Biochemical characterization and structural insight into aliphatic β‐amino acid adenylation enzymes IdnL1 and CmiS6

Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic β‐amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand‐alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic β‐amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3‐aminobutanoic acid and 3‐aminononanoic acid, respectively. We solved the X‐ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic β‐amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the β‐amino group of the substrates. However, the hydrophobic side‐chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3‐aminobutanoate of the trapped acyl‐adenylate intermediate to construct a shallow substrate‐binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3‐aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic β‐amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238–1247. © 2017 Wiley Periodicals, Inc.     

Culture medium refinement by dialysis for the expansion of human induced pluripotent stem cells in suspension culture

Human induced pluripotent stem cells (hiPSCs) secrete essential autocrine factors that are removed along with toxic metabolites when the growth medium is exchanged daily. In this study, after determining the minimum inhibitory level of lactic acid for hiPSCs, a medium refining system was constructed by which toxic metabolites were removed from used culture medium and autocrine factors as well as other growth factors were recycled. Specifically, about 87 % of the basic fibroblast growth factor and 80 % of transforming growth factor beta 1 were retained in the refined medium after dialysis. The refined medium efficiently potentiated the proliferation of hiPS cells in adherent culture. When the refining system was used to refresh medium in suspension culture, a final cell density of (1.1 ± 0.1) × 10⁶ cells mL^?1 was obtained, with 99.5 ± 0.2 % OCT 3/4 and 78.3 ± 1.1 % TRA-1-60 expression, on day 4 of culture. These levels of expression were similar to those observed in the conventional suspension culture. With this method, culture medium refinement by dialysis was established to remove toxic metabolites, recycle autocrine factors as well as other growth factors, and reduce the use of macromolecules for the expansion of hiPSCs in suspension culture.     

Mutagenic specificity of N-acetoxy-3-aminobenzanthrone, a major metabolically activated form of 3-nitrobenzanthrone, in shuttle vector plasmids propagated in human cells

3-Nitrobenzanthrone (3-NBA) is a potent environmental mutagen and a potential human carcinogen present in diesel exhaust and airborne particulates. N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) has been shown to be a major reactive metabolite of 3-NBA, which mainly produces adducts with guanine and adenine in cellular DNA. Here we analyzed mutations induced by N-Aco-ABA using supF shuttle vector plasmids to elucidate the mutagenic specificity of 3-NBA in human cells. Base sequence analysis of more than 100 plasmids with supF mutations induced in wildtype and DNA repair-deficient XP cells revealed that the major mutation was base substitutions of which the majority (42 and 38%, respectively) were G:C to T:A transversions. The next major mutation was G:C to A:T and A:T to G:C base substitutions in wildtype and XP cells, respectively. The DNA polymerase stop assay using N-Aco-ABA-treated plasmids as a template showed that most stop signals, i.e., adducted sites, appeared at G:C sites. These results suggest that N-Aco-ABA binds preferably to guanine rather than adenine, and adducted adenine is repaired more efficiently by the nucleotide excision repair. Error-prone DNA polymerases could insert adenine at sites opposite to N-Aco-ABA-adducted guanine, which leads to G:C to T:A transversion. These findings could be very important to evaluate the human lung cancer risk of environmental 3-NBA.     

Identification of a human monoclonal Fab with neutralizing activity against H3N2 influenza A strain from a newly constructed human Fab library

A combinatorial Fab library was constructed in pComb3H phagemid vectors, using RNA from peripheral blood lymphocytes of a healthy volunteer who had recovered from an influenza A virus infection. The library contained approximately 1.3 x 10(8)E. coli transformants. Bio-panning was carried out against an influenza vaccine containing components of influenza A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and B/Shandong/7/97 for the enrichment of phages displaying human Fab specific to the viral proteins. E. coli transformed with IF1A11, 1 of 94 randomly selected clones, displayed a human Fab antibody molecule (FabIF1A11) with efficient neutralizing activity against H3N2 influenza A virus strains. The purified FabIF1A11 demonstrated neutralizing activity against A/Okayama/6/01 (H3N2) and A/Kitakyushu/159/93 (H3N2) with 50% plaque reduction neutralization titers of 0.11 microg/ml (2.2 nM) and 1.4 microg/ml (28 nM) respectively. However, FabIF1A11 did not show neutralizing activity against the influenza A virus strain A/USSR/77 (H1N1) or the influenza B virus strain B/Kanagawa/73, even at a concentration of 20 microg/ml (400 nM). The Kd of FabIF1A11 was calculated as 3.6 x 10(-9) M. FabIF1A11 was estimated to recognize a conformational epitope on the hemagglutinin of A/Okayama/6/01 (H3N2). The human monoclonal Fab product FabIF1A11 may have potential as a therapeutic or short-term prophylactic molecule for humans with influenza A H3N2 infection.     

Paleotemperature response to monsoon activity in the Japan Sea during the last 160 kyr

Paleo-sea-surface temperatures in the northeastern- and southeastern-parts of the Japan Sea were reconstructed for the last 160 kyr using alkenone temperatures (U^K′₃₇-temperatures). U^K′₃₇-temperatures at two sites show distinct glacial–interglacial changes during the last 160 kyr except for the interval corresponding to middle MIS 3 to MIS 2. On orbital-timescales, U^K′₃₇-temperature tends to be high during MIS 5e, MIS 5c, and MIS 5a, which coincides with the intervals of stronger East Asian summer monsoon activity. The amplitude of temperature fluctuations in the Japan Sea is significantly higher than those in the neighboring seas. We suggest that the SST variation was amplified by the increasing source water (Kuroshio water) temperature and the changes in the volume transport of the Tsushima Warm Current (TWC) and/or the north–south oscillation of the sub-polar front position within the Japan Sea. Millennial-scale temperature fluctuations in the Japan Sea show that the temperature at the northern site was higher than that at the southern site during warmer periods of MIS 5, which is called “temperature reversal.” By analogy with modern oceanography, the temperature reversal could reflect the enhanced volume transport of the TWC and the spatial relationship between the studied site and the branches of the TWC, which is an essential factor in north–south temperature reversal around the eastern Japan Sea. Temperature drops were found at 114 ka, 111 ka, 93 ka, 87 ka, and 77 ka in MIS 5. Those events were associated with an increase in organic carbon and alkenone contents and can be correlated with the abundance peaks of ice-rafted debris (IRD) at Site GH05-1208 in the northern Japan Sea, suggesting that the surface water was cooled by enhanced mixing and consequent upwelling in a stronger winter monsoon regime.     

Dimethylfumarate inhibits tumor cell invasion and metastasis by suppressing the expression and activities of matrix metalloproteinases in melanoma cells

NF-κB acts as a signal transducer during tumor progression, cell invasion, and metastasis. Dimethylfumarate (DMF) is reported to inhibit tumor necrosis factor-α-induced nuclear entry of NF-κB/p65. However, only a few reports suggest that DMF inhibits tumor metastasis; also the molecular mechanisms underlying the inhibition of metastasis are poorly understood. We investigated the inhibition of tumor invasion and metastasis by DMF in a melanoma cell line, B16BL6. DMF inhibited B16BL6 cell invasion and metastasis by suppressing the expression and activities of MMPs. DMF also inhibited the nuclear entry of NF-κB/p65, thus inhibiting B16BL6 cell invasion and metastasis. These results suggest that DMF is potentially useful as an anti-metastatic agent for the treatment of malignant melanoma.     

Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci

Glycosylation of flagellin contributes to swimming and swarming motilities, adhesion ability, and consequently virulence in Pseudomonas syringae pv. tabaci 6605. Glycans attached to six serine residues are located in the central region of the flagellin polypeptide. The glycan structure at position Ser 201 was recently revealed to consist of two l-rhamnoses and one modified 4-amino-4,6-dideoxyglucose (viosamine). To clarify the mechanisms for glycosylation of modified viosamine, genes encoding dTDP-viosamine aminotransferase (vioA), dTDP-viosamine acetyltransferase (vioB), and viosamine-derivative transferase (vioT) were isolated and defective mutants were generated. MALDI-TOF–MS analysis of a lysyl endopeptidase-digested peptide including all six glycosylation sites from each flagellin indicated that the molecular masses of the three flagellin mutants were reduced with highly heterogeneous patterns at regular intervals of 146 Da in the mass range from m/z 13,819 to 15,732. The data indicated that the glycopeptides obtained from mutants had glycans consisting only of deoxyhexose instead of the flagellin glycans including the viosamine derivatives determined previously. The motility and virulence on host tobacco leaves were strongly impaired in the ΔvioA mutant and were weakly reduced in the ΔvioB and ΔvioT mutant strains. These results suggest that the genes vioA, vioB, and vioT are essential for glycosylation of flagellin, and accordingly are required for bacterial virulence.     

Dual wavelength imaging allows analysis of membrane fusion of influenza virus inside cells

Influenza virus hemagglutinin (HA) is a determinant of virus infectivity. Therefore, it is important to determine whether HA of a new influenza virus, which can potentially cause pandemics, is functional against human cells. The novel imaging technique reported here allows rapid analysis of HA function by visualizing viral fusion inside cells. This imaging was designed to detect fusion changing the spectrum of the fluorescence-labeled virus. Using this imaging, we detected the fusion between a virus and a very small endosome that could not be detected previously, indicating that the imaging allows highly sensitive detection of viral fusion.