Serum stability of selected decapeptide agonists of KISS1R using pseudopeptides

Metastin/kisspeptin, a 54-amino acid peptide, is the ligand of the G-protein-coupled receptor KISS1R which plays a key role in pathways that regulate reproduction and cell migration in many endocrine and gonadal tissues. The N-terminally truncated decapeptide, metastin(45–54), has 3–10 times higher receptor affinity and intracellular calcium ion-mobilizing activity but is rapidly inactivated in serum. In this study we designed and synthesized stable KISS1R agonistic decapeptide analogs with selected substitutions at positions 47, 50, and 51. Replacement of glycine with azaglycine (azaGly) in which the α-carbon is replaced with a nitrogen atom at position 51 improved the stability of amide bonds between Phe⁵⁰-Gly⁵¹ and Gly⁵¹-Leu⁵² as determined by in vitro mouse serum stability studies. Substitution for tryptophan at position 47 with other amino acids such as serine, threonine, β-(3-pyridyl)alanine, and d-tryptophan (d-Trp), produced analogs that were highly stable in mouse serum. d-Trp⁴⁷ analog 13 showed not only high metabolic stability but also excellent KISS1R agonistic activity. Other labile peptides may have increased serum stability using amino acid substitution.     

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.     

Biosynthetic mechanism of ribulose-1,5-bisphosphate carboxylase in the purple photosynthetic bacterium,Chromatium vinosum: II. Biosynthesis of constituent subunits

[³⁵S]Methionine-labeled free subunits A and B of RuBP carboxylase were present in barely detectable amounts; the radioactivity in the free subunit B was approximately 1/150th of that in the subunit B contained in the holoenzyme of RuBP carboxylase. The turnover rates of subunits A and B in the holoenzyme were equal at each time during the incubation period. The ratio of subunit A to subunit B was constant throughout the incubation time both in quantity and in the level of [³H]leucine and [³⁵S]methionine incorporated. CO₂ contained in the incubation medium suppressed [³⁵S]methionine incorporation into both subunits A and B equally. These results suggest that the biosynthesis of subunits A and B is completely synchronized and may be regulated by identical mechanisms.     

Analysis of the NH2-Terminal 83rd Amino Acid of Escherichia coli GyrA in Quinolone-Resistance

Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH₂-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues.     

Design and synthesis of 6-fluoro-2-naphthyl derivatives as novel CCR3 antagonists with reduced CYP2D6 inhibition

In our previous study on discovering novel types of CCR3 antagonists, we found a fluoronaphthalene derivative (1) that exhibited potent CCR3 inhibitory activity with an IC(50) value of 20 nM. However, compound 1 also inhibited human cytochrome P450 2D6 (CYP2D6) with an IC(50) value of 400 nM. In order to reduce its CYP2D6 inhibitory activity, we performed further systematic structural modifications on 1. In particular, we focused on reducing the number of lipophilic moieties in the biphenyl part of 1, using ClogD(7.4) values as the reference index of lipophilicity. This research led to the identification of N-{(3-exo)-8-[(6-fluoro-2-naphthyl)methyl]-8-azabicyclo[3.2.1]oct-3-yl}-3-(piperidin-1-ylcarbonyl)isonicotinamide 1-oxide (30) which showed comparable CCR3 inhibitory activity (IC(50)=23 nM) with much reduced CYP2D6 inhibitory activity (IC(50)=29,000 nM) compared with 1.     

Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN

We examined mechanisms of FITC-albumin uptake by alveolar type II epithelial cells using cultured RLE-6TN cells. Alkaline phosphatase activity and the expression of cytokeratin 19 mRNA, which are characteristic features of alveolar type II epithelial cells, were detected in RLE-6TN cells. The uptake of FITC-albumin by the cells was time and temperature dependent and showed the saturation kinetics of high- and low-affinity transport systems. FITC-albumin uptake was inhibited by native albumin, by chemically modified albumin, and by metabolic inhibitors and bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase. Confocal laser scanning microscopic analysis after FITC-albumin uptake showed punctate localization of fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by nystatin, indomethacin, or methyl-beta-cyclodextrin (inhibitors of caveolae-mediated endocytosis) but was inhibited by phenylarsine oxide and chlorpromazine (inhibitors of clathrin-mediated endocytosis) in a concentration-dependent manner. Uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. These results indicate that the uptake of FITC-albumin in cultured alveolar type II epithelial cells, RLE-6TN, is mediated by clathrin-mediated but not by caveolae-mediated endocytosis, and intracellular FITC-albumin is gradually degraded in lysosomes. Possible receptors involved in this endocytic system are discussed.     

Diversification of endosymbiosis: replacements,co-speciation and promiscuity of bacteriocyte symbionts in weevils

The processes and mechanisms underlying the diversification of host–microbe endosymbiotic associations are of evolutionary interest. Here we investigated the bacteriocyte-associated primary symbionts of weevils wherein the ancient symbiont Nardonella has experienced two independent replacement events: once by Curculioniphilus symbiont in the lineage of Curculio and allied weevils of the tribe Curculionini, and once by Sodalis-allied symbiont in the lineage of grain weevils of the genus Sitophilus. The Curculioniphilus symbiont was detected from 27 of 36 Curculionini species examined, the symbiont phylogeny was congruent with the host weevil phylogeny, and the symbiont gene sequences exhibited AT-biased nucleotide compositions and accelerated molecular evolution. These results suggest that the Curculioniphilus symbiont was acquired by an ancestor of the tribe Curculionini, replaced the original symbiont Nardonella, and has co-speciated with the host weevils over evolutionary time, but has been occasionally lost in several host lineages. By contrast, the Sodalis-allied symbiont of Sitophilus weevils exhibited no host–symbiont co-speciation, no AT-biased nucleotide compositions and only moderately accelerated molecular evolution. These results suggest that the Sodalis-allied symbiont was certainly acquired by an ancestor of the Sitophilus weevils and replaced the original Nardonella symbiont, but the symbiotic association must have experienced occasional re-associations such as new acquisitions, horizontal transfers, replacements and/or losses. We detected Sodalis-allied facultative symbionts in populations of the Curculionini weevils, which might represent potential evolutionary sources of the Sodalis-allied primary symbionts. Comparison of these newcomer bacteriocyte-associated symbiont lineages highlights potential evolutionary trajectories and consequences of novel symbionts after independent replacements of the same ancient symbiont.     

Copulatory courtship behavior and sine song as a mate recognition cue in Drosophila lini and its sibling species

Most Drosophila species sing species-specific pulse songs during their "precopulatory courtship." Three sibling species of the Drosophila montium species subgroup performed "copulatory courtship": males generated courtship songs by vibrating either wing only after mounting and during copulation. In these three species, strong sexual isolation was detected between D. ohnishii and D. lini and between D. ohnishii and D. ogumai, but not between D. lini and D. ogumai. Female showed strong repelling behavior when they were mounted by a heterospecific male in the species combinations including D. ohnishii, resulting in failure of the copulation attempt of the male. Acoustic analyses of courtship songs revealed that the pulse song was irregular, without any species-specific parameters, but that the frequency of the sine song was different among the three species in accordance with the modes of sexual isolation between them; it was significantly lower in D. ohnishii (mean ± SE = 193.0 ± 1.7 Hz) but higher in D. lini (253.4 ± 2.7 Hz) and D. ogumai (246.7 ± 5.3 Hz). We suggest that this difference in the sine song frequency is a sexual signal in the Specific Mate Recognition System (SMRS) among these three Drosophila species.