Changes In Intracellular Concentration Research Articles

Layered versatility atop circadian clocks

Circadian Rhythms The circadian clock evolved to allow cells or organisms to anticipate changes in physiological requirements associated with Earth's 24-hour light/dark cycle. Some activities, however, need to occur out of phase with the core clock. Liang et al. imaged changes in intracellular concentration of Ca2+ in populations of neurons in the fruit fly brain. Although the underlying clock was synchronous, the rhythms of Ca2+ changes corresponded with distinct timing of activities associated with activity of the particular neuronal populations. Proper coordination of these distinct phases required expression of the neuropeptide pigment-dispersing factor and its receptor. Science , this issue p. [976][1] [1]: /lookup/doi/10.1126/science.aad3997

Real-time functional characterization of cationic amino acid transporters using a new FRET sensor.

L-arginine is a semi-essential amino acid that serves as precursor for the production of urea, nitric oxide (NO), polyamines, and other biologically important metabolites. Hence, a fast and reliable assessment of its intracellular concentration changes is highly desirable. Here, we report on a genetically encoded Förster resonance energy transfer (FRET)-based arginine nanosensor that employs the arginine repressor/activator ahrC gene from Bacillus subtilis. This new nanosensor was expressed in HEK293T cells, and experiments with cell lysate showed that it binds L-arginine with high specificity and with a K d of ∼177 μM. Live imaging experiments showed that the nanosensor was expressed throughout the cytoplasm and displayed a half maximal FRET increase at an extracellular L-arginine concentration of ∼22 μM. By expressing the nanosensor together with SLC7A1, SLC7A2B, or SLC7A3 cationic amino acid transporters (CAT1-3), it was shown that L-arginine was imported at a similar rate via SLC7A1 and SLC7A2B and slower via SLC7A3. In contrast, upon withdrawal of extracellular L-arginine, intracellular levels decreased as fast in SLC7A3-expressing cells compared with SLC7A1, but the efflux was slower via SLC7A2B. SLC7A4 (CAT4) could not be convincingly shown to transport L-arginine. We also demonstrated the impact of membrane potential on L-arginine transport and showed that physiological concentrations of symmetrical and asymmetrical dimethylarginine do not significantly interfere with L-arginine transport through SLC7A1. Our results demonstrate that the FRET nanosensor can be used to assess L-arginine transport through plasma membrane in real time.

Alterations in cellular metabolome after pharmacological inhibition of Notch in glioblastoma cells.

Notch signaling can promote tumorigenesis in the nervous system and plays important roles in stem-like cancer cells. However, little is known about how Notch inhibition might alter tumor metabolism, particularly in lesions arising in the brain. The gamma-secretase inhibitor MRK003 was used to treat glioblastoma neurospheres, and they were subdivided into sensitive and insensitive groups in terms of canonical Notch target response. Global metabolomes were then examined using proton magnetic resonance spectroscopy, and changes in intracellular concentration of various metabolites identified which correlate with Notch inhibition. Reductions in glutamate were verified by oxidation-based colorimetric assays. Interestingly, the alkylating chemotherapeutic agent temozolomide, the mTOR-inhibitor MLN0128, and the WNT inhibitor LGK974 did not reduce glutamate levels, suggesting that changes to this metabolite might reflect specific downstream effects of Notch blockade in gliomas rather than general sequelae of tumor growth inhibition. Global and targeted expression analyses revealed that multiple genes important in glutamate homeostasis, including glutaminase, are dysregulated after Notch inhibition. Treatment with an allosteric inhibitor of glutaminase, compound 968, could slow glioblastoma growth, and Notch inhibition may act at least in part by regulating glutaminase and glutamate.

Effect of cryptotanshinone on imatinib sensitivity and P-glycoprotein expression of chronic myeloid leukemia cells

Cryptotanshinone (CPT), a lipid soluble active compound in Salvia miltiorrhiza, has a significant inhibitory effect on multiple malignant tumors, e. g. chronic myeloid leukemia (CML) cells and can effectively enhance imatinib's chemotherapeutic effect. However, its functional molecular mechanism remained unclear. In this experiment, the authors conducted a systematic study on the effect of CPT on the imatinib sensitivity and P-glycoprotein (P-gp) expression in CML cells by using CML cells K562 and imatinib persister K562-R. The MTT assays were performed to determine CPT's impact on the inhibitory effect of imatinib. Annexin V-FITC/PI staining analysis was used to detect the changes in the cell apoptosis rate. The active changes in apoptosis regulatory proteins Caspase-3, Caspase-9 and PARP were determined by Western blot. After the cells were pretreated with the gradient concentration of CPT, the expression of P-gp was analyzed by Western blot and flow cytometry. The changes in intracellular concentrations of imatinib were determined by HPLC analysis. The results indicated that the pretreatment with CPT significantly increased the proliferation inhibiting and apoptosis inducing effects of imatinib on K562 and K562-R cells as well as the degradation product expression of pro-apoptotic proteins Caspase-3, Caspase-9 and PARP, with a significant difference with the control group (P < 0.01). However, CPT showed no impact on the P-gp expression in CML cells and the intracellular concentrations of imatinib. In summary, the findings suggested that CPT enhanced the sensitivity of CML cells to imatinib. Its mechanism is not dependent on the inhibition in P-gp expression and the increase in intracellular drug concentration.

Mechanosensitive ion channel MscL controls ionic fluxes during cold and heat stress in Synechocystis.

Calcium plays an essential role in a variety of stress responses of eukaryotic cells; however, its function in prokaryotes is obscure. Bacterial ion channels that transport Ca(2+) are barely known. We investigated temperature-induced changes in intracellular concentration of Ca(2+), Na(+) and K(+) in the cyanobacterium Synechocystis sp. strain PCC 6803 and its mutant that is defective in mechanosensitive ion channel MscL. Concentration of cations rapidly and transiently increased in wild-type cells in response to cold and heat treatments. These changes in ionic concentrations correlated with the changes in cytoplasmic volume that transiently decreased in response to temperature treatments. However, no increase in ionic concentrations was observed in the MscL-mutant cells. It implies that MscL functions as a non-specific ion channel, and it participates in regulation of cell volume under temperature-stress conditions.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in &lt;em&gt;Streptococcus pneumoniae&lt;/em&gt; Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Intracellular changes of Na+, K+, Ca++ &amp; Mg++ in rat pancreatic islets in different glucose concentration and their relation with insulin secretion

Impairment of insulin secretion from pancreatic ?–cell constitutes an important pathophysiological factor in the development of diabetes mellitus. The changes of intracellular concentration of Na+, K+, Ca++ and Mg++ were observed in substimulatory and stimulatory different glucose concentrations. Pancreatic islets from Long-Evans rats were isolated by collagenase digestion. The concentrations of ions expressed in terms of islet protein in the homogenized islets were measured by using an ion-sensitive electrode based autoanalyzer. In the physiological medium, the islet content of all the four ions increased significantly in response to glucose with maximum level at 11 mM and no further increase at 20 mM. Initial depolarizing effect of glucose is due to reduction of K+ permeability. The reduction of K+ permeability by glucose in ?–cell is a major step in stimulus-secretion coupling for insulin release. DOI: http://dx.doi.org/10.3329/jdnmch.v18i2.16016 J. Dhaka National Med. Coll. Hos. 2012; 18 (02): 17-20

Determination of P-glycoprotein surface expression and functional ability after in vitro treatment with darunavir or raltegravir in lymphocytes of healthy donors

It has been shown that P-glycoprotein (P-gp) can greatly affect the cell uptake of antiretroviral drugs, thus hampering their access to HIV-1 replication sites. Lymphocytes are important sites of replication of HIV and target of other drugs, modification on these cells of P-gp could have an effect on pharmacokinetic of antiretrovirals and drug substrates. Blood samples from 16 healthy volunteers were used to determine the expression of P-gp on total, T and T helper lymphocytes after exposure to darunavir, a second generation protease inhibitor, and raltegravir, the first approved integrase inhibitor. Moreover, the effect of the drugs on P-gp functional activity was also studied by the rhodamine-123 efflux test. Darunavir, but not raltegravir, exposure caused a moderate, dose-dependent increment in P-gp expression in total, T and T helper lymphocytes, as demonstrated by the relative frequency of P-gp+ cells and by the amount of P-gp molecules present on cell surface. Functionally, incubation with darunavir led to a marked inhibition of P-gp activity measured by the efflux of rhodamine-123 similar to that observed by verapamil, a specific P-gp inhibitor. Raltegravir was not able to modify the efflux of rhodamine-123 level. Data show that darunavir, unlike raltegravir, may modify the expression and functionality of P-gp on human lymphocytes, thus leading to potential changes in intracellular concentrations of darunavir in patients treated with other drugs substrate of P-gp and vice versa. Our study highlights the need for studies on drug interactions via the P-gp modulation mechanism, especially with the current multi-drug regimens.

Effect of valproic acid on Ca2+ and cell apoptosis in spinal cord motor neurons after brachial plexus injury in rats

Objective To examine the effect of valproic acid (VPA) on concentration of Intracellular Ca2+ and on cell apoptosis in spinal cord motor neurons after brachial plexus injury in rats. Methods Totally 210 adult male Wistar rats were randomly divided into Sham operation group (disposed the brachial plexus nerve root, but not cutted it off), control group (rats with brachial plexus nerve root amputating wound)and VPA group(rats with brachial plexus nerve root amputating wound and fed by VPA water),with 70 rats in each group.The specimens were taken at 12,24,48,72 h,1,2 and 4 weeks after operation.Whole-cell patch-clamp recording techniques were used to assayed the L-type calcium channel of motoneuron and monitored the changes in intracellular concentration of Ca2+ with spectrofluorometer. The motoneruron apoptosis was detected by TUNEL. Results The set of indicators did not change in the sham group.From 12h to 1 weeks after the operation, the electrical current of L-type calcium channel and the intra-cellular Ca2+ concentration of the neuron were obviously more in control group than in sham operation group (P ＜0.05). From 12 h to 4 weeks after the injury, there were more apoptosis neurons in control group than in sham operation group (P ＜ 0.05). There was no obviously difference in electrical current of L-type calcium channel between the VPA group and the control group at each time point(P ＞ 0.05).Compared to the control group,the intra-cellular Ca2+ concentration was lower in VPA group from 48 h to 1 week after nerve injury (P ＜ 0.05) ; the number of apoptosis neurons were less in VPA group from 24 h to 2 weeks after the injury (P ＜ 0.05). Conclusions Brachial plexus nerve root amputating wound in rats can increase the intra-cellular Ca2+ concentration and apoptosis of the motor neuron.VPA can reduce the intra-cellular Ca2+ concentration and apoptosis,but has no effect on the L-type calcium channel of the motor neuron. Key words: Valproic acid; Motor neuron; Spinal cord; Brachial plexus injury; Ca2+; Apoptosis

Investigation of beta-endorphin reception in preimplantation development of a mouse embryo in vitro

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.

Magnesium and Vascular Changes in Hypertension

Many factors have been implicated in the pathogenesis of hypertension, including changes in intracellular concentrations of calcium, sodium, potassium, and magnesium. There is a significant inverse correlation between serum magnesium and incidence of cardiovascular diseases. Magnesium is a mineral with important functions in the body such as antiarrhythmic effect, actions in vascular tone, contractility, glucose metabolism, and insulin homeostasis. In addition, lower concentrations of magnesium are associated with oxidative stress, proinflammatory state, endothelial dysfunction, platelet aggregation, insulin resistance, and hyperglycemia. The conflicting results of studies evaluating the effects of magnesium supplements on blood pressure and other cardiovascular outcomes indicate that the action of magnesium in the vascular system is present but not yet established. Therefore, this mineral supplementation is not indicated as part of antihypertensive treatment, and further studies are needed to better clarify the role of magnesium in the prevention and treatment of cardiovascular diseases.

Oleaginous Yeasts: Biochemical Events Related with Lipid Synthesis and Potential Biotechnological Applications

The last years there has been a significant rise in the number of publications in the international literature that deal with the production of oils and fats deriving from microbial sources (the so called “single cell oils – SCOs”) that could be used as precursors for the synthesis of bio-diesel or as “tailor-made” lipids amenable for the replacement of expensive fatty materials found in the plant or animal kingdom [1,2]. These lipids are produced by the so-called “oleaginous” microorganisms (microorganisms principally belonging to yeasts, fungi and algae and to lesser extent bacteria, capable of storing quantities of lipids higher than 20%, wt/wt, in their dry weight) [1,3-5]. Remarkable differences in biochemical and kinetic level exist between the process of lipid accumulation when glucose or similarly metabolized compounds are used as substrates (“de novo” lipid synthesis) compared with that performed when hydrophobic materials are used as substrates (“ex novo” lipid synthesis). De novo lipid biosynthesis in the oleaginous microorganisms is non-growth associated process, conducted due to change of intra-cellular concentration of various metabolites after nitrogen depletion into the culture medium. Nitrogen exhaustion leads to a rapid decrease of the concentration of cellular AMP, which is further cleaved in order for nitrogen to be offered to the microorganism. Cellular AMP concentration decrease alters the Krebs cycle function; NAD + - (and in various cases NADP + isocitrate) dehydrogenase, allosterically activated by intracellular AMP, loses its activity and the carbon flow, hence, is directed towards the accumulation of intra-mitochondrial citric acid. When the concentration of citric acid inside the mitochondria becomes higher than a critical value, it is secreted inside the cytoplasm. Then, citric acid is cleaved by ATP-citrate lyase, enzyme-key showing the oleaginous character of the microorganisms, into acetyl-CoA and oxaloacetate and acetyl-CoA, by virtue of the action of fatty acid synthetase generates cellular fatty acids and subsequently triacylglycerols (TAGs), that are the most common form of lipophilic compounds found in the oleaginous microorganisms [1,3-5]. In the non-lipid producing microorganisms, nitrogen exhaustions provokes secretion of the previously hyper-synthesized citric acid into the growth medium (case of the fungus Aspergillus niger and many of the strains of the yeast Yarrowia lipolytica) or results in a block in the level of 6-phosphofructokinase (with mechanisms similar to the ones related with the decrease of activity of NAD

NMR based metabonomics study of NPY Y5 receptor activation in BT-549, a human breast carcinoma cell line

Overexpression of neuropeptide Y (NPY) and its receptors has been found in various cancers. In our previous study, we demonstrated expression of NPY Y5 receptor (Y5R) in various breast cancer cell lines along with Y1 receptor. In Y5R expressing BT-549 cells, NPY induced cell proliferation that was blocked by Y5R-selective antagonist CGP1683A (CGP). Here, NMR-based metabonomics was used to monitor the metabolic profile of BT-549 cells in the presence of NPY and CGP to assess the effect of Y5R activation and inhibition during NPY-induced cell proliferation. To study changes in intra and extra cellular metabolites in response to various treatments, 1D 1H-NMR spectra of both hydrophilic cell extracts and growth medium were recorded from BT-549 with three treatments: (1) NPY, (2) CGP, and (3) CGP followed by NPY (CGP/NPY). Principal component analysis and statistical significance analysis indicated changes in intracellular concentrations of seven metabolites in hydrophilic cell extracts with NPY treatment: decreases in lactate, succinate, myo-inositol, and creatine, and increases in acetate, glutamate, and aspartate. A significant increase in intracellular lactate level and attenuation of other metabolites to baseline was detected in CGP/NPY group. Also, significant decreases in lactate and increases in pyruvate were observed in growth medium from NPY treated cells. Based on the metabonomics analysis, Y5R activation induces cell proliferation by increasing the rate of glycolysis, glutaminolysis, and TCA cycle. Inhibition of Y5R by CGP counteracts NPY-induced changes in cellular metabolites. These changes may play a role in cell proliferation and migration by NPY through Y5R activation.

Statins Increase Antileukemic Potency of Imatinib Through the Inhibition of MDR/ABCB1 and BCRP/ABCG2 Drug Transporters Activity

Abstract 2742The treatment of chronic myeloid leukemia (CML) was revolutionized by the introduction of tyrosine kinase inhibitors (TKIs) to the clinical practice. However, there is still a significant group of patients who due to drug resistance do not fully benefit from the therapy. Among various reasons of drug resistance, the changes of intracellular concentration of TKIs caused by the activity of drug transporters may have profound effect on clinical activity of the drug. Intracellular concentration of imatinib is a result of: drug influx into the cell and drug efflux out of the cell. The latter is mediated by ATP-binding casette (ABC) transporters - MDR/ABCB1 and BCRP/ABCG2. According to the recent knowledge modulation of membrane cholesterol level may change the activity of ABC transporters by changing their conformation. Hence we decided to evaluate potential effect of statins, widely used hypocholesterolemic drugs, on antileukemic activity of imatinib in cell lines transformed with BCR/ABL oncoprotein and primary human CML CD34+ cells.In a set of experiments involving cytotoxic assays (i.e. clonogenic assay, XTT, trypan blue exclusion assay) we revealed that lovastatin, a member of statin family, is able to enhance imatinib cytotoxicity both in established cell lines (32Dcl3 BCR/ABL+, K562) as well as in primary CML CD34+ cells obtained from patients in various stages of the disease, including those clinically resistant to imatinib with no detectable ABL kinase domain mutations. In the same time, lovastatin alone and in combination with imatinib did not influence BCR/ABL negative cells (32Dcl3 wild type, HL-60, CD34+ cells obtained from healthy blood donors). As measured using specific, fluorescent-labeled substrates for ABC transporters (rhodamine-123 and Bodipy-FL-prazosin respectively), lovastatin significantly inhibits MDR/ABCB1- and BCRP/ABCG2-mediated efflux capacity. Similar effects were observed for other statins. The addition of cholesterol to the samples previously incubated with lovastatin completely reversed inhibitory activity of statins. To confirm these results we examined direct changes of intracellular concentration of imatinib itself using radiolabeled 14C-imatinib. In these settings, statins caused 2–3-fold increase in intracellular imatinib concentration in murine and human cell lines (32Dcl3 BCR/ABL+, K562) and also in primary CML CD34+ cells including clones resistant to imatinib (Figure 1). Statins did not influence initial concentration of imatinib what may suggest that their effects on imatinib concentration are not mediated by changes in OCT-1 activity. In addition, statins did not influence expression of MDR/ABCB1 and BCRP/ABCG2 drug transporters measured using qPCR nor ABC proteins levels measured using Wester blotting. Combination of imatinib and lovastatin induced cell cycle arrest and increased percentage of apoptotic leukemic cells measured using flow cytometric analysis after propidium iodide staining. Lovastatin also reduced imatinib-induced phosphorylation of the adaptor protein CrkL.In summary, we show that statins are able to increase therapeutic efficacy of imatinib through the inhibition of drug efflux mediated by MDR/ABCB1 and BCRP/ABCG2 transporters. Our results suggest that statin-mediated depletion of cholesterol in the cell may change conformational status of the efflux pumps, including MDR/ABCB1 and BCRP/ABCG2 pumps involved in the transport of imatinib, and in that way modulate intracellular concentration of the drug. The addition of statins may become attractive treatment modality for the selected group of patients in chronic phase and blast crisis. [Display omitted] CML-CP, CML-BP–CD34+ cells obtained from CML patient in chronic phase or blast crisis respectively; *p<0,05 Disclosures:No relevant conflicts of interest to declare.

Lipopolysaccharide modulates astrocytic S100B secretion: a study in cerebrospinal fluid and astrocyte cultures from rats

BackgroundInflammatory responses in brain are primarily mediated by microglia, but growing evidence suggests a crucial importance of astrocytes. S100B, a calcium-binding protein secreted by astrocytes, has properties of a neurotrophic or an inflammatory cytokine. However, it is not known whether primary signals occurring during induction of an inflammatory response (e.g. lipopolysaccharide, LPS) directly modulate S100B.MethodsIn this work, we evaluated whether S100B levels in cerebrospinal fluid (CSF) and serum of Wistar rats are affected by LPS administered by intraperitoneal (IP) or intracerebroventricular (ICV) injection, as well as whether primary astrocyte cultures respond directly to lipopolysaccharide.ResultsOur data suggest that S100B secretion in brain tissue is stimulated rapidly and persistently (for at least 24 h) by ICV LPS administration. This increase in CSF S100B was transient when LPS was IP administered. In contrast to these S100B results, we observed an increase in in TNFα levels in serum, but not in CSF, after IP administration of LPS. In isolated astrocytes and in acute hippocampal slices, we observed a direct stimulation of S100B secretion by LPS at a concentration of 10 μg/mL. An involvement of TLR4 was confirmed by use of specific inhibitors. However, lower levels of LPS in astrocyte cultures were able to induce a decrease in S100B secretion after 24 h, without significant change in intracellular content of S100B. In addition, after 24 h exposure to LPS, we observed a decrease in astrocytic glutathione and an increase in astrocytic glial fibrillary acidic protein.ConclusionsTogether, these data contribute to the understanding of the effects of LPS on astrocytes, particularly on S100B secretion, and help us to interpret cerebrospinal fluid and serum changes for this protein in neuroinflammatory diseases. Moreover, non-brain S100B-expressing tissues may be differentially regulated, since LPS administration did not lead to increased serum levels of S100B.

Some characteristics of membrane Cd2+ transport in rat thymocytes: an analysis using Fluo-3

Although cadmium-induced apoptosis of lymphocytes is one of common features in the immunotoxicity of cadmium, the membrane pathway for intracellular cadmium accumulation is not fully elucidated. To characterize membrane Cd(2+) transport of rat thymocytes, the change in intracellular Cd(2+) concentration under various conditions was examined by the use of Fluo-3, a fluorescent probe for monitoring the change in intracellular concentration of divalent metal cations. The membrane Cd(2+) transport was estimated by the augmentation of Fluo-3 fluorescence induced by bath application of CdCl(2). Lowering temperature strongly suppressed the augmentation of Fluo-3 fluorescence by CdCl(2), suggesting that the metabolic process can be involved in membrane Cd(2+) transport. External acidification (decreasing pH) and membrane depolarization by adding KCl attenuated the augmentation, indicating the requirement of electrochemical driving force for membrane Cd(2+) transport into the cells. Bath application of CaCl(2) and ZnCl(2) equally decreased the augmentation, suggesting their competition with Cd(2+) at the membrane transport. The augmentation by CdCl(2) was lesser in the cells treated with N-ethylmaleimide inducing chemical depletion of cellular thiols. The result suggests the contribution of sulfhydryl groups to membrane Cd(2+) transport. Taken together, it is suggested that the cells possess a temperature-sensitive membrane Cd(2+) pathway, driven by electrochemical gradient of Cd(2+) and transmembrane potential, with competitive binding site. Based on the characteristics described above, it is unlikely that the membrane Cd(2+) transport in rat thymocytes is attributed to a single transport system although it has characteristics that are similar to those of divalent cation transporter 1.

An Aptamer‐Based Nanobiosensor for Real‐Time Measurements of ATP Dynamics

How ATP! A nanosensor based on a new DNA aptamer can show changes in intracellular concentration of ATP of 0.5 to 8 mM and thereby the kinetics of ATP-consuming reactions in real-time. The biosensor was protected against nuclease attack by being buried in a polyacrylamide nanoparticle. This strategy can readily be applied to any metabolite of interest in cells.

Osmosensitivity through the PIP2availability: just add water

Osmosensitivity through the PIP₂availability: just add water

Effect of Sugars on Artemisinin Production in Artemisia annua L.: Transcription and Metabolite Measurements

The biosynthesis of the valuable sesquiterpene anti-malarial, artemisinin, is known to respond to exogenous sugar concentrations. Here young Artemisia annua L. seedlings (strain YU) were used to measure the transcripts of six key genes in artemisinin biosynthesis in response to growth on sucrose, glucose, or fructose. The measured genes are: from the cytosolic arm of terpene biosynthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), farnesyl disphosphate (FPS); from the plastid arm of terpene biosynthesis, 1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxyxylulouse 5-phosphate reductoisomerase (DXR); from the dedicated artemisinin pathway amorpha-4,11-diene synthase (ADS), and the P450, CYP71AV1 (CYP). Changes in intracellular concentrations of artemisinin (AN) and its precursors, dihydroartemisinic acid (DHAA), artemisinic acid (AA), and arteannuin B (AB) were also measured in response to these three sugars. FPS, DXS, DXR, ADS and CYP transcript levels increased after growth in glucose, but not fructose. However, the kinetics of these transcripts over 14 days was very different. AN levels were significantly increased in glucose-fed seedlings, while levels in fructose-fed seedlings were inhibited; in both conditions this response was only observed for 2 days after which AN was undetectable until day 14. In contrast to AN, on day 1 AB levels doubled in seedlings grown in fructose compared to those grown in glucose. Results showed that transcript level was often negatively correlated with the observed metabolite concentrations. When seedlings were gown in increasing levels of AN, some evidence of a feedback mechanism emerged, but mainly in the inhibition of AA production. Together these results show the complex interplay of exogenous sugars on the biosynthesis of artemisinin in young A. annua seedlings.

Calcium fingerprints induced by Calmodulin interactors in eukaryotic cells

Calcium (Ca 2+) is a ubiquitous second messenger which promotes cell responses through transient changes in intracellular concentrations. The prominent role of Ca 2+ in cell physiology is mediated by a whole set of proteins constituting a Ca 2+-signalling toolkit involved in Ca 2+-signal generation, deciphering and arrest. The different Ca 2+-signalosomes deliver Ca 2+-signals with spatial and temporal dynamics to control the function of specific cell types. Among the intracellular proteins involved in Ca 2+-signal deciphering, calmodulin (CaM) plays a pivotal role in controlling Ca 2+-homeostasis and downstream Ca 2+-based signalling events. Due to its ubiquitous expression in eukaryotic cells and the variety of proteins it interacts with, CaM is central in Ca 2+-signalling networks. For these reasons, it is expected that disrupting or modifying CaM interactions with its target proteins will affect Ca 2+-homeostasis and cellular responses. The resulting calcium response will vary depending on which interactions between CaM and target proteins are altered by the molecules and on the specific Ca 2+-toolkit expressed in a given cell, even in the resting state. In the present paper, the effect of six classical CaM interactors (W5, W7, W12, W13, bifonazole and calmidazolium) was studied on Ca 2+-signalling in tumor initiating cells isolated from human glioblastoma (TG1) and tobacco cells (BY-2) using the fluorescent Ca 2+-sensitive Indo-1 dye and aequorin, respectively. Various Ca 2+-fingerprints were obtained depending both on the CaM interactor used and the cell type investigated. These data demonstrate that interaction between the antagonists and CaM results in a differential inhibition of CaM-dependent proteins involved in Ca 2+-signal regulation. In addition, the distinct Ca 2+-fingerprints in tobacco and human tumor initiating glioblastoma cells induced by a given CaM interactor highlight the specificity of the Ca 2+-signalosome in eukaryotic cells.