On the origin of the 1602 cm–1 Raman band of yeasts; contribution of ergosterol

The 1602 cm^–1 Raman signature, which we call the “Raman spectroscopic signature of life” in yeasts, is a marker Raman band for cell metabolic activity. Despite the established fact that its intensity sensitively reflects the metabolic status of the cell, its molecular origin remained unclear. In this work, we propose ergosterol as the major contributor of the 1602 cm^–1 Raman signature. The theoretical isotope shift calculation for ergosterol agreed with previous observations. Furthermore, experiments showed that the Raman spectrum of ergosterol corresponds very well with the depleting spectral component in yeast that behaves together with the 1602 cm^–1 signature when the cells are under stress. This work implies that the 1602 cm^–1 Raman signature could serve as an intrinsic ergosterol marker in yeasts for the study of sterol metabolism in vivo and in a label‐free manner, which could not be done by any other techniques at the current stage. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Studies on anti-Helicobacter pylori agents. Part 2: new cephem derivatives

The synthesis and optimization of the anti-Helicobacter pylori activity of a novel series of cephem derivatives are described. Introduction of thio-heterocyclic groups containing N- and S-atoms to the 3-position and phenyl or thienyl acetamido groups to the 7-position of the cephem nucleus dramatically improved the activity. From this series of derivatives, compound 13i was found to have extremely potent in vitro anti-H. pylori activity, superior therapeutic efficacy compared to AMPC and CAM, no cross-resistance between CAM or MNZ and low potential for causing diarrhea due to instability to beta-lactamase.     

Effects of stilbene constituents from rhubarb on nitric oxide production in lipopolysaccharide-activated macrophages

Two new anthraquinone glucosides [chrysophanol 8-O-beta-D-(6'-galloyl)-glucopyranoside, aloe-emodin 1-O-beta-D-glucopyranoside] together with various known stilbenes and their glucosides, anthraquinone glucosides, and a naphthalene glucoside were isolated from the rhizome of Rheum undulatum L. Three stilbenes (rhapontigenin, piceatannol, resveratrol), a naphthalene glucoside (torachrysone 8-O-beta-D-glucopyranoside), and two stilbene glucoside gallates (rhaponticin 2'-O-gallate, rhaponticin 6'-O-gallate) showed inhibitory activity of NO production in lipopolysaccharide-activated macrophages, (IC50 = 11-69 microM). The oxygen functions (-OH,-OCH3) at the benzene ring were found to be essential to show the activity. Whereas, the glucoside moiety reduced the activity, while the alpha,beta-double bond did not affect the activity. Furthermore, the active stilbenes (rhapontigenin, piceatannol, resveratrol) inhibited iNOS induction.     

Adipose expression of catalase is regulated via a novel remote PPARgamma-responsive region

In adipose tissue of obese mice, the expression of catalase, an anti-oxidant enzyme, significantly decreases, which may cause insufficient elimination of hydrogen peroxide, but it does not in liver or skeletal muscle. However, the precise regulatory mechanism of catalase expression in adipocytes has not been fully defined. Here, we demonstrated that adipose tissues highly expressed catalase on the level comparable to liver and kidney, and treatment of mice with PPARγ agonist significantly enhanced catalase expression in adipose tissue but not in liver. In 3T3-L1 cells, mRNA expression of catalase was up-regulated by the induction for adipose differentiation, and down-regulated by TNFα, in parallel with alterations in PPARγ expression. PPARγ agonist significantly enhanced catalase mRNA and activity. Furthermore, we newly identified a remote enhancer region containing two functional PPARγ binding sites in mouse catalase gene. Collectively, our findings suggest that PPARγ plays a crucial role in the expression of catalase in adipocytes.     

XRASGRP2 expression during early development of Xenopus embryos

Previously, we described the DNA microarray screening of vascular endothelial cells that were formed by treatment of aggregates prepared from Xenopus animal cap cells with activin and angiopoietin-2. One of the genes identified in this screening showed homology to human RASGRP2 which plays a role in the regulation of GTP-GDP exchange of the Ras and Rap proteins, and was named XRASGRP2. In the present study, we analyzed the expression pattern of xrasgrp2 during Xenopus embryogenesis. The xrasgrp2 mRNA was expressed after stage 24, as assessed by stage PCR analysis. Whole-mount in situ hybridization showed that xrasgrp2 mRNA was located in the vascular region of the embryo. Loss-of-function analysis revealed that the formation of blood and endothelial cells in the explants transplanted into Xenopus embryos was inhibited by antisense morpholino oligonucleotides that block xrasgrp2 translation. These results suggest that XRASGRP2 plays a role in angiogenesis in Xenopus embryos.     

Inhibitory effect of chicken gonadotropin-releasing hormone II on food intake in the goldfish, Carassius auratus

Gonadotropin-releasing hormone (GnRH) is an evolutionarily conserved neuropeptide with 10 amino acid residues, which possesses some structural variants. A molecular form known as chicken GnRH II ([His⁵ Trp⁷ Tyr⁸] GnRH, cGnRH II) is widely distributed in vertebrates, and has recently been implicated in the regulation of sexual behavior and food intake in an insectivore, the musk shrew. However, the influence of cGnRH II on feeding behavior has not yet been studied in model animals such as rodents and teleost fish. In this study, therefore, we investigated the role of cGnRH II in the regulation of feeding behavior in the goldfish, and examined its involvement in food intake after intracerebroventricular (ICV) administration. ICV-injected cGnRH II at graded doses, from 0.1 to 10 pmol/g body weight (BW), induced a decrease of food consumption in a dose-dependent manner during 60 min after treatment. Cumulative food intake was significantly decreased by ICV injection of cGnRH II at doses of 1 and 10 pmol/g BW during the 60-min post-treatment observation period. ICV injection of salmon GnRH ([Trp⁷ Leu⁸] GnRH, sGnRH) at doses of 0.1-10 pmol/g BW did not affect food intake. The anorexigenic action of cGnRH II was completely blocked by treatment with the GnRH type I receptor antagonist, Antide. However, the anorexigenic action of cGnRH II was not inhibited by treatment with the corticotropin-releasing hormone (CRH) 1/2 receptor antagonist, α-helical CRH₍₉₋₄₁₎, and the melanocortin 4 receptor antagonist, HS024. These results suggest that, in the goldfish, cGnRH II, but not sGnRH, acts as an anorexigenic factor, as is the case in the musk shrew, and that the anorexigenic action of cGnRH II is independent of CRH- and melanocortin-signaling pathways.     

Structural insight into the specific interaction between murine SHPS-1/SIRP alpha and its ligand CD47

SRC homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1 or SIRPα/BIT) is an immunoglobulin (Ig) superfamily transmembrane receptor and a member of the signal regulatory protein (SIRP) family involved in cell-cell interaction. SHPS-1 binds to its ligand CD47 to relay an inhibitory signal for cellular responses, whereas SIRPβ, an activating member of the same family, does not bind to CD47 despite sharing a highly homologous ligand-binding domain with SHPS-1. To address the molecular basis for specific CD47 recognition by SHPS-1, we present the crystal structure of the ligand-binding domain of murine SHPS-1 (mSHPS-1). Folding topology revealed that mSHPS-1 adopts an I2-set Ig fold, but its overall structure resembles IgV domains of antigen receptors, although it has an extended loop structure (C′E loop), which forms a dimer interface in the crystal. Site-directed mutagenesis studies of mSHPS-1 identified critical residues for CD47 binding including sites in the C′E loop and regions corresponding to complementarity-determining regions of antigen receptors. The structural and functional features of mSHPS-1 are consistent with the human SHPS-1 structure except that human SHPS-1 has an additional β-strand D. These results suggest that the variable complementarity-determining region-like loop structures in the binding surface of SHPS-1 are generally required for ligand recognition in a manner similar to that of antigen receptors, which may explain the diverse ligand-binding specificities of SIRP family receptors.     

Plk3 phosphorylates topoisomerase IIalpha at Thr(1342), a site that is not recognized by Plk1

The Plk (polo-like kinase) family is involved in cell-cycle machinery. Despite the possible overlapping involvement of Plk1 and Plk3 in cell-cycle distribution, the precise role of each Plk might be different. To investigate mechanisms that may differentiate their physiological roles, we compared the substrate specificities of Plk1 and Plk3 using synthetic peptides. Among these substrate peptides, topoisomerase IIalpha EKT(1342)DDE-containing synthetic peptide was strongly phosphorylated by Plk3 but not by Plk1. By modulating the topoisomerase IIalpha peptide, we identified residues at positions +1, +2 and +4 as determinants of differential substrate recognition between Plk1 and Plk3. Acidic residues at positions +2 and +4 appear to be a positive determinant for Plk3 but not Plk1. Variation at position +1 appears to be tolerated by Plk3, while a hydrophobic residue at +1 is critical for Plk1 activity. The direct phosphorylation of Thr(1342) of topoisomerase IIalpha by Plk3 was demonstrated with an in vitro kinase assay, and overexpression of Plk3 induced the phosphorylation of Thr(1342) in cellular topoisomerase IIalpha. Furthermore, the physical interaction between Plk3 and topoisomerase IIalpha was also demonstrated in cells in addition to phosphorylation. These data suggest that topoisomerase IIalpha is a novel physiological substrate for Plk3 and that Plk1 and Plk3 play different roles in cell-cycle regulation.     

Enzymological analysis of mutant protein kinase Cgamma causing spinocerebellar ataxia type 14 and dysfunction in Ca2+ homeostasis

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cgamma (PKCgamma). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCgamma kinase activity. To determine the effect of these mutations on PKCgamma function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCgammas. We found that wild-type PKCgamma, but not C1 domain mutants, inhibits Ca2+ influx in response to muscarinic receptor stimulation. The sustained Ca2+ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCgamma. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca2+ influx regulated by PKCgamma. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCgammas was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca2+ entry. Such an alteration in Ca2+ homeostasis and Ca2+-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.