have common (public) and discrete (private) antigenic specificities; (d) human LT classes and subclasses do not appear to share Ag determinants with materials with LT activity released by lectin stimulated lymphoid cells from rabbit, rat, hamster, guinea pig, or mouse; and (e) human LT molecules are not immunologically related to cell toxins released by glass adherent human peripheral blood monocytes or PMN cells. These data indicate human LT molecules form a “discrete system” of lymphocyte derived cell toxins, which can associate together into various related but different MW forms in the supernatant.     

Aggregation of sponge cells: a novel mechanism of controlled intercerllular adhesion

From the marine sponge Geodia cydonium a series of macromolecules have been isolated and characterized which are involved in the control of aggregation and separation of sponge cells; these include aggregation factor, aggregation receptor, anti-aggregation receptor, β-glucuronidase, β-glucuronosyltransferase, β-galactosyltransferase, β-galactosidase and a lectin. These components might be linked in the following sequence: (a) activation of the aggregation receptor by its enzymic glucuronylaion; (b) adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated β-glucuronidase; (d) cell separation due either to the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is probably controlled by the Geodia lectin.