Increased production of antioxidative sesaminol glucosides from sesame oil cake through fermentation by Bacillus circulans strain YUS-2

Bacillus circulans strain YUS-2 was isolated as the strongest antioxidant-producer in fermentation of sesame oil cake (SOC, defatted residue yielded from sesame seed oil production). Two major strong antioxidants from fermented SOC were purified and identified as known sesaminol triglucoside and sesaminol diglucoside, however, our results demonstrated that the fermentation process with B. circulans YUS-2 was highly effective to gain the extraction efficiency of the sesaminol glucosides.     

Flagellar filament elongation can be impaired by mutations in the hook protein FlgE of Salmonella typhimurium: a possible role of the hook as a passage for the anti-sigma factor FlgM

Among motile revertants isolated from flagellar hook-deficient ( flgE ) mutants of Salmonella typhimurium one produced only short flagellar filaments in L broth, despite the fact that flagellin itself has the ability to polymerize into long filaments in vitro . This pseudorevertant has an intragenic suppressor, resulting in a two-amino-acid substitution (Asp-Gln→Ala-Arg) in the C-terminal region of the hook protein, FlgE. The flagellation of the pseudorevertant was greatly affected by the concentration of NaCl in the culture media: we observed no filaments in the absence of NaCl, short filaments in 1% NaCl and full-length filaments in 2% NaCl. Electron microscopy of osmotically shocked cells showed that the number of hook–basal bodies on cells was constant under various NaCl conditions. Furthermore, we found that the mutant hook was straight rather than curved. We monitored the cellular flagellin level of this pseudorevertant under various NaCl concentrations by immunoblotting. It was revealed that little flagellin was present under NaCl-free conditions in contrast with the ordinary amounts of flagellin present in 2% NaCl. As the expression of flagellin is regulated by competitive interaction of a sigma factor, FliA, and a corresponding anti-sigma factor, FlgM, we also observed the effect of NaCl on the secretion of FlgM. FlgM was secreted into the media in more than 1% NaCl but accumulated inside the cells in the absence of NaCl, indicating that the failure of secretion of FlgM in the absence of salt was the cause of the impaired elongation of filaments.     

Neuroepithelial cells supply an initial transient wave of MSC differentiation

Mesenchymal stem cells (MSCs) are defined as cells that undergo sustained in vitro growth and are able to give rise to multiple mesenchymal lineages. Although MSCs are already used in regenerative medicine little is known about their in vivo behavior and developmental derivation. Here, we show that the earliest wave of MSC in the embryonic trunk is generated from Sox1+ neuroepithelium but not from mesoderm. Using lineage marking by direct gfp knock-in and Cre-recombinase mediated lineage tracing, we provide evidence that Sox1+ neuroepithelium gives rise to MSCs in part through a neural crest intermediate stage. This pathway can be distinguished from the pathway through which Sox1+ cells give rise to oligodendrocytes by expression of PDGFRbeta and A2B5. MSC recruitment from this pathway, however, is transient and is replaced by MSCs from unknown sources. We conclude that MSC can be defined as a definite in vivo entity recruited from multiple developmental origins.     

Fermentative production of L-alanyl-L-glutamine by a metabolically engineered Escherichia coli strain expressing L-amino acid alpha-ligase

In spite of its clinical and nutritional importance, l-alanyl-l-glutamine (Ala-Gln) has not been widely used due to the absence of an efficient manufacturing method. Here, we present a novel method for the fermentative production of Ala-Gln using an Escherichia coli strain expressing l-amino acid alpha-ligase (Lal), which catalyzes the formation of dipeptides by combining two amino acids in an ATP-dependent manner. Two metabolic manipulations were necessary for the production of Ala-Gln: reduction of dipeptide-degrading activity by combinatorial disruption of the dpp and pep genes and enhancement of the supply of substrate amino acids by deregulation of glutamine biosynthesis and overexpression of heterologous l-alanine dehydrogenase (Ald). Since expression of Lal was found to hamper cell growth, it was controlled using a stationary-phase-specific promoter. The final strain constructed was designated JKYPQ3 (pepA pepB pepD pepN dpp glnE glnB putA) containing pPE167 (lal and ald expressed under the control of the uspA promoter) or pPE177 (lal and ald expressed under the control of the rpoH promoter). Either strain produced more than 100 mM Ala-Gln extracellularly, in fed-batch cultivation on glucose-ammonium salt medium, without added alanine and glutamine. Because of the characteristics of Lal, no longer peptides (such as tripeptides) or dipeptides containing d-amino acids were formed.     

cDNA microarray analyses reveal candidate marker genes for the detection of ascidian disease in Korea

A serious disease of the ascidian Halocynthia roretzi has been spread extensively among Korean aquaculture sites. To reveal the cause of the disease and establish a monitoring system for it, we constructed a cDNA microarray spotted with 2,688 cDNAs derived from H. roretzi hemocyte cDNA libraries to detect genes differentially expressed in hemocytes between diseased and non-diseased ascidians. We detected 21 genes showing increased expression and 16 genes showing decreased expression in hemocytes from diseased ascidians compared with those from non-diseased ascidians. RT-PCR analyses confirmed that the expression levels of genes encoding astacin, lysozyme, ribosomal protein PO, and ubiquitin-ribosomal protein L40e fusion protein were increased in hemocytes from diseased ascidians, while those of genes encoding HSP40, HSP70, fibronectin, carboxypeptidase and lactate dehydrogenase were decreased. These genes were expressed not only in hemocytes but also in various other tissues in ascidians. Furthermore, the expression of glutathione-S transferase omega, which is known to be up-regulated in H. roretzi hemocytes during inflammatory responses, was strongly increased in hemocytes from diseased ascidians. These gene expression profiles suggest that immune and inflammatory reactions occur in the hemocytes of diseased ascidians. These genes will be good markers for detecting and monitoring this disease of ascidians in Korean aquaculture sites.     

FliK regulates flagellar hook length as an internal ruler

The mechanism of length control of the flagellar hook is under debate between two theories. One claims that the FliK directly measures the hook length as a molecular ruler, while the other claims that the cytoplasmic substructure measures the amount of hook subunits to determine the hook length. Both agree that the FliK C-terminal domain catalyses the substrate-specificity switch to terminate hook elongation. In this study, we systematically created fliK mutants with deletions and insertions at various sites within the FliK N-terminal domain and analysed their effects on the final hook length. Insertions of peptide fragments from the Yersinia YscP into FliK gave rise to hooks with defined lengths, which was proportional to the molecular size of the FliK-YscP chimeras. Among fliK deletion mutants, only those with small truncations in three specific sites of FliK produced hooks of a defined, shortened length. For the majority of deletion mutants, FliK was secreted, but hook length was not controlled. On the other hand, for some deletion mutants FliK was not secreted, but the hook length was controlled, indicating that FliK secretion is not necessary for hook-length control. We conclude that FliK regulates hook length as an internal molecular ruler.