Genetic evidence that Arabidopsis ALTERED ROOT ARCHITECTURE encodes a putative dehydrogenase involved in homoserine biosynthesis

Key message

Genetic and molecular analysis of an Arabidopsis root development mutant identified a putative dehydrogenase gene involved in homoserine biosynthesis.

Abstract

In higher plants, homoserine (Hse) is derived from aspartate (Asp) and is an important intermediate for production of methionine (Met), threonine (Thr), and isoleucine (Ile). In Arabidopsis, six enzymes involved in the biosynthesis of Hse from Asp have been well characterized. It is not known, however, whether there exist other enzymes involved in this process. In this work, we characterized an Arabidopsis mutant, ara (a ltered r oot a rchitecture), with a short primary root and an increased number of lateral roots. Genetic and molecular analysis indicated that the ARA gene encodes a protein with a D-isomer specific 2-hydroxyacid dehydrogenase domain. ARA is expressed in all plant organs and is localized in the cell periphery. The ara mutant phenotypes can be rescued by exogenously applied Hse, Met, Ile and 2-oxobutanoate. Based on the results presented here, we propose that the ARA protein may be a dehydrogenase involved in homoserine biosynthesis.     

Identification and Characterization of PpLFL, a Homolog of FLORICAULA/LEAFY in Peach (Prunus persica)

A LEAFY/FLORICAULA (LFY/FLO) homolog PpLFL (P runus p ersica L EAFY/ F LORICAULA L ike) gene was isolated from axillary buds of peach (Prunus persica (L.) Batsch. cv. Bayuecui) during flower induction period. The open reading frame of PpLFL spanned 1,248 bp, encoding a putative protein of 415 amino acid residues, which was with high similarity (50.48 %–84.69 %) to other FLO/LFY inferred proteins from different species. The spatial expression patterns of PpLFL were detected in axillary buds during the periods of flower induction by using immunohistolocalisation. The results showed that PpLFL gene was mainly expressed during flower induction time, and also in leaf and petal promordia at the SAM. For further functional analysis, the PpLFL was constitutively expressed in the Arabidopsis lfy mutant background, and the results showed that overexpression of PpLFL under the control of CaMV 35S promoter can accelerate flowering and give rise to normal flower organs. Our results suggest that PpLFL might play an important role in flower induction, and could act as a functional flower meristem identity gene in peach.     

Acute ST-segment elevation myocardial infarction after amoxycillin-induced anaphylactic shock in a young adult with normal coronary arteries: a case report

Background

Sirolimus-eluting stents (CYPHER stents) demonstrated remarkable efficacy in reducing restenosis rates in patients with coronary artery disease. There is a concern of sub-acute and late stent thrombosis. Tissue factor (TF) is critical in thrombosis. This study investigated the effect of sirolimus on TF expression and activity in cultured human vascular smooth muscle cells (SMCs).

Methods

SMCs were cultured from human saphenous veins and aortas. Quiescent cells were stimulated with sirolimus (0.1 – 20 ng/ml) over 24 hours. Cellular TF expression and activity released into culture medium were measured. The effect of sirolimus on activation of mammalian target of rapamycin (mTOR) was measured by phosphorylation of the substrate p70s6k at T389, and activation of RhoA was measured by pull-down assay.

Results

Sirolimus increased TF protein level in cultured human SMCs in a concentration and time-dependent manner (about 2-fold, p < 0.01) reaching maximal effect at 5 ng/ml. The stimulation of TF expression by sirolimus was associated with inhibition of basal activity of mTOR. No effects of sirolimus on RhoA or p38mapk activation that are positive regulators of TF in vascular wall cells were observed. The stimulation of TF expression by sirolimus (20 ng/ml) was prevented by the HMG-CoA reductase inhibitor fluvastatin (1 μmol/L). However, no increase in TF activity released from SMC into culture medium was observed after sirolimus treatment.

Conclusion

Although sirolimus stimulates TF protein expression in human SMC associated with inhibition of mTOR, it does not enhance TF activity released from the cells, suggesting a relatively safe profile of CYPHER stents. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting.     

A proof of the DBRF-MEGN method, an algorithm for deducing minimum equivalent gene networks

Background

We previously developed the DBRF-MEGN (difference-based regulation finding-minimum equivalent gene network) method, which deduces the most parsimonious signed directed graphs (SDGs) consistent with expression profiles of single-gene deletion mutants. However, until the present study, we have not presented the details of the method's algorithm or a proof of the algorithm.

Results

We describe in detail the algorithm of the DBRF-MEGN method and prove that the algorithm deduces all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.

Conclusions

The DBRF-MEGN method provides all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

Developmental function of Nm23/awd: a mediator of endocytosis

The metastasis suppressor gene Nm23 is highly conserved from yeast to human, implicating a critical developmental function. Studies in cultured mammalian cells have identified several potential functions, but many have not been directly verified in vivo. Here, we summarize the studies on the Drosophila homolog of the Nm23 gene, named a bnormal w ing d iscs (awd), which shares 78% amino acid identity with the human Nm23-H1 and H2 isoforms. These studies confirmed that awd gene encodes a nucleoside diphosphate kinase, and provided strong evidence of a role for awd in regulating cell differentiation and motility via regulation of growth factor receptor signaling. The latter function is mainly mediated by control of endocytosis. This review provides a historical account of the discovery and subsequent analyses of the awd gene. We will also discuss the possible molecular function of the Awd protein that underlies the endocytic function.     

Identification and characterization of SHI family genes from Brassica rapa L. ssp. pekinensis

SHI (short internodes) is a negative regulator of gibberellin-induced cell elongation. Extensive searches in the Brassica rapa genome allowed for the prediction of at least six different SHI-related genes on six chromosomes in the genome. Genome structural examination revealed that these genes had one intron each in their corresponding open reading frames. Protein structure comparisons using the CLUSTALW program and based on alignments of all BrSRS (B. r apa SHI-related sequence) proteins revealed broad conservation of the RING finger-like zinc finger and IGGH motifs. According to the phylogenetic relationship based on deduced amino acid sequences, the six BrSRS proteins were most closely related to Arabidopsis SRS (AtSRS) proteins; however, BrSRS proteins were dispersed in the phylogenetic tree. Semi-quantitative RT-PCR analysis indicated that the six BrSRS genes exhibited different expression patterns in various tissues and responded differently to growth phytohormones. The differences among the six BrSRS genes with respect to gene structure and expression pattern suggest that these genes may play diverse physiological roles in the developmental process of B. rapa.     

Detection of d-amino acids in purified proteins synthesized in Escherichia coli

It has long been believed that amino acids comprising proteins of all living organisms are only of the l-configuration, except for Gly. However, peptidyl d-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of d-amino acids are naturally present in usual proteins. Thus we analyzed the d-amino acid contents of His-tag-purified β-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110°C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into d- and l-enantiomers. The contents of d-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index d/(d + l) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of d-amino acids were reproducibly detected, the d-amino acid profile being specific to an individual protein. This finding indicated the likelihood that d-amino acids are in fact present in the purified proteins. On the other hand, the d-amino acid contents of proteins were hardly influenced by the addition of d- or l-amino acids to the cultivation medium, whereas intracellular free d-amino acids sensitively varied according to the extracellular conditions. The origin of these d-amino acids detected in proteins was discussed.     

Notes on some African hepatic genera 1–5

The present study deals with five genera of hepatics in Africa, Isotachis Mitt., Anastrophyllum (Spruce) Steph., Tritomaria Schiffn. ex Loeske, Gymnocoleopsis (Schust.) Schust. and Lophozia (Dum.) Dum. All African populations of the genus Isotachis Mitt. are considered to be one species, I. aubertii (Schwaegr.) Mitt. Four species of Anastrophyllum (Spruce) Steph. (s.l.), A. auritum (Lehm.) Steph., A. piligerum (Nees) Spruce, A. subcomplicatum (Lehm. et Lindenb.) Steph. and A. minutum (Schreb.) Schust., and two species of Tritomaria Schiffn. et Loeske, T. camerunensis S. Arnell and T. exsecta (Schrad.) Schiffn. ex Loeske occur in Africa. Gymmocoleopsis multiflora (Steph.) Schust. represents a genus and species hitherto unreported for the African flora. Finally, five Lophozia (Dum.) Dum. species, L. argentina (Steph.) Schust., L. capensis S. Arnell, L. decolorans (Limpr.) Steph., L. hedbergii S. Arnell and L. tristaniana (S. Arnell) Váňa, are reported from central and southern Africa; two of these (L. argentina (Steph.) Schust. and L. decolorans (Limpr.) Steph.) represent the first reports from Africa.     

Upstream molecular signaling pathways of p27(Kip1) expression in human breast cancer cells in vitro: differential effects of 4-hydroxytamoxifen and deficiency of either D-(+)-glucose or L-leucine

Background

The objective of this study was to investigate whether the levels of glucose or certain amino acids could regulate the expression of a cell cycle repressor protein p27(Kip1), thereby dictating the risk of cancer in either obesity or caloric/dietary restriction. Previously, we identified and reported four different upstream molecular signaling pathways of p27 expression in human breast cancer cells. We called these four pathways as pathway #1, #2, #3 and #4. We found that 4-hydroxytamoxifen - but not tamoxifen - up-regulated the expression of p27 using pathway #1 which consisted mainly of receptor tyrosine kinases and mTORC1. We now investigate, using 4-hydroxytamoxifen as a reference anti-cancer agents, whether (a) the moderate increase in the concentration of D-(+)-glucose could down-regulate and, conversely, (b) the deficiency of D-(+)-glucose or certain L-amino acids could up-regulate the expression of p27 in these cells using pathway #2 which consists mainly of AMPK and mTORC1.

Results

Using human MDA-MB-231 breast cancer cells in vitro, these hypotheses were tested experimentally by performing p27-luciferase reporter transfection assays and western immunoblot analyses. The results obtained are consistent with these hypotheses. Furthermore, the results indicated that, although 4-hydroxytamoxifen used primarily pathway #1 to down-regulate the phosphorylation of 4E-BP1 and up-regulate the expression of p27, it also secondarily down-regulated the phosphorylation of S6K1. In contrast, the deficiency of D-(+)-glucose or L-leucine used primarily pathway #2 to down-regulate the phosphorylation of S6K1, but they also secondarily down-regulated the phosphorylation of 4E-BP1 and up-regulated the expression of p27. Finally, deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A and SIRT3.

Conclusions

(a) 4-Hydroxitamoxifen used primarily pathway #1 to up-regulate the expression of p27. (b) Moderate increase in the concentration of D-(+)-glucose used primarily pathway #2 to down-regulate the expression of p27. (c) Deficiency of D-(+)-glucose or L-leucine also used primarily pathway #2 to up-regulate the expression of p27. (d) Deficiency of D-(+)-glucose or L-leucine - but not 4-hydroxytamoxifen - up-regulated the expression of mitochondrial ATP5A in the Complex V of respiratory oxidation-phosphorylation chain and mitochondrial SIRT3. The SIRT3 is one of the seven mammalian anti-aging as well as anti-metabolic sirtuins.     

Taxonomic status of the belemnite genusBelemnopsis Bayle 1878

The Tethyan genus “Belemnopsis” of authors, notBayle 1878, includes 110–120 Bajocian to Hauterivian belemnite species and subspecies from Europe and the southern hemisphere. Its type species,Belemnites sulcatus Miller 1826 from the Callovian to Oxfordian of England, belongs to the Boreal belemnite genusLagonibelus Gustomesov 1956, since Phillips selected a lectotype for it in 1870. But the subsequent designation of the type species byDouvillé in 1879 is invalid according to Art. 67 (h) ICZN, and consequentlyBelemnopsis Bayle gets a nomen dubium. It is also a homonym ofBelemnopsis Edwards inGray 1849, and has, therefore, to be replaced byPachybelemnopsis Riegraf 1980, a junior subjective synonym, the family Belemnopseidae Naef 1922 by MesohibolitidaeNerodenko 1983, and the suborder BelemnopseinaJeletzky 1965 by PachybelemnopseinaRiegraf inRiegraf et al. 1998.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.     

Nomenklatorische Anmerkungen zur GattungPotentilla

Für folgende inkorrekte Epitheta schlägt der Autor richtige Namen vor, die in Klammern angeführt sind:Potentilla opizii Dom. (=P. lindackeri Tausch),P. dealbata Bunge (=P. virgata Lehm.),P. poterioides Franch. (=P. limprichtii J. Krause),P. fulgens Wall. exHook. (=P. siemersiana Lehm.),P. labradorica Lehm. (=P. flexuosa Raf.),P. ambigua Camb. (=P. cuneifolia Bertol.),P. anserina ssp.egedii (Wormsk.) Hitt. (=P. anserina ssp.groenlandica Tratt.),P. raddeana (Wolf) Juz. (=P. bertramii Aznav.) undP. bipinnatifida Dougl. exHook. (=P. normalis Bess. exSpreng.). FürP. nudicaulis Boiss. etBal.,P. eremica Wolf undP. sericata Wolf werden nomina nova veröffentlicht. Es werden einige neue Kombinationen angeführt:P. supina L. ssp.paradoxa (Nutt. exTorr. etGray) c.n.,P. hispanica Zimm. ssp.oreodoxa (Soják) c. n. undP. h. ssp.ibrahimiana (Maire) c. n. Strauchige Arten, oft in die GattungPotentilla eingereiht, betrachtet der Autor als eine selbständige GattungPentaphylloides und in dieser Gattung führt er einige neue Kombinationen (5 für Arten, 4 für Hybriden) an.     

Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites

Background

High throughput techniques have generated a huge set of biological data, which are deposited in various databases. Efficient exploitation of these databases is often hampered by a lack of appropriate tools, which allow easy and reliable identification of genes that miss functional characterization but are correlated with specific biological conditions (e.g. organotypic expression).

Results

We have developed a simple algorithm (DGSA = Database-dependent Gene Selection and Analysis) to identify genes with unknown functions involved in organ development concentrating on the heart. Using our approach, we identified a large number of yet uncharacterized genes, which are expressed during heart development. An initial functional characterization of genes by loss-of-function analysis employing morpholino injections into zebrafish embryos disclosed severe developmental defects indicating a decisive function of selected genes for developmental processes.

Conclusion

We conclude that DGSA is a versatile tool for database mining allowing efficient selection of uncharacterized genes for functional analysis.