Imidazoline-binding domains on monoamine oxidase B and subpopulations of enzyme.

A series of phenoxy-substituted methylimidazoline derivatives were synthesized and used to define the ligand recognition properties of the imidazoline-binding domain (IBD) on monoamine oxidase (MAO)-B and its role in substrate processing. The rank order of potency for selected compounds in competitive binding studies with the imidazoline [(3)H]idazoxan was different from that in enzyme activity assays, suggesting that the IBD and the site involved in enzyme inhibition are distinct. IC(50) values for inhibition of MAO-B activity by imidazoline/guanidinium ligands were one to two orders of magnitude greater than ligand concentrations that probably saturate the IBD, but were equal to the K(d) values of these ligands in competitive binding assays with the reversible MAO-B inhibitor [(3)H]Ro 19-6327. In addition, the degree of enzyme inhibition by these ligands was similar in platelet and liver, tissues exhibiting 10-fold differences in the amount of the IBD-accessible enzyme subpopulation. These data suggested that the inhibitory effect of these compounds on MAO-B activity involved a secondary interaction with the enzyme domain recognizing the inhibitor Ro 19-6327 and does not involve interaction with the IBD. Subsequent radioligand-binding studies indicated that human liver MAO-B actually existed as two distinct populations that differed in the accessibility of their IBD. The relatively small amounts of MAO-B possessing an accessible IBD ( approximately 5% in human liver) precludes determination of the functional consequences of ligand binding to the IBD. This subpopulation of MAO-B may be selectively regulated or generated in different individuals or tissues and targeted by pharmacologically active compounds in a cell type-specific manner.

Use of high affinity, radioiodinated probes for identification of imidazoline/guanidinium receptive sites.

Various pharmacologically active compounds with an imidazoline or guanidinium moiety are recognized by membrane bound proteins that appear structurally and functionally distinct from known hormone receptors. Such entities are termed imidazoline binding sites, I receptors, or imidazoline/guanidinium receptive sites (IGRS). To facilitate the identification and structural analysis of IGRS, we developed functionalized molecular probes exhibiting high affinity and selectivity for IGRS. The molecular probes are structurally related to cirazoline, and imidazoline that exhibits high affinity for IGRS in both central and peripheral tissues. The parent molecule 2-[3-aminophenoxy]methyl imidazoline (125I-AMIPI), which was used to identify IGRS in brain and peripheral tissues. 125I-AMIPI was converted to the photosensitive arylazide derivative (125I-AMIPI) and used to identify the M(r) of the ligand binding subunit of IGRS in various tissues including brain, pancreas, kidney, and liver. The results of these studies indicate that there are multiple binding proteins for these molecules that differ in their apparent molecular weight, tissue distribution, intratissue location, and ligand recognition properties.

Isomeric selectivity at dopamine D3 receptors.

Racemic 7-hydroxy-N,N-dipropylaminotetralin (7-OH-DPAT) shows greater affinity for limbic-selective dopamine D3 receptors than for more ubiquitous dopamine D2 receptors. R(+)-7-OH-DPAT was prepared and evaluated in radioreceptor assays using membranes of fibroblasts expressing the human dopamine D3 receptor as well as rat striatal membranes containing dopamine D2 receptors. This enantiomer had 2-fold greater D3 affinity than the racemate and similarly greater D3 vs. D2 selectivity (64-fold). The results may facilitate development of D3 selective agents and evaluation of functions of these receptors.

Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome.

The lymphadenopathy-associated virus (LAV) prototype strain of human T-lymphotropic virus type III/LAV was transmitted to juvenile chimpanzees with no prior immunostimulation by (i) intravenous injection of autologous cells infected in vitro, (ii) intravenous injection of cell-free virus, and (iii) transfusion from a previously infected chimpanzee. All five animals that received more than one 50% tissue culture infective dose were persistently infected with LAV or chimpanzee-passaged LAV for up to 18 months. During this time they developed no illnesses, but they exhibited various degrees of inguinal and axillary lymphadenopathy and significant reductions in rates of weight gain. Detailed blood chemistry and hematologic evaluations revealed no consistent abnormalities, with the exception of immunoglobulin G (IgG) hypergammaglobulinemia, which became apparent in one animal 6 months postinfection and continued at more than 1 year postinfection. Transient depressions followed by increases in the numbers of T4 cells to levels greater than normal were observed in all animals after virus inoculation. However, the number of LAV-infected peripheral blood cells decreased with time after infection. Results of enzyme immunoassays showed that all infected animals seroconverted to IgG anti-LAV within 1 month postinfection and that antibody titers remained high throughout the period of observation. In contrast, only three of the five LAV-infected chimpanzees had detectable IgM antibody responses, and these preceded IgG-specific serum antibodies by 1 to 2 weeks. Virus morphologically and serologically identical to LAV was isolated from peripheral blood mononuclear cells of all infected animals at all times tested and from bone marrow cells taken from one animal 8 months after infection. One chimpanzee that was exposed to LAV only by sharing a cage with an infected chimpanzee developed lymphadenopathy and an IgM response to LAV, both of which were transient; however, no persistent IgG antibody response to LAV developed, and no virus was recovered from peripheral blood cells during a year of follow-up. Thus, LAV readily infected chimpanzees following intravenous inoculation and persisted for extended periods despite the presence of high titers of antiviral antibodies. However, the virus was not easily transmitted from infected to uninfected chimpanzees during daily cage contact.

Continuous production of a cytopathic human T-lymphotropic virus in a permissive neoplastic T-cell line.

We developed cloned populations from the commonly available, well-characterized cell line HUT-78. These cloned cells grow permanently after infection with isolates of human T-lymphotropic virus type III, also called lymphadenopathy virus (HTLV-III/LAV), from patients with acquired immune deficiency syndrome and related syndromes. In contrast, activated human T cells are lysed after HTLV-III/LAV infection. The infected cloned cells have been in culture continuously for 6 months and have produced high levels of extracellular reverse transcriptase (400,000 cpm/ml). This level is comparable to that of similarly infected normal human T cells. Three weeks after infection with HTLV-III/LAV, more than 90% of the cloned HUT-78 cells lysed; the remaining cells continued to grow. Approximately 80% of these cells expressed HTLV-III/LAV antigens by immunofluorescence. The extracellular virus of the chronically infected cell line was shown to be similar to other HTLV-III/LAV isolates by competition radioimmunoassay, by reactivity with human serum, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This HTLV-III/LAV-infected immortalized cell line enables the continuous production of large amounts of virus.