High production of a class III lantipeptide AmfS in Streptomyces griseus

AmfS, a class III lantipeptide serves as a morphogen in Streptomyces griseus. Here, we constructed a high production system of AmfS in S. griseus. We isolated S. griseus Grd1 strain defective in glucose repression of aerial mycelium formation and found it suitable for the overproduction of AmfS. Two expression vectors carrying the strong and constitutive ermE2 promoter were constructed using a multicopy number plasmid, pIJ702. The use of the Grd1 strain combined with the expression vectors enabled high production of AmfS by S. griseus into its culture broth. The expression system was also effective for the generation of abundant AmfS derived from Streptomyces avermitilis. In addition, site-directed mutagenesis revealed the amino acid residues essential for the morphogen activity of AmfS. These results indicate that the constructed system enables efficient production of class III lantipeptides by Streptomyces.     

Biochemical characterization of thermostable β-1,4-mannanase belonging to the glycoside hydrolase family 134 from Aspergillus oryzae

Applied Microbiology and Biotechnology - A β-1,4-mannanase, termed AoMan134A, that belongs to the GH 134 family was identified in the filamentous fungus Aspergillus oryzae. Recombinant...     

Dynamics and local ordering of spin-labeled prion protein: an ESR simulation study of a highly PH-sensitive site

Valine 160 on beta-sheet-2 (S2) of mouse prion (moPrPC) has been previously identified as the most highly pH-sensitive site on moPrPC by ESR spectroscopy using site-directed spin labeling (SDSL) technique. However, no further theoretical analysis to reveal the molecular dynamics reported on the experimental ESR spectra is available. The X-band ESR spectra of R1 nitroxide spin label at V160 and four other sites are carefully analyzed over large pH and temperature ranges using a spectral simulation method based upon stochastic Liouville equation (SLE). The results clearly reveal the dynamics and ordering of the local environment of V160R1 showing that (i) molecular mobility of V160R1 on S2 gradually increases with a decrease of pH from 7.5 to 4.5; (ii) two distinctly different spectral components are simultaneously present in all spectra of V160R1 studied. The spectral components are, respectively, denoted as immobile (Im), characterized by lower molecular mobility and higher ordering, and mobile (Mb) component of high mobility and low ordering. The population ratio (Im/Mb) increases with increasing pH, while Im remains dominant in all V160R1 spectra. It suggests a more mobile and disordered dynamic molecular structure for mouse PrPC, which is very likely correlated with increased beta-sheet content at low pH, as the environment changes from neutral to acidic pH. Together with the results of the SLE-based analyses on the spectra of other sites that appear pH-insensitive, we suggest that the simultaneous presence of the spectral components for V160R1 is strongly correlated with the coexistence of multiple protein conformations in local structure of PrPC over the varied pH range. It demonstrates that the combined approach of the SDSL technique and the SLE-based analysis leads to a powerful method for unraveling the complexity of protein dynamics.     

Specific accumulation of gamma- and delta-tocotrienols in tumor and their antitumor effect in vivo

In contrast to extensive studies on tocopherols, very little is understood about tocotrienols (T3). We evaluated the antitumor activities of gamma-T3 and delta-T3 in murine hepatoma MH134 cells in vitro and in vivo. We found that delta-T3 inhibited the growth of MH134 cells more strongly than gamma-T3 by inducing apoptosis. In C3H/HeN mice implanted with MH134, it was found that gamma-T3 and delta-T3 feeding significantly delayed tumor growth. On the other hand, both T3 had no significant effect on body weight, normal-tissue weight and immunoglobulin levels. Intriguingly, we found that T3 was detected in tumor, but not in normal tissues. These results, to our knowledge, are the first demonstration of specific accumulation of gamma-T3 and delta-T3 in tumors and suggest that T3 accumulation is critical for the antitumor activities of T3.     

Laminin‐421 produced by lymphatic endothelial cells induces chemotaxis for human melanoma cells

Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC‐CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat‐treatment. The chemotactic activity of LEC‐CM was abolished by immunodepletion with anti‐laminin‐1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC‐CM contained laminin‐421. When melanoma C8161 cells were treated with function‐blocking antibodies to integrin α3 or α6, their chemotactic responses to LEC‐CM were markedly reduced. Furthermore, the knock‐down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC‐CM. These data suggest that laminin‐421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.     

ArfA recognizes the lack of mRNA in the mRNA channel after RF2 binding for ribosome rescue

Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3′ end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3′ end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding.