bluuebunny/crossref_metadata_2025_split huggingface dataset:
Carboxyl‐methylation of prenylated calmodulin CaM53 is required for efficient plasma membrane targeting of the protein
Authors: Rodríguez‐Concepción Manuel , Toledo‐Ortiz Gabriela , Yalovsky Shaul , Caldelari Daniela , Gruissem Wilhelm 
Published: August 2010

SummaryPrenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post‐prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP‐labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl‐methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl‐cysteine methyltransferase (PCM) activity that carboxyl‐methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.
Monosaccharide/proton symporter AtSTP1 plays a major role in uptake and response of Arabidopsis seeds and seedlings to sugars
Authors: Sherson Sarah M. , Hemmann Georg , Wallace Graham , Forbes Susan , Germain Véronique , Stadler Ruth , Bechtold Nicole , Sauer Norbert , Smith Steven M. 
Published: August 2010

SummaryThe aim of this study was to investigate the in vivo properties and function of the high‐affinity monosaccharide/proton symporter AtSTP1 of Arabidopsis. We isolated an Atstp1 knock‐out mutant and found that this plant grows and develops normally. The AtSTP1 gene is expressed in germinating seeds and seedlings, with AtSTP1 activity found mainly in the seedling root. The rate of uptake of [14C]‐3‐O‐methylglucose and [14C]‐d‐glucose is 60% less in Atstp1 seedlings than in the wild type, showing that AtSTP1 is the major monosaccharide transporter in Arabidopsis seedlings. Transport of d‐galactose and d‐mannose is also up to 60% less in Atstp1 seedlings compared to wild type, but transport of d‐fructose, l‐arabinose and sucrose is not reduced. Germination of Atstp1 seed shows reduced sensitivity to d‐mannose, demonstrating that AtSTP1 is active before germination. Atstp1 seedlings grow effectively on concentrations of d‐galactose that inhibit wild‐type growth, even at up to 100 mm d‐galactose, indicating that active transport by AtSTP1 plays a major role at very high concentrations of exogenous sugar. These findings provide insight into the physiological function of AtSTP1 and clearly establish its importance in the uptake of extracellular sugars by the embryo and in seedlings.
Double‐stranded RNA interferes with gene function at the single‐cell level in cereals
Authors: Schweizer Patrick , Pokorny Jana , Schulze‐Lefert Paul , Dudler Robert 
Published: August 2010

SummaryDouble‐stranded RNA (dsRNA) has been shown to specifically interfere with gene function in several organisms including tobacco and the model plant Arabidopsis. Here, we report on rapid and sequence‐specific interference of dsRNA with gene function in cereals. Delivery of cognate dsRNA into single epidermal cells of maize, barley or wheat by particle bombardment interfered with the function of co‐bombarded UidA (GUS) and TaGLP2a::GFP reporter genes. Cognate dsRNA was also found to specifically interfere with the function of the endogenous genes A1 and Ant18 encoding dihydroflavonol‐4‐reductase in maize and barley, respectively. Dihydroflavonol‐4‐reductase is an essential enzyme of the anthocyanin biosynthetic pathway in maize and barley. This pathway can be induced by transient expression of the C1‐ and b‐Peru genes that encode transcription factors. In the presence of dsRNA corresponding to the dihydroflavonol‐4‐reductase gene, C1‐ and b‐Peru‐dependent, cell‐autonomous accumulation of red anthocyanin pigments in bombarded cells of maize and barley was reduced. dsRNA was also demonstrated to negatively interfere with Mlo, which encodes a negative regulator of race non‐specific resistance to the powdery mildew fungus in barley. In the presence of Mlo dsRNA, transformed cells became more resistant, thereby phenocopying plants that carry a heritable loss‐of function mlo resistance allele. The results suggest that direct delivery of dsRNA to cereals leads to a rapid and sequence‐specific interference with gene function at the single‐cell level.
Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity
Authors: Gallego Maria E. , Jeanneau M. , Granier Fabienne , Bouchez David , Bechtold Nichole ,   Charles, White I. 
Published: August 2010

SummaryThe Rad50 protein is involved in the cellular response to DNA‐double strand breaks (DSBs), including the detection of damage, activation of cell‐cycle checkpoints, and DSB repair via recombination. It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice. Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene. A single copy of this gene is present in the Arabidopsis genome, located on chromosome II. Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells. The predicted protein presents strong conservation with the other known Rad50 homologues of the amino‐ and carboxy‐terminal regions. Mutant plants present a sterility phenotype which co‐segregates with the T‐DNA insertion. Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T‐DNA insertion. An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA‐damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double‐strand break repair in plant cells. This is the first report of a plant mutated in a protein of the Rad50–Mre11–Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.
Nod factor‐induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation
Authors: Den Hartog Martine , Musgrave Alan , Munnik Teun 
Published: August 2010

SummaryRhizobium‐secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [32P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.
The jasmonate‐inducible AP2/ERF‐domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate‐responsive promoter element
Authors: Van Der Fits Leslie , Memelink Johan 
Published: August 2010

SummaryThe AP2/ERF‐domain transcription factor ORCA3 is a master regulator of primary and secondary metabolism in Catharanthus roseus (periwinkle). Here we demonstrate that ORCA3 specifically binds to and activates gene expression via a previously characterized jasmonate‐ and elicitor‐responsive element (JERE) in the promoter of the terpenoid indole alkaloid biosynthetic gene Strictosidine synthase (Str). Functional characterization of different domains in the ORCA3 protein in yeast and plant cells revealed the presence of an N‐terminal acidic activation domain and a serine‐rich C‐terminal domain with a negative regulatory function. Orca3 mRNA accumulation was rapidly induced by the plant stress hormone methyljasmonate with biphasic kinetics. A precursor and an intermediate of the jasmonate biosynthetic pathway also induced Orca3 gene expression, further substantiating the role for ORCA3 in jasmonate signaling. The protein synthesis inhibitor cycloheximide did not inhibit jasmonate‐responsive expression of Orca3, nor of its target genes Str and Tryptophan decarboxylase (Tdc). In conclusion, ORCA3 regulates jasmonate‐responsive expression of the Str gene via direct interaction with the JERE. The activating activities of ORCA proteins do not seem to depend on jasmonate‐induced de novo protein synthesis, but presumably occur via modification of pre‐existing ORCA protein.
Spatial and temporal expression of two cytosolic glutamine synthetase genes in Scots pine: functional implications on nitrogen metabolism during early stages of conifer development
Authors: Avila Concepción , Suárez María Fernanda , Gómez‐Maldonado Josefa , Cánovas Francisco M. 
Published: August 2010

SummaryAmmonium assimilation during the initial stages of Scots pine growth involves two cytosolic glutamine synthetase (GS, EC: 6.3.1.2) isoenzymes encoded by separate genes, GS1a and GS1b. GS1a was most exclusively expressed in photosynthetic tissues of the seedling whereas GS1b was expressed ubiquitously showing higher levels in non‐photosynthetic tissues such as root and hypocotyl. Temporal expression analysis has shown that when germination starts GS1b is the predominant form in the embryo, however, its relative abundance in the tissue decreased in the postgerminative stages when green cotyledons are developed. In contrast GS1a was present at a low level in the embryo but its abundance increased markedly during germination and seedling growth. These data suggest that GS1a and GS1b genes display different and non‐redundant roles in the nitrogen metabolism of conifers. The precise localization of individual transcripts by in situ hybridization strongly supports this possibility. GS1 gene products are mainly expressed in different cellular types: GS1a in chlorophylic parenchyma and GS1b in the vascular bundles of all tissues examined in the seedling. Our data support that glutamine biosynthesis in pine seedlings follows a different pattern related to angiosperms involving two cytosolic GS proteins: one of them a typical cytosolic GS which may be involved in the generation of glutamine for N transport and a second cytosolic GS generating amino donors for the biosynthesis of major N compounds in photosynthetic tissues, a closer role to angiosperm chloroplastic GS. The results are discussed with regard to recent studies on N mobilization and metabolism during the initial stages of conifer development.
Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase
Authors: Mauch Felix , Mauch‐Mani Brigitte , Gaille Catherine , Kull Beatriz , Haas Dieter , Reimmann Cornelia 
Published: August 2010

SummarySalicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease‐resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate‐lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB‐A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c‐SAS plants) or to the chloroplast (p‐SAS plants). In p‐SAS plants, the amount of free and conjugated SA was increased more than 20‐fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P‐SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR‐1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c‐SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.
Interaction of pollen‐specific actin‐depolymerizing factor with actin
Authors: Smertenko Andrei P. , Allwood Ellen G. , Khan Safina , Jiang Chang‐Jie , Maciver Sutherland K. , Weeds Alan G. , Hussey Patrick J. 
Published: August 2010

SummaryWe have examined the interaction of recombinant lily pollen ADF, LlADF1, with actin and found that whilst it bound both G‐ and F‐actin, it had a much smaller effect on the polymerization and depolymerization rate constants than the maize vegetative ADF, ZmADF3. An antiserum specific to pollen ADF, antipADF, was raised and used to localize pollen ADF in daffodil – a plant in which massive reorganizations of the actin cytoskeleton have been seen to occur as pollen enters and exits dormancy. We show, for the first time, an ADF decorating F‐actin in cells that did not result from artificial increase in ADF concentration. In dehydrated pollen this ADF : actin array is replaced by actin : ADF rodlets and aggregates of actin, which presumably act as a storage form of actin during dormancy. In germinated pollen ADF has no specific localization, except when an adhesion is made at the tip where actin and ADF now co‐localize. These activities of pollen ADF are discussed with reference to the activities of ZmADF3 and other members of the ADF/cofilin group of proteins.
ISOLATION OF NON‐IDENTICAL POLYPEPTIDE CHAINS OF α‐CRYSTALLIN
Authors: Schoenmakers J. G. G. , Matze R. , Poppel M. van , Bloemendal H. 
Published: August 2010

α‐Crystallin–a water‐soluble lens protein–was dissociated into subunits by means of urea treatment at pH 3. The dissociated protein was resolved into three distinct polypeptide species by chromatography on SE‐Sephadex columns at pH 3.2 equilibrated with 7 M urea. In the presence of urea the isolated polypeptides, designated as I‐a, I‐b, and II, have different electrophoretic mobilities at acid and alkaline pH. It could be demonstrated that two cysteine residues are present in polypeptide I‐a whereas cysteine is absent in polypeptide II. Moreover, amino‐acid analysis reveals that both polypeptides have a different amino‐acid composition. The third polypeptide has shown to be a dimer of one of the polypeptides. This dimer arises during isolation by disulphide bond formation. The results indicate that a‐crystallin is composed of two distinctly different kinds of subunits. Evidence is presented that both types are built up by two different polypeptide chains.
STUDIES ON THE DENATURATION OF TROPOMYOSIN AND LIGHT MEROMYOSIN
Authors: Woods E. F. 
Published: August 2010

Optical rotatory dispersion has been used to study the heat denaturation of tropomyosin and light meromyosin fraction 1 (LMMFr I) as a function of pH and salt concentration. The unfolding of these highly a‐helical molecules takes place in stages indicating regions of varying stability along the molecule. At all pH values NaCl and KCl increase the stability and this is attributed to electrostatic screening of charged groups. There is a decrease in stability to heat at alkaline pH values and the molecules are most stable on the acid side of the iso‐electric point. Guanidine hydrochloride is more effective than urea as a denaturant. In the case of tropomyosin almost complete reversibility of the optical rotation changes by these denaturants can be achieved provided the ‐SH groups are prevented from forming disulphide bonds. For LMMFr I the rotation changes and reversibility are complicated by depolymerization into smaller units. Molecular weight determinations by sedimentation equilibrium indicate a critical urea concentration for depolymerization which is not evident from the optical rotation changes. Although guanidine hydrochloride is more effective than urea in abolishing the helical structure of LMM Fr 1, it is not as effective as a depolymerizing agent.
THE INTRODUCTION OF SYNTHETIC CROSS‐LINKS INTO TROPOCOLLAGEN AND THEIR EFFECTS ON THE THERMAL STABILITY OF THE MOLECULE
Authors: Woodlock Anne F. , Harrap B. S. 
Published: August 2010

Tropocollagen from rat‐tail tendon has been reacted with 33 per cent basic chromic sulphate (empirical formula CrOHSO4) to give soluble reaction products. Thermal denaturation of the products and examination in the ultracentrifuge has shown the appearance of ‐γ‐components suggesting that chrome cross‐links have been introduced into the collagen molecule. Further evidence for cross‐linking has been obtained from studies of renaturation of the thermally denatured reaction products. A study has been made of the effects of both temperature and chrome I collagen ratios on the thermal stability of the reaction products, characterized by their thermal transition temperatures, Tm, and on their contents of irreversibly bound chromium. The value of Tm decreased as the reaction proceeded in the initial stages but in most cases passed through a minimum to increase in the later stages. The amount of irreversibly bound chromium increased continuously to a plateau with time of reaction. These data are interpreted in terms of (A) the increase in net charge on the protein as the cationic chrome complex reacts with protein carboxylate groups and (B) the introduction of cross‐links into a previously ordered conformation (Flory 1956).
THE EFFECT OF ALIPHATIC ALCOHOLS ON THE THERMAL STABILITY OF TROPOCOLLAGEN UNDER ACIDIC CONDITIONS
Authors: Harrap B. S. 
Published: August 2010

Monohydric and dihydric alcohols have been added to aqueous solutions of rat tail tendon collagen at pH 3.0 and their effects on the thermal denaturation temperature, Tm, of the protein have been studied. At concentrations less than 2 M all the monohydric alcohols gave linear curves of Tm vs alcohol concentration with negative slopes. The extent of lowering of Tm increased with increasing Cn for the n‐alcohols from C1, to C4. An alcohol with its carbon chain branched at the α‐carbon atom lowered Tm less than its straight‐chain isomer but branching at the β‐carbon atom produced little effect. At high alcohol concentrations positive deviations from linearity were observed in the Tm vs c curves indicating a competitive stabilizing effect. Of the dihydric alcohols, ethylene glycol and propane‐1:3‐diol each increased Tm at all concentrations whereas propane‐J: 2‐diol decreased Tm except at very high concentration (< 10 M). The addition of ethanol (20% v/v) had only a small effect on the relations between both Tm and pH and Tm and concentration of added salts at pH 3.0. It is concluded from these data that addition of alcohols has only small effects on the contribution to the stability of tropocollagen from electrostatic interactions but major effects on the contributions from hydrogen‐ and hydrophobic‐bonding. The net effect appears to depend primarily on the hydrophobic‐hydrophilic balance and steric factors in the alcohol molecule.
STUDIES ON INHERITED ANTIGENIC VARIATION OF HUMAN SERUM β‐LIPOPROTEIN BY PASSIVE HEMAGGLUTINATION
Authors: Boman H. 
Published: August 2010

A methodological study on passive hemagglutination and passive hemagglutination inhibition reactions with human serum β‐lipoprotein coupled to human red cells by bis‐diazotized benzidine has been performed. Several variable factors have been studied, and a standardized procedure for optimal, reproducible results is described. A diluent containing dextrose which was found superior to several other diluents examined is described. Genetical differences in purified β‐lipoprotein were clearly demonstrated by the passive hemagglutination technique using human isoantisera, and routine typing can be performed by inhibition experiments. The passive hemagglutination and the hemagglutination inhibition tests described are specific and highly sensitive and are expected to become important supplements to the immunoprecipitation technique in the study of inherited β‐lipoprotein variants. This work was supported by grants from J. L. Tiedemanns Tobaksfabrik, Joh. H. Andresens medisinske fond, C. H. Homans legat og legatet til Henrik Homans Minde, and Dahls legat to the author, as well as by grants from The Norwegian Research Council for Science and the Humanities, and from Nordisk Insulinfond, to Professor Kare Berg.
HEMOGLOBIN ATAGO (α852 Tyr β2) A NEW ABNORMAL HUMAN HEMOGLOBIN FOUND IN NAGASAKI
Authors: Fujiwara Naoko , Maekawa Tomoyuki , Matsuda Genji 
Published: August 2010

An abnormal human hemoglobin was discovered in a woman living at Atago in Nagasaki and was designated Hb Atago. In the thin‐layer starch gel electrophoresis with Tris‐EDTA‐borate buffer, pH 8.6, the main component of this abnormal hemoglobin migrated towards the anode a little behind Hb F, and the minor abnormal component migrated to the same site as non‐hemoglobin protein did. These abnormal components were isolated by CM‐cellulose column chromatography. The hybridization method proved that the α chain of Hb Atago had the abnormality. The abnormal α chain was isolated by CM‐cellulose column chromatography in 8 M urea. The tryptic peptide map of the aminoethylated abnormal α chain showed that the abnormality existed in αT9. The abnormal αT9 was isolated and purified by high voltage paper electrophoresis. The abnormal chymotryptic peptides from αT9 were purified by high voltage paper electrophoresis and paper chromatography. The amino acid compositions of these peptides were analyzed. As a result, it was found that in Hb Atago, a tyrosine residue was substituted for the aspartic acid residue at α85 in the normal human hemoglobin.