Development of immunoassays for human interleukin 3 and interleukin 4, some of which discriminate between different recombinant DNA-derived molecules

Two panels of hybridomas were produced that secreted monoclonal antibodies (MAbs) against recombinant DNA-derived human interleukin 3 and interleukin 4 (rhIL-3 and rhIL-4). From each panel, sensitive immunoradiometric assays (IRMAs) were developed which were capable of detecting the recombinant molecule used as the immunogen but were unable to recognize natural or other recombinant forms of the same cytokine. Subsequent studies using the MAbs from each panel showed that a number of the MAbs appeared only to recognize that particular recombinant molecule used as immunogen, with little or no binding to other recombinant forms of the molecule. By using MAbs that were found to be unrestricted in their recognition for different recombinant forms of the cytokines, it was possible to develop an IRMA for IL-4 that was capable of detecting natural IL-4 as well as all the recombinant forms equally. An IRMA was also developed for IL-3 but was not of equivalent sensitivity in detecting the different recombinant forms of IL-3 used in the study. The recombinant DNA-derived cytokine molecules used to raise the two panels of MAbs contained amino acid substitutions relative to the natural sequences, and these findings indicate that caution should be exercised when using immunoassays to estimate natural sequence molecules if antibodies raised to modified rDNA-derived molecules are used.     

Inhibition of Microsomal Lipid Peroxidation and Cytochrome P-450-Catalyzed Reactions by Nitrofuran Compounds

(5-Nitro-2-furfuryliden)amino compounds bearing triazol-4-yl, benzimidazol-l-yl, pyrazol-l-yl, triazin-4-yl or related groups (a) stimulated superoxide anion radical generated by rat liver microsomes in the presence of NADPH and oxygen; (b) inhibited the NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (c) prevented the NADPH-dependent destruction of cytochrome P-450; (d) inhibited the NADPH-dependent microsomal aniline 4-hydroxylase activity; (e) failed to inhibit either the cumenyl hydroperoxide-dependent lipid peroxidation or the aniline-4-hydroxylase activity, except for the benzimidazol-l-yl and the substituted triazol-4-yl derivatives, which produced minor inhibitions. Reducing equivalents enhanced the benzimidazol-l-yl derivative inhibition of the cumenyl hydroperoxide-induced lipid peroxidation. The ESR spectrum of the benzimidazol-l-yl derivative, reduced anaerobically by NADPH-supplemented microsomes, showed characteristic spin couplings. Compounds bearing unsaturated nitrogen heterocycles were always more active than those bearing other groups, such as nifurtimox or nitrofurazone. The energy level of the lowest unoccupied molecular orbital was in fair agreement with the capability of nitrofurans for redox-cycling and related actions. It is concluded that nitrofuran inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions was mostly due to diversion of reducing equivalents from NADPH to dioxygen. Trapping of free radicals involved in propagating lipid peroxidation might contribute to the overall effect of the benzimidazol-l-yl and substituted triazol-4-yl derivitives.     

Expression of streptomycete cholesterol oxidase inEscherichia coli

Summary A streptomycete gene coding for extracellular cholesterol oxidase (choA) was subcloned and expressed inEscherichia coli. The pUCO series recombinants were obtained by inserting thechoA gene into the uniqueKpnI site of pUC19 vector. Expression was observed with pUCO192A and pUCO193 constructs in which the cloned gene(s) were aligned with the upstreamlacZ promoter. Isopropyl -d-thioglucopyranoside (IPTG) enhanced this expression up to 2.5-fold. Specific Cho activity in the cell extracts of the stable pUCO193 transformant were 0.004 U and 0.007 U per mg protein without and with IPTG induction, respectively. Cho activity was detected in the spent medium of this culture, suggesting possible secretion of the enzyme.     

Uptake and Distribution of Iron and Transferrin in the Adult Rat Brain

Brain uptake of iron-59 and iodine-125-labelled transferrin from blood in the adult rat has been investigated using graphical analysis to determine the blood-brain barrier permeability to these tracers in experiments that lasted between 5 min and 8 days. The blood-brain barrier permeability (K(in)) to 59Fe was 89 x 10(-5) ml/min/g compared to the value of 7 x 10(-5) ml/min/g for 125I-transferrin, which is similar to that of albumin, a plasma marker. The autoradiographic distribution of these tracers in brain was also studied to determine any regional variation in brain uptake after the tracers had been administered either systemically or applied in vitro. No regional uptake was seen for 125I-transferrin even after 24 h of circulation. In contrast, 59Fe showed selective regional uptake by the choroid plexus and extra-blood-brain barrier structures 4 h after administration. After 24 h of circulation, 59Fe distribution in brain was similar to the transferrin receptor distribution, as determined in vitro, but was unlike the distribution of nonhaem iron determined histochemically. The data suggest that brain iron uptake does not involve any significant transcytotic pathway of transferrin-bound iron into brain. It is proposed that the uptake of iron into brain involves the entry of iron-loaded transferrin to the cerebral capillaries, deposition of iron within the endothelial cells, followed by recycling of apotransferrin to the circulation. The deposited iron is then delivered to brain-derived transferrin for extracellular transport within the brain, and subsequently taken up via transferrin receptors on neurones and glia for usage or storage.     

Activation of Metabotropic Glutamate Receptors in the Hippocampus Increases Cyclic AMP Accumulation

The selective metabotropic glutamate receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulates phosphoinositide hydrolysis and elicits several physiological responses in rat hippocampal slices. However, recent studies suggest that the physiological effects of trans-ACPD in the hippocampus are mediated by activation of a receptor that is distinct from the phosphoinositide hydrolysis-linked receptor. Previous experiments indicate that cyclic AMP mimics many of the physiological effects of trans-ACPD in hippocampal slices. Furthermore, recent cloning and biochemistry experiments indicate that multiple metabotropic glutamate receptor subtypes exist, some of which are coupled to yet unidentified effector systems. Thus, we performed a series of experiments to test the hypothesis that ACPD increases cyclic AMP levels in hippocampal slices. We report that 1S,3R- and 1S,3S-ACPD (but not 1R,3S-ACPD) induce a concentration-dependent increase in cyclic AMP accumulation in hippocampal slices. This effect was blocked by the metabotropic glutamate receptor antagonist L-2-amino-3-phosphonoproprionic acid but not by selective antagonists of ionotropic glutamate receptors. Furthermore, our results suggest that 1S,3R-ACPD-stimulated increases in cyclic AMP accumulation are not secondary to increases in cell firing or to activation of phosphoinositide hydrolysis.     

The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP

Glibenclamide closes an ATP-sensitive K+ channel (K-ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonyl-ureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40-50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg-ATP (300 microM) reduced the number of high-affinity sites for [3H]-glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg-ATP was half-maximally effective at 41 microM. Alkaline phosphatase accelerated the reversal of Mg-ATP-induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.     

Effects of Monovalent and Divalent Cations on 3-(+)[125I]Iododizocilpine Binding to the N-Methyl-d-Aspartate Receptor of Rat Brain Membranes

We have investigated the binding of 3-[125I]iododizocilpine ([125I]iodo-MK-801) to the N-methyl-D-aspartate (NMDA) receptor in well-washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine greater than thienylphencyclidine greater than phencyclidine greater than ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS-19755, 7-chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 microM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.     

Ca2+ -Dependent Release from Rat Brain of Cannabinoid Receptor Binding Activity

As a result of the identification, pharmacological characterization, and localization of the cannabinoid receptor in the CNS, the existence of an endogenous ligand for this receptor can be hypothesized. Following the premise that such a substance could have the properties of a neuromodulator being stored in intracellular vesicles, we tested the ability of increased intracellular Ca2+ levels to stimulate release. We demonstrate here that the Ca2+ ionophore A23187 can induce release of cannabinoid-like binding activity in the presence but not in the absence of Ca2+. The effect of A23187 was maximal at 1.2 microM, consistent with vesicular release. It was necessary to increase the concentration of extracellular free Ca2+ to greater than 60 nM to evoke release. The released cannabinoid-like binding activity displaced [3H]CP-55940 binding to cannabinoid receptors in rat synaptosomal membranes in a concentration-dependent manner. This is the first report of a substance present endogenously in brain that can be released in a Ca(2+)-dependent manner and that binds to the cannabinoid receptor.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Solubilization and Purification of an α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding Protein from Bovine Brain

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X-100 in 0.5 M phosphate buffer and 20% glycerol at 37 degrees C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 nM and Bmax values of 670 and 1,073 pmol/mg of protein for the high- and low-affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [3H]AMPA binding activity at Mr approximately 500,000. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at Mr = 110,000. Previously, we have shown that a monoclonal antibody (KAR-B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at Mr approximately 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.