Fluorescence-detected assembly of the signal recognition particle: binding of the two SRP protein heterodimers to SRP RNA is noncooperative.

Protein-RNA and protein-protein interactions involved in the assembly of the signal recognition particle (SRP) were examined using fluorescence spectroscopy. Fluorescein was covalently attached to the 3'-terminal ribose of SRP RNA following periodate oxidation, and the resulting SRP RNA-Fl was reconstituted into a fluorescent SRP species that was functional in promoting translocation of secretory proteins across the membrane of the endoplasmic reticulum. Each of the two protein heterodimers purified from SRP elicited a substantial change in fluorescein emission upon association with the modified RNA. The binding of SRP9/14 to singly-labeled SRP RNA-Fl increased fluorescein emission intensity by 41% at pH 7.5 and decreased its anisotropy from 0.18 to 0.16. The binding of SRP68/72 increased the fluorescein anisotropy from 0.18 to 0.23 but did not alter the emission intensity of SRP RNA-Fl. These fluorescence changes did not result from a direct interaction between the dye and protein because the fluorescein remained accessible to both iodide ions and fluorescein-specific antibodies in the complexes. The spectral changes were elicited by specific SRP RNA-protein interactions, since (i) the SRP9/14- and SRP68/72-dependent changes were unique, (ii) an excess of unlabeled SRP RNA, but not of tRNA, blocked the fluorescence changes, and (iii) no emission changes were observed when SRP RNA-Fl was titrated with other RNA-binding proteins. Each heterodimer bound tightly to the RNA, since the Kd values determined spectroscopically and at equilibrium for the SRP9/14 and the SRP68/72 complexes with SRP RNA-Fl were less than 0.1 and 7 +/- 3 nM, respectively. The binding affinity of SRP68/72 for SRP RNA-Fl was unaffected by the presence of SRP9/14, and hence the binding of the heterodimers to SRP RNA is noncooperative in the absence of SRP54 and SRP19. The SRP protein heterodimers therefore associate randomly and independently with SRP RNA to form domains in the particle that are distinct both structurally and functionally. Any cooperativity in SRP assembly would have to be mediated by SRP54 and/or SRP19.     

Early after-depolarisations induced by noradrenaline may be initiated by calcium released from sarcoplasmic reticulum

We investigated the effect of 10^–8 M noradrenaline (NA) on [Ca²⁺], and electrical activity of single myocytes of guinea-pig ventricular myocardium loaded with Indo 1-AM. Membrane potential was recorded by means of the patch electrode and patch amplifier set to the current clamp mode. Cells were stimulated at a rate of 30/min by 3 ms pulses of the current injected through the recording electrode. Superfusion of NA resulted in slight shortening of action potentials (APs), increase in rate of rise and amplitude of the respective Ca²⁺ transients, and appearance of secondary Ca²⁺ transients of two kinds: 1. appearing before repolarisation of AP and decay of the preceding Ca²⁺ transient were completed and 2. appearing between the APs. We named them early after-transients (EAT) and delayed after-transients (DAT), respectively. Without any additional intervention EATS caused some prolongation of APs duration and DATs resulted in subthreshold delayed after-depolarisations (DADS). When sarcolemmal K⁺ conductance was decreased by tetraethylammonium (TEA) in the patch electrode or 20 M BaCl₂ in the Tyrode solution, EATs initiated early after depolarizations (EADs) and DATs initiated suprathreshold DADs triggering full-sized APs. Superfusion of 30.0 mM Na⁺ (replaced with LiCl) resulted in reduction of AP duration by -70% and appearance of DATs. Also, the frequent multiple oscillations of Ca ²⁺ concentration were often observed. Neither DATs nor the oscillations had any affect on electrical activity of the cells. Their electrogenicity could not be increased by TEA or 20.0 M Ba²⁺. EATs and DATs and their respective EADs and DADs could not be initiated by NA or low Na⁺ superfusion in the cells pretreated with 2 × 10^–7 M thapsigargin, a selective blocker of Ca²⁺-ATPase of sarcoplasmic reticulum (SR). We conclude that in contrast to the current hypothesis, EADs can be initiated by Ca²⁺ released early in the cardiac cycle from the overloaded SR, and that electrogenicity of both types of Ca²⁺ oscillations critically depends on the sarcolemmal K⁺ conductance.     

Interactions of cholesterol esters with phospholipids: cholesteryl myristate and dimyristoyl lecithin.

The ternary phase diagram of cholesteryl myristate--dimyristoyl lecithin--water has been determined by polarizing light microscopy, scanning calorimetry, and x-ray diffraction. Hydrated dimyristoyl lecithin forms a lamellar liquid--crystalline phase (L alpha) at temperatures greater than 23 degrees C into which limited amounts of cholesteryl myristate (less than 5 wt. %) can be incorporated. The amount of cholesterol ester incorporated is dependent upon the degree of hydration of the L alpha phase. Below 23 degrees C dimyristoyl lecithin forms ordered hydrocarbon chain structures (L beta' and P beta') which do not incorporate cholesterol ester. Comparison with other phospholipid--cholesterol ester--water phase diagrams suggests the following general principles: i) the incorporation of cholesterol ester occurs only into liquid crystalling phospholipid bilayers, ii) the extent of incorporation is temperature-dependent, with increasing amounts of cholesterol ester being incorporated at higher temperatures, and iii) unsaturated cholesterol esters induce increased disordering of the phospholipid bilayers.     

Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis

The ubiquity of elongation factor Tu (EF-Tu)-dependent conformational changes in amino-acyl-tRNA (aa-tRNA) and the origin of the binding energy associated with aa-tRNA.EF-Tu.GTP ternary complex formation have been examined spectroscopically. Fluorescein was attached covalently to the 4-thiouridine base at position 8 (s4U-8) in each of four elongator tRNAs (Ala, Met-m, Phe, and Val). Although the probes were chemically identical, their emission intensities in the free aa-tRNAs differed by nearly 3-fold, indicating that the dyes were in different environments and hence that the aa-tRNAs had different tertiary structures near s4U-8. Upon association with EF-Tu.GTP, the emission intensities increased by 244%, 57%, or 15% for three aa-tRNAs due to a change in tRNA conformation; the fourth aa-tRNA exhibited no fluorescence change upon binding to EF-Tu.GTP. Despite the great differences in the emission intensities of the free aa-tRNAs and in the magnitudes of their EF-Tu-dependent intensity increases, the emission intensity per aa-tRNA molecule was nearly the same (within 9% of the average) for the four aa-tRNAs when bound to EF-Tu-GTP. Thus, the binding of EF-Tu.GTP induced or selected a tRNA conformation near s4U-8 that was very similar, and possibly the same, for each aa-tRNA species. It therefore appears that EF-Tu functions, at least in part, by minimizing the conformational diversity in aa-tRNAs prior to their beginning the recognition and binding process at the single decoding site on the ribosome. Since an EF-Tu-dependent fluorescence change was also observed with fluorescein-labeled tRNA(Phe), the protein-dependent structural change is effected by direct interactions between EF-Tu and the tRNA and does not require the aminoacyl group. The Kd of the tRNA(Phe).EF-Tu.GTP ternary complex was determined, at equilibrium, to be 2.6 microM by the ability of the unacylated tRNA to compete with fluorescent Phe-tRNA for binding to the protein. Comparison of this Kd with that of the Phe-tRNA ternary complex showed that in this case the aminoacyl moiety contributed 4.3 kcal/mol toward ternary complex formation at 6 degrees C but that the bulk of the binding energy in the ternary complex was derived from direct protein-tRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification,Cloning and Functional Expression of hydroxyphenylpyruvate Reductase Involved in Rosmarinic Acid Biosynthesis in Cell Cultures of Coleus Blumei

Hydroxyphenylpyruvate reductase (HPPR) is an enzyme involved in the biosynthesis of rosmarinic acid in Lamiaceae reducing hydroxyphenylpyruvates in dependence of NAD(P)H to the corresponding hydroxyphenyllactates. The HPPR protein was purified from suspension cells of Coleus blumei accumulating high levels of rosmarinic acid by ammonium sulfate precipitation, anion exchange chromatography, hydroxylapatite chromatography, chromatography on 2',5'-ADP-Sepharose 4B and SDS-polyacrylamide gel electrophoresis. The protein was tryptically digested and the peptides sequenced. Sequence information was used to isolate a full-length cDNA-clone for HPPR (EMBL accession number AJ507733) by RT-PCR, screening of a C. blumei cDNA-library and 5'-RACE-PCR. The open reading frame of the HPPR-cDNA consists of 939 nucleotides encoding a protein of 313 amino acid residues. The sequence showed that HPPR belongs to the family of D-isomer-specific 2-hydroxyacid dehydrogenases. The HPPR-cDNA was heterologously expressed in Escherichia coli and the protein was shown to catalyse the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate and 3,4-dihydroxyphenylpyruvate to 3,4-dihydroxyphenyllactate.     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

Unexpected genetic diversity of Fallopia japonica from Central Europe revealed after AFLP analysis

Recently much attention has been paid to genetic aspects of invasive success in Japanese knotweed s.l. One hypothesis to explain the invasive spread of these species is a multiple introduction, which leads to a higher level of genetic diversity in the invaded range. Fallopia japonica is considered to be genetically uniform in Europe, introduced as a single female clone. However, there is some evidence suggesting that invasion history and dynamics differ between Western and Central-Eastern Europe. We used AFLP markers to characterize genetic diversity of three Fallopia taxa that occur in Poland: F. japonica, F. sachalinensis and their hybrid Fallopia × bohemica, growing in so-called ‘homogeneous’ populations, consisting of one taxon and ‘heterogeneous’ populations, composed of the three taxa cohabiting together. No polymorphism, resp. an insignificantly low variability was observed in the ‘homogeneous’ populations. In the ‘heterogeneous’ stands polymorphism was detected within each taxa, with one exception that concerns individuals of F. sachalinensis from a riparian habitat. The highest level of polymorphism was found among individuals of F. × bohemica. The most striking result of our study is the observation of polymorphism between individuals of F. japonica. The AFLP data also showed that F. × bohemica is most diverse when occurring in a heterogeneous configuration with F. japonica and F. sachalinensis in the same habitat. Our results are the first evidence of genetic diversity in F. japonica populations in Central Europe and can implicate the possibility of its multiple introduction in this region or the existence of sexual reproduction of this species.     

Epifaunal and algal assemblages associated with the native Chondrus crispus (Stackhouse) and the non-native Grateloupia turuturu (Yamada) in eastern Long Island Sound

Although the spread of non-native algae is rapidly escalating, relatively few ecological studies have been done to gauge the impacts incurred to native flora and fauna. A reduction in the dominance of a native habitat-forming macroalga due to the replacement by an introduced species can have adverse effects on the community. The non-native red alga Grateloupia turuturu, first reported in Rhode Island, USA in 1994, has since extended its southern range into eastern Long Island Sound. This large alga has the potential to impact coastal communities by altering the floristic composition important to associated flora and fauna. A comparison of algal and epifaunal assemblages was made during 2006 and 2007 between native and non-native algal communities dominated by either G. turuturu or the native, Chondrus crispus at two sites in Long Island Sound. We found that within Grateloupia-dominated habitat, there was a large decrease in overall macrophyte biomass as compared to native habitat. We also found that habitat dominated by the non-native alga reduced species richness and total abundance of invertebrates relative to nearby habitats dominated by C. crispus. In addition, we found that the dominant mesofaunal species, important to higher trophic level consumers, had greatly reduced densities in communities dominated by the non-native alga.     

A comparative analysis of proteins that accumulate during the initial stage of root hair development in barley root hair mutants and their parent varieties

The mechanisms of root hair formation have been studied extensively in Arabidopsis but knowledge about these processes in monocot species is still limited, especially in relation to the proteome level. The aim of this study was to identify the proteins that are involved in the initiation and the early stage of root hair tip growth in barley using two-dimensional (2D) electrophoresis and mass spectrometry. A comparison of proteins that accumulate differentially in two root hair mutants and their respective parent varieties resulted in the identification of 13 proteins that take part in several processes related to the root hair morphogenesis, such as the control of vesicular trafficking, ROS signalling and homeostasis, signal transduction by phospholipids metabolism and ATP synthesis. Among the identified proteins, two ATP synthases, two ABC transporters, a small GTPase from the SAR1 family, a PDI-like protein, a monodehydroascorbate reductase, a C2 domain-containing protein and a Wali7 domain-containing protein were found. This study is the first report on the proteins identified in the initial stage of root hair formation in barley and gives new insights into the mechanisms of root hair morphogenesis in a monocot species.