Regulation of Muscarinic Acetylcholine Receptor Concentration in Cloned Neuroblastoma Cells

Abstract: The concentration of muscarinic acetylcholine receptors in the neuroblastoma cell line NIE-115 is regulated by receptor activation. Muscarinic agonists cause a time and dose-dependent loss of [³H]quinuclidinyl benzilate binding sites from cultured cells. Muscarinic antagonists have no effect on receptor concentration and block agonist-induced regulation. The maximum decrease in steady state receptor levels is 80% and occurs within 9 h. The altered steady state concentration persists as long as agonist remains present. Upon withdrawal of agonist, the concentration of receptors returns to control levels. This increase requires protein synthesis. Kinetically, the increase in receptors following withdrawal of agonist is slower than the decrease caused by addition of agonist, suggesting that bursts of receptor activation could lower receptor levels. In harmony with this prediction, cycles in which receptors are active for 15 min and then inactive for 15 min cause a 50% decrease in receptor concentration in a 6-h period.     

Intermediate Filaments from Bovine, Rat, and Human CNS: Mapping Analysis of the Major Proteins

Abstract: Intermediate filaments were isolated by an axon-flotation method from bovine, rat, and human CNS. Gel electrophoresis showed four major proteins, having molecular weights of about 50,000, 70,000, 160,000, and 210,000, to be present in filaments of all three species. Small differences in molecular weights and major differences in relative distribution of the filament proteins were observed among species. In bovine and rat brain the predominant protein was the 50,000 band, but in human brain the 70,000 band was present in greatest amount. Each filament protein of the three species was studied by peptide mapping using limited proteolysis and cyanogen bromide cleavage. Within the same molecular weight group, filament proteins from different species gave similar maps with both techniques. Some degree of heterogeneity was also observed. However, filament proteins of different molecular weights of the same species gave distinctly different maps. These studies rule out the possibility that filament proteins from different molecular weight groups are related to each other by oligomerization; nor is it likely that the lower molecular weight proteins are derived from the subunit of molecular weight 210,000.     

Influence of Estrogen and Progesterone on Glutamic Acid Decarboxylase Activity in Discrete Regions of Rat Brain

Abstract: Female Charles River rats were ovariectomized and treated for three days with 17/8-estradiol benzoate (E) (1.0 /μg/day), progesterone (P) (500 μg/day), vehicle, or a combined treatment (2 days E, one day P). Animals were killed on day 3 and the brains were dissected by the micropunch technique. Glutamic acid decarboxylase (GAD) activity was measured by collection of ¹⁴CO². Estradiol benzoate and progesterone were potent inhibitors of GAD activity in regions such as the arcuate nucleus, ventromedial hypothalamus and corticomedial amygdala. Estrogen reduced the V_max of GAD for glutamate as a substrate without changing the K_m. Estrogen also failed to change the K_m for pyridoxal phosphate. Combined treatment with estrogen and progesterone did not reduce GAD from ovariectomized levels except in the septum, indicating an interaction of the two hormones at the level of GAD. The suggestion is made that under conditions that inhibit LH secretion GAD activity is low, but when LH secretion is stimulated GAD activity may be comparatively high.     

Catecholamine-Sensitive Guanylate Cyclase from Human Caudate Nucleus

Abstract: Partial purification of soluble guanylate cyclase on DEAE-Sephacel yields two separate peaks of guanylate cyclase activity. After 10-fold purification of the soluble enzyme, guanylate cyclase is markedly inhibited by micromolar concentrations of dopamine (I₅₀= 0.2 μm). Dopamine inhibition is observed whether the reaction is conducted with Mn²¹ or with Mg²⁺, under atmosphere or N₂(g), and using enzyme from either peak from the DEAESephacel column. Other catecholamines also inhibit partially purified guanylate cyclase with an order of potency at 1 μm of: dopamine =l -DOPA > norepinephrine = isoproterenol = adrenochrome > epinephrine. The structural requirements for inhibition are two free hydroxyl groups on the phenyl ring and an ethylamine side chain. Dopamine also inhibits the Triton X-100-solubilized microsomal guanylate cyclase after partial purification on DEAESephacel. Neither chlorpromazine, propranolol, nor phentolamine at 20 μm effectively block the dopamine inhibition of partially purified soluble guanylate cyclase. Micromolar concentrations of the reducing agents dithiothreitol and glutathione also inhibit partially purified guanylate cyclase, but unlike these agents, catecholamines can inhibit whether added in the reduced or the oxidized forms. Inhibition of enzyme activity by micromolar concentrations of dopamine, adrenochrome, or dithiothreitol is rapidly reversed by dilution and the dopamine inhibition is competitive with MgGTP. Inhibition does not appear to involve covalent binding or to result from the ability of catecholamines to reduce the concentrations of oxygen or free radicals in solution.     

Glutathione and Ascorbate During Ischemia and Postischemic Reperfusion in Rat Brain

Thirty minutes of total cerebral ischemia (decapitation) decreased total glutathione (GSH + GSSG) by 7% but had no detectable effect on the concentration of oxidized glutathione (GSSG), reduced ascorbate, or total ascorbate. In a model of reversible, bilateral hemispheric ischemia (four-vessel occlusion) no changes in glutathione or ascorbate were detected after 30 min of ischemia. During 24 h of reperfusion following such an insult no detectable change in total ascorbate, reduced ascorbate, or oxidized glutathione was noted; however, total brain glutathione declined by 25%. The findings are discussed in relation to the hypothesis that the deleterious effects of ischemia are due to an increase in free radical production which in turn leads to increased lipid peroxidation.     

Muscarinic receptor binding in rat brain using the agonist, [3H]cis methyldioxolane

Muscarinic receptor binding was measured in rat forebrain preparations using the muscarinic agonist, [³H]cis methyldioxolane ([³H]CD). The results of equilibrium binding studies using [³H]CD concentrations between 0.5–64 nM showed that [³H]CD binding did not saturate in this concentration range, although the binding isotherm was concave downward. Nonlinear regression analysis of the binding data revealed the presence of two populations of muscarinic receptors having dissociation constants of 1.83 and 123 nM and binding capacities of 85 and 1320 fmol/mg protein, respectively. Competitive inhibition experiments showed that [³H]CD binding was readily displaced by several muscarinic agonists and antagonists. The stereospecificity of [³H]CD binding was demonstrated in competitive inhibition experiments using the stereoisomers of benzetimide and acetyl-β-methylcholine. Dexetimide was 10,000 times more potent than levetimide and l-acetyl-β-methylcholine was 520 times more potent than d-acetyl-β-methylcholine. A variety of nonmuscarinic cholinergic drugs were not effective at inhibiting [³H]CD binding at a concentration of 10 μM.     

The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa

Summary The amidase genes of Pseudomonas aeruginosa were inserted into a replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant ami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with ami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of ami were made by introducing Q ^-, S ^- mutations. Cultures of E. coli infected with amiQ ^- S ^- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificity, thermal stability and immunological crossreaction.     

Wineries,drosophila, alcohol,and Adh

Summary Previous workers (McKenzie and Parsons, 1972, 1974; McKenzie, 1974; Briscoe et al., 1975) have found anomalous distributions of species of Drosophila, of sexes of D. melanogaster, and of Adh alleles in and around wineries in Australia and Spain. Field studies in California's Sonoma Valley provide evidence that the explanations advanced for these distributions may incorrect. The anomalous distribution of species was attributed to alcohol, either as a selective agent or as a behavioral stimulus. We find a virtually identical species distribution in the absence of environmental alcohol. The anomalous sex ratio was attributedd to differential survivall of the sexes when raised on alcohol. We present crude evidence thatehe difference may simply be a behavioral response to some product of fermentation, which need not be alcohol. Finally, the allele frequency difference reported from Spain was attributed to differential adult mortality on alcohol. We do not find an allele frequency difference even when alcohol is exposed, and therefore suggest that selection is occurring in pre-adult stages.     

Halogen chemistry of the red alga Bonnemaisonia

Complete accounts of the natural products chemistry of Bonnemaisonia nootkana, B. asparagoides, B. hamifera and Trailliella intricata are described. In contrast to the chemistry of the closely related alga Asparagopsis, Bonnemaisonia spp. do not produce halomethanes, but instead an array of C₇-C₉ halogen-containing ketones, alcohols and carboxylic acids. Biomimetic syntheses of these compounds suggest they are precursors and products of in vivo Favorsky rearrangements.     

Human plasma-derived immunosuppressive factors having anti-lymphoma activity

Summary Extraction of Cohn IV-1, an -globulin enriched fraction of human plasma, with a high-salt, low-pH solution, followed by sequential ultrafiltration steps yielded an immunosuppressive preparation (UM05R) of mol.wt. 500–10,000. UM05R inhibited antibody formation in the mouse in vivo and transformation in vitro of lymphocytes treated with either T-or B-cell stimulants. Suppression of lymphocyte transformation, indicated by inhibition of ³H-thymidine incorporation into DNA, was confirmed by inhibition of blast cell formation. From dose-response curves the UM05R concentration to produce 50% suppression of lymphocyte blast transformation was 15–50 g protein/ml. Selectivity for lymphoid cells was suggested by growth inhibition in vitro of L1210 and P1798 leukemias but not murine neuroblastoma or human fetal fibroblasts. This observation also rules out the presence of an agent which is broadly cytotoxic. Fractionation of UM05R on Sephadex G-25 in 10% acetic acid yielded an early-emerging fraction, mol. wt. 5,000–10,000, containing B-cell inhibitor, and a late fraction, mol. wt. 1,400, inhibitory for both T- and B-cell transformation and growth of L1210. The inhibitory activity for B cells was removed from the other two activities by 5% trichloroacetic acid (TCA). The possibility is raised that the inhibitory activity for T cells and L1210 may reside in the same molecule. Sensitivity of the early-emerging B-cell inhibitor to carboxypeptidase B suggests that it is a polypeptide, but resistance of the T-cell inhibitor to various treatments leaves its nature uncertain. The properties of these factors suggest consideration of them as lymphocyte chalones occurring in plasma complexed to high-molecular-weight components.