Simple and Rapid Voltammetric Method Using a Gold Microwire Electrode to Measure Inorganic Arsenic in Holopelagic <i>Sargassum</i> (Fucales, Phaeophyceae)

The valorization of massive strandings of holopelagic Sargassum spp. is strongly limited by high levels of inorganic arsenic (Asi) that are potentially above the limit of current regulations. Monitoring Asi in algal biomass is currently achieved using standard chromatographic separation followed by spectroscopic detection. Here, we propose an alternative simpler procedure based on the extraction of Asi from the freeze-dried algal powder in deionized water and the electroanalytical detection of the diluted extract at a gold-microwire electrode. The protocol was optimized both in terms of extraction (powder/water ratio, extraction time, temperature) and electrolyte used for the voltammetric detection. Two electrolytes were tested: one composed of citric acid, sulfamic acid and KCl (pH 2.0) and another composed of an acetate buffer (pH 4.7) and NaCl. We demonstrate here that Asi determination is possible with the first electrolyte but it is necessary to deal with a relative unstable signal. Measurement of Asi was best achieved with the second electrolyte (acetate buffer and NaCl) with the following optimized electrochemical conditions: deposition potential of −1.2 V, deposition time of 30 seconds and linear scan voltammetry. Voltammetric results were then compared to a reference method (HPLC-ICP-MS) using different morphotypes of holopelagic Sargassum spp. (S. natans VIII, S. natans I and S. fluitans III), using commercial extracts of brown seaweeds and using a Hijiki certified reference material. Very good agreement was obtained between our novel method and HPLC-ICP-MS. Both methods show that inorganic arsenic is almost entirely present as As(V) in Sargassum spp. extracts

PrCYP707A1, an ABA catabolic gene, is a key component of Phelipanche ramosa seed germination in response to the strigolactone analogue GR24

After a conditioning period, seed dormancy in obligate root parasitic plants is released by a chemical stimulus secreted by the roots of host plants. Using Phelipanche ramosa as the model, experiments conducted in this study showed that seeds require a conditioning period of at least 4 d to be receptive to the synthetic germination stimulant GR24. A cDNA-AFLP procedure on seeds revealed 58 transcript-derived fragments (TDFs) whose expression pattern changed upon GR24 treatment. Among the isolated TDFs, two up-regulated sequences corresponded to an abscisic acid (ABA) catabolic gene, PrCYP707A1, encoding an ABA 8\u27-hydroxylase. Using the rapid amplification of cDNA ends method, two full-length cDNAs, PrCYP707A1 and PrCYP707A2, were isolated from seeds. Both genes were always expressed at low levels during conditioning during which an initial decline in ABA levels was recorded. GR24 application after conditioning triggered a strong up-regulation of PrCYP707A1 during the first 18h, followed by an 8-fold decrease in ABA levels detectable 3 d after treatment. In situ hybridization experiments on GR24-treated seeds revealed a specific PrCYP707A1 mRNA accumulation in the cells located between the embryo and the micropyle. Abz-E2A, a specific inhibitor of CYP707A enzymes, significantly impeded seed germination, proving to be a non-competitive antagonist of GR24 with reversible inhibitory activity. These results demonstrate that P. ramosa seed dormancy release relies on ABA catabolism mediated by the GR24-dependent activation of PrCYP707A1. In addition, in situ hybridization corroborates the putative location of cells receptive to the germination stimulants in seeds

Selective Ion Changes during Spontaneous Mitochondrial Transients in Intact Astrocytes

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na+ concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na+ concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca2+ concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg2+ concentration accompanying mitochondrial Na+ spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na+-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication

Disrupted Membrane Structure and Intracellular Ca2+ Signaling in Adult Skeletal Muscle with Acute Knockdown of Bin1

Efficient intracellular Ca2+ ([Ca2+]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation

An integrated bioinformatics analysis reveals divergent evolutionary pattern of oil biosynthesis in high- and low-oil plants

Seed oils provide a renewable source of food, biofuel and industrial raw materials that is important for humans. Although many genes and pathways for acyl-lipid metabolism have been identified, little is known about whether there is a specific mechanism for high-oil content in high-oil plants. Based on the distinct differences in seed oil content between four high-oil dicots (20~50%) and three low-oil grasses (<3%), comparative genome, transcriptome and differential expression analyses were used to investigate this mechanism. Among 4,051 dicot-specific soybean genes identified from 252,443 genes in the seven species, 54 genes were shown to directly participate in acyl-lipid metabolism, and 93 genes were found to be associated with acyl-lipid metabolism. Among the 93 dicot-specific genes, 42 and 27 genes, including CBM20-like SBDs and GPT2, participate in carbohydrate degradation and transport, respectively. 40 genes highly up-regulated during seed oil rapid accumulation period are mainly involved in initial fatty acid synthesis, triacylglyceride assembly and oil-body formation, for example, ACCase, PP, DGAT1, PDAT1, OLEs and STEROs, which were also found to be differentially expressed between high- and low-oil soybean accessions. Phylogenetic analysis revealed distinct differences of oleosin in patterns of gene duplication and loss between high-oil dicots and low-oil grasses. In addition, seed-specific GmGRF5, ABI5 and GmTZF4 were predicted to be candidate regulators in seed oil accumulation. This study facilitates future research on lipid biosynthesis and potential genetic improvement of seed oil content