Nod Factor/Nitrate-Induced CLE Genes that Drive HAR1-Mediated Systemic Regulation of Nodulation

Host legumes control root nodule numbers by sensing externaland internal cues. A major external cue is soil nitrate, whereasa feedback regulatory system in which earlier formed nodulessuppress further nodulation through shoot–root communicationis an important internal cue. The latter is known as autoregulationof nodulation (AUT), and is believed to consist of two long-distancesignals: a root-derived signal that is generated in infectedroots and transmitted to the shoot; and a shoot-derived signalthat systemically inhibits nodulation. In Lotus japonicus, theleucine-rich repeat receptor-like kinase, HYPERNODULATION ABERRANTROOT FORMATION 1 (HAR1), mediates AUT and nitrate inhibitionof nodulation, and is hypothesized to recognize the root-derivedsignal. Here we identify L. japonicus CLE-Root Signal 1 (LjCLE-RS1)and LjCLE-RS2 as strong candidates for the root-derived signal.A hairy root transformation study shows that overexpressingLjCLE-RS1 and -RS2 inhibits nodulation systemically and, furthermore,that the systemic suppression depends on HAR1. Moreover, LjCLE-RS2expression is strongly up-regulated in roots by nitrate addition.Based on these findings, we propose a simple model for AUT andnitrate inhibition of nodulation mediated by LjCLE-RS1, -RS2peptides and the HAR1 receptor-like kinase.     

Improvement of isoflavone aglycones production using β-glucosidase secretory produced in recombinant Aspergillus oryzae

β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides.     

A Novel Highly Potent Autotaxin/ENPP2 Inhibitor Produces Prolonged Decreases in Plasma Lysophosphatidic Acid Formation In Vivo and Regulates Urethral Tension

Autotaxin, also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted enzyme that has lysophospholipase D activity, which converts lysophosphatidylcholine to bioactive lysophosphatidic acid. Lysophosphatidic acid activates at least six G-protein coupled recpetors, which promote cell proliferation, survival, migration and muscle contraction. These physiological effects become dysfunctional in the pathology of cancer, fibrosis, and pain. To date, several autotaxin/ENPP2 inhibitors have been reported; however, none were able to completely and continuously inhibit autotaxin/ENPP2 in vivo. In this study, we report the discovery of a highly potent autotaxin/ENPP2 inhibitor, ONO-8430506, which decreased plasma lysophosphatidic acid formation.The IC₅₀ values of ONO-8540506 for lysophospholipase D activity were 6.4–19 nM for recombinant autotaxin/ENPP2 proteins and 4.7–11.6 nM for plasma from various animal species. Plasma lysophosphatidic acid formation during 1-h incubation was almost completely inhibited by the addition of >300 nM of the compound to human plasma. In addition, when administered orally to rats at a dose of 30 mg/kg, the compound demonstrated good pharmacokinetics in rats and persistently inhibited plasma lysophosphatidic acid formation even at 24 h after administration.Smooth muscle contraction is a known to be promoted by lysophosphatidic acid. In this study, we showed that dosing rats with ONO-8430506 decreased intraurethral pressure accompanied by urethral relaxation. These findings demonstrate the potential of this autotaxin/ENPP2 inhibitor for the treatment of various diseases caused by lysophosphatidic acid, including urethral obstructive disease such as benign prostatic hyperplasia.     

Amino acid sequence of the calcium-binding light chain of myosin from the lower eukaryote, Physarum polycephalum

We have established a new method for preparing Physarum myosin whose actin-activated ATPase activity is inhibited by micromolar levels of Ca2+. This Ca2+-inhibition is mediated by the Ca2+ binding to the myosin rather than by the Ca2+-dependent modification of the phosphorylated state of the myosin (Kohama, K., and Kendrick-Jones, J. (1986) J. Biochem. (Tokyo) 99, 1433-1446). Ca2+-binding light chain (CaLC) has been suggested to be primary importance in this Ca2+ inhibition (Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, T., and Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). The amino acid sequence of CaLC was determined; it was composed of 147 amino acid residues and the N terminus was acetylated. The molecular weight was calculated to be 16,131. The homology of CaLC in the amino acid sequence with 5,5'-dithiobis-(2-nitrobenzoic acid) light chain and alkali light chain of skeletal muscle myosin were rather low, i.e., 25% and 30%, respectively. Interestingly, however, the CaLC sequence was 40% homologous with brain calmodulin. This amino acid sequence was confirmed by sequencing the cloned phage DNA accommodating cDNA coding CaLC. Northern and Southern blot analysis indicated that 0.8-kilobase pair mRNA was transcribed from a single CaLC gene. This is the first report on the amino acid sequence of myosin light chain of lower eukaryotes and nucleotide sequence of its mRNA.     

Characterization of a dihydrolipoyl dehydrogenase having diaphorase activity of Clostridium kluyveri

The Clostridium kluyveri bfmBC gene encoding a putative dihydrolipoyl dehydrogenase (DLD; EC 1.8.1.4) was expressed in Escherichia coli, and the recombinant enzyme rBfmBC was characterized. UV-visible absorption spectrum and thin layer chromatography analysis of rBfmBC indicated that the enzyme contained a noncovalently but tightly attached FAD molecule. rBfmBC catalyzed the oxidation of dihydrolipoamide (DLA) with NAD(+) as a specific electron acceptor, and the apparent K(m) values for DLA and NAD(+) were 0.3 and 0.5 mM respectively. In the reverse reaction, the apparent K(m) values for lipoamide and NADH were 0.42 and 0.038 mM respectively. Like other DLDs, this enzyme showed NADH dehydrogenase (diaphorase) activity with some synthetic dyes, such as 2,6-dichlorophenolindophenol and nitro blue tetrazolium. rBfmBC was optimally active at 40 degrees C at pH 7.0, and the enzyme maintained some activity after a 30-min incubation at 60 degrees C.     

Persistent and stable gene expression by a cytoplasmic RNA replicon based on a noncytopathic variant Sendai virus

Persistent and stable expression of foreign genes has been achieved in mammalian cells by integrating the genes into the host chromosomes. However, this approach has several shortcomings in practical applications. For example, large scale production of protein pharmaceutics frequently requires laborious amplification of the inserted genes to optimize the gene expression. The random chromosomal insertion of exogenous DNA also results occasionally in malignant transformation of normal tissue cells, raising safety concerns in medical applications. Here we report a novel cytoplasmic RNA replicon capable of expressing installed genes stably without chromosome insertion. This system is based on the RNA genome of a noncytopathic variant Sendai virus strain, Cl.151. We found that this variant virus establishes stable symbiosis with host cells by escaping from retinoic acid-inducible gene I-interferon regulatory factor 3-mediated antiviral machinery. Using a cloned genome cDNA of Sendai virus Cl.151, we developed a recombinant RNA installed with exogenous marker genes that was maintained stably in the cytoplasm as a high copy replicon (about 4 x 10(4) copies/cell) without interfering with normal cellular function. Strong expression of the marker genes persisted for more than 6 months in various types of cultured cells and for at least two months in rat colonic mucosa without any apparent side effects. This stable RNA replicon is a potentially valuable genetic platform for various biological applications.     

The Waste Input-Output Approach to Materials Flow Analysis

Abstract: A general analytical model of materials flow analysis (MFA) incorporating physical waste input-output is proposed that is fully consistent with the mass balance principle. Exploiting the triangular nature of the matrix of input coefficients, which is obtained by rearranging the ordering of sectors according to degrees of fabrication, the material composition matrix is derived, which gives the material composition of products. A formal mathematical definition of materials (or the objects, the flow of which is to be accounted for by MFA) is also introduced, which excludes the occurrence of double accounting in economy-wide MFAs involving diverse inputs. By using the model, monetary input-output (IO) tables can easily be converted into a physical material flow account (or physical input-output tables [PIOT]) of an arbitrary number of materials, and the material composition of a product can be decomposed into its input origin. The first point represents substantial saving in the otherwise prohibitive cost that is associated with independent compilation of PIOT. The proposed methodology is applied to Japanese IO data for the flow of 11 base metals and their scrap (available as e-supplement on the JIE Web site).     

Specific enzyme complex of beta-1,4-galactosyltransferase-II and glucuronyltransferase-P facilitates biosynthesis of N-linked human natural killer-1 (HNK-1) carbohydrate

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.     

Efficient induction of formate hydrogen lyase of aerobically grown <Emphasis Type="Italic">Escherichia coli</Emphasis> in a three-step biohydrogen production process

A three-step biohydrogen production process characterized by efficient anaerobic induction of the formate hydrogen lyase (FHL) of aerobically grown Escherichia coli was established. Using E. coli strain SR13 (fhlA ⁺⁺, ΔhycA) at a cell density of 8.2 g/l medium in this process, a specific hydrogen productivity (28.0 ± 5.0 mmol h⁻¹ g⁻¹ dry cell) of one order of magnitude lower than we previously reported was realized after 8 h of anaerobic incubation. The reduced productivity was attributed partly to the inhibitory effects of accumulated metabolites on FHL induction. To avoid this inhibition, strain SR14 (SR13 ΔldhA ΔfrdBC) was constructed and used to the effect that specific hydrogen productivity increased 1.3-fold to 37.4 ± 6.9 mmol h⁻¹ g⁻¹. Furthermore, a maximum hydrogen production rate of 144.2 mmol h⁻¹ g⁻¹ was realized when a metabolite excretion system that achieved a dilution rate of 2.0 h⁻¹ was implemented. These results demonstrate that by avoiding anaerobic cultivation altogether, more economical harvesting of hydrogen-producing cells for use in our biohydrogen process was made possible.