Artificial dimers of native actin: Preparation and properties in biological functions

With the aid of tartryl-bis--aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited. The dimers are able to activate subfragment I ATPase of myosin and bind to DNase I with inactivation of the enzyme in the same manner as native G actin. Within the dimer, one ADP is immobilized and will exchange against ATP extremely slowly. The dimers do not bind to the mushroom toxin phalloidin.     

Fusicocca-3(16),10(14)-diene, and beta- and delta-araneosenes, new fusicoccin biosynthesis-related diterpene hydrocarbons from Phomopsis amygdali

Further isolation and examination of fusicoccane hydrocarbons biosynthetically related to fusicoccin from Phomopsis amygdali allowed us to identify new fungal diterpene hydrocarbons of fusicoccadiene and araneosene. These were assigned as (+)-fusicocca-3(16),10(14)-diene, and (+)-beta- and (+)-delta-araneosenes. These findings led to the experimental clarification of the structures of the biosynthetic hydrocarbon intermediates presumed earlier.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Classification of RNA structures based on hydrogen bond and base-base stacking patterns: application for NMR structures

A computational system, CSNA, for classifying RNA structures according to structural characters was developed. CSNA lists up all the hydrogen bonds and base-base stackings in the structures, and classifies the structures into sub-groups based on their patterns as the first step grouping. The frequency of each hydrogen bond or base-base stacking is calculated, the frequency score being defined as the sum of the frequency of existing hydrogen bonds or base-base stackings for each sub-group. Finally, the sub-groups are further classified into groups based on the frequency score defined in this study and the difference between the patterns. According to the frequency score, CSNA suggests a group that shares most frequently appearing hydrogen bonds and base-base stackings. CSNA was applied to the classification of the results of two individual simulated annealing calculations based on NMR information. It was found that CSNA could extract structures with lower energy without checking any energy term and could provide well converged groups as the lowest energy structures. Thus, CSNA could be a new tool for structural determination of nucleic acids.     

Molecular cloning and overexpression of fleA gene encoding a fucose-specific lectin of Aspergillus oryzae

A protein from the cell lysate of Aspergillus oryzae was purified by column chromatography immobilized with a ferrichrysin (Fcy), which is one of the siderophores of A. oryzae. It is produced only in an iron-deficient culture and its molecular weight is estimated as 35,000 by SDS-PAGE. Two internal amino acid sequences of the protein obtained by lysylendopeptidase digestion were analyzed. Molecular cloning shows that it encodes 310 putative amino acid residues separated by 4 introns and is designated as fleA. It shows approximately 26% similarity with the gene encoding a fucose-specific lectin of Aleuria aurantia (AAL). The gene was overexpressed under control of the melO promoter in a submerged culture of A. oryzae. The fleA gene product showed hemagglutination activity against rabbit erythrocytes. A hemagglutination inhibition assay of monosaccharides showed that this lectin specifically binds to L-fucose and weakly reacts with mannose and N-acetyl-neuraminic acid.     

Novel regulation of MHC class II function in B cells

The presence of post-translational regulation of MHC class II (MHC II) under physiological conditions has been demonstrated recently in dendritic cells (DCs) that potently function as antigen-presenting cells (APCs). Here, we report that MARCH-I, an E3 ubiquitin ligase, plays a pivotal role in the post-translational regulation of MHC II in B cells. MARCH-I expression was particularly high in B cells, and the forced expression of MARCH-I induced the ubiquitination of MHC II. In B cells from MARCH-I-deficient mice (MARCH-I KO), the half-life of surface MHC II was prolonged and the ubiquitinated form of MHC II completely disappeared. In addition, MARCH-I-deficient B cells highly expressed exogenous antigen-loaded MHC II on their surface and showed high ability to present exogenous antigens. These results suggest that the function of MHC II in B cells is regulated through ubiquitination by MARCH-I.