Expression of the alpha3/beta1 isoform of human Na,K-ATPase in the methylotrophic yeast Pichia pastoris

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.     

Nitrogen Form Alters Hormonal Balance in Salt-treated Tomato (Solanum lycopersicum L.)

Mixed nitrate/ammonium fertilization can partially alleviate the negative effects of salinity on growth of some plant species compared to all-nitrate or all-ammonium fertilization. To gain insights about the mechanisms involved, tomato (Solanum lycopersicum L. cv Moneymaker) plants were grown hydroponically for 3 weeks with two NO₃ ⁻/NH₄ ⁺ fertilization regimes (6/0.5 and 5/1.5; N_total = 6.5 mM) in the absence (control) or presence of salt stress (100 mM NaCl). Ammonium enrichment had no effect on growth and other parameters under control conditions. Under salinity, however, ammonium enrichment improved shoot and root biomass by 20% and maintained leaf PSII efficiency close to control levels. These changes were related to higher leaf K⁺, NO₃ ⁻, and NH₄ ⁺ concentrations and activities of the N-assimilatory enzymes glutamate synthase (GOGAT) and glutamine synthase (GS) in the leaves. Ammonium enrichment also attenuated the salt-induced increase in leaf abscisic acid (ABA) concentration and decrease in leaf concentrations of indole 3-acetic acid (IAA) and the cytokinins trans-zeatin (tZ) and trans-zeatin riboside (tZR). Enhanced cytokinin status was probably due to maintenance of root-to-shoot cytokinin transport and decreased leaf induction of the cytokinin-degrading enzyme cytokinin oxidase/dehydrogenase (CKX) under ammonium-enriched conditions. It is concluded that nitrogen form modifies salinity-induced physiological responses and that these modifications are associated with changes in plant hormone status.     

Landscape genetics of a tropical rescue pollinator

Pollination services are increasingly threatened by the loss and modification of natural habitats, posing a risk to the maintenance of both native plant biodiversity and agricultural production. In order to safeguard pollination services, it is essential to examine the impacts of habitat degradation on the population dynamics of key pollinators and identify potential “rescue pollinators” capable of persisting in these human-altered landscapes. Using a landscape genetic approach, we assessed the impact of landscape structure on genetic differentiation in the widely-distributed tropical stingless bee Trigona spinipes (Apidae: Meliponini) across agricultural landscape mosaics composed of coffee plantations and Atlantic forest fragments in southeastern Brazil. We genotyped 115 bees at 16 specific and highly polymorphic microsatellite loci, developed using next-generation sequencing. Our results reveal that T. spinipes is capable of dispersing across remarkably long distances, as we did not find genetic differentiation across a 200 km range, nor fine-scale spatial genetic structure. Furthermore, gene flow was not affected by forest cover, land cover, or elevation, indicating that reproductive individuals are able to disperse well through agricultural landscapes and across altitudinal gradients. We also found evidence of a recent population expansion, suggesting that this opportunistic stingless bee is capable of colonizing degraded habitats. Our results thus suggest that T. spinipes can persist in heavily-altered landscapes and can be regarded as a rescue pollinator, potentially compensating for the decline of other native pollinators in degraded tropical landscapes.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Cannabinoids and ceramide: two lipids acting hand-by-hand

Cannabinoids, the active components of Cannabis sativa (marijuana) and their endogenous counterparts, exert their effects by binding to specific G-protein-coupled receptors that modulate adenylyl cyclase and ion channels. Recent research has shown that the CB1 cannabinoid receptor is also coupled to the generation of the lipid second messenger ceramide via two different pathways: sphingomyelin hydrolysis and ceramide synthesis de novo. Sustained ceramide accumulation in tumor cells mediates cannabinoid-induced apoptosis, as evidenced by in vitro and in vivo studies. This effect seems to be due to the impact of ceramide on key cell signalling systems such as the extracellular signal-regulated kinase cascade and the Akt pathway. These findings provide a new conceptual view on how cannabinoids act, and raise interesting physiological and therapeutic questions.     

CD38 expression enhances sensitivity of lymphoma T and B cell lines to biochemical and receptor-mediated apoptosis

CD38 has been widely characterised both as an ectoenzyme and as a receptor. In the present paper, we investigated the role of CD38 as possible modulator of apoptosis. CD38-positive (CD38(+)) and negative (CD38(-)) fractions, obtained by sorting CD38(+) cells from lymphoma T (Jurkat) and lymphoma B (Raji) and by transfecting lymphoma LG14 and myeloid leukemia K562 cell lines, were used. Cellular subpopulations were exposed to different triggers (H(2)O(2), UV-B, alpha-TOS and hrTRAIL) and the extent of apoptosis was determined by Annexin V-FITC/PI assay. Our data showed that, in lymphoma cells, propensity to apoptosis was significantly linked to CD38 expression and that, remarkably, such response was independent of the nature of the trigger used. Inhibition of CD38 expression by antisense oligonucleotides treatment resulted in CD38-silenced fractions which were as prone to apoptosis as CD38(-) ones. Notably, susceptibility of K562 to apoptosis-inducing challenges was not affected by CD38 expression.