Metabolic engineering of bread wheat improves grain iron concentration and bioavailability

Bread wheat (Triticum aestivum L.) is cultivated on more land than any other crop and produces a fifth of the calories consumed by humans. Wheat endosperm is rich in starch yet contains low concentrations of dietary iron (Fe) and zinc (Zn). Biofortification is a micronutrient intervention aimed at increasing the density and bioavailability of essential vitamins and minerals in staple crops; Fe biofortification of wheat has proved challenging. In this study we employed constitutive expression (CE) of the rice (Oryza sativa L.) nicotianamine synthase 2 (OsNAS2) gene in bread wheat to up‐regulate biosynthesis of two low molecular weight metal chelators – nicotianamine (NA) and 2′‐deoxymugineic acid (DMA) – that play key roles in metal transport and nutrition. The CE‐OsNAS2 plants accumulated higher concentrations of grain Fe, Zn, NA and DMA and synchrotron X‐ray fluorescence microscopy (XFM) revealed enhanced localization of Fe and Zn in endosperm and crease tissues, respectively. Iron bioavailability was increased in white flour milled from field‐grown CE‐OsNAS2 grain and positively correlated with NA and DMA concentrations.     

Leucine-responsive regulatory protein, IS 1 insertions, and the negative regulator FaeA control the expression of the fae (K88) operon in Escherichia coli

Nucleotide sequence analysis of the fae operon encoding the biosynthesis of K88 fimbriae revealed the presence of two divergently transcribed regulatory genes, faeA and faeB, separated by two inverted iS 1 insertions. The amino acid sequences of the regulatory proteins FaeA and FaeB show similarity to the primary structure of corresponding regulatory proteins involved in the biosynthesis of Pap and S fimbriae. Expression of faeA is positively controlled by the FaeA protein, whereas K88 fimbriae production is negatively controlled by the co-operative activity of FaeA and the leucine-responsive regulatory protein (Lrp). Exchange of FaeA for Papl, a positive regulator of Pap fimbriae expression, also represses K88 production indicating that the combination Papl/Lrp has opposite effects on fae and pap expression. Mutations in faeB had no effect on the biosynthesis of K88 fimbriae. The presence of the two iS 1 insertions is hypothesized to neutralize part of the repression of K88 biosynthesis by FaeA/Lrp. Like pap, the fae operon does not respond to exogenous leucine.     

Discovery of S1P agonists with a dihydronaphthalene scaffold

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P₁ receptor agonist with high selectivity against S1P₃ and enhanced efficacy in lowering peripheral lymphocyte counts in mice.     

Investigation on hyperuricemia in children with obesity or various pediatric disorders

The present study aims at investigating the frequency and characteristics of hyperuricemia in both obese and sick children. First, we established our own reference values for serum uric acid (UA), since UA values are highly dependent upon age. In the analysis of 328 samples consisting of six different age groups: <1, 1-3, 4-6, 7-9, 10-12, and 13-15 years, the mean values for UA were found to increase significantly with an increase of age. A significant sex difference was observed only in the age group of 13-15 years. Hyperuricemia was defined as the values over the mean value plus 2 standard deviations for each age group. Next, we examined the frequency of hyperuricemia in 1,687 obese children aged 6-15 years and its relation to metabolic syndrome (MetS). A total of 328 children (19.4%) were found to have hyperuricemia. Among them, 98 children (29.9%) had MetS, whereas 197 (14.5%) out of 1,359 children without hyperuricemia had MetS. Finally, the frequency of hyperuricemia in sick patients was investigated using 13,675 samples from 9,405 patients. Hyperuricemia was seen in 348 (3.7%) patients after excluding redundant samples. The number of patients with hyperuricemia was higher in males than in females. The most common disorder causing hyperuricemia was gastroenteritis, followed by respiratory tract infection and cardiac diseases. This first comprehensive study of childhood hyperuricemia is useful for considering its relationship with hyperuricemia and life-style-related disorders occurring in adulthood.     

Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.     

Discovery of 3H-imidazo[4,5-c]quinolin-4(5H)-ones as potent and selective dipeptidyl peptidase IV (DPP-4) inhibitors

In recent years, dipeptidyl peptidase IV inhibitors have been noted as valuable agents for treatment of type 2 diabetes. Herein, we report the discovery of a novel potent DPP-4 inhibitor with 3H-imidazo[4,5-c]quinolin-4(5H)-one as skeleton. After efficient optimization of the lead compound 2a at the 7- and 8-positions using a docking study, we found 28 as a novel DPP-4 inhibitor with excellent selectivity against various DPP-4 homologues. Compound 28 showed strong DPP-4 inhibitory activity compared to marketed DPP-4 inhibitors. We also found that a carboxyl group at the 7-position could interact with the residue of Lys554 to form a salt bridge. Additionally, introduction of a carboxyl group to 7-position led to both activity enhancement and reduced risk for hERG channel inhibition and induced phospholipidosis. In our synthesis of compounds with 7-carboxyl group, we achieved efficient regioselective synthesis using bulky ester in the intramolecular palladium coupling reaction.     

Complete amino acid sequence of jack bean urease

The subunit structure of jack bean urease has been unresolved in spite of many investigations. Thus far, the molecular weight for the native urease seem to range from 480,000 to 590,000 and the values for the monomer range from 30,000 to 97,000. The complete amino acid sequence of jack bean urease has been determined primarily by sequencing cyanogen bromide peptides, which were aligned by overlapping peptides obtained by lysylendopeptidase digestion of the protein and tryptic digestion of the citraconylated protein. The protein contains 840 amino acid residues in a single polypeptide chain and the subunit molecular weight calculated from the sequence is 90,790. The value of 544,740 for the hexamer, consistent with the value of 580,000 determined for intact urease by centrifugal analyses, indicated that urease consists of six subunits. Thirteen of 25 histidine residues in the urease subunit are crowded in the region between residues 479 and 607. Urease is a nickel metalloenzyme and the nickel has an essential role in catalysis by this enzyme. It is noteworthy that cysteine-592, which is recognized as essential for enzymatic activity and is related to the nickel ion in the active center, is located on this histidine-rich sequence.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway

Claudin-16 (CLDN-16) is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. The tight junctional localization and Mg(2+) transport of CLDN-16 are regulated by cAMP/PKA-dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN-16 in a direct or indirect manner. CLDN-16 was stably expressed in Madin-Darby canine kidney (MDCK) cells using a Tet-OFF system. The phosphorylation of CLDN-16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN-16 in a concentration-dependent manner. The phosphorylated CLDN-16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg(2+). Vasodilator-stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione-S-transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN-16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN-16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser160Ala) of VASP, CLDN-16 was phosphorylated by DBcAMP and was associated with ZO-1, a tight junctional-scaffolding protein, without integral cell-cell junctions. We suggest that PKA directly phosphorylates CLDN-16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg(2+) reabsorption.