Synthesis and evaluation of substituted 4-alkoxy-2-aminopyridines as novel neuropeptide Y1 receptor antagonists

A series of substituted 4-alkoxy-2-aminopyridines 2, which were formally derived from neuropeptide Y1 antagonist 1 by replacing the morpholino portion with alkoxy groups, were synthesized and evaluated as neuropeptide Y Y1 receptor antagonists. Primary structure-activity relationships and identification of potent 4-alkoxy derivatives are described.     

Size-dependent male–male competition for a spawning substrate between <Emphasis Type="Italic">Pseudorasbora parva</Emphasis> and <Emphasis Type="Italic">Pseudorasbora pumila</Emphasis>

Pseudorasbora parva, a species native to western Japan, has been accidentally introduced into eastern Japan, where P. pumila is indigenous. We investigated inter- and intraspecific, male–male competition between P. parva and P. pumila for acquisition of spawning substrates in an experimental setting. Within each species, males of larger standard length and heavier body weight were more successful in acquiring a substrate. Males of the two species competed, but the outcome was determined primarily by body weight. This interspecific, size-dependent, male–male competition might be an important factor in the species replacement of P. pumila by P. parva.     

Composition and structure of the centromeric region of rice chromosome 8

Understanding the organization of eukaryotic centromeres has both fundamental and applied importance because of their roles in chromosome segregation, karyotypic stability, and artificial chromosome-based cloning and expression vectors. Using clone-by-clone sequencing methodology, we obtained the complete genomic sequence of the centromeric region of rice (Oryza sativa) chromosome 8. Analysis of 1.97 Mb of contiguous nucleotide sequence revealed three large clusters of CentO satellite repeats (68.5 kb of 155-bp repeats) and >220 transposable element (TE)-related sequences; together, these account for approximately 60% of this centromeric region. The 155-bp repeats were tandemly arrayed head to tail within the clusters, which had different orientations and were interrupted by TE-related sequences. The individual 155-bp CentO satellite repeats showed frequent transitions and transversions at eight nucleotide positions. The 40 TE elements with highly conserved sequences were mostly gypsy-type retrotransposons. Furthermore, 48 genes, showing high BLAST homology to known proteins or to rice full-length cDNAs, were predicted within the region; some were close to the CentO clusters. We then performed a genome-wide survey of the sequences and organization of CentO and RIRE7 families. Our study provides the complete sequence of a centromeric region from either plants or animals and likely will provide insight into the evolutionary and functional analysis of plant centromeres.     

Polycation liposome-mediated gene transfer in vivo

The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined.     

Adaptor protein Shc is an isoform-specific direct activator of the tyrosine kinase c-Src

The activity of c-Src protein-tyrosine kinase is up-regulated under a number of receptor signaling pathways. However, the activation mechanism of c-Src under physiological conditions has remained unclear. We show here that the Shc adaptor protein is a novel direct activator of c-Src in epidermal growth factor receptor signaling in A431 human epidermoid carcinoma cells. Among the three Shc isoforms, P66 and P52, but not P46, were found to interact with and activate c-Src in vitro and in vivo. Activation of c-Src accompanied autophosphorylation of c-Src in the activation segment, but the carboxyl-terminal dephosphorylation was not observed. We have identified the interaction sites between Shc and c-Src and constructed a point mutant of Shc that abolishes the c-Src activation. Using this mutant, we have confirmed that the Shc-mediated c-Src activation triggers Stat-p21/WAF1/Cip1 pathway that has been implicated in the cell cycle arrest and apoptosis of epidermal growth factor-stimulated A431 cells.     

Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa belladonna after treatment with exogeneously added salicylic acid

In Atropa belladonna hairy roots, exogeneously added salicylic acid (SA) is converted to methyl salicylate (MSA) through the reaction, which might be catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT). Here we cloned a cDNA for A. belladonna SAMT (AbSAMT1), which consisted of 357 aa residues. It was expressed in E. coli, and the recombinant AbSAMT1 showed SAMT activity. When A. belladonna hairy roots were exposed to a high concentration of SA, AbSAMT1 mRNA begins to be expressed 12 h after the exposure, and steady expression continued over 144 h.     

NADPH-diaphorase and calcium binding proteins in the trigeminal nucleus oralis of rats

We have examined the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and the calcium binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV), in the trigeminal nucleus oralis (Sp5O). NADPH-d was detected by histochemistry while CBP was detected by immunohistochemistry. NADPH-d-positive neurons were distributed in the medial rostro-dorsomedial part (RDMsp5O) and dorsomedial part (DMsp5O) of Sp5O, and the rostrolateral part of the nucleus of the solitary tract (NTS). CB- and CR-positive neurons were mainly distributed in the dorsal part of Sp5O. In contrast, PV-positive neurons were mainly distributed in the ventral part of Sp5O. NADPH-d colocalized with CB (40%) and CR (20%) but not with PV in neurons of DMsp5O/ NTS. The mean cell sizes of neurons in RDMsp5O were larger than those in DMsp5O/NTS. PV-positive neurons were larger than NADPH-d-positive neurons. NADPH-d-, CB- and CR-positive neurons were generally small in RDMsp5O and DMsp5O/NTS. Few neurons were retrogradely labeled in RDMsp5O and DMsp5O from the thalamus, when numerous labeled neurons were in the principal and interpolar nuclei. These data indicate that NADPH-d histochemistry and CB, CR and PV immunohistochemistry identify a discrete cell population in Sp5O. Those labeled neurons in RDMsp5O and DMsp5O/NTS were considered to be involved in sensorimotor reflexive function of the intra-oral structures.     

Effects of oolong tea on metabolism of plasma fat in mice under restraint stress

We investigated the effects of oolong tea on the basic metabolism of plasma lipids in mice under restraint stress. When a lipid emulsion (Intralipid 20%; a lipid emulsion containing 20% soybean oil) was injected intravenously into mice, the restraint stress prolonged the half-life (T 1/2) of elimination for plasma triglyceride (TG) from 28.7 to 55.5 min. The elimination rate per minute was 48.2% in stressed mice with the rate in starved control mice as 100%. Therefore, TG metabolism was disrupted by the stress, and the use of TG as an energy source decreased. We found that the metabolism of lipids significantly response to the restrained stress in the present study. Plasma TG was 515.9 +/- 29.9mg/dl 35min after Intralipid administration in control stressed mice, 478.7 +/- 26.7 mg/dl in the stressed group given caffeine 100 mg/kg of body weight, and 418.3 +/- 18.4 mg/dl in the stressed group given 1,000 mg/kg oolong tea, an improvement by 7.2% and 18.9%, respectively, with the value for the untreated control group. The intake of oolong tea alleviated the stress-induced decrease in the rate of blood lipid metabolism; this effect may have arisen from some non-specific stress-relieving property of the tea or from acceleration of lipid metabolism by properties of polyphenols, etc. in tea. Oolong tea had anti-stress effects on plasma TG metabolism, and the effects did not depend on caffeine.