Is intracytoplasmic membrane structure a generic criterion? It does not coincide with phylogenetic interrelationships among phototrophic purple nonsulfur bacteria

The 16S rRNA or rRNA gene sequences of the type strains of 5 species of Rhodobacter, Rhodopseudomonas blastica and Paracoccus denitrificans were determined. The sequence analysis revealed that Rhodobacter species, whose intracytoplasmic membrane systems were characteristically vesicular, composed a sole cluster. Rhodopseudomonas blastica, whose intracytoplasmic membrane system was lamellar, was included in the cluster of Rhodobacter. The phylogenetic co-clustering of these bacteria conformed to their possessing of the identical types of carotenoids. Paracoccus denitrificans, which is nonphototrophic, is a right member of the Rhodobacter cluster. Rhodobacter species, Rhodopseudomonas blastica and Paracoccus denitrificans are apart from the other phototrophic bacteria and have the common deletions of 21 bases at the positions 1258 to 1278 (Escherichia coli numbering system). It was demonstrated that the morphological character intracyto-plasmic membrane structure, that has been regarded as a generic criterion does not reflect the phylogeny in the phototrophic bacteria. The transfer of Rhodopseudomonas blastica to the genus Rhodobacter is proposed.     

Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-glucan synthase.     

A Systematic Evaluation of the Function of the Protein-Remodeling Factor Hsp104 in [PSI+] Prion Propagation in S. cerevisiae by Comprehensive Chromosomal Mutations

The yeast prion [PSI⁺] represents an aggregated state of the translational release factor Sup35 (eRF3) and deprives termination complexes of functional Sup35, resulting in nonsense codon suppression. Protein-remodeling factor Hsp104 is involved in thermotolerance and [PSI⁺] propagation, however the structure-and-function relationship of Hsp104 for [PSI⁺] remains unclear. In this study, we engineered 58 chromosomal hsp104 mutants that affect residues considered structurally or functionally relevant to Hsp104 remodeling activity, yet most remain to be examined for their significance to [PSI⁺] in the same genetic background. Many of these hsp104 mutants were affected both in thermotolerance and [PSI⁺] propagation. However, nine mutants were impaired exclusively for [PSI⁺], while two mutants were impaired exclusively for thermotolerance. Mutations exclusively affecting [PSI⁺] are clustered around the lateral channel of the Hsp104 hexamer. These findings suggest that Hsp104 possesses shared as well as distinct remodeling activities for stress-induced protein aggregates and [PSI⁺] prion aggregates and that the lateral channel plays a role specific to [PSI⁺] prion propagation.Key Words: Hsp104, reverse genetics, hexamer, nonsense suppression, yeast prion [PSI⁺], thermotolerance     

Discovery and structure-activity relationships of urea derivatives as potent and novel CCR3 antagonists

The synthesis and structure-activity relationships of ureas as CCR3 antagonists are described. Optimization starting with lead compound 2 (IC(50)=190 nM) derived from initial screening hit compound 1 (IC(50)=600 nM) led to the identification of (S)-N-((1R,3S,5S)-8-((6-fluoronaphthalen-2-yl)methyl)-8-azabicyclo[3.2.1]octan-3-yl)-N-(2-nitrophenyl)pyrrolidine-1,2-dicarboxamide 27 (IC(50)=4.9 nM) as a potent CCR3 antagonist.     

Alstiphyllanines I-O, ajmaline type alkaloids from Alstonia macrophylla showing vasorelaxant activity

Seven new ajmaline type alkaloids, alstiphyllanines I-O (1-7) were isolated from the leaves of Alstonia macrophylla together with six related alkaloids (8-13). Structures and stereochemistry of 1-7 were fully elucidated and characterized by 2D NMR analysis. A series of alstiphyllanines I-O (1-7) as well as the known ajmaline type alkaloids (8-13) showed that they relaxed phenylephrine (PE)-induced contractions against rat aortic ring. Among them, vincamedine (10) showed potent vasorelaxant activity, which may be mediated through inhibition of Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCs) and/or receptor-operated Ca(2+) channels (ROCs) as well as partially mediated the NO release from endothelial cells. The presence of substituents at both N-1 and C-17 may be important to show vasorelaxation activity.     

The Phytochrome-Interacting VASCULAR PLANT ONE–ZINC FINGER1 and VOZ2 Redundantly Regulate Flowering in Arabidopsis

The timing of the transition to flowering in plants is regulated by various environmental factors, including daylength and light quality. Although the red/far-red photoreceptor phytochrome B (phyB) represses flowering by indirectly regulating the expression of a key flowering regulator, FLOWERING LOCUS T (FT), the mechanism of phyB signaling for flowering is largely unknown. Here, we identified two Arabidopsis thaliana genes, VASCULAR PLANT ONE–ZINC FINGER1 (VOZ1) and VOZ2, which are highly conserved throughout land plant evolution, as phyB-interacting factors. voz1 voz2 double mutants, but neither single mutant, showed a late-flowering phenotype under long-day conditions, which indicated that VOZ1 and VOZ2 redundantly promote flowering. voz1 voz2 mutations suppressed the early-flowering phenotype of the phyB mutant, and FT expression was repressed in the voz1 voz2 mutant. Green fluorescent protein–VOZ2 signal was observed in the cytoplasm, and interaction of VOZ proteins with phyB was indicated to occur in the cytoplasm under far-red light. However, VOZ2 protein modified to localize constitutively in the nucleus promoted flowering. In addition, the stability of VOZ2 proteins in the nucleus was modulated by light quality in a phytochrome-dependent manner. We propose that partial translocation of VOZ proteins from the cytoplasm to the nucleus mediates the initial step of the phyB signal transduction pathway that regulates flowering.     

PTEN is involved in the signal transduction pathway of contact inhibition in endometrial cells

PTEN is involved in the regulation of normal cellular functions in addition to its well–known role as a tumor suppressor. In the present study, we have shown that stable transfection of the PTEN gene into PTEN–mutated endometrial carcinoma cells leads to contact inhibition accompanied by a decreased level of phosphorylated–Akt (p–Akt) expression, an increase in p27^Kip1, and a decrease in β–catenin. PTEN–induced cells with contact inhibition exhibit G0–G1 cell-cycle arrest, and the Ki–67 labeling index is reduced. These changes are canceled by transfection of a double–stranded short–interfering RNA against the PTEN gene. Normal endometrial stromal cells increase their PTEN expression when reaching confluence; this is followed by changes in the expression of Akt–related proteins in the same way as in tumor cells. These results indicate that PTEN, p–Akt, p27, and β–catenin are involved in the signal transduction of contact inhibition and suggest that PTEN may, in part, control the proliferation of endometrial carcinoma cells through the induction of contact inhibition.     

Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis

Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130–160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.