Cannabinoid ligand-receptor signaling in the mouse uterus.

Using RNA (Northern) blot hybridization and reverse transcription-PCR, we demonstrate that the brain-type cannabinoid receptor (CB1-R) mRNA, but not the spleen-type cannabinoid receptor (CB2-R) mRNA, is expressed in the mouse uterus and that this organ has the capacity to synthesize the putative endogenous cannabinoid ligand, anandamide (arachidonylethanolamide). The psychoactive cannabinoid component of marijuana--delta 9-tetrahydrocannabinol (THC)--or anandamide, but not the inactive and nonpsychoactive cannabidiol (CBD), inhibited forskolin-stimulated cyclic AMP formation in the mouse uterus, which was prevented by pertussis toxin pretreatment. These results suggest that uterine CB1-R is coupled to inhibitory guanine nucleotide-binding protein and is biologically active. Autoradiographic studies identified ligand binding sites ([3H]anandamide) in the uterine epithelium and stromal cells, suggesting that these cells are perhaps the targets for cannabinoid action. Scatchard analysis of the binding of [3H]WIN 55212-2, another cannabinoid receptor ligand, showed a single class of high-affinity binding sites in the endometrium with an apparent Kd of 2.4 nM and Bmax of 5.4 x 10(9) molecules per mg of protein. The gene encoding lactoferrin is an estrogen-responsive gene in the mouse uterus that was rapidly and transiently up-regulated by THC, but not by CBD, in ovariectomized mice in the absence of ovarian steroids. This effect, unlike that of 17 beta-estradiol (E2), was not influenced by a pure antiestrogen, ICI 182780, suggesting that the THC-induced uterine lactoferrin gene expression does not involve estrogen receptors. We propose that the uterus is a new target for cannabinoid ligand-receptor signaling.

On the magnitude of the electrostatic contribution to ligand-DNA interactions.

A model based on the nonlinear Poisson-Boltzmann equation is used to study the electrostatic contribution to the binding free energy of a simple intercalating ligand, 3,8-diamino-6-phenylphenanthridine, to DNA. We find that the nonlinear Poisson-Boltzmann model accurately describes both the absolute magnitude of the pKa shift of 3,8-diamino-6-phenylphenanthridine observed upon intercalation and its variation with bulk salt concentration. Since the pKa shift is directly related to the total electrostatic binding free energy of the charged and neutral forms of the ligand, the accuracy of the calculations implies that the electrostatic contributions to binding are accurately predicted as well. Based on our results, we have developed a general physical description of the electrostatic contribution to ligand-DNA binding in which the electrostatic binding free energy is described as a balance between the coulombic attraction of a ligand to DNA and the disruption of solvent upon binding. Long-range coulombic forces associated with highly charged nucleic acids provide a strong driving force for the interaction of cationic ligands with DNA. These favorable electrostatic interactions are, however, largely compensated for by unfavorable changes in the solvation of both the ligand and the DNA upon binding. The formation of a ligand-DNA complex removes both charged and polar groups at the binding interface from pure solvent while it displaces salt from around the nucleic acid. As a result, the total electrostatic binding free energy is quite small. Consequently, nonpolar interactions, such as tight packing and hydrophobic forces, must play a significant role in ligand-DNA stability.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.

Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development.

The development of megakaryocytes (MKs) from their marrow precursors is one of the least understood aspects of hematopoiesis. Current models suggest that early-acting MK colony-stimulating factors, such as interleukin (IL) 3 or c-kit ligand, are required for expansion of hematopoietic progenitors into cells capable of responding to late-acting MK potentiators, including IL-6 and IL-11. Recently, the Mp1 ligand, or thrombopoietin (Tpo), has been shown to display both MK colony-stimulating factor and potentiator activities, at potencies far greater than that of other cytokines. In light of these findings, we tested the hypothesis that Tpo is absolutely necessary for MK development. In this report we demonstrate that neutralizing the biological activity of Tpo eliminates MK formation in response to c-kit ligand, IL-6, and IL-11, alone and in combination, but that these reagents only partially reduce MK formation in the presence of combinations of cytokines including IL-3. However, despite the capacity of IL-3 to support the proliferation and initial stages of MK differentiation, elimination of Tpo prevents the full maturation of IL-3-induced MK. These data indicate that two populations of MK progenitors can be identified: one that is responsive to IL-3 but can fully develop only in the presence of Tpo and a second that is dependent on Tpo for both proliferation and differentiation. Thus, our results strongly suggest that Tpo is the primary regulator of MK development and platelet production.

Infection of cells by varicella zoster virus: inhibition of viral entry by mannose 6-phosphate and heparin.

Envelope glycoproteins of varicella zoster virus (VZV) contain mannose 6-phosphate (Man6P) residues. We now report that Man6P competitively and selectively inhibits infection of cells in vitro by cell-free VZV; furthermore, dephosphorylation of VZV by exposure to alkaline phosphatase rapidly destroys infectivity. Cells are also protected from VZV in a concentration-dependent manner by heparin (ED50 = 0.23 micrograms/ml; 95% confidence limits = 0.16-0.26 microgram/ml) but not by chondroitin sulfate. Both heparin and Man6P are protective only when present about the time of inoculation. Heparin but not Man6P interferes with the attachment of VZV to cell surfaces; moreover, VZV binds to heparin-affinity columns. These data are compatible with a working hypothesis, whereby VZV attaches to cell surfaces by binding to a heparin sulfate proteoglycan. This binding stabilizes VZV, making possible a low-affinity interaction with another Man6P-dependent receptor, which is necessary for viral entry.