Transient modulation and internalization of T4 antigen induced by phorbol esters

Phorbol esters are known to alter the expression of surface antigens and receptors on a variety of mammalian cell types. On T lymphoblastoid cell lines and peripheral blood T cells, phorbol esters have been shown to selectively reduce the expression of the T4 antigen. To more fully characterize this process, we have examined the metabolic requirements for this phorbol ester effect, and have evaluated the relationship between phorbol ester-induced T4 loss and the expression of receptors for phorbol-12,13-dibutyrate (PDB) on purified peripheral blood T4 cells. We observed that the loss of T4 on peripheral blood lymphocytes (PBL) occurred at PDB concentrations at which 10 to 15% of phorbol ester binding sites were occupied. The loss of T4 was inhibited at 4 degrees C, and by azide, methylamine, and sodium fluoride, but not by inhibitors of DNA synthesis. When cells were exposed to phorbol esters for greater than 2 days, the T4 antigen was again expressed on the cell surface despite the continued presence of phorbol esters. Cells which had recovered T4 were resistant to the effects of freshly added PDB on this antigen, and this resistance correlated with a 55% reduction in phorbol ester binding sites. Studies on fixed PBL T4 cells and MOLT-4 cells by immunofluorescence microscopy demonstrated that the decreased expression of T4 from the cell surface correlated with a bright clustering of T4 within the cytoplasm, indicating that PDB had induced an internalization of this antigen. These observations demonstrate that the binding of phorbol esters to specific receptors on lymphocytes initiates metabolically dependent events which result in the internalization of the T4 antigen. These findings may be relevant to mechanisms by which T4 functions as a signal-transducing molecule in vivo.     

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.     

Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.     

Effects of plant spinescence on large mammalian herbivores

Summary Plant thorns and spines had these effects on the feeding behaviour of the three species of browsing ungulate that we studied, kudu, impala and domestic goats: (i) bite sizes were restricted, in most cases to single leaves or leaf clusters; (ii) hooked thorns retarded biting rates; (iii) the acceptability of those plant species offering small leaf size in conjunction with prickles was lower, at least for the kudus, than those of other palatable plant species; (iv) the inhibitory effect of prickles on feeding was much less for the smaller impalas and goats than for the larger kudus; (v) from certain hook-thorned species the kudus bit off shoot ends despite their prickles; (vi) for certain straight-thorned species the kudus compensated partially for the slow eating rates obtained by extending their feeding durations per encounter. Most spinescent species were similar in their acceptability to the ungulates to unarmed palatable species, despite higher crude protein contents in their foliage than the latter. Such structural features furthermore reduce the tissue losses incurred by plants per encounter by a large ungulate herbivore, by restricting the eating rates that the animals obtain. In this way prickles function to restrict foliage losses to large herbivores below the levels that might otherwise occur.     

Nitrogen assimilation in field-grown winter wheat: Direct measurements of nitrate reduction in roots using15N

Summary Nitrate fertiliser labelled with¹⁵N was applied to a field grown crop of winter wheat. Uptake and assimilation of fertiliser nitrate was studied by monitoring the appearance of labelled nitrate and labelled amino acids in the xylem sap. Shortly after applying¹⁵N-nitrate to the soil about 30 per cent of recently absorbed¹⁵N was in the reduced form, indicating that roots of cereal crops can make a substantial contribution in reducing nitrate. Seasonal changes in crop growth andin vivo NRA are also described.     

Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii

Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.     

Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction

Denervation of vertebrate muscle causes an acceleration of acetylcholine receptor turnover at the neuromuscular junction. This acceleration reflects the composite behavior of two populations of receptors: "original receptors" present at the junction at the time of denervation, and "new receptors" inserted into the denervated junction to replace the original receptors as they are degraded (Levitt, T. A., and M. M. Salpeter, 1981, Nature (Lond.), 291:239-241). The present study examined the degradation rate of original receptors to determine whether reinnervation could reverse the effect of denervation. Sternomastoid muscles in adult mice were denervated by either cutting or crushing the nerve, and the nerves either allowed to regenerate or ligated to prevent regeneration. The original receptors were labeled with 125I-alpha-bungarotoxin at the time of denervation, and their degradation rate followed by gamma counting. We found that when the nerve was not allowed to regenerate, the degradation decreased from a t1/2 of approximately 8-10 d to one of approximately 3 d (as reported earlier for denervated original receptors) and remained at that half-life throughout the experiment (approximately 36 d). If the axons were allowed to regenerate (which occurred asynchronously between day 14 and day 30 after nerve cut and between day 7 and 13 after nerve crush), the accelerated degradation rate of the original receptors reverted to a t1/2 of approximately 8 d. Our data lead us to conclude that the effect of denervation on the degradation rate of original receptors can be reversed by reinnervating. The nerve can thus slow the degradation rate of receptors previously inserted into the postsynaptic membrane.     

Organization and structure of two mixed micellar phases of the sphingomyelin/Triton X-305 system

Properties of mixed dispersions of sphingomyelin and the nonionic detergent, Triton X-305, were investigated by analytical ultracentrifugation and by autocorrelation spectroscopy of scattered laser light. These properties were compared with those of the sphingomyelin/Triton X-100 mixed micellar system reported previously [S. Yedgar, Y. Barenholz, and V. G. Cooper (1974) Biochim. Biophys. Acta 363, 98-111]. The substitution of the 30-unit ethylene oxide chain of Triton X-305 for the 10-unit chain of the Triton X-100 resulted in the appearance of two micellar phases at all detergent/lipid mixture ratios studied, whereas only a single mixed micellar phase was observed using Triton X-100. Despite this difference, the properties of the mixed lipid/detergent micelles obtained using Triton X-100 have been verified in the following respects: The detergent aggregation numbers in the mixed micelles are quite constant over a wide range of detergent molar fractions, being about 70 and 400 for the lighter and heavier mixed micellar phases, respectively. The detergent aggregation numbers are larger in the mixed micelle than in the pure detergent micelle. Very large sphingomyelin aggregation numbers can be accommodated within the mixed micelles, apparently by the critical intervention of the detergent molecules to produce a stable micellar structure.     

Human lymphocyte differentiation antigens HB-10 and HB-11. II. Differential production of B cell growth and differentiation factors by distinct helper T cell subpopulations

Two monoclonal antibodies (HB-10 and HB-11), which react with human T, B, and NK cells, identify approximately 50% of the Leu-3+ T helper (TH) cells in adult blood. In the present studies, the functional capabilities of the HB-11+ and HB-11-TH cell subpopulations were examined after purification by fluorescence-activated cell sorting. Both subpopulations proliferated in response to PHA, Con A, PWM, and OKT-3 antibodies. The HB-11+ TH cells gave a minimal proliferative response to soluble tetanus toxoid antigen, whereas HB-11-TH cells responded well. After mitogen activation, both HB-11+ and HB-11-TH cells and to produce soluble factors which induce large B cells to proliferate. However, PWM-stimulated HB-11+TH cells were incapable of inducing B cells to differentiate into antibody-secreting plasma cells, whereas HB-11-TH cells were efficient in this regard. The results suggest that the HB-11 antigen is expressed on a subpopulation of virgin TH cells that can produce B cell growth factors but are deficient in the ability to produce B cell differentiation factors.