Common and specific responses to iron and phosphorus deficiencies in roots of apple tree (Malus × domestica)

Plant Molecular Biology - Iron and phosphorus are abundant elements in soils but poorly available for plant nutrition. The availability of these two nutrients represents a major constraint for...     

Proline-specific aminopeptidase P prevents replication-associated genome instability

Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1’s role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication.     

Oestrogens down-regulate type I but not type II adrenal corticoid receptors in rat anterior pituitary

We have studied type I and type II adrenal cortical steroid receptors in the anterior (AL), intermediate (IL) and posterior (PL) lobes of the pituitary and in the hippocampus of ovariectomized-adrenalectomized female rats and in castrated-adrenalectomized male animals, with or without oestrogen treatment. Using [3H]dexamethasone as ligand and conditions suitable for determination of its binding to type I and type II receptors, we found that 4 or 15 days of oestrogen reduced type I receptors in AL by 50-60% without changes in IL, PL or hippocampus, or in type II sites in any of the four neuroendocrine tissues studied. This down-regulatory effect was seen only in female rats and no change was found for males. The reduction in type I sites in AL in oestrogenized female rats was confirmed by labelling type I sites with the synthetic antimineralocorticoid [3H]ZK 91587. Saturation analysis with [3H]ZK 91587 demonstrated that the reduction was due to a reduction in Bmax without change in Kd. We conclude that: (a) type I receptors in the anterior pituitary are under oestrogenic control; (b) there is a sex difference in the response to oestrogen of AL type I sites; and (c) this demonstration may be useful in determining the role of type I receptors in neuroendocrine regulation of the anterior pituitary by hormones derived from the adrenal cortex, and the participation of sex hormones in this process.     

The macronutrient composition of wild and cultivated plant foods of West African chimpanzees (Pan troglodytes verus) inhabiting an anthropogenic landscape

Agricultural expansion encroaches on tropical forests and primates in such landscapes frequently incorporate crops into their diet. Understanding the nutritional drivers behind crop-foraging can help inform conservation efforts to improve human-primate coexistence. This study builds on existing knowledge of primate diets in anthropogenic landscapes by estimating the macronutrient content of 24 wild and 11 cultivated foods (90.5% of food intake) consumed by chimpanzees (Pan troglodytes verus) at Bossou, Guinea, West Africa. We also compared the macronutrient composition of Bossou crops to published macronutrient measures of crops from Bulindi, Uganda, East Africa. The composition of wild fruits, leaves, and pith were consistent with previous reports for primate diets. Cultivated fruits were higher in carbohydrates and lower in insoluble fiber than wild fruits, while wild fruits were higher in protein. Macronutrient content of cultivated pith fell within the ranges of consumed wild pith. Oil palm food parts were relatively rich in carbohydrates, protein, lipids, and/or fermentable fiber, adding support for the nutritional importance of the oil palm for West African chimpanzees. We found no differences in the composition of cultivated fruits between Bossou and Bulindi, suggesting that macronutrient content alone does not explain differences in crop selection. Our results build on the current understanding of chimpanzee feeding ecology within forest-agricultural mosaics and provide additional support for the assumption that crops offer primates energetic benefits over wild foods.     

Comparative study of the O(2), CO(2) and temperature effect on respiration between "Conference" pear cell protoplasts in suspension and intact pears

The influence of the O(2) and CO(2) concentration and the temperature on the O(2) uptake rate of cool-stored intact pears and pear cell protoplasts in suspension was compared. Protocols to isolate pear cell protoplasts from pear tissue and two methods to measure protoplast respiration have been developed. Modified Michaelis-Menten kinetics were applied to describe the effect of the O(2) and the CO(2) concentration on the O(2) uptake rate and temperature dependence was analysed with an Arrhenius equation. Both systems were described with a non-competitive type of CO(2) inhibition. Due to the inclusion of gas diffusion properties, the Michaelis-Menten constant for intact pears (2.5 mM) was significantly larger than the one for protoplasts in suspension (3 microM), which was in turn larger than the Michaelis-Menten constant obtained in mitochondrial respiration measurements described in the literature. It was calculated that only 3.6% of the total diffusion effect absorbed in the Michaelis-Menten constant for intact pears, could be attributed to intracellular gas diffusion. The number of cells per volume of tissue was counted microscopically to establish a relationship between the pear cell protoplast and intact pear O(2) uptake rate. A remarkable similarity was observed: values of 61.8 nmol kg(-1) s(-1) for protoplasts and 87.1 nmol kg(-1) s(-1) for intact pears were obtained. Also, the inhibitory effect of CO(2) on the respiration rate was almost identical for protoplasts and intact pears, suggesting that protoplast suspensions are useful for the study of other aspects of the respiration metabolism.     

High expression level of a gene coding for a chloroplastic amino acid selective channel protein is correlated to cold acclimation in cereals

A cold-regulated gene (cor tmc-ap3) coding for a putative chloroplastic amino acid selective channel protein was isolated from cold-treated barley leaves combining the differential display and the 5-RACE techniques. Cor tmc-ap3 is expressed at low level under normal growing temperature, and its expression is strongly enhanced after cold treatment. A positive correlation between the expression of cor tmc-ap3 and frost tolerance was found both among barley cultivars and among cereal species. The COR TMC-AP3 protein was expressed in vitro, purified and used to raise a polyclonal antibody. Western analysis showed that the cor tmc-ap3 gene product is localized to the chloroplastic outer envelope fraction, supporting its putative function. The frost-resistant winter cultivar Onice accumulated COR TMC-AP3 more rapidly and at a higher level than the frost-susceptible spring cultivar Gitane. After 28 days of cold acclimation the winter cultivar had about 2-fold more protein than the spring genotype. All these results suggest that an increased amount of a chloroplastic amino acid selective channel protein could be required for cold acclimation in cereals. Hypotheses about the role of COR TMC-AP3 during the hardening process are discussed.     

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.     

Nitric oxide and superoxide impair human placental amino acid uptake and increase Na+ permeability: implications for fetal growth

Based on evidence that thiol and tyrosine reagents inhibit some amino acid transporters, we tested the hypothesis that NO- and O2- -derived free radicals would impair nutrient uptake by the human placenta. Syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments were exposed to the drug SIN-1 in the presence or absence of superoxide dismutase (SOD) and hemoglobin (Hb). The uptake of [3H]arginine, [3H]taurine, and [3H]leucine; [14C]MeAIB; and 22Na was studied in MVM, whereas the uptake of [3H]taurine was examined in villous fragments. Nitrotyrosine formation was assessed by Western blotting and quantified by ELISA. In MVM, SIN-1 caused an inhibition of [3H]arginine, [3H]taurine, and [14C]MeAIB uptake but had no significant effect on equilibrium [3H]leucine uptake. These effects were prevented by SOD or Hb, implying that both NO and O2- radicals were essential. In contrast, 22Na+ uptake was significantly increased, and this effect was prevented by SOD. In villous fragments, SIN-1 impaired Na+-dependent [3H]taurine uptake, with no effect on Na+-independent uptake. Increased nitrotyrosine formation was observed in MVM after SIN-1 treatment. Endogenous NO- and O2- -derived free radicals may alter human placental nutrient transfer in vivo, with implications for fetal growth.     

Cellular mechanisms involved in CO(2) and acid signaling in chemosensitive neurons

An increase in CO₂/H⁺ is a major stimulus for increased ventilation and is sensed by specialized brain stem neurons called central chemosensitive neurons. These neurons appear to be spread among numerous brain stem regions, and neurons from different regions have different levels of chemosensitivity. Early studies implicated changes of pH as playing a role in chemosensitive signaling, most likely by inhibiting a K⁺ channel, depolarizing chemosensitive neurons, and thereby increasing their firing rate. Considerable progress has been made over the past decade in understanding the cellular mechanisms of chemosensitive signaling using reduced preparations. Recent evidence has pointed to an important role of changes of intracellular pH in the response of central chemosensitive neurons to increased CO₂/H⁺ levels. The signaling mechanisms for chemosensitivity may also involve changes of extracellular pH, intracellular Ca²⁺, gap junctions, oxidative stress, glial cells, bicarbonate, CO₂, and neurotransmitters. The normal target for these signals is generally believed to be a K⁺ channel, although it is likely that many K⁺ channels as well as Ca²⁺ channels are involved as targets of chemosensitive signals. The results of studies of cellular signaling in central chemosensitive neurons are compared with results in other CO₂- and/or H⁺-sensitive cells, including peripheral chemoreceptors (carotid body glomus cells), invertebrate central chemoreceptors, avian intrapulmonary chemoreceptors, acid-sensitive taste receptor cells on the tongue, and pain-sensitive nociceptors. A multiple factors model is proposed for central chemosensitive neurons in which multiple signals that affect multiple ion channel targets result in the final neuronal response to changes in CO₂/H⁺. hypercapnia; brain stem; ventilation; peripheral chemoreceptor; glia; gap junction; glomus; channel; calcium; potassium; carbonic anhydrase; taste receptor; nociception     

The lys5 mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters for starch synthesis in cereal endosperm

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.