Excitatory and inhibitory actions of norepinephrine on the Ba2+ current through L-type Ca2+ channels of smooth muscle cells of guinea-pig vas deferens

The effect of norepinephrine (NE) was examined on the whole-cell Ba²⁺ current through L-type Ca²⁺ channels of freshly isolated smooth muscle cells of guinea-pig vas deferens. The magnitude of maximum Ba²⁺ current [IBa(max)] varied in different cells, although the capacitance of the cell membrane was similar (∼50 pF). Application of dbcAMP augmented IBa(max) by 37%, which was canceled by Rp-cAMPs, while PMA decreased the current by 32%, which was canceled by staurosporine. NE increased IBa(max) of the cells which originally showed relatively small IBa(max), and decreased the current of the cells which showed larger IBa(max). In the presence of phentolamine, NE increased IBa(max), and this effect was remarkable in cells showed smaller IBa(max). In the presence of propranolol, NE decreased IBa(max). The excitatory β-adrenoceptor activation was canceled by Rp-cAMPs, and the inhibitory α-adrenoceptor effect was canceled by staurosporine. It is suggested that NE shows dual (excitatory and inhibitory) actions on the L-type Ca²⁺ channels of smooth muscle of guinea-pig vas deferens. The excitatory β-adrenoceptor action mediated through cAMP/PKA is predominant in cells with lower density of the Ca²⁺ channels, while inhibitory α-adrenoceptor action mediated through PKC is predominant in cells with higher channel density. © 1996 Wiley-Liss, Inc.     

Herquline A,produced by Penicillium herquei FKI-7215, exhibits anti-influenza virus properties

In the course of screening for new anti-influenza virus antibiotics, we isolated herquline A from a culture broth of the fungus, Penicillium herquei FKI-7215. Herquline A inhibited replication of influenza virus A/PR/8/34 strain in a dose-dependent manner without exhibiting cytotoxicity against several human cell lines. It did not inhibit the viral neuraminidase.     

Initiating translation with D-amino acids

Here we report experimental evidence that the translation initiation apparatus accepts D-amino acids ((D)aa), as opposed to only L-methionine, as initiators. Nineteen (D)aa, as the stereoisomers to their natural L-amino acids, were charged onto initiator tRNA(fMet)(CAU) using flexizyme technology and tested for initiation in a reconstituted Escherichia coli translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to (D)aa. Remarkably, all (D)aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with (D)aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding (D)aa-tRNA(fMet)(CAU). Although the inefficient formylation of (D)aa-tRNA(fMet)(CAU) resulted in modest expression of the corresponding peptide, preacetylation of (D)aa-tRNA(fMet)(CAU) dramatically increased expression level, implying that the formylation efficiency is one of the critical determinants of initiation efficiency with (D)aa. Our findings provide not only the experimental evidence that translation initiation tolerates (D)aa, but also a new means for the mRNA-directed synthesis of peptides capped with (D)aa or acyl-(D)aa at the N terminus.     

Design, syntheses, and structure-activity relationships of novel NPY Y5 receptor antagonists: 2-{3-Oxospiro[isobenzofuran-1(3H),4'-piperidin]-1'-yl}benzimidazole derivatives

Design, syntheses, and structure-activity relationships of a novel class of 2-{3-oxospiro[isobenzofuran-1(3H),4'-piperidin]-1'-yl}benzimidazole NPY Y5 receptor antagonists are described. The benzimidazole structures were newly designed based on the urea linkage of our prototype Y5 receptor antagonists (2 and 3). By optimizing substituents on the benzimidazole core part of the lead compound 5a, we were able to develop a potent, orally available, and brain-penetrable Y5 selective antagonist (5k).     

Ancient divergence of animal protein tyrosine kinase genes demonstrated by a gene family tree including choanoflagellate genes

Animal-specific gene families involved in cell-cell communication and developmental control comprise many subfamilies with distinct domain structures and functions. They diverged by subfamily-generating duplications and domain shufflings before the parazoan-eumetazoan split. Here, we have cloned 40 PTK cDNAs from choanoflagellates, Monosiga ovata, Stephanoeca diplocostata and Codosiga gracilis, the closest relatives to animals. A phylogeny-based analysis of PTKs revealed that 40 out of 47 subfamilies analyzed have unique domain structures and are possibly generated independently in animal and choanoflagellate lineages by domain shufflings. Seven cytoplasmic subfamilies showed divergence before the animal-choanoflagellate split originated by both duplications and shufflings.     

Differential roles of MAP kinases in atorvastatin-induced VEGF release in cardiac myocytes

Statins, specific inhibitors of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, are now widely used for treatment of patients with hypercholesterolemia. In addition to the reduction of cholesterol biosynthesis, accumulating evidence indicates that statins have several pleiotropic effects especially on cardiovascular system. However, the exact role of statin in cardiac myocytes remains unclear. In the present study, we investigated whether atorvastatin induces vascular endothelial growth factor (VEGF) release in cardiac myocytes, and the underlying mechanism. We observed that atorvastatin significantly stimulated VEGF release in a dose-dependent manner. It induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase but not SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). The atorvastatin-induced VEGF release was enhanced by PD98059, which is a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Further, it was significantly reduced by SB203580, a specific inhibitor of p38 MAP kinase. Furthermore, the atorvastatin-induced phosphorylation of p38 MAP kinase was attenuated by SB203580, whereas it was enhanced by PD98059. Taken together, these results suggest that the atorvastatin-induced VEGF release in cardiac myocytes is positively regulated by p38 MAP kinase and negatively regulated byp44/p42 MAP kinase and that the atorvastatin-induced phosphorylation of p38 MAP kinase is regulated by p44/p42 MAP kinase in these cells.     

Conformational change of single-stranded RNAs induced by liposome binding

The interaction between single-stranded RNAs and liposomes was studied using UV, Fourier Transform Infrared spectroscopy (FTIR) and Circular Dichroism spectroscopy (CD). The effect of the surface characteristics of liposomes, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and modified with cholesterol (Ch) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), on the liposome–RNA interaction was investigated. The fluorescence of 6-(p-toluidino)naphthalene-2-sulfonate (TNS) embedded in the liposome surface (ε = 30–40) was decreased in the presence of tRNA, suggesting that single-stranded tRNA could bind onto the liposome. The dehydration of –PO₂⁻ –, guanine (G) and cytosine (C) of tRNA molecules in the presence of liposomes suggested both an electrostatic interaction (phosphate backbone of tRNA and trimethylammonium group of POPC, DOTAP) and a hydrophobic interaction (guanine or cytosine of tRNA and aliphatic tail of lipid). The tRNA conformation on the liposome was determined by CD spectroscopy. POPC/Ch (70/30) maintained tRNA conformation without any denaturation, while POPC/DOTAP(70/30) drastically denatured it. The mRNA translation was evaluated in an Escherichia coli cell-free translation system. POPC/Ch(70/30) enhanced expression of green fluorescent protein (GFP) (116%) while POPC/DOTAP(70/30) inhibited (37%), suggesting that the conformation of RNAs was closely related to the translation efficiency. Therefore, single-stranded RNAs could bind to liposomal membranes through electrostatic and hydrophobic attraction, after which conformational changes were induced depending on the liposome characteristics.