Calcium signaling by cyclic ADP-ribose and NAADP

Ca²⁺ mobilization as a signaling mechanism has been placed on center stage with the discovery of the first Ca²⁺ messenger, inositol trisphosphate (IP₃). This article focuses on two new Ca²⁺ release activators, which mobilize internal Ca²⁺ stores via mechanisms totally independent of IP₃. They are cyclic ADP-ribose (cADPR) and nicotinic acid dinucleotide phosphate (NAADP), metabolites derived respectively from NAD and NADP. Major advances in the past decade in the understanding of these two novel signaling mechanisms are chronologically summarized.     

Gonadal Steroid Hormones and Hypothalamic Opioid Circuitry

Endogenous opioid peptides derived from several gene families are localized within hypothalamic regions known to be involved in the regulation of reproduction. For example, the proenkephalin gene products, met- and leu-enkephalin, and the proopiomelanocortin (POMC) gene product, β-endorphin, are found in the rat medial preoptic area (MPOA). Moreover, the expression of these peptides and their receptors varies across the estrous cycle in the female rat. We have examined the gonadal steroid regulation of μ-opiate receptors and opioid peptides in the MPOA, and POMC mRNA expression in neurons that innervate the MPOA. μ-Opiate receptors in the MPOA are sexually dimorphic and gonadal steroid hormone-dependent. Hormonal priming of ovariectomized rats with estrogen and progesterone (P) upregulates MPOA μ-receptors 27, but not 3, hr after P treatment. Inhibition of protein synthesis during the first 6 hr after P prevents receptor upregulation, The density of β-endorphin fibers in the MPOA also increases following hormone treatment, and POMC mRNA expression in neurons that innervate the MPOA is induced by hormone treatment beginning 13 hr after P treatment. This delayed response might be ubiquitous among POMC neurons, as those innervating the median eminence also exhibit increased POMC mRNA expression along a similar time course. The results suggest that hormonal feedback regulates opioid peptides which act at μ-receptors in the MPOA to influence reproductive behavior and cyclicity. These opioid functions represent an important component in the complex regulatory processes which control reproduction.     

A cell surface protein that binds avian hepatitis B virus particles.

We have identified a 180-kDa cellular glycoprotein (gp180) that binds with high affinity to duck hepatitis B virus (DHBV) particles. The protein was detected by coprecipitating labeled duck hepatocyte proteins with virions or recombinant DHBV envelope proteins, using nonneutralizing monoclonal antibodies to the virion envelope. Binding of gp180 requires only the pre-S region of the viral large envelope protein, since recombinant fusion proteins bearing only this region efficiently coprecipitate gp180. The DHBV-gp180 interaction is blocked by two independent neutralizing monoclonal antibodies. The protein is found on both internal and surface membranes of the cell, and the species distribution of gp180 binding activity mirrors the known host range of DHBV infection. Functional gp180 is expressed in a wide variety of tissues in susceptible ducks.     

Key Features of Cereal Genome Organization as Revealed by the Use of Cytosine Methylation-Sensitive Restriction Endonucleases

Unlike mammalian genomes, cereal (Gramineae) genomes exhibit little suppression of CpG dinucleotides. In cereal genomes, however, most of the numerous potential recognition sites for CpG methylation-sensitive restriction enzymes are methylated. Analysis of cereal genomic libraries and of regions flanking genes indicates that unmethylated NotI sites are useful landmarks for regions containing genes/single-copy sequences. Studies of a rye chromosome arm indicate that its pericentromeric region has a reduced density of unmethylated NotI (and MluI) sites and therefore of genes. Unmethylated MluI and NruI sites are distributed nonrandomly in the genomes of wheat, barley, and rice. Analysis of the genomic blocks defined by these sites in wheat and barley indicates that they are most likely to have arisen by amplification. These observations form the basis of a proposed model for the organization and evolution of the wheat, barley, and rice genomes.     

Comparative analysis of the N-glycans of rat, mouse and human Thy-1. Site-specific oligosaccharide patterns of neural Thy-1, a member of the immunoglobulin superfamily

Protein structure and tissue type are known to influence glycosylationof proteins. We have previously investigated the N-glycans ateach of the three glycosylation sites of the cell surface glycoproteinThy-1 when isolated from rat brain and thymocytes. Here we reporta comparative analysis of the site-specific N-glycosylationpatterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) andhuman (Asn 23, 60, 100) neural Thy-1. Despite considerable differencesin amino acid sequence, the results show a remarkable conservationof the pattern of N-glycans at corresponding sites between thethree species, as judged by chromatographic comparisons andglycosidase susceptibility. This is particularly marked forsites at Asn 74/75 in rat/mouse and the equivalent site at 60in human Thy-1, as well as for sites at Asn 98/99 and 100, respectively.The sites at Asn 23 in rat/mouse also contained almost identicalglycosylation paterns, but at this site human Thy-1 showed significantlydifferent glycosylation patterns. These site glycosylation patternsare discussed in relation to the likely accessibility of theoligosaccharides for processing. It is known that within a species,the glycosylation of Thy-1 is tissue specific; therefore, thisdegree of conservation of glycosylation of Thy-1 expressed inthe same tissue in different species is all the more striking,given the known variation between species in the amino acidsequence of Thy-1. It is therefore proposed that neural cellshave a particular requirement for specific surface carbohydratesand that the Thy-1 polypep-tide serves as an appropriate carrierfor these structures. glycosylation site-specific Thy-1     

Cyclic ADP-ribose: A calcium mobilizing metabolite of NAD+

Mobilization of Ca⁺² from intracellular stores is a signalling mechanism that is of fundamental importance to many cellular processes. It is mediated by two major mechanisms, the inositol 1,4,5-trisphosphate pathway and the Ca⁺²-induced Ca⁺² release process. A naturally occurring metabolite of NAD⁺ called cyclic ADP-ribose has been discovered recently and shown to be as effective as inositol 1,4,5-trisphosphate in mobilizing Ca⁺² stores in sea urchin eggs, a marine invertebrate cell, as well as several mammalian cells. This article reviews the accumulating evidence that indicates cyclic ADP-ribose may function as a physiological regulator of the Ca⁺²-induced Ca⁺² release process and the current knowledge about its receptor as well as the enzymes involved in its metabolism.     

Synthetic peptide analogs of skeletal troponin C: fluorescence studies of analogs of the low-affinity calcium-binding site II

Two 12-residue peptides were synthesized by the solid-phase method as structural analogs of a Ca2+-binding loop of rabbit skeletal troponin C. The sequence of the analogs corresponds to the binding loop of the Ca2+-specific low affinity binding site II (residues 63-74) but with two amino acid substitutions. In one analog, Phe-72 was replaced by tyrosine. In the other Gly-66 was substituted by serine and Phe-72 by tyrosine. The intrinsic fluorescence of the peptides was enhanced upon addition of Tb3+ or large excess of Ca2+. From the enhancement of Tb3+ emission association constants in the range (2-3) X 10(5) M-1 and a binding stoichiometry of 1 were determined for Tb3+ binding to the peptides. Large excess of Ca2+ displaced Tb3+ from the Tb3+-peptide complexes and from these results apparent stability constants of 500-700 M-1 were deduced for Ca2+ binding. Preliminary proton nuclear magnetic resonance results on one of the peptides indicated that La3+ induced considerable perturbation of the amide proton resonances of several residues, including the aspartate at position 3, the tyrosine at position 10, and the two glutamates at the C-terminus. The results suggest involvement of these residues in cation coordination.     

Effects of prostaglandins on the spreading, adhesion and migration of mouse peritoneal macrophages

The effects of prostaglandins on the properties of mouse peritoneal macrophages namely spreading, adhesion and migration were investigated. PGE₁ and PGE₂ inhibit the spreading and adhesion of complete Freund's Adjuvant induced peritoneal macrophages significantly at concentrations of 1 ng per ml and above whereas they enhance the migration of these cells at concentrations of 100 ng per ml and above. PGA₂ and PGB₂ are less potent as they inhibit spreading and adhesion only at a concentration of 1 μg per ml. At this concentration PGB₂ enhances migration whereas PGA₂ has no effect. PGF_2α has no effect on the spreading, adhesion and migration of macrophages in the concentration range of 0.1 ng to 1,000 ng per ml.     

Quantitative extraction of microgram amounts of lipid from cultured human cells

A simple and efficient procedure based on the solvent system, ethyl acetate:acetone, was developed to extract the principal cellular lipids quantitatively from monolayer cultures of human fibroblasts with populations of even less than one-third million cells. The method was suitable for both radioisotope incorporation studies and studies involving quantitative analysis of fatty acid composition by gas-liquid chromatography. In a modified form the extraction procedure also allowed a 99% recovery of radioactive cell lipid from serum-containing growth medium. Included are results and discussion on the validity of cell number data obtained from monolayer cell cultures which are detached by trypsin and counted by a hemocytometer and a Coulter counting apparatus.