Identification and partial characterization of a glycoprotein species with high affinity for methylmercury in peripheral nervous tissues of man and experimental animals

A small amount of a glycoprotein species (21-kDa glycoprotein) with high affinity for methylmercury (MeHg) was detected in the post-nuclear or post-mitochondrial supernatant fraction of the homogenate of rat sciatic nerve on electrophoresis and autoradiography after binding of Me203Hg to the fraction. The 21-kDa glycoprotein was also found in the subcellular fractions of mouse, hamster, guinea pig, rabbit and human peripheral nervous tissues. Experiments with the cellular fractions of the tissues revealed that the 21-kDa glycoprotein is localized mainly in the myelin fraction, whereas it was not found in the cellular fractions of brain, spinal cord and nonneural tissues, such as kidney and liver. The specific binding activity of the 21-kDa glycoprotein with MeHg was 12-15 fold that of the major myelin protein, Po. It was shown that the interaction of the 21-kDa glycoprotein with MeHg was mediated through sulfhydryl groups in experiments with iodoacetamide and dithiothreitol. The amino acid compositions of the rat and human 21-kDa glycoproteins were similar but very different from that of a typical metallothionein. The N-terminal amino acid sequences of the two components of the rat 21-kDa glycoprotein were identical to those of P0 and PMP-22, respectively. The in vitro binding of MeHg was also observed in the myelin fraction obtained from the sciatic nerves of MeHg-dosed rats.     

Molecular alterations of canalicular transport systems in experimental models of cholestasis: possible functional correlations

Ajzen's (1988) theory of planned behavior (TOPB) was used to examine psychological determinants of high-risk UV radiation exposure-related behaviors (sunbathing, tanning salon use, and sunscreen use). Undergraduates at a midsized southeastern university were assessed on their psychological and behavioral tendencies toward high-risk UV radiation exposure-related behaviors. The results generally supported the utility of the TOPB as an explanatory model for high-risk behavior. Attitudes were strongly associated with high-risk intentions (e.g., not utilize sunscreen, use salons), whereas subjective norms were less so. Perceived behavioral control was found to moderate the relationship among attitudes, norms, and intentions to sunbathe and tan at a salon. Implications for intervention strategies and future model building in this area are discussed.     

The ratio of reduced glutathione/oxidized glutathione is maintained in the liver during short-term hepatic hypoxia

Controversy persists as to whether reperfusion-induced injuries actually occur in the hepatocyte. The liver is the major source of glutathione, a scavenger of hydrogen peroxide. The aim of this study was to evaluate the sensitivity of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) [GSH:GSSG] as an index of hepatic metabolic stress. A total of 121 rats were studied. The superior mesenteric vein (SMV) was occluded for 30 min, and this was followed by 0, 10, or 120 min of reperfusion. Total glutathione and GSSG levels in the liver, bile, and plasma were quantified, using glutathione reductase-coupled enzymatic assays. Results indicated that the hepatic GSH/GSSG ratio was maintained after an occlusion of the SMV, despite a decrease in adenosine triphosphate (ATP) level and energy charge potential. However, plasma levels of total glutathione and GSSG in the inferior vena cava increased after SMV occlusion and continued to increase after reperfusion. Biliary GSSG efflux decreased during 30-min occlusion of the SMV, and remained low even after reperfusion. The liver maintains homeostasis despite a decrease in biliary GSSG efflux, probably by secreting excess GSSG into the hepatic vein when the SMV is occluded. We conclude that the total amount of glutathione and GSSG in the plasma is directly correlated with oxidative stress in the liver.     

Alpha,beta-unsaturated carbonyl compounds: inhibition of rat liver glutathione S-transferase isozymes and chemical reaction with reduced glutathione

Monoclonal antibodies have been obtained by fusing mouse myeloma cells with spleen cells of mice immunized with crude thyroid membranes. Among the antibodies reactive with different thyroid antigenic components, three were found to specifically react with TSH receptor molecules. These antibodies displayed characteristic staining patterns on frozen sections of thyroid tissue from patients with various thyroid diseases upon identification of antibody binding by indirect peroxidase staining. No specific reactivity was detected with tissue from other human organs, such as pancreas, liver, fat, and muscle. The results demonstrate that the immunoperoxidase technique and the specificity of the monoclonal antibodies produced permitted the identification of cellular constituents that might be important antigens in autoimmune thyroid disease.     

Identification of high affinity binding sites for inhibin on ovine pituitary cells in culture

BACKGROUND AND OBJECTIVE: The clinical picture, response to therapy, and prognosis of women with diffuse malignant peritoneal mesotheliomas (DMPM) are ill defined. The purpose of this study is to report on the clinical picture, response to therapy, and survival of women with DMPM. METHODS: The study is a retrospective review of 15 women with the confirmed pathologic diagnosis of DMPM treated between 1964 and 1996. Survival curves were constructed according to the Kaplan-Meier method. The effect of different factors on survival was studied using the log-rank test. Two-tailed P values < 0.05 were considered significant. RESULTS: Clinical features included abdominal distension (11/15, 73%), abdominal pain (6/15, 40%), ascites (9/15, 60%), abdominal or pelvic masses (14/15, 93%), elevated CA-125 (4/4, 100%), thrombocytosis (4/ 15, 27%), and thrombo-embolic manifestations (3/15, 20%). The response rate to all first-line chemotherapy regimens was 30%. The response rate to paclitaxel/cisplatin was 66.7% and the toxicity was tolerable. The median survival of all patients was 12.5 months. Patients who underwent cytoreductive surgery survived longer than those who underwent biopsy only (median survival 13.5 vs. 6.0 months, P = 0.24). Patients who received chemotherapy survived significantly longer than those who did not receive chemotherapy (29.0 vs. 1.0 months, P = 0.03). Patients who responded to first-line chemotherapy survived significantly longer than those who did not respond (P = 0.04). CONCLUSIONS: Cytoreductive surgery and chemotherapy, especially with paclitaxel and cisplatin, might be of benefit in women with DMPM.     

Low affinity interaction of human or rat T cell adhesion molecule CD2 with its ligand aligns adhering membranes to achieve high physiological affinity

The mechanism by which low affinity adhesion molecules function to produce stable cell-cell adhesion is unknown. In solution, the interaction of human CD2 with its ligand CD58 is of low affinity (500 mM-1) and the interaction of rat CD2 with its ligand CD48 is of still lower affinity (40 mM-1). At the molecular level, however, the two systems are likely to be topologically identical. Fluorescently labeled glycosylphosphatidylinositol-anchored CD48 and CD58 were prepared and incorporated into supported phospholipid bilayers, in which the ligands were capable of free lateral diffusion. Quantitative fluorescence imaging was used to study the binding of cell surface human and rat CD2 molecules to the fluorescent ligands in contact areas between Jurkat cells and the bilayers. These studies provide two major conclusions. First, CD2/ligand interactions cooperate to align membranes with nanometer precision leading to a physiologically effective two-dimensional affinity. This process does not require the intact cytoplasmic tail of CD2. Second, the degree of membrane alignment that can be achieved by topologically similar receptors deteriorates with decreasing affinity. This suggests an affinity limit for the ability of this mode of cooperativity to achieve stable cell-cell adhesion at approximately 10 mM-1.     

Identification of the high affinity receptor binding region in human immunoglobulin E

We have investigated the capacity of N- and C-terminally truncated and chimeric human (h) IgE-derived peptides to inhibit the binding of 125I-labeled hIgE, and to engage cell lines expressing high and low affinity receptors (Fc-epsilon-RI/II). The peptide sequence Pro343-Ser353 of the hC-epsilon-3 domain is common to all h-epsilon-chain peptides that recognize hFc-epsilon-RI. This region in IgE is homologous to the A loop in C-gamma-2 that engages the rat neonatal IgG receptor. Optimum Fc-epsilon-RI occupancy by hIgE occurs at pH 6.4, with a second peak at 7.4. N- or C-terminal truncation has little effect on the association rate of the ligands with this receptor. Dissociation markedly increases following C-terminal deletion, and hFc-epsilon-RI occupancy at pH 6.4 is diminished. His residue(s) in the C-terminal region of the epsilon-chain may thus contribute to the high affinity of interaction. Grafting the homologus rat epsilon-chain sequence into hIgE maintains hFc-epsilon-RI interaction without conferring binding to rat Fc-epsilon-RI. hFc-epsilon-RII interaction is lost, suggesting that these residues also contribute to hFc-epsilon RII binding. h-epsilon-chain peptides comprising only this sequence do not block hIgE/hFc-epsilon-RI interaction or engage the receptor. Therefore, sequences N- or C-terminal to this core peptide provide structures necessary for receptor recognition.     

4-Azidophlorizin, a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative (2'-O-(beta-D-glucopyranosyl)-4-azidophloretin, 4-azidophlorizin) has been synthesized and its affinity for the D-glucose, Na+ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) Ki', the constant for fully-competitive inhibition of (Na+, delta psi)-dependent D-glucose uptake, (2) Kd', the dissociation constant of 4-azido[3H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) Ki", the constant for fully-competitive inhibition of the specific ((Na+, delta psi)-dependent, D-glucose protectable) high-affinity [3H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (Ki', Kd' and Ki") were essentially equal and ranged between 3.2 and 5.2 microM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its Ki' values are some 15-20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244-248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.     

Evidence for a high affinity, saturable, prenylation-dependent p21Ha-ras binding site in plasma membranes

Oncogenic p21ras proteins can only exert their stimulation of cellular proliferation when plasma membrane-associated. This membrane association has an absolute requirement for post-translational modification with isoprenoids. The mechanism by which isoprenoids participate in the specific association of p21ras with plasma membranes is the subject of this report. We present in vitro evidence for a plasma membrane binding protein for p21(ras) that can recognize the isoprenoid substituent and, therefore, may facilitate the localization of p21ras.     

Identification of glutathione S-transferase isozymes and gamma-glutamylcysteine synthetase as negative acute-phase proteins in rat liver

Because acute infection and inflammation affect drug metabolism and drug-metabolizing enzymes, the effect of the acute-phase response on the expression of glutathione S-transferase (GST) isoenzymes, glutathione synthesis, and several antioxidant enzymes was investigated. Hepatic expression of GST isozymes, positive and negative acute-phase reactants, and antioxidant enzymes were determined by Northern blotting and hybridization with gene-specific oligonucleotide probes after lipopolysaccharide treatment of rats. Lipopolysaccharide caused the expected acute-phase response as judged by the increased expression of positive and decreased expression of negative acute-phase proteins. The messenger RNA (mRNA) expression of the major hepatic rat GST isozymes A1, A2, A3, M1, and M2 was decreased 50% to 90%. Total hepatic GST activity toward 1-chloro-2,4-dinitrobenzene was also significantly decreased. mRNA expression of gamma-glutamylcysteine synthetase (GCS) large subunit and catalase was reduced by approximately 60%. GCS enzyme activity was also decreased, resulting in a 35% decrease in the hepatic content of reduced glutathione 4 days after lipopolysaccharide challenge. Mn-Superoxide dismutase expression was increased 13-fold, and thioredoxin level was elevated 3-fold after lipopolysaccharide challenge. The expression of all parameters determined returned to near control levels 7 days after treatment. Together, these data show that GSTs and GCS are negative acute-phase proteins and that decreased GCS activity results in a decrease in hepatic glutathione content. Thus, in addition to the phase I drug-metabolizing enzymes known to be decreased during the acute-phase response, some phase II enzymes involved in the elimination of xenobiotics and carcinogens are also decreased.     

Stimulatory effect of regucalcin on ATP-dependent calcium transport in rat liver plasma membranes

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca(2+)- Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 microM) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 microM) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 microM regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca(2+)-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes.     

Surface makers for mast cell subtypes: low affinity IgG receptors and gp49 family

Members of the Immunoglobulin Superfamily (Ig) present in the surface of rodent mast cells include the high affinity IgE receptor (Fc epsilon RI), the low affinity receptors for the Fc portion of IgG, the Fc gamma RII family and Fc gamma RIII as well as the recently cloned gp49 family that includes three members gp49A, gp49B1 and gp49B2. Fc epsilon RI and Fc gamma RIII are members of the multi-chain immune recognition receptor (MIRR) family since they possess a multimeric structure in which the signal transducing chains (gamma chains) contain six acids that conform the Antigen Homology Receptor 1 Motif (ARH1M), also present in the T cell receptor (TCR) transducing chains. gp49B1, gp49B2 and the FC gamma R family are monomeric chains that also contain the partial of full AHR1M motif in their cytoplasmic domain indicating the capability for signal transduction through tyrosine phosphorylation and the possibility of cell activation with mediator (s) or cytokine (s) release. Distribution of the Fc gamma R receptors and gp49 family members varies in the different stages of mast cell differentiation and maturation.     

Identification of a unique ligand which has high affinity for all four bombesin receptor subtypes

Four subtypes of bombesin receptors are identified (gastrin-releasing peptide receptor, neuromedin B receptor, the orphan receptor bombesin receptor subtype 3 (BB3 or BRS-3) and bombesin receptor subtype 4 (BB4)), however, only the pharmacology of the gastrin-releasing peptide receptor has been well studied. This lack of data is due in part to the absence of a general ligand. Recently we have discovered a ligand, 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) that binds to BRS-3 receptors. In this study we investigate its ability to interact with all four bombesin receptor subtypes. In rat pancreatic acini containing only gastrin-releasing peptide receptor and in BB4 transfected BALB cells, this ligand and 125I-[Tyr4]bombesin, the conventional gastrin-releasing peptide receptor ligand, gave similar results for receptor number, affinity for bombesin and affinity for the unlabeled ligand. In neuromedin B receptor transfected BALB cells, this ligand and 125I-[D-Tyr0]neuromedin B, the generally used neuromedin B receptor ligand, gave similar results for receptor number, neuromedin B affinity or the unlabeled ligand affinity. Lastly, in BRS-3 transfected BALB cells, only this ligand had high affinity. For all four bombesin receptors this ligand had an affinity of 1-8 nM and was equal or greater in affinity than any other specific ligands for any receptor. The unlabeled ligand is specific for gastrin-releasing peptide receptors on rat pancreatic acini and did not inhibit binding of 125I-cholecystokinin octapeptide (125I-CCK-8), 125I-vasoactive intestinal peptide (125I-VIP) or 125I-endothelin to their receptors. The unlabeled ligand was an agonist only at the gastrin-releasing peptide receptor in rat acini and did not interact with CCK(A) receptors or muscarinic M3 acetylcholine receptors to increase [3H]inositol phosphates. These results demonstrate 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) is a unique ligand with high affinity for all subtypes of bombesin receptors. Because of the specificity for bombesin receptors, this ligand will be a valuable addition for such pharmacological studies as screening for bombesin receptor agonists or antagonists and, in particular, for investigating BRS-3 cell biology, a receptor for which no ligand currently exists.     

Facile synthesis of high affinity styrylpyridine systems as inherently fluorescent ligands for the estrogen receptor

A series of styrylpyridine derivatives containing two phenols was prepared via an efficient two-step synthesis. These compounds were designed to maximize the estrogen receptor binding affinity of a known series of inherently fluorescent styrylpyridines. While significant improvements were achieved in receptor affinity, the fluorescence intensity of this series of compounds is poor.