Separation and characterization of two related giardiaviruses in the parasitic protozoan Giardia lamblia

Giardiavirus encapsidates a 6.2-kb double-stranded (ds) RNA within a capsid that consists of a major 100-kDa capsid protein (p100) and a minor 190-kDa protein (p190). In this study, two nonhomologous 6.2-kb ds RNAs cohabiting in Giardia lamblia trophozoites were found to be separately encapsidated into two distinct virions, one (designated GLV[p100]) whose capsid consists of p100 and p190, and the other (designated GLV[p95]) whose capsid consists of a 95-kDa protein (p95) and a minor p190-equivalent protein. Both types of virions were enriched in the membranous fraction of a lysate from virus-infected G. lamblia cells. Separation of these virions was achieved by CsCl gradient centrifugation following osmotic rupture of the viral particles. By these treatments, the 6.2-kb ds RNA was removed from GLV[p100] whereas that in GLV[p95] remained unchanged, and the two 6.2-kb ds RNAs that had been purified by this protocol displayed differential hybridization properties to viral cDNA probes. Western blotting and peptide mapping experiments show that p100 and p95 were closely related proteins, but each had distinct amino acid sequences. Virus purification and pulse-chase experiments show that GLV[p100] was selectively secreted into the medium whereas GLV[p95] remained within the trophozoites of G. lamblia toward the late phase of cell growth. Secretion of GLV[p100] was not inhibited by Brefeldin A. These findings demonstrate the cohabitation of multiple Giardiavirus species in G. lamblia.     

Predominant expression of monoamine oxidase B isoform in rabbit renal proximal tubule: regulation by I2 imidazoline ligands in intact cells

Previous studies have shown that a subpopulation of the catecholamine-degrading enzymes monoamine oxidase (MAO) A and B holds a previously unknown regulatory site, the I2-imidazoline binding site (I2BS). In the present work, we characterized the isoforms of monoamine oxidases expressed in the rabbit renal proximal tubule, defined their relationship with I2BS, and investigated the ability of I2BS ligands to inhibit enzyme activity in intact cells. Two findings indicate that MAO-B is the predominant isoform expressed in the renal proximal tubule cells: 1) Western blot performed with an anti-MAO-A/MAO-B polyclonal antiserum revealed a single 55-kDa band corresponding to MAO-B; 2) enzyme assays showed an elevated MAO-B activity ([14C]beta-phenylethylamine oxidation: Vmax = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([14C]5-HT oxidation: Vmax = 80.3 +/- 19 pmol/min/mg protein). Photoaffinity labeling with the I2BS ligand [125I]2-(3-azido-4-iodophenoxy)-methylimidazoline revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I2 imidazoline binding site. [3H]Idazoxan binding studies and enzyme assays showed that, in intact cells, I2BS ligands bind to and inhibit MAO-B. Indeed, the increase in the accessibility of intracellular compartment by cell permeabilization did not enhance [3H]idazoxan binding, which indicates that, in intact cells, intracellular I2BS are fully occupied by imidazoline ligands. In addition, enzyme assays showed that incubation of proximal tubule cells with imidazoline ligands leads to a complete, dose-dependent inhibition of MAO activity. These data show the predominant expression of MAO-B in rabbit renal proximal tubule and its regulation by imidazoline ligands in intact cells.     

The partnership model: working with individuals, families, and communities toward a new vision of health

A novel irreversible 5-HT1A receptor binding ligand, NCS-MPP (4-(2'- methoxy-phenyl)-1-[2'-(N-2"-pyridyl)-p-isothiocyanobenzamido]- ethyl-piperazine), based on the new 5-HT1A receptor antagonist p-MPPI (4-(2'-methoxy-phenyl)-1-[2'-(N-2"-pyridyl)-p-iodobenzamido]-ethyl -piperazine ), was synthesized, and its binding characteristics were evaluated using in vitro homogenate binding with rat hippocampal membranes. The Ki value of NCS-MPP was estimated to be 1.8 +_ 0.2 nM using analysis of concentration-dependent inhibition for the binding of [125I]p-MPPI to 5-HT1A receptors. NovaScreen of NCS-MPP showed low to moderate binding affinities to alpha-1, alpha-2-adrenergic and 5-HT2 receptors, with Ki values of 350, 420, and 103 nM, respectively. These data strongly suggest that the ligand bound to 5-HT1A receptors with high affinity and high selectivity. Irreversible inhibition of [125I]p-MPPI binding by NCS-MPP following a 5 min incubation at room temperature was concentration dependent; the inhibition increased to 50% at a concentration less than 10 nM, and became more pronounced (90%) at 400 nM. Under similar assay conditions, NCS-MPP was significantly less efficient in irreversibly inhibiting agonist ligand [125I]8-OH-PIPAT binding to 5-HT1A receptors at lower concentrations (<10nM). After pretreatment of membranes with a low concentration of NCS-MPP (2nM), there was an apparent loss of [125I]p-MPPI binding sites, as expected, but no change in the binding affinity (Kd) was observed. However, the significant increase in Kd at a higher concentration of NCS-MPP (50 nM) indicated that there may be a secondary alkylation site, which may not be directly involved in p-MPPI binding to receptors; nevertheless, it would lead to an increased Kd value. The availability of an irreversible ligand, NCS-MPP, may provide a useful tool for studies of 5-HT1A receptors in the central nervous system.     

Biological and conformational examination of stereochemical modifications using the template melanotropin peptide, Ac-Nle-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2, on human melanocortin receptors

Examination of conformationally constrained melanotropin peptide (Ac-Nle4-c[Asp5-His-Phe7-Arg-Trp9-Ala-Lys]-NH2) on four human melanotropin receptors (hMC1R, hMC3R, hMC4R, and hMC5R) resulted in identifying the importance of ligand stereochemistry at positions 5, 7, and 9 for agonist binding affinity and receptor selectivity. A trend in ligand structure-activity relationships emerged for these peptides, with the hMC1R and hMC4R possessing similar tendencies, as did the hMC3R and hMC5R. alpha-MSH (Ac-Ser-Tyr-Ser-Met4-Glu-His-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-D-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), and MTII (Ac-Nle4-c[Asp5,D-Phe7,Lys10]-alpha-MSH(4-10)-NH2) were also examined at each of these melanocortin receptors. Interestingly, the linear NDP-MSH possessed greater binding affinity for the hMC3R and hMC5R than did the cyclic analogue MTII. The peptide Ac-Nle-c[Asp-His-Phe-Arg-D-Trp9-Ala-Lys]-NH2 demonstrated the greatest differentiation in binding affinity between the hMC1R and hMC4R (78-fold). Analogue Ac-Nle-c[Asp-His-Phe7-Arg-Trp-Ala-Lys]-NH2 resulted in micromolar binding affinity (or greater) at the hMC3R and hMC5R, demonstrating the importance of D-Phe7 for ligand binding potency at these receptors. Ac-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2 resulted in loss of binding affinity at the hMC5R, implicating the importance of Nle4 (or a hydrophobic residue in this position) for binding to this receptor. Ac-Nle-c[D-Asp5-His-Phe-Arg-Trp-Ala-Lys]-NH2 was unable to competitively displace [125I]NDP-MSH binding at micromolar concentrations on the hMC3R and hMC5R, suggesting the importance of chirality of Asp5 either for ligand-receptor interactions or for orientation of the side chain lactam bridge and the structural integrity of the peptide conformation. Energy calculations performed for these peptides resulted in the identification of a low-energy ligand conformer family that is common to all the ligands. The differences in ligand binding affinities observed in this study are postulated to be a result of different ligand-receptor complexed interactions and not solely to the ligand structure.     

Binding of the novel nonxanthine A2A adenosine receptor antagonist [3H]SCH58261 to coronary artery membranes

This study demonstrates quantification of A2A adenosine receptors (A2AAdoRs) in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells. Radioligand binding assays were performed using the new selective A2AAdoR antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-epsilon]-1,2,4-triazolo[1,5-c)pyrimidine ([3H]SCH58261). Binding of the radioligand to membranes was rapid, reversible, and saturable. The densities of A2AAdoRs in membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells were 900 +/- 61, 892 +/- 35, and 959 +/- 76 fmol/mg protein, respectively. Equilibrium dissociation constants (Kd values) calculated from results of saturation binding assays were 2.19, 1.20, and 0.81 nmol/L, and Kd values calculated from results of association and dissociation assays were 2.42, 1.01, and 0.40 nmol/L for [3H]SCH58261 binding to membranes prepared from porcine coronary arteries, porcine striatum, and PC12 cells, respectively. The specific binding of [3H]SCH58261 as a percentage of total binding at a radioligand concentration equal to the Kd value was 65% to 90% in the three membrane preparations. The order of ligand potencies determined by assay of competition binding to sites in porcine coronary membranes using [3H]SCH58261, unlabeled antagonists (SCH58261, 8-(3-chlorostyryl)caffeine [CSC], and xanthine amine congener [XAC]), and unlabeled agonists ([3H]2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoaden osine [CGS 21680], 2-hexynyl-5'-N-ethylcarboxamidoadenosine [HE-NECA], [3H]5'-N-ethylcarboxamidoadenosine [NECA], and R(-)N6-(2-phenylisopropyl)adenosine [R-PIA]) was SCH58261 > HE-NECA = CSC = CGS 21680 = XAC > NECA = R-PIA. The Hill coefficients of displacement by A2AAdoR ligands of [3H]SCH58261 binding were not significantly different from unity, indicating that [3H]SCH58261 bound to a group of homogeneous noninteracting sites in all membrane preparations. The order of ligand potencies to compete for [3H]SCH58261 binding sites in porcine striatal and PC12 cell membranes was, in part, different from that for porcine coronary arterial membranes. The different rank orders of potencies for agonists and antagonists at A2A receptors of porcine coronary arteries, striatum, and PC12 cells and significant differences in absolute values of potency of ligands in the three preparations may indicate the existence of different subtypes of A2AAdoRs. The antagonist radio-ligand [3H]SCH58261 should be of value for pharmacological characterization of A2A adenosine receptors in other preparations.     

Characterization of desamino-5-[125I]iodo-3-methoxy-zacopride ([125I]MIZAC) binding to 5-HT3 receptors in the rat brain

1. Antagonists at 5-HT3 receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT3 ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT3 antagonist, zacopride, binds with high affinity (1.3-1.5 nM) to CNS 5-HT3 sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates. 2. Ninety-seven percent of total specific binding of [125I]MIZAC (0.1 nM) of was displaced by bemesetron (3 microM), a selective 5-HT3 antagonist. Competition studies using ligands with known affinities for 5-HT3 sites give a high correlation with reported pKi values (r2 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT3 receptor, i.e. highest in cortex and hippocampus, and lowest in striatum and cerebellum. 3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [125I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT4, 5-HT6, and 5-HT7 receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [125I]MIZAC for the 5-HT3 receptor. 4. [125I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [125I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT3 receptor. Given its high selectivity for 5-HT3 binding sites, [125I]MIZAC appears to be a promising ligand for labeling 5-HT3 receptors in vitro and in vivo.     

Nonclassical 2,4-diamino-8-deazafolate analogues as inhibitors of dihydrofolate reductases from rat liver, Pneumocystis carinii, and Toxoplasma gondii

The synthesis and biological activity of 42 6-substituted-2,4-diaminopyrido[3,2-d]pyrimidines (2,4-diamino-8-deazafolate analogues) are reported. The compounds were synthesized in improved yields compared to previous classical analogues using modifications of procedures reported previously by us. Specifically, the S-phenyl-; mono-, di-, and trimethoxyphenyl-; and mono-, di-, and trichlorophenyl-substituted analogues with H or CH3 at the N10 position and methyl and trifluoromethyl phenyl ketone analogues with H, CH3, and CH2C identical to CH at the N10 position were synthesized. The S10 and N10 alpha- and beta-naphthyl analogues along with the N10 CH3 analogues were also synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii (pc) and Toxoplasma gondii (tg); selectivity ratios were determined against rat liver (rl) DHFR as the mammalian reference enzyme. Against pcDHFR the IC50 values ranged from 0.038 x 10-6 M for 2,4-diamino-6-[(N-methyl-2'-naphthylamino)methyl]pyrido[3,2-d]pyrimidine (28) to 5.5 x 10(-6) M for 2,4-diamino-6[(2',4'-dimethoxyanilino)methyl]pyrido[3,2-d]pyrim idi ne (15). N10 methylation in all instances increased potency. None of the analogues were selective for pcDHFR. Against tgDHFR the most potent analogue was 2,4-diamino-6-[(N-methylanilino)methyl]pyrido[3,2-d]pyrimidine (5) (IC50 0.0084 x 10(-6) M) and the least potent was 2,4-diamino-6[(2'-naphthylamino)methyl]-pyrido[3,2-d]pyrimidine (37) (IC50 0.16 x 10-6 M). N10 methylation afforded an increase in potency up to 10-fold. In contrast to pcDHFR, several of the 8-deaza analogues were significantly selective for tgDHFR, most notably 2,4-diamino-6-[(2'-chloro-N-methylanilino)-methyl]pyrido[3,2-d] pyrimidine (13), 2,4-diamino-6-[(3',4',5'-trimethoxyanilino)methyl]pyrido[3,2-d]pyr pyrimidine (29), and 2,4-diamino-6-[(2',4',6'-trichloroanilino)methyl]pyrido[3,2-d] pyrimidine (32) which combined high potency at 10-8 M along with selectivities of 8.0, 5.0, and 12.4, respectively. The potency of these three analogues are comparable to the clinically used agent trimetrexate while their selectivities for tgDHFR are 17-43-fold better than trimetrexate.     

Purification and characterization of a guanine nucleotide-exchange protein for ADP-ribosylation factor from spleen cytosol

ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins and are active in the GTP-bound state and inactive with GDP bound. ARF-GTP has a critical role in vesicular transport in several cellular compartments. Conversion of ARF-GDP to ARF-GTP is promoted by a guanine nucleotide-exchange protein (GEP). We earlier reported the isolation from bovine brain cytosol of a 700-kDa protein complex containing GEP activity that was inhibited by brefeldin A (BFA). Partial purification yielded an approximately 60-kDa BFA-insensitive GEP that enhanced binding of ARF1 and ARF3 to Golgi membranes. GEP has now been purified extensively from rat spleen cytosol in a BFA-insensitive, approximately 55-kDa form. It activated class I ARFs (ARFs 1 and 3) that were N-terminally myristoylated, but not nonmyristoylated ARFs from class-I, II, or III. GEP activity required MgCl2. In the presence of 0.6-0.8 mM MgCl2 and 1 mM EDTA, binding of guanosine 5'-[gamma[35S]thio]triphosphate ([35S]GTP gamma S) by ARF1 and ARF3 was equally high without and with GEP. At higher Mg2+ concentrations, binding without GEP was much lower; with 2-5 mM MgCl2, GEP-stimulated binding was maximal. The rate of GDP binding was much less than that of GTP gamma S with and without GEP. Phospholipids were necessary for GEP activity; phosphatidylinositol was more effective than phosphatidylserine, and phosphatidic acid was less so. Other phospholipids tested were ineffective. Maximal effects required approximately 200 microM phospholipid, with half-maximal activation at 15-20 microM. Release of bound [35S]GTP gamma S from ARF3 required the presence of both GEP and unlabeled GTP or GTP gamma S; GDP was much less effective. This characterization of the striking effects of Mg2+ concentration and specific phospholipids on the purified BFA-insensitive ARF GEP should facilitate experiments to define its function in vesicular transport.     

Identification and isolation of a 45-kDa calcium-dependent lactoferrin receptor from rat hepatocytes

Isolated rat hepatocytes bind, internalize, and degrade bovine lactoferrin (Lf) via high-affinity Ca2+-dependent sites [<10(6) sites/cell; McAbee et al., (1993) Biochemistry 32, 13749-13760]. In this study, we identified a 45-kDa Ca2+-dependent Lf binding protein on rat hepatocytes by three independent approaches. First, dithiobis(sulfosuccimidylproprionate) (DTSSP) cross-linked 125I-Lf to a 45-kDa adduct in a Ca2+-dependent manner on intact cells. The 125I-labeled cross-linked complexes were absent when either surface-bound 125I-Lf was stripped prior to cross-linking or an excess of unlabeled Lf was included in the DTSSP reaction. Second, 125I-Lf bound to a 45-kDa hepatocyte polypeptide in a Ca2+-dependent fashion following incubation with SDS-PAGE fractioned hepatocyte membrane proteins absorbed on nitrocellulose. Third, when Triton X-100 extracts of hepatocyte membrane ghosts were chromatographed on Lf-agarose, a 45-kDa polypeptide (p45) was eluted by EGTA. Column fractions enriched in p45--but not those depleted of p45--possessed soluble Lf receptor activity as determined by competition binding assay. Monospecific polyclonal anti-p45 IgG detected p45 in crude hepatocyte ghost homogenates and blocked vigorously 125I-Lf binding and endocytosis to intact rat hepatocytes. We conclude, therefore, that p45 constitutes the Ca2+-dependent Lf receptor on isolated rat hepatocytes.     

The ligand binding site of NPY at the rat Y1 receptor investigated by site-directed mutagenesis and molecular modeling

The ligand binding site of neuropeptide Y (NPY) at the rat Y1 (rY1,) receptor was investigated by construction of mutant receptors and [3H]NPY binding studies. Expression levels of mutant receptors that did not bind [3H]NPY were examined by an immunological method. The single mutations Asp85Asn, Asp85Ala, Asp85Glu and Asp103Ala completely abolished [3H]NPY binding without impairing the membrane expression. The single mutation Asp286Ala completely abolished [3H]NPY binding. Similarly, the double mutation Leu34Arg/Asp199Ala totally abrogated the binding of [3H]NPY, whereas the single mutations Leu34Arg and Asp199Ala decreased the binding of [3H]NPY 2.7- and 5.2-fold, respectively. The mutants Leu34Glu, Pro35His as well as Asp193Ala only slightly affected [3H]NPY binding. A receptor with a deletion of the segment Asn2-Glu20 or with simultaneous mutations of the three putative N-terminal glycosylation sites, displayed no detectable [3H]NPY binding, due to abolished expression of the receptor at the cell surface. Taken together, these results suggest that amino acids in the N-terminal part as well as in the first and second extracellular loops are important for binding of NPY, and that Asp85 in transmembrane helix 2 is pivotal to a proper functioning of the receptor. Moreover, these studies suggest that the putative glycosylation sites in the N-terminal part are crucial for correct expression of the rY1 receptor at the cell surface.     

Requirement for the CD95 receptor-ligand pathway in c-Myc-induced apoptosis

Induction of apoptosis by oncogenes like c-myc may be important in restraining the emergence of neoplasia. However, the mechanism by which c-myc induces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc-induced apoptosis was shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. Moreover, IGF-I signaling and Bcl-2 suppress c-myc-induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity.     

Purification of a cell-surface receptor for surfactant protein A

In the present report we have characterized the binding of surfactant protein A (SP-A) to bone marrow-derived macrophages, U937 cells, alveolar macrophages, and type II epithelial cells. The binding of SP-A to all cell types is Ca2+-dependent and trypsin-sensitive, but type II cells express distinct Ca2+-independent binding sites. The binding of SP-A to macrophages is independent of known cell surface carbohydrate-specific receptors and of glycoconjugate binding sites on the surface of the cells and is distinct from binding to C1q receptors. Based on ligand blot analysis, both type II cells and macrophages express a 210-kDa SP-A-binding protein. The 210-kDa protein was purified to apparent homogeneity from U937 macrophage membranes using affinity chromatography with noncovalently immobilized surfactant protein A, and was purified from rat lung by differential detergent and salt extraction of isolated rat lung membranes. Polyclonal antibodies against the rat lung SP-A-binding protein inhibit binding of SP-A to both type II cells and macrophages, indicating that the 210-kDa protein is expressed on the cell surface. The polyclonal antibodies also block the SP-A-mediated inhibition of phospholipid secretion by type II cells, indicating that the 210-kDa protein is a functional cell-surface receptor on type II cells. In a separate report we have determined that antibodies to the SP-A receptor block the SP-A-mediated uptake of Mycobacterium bovis, indicating that the macrophage SP-A receptor is involved in SP-A-mediated clearance of pathogens.     

Taxol-mediated augmentation of CD95 ligand-induced apoptosis of human malignant glioma cells: association with bcl-2 phosphorylation but neither activation of p53 nor G2/M cell cycle arrest

The anti-tumour alkaloid taxol shows strong cytotoxic and antiproliferative activity in two human malignant glioma cell lines, T98G and LN-229. CD95 (Fas/APO-1) ligand is a novel cytotoxic cytokine of the tumour necrosis factor (TNF) family that exerts prominent antiglioma activity. At clinically relevant taxol concentrations of 5-100 nM, taxol and CD95 ligand showed significant synergistic cytotoxicity and growth inhibition. High concentrations of taxol induced G/M cell cycle arrest in both cell lines. The synergy of taxol and CD95 ligand was independent of cell cycle effects of taxol as synergy was achieved at much lower taxol concentrations than G2/M arrest and as cell cycle effects of taxol were unaffected by co-exposure to CD95 ligand. Similarly, high concentrations of taxol were required to induce p53 activity in the p53 wild-type cell line LN-229. This effect was not modulated by CD95 ligand, suggesting that synergy is also independent of p53 activation. However, taxol induced a mobility shift of the bcl-2 protein on immunoblot analysis, indicative of bcl-2 phosphorylation. Bcl-2 phosphorylation on serine was confirmed by immunoprecipitation and phosphoserine immunoblot analysis. Considering (1) that phosphorylation of bcl-2 interferes with its heterodimerization with bax and (2) the inhibition of CD95-mediated apoptosis by bcl-2, we propose that taxol sensitizes malignant glioma cells to CD95 ligand by increasing the functional bax/bcl-2 rheostat in favour of bax and thus cell death.     

Heterogeneity of motilin receptors in the gastrointestinal tract of the rabbit

Motilin, a 22-amino acid peptide synthesized in endocrine cells of intestinal mucosa, stimulates GI smooth muscle contractility. To elucidate the mode of action of motilin, we attempted to determine whether motilin receptors are localized on nerve cells or on smooth muscle cells of the GI tract. Mucosa-free tissues from rabbit antrum and duodenum were homogenized separately with a Polytron prior to differential centrifugation to obtain synaptosome or plasma membrane-enriched fractions, as determined by the distribution of [3H]saxitoxin (SAX) binding (neural membranes) and 5' nucleotidase (5'N) activity (smooth muscle plasma membranes). Motilin binding was evaluated by the displacement of [125I]motilin by motilin (1-22) on the various membrane fractions. In the antrum, motilin binding was highly correlated with SAX binding (r = 0.81, p < 0.0005), and also significantly with 5'N activity (r = 0.54, p < 0.05). In the duodenum, motilin binding correlated significantly with 5'N activity (r = 0.67, p < 0.005), but not with SAX binding (r = -0.11, NS). Receptor affinity, for the motilin antagonist MOT(1-12)[CH2NH]10-11, for motilin(1-22), and for the motilin agonist erythromycin lactobionate was significantly (p < 0.001, p < 0.001, and p < 0.05, respectively) higher in SAX-enriched fractions from the antrum than in 5'N-enriched fractions from the duodenum. Therefore, in the rabbit: 1) motilin receptors appear to be predominantly located on nerve tissues in the antrum and restricted to smooth muscle cells in the duodenum, and 2) antral receptors and duodenal receptors displayed different pharmacological characteristics, probably corresponding to two specific and heterogeneous motilin receptor subtypes.     

Selective visualization of rat brain 5-HT2A receptors by autoradiography with [3H]MDL 100,907

The recently developed 5-HT2A receptor selective antagonist [3H]MDL100,907 ((+/-)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic visualization of these receptors. [3H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd = 0.2-0.3 nM), and presented a pharmacological profile consistent with its binding to 5-HT2A receptors (rank order of affinity for [3H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled by [3H]MDL100,907 was compared to the autoradiographical patterns obtained with [3H]Ketanserin, [3H]Mesulergine, and [3H]RP62203 (N-[3-[4-(4-fluorophenyl)piperazin-1-y1]propyl]-1,8-naphtalenes ultam) and to the distribution of 5-HT2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [3H]MDL100,907 labelled a single population of sites (5-HT2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [3H]MDL100,907-labelled receptors and 5-HT2A mRNA further supports the selectivity of this radioligand for 5-HT2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [3H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT2A receptors.     

Specificities and rates of binding of anti-(6-4) photoproduct antibody fragments to synthetic thymine photoproducts

Pyrimidine (6-4) pyrimidone photoproducts are some of the major DNA photolesions induced by ultraviolet (UV) light. A monoclonal antibody (64M5) specific to a (6-4) photoproduct has been established and the corresponding single-chain antibody (64M5scFv) has been prepared. In this study, we characterized the ligand selectivities of 64M5 and 64M5scFv using synthetic octadeoxynucleotides containing either a central cis-syn cyclobutane thymine dimer (T[c,s]T), the (6-4) photoproduct of TpT (T[6-4]T), or its Dewar isomer (T[Dewar]T) by means of enzyme-linked immunosorbent assays (ELISA). Both 64M5 and 64M5scFv recognized T[6-4]T, but not the other photoproducts. We synthesized several biotinylated oligonucleotides of different lengths containing (T[6-4]T) to analyze the effects of the antigen size on the binding rates of an antigen binding fragment (64M5Fab) and 64M5scFv by means of surface plasmon resonance. The association rate constants for oligonucleotides of different sizes containing T[6-4]T as to 64M5Fab were found to be almost the same (1.9-5.6 x 10(5) M(-1) x s(-1)), while the dissociation rate constant for the largest oligonucleotide (d8mer, 8.0 x 10(-5) s(-1)) was significantly smaller than that for the d2mer (4.2 x 10(-2) s(-1)). These results indicate that 64M5Fab recognized the d2mer as the epitope and that the binding affinity for T[6-4]T depended on the flanking oligonucleotides. The dissociation rate constants for 64M5scFv as to the antigen analogs were almost the same as those for the various T[6-4]T-oligonucleotides as to 64M5Fab, suggesting that the conformations of these antibody binding regions are pretty similar to each other.     

Agonist-induced up-regulation of alpha4beta2 nicotinic acetylcholine receptors in M10 cells: pharmacological and spatial definition

Chronic nicotine up-regulates the number of high affinity nicotinic acetylcholine receptors (nAChRs) in mammalian brain. Here, we studied up-regulation of the nAChR composed of alpha4 and beta2 subunits in the M10 cell line by using [3H]epibatidine to measure nAChR in cells in situ and in membrane preparations. Cultures were exposed to drugs for 2 days before assay. All agonists up-regulated [3H]epibatidine binding sites with EC50 values typically 10-100-fold higher than their respective Ki values from competition binding assays. Maximum up-regulation ranged from 40% to 250% above control values. Maximally effective concentrations of the less efficacious agonists methylcarbamylcholine or (+/-)-epibatidine together with nicotine resulted in less up-regulation than that produced by nicotine alone, showing that they are partial up-regulatory agonists. The antagonists dihydro-beta-erythroidine, methyllycaconitine, d-tubocurarine, hexamethonium, decamethonium, and mecamylamine either failed to up-regulate [3H]epibatidine binding sites or up-regulated mildly at high concentrations. When tested at non-up-regulating concentrations, only d-tubocurarine significantly inhibited agonist-induced up-regulation; this inhibition seemed to be noncompetitive. Comparison of [3H]epibatidine displacement in intact M10 cells and membrane preparations by membrane-impermeant ligands indicated that 85% of [3H]epibatidine binding sites are intracellular. On chronic treatment with agonist, the proportion of surface receptors did not change significantly, indicating that most up-regulated [3H]epibatidine binding sites are internal. However, up-regulation is mediated at the cell surface because the impermeant ligand tetramethylammonium was as efficacious as nicotine in eliciting up-regulation, and methylcarbamylcholine (i.e., impermeant but with low efficacy) blocked nicotine induced up-regulation. Thus, agonists elicit up-regulation (mainly of intracellular receptors) by interacting with cell surface nAChRs that are not compatible with either an active or high affinity desensitized conformation.     

Antimutagenicity and the influence of physical factors in binding Lactobacillus gasseri and Bifidobacterium longum cells to amino acid pyrolysates

Antimutagenic and binding properties of 28 strains of Lactobacillus gasseri and 2 strains of Bifidobacterium longum on the mutagenicity of amino acid pyrolysates were investigated in vitro using a streptomycin-dependent (SD510) strain of Salmonella typhimurium TA 98. Four strains of L. acidophilus (SBT0274, SBT1703, SBT10239, and SBT10241) and 1 strain of B. longum (SBT 2928) exhibited the highest percentage of antimutagenicity and binding. These 5 strains were further optimized for other physical factors influencing the mechanism of binding, such as cell and mutagen concentration, pH, and incubation time. In all of the selected strains, 2 mg of cells bound with 88 to 95% of 0.2 mg of 3-amino-1,4 dimethyl-5H-pyrido[4,3-b]indole in 30 min at pH 7.0. Other amino acid pyrolysates, such as 3-amino-1-methyl-5H-pyrido[4,3-b]indole, 2-amino-6-methyldi-pyrido[1,2-a:3',2'-d]imidazole, 2-amino-3-methyl-imidazo[4,5,f]quinoline, and 2-amino-3,4-dimethyl-imidazo[4,5,f]quinoline were also tested for the binding ability of these strains. We observed that the complexity of the mutagens greatly influenced the binding properties. The binding of 3-amino-1,4 dimethyl-5H-pyrido[4,3-b]indole to the purified cell walls was very high compared with that of the crude cell wall, peptidoglycan, or the cell extract. Binding was inhibited when the cell walls were subjected to treatment with metaperiodate or trichloroacetic acid but not when they were subjected to treatment with lysozyme, trypsin, or proteinase K, reflecting the role of the carbohydrate component as a binding site.     

The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor

In this work, the 100-kDa neurotensin (NT) receptor previously purified from human brain by affinity chromatography (Zsürger, N., Mazella, J., and Vincent, J. P. (1994) Brain Res. 639, 245-252) was cloned from a human brain cDNA library. This cDNA encodes a 833-amino acid protein 100% identical to the recently cloned gp95/sortilin and was then designated NT3 receptor-gp95/sortilin. The N terminus of the purified protein is identical to the sequence of the purified gp95/sortilin located immediately after the furin cleavage site. The binding of iodinated NT to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized extracts of COS-7 cells transfected with the cloned cDNA was saturable and reversible with an affinity of 10-15 nM. The localization of the NT3 receptor-gp95/sortilin into intracellular vesicles was in agreement with previous results obtained with the purified receptor and with gp95/sortilin. Affinity labeling and binding experiments showed that the 110-kDa NT3 receptor can be partly transformed into a higher affinity (Kd = 0.3 nM) 100-kDa protein receptor by cotransfection with furin. This 100-kDa NT receptor corresponded to the mature form of the receptor. The NT3/gp95/sortilin protein is the first transmembrane neuropeptide receptor that does not belong to the superfamily of G-protein-coupled receptors.