Identification of the actinorhodin monomer and its related compound from a deletion mutant of the actVA-ORF4 gene of Streptomyces coelicolor A3(2)

An oxygenated derivative of dihydrokalafungin (DHK) was isolated from a deletion mutant of the actVA-ORF4 gene involved in the biosynthesis of a dimeric benzoisochromanequinone (BIQ) antibiotic, actinorhodin (ACT), in Streptomyces coelicolor A3(2). Spectroscopic analysis elucidated its structure as 8-hydroxy-DHK, corresponding to the monomeric unit of ACT. Further metabolite analysis identified its related compound, clearly derived from the reduction of 8-hydroxy-DHK. The structures of these metabolites indicate the essential role of ActVA-ORF4 in ACT biosynthesis, specifically in dimerization of a BIQ intermediate via C-C bond formation.     

Specific expression of annexin III in rat-small-hepatocytes

Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.     

Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and 42.5 degrees C, and the enzyme was stable under 40 degrees C. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a beta-agarase.     

HLA-B51 and HLA-Bw52 differ by only two amino acids which are in the helical region of the alpha 1 domain

Genes encoding the serologically cross-reactive HLA-B51 and HLA-Bw52 molecules were isolated and the exons sequenced. HLA-B51 genes obtained from Caucasian and Oriental individuals were identical. HLA-Bw52 differs from HLA-B51 by four nucleotide substitutions in exon 2 encoding the alpha 1 domain. These comprise one isolated silent substitution in codon 23 and a cluster of three coding substitutions in codons 63 and 67. Amino acid substitutions of N----E at position 63 and F----S at position 67 are the only differences between HLA-B51 and HLA-Bw52 and these residues are postulated to form HLA-B51 specific epitopes. HLA-B51 could have been formed from HLA-Bw52 by the combination of a genetic exchange with HLA-B8 and a point mutation. Similarity of HLA-B51 and HLA-Bw52 with HLA-Bw58 suggest they also share a common ancestor.     

Association of PAP-1 and Prp3p, the products of causative genes of dominant retinitis pigmentosa, in the tri-snRNP complex

PAP-1 has been identified by us as a Pim-1-binding protein and has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP). We have then shown that PAP-1 plays a role in pre-mRNA splicing. Because four causative genes for adRP, including PAP-1, Prp31, Prp8, and Prp3, encode proteins that function as splicing factors or splicing-modulating factors, we investigated the interaction of PAP-1 with Prp3p and Prp31p in this study. The results showed that PAP-1 interacted with Prp3p but not Prp31p in human cells and yeast, and that the basic region of PAP-1 and the C-terminal region of Prp3p, regions beside spots found in adRP mutations, were needed for binding. Furthermore, both Prp3p and a part of PAP-1 were found to be components of the U4/U6.U5-tri-snRNP complex, one form of the spliceosome, in Ba/F3 and K562 cells by analysis of sucrose density gradients, suggesting that PAP-1 is weakly associated with the spliceosome. These results also suggest that splicing factors implicated in adRP contribute alone or mutually to proper splicing in the retina and that loss of their functions leads to onset of adRP.     

Effects of Amyloid β-Peptides and Gangliosides on Mouse Neural Stem Cells

The interaction of amyloid β-proteins (Aβs) with membrane lipids has been postulated as an early event in Aβ fibril formation in Alzheimer’s disease. We evaluated the effects of several putative bioactive Aβs and gangliosides on neural stem cells (NSCs) isolated from embryonic mouse brains or the subventricular zone of adult mouse brains. Incubation of the isolated NSCs with soluble Aβ1–40 alone did not cause any change in the number of NSCs, but soluble Aβ1–42 increased their number. Aggregated Aβ1–40 and Aβ1–42 increased the number of NSCs but soluble and aggregated Aβ25–35 decreased the number. Soluble Aβ1–40 and Aβ1–42 did not affect the number of apoptotic cells but aggregated Aβ1–40 and Aβ1–42 did. When NSCs were treated with a combination of GM1 or GD3 and soluble Aβ1–42, cell proliferation was enhanced, indicating that both GM1 and GD3 as well as Aβs are involved in promoting cell proliferation and survival of NSCs. These observations suggest the potential of beneficial effects of using gangliosides and Aβs for promoting NSC proliferation.