Comparison of Soybean Tissue Extracted with Different Solvents

The enantioselective hydrolysis of (R,S)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine (I) with enzymes was investigated. Optically active I and its hydrolyzate, 7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine (II), are the intermediates for preparing optically active ofloxacins, whose racemate is known to be an excellent antibacterial agent. Lipoprotein lipase from Pseudomonas fluorescens (LPL Amano 3) was found to predominantly hydrolyze (S)-I, giving (R)-I in 54% e.e. and (R)-II in 44% e.e. On the other hand, lipase from Candida cylindracea was found to predominantly hydrolyze (R)-I, giving (S)-I in 24% e.e. and (S)-II in 20% e.e. Since, the optical purities of I and II thus obtained were not particularly high, these optically active I and II were converted into 3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4-(3,5-dinitrobenzoyl)-4H-[1,4]benzoxazine (IV). After recrystallizing IV from ethyl acetate-hexane, (S)- and (R)-II were obtained with high enantiomeric excess by removing the crystallized racemic IV and subsequently hydrolyzing the resulting optically active IV with alkali. The reduction of II afforded 7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine (III), for which the optical purity was estimated to be >96%e.e. by HPLC analysis. (R)- and (S)-ofloxacin were prepared from (R)- and (S)-III with retention of their configuration.     

Simple Synthesis of (R)-5-Hexadecanolide

Chemical modification of tryptophan residues in abrin-a with N-bromosuccinimide (NBS) was studied with regard to saccharide-binding. The number of tryptophan residues available for NBS oxidation increased with lowering pH, and 11 out of the 13 tryptophan residues in abrin-a were eventually modified with NBS at pH 4.0, while 6 tryptophan residues were modified at pH 6.0 in the absence of specific saccharides. Modification of tryptophan residues at pH 6.0 greatly decreased the saccharide-binding ability of abrin-a, and only 2% of the hemagglutinating activity was retained after modification of 3 residues/mol. When the modification was done in the presence of lactose or galactose, 1 out of 3 residues/mol remained unmodified with a retention of a fairly high hemagglutinating activity. However, GalNAc did not show such a protective effect. NBS-oxidation led to a great loss of the fluorescence of abrin-a, and after modification of 3 tryptophan residues/mol, the fluorescence intensity at 345 nm was only 38% of that of the unmodified abrin-a. The binding of lactose to abrin-a altered the environment of the tryptophan residue at the saccharide-binding site of abrin-a, leading to a blue shift of the fluorescence spectrum. The ability to generate such fluorescence spectroscopic changes induced by lactose-binding was retained in the derivative in which 2 tryptophan residues/mol were oxidized in the presence of lactose, but not in the derivative in which 3 tryptophan residues/mol were oxidized in the absence of lactose. Importance of the tryptophan residue(s) in the saccharide-binding of abrin-a is suggested.     

Food Processing Characteristics of Soybean Proteins

Relation between sulfhydryl groups in soybean proteins and the physical properties of tofu was studied. Changes in the amount of sulfhydryl groups by heating or treatment with urea were more rapid in 11S protein as compared with 7S protein. Moreover, by changing the amount of sulfhydryl groups in proteins by N-ethylmaleimide, 2-mercapto-ethanol and dithiothreitol, the physical properties of tofu from 11S protein were more significantly effected than that from 7S protein. Namely, tofu-gel from 11S. protein got harder and stronger as the amount of sulfhydryl groups increased.

The results may suggest that tofu prepared from IIS protein has more disulfide bonds in its gel than that from 7S protein.     

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.     

Suppression of Hepcidin Expression and Iron Overload Mediate Salmonella Susceptibility in Ankyrin 1 ENU-Induced Mutant

Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1^Ity16/Ity16 mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1^Ity16/Ity16 mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1^Ity16/Ity16, the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1^+/Ity16 mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1^Ity16/Ity16 and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1^+/Ity16 mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1 ^+/Ity16 heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.     

Oceanic Distribution,Behaviour, and a Winter Aggregation Area of Adult Atlantic Sturgeon,Acipenser oxyrinchus oxyrinchus,in the Bay of Fundy,Canada

Seasonal distribution of adult Atlantic sturgeon was examined using pop-up satellite archival tags (PSATs) and ultrasonic transmitters deployed in the Saint John River, New Brunswick, Canada. Seven MK10 PSATs programmed for release in June 2012 and seven MiniPAT PSATs programmed for release in February and April 2013 were deployed in August 2011 and 2012, respectively. Eleven of 14 PSATs surfaced and transmitted depth and temperature data archived for the duration of their deployment (121–302 days). Among these eleven PSATs, five were recovered and 15-sec archival data was downloaded. Following exit from the Saint John River in the fall, tagged fish occupied a mean monthly depth of 76.3–81.6 m at temperatures as low as 4.9˚C throughout the winter before returning to shallower areas in the spring. The majority of ultrasonic detections occurred in the Bay of Fundy, but fish were detected as far as Riviere Saint-Jean, Quebec, approximately 1500 km from the Bay of Fundy (representing long-distance migratory rates of up to 44 km/day). All PSATs were first detected in the Bay of Fundy. Tags that released in February and April were found 5–21 km offshore of the Saint John Harbour, while tags that released in June were first detected in near shore areas throughout the Bay of Fundy. The substrate at winter tag release locations (estimated from backward numerical particle-tracking experiments) consisted primarily of moraines and postglacial mud substrate with low backscatter strength, indicative of soft or smooth seabed. Based on the proximity of winter tag release locations, the consistent depths observed between fish, and previous research, it is suspected that a winter aggregation exists in the Bay of Fundy. This study expands the understanding of the marine distribution and range of Atlantic sturgeon on the east coast of Canada.     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.