Molecular basis for the interaction of histamine with the histamine H2 receptor.

We undertook these studies to characterize the molecular basis of the interaction of histamine with the H2 receptor. Key areas of homology in the structures of the histamine H2 and beta 2 adrenergic receptor suggested specific transmembrane amino acids that might be important for binding of histamine. A third transmembrane aspartic acid of the histamine receptor (Asp98), thought to serve as a counter anion that interacts with the cationic amine moiety of histamine, was mutated to Asn98, and the mutated receptor was expressed in Hepa cells. Removal of the negatively charged amino acid abolished both binding of the H2 receptor antagonist [methyl-3H]tiotidine and histamine stimulated increases in cellular cAMP content. Mutation of a fifth transmembrane aspartic acid (Asp186) to Ala186 or Asn186 by itself or in conjunction with mutation of another fifth transmembrane amino acid (Thr190 to Ala190) resulted in a loss of [methyl-3H] tiotidine binding, although the generation of cAMP in response to histamine was maintained. The histamine receptor with only a Thr190 to Ala190 or Cys190 mutation retained the ability to bind [methyl-3H]tiotidine, but both the affinity and efficacy of binding were reduced. These data lead us to propose a model for histamine binding in which Asp98 is essential for histamine binding and action, Asp186 defines H2 selectivity, and Thr190 is important in establishing the kinetics of histamine binding, but is not essential for H2 selectivity.     

A multifunctional cell surface developmental stage-specific antigen in the cockroach embryo: involvement in pathfinding by CNS pioneer axons

mAb DSS-8 binds to a 164-kD developmental stage-specific cell surface antigen in the nervous system of the cockroach, Periplaneta americana. The antigen is localized to different subsets of cells at various stages of development. The spatial and temporal distributions of DSS-8 binding were determined and are consistent with this antigen playing multiple roles in the development of the nervous system. Direct identification of some of these functions was made by perturbation experiments in which pioneer axon growth occurs in embryos that are cultured in vitro in the presence of mAb DSS-8 or its Fab fragment. Under these conditions the pioneer axons of the median fiber tract grow but follow altered pathways. In a smaller percentage of the ganglia, the immunoreagents additionally produce defasciculation of a subset of DSS-8 labeled axons. Therefore, direct roles for the DSS-8 antigen in both the guidance of pioneer axons and selective fasciculation have been demonstrated.     

Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Ca(2+)-dependent release of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-7-nitroquinoxaline-2,3- dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.     

Nuclear entry and nucleolar localization of the Newcastle disease virus (NDV) matrix protein occur early in infection and do not require other NDV proteins.

A large proportion of the Newcastle disease virus (NDV) matrix (M) protein is found in the nuclei of infected chicken embryo cells. Kinetic analysis indicated that much of the M protein enters the nucleus early in infection, concentrating in discrete regions of the nucleus and remaining there throughout infection. The M protein was found in localized regions of the nuclei of a variety of cell lines infected with NDV. Immunostaining for both M protein and nucleolar antigens indicated that most of these regions represent nucleoli. Moreover, this nucleolar localization of the M protein was observed in chicken embryo cells infected with 11 different strains of NDV. Only the M protein of strain HP displayed a modified pattern, concentrating in the nucleolus early in infection but in the cytoplasm late in infection. M protein transiently expressed in COS-1 cells also localized to the nucleus and nucleolus, indicating that the M protein does not require other NDV proteins for this localization.     

Conformational perturbation of the anticancer nucleotide arabinosylcytosine on Z-DNA: molecular structure of (araC-dG)3 at 1.3 A resolution.

The left-handed Z-DNA structure of an araC-containing (where araC stands for arabinosylcytosine) hexamer, (araC-dG)3, has been solved by x-ray diffraction analysis at 1.3 A resolution. This hexamer was crystallized in the hexagonal P6(5)22 (a = b = 17.96 A, c = 43.22 A) space group in which the hexamers have statistically disordered packing arrangement along the 6(5) screw axis, yet the crystals diffract x-rays to high resolution. Its structure has been refined by the constrained least square refinement to a final R factor of 0.287 using 737 [> 3.0 sigma(F)] observed reflections. The asymmetric unit of the unit cell contains only a dinucleotide, 5'-p (araC)p(dG). The overall conformation resembles that of the canonical Z-DNA, but with some differences in details. The O2' hydroxyl groups of the araC residues form intramolecular hydrogen bonds with N2 of the 5'-guanine residues. In the deep groove of Z-DNA, these hydroxy groups replace the bridging water molecules that stabilize the guanine in the syn conformation. The results reinforce the earlier observation made by the structural analysis of another hexamer, d(CG[araC]GCG), with a mono-substitution of araC [M.-K. Teng, Y.-C. Liaw, G. A. van der Marel, J. H. van Boom, and A. H.-J. Wang (1989) Biochemistry, vol. 28, pp. 4923-4928]. These two structures show that araC residue can be incorporated readily into the Z structure and probably facilitates the B to Z transition, as supported by uv absorption spectroscopic studies in a number of araC-containing oligonucleotides. The potential biological roles of the araC-modified Z-DNA are discussed.     

Effect of adsorbents on degradation of toxic organic compounds by coimmobilized systems

The use of coimmobilized systems for treatment of toxic organic compounds has been proposed. The proposed approach combines the use of adsorbents and laboratory identified microorganisms immobilized in a protective permeable barrier to achieve a greater degree of control over the remediation process. This study was launched to understand the effect of adsorbents and changes in adsorption on the degradation of toxic compounds by coimmobilized systems. The specific case studied involved the degradation of pentachlorophenol (PCP) by Arthrobacter (ATCC 33790) coimmobilized with powdered activated carbon within calcium alginate capsules.The design parameters studied included adsorbent content and type as well as the effect of solution pH and surfactant concentration on adsorption and biodegradation. It was found that the equilibrium adsorption behavior of PCP was strongly influenced by solution pH and surfactant concentration. A mathematical model was developed that combined the physical processes of mass transfer and adsorption with biological degradation of PCP. The model was used to predict the effect of various parameters on the degradation of PCP. Based on model predictions, the degradation of PCP. Based on model predictions, the degradation of PCP was strongly dependent on variations in adsorbent capacity and affinity for this contaminant.     

A comparison of neural networks and partial least squares for deconvoluting fluorescence spectra

This article compares backpropagation neural networks (BNN) with partial least squares (PLS) techniques in terms of their ability to deconvolute fluorescence spectra. Both actual experimental and simulated spectral data are studied for 2 binary systems. These systems consist of mixtures of tryptophan and tyrosine, and NADH and tryptophan over a total concentration range of 10(-7) to 10(-4) M. It is shown that BNN is superior to PLS for both systems.