Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors

N-[3H]Methylscopolamine has been used to characterize muscarinic receptors in crude homogenates prepared from hearts of Syrian golden hamsters. The Hill coefficient is one for specific binding of the radioligand itself and for its inhibition by muscarinic antagonists; markedly lower values are obtained for its inhibition by muscarinic agonists. The binding patterns of agonists have been analyzed in terms of a mixture of sites differing in affinity for the drug and reveal the following. All agonists discern at least two classes of receptor in atrial and ventricular homogenates. The number of classes and the relative size of each differ for different agonists in the same region and for the same agonist in different regions. Atrial and ventricular affinities are in good agreement for some agonists but differ for others. Guanylyl imidodiphosphate (GMP-PNP) is without effect on the specific binding of the radioligand but alters the binding of carbachol via an apparent redistribution of receptors from one class to another; the apparent affinity at either class remains unchanged. Carbachol reveals two classes of sites in ventricular preparations, and the nucleotide mediates an interconversion from higher to lower affinity; three classes are revealed in atrial preparations, and the nucleotide eliminates the sites of highest affinity with a concomitant increase in the number of sites of lowest affinity. Taken together, the data are incompatible with the notion of different, noninterconverting sites; rather, there appear to be several possible states of affinity such that the equilibrium distribution of receptors among the various states is determined by the tissue, by the agonist, and by neurohumoral modulators such as guanylyl nucleotides. The effects of agonists and GMP-PNP cannot be rationalized in terms of a ternary complex model in which the low Hill coefficients arise from a spontaneous equilibrium between receptor (R) and G protein (G) and in which agonists bind preferentially to the RG complex.     

A spectrin-like protein in retinal rod outer segments

Biochemical and immunochemical studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the alpha-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghosts and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labeled the alpha-chain of RBC spectrin having an apparent molecular weight of 240,000. Polyclonal anti-spectrin antibody that bound to both the alpha- and beta-chain of RBC spectrin predominantly labeled a Mr 240,000 polypeptide of ROS membranes. Two faintly labeled bands in the molecular weight range of 210,000-220,000 were also observed. These components may represent variants of the beta-chain of spectrin that are weakly cross-reacting or present in smaller quantities than the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regioselectivity in rat microsomal metabolism of benzo[a]pyrene: evidence for involvement of two distinct binding sites

The metabolic profile of benzo[a]pyrene (BP) in cumene hydroperoxide-(CHP)-dependent reaction by male rat liver microsomes was dependent on CHP concentration. At 0.05 mM CHP, 3-hydroxy-BP was the major metabolite. Increase in CHP reduced 3-hydroxy-BP formation but increased BP quinone formation simultaneously. This change in metabolic profile was reversed by preincubation with pyrene. Pyrene (PY) selectively inhibited quinone formation but enhanced 3-hydroxy-BP formation. Naphthalene (NP) had no effect on BP quinone formation but inhibited BP 3-hydroxylation. Phenanthrene (PA) and benz[a]anthracene (BA) inhibited effectively 3-hydroxy-BP formation but only slightly quinone formation. BP binding to microsomal protein correlated to quinone formation and not BP 3-hydroxylation. BP metabolism by female rat liver microsomes also depended on CHP concentration but was much less efficient than the male. Quinones were consistently predominant metabolites and their formation was also inhibited by pyrene. Our data provide evidence that regioselectivity in BP metabolism involves at least two distinct binding sites. One site recognizes the benzo region of BP in BP 3-hydroxylation and the other recognizes the pyrene region in quinone formation. The different ratios of 3-hydroxy-BP to quinone formation by male and female rat liver microsomes suggest that the two binding sites are probably located at separate cytochrome P-450 isozymes.     

Conjugational fertility and location of chloramphenicol biosynthesis genes on the chromosomal linkage map of Streptomyces venezuelae

In Streptomyces venezuelae fertility, defined as chromosomal gene recombination, was enhanced over 1000-fold when one parent in a biparental conjugational cross lacked the physically-undetected plasmid SVP1, as compared with crosses in which both parents carried SVP1. The existence of SVP1 and at least two other fertility plasmids, SVP2 and SVP3, was detected in S. venezuelae by 'lethal zygosis' elicited by a plasmid-plus mycelium in contact with a plasmid-minus mycelium. Conjugational crosses were used to construct a linkage map of S. venezuelae which was highly consistent with the map of analogous loci in S. coelicolor A3(2). A cluster of genes governing chloramphenicol biosynthesis was located near arg, cys and pdxB genes at a position roughly equivalent to the 1-2 o'clock region of the S. coelicolor A3(2) map.     

Mutant allele frequencies in cats of Minneapolis-St. Paul

Coat color phenotype frequencies were determined in the cat population of Minneapolis and St. Paul. Mutant allele frequencies are estimated to be p (O) = 0.287, q(a) = 0.742, q(d) = 0.635, q(l) = 0.507, p(S) = 0.288. q(tb) = 0.472, p(W) = 0.016, and q(cs) = 0.214. A substantial number of cats displaying the Siamese coat pattern were found. These cats have a long history in the population.     

Suppressor T lymphocytes from lepromatous leprosy skin lesions

The immune response in leprosy forms a spectrum with lepromatous leprosy patients exhibiting specific unresponsiveness to antigens of Mycobacterium leprae. This unresponsiveness is thought to be related to the prevalence of T8-positive lymphocyte in these lepromatous lesions. To analyze the immunoregulatory function of these T8 cells, we developed simple procedures to extract lymphocytes from skin biopsy specimens of patients with leprosy. These lymphocytes were sorted for T8 and T4 positive cells, and cell lines were established by expansion with interleukin 2 (IL 2) and irradiated feeder cells. All T8 positive lines tested were positive for IL 2 receptors and HLA-DR determinants. These lines were additionally assayed for lepromin-induced suppression of the normal peripheral blood lymphocyte Con A proliferative response. Thirteen of 32 lines from six lepromatous patients showed significant suppressor activity, whereas nine lines from six tuberculoid patients and one line from normal peripheral blood failed to show suppression (p less than 0.001). Taken together, the finding of M. leprae-triggered suppressor cells within lepromatous skin lesions may in part explain the M. leprae unresponsiveness of lepromatous leprosy patients.     

Identification and mapping of Epstein-Barr virus early antigens and demonstration of a viral gene activator that functions in trans.

The BamHI M DNA fragment of the Epstein-Barr virus (EBV) genome was inserted in two orientations into a simian virus 40-based expression vector, and the EBV-specific proteins produced in COS-7 monkey cells were examined. In one orientation, termed BamHI-M rightward reading frame 1 (BMRF1), a set of phosphoproteins ranging in size from 47,000 to 54,000 daltons was synthesized. These proteins reacted with monoclonal and polyclonal antisera, defining them as components of the EBV early antigen diffuse set of proteins (EA-D). The BamHI M DNA fragment in the opposite orientation, termed BamHI-M leftward reading frame 1 (BMLF1), directed the synthesis of a nuclear antigen detected by antibodies in serum from a patient with nasopharyngeal carcinoma. The BMLF1 antigen was not detected by monoclonal or polyclonal antibodies directed against the EA-D complex. A series of deletion mutants were constructed in the BamHI M DNA fragment, and the EA-D complex and BMLF1 antigen were mapped to discrete open reading frames in this DNA fragment. A test for several possible functions of these antigens showed that the BMLF1 antigen had the ability to activate or enhance, in trans, the level of expression of a gene under the control of the adenovirus early region 3 promoter or the simian virus 40 early promoter in the absence of its cis-acting enhancer. These experiments demonstrate a new gene function, encoded by EBV, that may be important in the positive regulation of viral or cellular genes.     

Inter-relationship between shoot weight,severity of root rot and survival rate of subterranean clover inoculated with certain pathogenic fungi

Summary Plant survival rate, root disease index and fresh shoot weight of subterranean clover seedlings inoculated with fungal pathogens (Fusarium avenaceum, F. oxysporum, Phoma medicaginis, Pythium irregulare andRhizoctonia solani, both singly and in combinations) were generally inter-correlated over a wide range of temperature (10, 15, 20 and 25°C) and moisture conditions (45, 65% water holding capacity and flooding). There was a negative correlation between root disease index and shoot weight for all treatments exceptF. avenaceum + P. irregulare. Root disease index and seedling survival rate were negatively correlated except forF. oxygsporum, Phoma medicaginis, P. irregulare andF. oxysporum + F. avenaceum. However, a good positive correlation was found between the survival rate and shoot weight for all treatments with the exception ofPhoma medicaginis.     

Simultaneous Measurements of Steady State Chlorophyll a Fluorescence and CO(2) Assimilation in Leaves: The Relationship between Fluorescence and Photosynthesis in C(3) and C(4) Plants

Rates of CO₂ assimilation and steady state chlorophyll a fluorescence were measured simultaneously at different intercellular partial pressures of CO₂ in attached cotton (Gossypium hirsutum L. cv Deltapine 16) leaves at 25°C. Electron transport activity for CO₂ assimilation plus photorespiration was calculated for these experiments. Under light saturating (1750 microeinsteins per square meter per second) and light limiting (700 microeinsteins per square meter per second) conditions there was a good correlation between fluorescence and the calculated electron transport activity at 19 and 200 millibars O₂, and between fluorescence and rates of CO₂ assimilation at 19 millibars but not 200 millibars O₂. The values of fluorescence measured at about 220 microbars intercellular CO₂ were not greatly affected by increasing O₂ from 19 to 800 millibars. Fluorescence increased with light intensity at any one intercellular CO₂ partial pressure. But the values obtained for fluorescence, expressed as a ratio of the maximum fluorescence obtained in DCMU-treated tissue, over the same range of CO₂ partial pressure at 500 microeinsteins per square meter per second were similar to those obtained at 1000 and 2000 microeinsteins per square meter per second. There were two phases in the observed correlation between fluorescence and calculated electron transport activity: an initial inverse relationship at low CO₂ partial pressures which reversed to a positive correlation at higher values of CO₂ partial pressures. Similar results were observed in the C₃ species Helianthus annuus L., Phaseolus vulgaris L., and Brassica chinensis. In all C₄ species (Zea mays L., Sorghum bicolor L., Panicum maximum Jacq., Amaranthus edulis Speg., and Echinochloa frumentacea [Roxb.] Link) examined changes in fluorescence were directly correlated with changes in CO₂ assimilation rates. The nature and the extent to which Q (primary quencher) and high-energy state (q_E) quenching function in determining the steady state fluorescence obtained during photosynthesis in leaves is discussed.