Ionophore-mediated Ca2+ countertransport: role of Na+, Li+ or H+ gradient

Summary In an artificial system, the ionophore A23187, which transports Ca²⁺ but not Na⁺, is able to mediate the uphill translocation of Ca²⁺ from one aqueous medium to another across an organic immiscible phase, provided that a Na⁺, Li⁺ or H⁺ gradient is imposed on the system. Therefore, in the process known as Na-Ca countertransport, the downhill influx of Na⁺ may not be necessary for causing Ca²⁺ extrusion against its electrochemical gradient.     

Ionomycin-mediated calcium transport in rigid and fluid liposomes

The antibiotic ionophore ionomycin translocates Ca from an aqueous medium into or across an organic immiscible phase. At pH 8.0, ionomycin translocates less Ca than A23187, the effects of these ionophores being additive to one another. The capacity of ionomycin to translocate Ca across the organic phase is dramatically decreased when the pH of the aqueous media is reduced from 8.0 to 7.5 or lower values. Ionomycin also mediates Ca exchange-diffusion in liposomes, the magnitude of such a process being greater in fluid than in rigid liposomes. At a physiological pH (7.4), ionomycin is unexpectedly as potent as A23187 in mediating Ca transport in fluid liposomes. These findings suggest that the capacity of ionophores to translocate Ca across model membranes depends on both the transverse and lateral mobility of the ionophoretic molecules. The relative importance of the latter phenomenon itself largely depends on the stoichiometry of the Ca-ionophore complex.     

In vivo activation of blood and arterial prophospholipase by an activator of blood platelet origin]

When partially purified platelet-rat lysate is injected in the rat aorta, transformation of prophospholipase into phospholipase is observed. Blood prophospholipases are activated almost entirely during about 15 minutes; aortic phospholipase are entirely activated for a longer time.     

Purification of rat adipose tissue lipoprotein lipase by affinity chromatography.

Lipoprotein lipase (EC 3.1.1.3) from rat adipose tissue was purified by affinity chromatography with heparin-Sepharose. Elution was carried out with buffered solutions of increasing NaCl molarity. Proteins without affinity for heparin were eluted with 0.5 M NaCl, while lipoprotein lipase activity was eluted as two peaks with 1.16 M NaCl (In earlier work on human adipose tissue (Etienne et al. (1974) C.R. Acad. Sc. Paris 279, 1487-1490) two fractions with lipoprotein lipase activity were also obtained). Phospholipase activity was detected in the fraction eluted with buffered 0.5 M NaCl and containing proteins without affinity for heparin. On feeding the fasting rats with fresh cream or glucose two peaks were also obtained, but the first peak had clearly increased while the second one had remained virtually unchanged.     

Coagulase-negative staphylococci isolated from two cases of toxic shock syndrome lack superantigenic activity, but induce cytokine production

Abstract Two strains of Staphylococcus epidermidis isolated from patients with toxic shock symptoms have been reported to carry genes related to S. aureus enterotoxins B and C by dot-blot hybridisation, although the corresponding superantigenic toxins were not detected immunologically. We here show that these strains produce no superantigens capable of stimulating proliferation of human mononuclear leukocytes or rabbit splenocytes, and that no DNA homologous to the seb or sec genes can be detected by PCR. However, stimulation of human monocytes by whole killed bacteria induced dose-dependent production of the cytokines TNFα, IL-1 β and IL-6, which may be responsible for the clinical symptoms in these patients.     

Shifts in symbiotic associations in plants capable of forming multiple root symbioses across a long‐term soil chronosequence

Changes in soil nutrient availability during long‐term ecosystem development influence the relative abundances of plant species with different nutrient‐acquisition strategies. These changes in strategies are observed at the community level, but whether they also occur within individual species remains unknown. Plant species forming multiple root symbioses with arbuscular mycorrhizal (AM) fungi, ectomycorrhizal (ECM) fungi, and nitrogen‐(N) fixing microorganisms provide valuable model systems to examine edaphic controls on symbioses related to nutrient acquisition, while simultaneously controlling for plant host identity. We grew two co‐occurring species, Acacia rostellifera (N₂‐fixing and dual AM and ECM symbioses) and Melaleuca systena (AM and ECM dual symbioses), in three soils of contrasting ages (c. 0.1, 1, and 120 ka) collected along a long‐term dune chronosequence in southwestern Australia. The soils differ in the type and strength of nutrient limitation, with primary productivity being limited by N (0.1 ka), co‐limited by N and phosphorus (P) (1 ka), and by P (120 ka). We hypothesized that (i) within‐species root colonization shifts from AM to ECM with increasing soil age, and that (ii) nodulation declines with increasing soil age, reflecting the shift from N to P limitation along the chronosequence. In both species, we observed a shift from AM to ECM root colonization with increasing soil age. In addition, nodulation in A. rostellifera declined with increasing soil age, consistent with a shift from N to P limitation. Shifts from AM to ECM root colonization reflect strengthening P limitation and an increasing proportion of total soil P in organic forms in older soils. This might occur because ECM fungi can access organic P via extracellular phosphatases, while AM fungi do not use organic P. Our results show that plants can shift their resource allocation to different root symbionts depending on nutrient availability during ecosystem development.     

PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.     

CRISPR/Cas9-mediated efficient targeted mutagenesis has the potential to accelerate the domestication of <Emphasis Type="Italic">Coffea canephora</Emphasis>

Genome editing, which is an unprecedented technological breakthrough, has provided a valuable means of creating targeted mutations in plant genomes. In this study, we developed a genomic web tool to identify all gRNA target sequences in the coffee genome, along with potential off-targets. In all, 8,145,748 CRISPR guides were identified in the draft genome of Coffea canephora corresponding to 5,338,568 different sequences and, of these, 4,655,458 were single, and 514,591 were covering exons. The proof of concept was established by targeting the phytoene desaturase gene (CcPDS) using the Agrobacterium tumefaciens transformation technique and somatic embryogenesis as the plant regeneration method. An analysis of the RNA-guided genome-editing events showed that 22.8% of the regenerated plants were heterozygous mutants and 7.6% were homozygous mutants. Mutation efficiency at the target site was estimated to be 30.4%. We demonstrated that genome editing by the CRISPR/Cas9 method is an efficient and reliable way of knocking out genes of agronomic interest in the coffee tree, opening up the way for coffee molecular breeding. Our results also showed that the use of somatic embryogenesis, as the method for regenerating genome-edited plants, could restrict the choice of targeted genes to those that are not essential to the embryo development and germination steps.