Plant proton pumping pyrophosphatase: the potential for its pyrophosphate synthesis activity to modulate plant growth

Cellular pyrophosphate (PPi) homeostasis is vital for normal plant growth and development. Plant proton‐pumping pyrophosphatases (H⁺‐PPases) are enzymes with different tissue‐specific functions related to the regulation of PPi homeostasis. Enhanced expression of plant H⁺‐PPases increases biomass and yield in different crop species. Here, we emphasise emerging studies utilising heterologous expression in yeast and plant vacuole electrophysiology approaches, as well as phylogenetic relationships and structural analysis, to showcase that the H⁺‐PPases possess a PPi synthesis function. We postulate this synthase activity contributes to modulating and promoting plant growth both in H⁺‐PPase‐engineered crops and in wild‐type plants. We propose a model where the PPi synthase activity of H⁺‐PPases maintains the PPi pool when cells adopt PPi‐dependent glycolysis during high energy demands and/or low oxygen environments. We conclude by proposing experiments to further investigate the H⁺‐PPase‐mediated PPi synthase role in plant growth.     

Enzymatic systems of inorganic pyrophosphate bioenergetics in photosynthetic and heterotrophic protists: remnants or metabolic cornerstones?

An increasing body of biochemical and genetic evidence suggests that inorganic pyrophosphate (PPi) plays an important role in protist bioenergetics. In these organisms, two types of inorganic pyrophosphatases [EC 3.6.1.1, namely soluble PPases (sPPases) and proton-translocating PPases (H+-PPases)] that hydrolyse the PPi generated by cell anabolism, thereby replenishing the orthophosphate pool needed for phosphorylation reactions, are present in different cellular compartments. Photosynthetic and heterotrophic protists possess sPPases located in cellular organelles (plastids and mitochondria), where many anabolic and biosynthetic reactions take place, in addition to H+-PPases, which are integral membrane proteins of the vacuolysosomal membranes and use the chemical energy of PPi to generate an electrochemical proton gradient useful in cell bioenergetics. This last category of proton pumps was considered to be restricted to higher plants and some primitive photosynthetic bacteria, but it has been found recently in many protists (microalgae and protozoa) and bacteria, thus indicating that H+-PPases are much more widespread than previously thought. No cytosolic sPPase (in bacteria, fungi and animal cells) has been shown to occur in these lower eukaryotes. The widespread occurrence of these key enzymes of PPi metabolism among evolutionarily divergent protists strongly supports the ancestral character of the bioenergetics based on this simple energy-rich compound, which may play an important role in survival under different biotic and abiotic stress conditions.     

Altered sarcoplasmic reticulum calcium transport in the presence of the heavy metal chelator TPEN

TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylenediamine) is a membrane-permeable heavy-metal ion chelator with a dissociation constant for Ca²⁺ comparable to the Ca²⁺ concentration ([Ca²⁺]) within the intracellular Ca²⁺ stores. It has been used as modulator of intracellular heavy metals and of free intraluminal [Ca²⁺], without influencing the cytosolic [Ca²⁺] that falls in the nanomolar range. In our previous studies, we gave evidence that TPEN modifies the Ca²⁺ homeostasis of striated muscle independent of this buffering ability. Here we describe the direct interaction of TPEN with the ryanodine receptor (RyR) Ca²⁺ release channel and the sarcoplasmic reticulum (SR) Ca²⁺ pump (SERCA). In lipid bilayers, at negative potentials and low [Ca²⁺], TPEN increased the open probability of RyR, while at positive potentials it inhibited channel activity. On permeabilized skeletal muscle fibers of the frog, but not of the rat, 50 μM TPEN increased the number of spontaneous Ca²⁺ sparks and induced propagating events with a velocity of 273 ± 7 μm/s. Determining the hydrolytic activity of the SR revealed that TPEN inhibits the SERCA pump, with an IC₅₀ = 692 ± 62 μM and a Hill coefficient of 0.88 ± 0.10. These findings provide experimental evidence that TPEN directly modifies both the release of Ca²⁺ from and its reuptake into the SR.     

Pharmacological investigation of the bioluminescence signaling pathway of the dinoflagellate Lingulodinium polyedrum: evidence for the role of stretch‐activated ion channels

Dinoflagellate bioluminescence serves as a whole‐cell reporter of mechanical stress, which activates a signaling pathway that appears to involve the opening of voltage‐sensitive ion channels and release of calcium from intracellular stores. However, little else is known about the initial signaling events that facilitate the transduction of mechanical stimuli. In the present study using the red tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge, two forms of dinoflagellate bioluminescence, mechanically stimulated and spontaneous flashes, were used as reporter systems to pharmacological treatments that targeted various predicted signaling events at the plasma membrane level of the signaling pathway. Pretreatment with 200 μM Gadolinium III (Gd³⁺), a nonspecific blocker of stretch‐activated and some voltage‐gated ion channels, resulted in strong inhibition of both forms of bioluminescence. Pretreatment with 50 μM nifedipine, an inhibitor of L‐type voltage‐gated Ca²⁺ channels that inhibits mechanically stimulated bioluminescence, did not inhibit spontaneous bioluminescence. Treatment with 1 mM benzyl alcohol, a membrane fluidizer, was very effective in stimulating bioluminescence. Benzyl alcohol‐stimulated bioluminescence was inhibited by Gd³⁺ but not by nifedipine, suggesting that its role is through stretch activation via a change in plasma membrane fluidity. These results are consistent with the presence of stretch‐activated and voltage‐gated ion channels in the bioluminescence mechanotransduction signaling pathway, with spontaneous flashing associated with a stretch‐activated component at the plasma membrane.     

Chemical Composition,Antibacterial, Schistosomicidal,and Cytotoxic Activities of the Essential Oil of Dysphania ambrosioides (L.) Mosyakin & Clemants (Chenopodiaceae)

We have investigated the chemical composition and the antibacterial activity of the essential oil of Dysphania ambrosioides (L.) Mosyakin & Clemants (Chenopodiaceae) (DA‐EO) against a representative panel of cariogenic bacteria. We have also assessed the in vitro schistosomicidal effects of DA‐EO on Schistosoma mansoni and its cytotoxicity to GM07492‐A cells in vitro. Gas chromatography (GC) and gas chromatography‐mass spectrometry (GC/MS) revealed that the monoterpenes cis‐piperitone oxide (35.2%), p‐cymene (14.5%), isoascaridole (14.1%), and α‐terpinene (11.6%) were identified by as the major constituents of DA‐EO. DA‐EO displayed weak activity against Streptococcus sobrinus and Enterococcus faecalis (minimum inhibitory concentration (MIC) = 1000 μg/ml). On the other hand, DA‐EO at 25 and 12.5 μg/ml presented remarkable schistosomicidal action in vitro and killed 100% of adult worm pairs within 24 and 72 h, respectively. The LC₅₀ values of DA‐EO were 6.50 ± 0.38, 3.66 ± 1.06, and 3.65 ± 0.76 μg/ml at 24, 48, and 72 h, respectively. However, DA‐EO at concentrations higher than 312.5 μg/ml significantly reduced the viability of GM07492‐A cells (IC₅₀ = 207.1 ± 4.4 μg/ml). The selectivity index showed that DA‐EO was 31.8 times more toxic to the adult S. mansoni worms than GM07492‐A cells. Taken together, these results demonstrate the promising schistosomicidal potential of the essential oil of Dysphania ambrosioides.     

Effect of tetramethylpyrazine (TMP) on Ca2+ signal transduction and cell viability in a model of renal tubular cells

Tetramethylpyrazine (TMP) is a compound purified from herb. Its effect on Ca²⁺ concentrations ([Ca²⁺]_i) in renal cells is unclear. This study examined whether TMP altered Ca²⁺ signaling in Madin‐Darby canine kidney (MDCK) cells. TMP at 100–800 μM induced [Ca²⁺]_i rises, which were reduced by Ca²⁺ removal. TMP induced Mn²⁺ influx implicating Ca²⁺ entry. TMP‐induced Ca²⁺ entry was inhibited by 30% by modulators of protein kinase C (PKC) and store‐operated Ca²⁺ channels. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 93% of TMP‐evoked [Ca²⁺]_i rises. Treatment with TMP abolished BHQ‐evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) abolished TMP‐induced responses. TMP at 200–1000 μM decreased viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester. Together, in MDCK cells, TMP induced [Ca²⁺]_i rises by evoking PLC‐dependent Ca²⁺ release from endoplasmic reticulum and Ca²⁺ entry via PKC‐sensitive store‐operated Ca²⁺ entry. TMP also caused Ca²⁺‐independent cell death.     

Ca2+ Signaling and Cell Death Induced by Protriptyline in HepG2 Human Hepatoma Cells

The effect of protriptyline on Ca²⁺ physiology in human hepatoma is unclear. This study explored the effect of protriptyline on [Ca²⁺]_i and cytotoxicity in HepG2 human hepatoma cells. Protriptyline (50–150 μM) evoked [Ca²⁺]_i rises. The Ca²⁺ entry was inhibited by removal of Ca²⁺. Protriptyline‐induced Ca²⁺ entry was confirmed by Mn²⁺‐induced quench of fura‐2 fluorescence. Except nifedipine, econazole, SKF96365, GF109203X, and phorbol 12‐myristate 13 acetate did not inhibit Ca²⁺ entry. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 40% of protriptyline‐induced response. Treatment with protriptyline abolished BHQ‐induced response. Inhibition of phospholipase C (PLC) suppressed protriptyline‐evoked response by 70%. At 20–40 μM, protriptyline killed cells which was not reversed by the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester (BAPTA/AM). Together, in HepG2 cells, protriptyline induced [Ca²⁺]_i rises that involved Ca²⁺ entry through nifedipine‐sensitive Ca²⁺ channels and PLC‐dependent Ca²⁺ release from endoplasmic reticulum. Protriptyline induced Ca²⁺‐independent cell death.     

Alpha‐synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation

Aggregation of α‐synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca²⁺ and α‐synuclein aggregation. Analyses of cell lines and primary culture models of α‐synuclein cytopathology reveal an early phase with reduced cytosolic Ca²⁺ levels followed by a later Ca²⁺ increase. Aggregated but not monomeric α‐synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca²⁺. CPA protects the cells against α‐synuclein‐aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo. Proximity ligation assays also reveal an increased interaction between α‐synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α‐synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down‐stream processes may be therapeutic targets for treating α‐synucleinopathies.     

L-type channel inhibition by CB1 cannabinoid receptors is mediated by PTX-sensitive G proteins and cAMP/PKA in GT1-7 hypothalamic neurons

Using immortalized hypothalamic GT1-7 neurons, which express the CB1 cannabinoid receptor (CB1R) and three Ca²⁺ channel types (T, R and L), we found that the CB1R agonist WIN 55,212-2 inhibited the voltage-gated Ca²⁺ currents by about 35%. The inhibition by WIN 55,212-2 (10 μM) was reversible and prevented by nifedipine (3 μM), suggesting a selective action on L-type Ca²⁺ channels (LTCCs). WIN 55,212-2 action exhibited all the features of voltage-independent Ca²⁺ channel modulation: (1) no changes of the activation kinetics, (2) equal depressive action at all potentials and (3) no facilitation following strong prepulses. At variance with WIN 55,212-2, the CB1R inverse agonist AM-251 (10 μM) caused 20% increase of Ca²⁺ currents. The inhibition of LTCCs by WIN 55,212-2 was prevented by overnight PTX-incubation and by intracellular perfusion with GDP-β-S. The latter caused also a 20% Ca²⁺ current up-regulation. WIN 55,212-2 action was also prevented by application of the PKA-blocker H89 or by loading the neurons with 8-CPT-cAMP. Our results suggest that LTCCs in GT1-7 neurons are partially inhibited at rest due to a constitutive CB1R activity removed by AM-251 and GDP-β-S. Activation of CB1R via PTX-sensitive G proteins and cAMP/PKA pathway selectively depresses LTCCs that critically control the synchronized spontaneous firing and pulsatile release of gonadotropin-releasing hormone in GT1-7 neurons.     

Intracellular calcium plays a critical role in the alcohol‐mediated death of cerebellar granule neurons

Alcohol is a potent neuroteratogen that can trigger neuronal death in the developing brain. However, the mechanism underlying this alcohol‐induced neuronal death is not fully understood. Utilizing primary cultures of cerebellar granule neurons (CGN), we tested the hypothesis that the alcohol‐induced increase in intracellular calcium [Ca²⁺]_i causes the death of CGN. Alcohol induced a dose‐dependent (200–800 mg/dL) neuronal death within 24 h. Ratiometric Ca²⁺ imaging with Fura‐2 revealed that alcohol causes a rapid (1–2 min), dose‐dependent increase in [Ca²⁺]_i, which persisted for the duration of the experiment (5 or 7 min). The alcohol‐induced increase in [Ca²⁺]_i was observed in Ca²⁺‐free media, suggesting intracellular Ca²⁺ release. Pre‐treatment of CGN cultures with an inhibitor (2‐APB) of the inositol‐triphosphate receptor (IP₃R), which regulates Ca²⁺ release from the endoplasmic reticulum (ER), blocked both the alcohol‐induced rise in [Ca²⁺]_i and the neuronal death caused by alcohol. Similarly, pre‐treatment with BAPTA/AM, a Ca²⁺‐chelator, also inhibited the alcohol‐induced surge in [Ca²⁺]_i and prevented neuronal death. In conclusion, alcohol disrupts [Ca²⁺]_i homeostasis in CGN by releasing Ca²⁺ from intracellular stores, resulting in a sustained increase in [Ca²⁺]_i. This sustained increase in [Ca²⁺]_i may be a key determinant in the mechanism underlying alcohol‐induced neuronal death.     

Structural and mechanistic insights into MICU1 regulation of mitochondrial calcium uptake

Mitochondrial calcium uptake is a critical event in various cellular activities. Two recently identified proteins, the mitochondrial Ca²⁺ uniporter (MCU), which is the pore‐forming subunit of a Ca²⁺ channel, and mitochondrial calcium uptake 1 (MICU1), which is the regulator of MCU, are essential in this event. However, the molecular mechanism by which MICU1 regulates MCU remains elusive. In this study, we report the crystal structures of Ca²⁺‐free and Ca²⁺‐bound human MICU1. Our studies reveal that Ca²⁺‐free MICU1 forms a hexamer that binds and inhibits MCU. Upon Ca²⁺ binding, MICU1 undergoes large conformational changes, resulting in the formation of multiple oligomers to activate MCU. Furthermore, we demonstrate that the affinity of MICU1 for Ca²⁺ is approximately 15–20 μM. Collectively, our results provide valuable details to decipher the molecular mechanism of MICU1 regulation of mitochondrial calcium uptake.     

Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei

Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca²⁺) transport because of molting-related events and the deposition of CaCO₃ in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca²⁺ transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca²⁺ change was monitored by adding Ca as CaCl₂ (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca²⁺ transport followed Michaelis–Menten kinetics with K _m = 0.42 ± 0.04 mM and V _max = 0.50 ± 0.02 μM (Ca²⁺ change × initial intracellular Ca⁻¹ × 180 s⁻¹; N = 14, r ² = 0.99). Verapamil (a Ca²⁺ channel inhibitor) and amiloride (a Na⁺/Ca²⁺ exchanger [NCX] inhibitor) completely reduced intracellular Ca²⁺ transport, while nifedipine, another Ca²⁺ channel inhibitor, did not. Vanadate, a plasma membrane Ca²⁺-ATPase inhibitor (PMCA), increased intracellular Ca²⁺ in gill cells through a decrease in the efflux of Ca²⁺. Ouabain increased intracellular Ca²⁺, similar to the effect of KB-R, a specific NCX inhibitor for Ca²⁺ in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca²⁺ transport. Caffeine, responsible for inducing Ca release from sarcoplasmic reticulum in vertebrate muscle, increased intracellular Ca²⁺ compared to control, suggesting an effect of this inhibitor in gill epithelial cells of Dilocarcinus pagei, probably through release of intracellular stores. We also demonstrate here that intracellular Ca²⁺ in gill cells of Dilocarcinus pagei was kept relatively constant in face of an extracellular Ca concentration of 50-fold, suggesting that crustaceans are able to display Ca²⁺ homeostasis through various Ca²⁺ intracellular sequestration mechanisms and/or plasma membrane Ca²⁺ influx and outflux that are highly regulatory. In summary, studies using whole gill cells are an interesting approach for working with real regulatory Ca²⁺ mechanisms in intact cells under physiological Ca levels (mM range), compared to earlier work using isolated vesicles of various epithelial cells.     

N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca²⁺]_i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca²⁺]_i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca²⁺]_i mobilization using several extraction methods. The spatial distribution pattern of [Ca²⁺]_i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 μM and 50 μM AHL triggered opposing osteoblast fates. At 50 μM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 μM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca²⁺]_i levels in osteoblasts treated with 50 μM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 μM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 μM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca²⁺]_i changes. Thus, AHL acts as a double-edged sword for osteoblast function.     

Physiologically Relevant Free Ca2+ Ion Concentrations Regulate STRA6-Calmodulin Complex Formation via the BP2 Region of STRA6

The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca²⁺, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg²⁺-bound CaM (^MgCaM). Upon titration of Ca²⁺ into ^MgCaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca²⁺-binding domains, EF3 and EF4 (^CaK_D = 60 ± 7 nM). As higher concentrations of free Ca²⁺ were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (^CaK_D = 1000 ± 160 nM). Thermodynamic and kinetic Ca²⁺ binding studies showed that BP2 addition increased the Ca²⁺-binding affinity of CaM and slowed its Ca²⁺ dissociation rates (k_off) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca²⁺ concentrations (<100 nM) like those found at resting intracellular levels. As free Ca²⁺ levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca²⁺-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca²⁺-loaded CaM (^CaCaM-BP2). Because this structural rearrangement observed for the ^CaCaM-BP2 complex occurs as intracellular free Ca²⁺ concentrations approach those typical of a Ca²⁺-signaling event (^CaK_D = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length ^CaCaM-STRA6.     

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis

Endoplasmic reticulum (ER)–plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca²⁺ levels has been uncovered in recent years, the functional significance of ER‐PM junctions in non‐excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane‐localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α‐melanocyte‐stimulating hormone (αMSH) depletes ER Ca²⁺ stores, thus recruiting STIM1 to ER‐PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation in vivo. STIM1 domain deletion studies reveal the importance of Ser/Pro‐rich C‐terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH‐cAMP‐MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions.     

In vivo NMR studies of regional cerebral energetics in MPTP model of Parkinson's disease: recovery of cerebral metabolism with acute levodopa treatment

In this study, we have evaluated cerebral atrophy, neurometabolite homeostasis, and neural energetics in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridin (MPTP) model of Parkinson's disease. In addition, the efficacy of acute l ‐DOPA treatment for the reversal of altered metabolic functions was also evaluated. Cerebral atrophy and neurochemical profile were monitored in vivo using MRI and ¹H MR Spectroscopy. Cerebral energetics was studied by ¹H‐[¹³C]‐NMR spectroscopy in conjunction with infusion of ¹³C labeled [1,6^‐13C₂]glucose or [2‐¹³C]acetate. MPTP treatment led to reduction in paw grip strength and increased level of GABA and myo‐inositol in striatum and olfactory bulb. ¹³C Labeling of glutamate‐C4 (1.93 ± 0.24 vs. 1.48 ± 0.06 μmol/g), GABA‐C2 (0.24 ± 0.04 vs. 0.18 ± 0.02 μmol/g) and glutamaine‐C4 (0.26 ± 0.04 vs. 0.20 ± 0.04 μmol/g) from [1,6‐¹³C₂]glucose was found to be decreased with MPTP exposure in striatum as well as in other brain regions. However, glutamine‐C4 labeling from [2‐¹³C]acetate was found to be increased in the striatum of the MPTP‐treated mice. Acute l ‐DOPA treatment failed to normalize the increased ventricular size and level of metabolites but recovered the paw grip strength and ¹³C labeling of amino acids from [1,6‐¹³C₂]glucose and [2‐¹³C]acetate in MPTP‐treated mice. These data indicate that brain energy metabolism is impaired in Parkinson's disease and acute l ‐DOPA therapy could temporarily recover the cerebral metabolism.

     

Molecular characterisation of a calmodulin gene,VcCaM1, that is differentially expressed under aluminium stress in highbush blueberry

Calmodulin (CaM), a small acidic protein, is one of the best characterised Ca²⁺ sensors in eukaryotes. This Ca²⁺‐regulated protein plays a critical role in decoding and transducing environmental stress signals by activating specific targets. Many environmental stresses elicit changes in intracellular Ca²⁺ activity that could initiate adaptive responses under adverse conditions. We report the first molecular cloning and characterisation of a calmodulin gene, VcCaM1 (Vaccinium corymbosum Calmodulin 1), in the woody shrub, highbush blueberry. VcCaM1 was first identified as VCAL19, a gene induced by aluminium stress in V. corymbosum L. A full‐length cDNA of VcCaM1 containing a 766‐bp open reading frame (ORF) encoding 149 amino acids was cloned from root RNA. The sequence encodes four Ca²⁺‐binding motifs (EF‐hands) and shows high similarity (99%) with the isoform CaM 201 of Daucus carota. Expression analyses showed that following Al treatment, VcCaM1 message level decreased in roots of Brigitta, an Al‐resistant cultivar, and after 48 h, was lower than in Bluegold, an Al‐sensitive cultivar. VcCAM1 message also decreased in leaves of both cultivars within 2 h of treatment. Message levels in leaves then increased by 24 h to control levels in Brigitta, but not in Bluegold, but then decreased again by 48 h. In conclusion, VcCaM1 does not appear to be directly involved in Al resistance, but may be involved in improved plant performance under Al toxicity conditions through regulation of Ca²⁺ homeostasis and antioxidant systems in leaves.     

Schistosomicidal Activity of Dihydrobenzofuran Neolignans

We have evaluated the antischistosomal activity of synthetic dihydrobenzofuran neolignans (DBNs) derived from (±)‐trans‐dehydrodicoumaric acid dimethyl ester ( 1 ) and (±)‐trans‐dehydrodiferulic acid dimethyl ester ( 2 ) against adult Schistosoma mansoni worms in vitro. Compound 4 ((±)‐trans‐4‐O‐acetyldehydrodiferulic acid dimethyl ester) displayed the most promising activity; at 200 μm , it kills 100 ± 0% of worms after 24 h, which resembles the result achieved with praziquantel (positive control) at 1.56 μm . The hydrogenation of the double bond between C7′ and C8′, the introduction of an additional methyl group at C3′, and a double bond between C7 and C8 decreased the schistosomicidal activity of DBNs. On the other hand, the presence of the acetoxy group at C4 played an interesting role in this activity. These results demonstrated the interesting schistosomicidal potential of DBNs, which could be further exploited.     

Uptake kinetics and storage capacity of dissolved inorganic phosphorus and corresponding dissolved inorganic nitrate uptake in Saccharina latissima and Laminaria digitata (Phaeophyceae)

Uptake rates of dissolved inorganic phosphorus and dissolved inorganic nitrogen under unsaturated and saturated conditions were studied in young sporophytes of the seaweeds Saccharina latissima and Laminaria digitata (Phaeophyceae) using a “pulse‐and‐chase” assay under fully controlled laboratory conditions. In a subsequent second “pulse‐and‐chase” assay, internal storage capacity (ISC) was calculated based on V_M and the parameter for photosynthetic efficiency F_v/F_m. Sporophytes of S. latissima showed a V_S of 0.80 ± 0.03 μmol · cm^?2 · d^?1 and a V_M of 0.30 ± 0.09 μmol · cm^?2 · d^?1 for dissolved inorganic phosphate (DIP), whereas V_S for DIN was 11.26 ± 0.56 μmol · cm^?2 · d^?1 and V_M was 3.94 ± 0.67 μmol · cm^?2 · d^?1. In L. digitata, uptake kinetics for DIP and DIN were substantially lower: V_S for DIP did not exceed 0.38 ± 0.03 μmol · cm^?2 · d^?1 while V_M for DIP was 0.22 ± 0.01 μmol · cm^?2 · d^?1. V_S for DIN was 3.92 ± 0.08 μmol · cm^?2 · d^?1 and the V_M for DIN was 1.81 ± 0.38 μmol · cm^?2 · d^?1. Accordingly, S. latissima exhibited a larger ISC for DIP (27 μmol · cm^?2) than L. digitata (10 μmol · cm^?2), and was able to maintain high growth rates for a longer period under limiting DIP conditions. Our standardized data add to the physiological understanding of S. latissima and L. digitata, thus helping to identify potential locations for their cultivation. This could further contribute to the development and modification of applications in a bio‐based economy, for example, in evaluating the potential for bioremediation in integrated multitrophic aquacultures that produce biomass simultaneously for use in the food, feed, and energy industries.     

Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis‐like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin‐elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca²⁺ activity as well as Ca²⁺‐dependent proteolytic processing of μ‐calpain. Pyocyanin further up‐regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin‐induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl‐ester labelling, pyocyanin‐treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis‐inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection.