Flavonoids are group of plant‐derived hydroxylated polycyclic molecules found in fruit and vegetables. They are known to bio‐accumulate within humans and are considered to have beneficial health ...effects, including cancer chemoprotection. One mechanism proposed to explain this is that they are able to induce apoptosis in cancer cells by inhibiting a variety of kinases and also the Ca2+ ATPase. An investigation was undertaken with respect to the mechanism of inhibition for three flavonoids: quercetin, galangin and 3,6 dihydroxyflavone (3,6‐DHF). Each inhibited the Ca2+ ATPase with Ki values of 8.7, 10.3 and 5.4 μm, respectively, showing cooperative inhibition with n ~ 2. Given their similar structures, the flavonoids showed several differences in their mechanisms of inhibition. All three flavonoids stabilized the ATPase in the E1 conformation and reduced 32P‐ATP binding. However, both galangin and 3,6‐DHF increased the affinity of Ca2+ for the ATPase by decreasing the Ca2+‐dissociation rate constant, whereas quercetin had little effect. Ca2+‐induced changes in tryptophan fluorescence levels were reduced in the presence of 3,6‐DHF and galangin (but not with quercetin), indicating that Ca2+‐associated changes within the transmembrane helices are altered. Both galangin and quercetin reduced the rates of ATP‐dependent phosphorylation and dephosphorylation, whereas 3,6‐DHF did not. Modelling studies suggest that flavonoids could potentially bind to two sites: one directly where nucleotides bind within ATP binding site and the other at a site close by. We hypothesize that interactions of these two neighbouring sites may account for both the cooperative inhibition and the multimode mechanisms of action seen with related flavonoids.
Flavonoids are cancer chemo‐protective agents that target the Ca2+ ATPase. Here, we show that flavonoids cause cooperative inhibition (n~2) and that related flavonoids inhibit different steps, including; ATP binding, phosphorylation and the E2/ E1 transition. From enzymatic and modeling studies, we postulate that flavonoids are likely to bind to two sites which are located close together within the ATP binding domain.
TBBPA (tetrabromobisphenol A) is currently the most widely used type of BFR (brominated flame retardant) employed to reduce the combustibility of a large variety of electronic and other manufactured ...products. Recent studies have indicated that BFRs, including TBBPA, are bio-accumulating within animal and humans. BFRs including TBBPA have also been shown to be cytotoxic and potentially endocrine-disrupting to a variety of cells in culture. Furthermore, TBBPA has specifically been shown to cause disruption of Ca2+ homoeostasis within cells, which may be the underlying cause of its cytotoxicity. In this study, we have demonstrated that TBBPA is a potent non-isoform-specific inhibitor of the SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) (apparent K(i) 0.46-2.3 microM), thus we propose that TBBPA inhibition of SERCA contributes in some degree to Ca2+ signalling disruption. TBBPA binds directly to the SERCA without the need to partition into the phospholipid bilayer. From activity results and Ca2+-induced conformational results, it appears that the major effect of TBBPA is to decrease the SERCA affinity for Ca2+ (increasing the K(d) from approx. 1 microM to 30 microM in the presence of 10 microM TBBPA). Low concentrations of TBBPA can quench the tryptophan fluorescence of the SERCA and this quenching can be reversed by BHQ 2,5-di-(t-butyl)-1,4-hydroquinone and 4-n-nonylphenol, but not thapsigargin, indicating that TBBPA and BHQ may be binding to similar regions in the SERCA.
The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca2+ stores remains controversial. It is open to ...question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca2+ uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca2+-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca2+ stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca2+ signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca2+ concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca2+ transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca2+ transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca2+ stores by bafilomycin. By contrast, NAADP failed to evoke a Ca2+ transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca2+ transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca2+ release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.
Multivariate analysis is based on a statistical principle involving observation and analysis of more than one statistical variable at a time. The variability of 434 accessions of rice (Oryza sativa ...L.) germplasm from Côte d’Ivoire was evaluated for 10 agro-morphological traits in upland conditions at M’bé, Côte d’Ivoire (7°5′ N, 5°1′ W) in 2002 using augmented experimental design and analyzed with multivariate methods. The unweighted pair group method of the average linkage (UPGMA) cluster analysis, canonical discriminant analysis, and principal component analysis (PCA) were used to analyze the data obtained. This enabled the assessment of the extent and pattern of variation of the germplasm and identification of the major traits contributing to the diversity. Seven cluster groups were obtained from the 10 agro-botanical traits using the UPGMA. Canonical discriminant analysis showed the contribution of each trait to the classification of the rice accessions into different cluster groups. The first three principal components explained about 72.4% of the total variation among the 10 characters. The results of canonical discriminant analysis and PCA suggested that traits such as plant height, leaf length, number of days to heading and maturity, tillering ability, and grain size (weight, length, width, and shape) were the principal discriminatory characteristics. It was concluded that variation exists in the germplasm, which provides opportunities for this collection to be useful for genetic improvement.
This study aimed at determining shoot and root characteristics of cassava as affected by root yield and the influence of soil moisture on vegetative growth and yield. Thirty cassava genotypes were ...evaluated for morphological and physiological characterization in three locations in Nigeria: Ibadan, Mokwa and Zaria. Randomized complete block design was used with four replicates. Studies on the pasting properties of the genotypes were also carried out. Data were collected on plant height, stem girth, stay-green ability, garri and fresh root weight. Genotypes differed significantly (
P
< 0.05) across and within locations for shoot and root characteristics. Across locations, genotype 011663 had the highest plant height (132.4 cm); 30572 had the largest stem girth (8.6 cm); and 010040 was the best stay-green (2.2). Genotype 011086 had the highest number of roots per plot (95.7), 950289 had the highest fresh root yield (24.3 t/ha), and 990554 had the highest percentage of dry matter (35.2). Trends in root yields across locations were Ibadan (28.9 t/ha), Mokwa (20.3 t/ha), and Zaria (8 t/ha). Five genotypes IITA-TMS-IBA950289, 010034, 990554, 011807, and 980581 had negligible interactions with the environment and so have broad adaptation and are considered stable; and two clones 011807 and 950166 were found to be the best for pasting properties. Breeding strategies that consider root size, total root number, harvest index, dry matter, with applications for household foods and industrial uses, will be an effective and efficient way to select genotypes for high yield.
Calcium signals determine, for example, smooth muscle contraction and changes in gene expression. How calcium signals select for these processes is enigmatic. We build on the “panjunctional ...sarcoplasmic reticulum” hypothesis, describing our view that different calcium pumps and release channels, with different kinetics and affinities for calcium, are strategically positioned within nanojunctions of the SR and help demarcate their respective cytoplasmic nanodomains. SERCA2b and RyR1 are preferentially targeted to the sarcoplasmic reticulum (SR) proximal to the plasma membrane (PM), i.e., to the superficial buffer barrier formed by PM-SR nanojunctions, and support vasodilation. In marked contrast, SERCA2a may be entirely restricted to the deep, perinuclear SR and may supply calcium to this sub-compartment in support of vasoconstriction. RyR3 is also preferentially targeted to the perinuclear SR, where its clusters associate with lysosome-SR nanojunctions. The distribution of RyR2 is more widespread and extends from this region to the wider cell. Therefore, perinuclear RyR3s most likely support the initiation of global calcium waves at L-SR junctions, which subsequently propagate by calcium-induced calcium release via RyR2 in order to elicit contraction. Data also suggest that unique SERCA and RyR are preferentially targeted to invaginations of the nuclear membrane. Site- and function-specific calcium signals may thus arise to modulate stimulus-response coupling and transcriptional cascades.
Tetrabromobisphenol A (TBBPA) is a commonly used brominated flame retardant (BFR) utilized to reduce the flammability of a variety of products. Studies have indicated that a number of BFRs are ...becoming widely distributed within the environment and are bio-accumulating within organisms. There has been much speculation that a variety of phenolic pollutants (including compounds chemically related to TBBPA, such as bisphenol A) may cause endocrine disruption and Ca2+ dysregulation in cells involved in spermatogenesis. In this study we therefore investigate the effects of TBBPA on mouse TM4 Sertoli cells (essential for sperm development). Results show that TBBPA increases Ca2+ within these cells in the 5–60μM concentration range (EC50, 21μM). TBBPA also causes cell death (LC50, 18μM) partly via apoptosis, involving Ca2+-dependent mitochondrial depolarisation. Studies on intracellular Ca2+ transporters shows that TBBPA can inhibit sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) at low concentrations (IC50, 0.4 to 1.2μM) and also activate the Ryanodine receptor Ca2+ channel within the 0.4–4μM concentration range. Therefore these studies suggest that the cytotoxic effects of TBBPA on cells is partly due to dysregulation of Ca2+ signalling, by directly affecting Ca2+ transport proteins.
Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca ...(2+) waves. In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca (2+) via L-SR junctions, in a manner that requires initial Ca (2+) release from lysosomes and subsequent Ca (2+)-induced Ca (2+) release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca (2+) signal information as input data. Simulations of NAADP-dependent junctional Ca (2+) transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca (2+) signals and simulated Ca (2+) transients within L-SR junctions, we estimate that "trigger zones" comprising 60-100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional Ca (2+) such that there is a failure to breach the threshold for CICR via RyR3. L-SR junctions are therefore a pre-requisite for efficient Ca (2+)signal coupling and may contribute to cellular function in health and disease.
A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or ...AMP-insensitive (R531G) γ2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca2+ ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca2+, and their effects were inhibited by STO609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.
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► AMPK is activated by a wide variety of different stresses, drugs, and xenobiotics ► We have developed a sensitive method to test which activators are AMP mediated ► Most, e.g., metformin and resveratrol, inhibit mitochondrial function and elevate AMP ► However, we can now distinguish six different mechanisms for AMPK activation
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