Optimization of a series of 2,4-diaminopyridines as neuropeptide Y Y1 receptor antagonists with reduced hERG activity

The synthesis and evaluation of a series of 2,4-diaminopyridine-based neuropeptide Y Y1 (NPY Y1) receptor antagonists are described. Compound 1 was previously reported by our laboratory to be a potent and selective Y1 antagonist; however, 1 was also found to have potent hERG inhibitory activity. The main focus of this communication is structure–activity relationship development aimed at eliminating the hERG activity of 1. This resulted in the identification of compound 3d as a potent and selective NPY Y1 antagonist with reduced hERG liability.     

Hormonal control of pupal coloration in the painted lady butterfly Vanessa cardui

Pupae of the painted lady butterfly Vanessa cardui exhibit pupal color polyphenism consisting of white, dark and intermediate types. We investigated environmental factors affecting pupal coloration and the physiological mechanisms underlying the control of pupal color polyphenism in this species. Over 80% of larvae reared at 16 °C developed into pupae of dark types, whereas over 82% of larvae at 32 °C developed into pupae of white types irrespective of long/short-day photoperiod conditions. When mature larvae reared at 32 °C were ligatured between thoracic and abdominal parts at three different pharate pupal stages, all of the head-thoracic parts developed into white pupae regardless of pupal stage, but all abdominal parts ligatured at the early pharate pupal stage only developed into dark pupae. These results indicate that temperature during larval stages is an important element affecting pupal coloration as an environmental cue in V. cardui, and that a factor(s) inducing white pupae is released from head-thoracic parts under conditions of high temperature. Additionally, when ligatured abdomens destined to develop into dark pupae were treated with crude extracts prepared from the central nervous system, all of the ligatured abdomens developed into white pupae at a level dependent on dose and pupal stage. These results suggest that the factor inducing white pupae is a key molecule controlling pupal color polyphenism in V. cardui.     

High‐throughput protein refolding screening method using zeolite

We established a 96‐well‐plate‐based refolding screening system using zeolite. In this system, protein denatured and solubilized with 6 M guanidine hydrochloride is adsorbed onto zeolite placed in a 96‐well plate. The refolding conditions can be tested by incubating the samples with refolding buffers under various conditions of pH, salts, and additives. In this study, we chose green fluorescent protein as the model protein. Green fluorescent protein was expressed as inclusion bodies, and we tested the effects of four pH conditions and six additives on its refolding. The results demonstrate that green fluorescent protein was more efficiently refolded with zeolite than with the conventional dilution method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Inhibition of cellular proliferation by diterpenes, topoisomerase II inhibitor

We examined the effects of 12 terpene compounds derived from the roots of Euphorbia kansui on the proliferative activity of Xenopus embryo cells. Eight of these compounds showed significant inhibition of cellular proliferation even at low concentrations, while four of them needed to be present at higher concentrations to inhibit cellular proliferation. In order to define the mechanism of inhibition of cellular proliferation by these compounds, the effects of diterpene compounds on the activity of topoisomerase II were measured. Most of the diterpene compounds that inhibited cellular proliferation also inhibited topoisomerase II activity.     

Involvement of both the N-glycan-relevant and N-glycan-irrelevant structural elements in the recognition of human milk lactoferrin by the 1CF11 monoclonal antibody

Monoclonal antibody 1CF11 has been suggested to specifically recognize a certain carbohydrate epitope shared by glycoproteins in human external secretions. We examined the effect of cleaving the polypeptide backbone and removing N-linked oligosaccharides on the reactivity with 1CF11 of human milk lactoferrin (hLf) to elucidate the structural features of the 1CF11 epitope. We reveal by treating hLF with trypsin and/or N-glycosidase that both the N-glycan-relevant and N-glycan-irrelevant structural elements were involved in the recognition of hLf by 1CF11.     

A small nucleolar RNA functions in rRNA processing in Caenorhabditis elegans

CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.     

Geraniol and linalool synthases from wild species of perilla

Geraniol and linalool synthases were isolated from three pure strains of Perilla hirtella and Perilla setoyensis, which are wild species of perilla. Their amino acid sequences were very similar to those of Perilla citriodora and Perilla frutescens that were reported previously. However, comparison of the sequences of the same functional synthases derived from different species of Perilla demonstrated that the similarities were high among P. citriodora, P. hirtella and P. frutescens, but low between P. setoyensis and any of the others. This result corresponds well with our previous results showing that P. setoyensis is remotely related to the other perilla species. Both geraniol and linalool synthases utilize geranyl diphosphate (GDP) as their catalytic substrate and they were expressed simultaneously in perilla. The linalool synthase is considered to be the enzyme whose metabolite seems not to be oxidized nor reduced in the plant body and the geraniol and limonene synthases are the initial-step-catalyzing enzymes for a variety of oil compounds. The regulation of the substrate flow between them would be interesting for further study.     

Environmental Transition of Signal-Anchor Sequences during Membrane Insertion via the Endoplasmic Reticulum Translocon

In biogenesis of membrane proteins on the endoplasmic reticulum, a protein-conducting channel called the translocon functions in both the membrane translocation of lumenal domains and the integration of transmembrane segments. Here we analyzed the environments of polypeptide chains during the processes by water-dependent alkylation of N-ethylmaleimide at site-directed Cys residues. Using the technique, the region embedded in the hydrophobic portion of the membrane within a signal-anchor sequence and its shortening by insertion of a Pro residue could be detected. When translocation of the N-terminal domain of the signal-anchor was arrested by trapping an N-terminally fused affinity tag sequence, the signal-anchor was susceptible to alkylation, indicating that its migration into the hydrophobic environment was also arrested. Furthermore, when the tag sequence was separated from the signal-anchor by insertion of a hydrophilic sequence, the signal-anchor became inaccessible to alkylation even in the N-terminally trapped state. This suggests that membrane integration of the signal-anchor synchronizes with partial translocation of its N-terminal domain. Additionally, in an integration intermediate of a membrane protein, both of the two translocation-arrested hydrophilic chains were in an aqueous environment flanking the translocon, suggesting that the translocon provides the hydrophilic pathway capable of at least two translocating chains.