Functional Characterization of 3-Aminobenzoic Acid Adenylation Enzyme PctU and UDP-N-Acetyl-d-Glucosamine: 3-Aminobenzoyl-ACP Glycosyltransferase PctL in Pactamycin Biosynthesis

Pactamycin is an antibiotic produced by Streptomyces pactum with antitumor and antimalarial properties. Pactamycin has a unique aminocyclitol core that is decorated with 3-aminoacetophenone, 6-methylsaliciate, and an N,N-dimethylcarbamoyl group. Herein, we show that the adenylation enzyme PctU activates 3-aminobenzoic acid (3ABA) with adenosine triphosphate and ligates it to the holo form of the discrete acyl carrier protein PctK to yield 3ABA-PctK. Then, 3ABA-PctK is N-glycosylated with uridine diphosphate-N-acetyl-d -glucosamine (UDP-GlcNAc) by the glycosyltransferase PctL to yield GlcNAc-3ABA-PctK. Because 3ABA is known to be a precursor of the 3-aminoacetophenone moiety, PctU appears to be a gatekeeper that selects the appropriate 3-aminobenzoate starter unit. Overall, we propose that acyl carrier protein-bound glycosylated 3ABA derivatives are biosynthetic intermediates of pactamycin biosynthesis.     

Inference of gene regulation via miRNAs during ES cell differentiation using MiRaGE method

MicroRNA (miRNA) is a critical regulator of cell growth, differentiation, and development. To identify important miRNAs in a biological process, many bioinformatical tools have been developed. We have developed MiRaGE (MiRNA Ranking by Gene Expression) method to infer the regulation of gene expression by miRNAs from changes of gene expression profiles. The method does not require precedent array normalization. We applied the method to elucidate possibly important miRNAs during embryonic stem (ES) cell differentiation to neuronal cells and we infer that certain miRNAs, including miR-200 family, miR-429, miR-302 family, and miR-17-92 cluster members may be important to the maintenance of undifferentiated status in ES cells.     

Formation of macroporous organic‐inorganic polymer hybrids using tea extract

Macroporous organic–inorganic polymer hybrids were prepared from poly(vinyl pyrrolidone), and inorganic alkoxides. To a reaction mixture of poly(vinyl pyrrolidone) and tetramethoxysilane, extract from tea leafs and HCl aqueous solution in methanol were added. The resulting mixture was constantly stirred at room temperature for 1 h and heated at 60°C for two weeks. Consequently, the corresponding polymer hybrid became a macroporous material having a pore size from 3.26 to 20.86 μm. We succeeded in finding that the pruned tea leafs were able to utilize the synthesis of novel macroporous materials. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

NAD+‐Dependent Dehydrogenase PctP and Pyridoxal 5′‐Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis

The unique five‐membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d ‐glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar‐derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD⁺‐dependent alcohol dehydrogenase PctP and pyridoxal 5′‐phosphate dependent aminotransferase PctC converted N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophonone into 3′‐amino‐3′‐deoxy‐N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophenone. Further, N‐acetyl‐d ‐glucosaminyl‐3‐aminophenyl‐β‐oxopropanoic acid ethyl ester was converted into the corresponding 3′‐amino derivative. However, PctP did not oxidize most of the tested d ‐glucose derivatives, including UDP‐GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.     

Mechanism‐Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP‐Dependent 3‐Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin

Mutational analysis of the pyridoxal 5′‐phosphate (PLP)‐dependent enzyme PctV was carried out to elucidate the multi‐step reaction mechanism for the formation of 3‐aminobenzoate (3‐ABA) from 3‐dehydroshikimate (3‐DSA). Introduction of mutation K276R led to the accumulation of a quinonoid intermediate with an absorption maximum at 580 nm after the reaction of pyridoxamine 5′‐phosphate (PMP) with 3‐DSA. The chemical structure of this intermediate was supported by X‐ray crystallographic analysis of the complex formed between the K276R mutant and the quinonoid intermediate. These results clearly show that a quinonoid intermediate is involved in the formation of 3‐ABA. They also indicate that Lys276 (in the active site of PctV) plays multiple roles, including acid/base catalysis during the dehydration reaction of the quinonoid intermediate.     

Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2

ALG-2, a 22-kDa penta-EF-hand protein, is involved in cell death, signal transduction, membrane trafficking, etc., by interacting with various proteins in mammalian cells in a Ca²⁺-dependent manner. Most known ALG-2-interacting proteins contain proline-rich regions in which either PPYPXnYP (type 1 motif) or PXPGF (type 2 motif) is commonly found. Previous X-ray crystal structural analysis of the complex between ALG-2 and an ALIX peptide revealed that the peptide binds to the two hydrophobic pockets. In the present study, we resolved the crystal structure of the complex between ALG-2 and a peptide of Sec31A (outer shell component of coat complex II, COPII; containing the type 2 motif) and found that the peptide binds to the third hydrophobic pocket (Pocket 3). While amino acid substitution of Phe⁸⁵, a Pocket 3 residue, with Ala abrogated the interaction with Sec31A, it did not affect the interaction with ALIX. On the other hand, amino acid substitution of Tyr¹⁸⁰, a Pocket 1 residue, with Ala caused loss of binding to ALIX, but maintained binding to Sec31A. We conclude that ALG-2 recognizes two types of motifs at different hydrophobic surfaces. Furthermore, based on the results of serial mutational analysis of the ALG-2-binding sites in Sec31A, the type 2 motif was newly defined.     

In Situ Observation of Phase Separation of a Barium Borate Melt in a Stable Immiscibility Region under Microgravity

The precipitation of droplets was directly observed on a BaO–B₂O₃ melt in a drop shaft experiment. This is the first time that precipitation of droplets has been observed in a 4.5 s drop test. The melt film of 4BaOz96B₂O₃ (mol%) held on a platinum wire loop was heated above the critical tem-perature to produce uniformity and was cooled down to the phase separation temperature range. Phase separation of the melt was observed directly with a video camera. The IR image of the melt was simultaneously detected with a CCD array and was converted into a two-dimensional thermograph.     

Effect of the Helical Tether of a Resin‐Supported Peptide Catalyst for Friedel–Crafts‐Type Alkylation in Water

A detailed investigation of the helical part of the resin‐supported peptide catalyst possessing a turn motif and helical unit was conducted to clarify the structure‐activity relationship. The peptide catalyst with an α‐ or 3₁₀‐helical tether was effective for an enantioselective Friedel–Crafts‐type alkylation in water. From the spectral analysis of the peptide with an optimum sequence, it was demonstrated that the helical moiety of the peptide catalyst played a role for stabilizing a terminal turn structure.     

Submicron laser-textured vents for self-cleaning injection molds

Clogging of venting slots in injection molds is a common maintenance problem caused by the degradation and the accumulation of gaseous and volatiles by-products of polymer melting. In this work, the effect of laser-induced periodic surface structures on the self-cleaning properties of venting slots is investigated. The degradation of poly(ethylene terephthalate) (PET) over different surfaces is characterized by reproducing the mechanisms that occurs in mold cavities when the air is pushed through the venting channel. An imaging technique is developed for the quantification of the sediment that deposits on sample surfaces due to condensation of by-products of PET melting. The experimental results indicate that the use of a multiscale texture minimizes the deposition of residues on the vent surface reducing it from 17.2 to 3.1%. A linear dependency between contact angle and clogging ratio indicates the efficacy of the model that explains vent self-cleaning properties considering their wetting properties.     

Supramolecular Architectures of Nucleic Acid/Peptide Hybrids

Supramolecular architectures that are built artificially from biomolecules, such as nucleic acids or peptides, with structural hierarchical orders ranging from the molecular to nano-scales have attracted increased attention in molecular science research fields. The engineering of nanostructures with such biomolecule-based supramolecular architectures could offer an opportunity for the development of biocompatible supramolecular (nano)materials. In this review, we highlighted a variety of supramolecular architectures that were assembled from both nucleic acids and peptides through the non-covalent interactions between them or the covalently conjugated molecular hybrids between them.