Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

Crystal structures of aprotinin and its complex with sucrose octasulfate reveal multiple modes of interactions with implications for heparin binding

The crystal structures of aprotinin and its complex with sucrose octasulfate (SOS), a polysulfated heparin analog, were determined at 1.7-2.6 Å resolutions. Aprotinin is monomeric in solution, which associates into a decamer at high salt concentrations. Sulfate ions serve to neutralize the basic amino acid residues of aprotinin to stabilize the decameric aprotinin. Whereas SOS interacts with heparin binding proteins at 1:1 molar ratio, SOS was surprisingly found to induce strong agglutination of aprotinins. Five molecules of aprotinin interact with one molecule of the sulfated sugar, which is stabilized by electrostatic interactions between the positively charged residues of aprotinin and sulfate groups of SOS. The multiple binding modes of SOS with five individual aprotinin molecules may represent the diverse patterns of potential heparin binding to aprotinin, reflecting the interactions of densely packed protein molecules along the heparin polymer.     

Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity of P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.     

Building an understanding of cystic fibrosis on the foundation of ABC transporter structures

Cystic fibrosis (CF) is a fatal disease affecting the lungs and digestive system by impairment of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). While over 1000 mutations in CFTR have been associated with CF, the majority of cases are linked to the deletion of phenylalanine 508 (ΔF508). F508 is located in the first nucleotide binding domain (NBD1) of CFTR. This mutation is sufficient to impair the trafficking of CFTR to the plasma membrane and, thus, its function. As an ABC transporter, recent structural data from the family provide a framework on which to consider the effect of the ΔF508 mutation on CFTR. There are fifty-seven known structures of ABC transporters and domains thereof. Only six of these structures are of the intact transporters. In addition, modern bioinformatic tools provide a wealth of sequence and structural information on the family. We will review the structural information from the RCSB structure repository and sequence databases of the ABC transporters. The available structural information was used to construct a model for CFTR based on the ABC transporter homologue, Sav1866, and provide a context for understanding the molecular pathology of Cystic Fibrosis.     

Axillary proliferation and tuberisation of Dioscorea cayenensis–D. rotundata complex

Yams (Dioscorea spp) are tuber crops used as staple food in Africa because of their nutritional value. However agronomic constraints, phytosanitary problems and the lack of good healthy planting material restrict their production. In contrast to the inefficiency of traditional method of planting, tissue culture techniques allow to increase the multiplication and the rapid production of pathogen- free plant material. This work was undertaken to provide farmers in African countries with healthy microplants and microtubers as seeds. In vitro nodal segments of two varieties of local yams D. cayenensis–D. rotundata complex (cv. ‘Singo’, cv. ‘Singou’ and cv. ‘Gnidou’) were micropropagated on the modified medium of Murashige and Skoog. The morphogenesis, the growth of microplants and microtuber formation have been found to be controlled by external factors that act individually and synergistically. Addition of kinetin (2 mg l⁻¹) to the culture media could reduce multiplication rate (node number) of some clones. An increase of the sucrose concentration from 3% to 5% induced no change in the multiplication and tuberisation parameters. An important reduction of the multiplication (shoot number, height and node number) and the tuberisation (tuber number and length) was observed with 8% sucrose. Multiplication (shoot and node number) was increased in the presence of jasmonic acid (10 μM). JA also induced an increase of tuber number in the absence of Kin. Multiplication of yam by in vitro growth of nodal segments is a way for rapid clonal multiplication and could allow solving the problem of lack of seed material faced by farmers. This method could also be used for multiplication of elite cultivars, independently of the growing season.     

Carbon and nitrogen metabolism in the seagrass, Zostera marina L.: Environmental control of enzymes involved in carbon allocation and nitrogen assimilation

This study experimentally examined influences of environmental variables on the activities of key enzymes involved in carbon and nitrogen metabolism of the submersed marine angiosperm, Zostera marina L. Nitrate reductase activity in leaf tissue was correlated with both water-column nitrate concentrations and leaf sucrose levels. Under elevated nitrate, shoot nitrate reductase activity increased in both light and dark periods if carbohydrate reserves were available. When water-column nitrate was low, glutamine synthetase activity in leaf tissue increased with environmental ammonium. In contrast, glutamine synthetase activity in belowground tissues was statistically related to both nitrate and temperature. At the optimal growth temperature for this species (ca. 25 °C), increased water-column nitrate promoted an increase in glutamine synthetase activity of belowground tissues. As temperatures diverged from the optimum, this nitrate effect on glutamine synthetase was no longer evident. Activities of both sucrose synthase and sucrose-P synthase were directly correlated with temperature. Sucrose-P synthase activity also was correlated with salinity, and sucrose synthase activity was statistically related to tissue ammonium. Overall, the enzymatic responses that were observed indicate a tight coupling between carbon and nitrogen metabolism that is strongly influenced by prevailing environmental conditions, especially temperature, salinity, and environmental nutrient levels.     

Plant KT/KUP/HAK potassium transporters: single family - multiple functions

BACKGROUND AND AIMS: Potassium transporters belonging to the KT/KUP/HAK family are important for various aspects of plant life including mineral nutrition and the regulation of development. Genes encoding these transporters are present in the genomes of all plants, but have not been found in the genomes of Protista or Animalia. The aim of this Botanical Briefing is to analyse the function of KT/KUP/HAK transporters from evolutionary, molecular and physiological perspectives. SCOPE: This Briefing covers the phylogeny and evolution of KT/KUP/HAK transporters, the role of transporters in plant mineral nutrition and potassium homeostasis, and the role of KT/KUP/HAK transporters in plant development.     

Transport and transporters in the endoplasmic reticulum

Enzyme activities localized in the luminal compartment of the endoplasmic reticulum are integrated into the cellular metabolism by transmembrane fluxes of their substrates, products and/or cofactors. Most compounds involved are bulky, polar or even charged; hence, they cannot be expected to diffuse through lipid bilayers. Accordingly, transport processes investigated so far have been found protein-mediated. The selective and often rate-limiting transport processes greatly influence the activity, kinetic features and substrate specificity of the corresponding luminal enzymes. Therefore, the phenomenological characterization of endoplasmic reticulum transport contributes largely to the understanding of the metabolic functions of this organelle. Attempts to identify the transporter proteins have only been successful in a few cases, but recent development in molecular biology promises a better progress in this field.     

Heterodimerization of yLAT-1 and 4F2hc visualized by acceptor photobleaching FRET microscopy

y⁺LAT-1 and 4F2hc are the subunits of a transporter complex for cationic amino acids, located mainly in the basolateral plasma membrane of epithelial cells in the small intestine and renal tubules. Mutations in y⁺LAT-1 impair the transport function of this complex and cause a selective aminoaciduria, lysinuric protein intolerance (LPI, OMIM #222700), associated with severe, complex clinical symptoms. The subunits of an active transporter co-localize in the plasma membrane, but the exact process of dimerization is unclear since direct evidence for the assembly of this transporter in intact human cells has not been available. In this study, we used fluorescence resonance energy transfer (FRET) microscopy to investigate the interactions of y⁺LAT-1 and 4F2hc in HEK293 cells expressing y⁺LAT-1 and 4F2hc fused with ECFP or EYFP. FRET was quantified by measuring fluorescence intensity changes in the donor fluorophore (ECFP) after the photobleaching of the acceptor (EYFP). Increased donor fluorescence could be detected throughout the cell, from the endoplasmic reticulum and Golgi complex to the plasma membrane. Therefore, our data prove the interaction of y⁺LAT-1 and 4F2hc prior to the plasma membrane and thus provide evidence for 4F2hc functioning as a chaperone in assisting the transport of y⁺LAT-1 to the plasma membrane.     

Signs of attenuated depression-like behavior in vasopressin deficient Brattleboro rats

Vasopressin, a peptide hormone functioning also as a neurotransmitter, neuromodulator and regulator of the stress response is considered to be one of the factors related to the development and course of depression. In the present study, we have tested the hypothesis that congenital deficit of vasopressin in Brattleboro rats leads to attenuated depression-like behavior in tests modeling different symptoms of depression. In addition, hypothalamic-pituitary-adrenocortical axis activity was investigated. Vasopressin deficient rats showed signs of attenuated depression-like behavior in forced swimming and sucrose preference tests, while their behavior on elevated plus maze was unchanged. Vasopressin deficiency had no influence on basal levels of ACTH and corticosterone and had only mild impact on hormonal activation in response to forced swimming and plus-maze exposure. However, vasopressin deficient animals showed higher level of dexamethasone induced suppression of corticosterone response to restraint stress and higher basal levels of corticotropin-releasing hormone mRNA in the hypothalamic paraventricular nucleus. In conclusion, present data obtained in vasopressin deficient rats show that vasopressin is involved in the development of depression-like behavior, in particular of the coping style and anhedonia. Moreover, behavioral and endocrine responses were found to be dissociated. We suggest that brain vasopressinergic circuits distinct from those regulating the HPA axis are involved in generating depression-like behavior.