Independent inhibition of DNA synthesis by protein kinase C, cyclic AMP and interferon alpha/beta in rabbit aortic smooth muscle cells

In quiescent cultures of rabbit aortic smooth muscle cells, whole blood serum-induced DNA synthesis was inhibited markedly by protein kinase C-activating 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12, 13-dibutyrate (PDBu), cyclic AMP-derivatives, such as dibutyryl cyclic AMP (Bt2cAMP) and 8-bromo-cyclic AMP, and interferon alpha/beta. Neither TPA nor interferon alpha/beta elevated the cellular cyclic AMP level. Neither Bt2cAMP nor interferon alpha/beta induced the phospholipase C-mediated hydrolysis of phosphoinositides. The down-regulation of protein kinase C by prolonged treatment with PDBu abolished the antiproliferative action of TPA but did not affect that of Bt2cAMP or interferon alpha/beta. TPA and Bt2cAMP inhibited the serum-induced DNA synthesis when added within 12 h after the addition of the serum, while interferon alpha/beta was active only when added within 6 h. These results suggest that there are at least three independent signaling systems, protein kinase C- and cyclic AMP-mediated systems and an unidentified system for interferon alpha/beta, which are involved in the antiproliferative mechanisms in rabbit aortic smooth muscle cells.     

Socioeconomic factors affecting the longevity of the Japanese population: a study for 1980 and 1985.

The effects of urbanisation, low income and rejuvenation of the population on life expectancy at birth and at 20, 40 and 65 years of age for males and females in Japan were examined twice, in 1980 and 1985. For males, urbanisation was the major factor determining life expectancy at birth and at age 20 years, and low income was the key determinant of decreased life expectancy except at 65 years of age. For females high income was the factor significantly decreasing life expectancy at 65 years of age in 1980, and rejuvenation of the population inversely influenced life expectancy except at birth in 1985. Life expectancy for all age groups in 1985 was significantly longer than in 1980 for both males and females.     

Characterization of two novel pyruvylated glycosphingolipids containing 2'-aminoethylphosphoryl(-->6)-galactose from the nervous system of Aplysia kurodai.

Two novel acidic glycosphingolipids containing pyruvylated galactose were purified from the nervous tissue of Aplysia kurodai by successive Iatrobeads column chromatographies. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structures of these acidic glycosphingolipids, named F-9 and FGL-I, were determined to be: [3,4-O-(S-1-carboxyethylidene)]Gal beta 1-->3 GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2] (2-aminoethylphosphoryl 1-->6)Gal beta 1-->4Glc beta 1-->1ceramide and [3,4-O-(S-1-carboxyethylidene)] Gal beta 1-->3GalNAc alpha 1-->3(Fuc alpha 1-->2)(2-aminoethylphosphonyl-->6 Gal beta 1-->4Glc beta 1-->1ceramide, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, pyruvylated glycosphingolipids containing phosphoethanolamine in addition to or in place of 2-aminoethylphosphonate are present in the nervous system of Aplysia.     

Cloning, expression and modulation of a mouse NMDA receptor subunit.

The primary structure and presence of two forms of the mouse N-methyl-D-aspartate (NMDA) receptor channel subunit zeta 1 have been disclosed by cloning and sequencing the cDNAs. The zeta 1 subunit shows approximately 20% amino acid sequence identities with the rodent alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)- or kainate-selective GluR subunits and has structural features common to neurotransmitter-gated ion channels. Functional homomeric zeta 1 channels expressed in Xenopus oocytes by injection of the subunit-specific mRNA exhibit current responses characteristic for the NMDA receptor channel such as activation by glycine, Ca2+ permeability, blocking by Mg2+ and activation by polyamine. It has been found that the zeta 1 channel activity is positively modulated by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA).     

Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase

N-acyl-phosphatidylethanolamine (NAPE) is known to be a precursor for various bioactive N-acylethanolamines including the endocannabinoid anandamide. NAPE is produced in mammals through the transfer of an acyl chain from certain glycerophospholipids to phosphatidylethanolamine (PE) by Ca²⁺-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A₂ (cPLA₂ε) was recently identified as a Ca²⁺-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA₂ε function as Ca-NAT. We next purified both mouse recombinant cPLA₂ε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca²⁺ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1?mM CaCl₂ and lowered the EC₅₀ value of Ca²⁺ >8-fold. Using a PS probe, we showed that cPLA₂ε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA₂ε with PS in living cells. Finally, we found that the Ca²⁺-ionophore ionomycin increased [¹⁴C]NAPE levels >10-fold in [¹⁴C]ethanolamine-labeled cPLA₂ε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca²⁺-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca²⁺-dependent activity and human cPLA₂ε isoforms also functioned as Ca-NAT.     

Design,synthesis and evaluation of γ-turn mimetics as LSD1-selective inhibitors

Lysine-specific demethylase 1 (LSD1) is an attractive molecular target for cancer therapy. We have previously reported potent LSD1-selective inhibitors (i.e., NCD18, NCD38, and their analogs) consisting of trans-2-phenylcyclopropylamine (PCPA) or trans-2-arylcyclopropylamine (ACPA) and a lysine moiety that could form a γ-turn structure in the active site of LSD1. Herein we report the design, synthesis and evaluation of γ-turn mimetic compounds for further improvement of LSD1 inhibitory activity and anticancer activity. Among a series of γ-turn mimetic compounds synthesized by a Mitsunobu-reaction-based amination strategy, we identified 1n as a potent and selective LSD1 inhibitor. Compound 1n induced cell cycle arrest and apoptosis through histone methylation in human lung cancer cells. The γ-turn mimetics approach should offer new insights into drug design for LSD1-selective inhibitors.     

Preparation and Characterization of Monoclonal Antibodies Specific for Lauroylated Isoform of Bovine Transducin α-Subunit: Immunohistochemical Analysis of Bovine Retinas

Abstract: The photoreceptor G protein transducin [α- and βγ-subunits (Tα/Tβγ)] plays a central role in the visual transduction process. The amino-terminus of bovine Tα is modified by one of four distinct fatty acids—laurate (C12:0), myristate (C14:0), C14:1 (5- cis ), and C14:2 (5- cis , 8- cis )—but the biological significance and the localization of the four isoforms of Tα are poorly understood. To investigate the cellular distribution of each isoform, we prepared monoclonal antibodies against a synthetic C12:0-, C14:0-, C14:1-, or C14:2-nonapeptide corresponding to the N-terminal region of Tα. Among several types of antibodies isolated, only one type, represented by LA4, reacted specifically with the C12:0-peptide as well as purified Tα but not with the other proteins in bovine retinal homogenate, including recoverin, indicating that the epitope comprises both C12:0 and the N-terminal amino acids of Tα. Immunohistochemical analyses of bovine retinal sections by LA4 showed the uniform distribution of C12:0-Tα in almost all the rod outer segments. Hence, it seemed unlikely that each isoform of Tα was localized in specific cells. This observation, together with evidence for a possible functional diversity among the isoforms, suggests that the four isoforms of Tα in a single rod cell may contribute simultaneously to a fine tuning of the photon-signal transduction process.