TectoRNP: self‐assembling RNAs with peptide recognition motifs as templates for chemical peptide ligation

TectoRNA, an artificial RNA with self‐assembling ability, has been employed as a structural platform for RNA nanotechnology and RNA synthetic biology. In this study, tectoRNA was applied as a specific template for chemical peptide ligation. On the basis of a self‐assembling tectoRNA, we designed and constructed a template RNA that facilitates peptide ligation depending on controlled dimer formation. Two RNA‐binding peptides were recognized by two peptide‐binding RNA motifs embedded in the template RNA, and chemical ligation was promoted because of the entropic effect of Mg²⁺‐dependent dimerization. In a series of biochemical analyses, we determined the relationship between the structures of the tectoRNA‐based templates and the extent of acceleration in peptide ligation. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Calcium-Sensitive Nonselective Cation Channel Identified in the Epithelial Cells Isolated from the Endolymphatic Sac of Guinea Pigs

We identified a Ca²⁺-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K⁺= Na⁺ > Ca²⁺≫ Cl⁻. This channel was weakly voltage-dependent but was strongly activated by Ca²⁺ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca²⁺ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), activated the channel at an estimated [Ca²⁺]_i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca²⁺ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na⁺ under certain pathological conditions that will increase [Ca²⁺]_i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells. Received: 24 October 2000/Revised: 10 April 2001     

Stimulation of phagocytosis and phagosome—lysosome (P-L) fusion of human polymorphonuclear leukocytes by sulfatide (galactosylceramide-3-sulfate)

Abstract Recently, extensive attention has been paid to the physiological function of glycosphingolipids (GSLs) of mammalian cell membranes. Among a variety of GSLs, sulfatide (galactosylceramide-3-sulfate) has been proposed to be a specific receptor or binding molecule to microorganisms. However, no report has appeared on the direct stimulation by sulfatide for cellular function differentiation in phagocytic cells. We found that sulfatide showed a marked stimulation for phagocytic processes of human peripheral polymorphonuclear leukocytes (PMN) using heat-killed cells of Staphylococcus aureus coated with isolated lipid. Among mammalian acidic GSLs, sulfatide showed the highest stimulative activity for adhesion, phagocytosis and phagosome—lysosome (P-L) fusion by PMN. On the other hand, neutral GSLs did not stimulate essentially. Relative phagocytic rate of sulfatide-coated staphylococci was six times higher than that of the non-coated control and P-L fusion rate was ten times at maximum, respectively. Although the promotion mechanism of sulfatide for such phagocytosis or P-L fusion is not clear, it was strongly suggested that the existence of negative charges on carbohydrate moiety may be essential for the induction of differentiation of phagocytic cell function via signal transduction systems.     

Isolation and characterization of histamine-releasing peptides from human parotid saliva

Peptides responsible for releasing histamine were purified from human parotid saliva. The amino acid composition of the peptides showed a high proportion of histidine, lysine and arginine. Molecular weights of these peptides were between 3000 and 5000 as determined by SDS-acrylamide gel electrophoresis. These peptides induced histamine release from rat-isolated mast cells accompanied with degranulation in a dose-dependent manner over the concentration range 5-50 micrograms/ml.     

35S]S-[5-(4-benzoylphenyl)pentyl]glutathione: a versatile radioligand targeting GS-X pumps with the ability of photoaffinity labeling.

[35S]S-[5-(4-benzoylphenyl)pentyl]glutathione (GIF-0017) as a biochemical probe targeting the ATP-dependent organic anion transporters GS-X pumps was synthesized by the reaction of [35S]glutathione and excess 4-(5-bromo)pentylbenzophenone under alkaline conditions, with the radiochemical yield of 24-33% after HPLC purification. Photolysis of the mixture of [35S]GIF-0017 and plasma membrane vesicles prepared from the MRP1 cDNA-transfected LLC-PK1 cells resulted in radio-labeling of a 180-kDa membrane protein. Immunoprecipitation and western blotting using an anti-MRP1 monoclonal antibody confirmed that the [35S]GIF-0017-labeled protein was the MRPI/GS-X pump.     

A Duplicated NUCLEOLIN Gene with Antagonistic Activity Is Required for Chromatin Organization of Silent 45S rDNA in Arabidopsis

In plants as well as in animals, hundreds to thousands of 45S rRNA gene copies localize in Nucleolus Organizer Regions (NORs), and the activation or repression of specific sets of rDNA depends on epigenetic mechanisms. Previously, we reported that the Arabidopsis thaliana nucleolin protein NUC1, an abundant and evolutionarily conserved nucleolar protein in eukaryotic organisms, is required for maintaining DNA methylation levels and for controlling the expression of specific rDNA variants in Arabidopsis. Interestingly, in contrast with animal or yeast cells, plants contain a second nucleolin gene. Here, we report that Arabidopsis NUC1 and NUC2 nucleolin genes are both required for plant growth and survival and that NUC2 disruption represses flowering. However, these genes seem to be functionally antagonistic. In contrast with NUC1, disruption of NUC2 induces CG hypermethylation of rDNA and NOR association with the nucleolus. Moreover, NUC2 loss of function triggers major changes in rDNA spatial organization, expression, and transgenerational stability. Our analyses indicate that silencing of specific rRNA genes is mostly determined by the active or repressed state of the NORs and that nucleolin proteins play a key role in the developmental control of this process.     

'Lysobacter enzymogenes ssp. cookii ' Christensen 1978 should be recognized as an independent species, Lysobacter cookii sp. nov.

' Lysobacter enzymogenes ssp. cookii ' was proposed by Christensen and Cook in 1978; however, this subspecies name has not been cited in the Approved Lists of Bacterial Names and therefore the nomenclature has not been validated. In our genetic approach to clarify the relationships of the designated type strain of ' L. enzymogenes ssp. cookii ' PAGU 1119 (GenBank accession number ATCC29488 ) within the genus Lysobacter revealed that the strain was closely related to Lysobacter capsici YC5194 ^T (99.4%) rather than L. enzymogenes DSM2043 ^T (97.2%). The value for whole genome DNA–DNA relatedness between strain PAGU 1119 and L. enzymogenes DSM 2043^T or L. capsici YC5194 ^T was 20.7–26.1% or 60.9–62.0%, respectively. Although PAGU 1119 and L. capsici YC5194 ^T showed relatively high DNA relationships, the fatty acid profiles and some phenotypic characteristics were different, and we concluded that PAGU 1119 should be placed in a new species. We therefore propose a new species with the name Lysobacter cookii sp. nov. The type strain is PAGU 1119^T ( ATCC29488 ).     

Determination of naringin and naringenin in human urine by high-performance liquid chromatography utilizing solid-phase extraction

An HPLC method for determining a flavonoid naringin and its metabolite, naringenin, in human urine is presented for application to the pharmacokinetic study of naringin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using hesperidin for naringin or hesperetin for naringenin as internal standard and solid-phase extraction using a strong anion exchanger, Sep-Pak Accell QMA cartridge. The HPLC assay was carried out using an Inertsil ODS-2 column (250×4.6 mm I.D., 5 μm particle size). The mobile phases were acetonitrile–0.1 M ammonium acetate–acetic acid (18:81:1, v/v; pH 4.7) for naringin and acetonitrile–0.1 M ammonium acetate–triethylamine (25:75:0.05; v/v; pH 8.0) for naringenin. The flow-rate was 1.0 ml min⁻¹. The analyses were performed by monitoring the wavelength of maximum UV absorbance at 282 nm for naringin and at 324 nm for naringenin. The lower limits of quantification were ca. 25 ng/ml for naringin and naringenin with R.S.D. less than 10%. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 5 ng for naringin and 1 ng for naringenin. A preliminary experiment to investigate the urinary excretion of naringin, naringenin and naringenin glucuronides after oral administration of 500 mg of naringin to a healthy volunteer demonstrated that the present method was suitable for determining naringin and naringenin in human urine.