Characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate-binding module

We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 α-l-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (M _r) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.     

Efficient soluble protein production on transgenic silkworms expressing cytoplasmic chaperones

Baculovirus expression systems (BES) are widely used for recombinant protein production in lepidopteran cells or larvae. However, even in BES, the insolubility of recombinant proteins sometimes makes their expression difficult. In this study, to improve the solubility and yield of foreign proteins, we constructed transgenic silkworms using silkworm heat-shock proteins, Hsp70 and Hsp40, or Hsc70 and Hsp90 co-chaperone Hop. In these transgenic silkworms, the expression levels of the transgenes were under the control of a UAS·hsp mini-promoter driven by a Gal4NFkBp65 activator. When the transgenic silkworm with HSP70 and 40 (TGS-HSP70/40) was infected with BmNPV carrying mC3d and Gal4NFkBp65 under the control of baculovirus polyhedrin or p10 promoters, respectively, the soluble fraction of the His- or His·GST-tagged mC3d increased significantly. Similarly, the transgenic silkworm with HSC70 and HOP (TGS-HOP7) was effective for the expression of a steroid hormone receptor, USP2. In conclusion, the His-tagged baculovirus expression system featuring the chaperone effect TGS-HSP70/40 and TGS-HOP7 silkworms is effective for increasing the yields of soluble and functional foreign gene products.     

Structure-based drug design of tricyclic 8H-indeno[1,2-d][1,3]thiazoles as potent FBPase inhibitors

With the goal of improving metabolic stability and further enhancing FBPase inhibitory activity, a series of tricyclic 8H-indeno[1,2-d][1,3]thiazoles was designed and synthesized with the aid of structure-based drug design. Extensive SAR studies led to the discovery of 19a with an IC₅₀ value of 1 nM against human FBPase. X-ray crystallographic studies revealed that high affinity of 19a was due to the hydrophobic interaction arising from better shape complementarity and to the hydrogen bonding network involving the side chain on the tricyclic scaffold.     

Discovery of potent and orally active tricyclic-based FBPase inhibitors

With the aim of exploring the effect of tricyclic-based FBPase inhibitors in cells and in vivo, a series of prodrugs of tricyclic phosphonates was designed and synthesized. Introducing prodrug moieties into tricyclic-based phosphonates led to the discovery of prodrug 15c, which strongly inhibited glucose production in monkey hepatocytes. Furthermore, prodrug 15c lowered blood glucose levels in fasted cynomolgus monkeys.     

The ODN probes conjugating the Cu(II) complex enhance the luminol chemiluminescence by assembling on the DNA template

Potent peroxidase-like activity of the β-ketoenamine (1)-dicopper (II) complex (2) for the chemiluminescence (CL) of luminol either in the presence or absence of H(2)O(2) has been previously demonstrated by our group. In this study, the β-ketoenamine (1) as the ligand unit for copper(II) was incorporated into the oligonucleotide (ODN) probes. It has been shown that the catalytic activity of the ODN probes conjugating the ligand-Cu(II) complex is activated by hybridization with the target DNA with the complementary sequence. Thus, this study has successfully demonstrated the basic concept for the sensitive detection of nucleic acids by CL based on the template-inductive activation of the catalytic unit for CL.     

Distinct Regulation of Adaxial-Abaxial Polarity in Anther Patterning in Rice

Establishment of adaxial-abaxial polarity is essential for lateral organ development. The mechanisms underlying the polarity establishment in the stamen remain unclear, whereas those in the leaf are well understood. Here, we investigated a rod-like lemma (rol) mutant of rice (Oryza sativa), in which the development of the stamen and lemma is severely compromised. We found that the rod-like structure of the lemma and disturbed anther patterning resulted from defects in the regulation of adaxial-abaxial polarity. Gene isolation indicated that the rol phenotype was caused by a weak mutation in SHOOTLESS2 (SHL2), which encodes an RNA-dependent RNA polymerase and functions in trans-acting small interfering RNA (ta-siRNA) production. Thus, ta-siRNA likely plays an important role in regulating the adaxial-abaxial polarity of floral organs in rice. Furthermore, we found that the spatial expression patterns of marker genes for adaxial-abaxial polarity are rearranged during anther development in the wild type. After this rearrangement, a newly formed polarity is likely to be established in a new developmental unit, the theca primordium. This idea is supported by observations of abnormal stamen development in the shl2-rol mutant. By contrast, the stamen filament is likely formed by abaxialization. Thus, a unique regulatory mechanism may be involved in regulating adaxial-abaxial polarity in stamen development.     

12′-Hydroxyl group remarkably reduces Roridin E cytotoxicity

Roridin E is a well-known macrocyclic trichothecene mycotoxin possessing potent antiproliferative activity against cancer cell lines. 12′-Hydroxyroridin E was isolated from a marine-derived fungus, Myrothecium roridum 98F42. The cytotoxicities of these two compounds were tested against human monocytic THP-1, human promyelocytic leukemia HL-60, and Chinese hamster V79 cells, and roridin E exhibited more than 1000-fold stronger cytotoxicity than its 12′-OH derivative; therefore, it was suggested that the 12′-position is closely involved in the cytotoxicity of these compounds.     

Structural Basis for Translation Factor Recruitment to the Eukaryotic/Archaeal Ribosomes

The archaeal ribosomal stalk complex has been shown to have an apparently conserved functional structure with eukaryotic pentameric stalk complex; it provides access to eukaryotic elongation factors at levels comparable to that of the eukaryotic stalk. The crystal structure of the archaeal heptameric (P0(P1)₂(P1)₂(P1)₂) stalk complex shows that the rRNA anchor protein P0 consists of an N-terminal rRNA-anchoring domain followed by three separated spine helices on which three P1 dimers bind. Based on the structure, we have generated P0 mutants depleted of any binding site(s) for P1 dimer(s). Factor-dependent GTPase assay of such mutants suggested that the first P1 dimer has higher activity than the others. Furthermore, we constructed a model of the archaeal 50 S with stalk complex by superposing the rRNA-anchoring domain of P0 on the archaeal 50 S. This model indicates that the C termini of P1 dimers where translation factors bind are all localized to the region between the stalk base of the 50 S and P0 spine helices. Together with the mutational experiments we infer that the functional significance of multiple copies of P1 is in creating a factor pool within a limited space near the stalk base of the ribosome.