Preparation of reminiscent aroma mixture of Japanese soy sauce

To prepare an aroma mixture of Japanese soy sauce by fewest components, the aroma concentrate of good sensory attributes was prepared by polyethylene membrane extraction, which could extract only the volatiles with diethyl ether. GC-MS-Olfactometry was done with the aroma concentrate, and 28 odor-active compounds were detected. Application of aroma extract dilution analysis to the separated fraction revealed high flavor dilution factors with respect to acetic acid, 4-hydroxy-2(or5)-ethyl-5(or2)-methyl-3(2H)-furanone (HEMF), 3-methyl-1-butanol (isoamyl alcohol), and 3-(methylsulfanyl)propanal (methional). A model aroma mixture containing above four odorants showed a good similarity with the aroma of the soy sauce itself. Consequently, the reminiscent aroma mixture of soy sauce was prepared in water. The ratio of acetic acid, HEMF, isoamyl alcohol, and methional was 2500:300:100:1.     

Characterization of NADP+-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium,Colwellia psychrerythraea strain 34H

NADP⁺-dependent isocitrate dehydrogenase (IDH) isozymes of a psychrophilic bacterium, Colwellia psychrerythraea strain 34H, were characterized. The coexistence of monomeric and homodimeric IDHs in this bacterium was confirmed by Western blot analysis, the genes encoding two monomeric (IDH-IIa and IDH-IIb) and one dimeric (IDH-I) IDHs were cloned and overexpressed in Escherichia coli, and the three IDH proteins were purified. Both of the purified IDH-IIa and IDH-IIb were found to be cold-adapted enzymes while the purified IDH-I showed mesophilic properties. However, the specific activities of IDH-IIa and IDH-IIb were lower even at low temperatures than that of IDH-I. Therefore, IDH-I was suggested to be important for the growth of this bacterium. The results of colony formation of E. coli transformants carrying the respective IDH genes and IDH activities in their crude extracts indicated that the expression of the IDH-IIa gene is cold-inducible in the E. coli cells.     

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-d-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-d-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.     

Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301)

Monoclonal antibodies (mAbs) specific for the human macrophage galactose-type calcium-type lectin (MGL) were established. The recombinant extracellular domain of MGL was used to immunize a mouse, and 10 hybridoma clones were obtained. Binding of recombinant MGL to asialo-bovine submaxillary mucin was shown to be blocked by mAbs MLD-1, 4 and 6. Immunoprecipitation of MGL from lysates of COS-1 cells transfected with MGL cDNA (form 6A) was achieved with mAbs MLD-1, 4, 7, 8 and 16. Chimeric recombinant proteins between human MGL and mouse MGL1 were used to determine the location of the epitopes for these mAbs. mAbs MLD-8, 13, 15 and 16 interacted with the amino terminal side of the conserved WVDGTD sequence immediately upstream of QPD, whereas mAbs MLD-7, 12 and 17 interacted with the other side. mAbs MLD-1, 4, and 6 apparently required both sides of this boundary. mAbs MLD-15 and 16 were shown to recognize the protein products of alternatively spliced mRNA 6A/8A and 6C/8A, having deletions at the boundary of exons 7 and 8, in addition to full length and other spliced forms of MGL (6A, 6B and 6C), whereas the other mAbs bound only full length and forms 6A, 6B and 6C.     

Backbone 1H, 13C and 15N assignments of a 59 kDa Salmonella typhimurium periplasmic oligopeptide binding protein, OppA

Salmonella typhimurium OppA is the periplasmic oligopeptide-binding protein. Backbone resonances of OppA(D419N) on its own were assigned for ∼90% of residues. Missing residues are localised around the ligand-binding site, suggesting conformational flexibility in the unliganded state.     

Telomere protection by mammalian Pot1 requires interaction with Tpp1

The shelterin complex at mammalian telomeres contains the single-stranded DNA-binding protein Pot1, which regulates telomere length and protects chromosome ends. Pot1 binds Tpp1, the shelterin component that connects Pot1 to the duplex telomeric DNA-binding proteins Trf1 and Trf2. Control of telomere length requires that Pot1 binds Tpp1 as well as the single-stranded telomeric DNA, but it is not known whether the protective function of Pot1 depends on Tpp1. Alternatively, Pot1 might function similarly to the Pot1-like proteins of budding and fission yeast, which have no known Tpp1-like connection to the duplex telomeric DNA. Using mutant mouse cells with diminished Tpp1 levels, RNA interference directed to mouse Tpp1 and Pot1, and complementation of mouse Pot1 knockout cells with human and mouse Pot1 variants, we show here that Tpp1 is required for the protective function of mammalian Pot1 proteins.     

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.     

Contribution of Neuropilin-1 in Radiation-Survived Subclones of NSCLC Cell Line H1299

Non-small cell lung cancer (NSCLC) is an aggressive lung cancer accounting for approximately 85% of all lung cancer patients. For the patients with Stages IIIA, IIIB, and IIIC, the 5-year survival is low though with the combination with radiotherapy and chemotherapy. In addition, the occurrence of tumor cells (repopulated tumors) that survive irradiation remains a challenge. In our previous report, we subcloned the radiation-surviving tumor cells (IR cells) using the human NSCLC cell line, H1299, and found that the expression of neuropilin-1 (NRP-1) was upregulated in IR cells by the microarray analysis. Here, we investigated the contribution of neuropilin-1 to changes in the characteristics of IR cells. Although there were no differences in angiogenic activity in the tube formation assay between parental and IR cells, the cell motility was increased in IR cells compared to parental cells in the cell migration assay. This enhanced cell motility was suppressed by pretreatment with anti-NRP-1 antibody. Although further studies are necessary to identify other molecules associated with NRP-1, the increase in cellular motility in IR cells might be due to the contribution of NRP-1. Inhibition of NRP-1 would help control tumor malignancy in radiation-surviving NSCLC.