Combined Hybridization and Evaluation of High-Lysine Rice: Nutritional and Physicochemical Qualities and Field Performance.

Rice, as a major food crop, provides necessary energy and nutrition for humans and livestock. However, its nutritional value is affected by lysine. Using point mutation, we previously obtained AK2 (aspartokinase) and DHDPS1 (dihydrodipicolinate synthase) genes insensitive to lysine feedback inhibition and constructed transgenic lines AK2-52 and DHDPS1-22, which show increased lysine synthesis, as well as Ri-12, which shows decreased lysine degradation by inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) activity. In this study, further transgenic lines were hybridized and evaluated. The lysine content of mature seeds from pyramid lines PRD and PRA increased 32.5- and 29.8-fold, respectively, compared with the wild-type, while the three-gene pyramiding line PRDA had a moderate lysine content. The total lysine, total free lysine, and total protein contents of PRD and PRA also increased and had no obvious impact on the physical and chemical quality, seed appearance, and main agronomic traits. Meanwhile, comparative analysis with polygenic polymeric lines GR containing bacterial AK (lysC) and DHDPS (dapA) genes revealed differences in the way bacterial and endogenous rice AK and DHDPS regulate lysine biosynthesis. These results provide a reference for further evaluation and commercialization of high-lysine transgenic rice.

The 1.3 isoform of Na+–Ca2+ exchanger expressed in guinea pig tracheal smooth muscle is less sensitive to KB-R7943

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The sodium–calcium exchanger (NCX) plays a major role in the regulation of cytosolic Ca2+ in muscle cells. In this work, we performed force experiments to explore the role of NCX during contraction and relaxation of Cch-stimulated guinea pig tracheal smooth muscle strips. This tissue showed low sensitivity to NCX inhibitor KB-R7943 (IC50, 57 ± 2 µM), although a complete relaxation was obtained by NCX inhibition at 100 µM. Interestingly, relaxation after washing the agonist was prolonged in the absence of external Na+, whereas washing without Na+ and in the presence of KB-R7943 resembled control conditions with physiological solution. Altogether, this suggests the reversal of NCX to a Ca2+ influx mode by the manipulation on the Na+ gradient, which can be inhibited by KB-R7943. In order to understand the low sensitivity to KB-R7943, we studied the molecular aspects of the NCX expressed in this tissue and found that the isoform of NCX expressed is 1.3, similar to that described in human tracheal smooth muscle. Sequencing revealed that amino acid 19 in exon B is phenylalanine, whereas in its human counterpart is leucine, and that the first amino acid after exon D is aspartate instead of glutamate in humans. Results herein presented are discussed in term of their possible functional implications in the exchanger activity and thus in airway physiology (AU)

Cohesion group approach for evolutionary analysis of aspartokinase, an enzyme that feeds a branched network of many biochemical pathways.

Aspartokinase (Ask) exists within a variable network that supports the synthesis of 9 amino acids and a number of other important metabolites. Lysine, isoleucine, aromatic amino acids, and dipicolinate may arise from the ASK network or from alternative pathways. Ask proteins were subjected to cohesion group analysis, a methodology that sorts a given protein assemblage into groups in which evolutionary continuity is assured. Two subhomology divisions, ASK(alpha) and ASK(beta), have been recognized. The ASK(alpha) subhomology division is the most ancient, being widely distributed throughout the Archaea and Eukarya and in some Bacteria. Within an indel region of about 75 amino acids near the N terminus, ASK(beta) sequences differ from ASK(alpha) sequences by the possession of a proposed ancient deletion. ASK(beta) sequences are present in most Bacteria and usually exhibit an in-frame internal translational start site that can generate a small Ask subunit that is identical to the C-terminal portion of the larger subunit of a heterodimeric unit. Particularly novel are ask genes embedded in gene contexts that imply specialization for ectoine (osmotic agent) or aromatic amino acids. The cohesion group approach is well suited for the easy recognition of relatively recent lateral gene transfer (LGT) events, and many examples of these are described. Given the current density of genome representation for Proteobacteria, it is possible to reconstruct more ancient landmark LGT events. Thus, a plausible scenario in which the three well-studied and iconic Ask homologs of Escherichia coli are not within the vertical genealogy of Gammaproteobacteria, but rather originated via LGT from a Bacteroidetes donor, is supported.

Characterization of aspartate kinase III of Bacillus subtilis.

A search in the Bacillus subtilis genome sequence found that the gene designated yclM encode(s) a protein showing significant identity in amino acid sequence to aspartate kinases. When yclM was introduced into Escherichia coli cells deficient in all three aspartate kinase genes, production of a protein with molecular size 50 kDa, which was similar to the value deduced from the nucleotide sequence of the gene, was observed. Expectedly, the protein purified to homogeneity had aspartate kinase activity. The enzyme was significantly inhibited by simultaneous addition of both threonine and lysine, which is a typical feature of aspartate kinase III of B. subtilis. The enzyme was very unstable in 10 mM tris-HCl (pH 7.5) buffer, but was stabilized by addition of 500 mM ammonium sulfate. Although all the aspartate kinases so far investigated are oligomeric enzymes, this aspartate kinase was suggested to be a monomer.

Bacterial aspartate kinase-like activity in human platelet.

One form of a group of enzymes known as aspartate kinases, primarily reported in prokaryotes and plants, might also exist in animal cells. Here we report the immunodetection of an aspartate kinase-like activity in human platelets using antibodies against the pure form of the enzyme purified from Escherichia coli. Moreover, the enrichment of platelet extracts with the bacterial kinase results in the phosphorylation of discrete forms mainly of membrane-bound endogenous polypeptides.

[The effect of amino acids in the asparagine family on the aspartate kinase and homoserine dehydrogenase of ethionine-resistant mutants of Pseudomonas putida]. / Vliianie aminokislot asparaginovogo semeistva na aktivnost' aspartatkinazy i gomoserindegidrogenazy étioninrezistentnykh mutantov Pseudomonas putida.

The activity of aspartate kinase and homoserin dehydrogenase from ethionine resistant mutants Pseudomonas putida 25 and 6 have been studied as affected by amino acids from the family of asparagine. They are characterized by a capacity to the surplus synthesis of methionine. It is shown that mutants have negative regulation of the level of activity of the studied enzymes. It is supposed that the mutations (or mutation) could take place which affected properties of enzymes, which participated directly in the biosynthesis of methionine, in the analogue resistant clones 25 and 6.

Aspartokinase III, a new isozyme in Bacillus subtilis 168.

A previously undetected Bacillus subtilis aspartokinase isozyme, which we have called aspartokinase III, has been characterized. The new isozyme was most readily detected in extracts of cells grown with lysine, which repressed aspartokinase II and induced aspartokinase III, or in extracts of strain VS11, a mutant lacking aspartokinase II. Antibodies against aspartokinase II did not cross-react with aspartokinase III. Aspartokinases II and III coeluted on gel filtration chromatography at Mr 120,000, which accounts for the previous inability to detect it. Aspartokinase III was induced by lysine and repressed by threonine. It was synergistically inhibited by lysine and threonine. Aspartokinase III activity, like aspartokinase II activity, declined rapidly in B. subtilis cells that were starved for glucose. In contrast, the specific activity of aspartokinase I, the diaminopimelic acid-inhibitable isozyme, was constant under all growth conditions examined.

Detection of aspartate kinase in rat liver and its activation by Ca++ and calmodulin.

The enzyme aspartate kinase (EC 2.7.2.4) has been detected in rat liver (animal tissue) for the first time. This enzyme, like the aspartate kinase from bacteria and plants is inhibited by lysine and threonine. Further, the activity of the enzyme is stimulated over two fold by Ca++ and calmodulin and inhibited by EGTA, a Ca++ chelator and trifluoperazine, an anti-calmodulin compound.

Regulation of cephamycin C synthesis, aspartokinase, dihydrodipicolinic acid synthetase, and homoserine dehydrogenase by aspartic acid family amino acids in Streptomyces clavuligerus.

The effect of the cephalosporin precursors and amino acids of the aspartic acid family on antibiotic production by Streptomyces clavuligerus was investigated DL-meso-Diaminopimelate and L-lysine each stimulated specific antibiotic production by 75%. A fourfold increase in specific production was obtained by simultaneous addition of the two compounds. The stimulation could be further increased by adding valine to the two effectors. In the streptomycetes the alpha-aminoadipyl side chain of the cephalosporin antibiotics is derived from lysine. Streptomycetes, like other bacteria, are expected to produce lysine from aspartic acid; therefore, the feedback control mechanisms operating in the aspartic acid family pathway of S. clavuligerus, which may affect the flow of carbon to alpha-aminoadipic acid, were investigated. Threonine inhibited antibiotic production by 41% when added to minimal medium at a concentration of 10 mM. Simultaneous addition of 10 mM lysine completely reversed this inhibition. The aspartokinase of S. clavuligerus was found to be subject to concerted feedback inhibition by threonine and lysine. Threonine may act to limit the supply of lysine available for cephamycin C biosynthesis via this concerted mechanism. Single or simultaneous addition of any other amino acid of the aspartate family in the in vitro assay did not inhibit aspartokinase activity. Activity was stimulated by lysine. Aspartokinase biosynthesis was partially repressed by methionine or isoleucine at concentrations higher than 10 mM. Methionine, but not isoleucine, inhibited cephamycin C synthesis by 27% when added to minimal medium at a concentration of 10 mM. Dihydrodipicolinate synthetase, the first specific enzyme of the lysine branch, was not inhibited by lysine but was partially inhibited by high concentrations of 2,6-diaminopimelate and alpha-aminoadipate; it was slightly repressed by diaminopimelic acid. Homoserine dehydrogenase activity was inhibited by threonine and partially repressed by isoleucine. It appears that S. clavuligerus aspartokinase is a key step in the control of carbon flow toward alpha-aminoadipic acid.