Evaluation of amino acid content and nutritional quality of transgenic soybean seeds with high-level tryptophan accumulation

Anthranilate synthase (AS) is a key regulatory enzyme in tryptophan (Trp) biosynthesis and is subject to feedback inhibition by Trp. The gene encoding a mutated feedback-resistant α subunit of rice AS (OASA1D) under the control of either a soybean glycinin gene promoter or the 35S promoter of cauliflower mosaic virus for seed-specific or constitutive expression, respectively, was introduced into soybean [Glycine max (L.) Merrill] by particle bombardment. A total of seven different transgenic lines that showed markedly increased accumulation of free Trp in their seeds were developed. The overproduction of free Trp was stably inherited in subsequent generations without any apparent detrimental effect on plant growth or reproduction. The total Trp content of transgenic seeds was also about twice that of nontransgenic seeds, whereas the amount of protein-bound Trp was not substantially affected by OASA1D expression. In spite of the marked increase in free Trp content, metabolic profiling by high-performance liquid chromatography coupled with mass spectrometry revealed little change in the amounts of other aromatic compounds in the transgenic seeds. We developed a rapid and feasible system based on farmed rainbow trout to evaluate the nutritional quality of a limited quantity of transgenic soybean seeds. Supplementation of fish food with OASA1D transgenic soybean seeds or with nontransgenic seeds plus crystalline Trp increased the growth rate of the farmed fish. These results indicate transformation with OASA1D is a reliable approach to improve the nutritional quality of soybean (or of other grain legumes) for human and animal food.     

Comparative Analysis of argK-tox Clusters and Their Flanking Regions in Phaseolotoxin-Producing Pseudomonas syringae Pathovars

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181–10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064–11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named “tox islands.” The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437–457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Analgesic action of nicotine on tibial nerve transection (TNT)-induced mechanical allodynia through enhancement of the glycinergic inhibitory system in spinal cord

The activation of cholinergic pathways by nicotine elicits various physiological and pharmacological effects in mammals. For example, the stimulation of nicotinic acetylcholine receptors (nAChRs) leads to an antinociceptive effect. However, it remains to be elucidated which subtypes of nAChR are involved in the antinociceptive effect of nicotine on nerve injury-induced allodynia and the underlying cascades of the nAChR-mediated antiallodynic effect. In this study, we attempted to characterize the actions of nicotine at the spinal level against mechanical allodynia in an animal model of neuropathic pain, tibial nerve transection (TNT) in rats. It was found that the intrathecal injection of nicotine, RJR-2403, a selective alpha4beta2 nAChR agonist, and choline, a selective alpha7 nAChR agonist, produced an antinociceptive effect on the TNT-induced allodynia. The actions of nicotine were almost completely suppressed by pretreatment with mecamylamine, a non-selective nicotinic antagonist, or dihydro-beta-erythroidine, a selective alpha4beta2 nAChR antagonist, and partially reversed by pretreatment with methyllycaconitine, a selective alpha7 nAChR antagonist. Furthermore, pretreatment with strychnine, a glycine receptor antagonist, blocked the antinociception induced by nicotine, RJR-2403, and choline. On the other hand, the GABAA antagonist bicuculline did not reverse the antiallodynic effect of nicotine. Together, these results indicate that the alpha4beta2 and alpha7 nAChR system, by enhancing the activities of glycinergic neurons at the spinal level, exerts a suppressive effect on the nociceptive transduction in neuropathic pain.     

IRS-2 deficiency in macrophages promotes their accumulation in the vascular wall

The aim of this study was to investigate the role of insulin receptor substrate-2 (IRS-2) mediated signal in macrophages on the accumulation of macrophages in the vascular wall. Mice transplanted with IRS-2^−/− bone marrow, a model of myeloid cell restricted defect of IRS-2, showed accumulation of monocyte chemoattractant protein-1-expressing macrophages in the vascular wall. Experiments using cultured peritoneal macrophages showed that IRS-2-mediated signal pathway stimulated by physiological concentrations of insulin, not by IL-4, contributed to the suppression of monocyte chemoattractant protein-1 expression induced by lipopolysaccharide. Our data indicated that IRS-2 deficiency in macrophages enhanced their accumulation in the vascular wall accompanied by increased expression of proinflammatory mediators in macrophages. These results suggest a role for insulin resistance in macrophages in early atherosclerogenesis.     

Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming

Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed. The present findings show that fluid flow stress exerts a prolonged bioactive effect on mesothelial cells after termination of fluid streaming. These findings support the hypothesis that a history of PD for a certain period could serve as a trigger of EPS after stoppage of PD.     

Systematic characterization of Escherichia coli genes/ORFs affecting biofilm formation

To understand the nature and function of bacterial biofilm and the process of its formation, we have performed systematic screening of a complete set of Escherichia coli genes/open reading frames (ORFs) to identify those that affect biofilm development upon over-expression. In contrast to the biofilm of strain AG1 used as a control, some of the genes/ORFs when over-expressed led to the formation of an abnormal biofilm such as thin, mat-like, filamentous or one easily detaching from various surfaces. Disruptants of selected genes were constructed in order to clarify their roles in the different stages of biofilm formation. Our results suggest that diverse metabolic pathways contribute to the development of biofilm.     

Oxidation of 3-C-(2-amino-2-deoxy-D-glucopyranosyl)-1-propene compounds and the structure of 3-C-(2-amino-2-deoxy-D-glucopyranosyl)-1,2-propanediol derivatives for a synthesis of 2,3-didehydro-2,7-dideoxy-N-acetylneuraminic acid

Oxidation of 5-acetamido-4,8-anhydro-1,2,3,5-tetradeoxy-D-glycero-D-ido-non-1-enitol [3-C-(2-amino-2-deoxy-beta-D-glucopyranosyl)-1-propene] was studied to search for preparative routes to aminodeoxy didehydro nonulosonic acid derivatives. Since only moderate chiral induction was observed with osmium tetroxide dihydroxylation as well as with peracid epoxidation, the catalytic asymmetric dihydroxylation conditions were applied to give the stereocontrolled formation of 1,2-propanediol derivatives. The structures of these diastereoisomeric 1,2-propanediol derivatives were determined by X-ray crystallographic analyses. The formation of diastereoisomeric 1,2-propanediols also varied with the nature of 2-substituent on the aminodoexy glycosyl moiety. Thus 5-acetamido-4,8-anhydro-3,5-dideoxy-D-erythro-L-ido-nonitol [(2S)-3-C-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-1,2-propanediol] was obtained predominantly up to 70% from 3-C-(2-acetamido-2-deoxyglycosyl)-1-propene by the use of ADmixbeta reagent. The (2S)-propanediol derivative was transformed in a five-step reaction sequence to 2,3-didehydro-2,7-dideoxy-N-acetylneuraminic acid.     

Achromobacter denitrificans Strain YD35 Pyruvate Dehydrogenase Controls NADH Production To Allow Tolerance to Extremely High Nitrite Levels

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO₂⁻) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO₂⁻-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO₂⁻ tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO₂⁻. Nitrite decreased the cellular NADH/NAD⁺ ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO₂⁻-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO₂⁻. These findings demonstrate a link between NO₂⁻ tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO₂⁻-tolerating mechanism in this strain.