Synthesis of an Oligonucleotide Bearing a Phosphate Function at the 5′-Terminus Using a Novel Protecting Group in Terms of a Cellulose Acetate Derivative as a Polymer-Support

Abstract

Synthesis of the title oligonucleotide bearing a phosphate function at the 5′-terminus, i.e., pCpUpCpGpUpCpCpApCpCpA, by the use of the terminal cytidylic acid unit involving a novel protecting group of 2-[2-(monomethoxytrityloxy)ethylthio]ethyl group on its 5′-phosphoryl function in terms of a cellulose acetate polymer-support is described.     

Structure-based design and synthesis of a bivalent iminobiotin analog showing strong affinity toward a low immunogenic streptavidin mutant

The streptavidin/biotin interaction has been widely used as a useful tool in research fields. For application to a pre-targeting system, we previously developed a streptavidin mutant that binds to an iminobiotin analog while abolishing affinity for natural biocytin. Here, we design a bivalent iminobiotin analog that shows 1000-fold higher affinity than before, and determine its crystal structure complexed with the mutant protein.     

Photoreactivities of 5-Bromouracil-containing RNAs

5-Bromouracil (^BrU) was incorporated into three types of synthetic RNA and the products of the photoirradiated ^BrU-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA^BrUGC)₂ and r(CGAA^BrUUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2′-keto adenosine (^ketoA) product at the 5′-neighboring nucleotide, such as r(GC^ketoAUGC) and r(CGA^ketoAUUGC), respectively. The photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in Z-form RNA produced the 2′-keto guanosine (^ketoG) product r(CGC^ketoGUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG^BrUGCG)/r(C^mGCAC^mGCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2′ position to provide a 2′-keto RNA product.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Direct observation of poly(3-hydroxybutyrate) depolymerase adsorbed on polyester thin film by atomic force microscopy

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerases adsorbed on poly(L-lactide) (PLLA) thin film were directly observed by atomic force microscopy (AFM). A PLLA thin film of 100 nm thickness was prepared on a silicon wafer by spin-cast method. The PLLA thin film was treated at 220 degrees C and quenched to room temperature, resulting in the formation of a completely amorphous film with a smooth surface. Then, the PHB depolymerases from Pseudomonas stutzeri YM1006 and Ralstonia pickettii T1 were dispersed on the amorphous PLLA thin film. Direct AFM observation has revealed that the PHB depolymerases bind in an elliptic shape on the surface of the PLLA thin film and that a small ridge is created around each enzyme molecule. After removal of the enzymes with 40% ethanol aqueous solution, small hollows were found on the PLLA thin film. These results suggest that a PHB depolymerase interacts with polyester molecules during their adsorption to make a hollow on the substrate surface.     

Establishment of laurel forest vegetation in adjacent secondary forest in central Japan

Distributions of lucidophyllous species are limited due to the fragmentation of laurel forest. On Komayama Hill in central Japan, we evaluated the colonization of typical lucidophyllous vascular plants from a 350-year-old laurel forest into adjacent abandoned secondary forest for conservation and restoration purposes. A total of 14 consecutive subplots were established along the vegetation border between the two forests (length, 30 m; width, 5 m), extending 70 m into the secondary forest; 18 quadrats of old-growth forest were surveyed. Edge effects of old-growth forest were found to play an important role in re-establishing lucidophyllous saplings and seedlings in the secondary forest. In particular, the abundances of the four dominant canopy species of the old-growth forest significantly decreased with increasing distance. Hence, they are expected to colonize further into the secondary forest and, ultimately, to dominate the canopy. However, the number of lucidophyllous species did not change with distance. Species such as Ficus nipponica, Damnacanthus indicus, Ilex integra, and Lemmaphyllum microphyllum were near-completely or completely limited to the old-growth forest. They are known to be negatively affected by forest fragmentation and were observed to be struggling to colonize the exterior of the old-growth forest even after 60 years of abandonment. Their absence highlighted the limited colonization capacities of some old-growth forest species and underlined the time required for habitat restoration following human disturbance. We conclude that it is important to consider the population dynamics of dominant canopy species and the colonization of these interior species when assessing the habitat expansion of lucidophyllous species and hence the restoration of degraded lands.     

Morphological behavior of acidic and neutral liposomes induced by basic amphiphilic alpha-helical peptides with systematically varied hydrophobic-hydrophilic balance

Lipid-peptide interaction has been investigated using cationic amphiphilic alpha-helical peptides and systematically varying their hydrophobic-hydrophilic balance (HHB). The influence of the peptides on neutral and acidic liposomes was examined by 1) Trp fluorescence quenched by brominated phospholipid, 2) membrane-clearing ability, 3) size determination of liposomes by dynamic light scattering, 4) morphological observation by electron microscopy, and 5) ability to form planar lipid bilayers from channels. The peptides examined consist of hydrophobic Leu and hydrophilic Lys residues with ratios 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively; Kiyota, T., S. Lee, and G. Sugihara. 1996. Biochemistry. 35:13196-13204). The most hydrophobic peptide (Hel 13-5) induced a twisted ribbon-like fibril structure for egg PC liposomes. In a 3/1 (egg PC/egg PG) lipid mixture, Hel 13-5 addition caused fusion of the liposomes. Hel 13-5 formed ion channels in neutral lipid bilayer (egg PE/egg PC = 7/3) at low peptide concentrations, but not in an acidic bilayer (egg PE/brain PS = 7/3). The peptides with hydrophobicity less than Hel 13-5 (Hels 11-7 and Hel 9-9) were able to partially immerse their hydrophobic part of the amphiphilic helix in lipid bilayers and fragment liposome to small bicelles or micelles, and then the bicelles aggregated to form a larger assembly. Peptides Hel 11-7 and Hel 9-9 each formed strong ion channels. Peptides (Hel 7-11 and Hel 5-13) with a more hydrophilic HHB interacted with an acidic lipid bilayer by charge interaction, in which the former immerses the hydrophobic part in lipid bilayer, and the latter did not immerse, and formed large assemblies by aggregation of original liposomes. The present study clearly showed that hydrophobic-hydrophilic balance of a peptide is a crucial factor in understanding lipid-peptide interactions.     

Levamisole in postoperative adjuvant immunochemotherapy for gastric cancer

Summary The usefulness of LMS in postoperative immunochemotherapy of gastric cancer was investigated. In compliance with the protocol, MMC was given at a dose of 20 mg on the day of gastrectomy, and an additional 10 mg on the next day IV. The patients receiving 600 mg Tegafur daily were then divided into two groups according to whether LMS was also given or not. LMS was administered for 3 days before the operation in a daily dose of 150 mg and for 1 year or more after operation according to a schedule of 3 days' administration followed by an 11-day interval. The 2-year follow-up demonstrated that in stage III patients, the LMS (+) regimen was superior to the LMS (–) regimen, since the former prolonged the relapse-free interval significantly. The survival rate for stage III disease was also significantly higher in the LMS (+) than in the LMS (–) group. There was no significant difference in the incidence of subjective or objective side-effects between two groups. The incidence of agranulocytosis was comparable in the two groups.Gastrointestinal Cancer Research Group, Japan Levamisole Research AssociationChairmen of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationController of the Gastrointestinal Cancer Research Group, Japan LMS Research AssociationMembers of the Data Collection and Analysis SubcommitteeThis study was carried out by the Gastrointestinal Cancer Research Group, Japan LMS Research Association (directed by Prof. Kiyoshi Inokuchi, Dept. of Surgery, Kyushu University and Prof. Eiro Tsubura, Dept. of Internal Medicine, Tokushima University). The results were presented in part at the 19th General Meeting of the Japanese Society for Gastroenterological Surgery in February, 1982