Transmembrane distribution of phospholipids and their involvement in electron transport,as revealed by phospholipase A2 treatment of spinach thylakoids

Thylakoid membranes were treated with either pancreatic or snake venom phospholipase A₂, and the residual phospholipid content of these membranes was determined and compared to the rates of Photosystem II and/or Photosystem I electron transports. The hydrolysis curves of both phosphatidylglycerol and phosphatidylcholine displayed a first, rapid phase which was almost temperature-insensitive, followed by a second, slower phase which depended strongly on the temperature. When pancreatic phospholipase A₂ had access either to the outer face or to both faces of the thylakoid membrane, either only part of or all the phospholipids, respectively, could be hydrolysed. These results were interpreted as indicating an asymmetric distribution of phospholipids across the thylakoid membrane, phosphatidylglycerol and phosphatidylcholine being preferentially located in the outer and the inner layer, respectively. When acting on uncoupled thylakoid membranes, phospholipase A₂ exerted an inhibitory effect on Photosystem II activity and a stimulatory effect on Photosystem I activity. The involvement of phosphatidylcholine and of phosphatidylglycerol in electron transport activities of Photosystem II and of Photosystem I are discussed with special reference to the role of the external and internal pools of these phospholipids.     

Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.

Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta.     

Domains of cellular retinoic acid-binding protein I (CRABP I) expression in the hindbrain and neural crest of the mouse embryo.

We describe here the distribution of cellular retinoic acid-binding protein I (CRABP I) in the head of the early mouse embryo from day 8 to day 13 of gestation, using both in situ hybridisation to localise mRNA and immunocytochemistry to localise protein. The distribution of mRNA and protein was found to be identical. CRABP I first appeared in part of the presumptive hindbrain of the presomite embryo and then became localised to rhombomeres 2, 4, 5 and 6. The only other area of expression in the cephalic neuroepithelium was in a part of the midbrain roof. The neural crest and its mesenchymal derivatives, the branchial arches, expressed CRABP I and crest could be seen streaming from the neuroepithelium of individual rhombomeres into particular branchial arches. This suggested a fate map could be constructed describing the rhombomeric origin of branchial arch mesenchyme. Later in development, axons throughout the hindbrain expressed CRABP I. The results are considered in terms of the role of retinoic acid in the specification of neuronal phenotype in the hindbrain and in axon outgrowth.     

Night-day variations of renin-activity in primary aldosteronism

To detect changes in previously unmeasurable low renin activity plasma specimens of 20 patients with primary aldosteronism (12 with an unilateral adenoma and 8 with idiopathic bilateral adrenal hyperplasia), obtained at short term intervals between 20.00 and 8.00, were incubated over a prolonged period of 18 hours. 6 of 12 patients with an aldosterone producing adenoma (APA) and 3 of 8 patients with idiopathic bilateral adrenal hyperplasia (IAH) showed typical night-day variations of PRA with lower values before and higher values after midnight. 7 of these 9 patients with night-day rhythmicity of PRA simultaneously showed secretory episodes. In 2 patients (1 with APA, 1 with IAH) PRA was constantly undetectable (less than 0.2 ng/ml . 18 h) and in 2 patients with APA a fixed secretion of renin was observed. We failed to demonstrate typical night-day variations of PRA in 3 patients with APA and in 4 with IAH, although in 5 of these 7 patients secretory episodes of PRA were found. Our results show that different patterns of PRA curves may be observed both in patients with APA and IAH. Thus, analysis of PRA curves is of no value to differentiate patients with APA from those with IAH.     

Three distinct lipid kinase activities are present in spinach chloroplast envelope membranes: phosphatidylinositol phosphorylation is sensitive to wortmannin and not dependent on chloroplast ATP.

Chloroplast envelope membranes display properties that are important in lipid synthesis, regulation of metabolites, and protein transport, as well as in signal transduction. The recent discovery showing that phosphorylation of lipids occurs in envelope membranes provides a new approach for understanding the role of chloroplast lipids in these processes. The present investigation shows that three major lipid kinase activities are at least present in envelope membranes. These activities greatly depend on external conditions, such as pH, ATP concentrations, temperature, and chloroplast ATP and wortmannin sensitivity. Two types of phosphorylated lipid couples displayed similar intrinsic responses toward these biochemical parameters, namely phosphatidic acid (PA) and its lysoderivative (LPA) and monogalactosyl-phosphate-diacylglycerol (MGpDG) and its lysoderivative (LMGpDG), but not phosphatidylinositol-monophosphate (PIP) and its lysoderivative (LPIP). Phosphorylation of phosphatidylinositol was not dependent on chloroplast ATP, but was sensitive toward wortmannin in intact chloroplasts and outer envelope membrane vesicles.     

The <Emphasis Type="Italic">transformer2</Emphasis> gene in <Emphasis Type="Italic">Musca domestica</Emphasis> is required for selecting and maintaining the female pathway of development

We present the isolation and functional analysis of a transformer2 homologue Mdtra2 in the housefly Musca domestica. Compromising the activity of this gene by injecting dsRNA into embryos causes complete sex reversal of genotypically female individuals into fertile males, revealing an essential function of Mdtra2 in female development of the housefly. Mdtra2 is required for female-specific splicing of Musca doublesex (Mddsx) which structurally and functionally corresponds to Drosophila dsx, the bottom-most regulator in the sex-determining pathway. Since Mdtra2 is expressed in males and females, we propose that Mdtra2 serves as an essential co-factor of F, the key sex-determining switch upstream of Mddsx. We also provide evidence that Mdtra2 acts upstream as a positive regulator of F supporting genetic data which suggest that F relies on an autocatalytic activity to select and maintain the female path of development. We further show that repression of male courtship behavior by F requires Mdtra2. This function of F and Mdtra2 appears not to be mediated by Mddsx, suggesting that bifurcation of the pathway at this level is a conserved feature in the genetic architecture of Musca and Drosophila.Edited by D. Tautz     

Thermosensor action of GrpE. The DnaK chaperone system at heat shock temperatures

Temperature directly controls functional properties of the DnaK/DnaJ/GrpE chaperone system. The rate of the high to low affinity conversion of DnaK shows a non-Arrhenius temperature dependence and above approximately 40 degrees C even decreases. In the same temperature range, the ADP/ATP exchange factor GrpE undergoes an extensive, fully reversible thermal transition (Grimshaw, J. P. A., Jelesarov, I., Sch?nfeld, H. J., and Christen, P. (2001) J. Biol. Chem. 276, 6098-6104). To show that this transition underlies the thermal regulation of the chaperone system, we introduced an intersubunit disulfide bond into the paired long helices of the GrpE dimer. The transition was absent in disulfide-linked GrpE R40C but was restored by reduction. With disulfide-stabilized GrpE, the rate of ADP/ATP exchange and conversion of DnaK from its ADP-liganded high affinity R state to the ATP-liganded low affinity T state continuously increased with increasing temperature. With reduced GrpE R40C, the conversion became slower at temperatures >40 degrees C, as observed with wild-type GrpE. Thus, the long helix pair in the GrpE dimer acts as a thermosensor that, by decreasing its ADP/ATP exchange activity, induces a shift of the DnaK.substrate complexes toward the high affinity R state and in this way adapts the DnaK/DnaJ/GrpE system to heat shock conditions.     

Factors modulating cottongrass seedling growth stimulation to enhanced nitrogen and carbon dioxide: compensatory tradeoffs in leaf dynamics and allocation to meet potassium-limited growth

Eriophorum vaginatum is a characteristic species of northern peatlands and a keystone plant for cutover bog restoration. Understanding the factors affecting E. vaginatum seedling establishment (i.e. growth dynamics and allocation) under global change has practical implications for the management of abandoned mined bogs and restoration of their C-sequestration function. We studied the responses of leaf dynamics, above- and belowground biomass production of establishing seedlings to elevated CO₂ and N. We hypothesised that nutrient factors such as limitation shifts or dilutions would modulate growth stimulation. Elevated CO₂ did not affect biomass, but increased the number of young leaves in spring (+400 %), and the plant vitality (i.e. number of green leaves/total number of leaves) (+3 %), both of which were negatively correlated to [K⁺] in surface porewater, suggesting a K-limited production of young leaves. Nutrient ratios in green leaves indicated either N and K co-limitation or K limitation. N addition enhanced the number of tillers (+38 %), green leaves (+18 %), aboveground and belowground biomass (+99, +61 %), leaf mass-to-length ratio (+28 %), and reduced the leaf turnover (?32 %). N addition enhanced N availability and decreased [K⁺] in spring surface porewater. Increased tiller and leaf production in July were associated with a doubling in [K⁺] in surface porewater suggesting that under enhanced N production is K driven. Both experiments illustrate the importance of tradeoffs in E. vaginatum growth between: (1) producing tillers and generating new leaves, (2) maintaining adult leaves and initiating new ones, and (3) investing in basal parts (corms) for storage or in root growth for greater K uptake. The K concentration in surface porewater is thus the single most important factor controlling the growth of E. vaginatum seedlings in the regeneration of selected cutover bogs.     

Tuning of DnaK chaperone action by nonnative protein sensor DnaJ and thermosensor GrpE

DnaK, an Hsp70 molecular chaperone, processes its substrates in an ATP-driven cycle, which is controlled by the co-chaperones DnaJ and GrpE. The kinetic analysis of substrate binding and release has as yet been limited to fluorescence-labeled peptides. Here, we report a comprehensive kinetic analysis of the chaperone action with protein substrates. The kinetic partitioning of the (ATP x DnaK) x substrate complexes between dissociation and conversion into stable (ADP x DnaK) x substrate complexes is determined by DnaJ. In the case of substrates that allow the formation of ternary (ATP x DnaK) x substrate x DnaJ complexes, the cis-effect of DnaJ markedly accelerates ATP hydrolysis. This triage mechanism efficiently selects from the (ATP x DnaK) x substrate complexes those to be processed in the chaperone cycle; at 45 degrees C, the fraction of protein complexes fed into the cycle is 20 times higher than that of peptide complexes. The thermosensor effect of the ADP/ATP exchange factor GrpE retards the release of substrate from the cycle at higher temperatures; the fraction of total DnaK in stable (ADP x DnaK) x substrate complexes is 2 times higher at 45 degrees C than at 25 degrees C. Monitoring the cellular situation by DnaJ as nonnative protein sensor and GrpE as thermosensor thus directly adapts the operational mode of the DnaK system to heat shock conditions.     

A Parallel Study of Pigment Bleaching and Cytochrome Breakdown during Aging of Thylakoid Membranes

Aging of freshly isolated thylakoid membranes from spinach leaves(Spinacia oleracea L.) leads to dramatic alterations in boththe cytochrome (b_{559 (HP)} and f) composition and pigment (chlorophyllsa and b and ß-carotene) content. These changes occurat a faster rate under anaerobic conditions or after heatingthylakoid membranes, and in light as well as in darkness. Inaddition, when thylakoid membranes are heated at 78°C for8 min, or incubated in the presence of an emulsion of linoleicacid, a huge decrease in both cytochrome (particularly cyt.b_{559 (HP)}) and pigment contents occur. Whatever the experimentalconditions, cytochrome b_{559 (HP)} destruction occurs as soonthe aging process starts. Conversely, pigment bleaching is detectableafter an initial lag phase of about 60–70 min. Then, thetwo processes (cytochrome breakdown and bleaching of pigments)appear to take place in parallel. The addition of salicylhydroxamicacid or 8-hydroxy-quinoline, two radical scavenger components,to the aging medium strongly reduces the rate and extent ofcytochrome breakdown and pigment bleaching. On the basis of these results, a tentative scheme accountingfor the bleaching of pigments and the breakdown of cytochromesduring aging in vitro of thylakoid membranes is proposed. Itis suggested that these changes are mediated via a non-enzymaticmechanism in which free radicals could be implicated. The possiblerole of free radicals inducing ultrastructural changes at thelevel of chloroplast membranes in senescent leaves is also considered. (Received October 11, 1985; Accepted January 24, 1986)