A plasma membrane Ca<sup>2+-ATPase (PMCA) from the liver fluke, Fasciola hepatica</em>

Fasciolosis is a parasitic infection by the liver fluke Fasciola hepatica, which costs the global agricultural community over US $2 billion per year. Its prevalence is rising due to factors such as climate change and drug resistance. ATP-dependent membrane transporters are considered good potential drug targets as they are essential for cellular processes and are in an exposed, accessible position in the cell. Immunolocalisation studies demonstrated that a plasma membrane calcium ATPase (PMCA) was localised to the parenchymal tissue in F. hepatica. The coding sequence for a F. hepatica PMCA (FhPMCA) has been obtained. This sequence encodes a 1,163 amino acid protein which contains motifs which are commonly conserved in PMCAs. Molecular modelling predicted that the protein has 10 transmembrane segments which include a potential calcium ion binding site and phosphorylation motif. FhPMCA interacts with the calmodulin-like protein FhCaM1, but not the related proteins FhCaM2 or FhCaM3, in a calcium-ion dependent manner. This interaction occurs through a region in the C-terminal region of FhPMCA which most likely adopts an a-helical conformation. When FhPMCA was heterologously expressed in a budding yeast strain deleted for its PMCA (Pmc1p), it restored viability. Microsomes prepared from these yeast cells had calcium ion stimulated ATPase activity which was inhibited by the known PMCA inhibitors, bisphenol and eosin. The potential of FhPMCA as a new drug target is discussed.

INFLUENCE OF CORE POLARIZATION ON THE ELECTRON-AFFINITY OF CA

We have developed a method, based on the use of B-spline basis sets and model potentials, for determining properties of systems with two or three electrons outside a polarizable closed-shell core. It is applied to the calculation of the electron affinity of Ca and the resulting value of 17.7 meV is in excellent agreement with the most recent experiments. It is found that the dielectronic core-valence interaction reduces the electron affinity by 39.5 meV.

Pharmacological profiling of store-operated Ca(2+) entry in retinal arteriolar smooth muscle

Objective: Pharmacological profiling of store-operated Ca(2+) entry (SOCE) and molecular profiling of ORAI and TRPC expression in arterioles. 
Methods: Fura-2 based microfluorimetry was used to assess CPA-induced SOCE in rat retinal arteriolar myocytes. Arteriolar ORAI and TRP transcript expression were screened using RT-PCR. 
Results: SKF96365 and LOE908 blocked SOCE (IC(50) s of 1.2µM and 1.4µM, respectively). Gd(3+) and La(3+) potently inhibited SOCE (IC(50) s of 21nM and 42nM, respectively), but Ni(2+) showed lower potency (IC(50) = 11.6µM). 2-aminoethyldiphenyl borate (2APB) inhibited SOCE (IC(50) = 3.7µM) but enhanced basal influx (>100µM). Verapamil and nifedipine had no effect at concentrations that inhibit L-type Ca(2+) channels, but diltiazem inhibited SOCE by approximately 40% (=0.1µM). RT-PCR demonstrated transcript expression for ORAI 1, 2 and 3, and TRPC1, 3, 4 and 7. Transcripts for TRPV1 and 2, which are activated by 2APB, were also expressed. 
Conclusion: The pharmacological profile of SOCE in retinal arteriolar smooth muscle appears unique when compared to other vascular tissues. This suggests that the molecular mechanisms underlying SOCE can differ, even in closely related tissues. Taken together, the pharmacological and molecular data are most consistent with involvement of TRPC1 in SOCE, although involvement of ORAI or other TRPC channels cannot be excluded. © 2012 John Wiley & Sons Ltd.

Ca(2+) sparks promote myogenic tone in retinal arterioles

Background and Purpose: Ca(2+) imaging reveals subcellular Ca(2+) sparks and global Ca(2+) waves/oscillations in vascular smooth muscle. It is well established that Ca(2+) sparks can relax arteries, but we have previously reported that sparks can summate to generate Ca(2+) waves/oscillations in unpressurized retinal arterioles, leading to constriction. We have extended these studies to test the functional significance of Ca(2+) sparks in the generation of myogenic tone in pressurized arterioles.

Experimental Approach: Isolated retinal arterioles (25-40 μm external diameter) were pressurized to 70 mmHg, leading to active constriction. Ca(2+) signals were imaged from arteriolar smooth muscle in the same vessels using Fluo4 and confocal laser microscopy.

Key Results: Tone development was associated with an increased frequency of Ca(2+) sparks and oscillations. Vasomotion was observed in 40% of arterioles and was associated with synchronization of Ca(2+) oscillations, quantifiable as an increased cross-correlation coefficient. Inhibition of Ca(2+) sparks with ryanodine, tetracaine, cyclopiazonic acid or nimodipine, or following removal of extracellular Ca(2+) , resulted in arteriolar relaxation. Cyclopiazonic acid-induced dilatation was associated with decreased Ca(2+) sparks and oscillations but with a sustained rise in the mean global cytoplasmic [Ca(2+) ] ([Ca(2+) ]c ), as measured using Fura2 and microfluorimetry.

Conclusions and Implications: This study provides direct evidence that Ca(2+) sparks can play an excitatory role in pressurized arterioles, promoting myogenic tone. This contrasts with the generally accepted model in which sparks promote relaxation of vascular smooth muscle. Changes in vessel tone in the presence of cyclopiazonic acid correlated more closely with changes in spark and oscillation frequency than global [Ca(2+) ]c , underlining the importance of frequency-modulated signalling in vascular smooth muscle.

Essential and toxic elements in infant foods from Spain, UK, China and USA

Spanish gluten-free rice, cereals with gluten, and pureed baby foods were analysed for essential macro-elements (Ca and Na), essential trace elements (Fe, Cu, Zn, Mn, Se, Cr, Co and Ni) and non-essential trace elements (As, Pb, Cd and Hg) using ICP-MS and AAS. Baby cereals were an excellent source of most of the essential elements (Ca, Fe, Cu, Mn and Zn). Sodium content was high in pureed foods to improve their flavour; fish products were also rich in Se. USA pure baby rice samples had the highest contents of all studied essential elements, showing a different nutrient pattern compared to those of other countries. Mineral fortification was not always properly stated in the labelling of infant foods. Complementary infant foods may also contain significant amounts of contaminants. The contents of Hg and Cd were low enough to guarantee the safety of these infant foods. However, it will be necessary to identify the source and reduce the levels of Pb, Cr and As in Spanish foods. Pure baby rice samples contained too much: Pb in Spain; As in UK; As, Cr and Ni in USA; and Cr and Cd in China.

Knowledge Formation and the Great Divergence between China and Europe: Manuscripts and Printed Books, ca. 581–1840

Literature dealing with the history of Chinese printed books and printing is voluminous. Yet studies of how knowledge in general and utilitarian forms of knowledge in particular were generated, accumulated and circulated by printed books and their relationship with the long-term socio-economic transformation of China are rare. This paper aims to open up the subject by examining the long-term trends in the production of manuscripts and books and focusing on the connections between the generation and dissemination of useful knowledge in China and the production and circulation of printed books over the centuries and dynasties from circa 581 to 1840 compared to Europe. It connects trends in this indicator for knowledge formation and diffusion to economic growth, urbanization, changes in higher forms of education, the rise of literacy, the development of printing technologies, and changes in perceptions of the natural world. It concludes that human capital formation in China probably proceeded at a slower rate,which is relevant for narratives of the “divergence” between China and Europe.

Switching between molecular switch types by module rearrangement:Ca -enabled, H-driven 'off-on-off', H-driven YES and PASS 0 as well as H, Ca-driven AND logic operations

Several logic gates and switches can be accessed from two different combinations of a single set of fluorophore, receptor and spacer components.

Electron excitation of Ca XVII

Electron-excitation collision strengths have been calculated for transitions between the ten lowest levels of Ca XVII (2sS, 2s2p P, 2s2p P, 2pP 2p D, 2pS ). At high impact energies, where all the channels are open, the calculation was carried out in the LS-coupling approximation by means of the R-matrix method. Transitions between the fine structure levels were then determined by application of a unitary transformation to the LS-coupled K-matrices. At low impact energies, where some of the channels may be closed, an extension of the R-matrix method was employed to take account of relativistic effects directly in the scattering equations. In general, results are in good agreement with recent distorted-wave calculations. Electron-excitation rates are given for a range of electron temperatures.

Turbulent Fluctuations in G-band and K-line Intensities Observed with the Rapid Oscillations in the Solar Atmosphere (ROSA) Instrument

Using the Rapid Oscillation in the Solar Atmosphere (ROSA) instrument at the Dunn Solar Telescope we have found that the spectra of fluctuations of the G-band (cadence 1.05 s) and Ca II K-line (cadence 4.2 s) intensities show correlated fluctuations above white noise out to frequencies beyond 300 mHz and up to 70 mHz, respectively. The noise-corrected G-band spectrum presents a scaling range (Ultra High Frequency “UHF”) for f = 25-100 mHz, with an exponent consistent with the presence of turbulent motions. The UHF power, is concentrated at the locations of magnetic bright points in the intergranular lanes, it is highly intermittent in time and characterized by a positive kurtosis κ. Combining values of G-band and K-line intensities, the UHF power, and κ, reveals two distinct “states” of the internetwork solar atmosphere. State 1, with κ ≍ 6, which includes almost all the data, is characterized by low intensities and low UHF power. State 2, with κ ≍ 3, including a very small fraction of the data, is characterized by high intensities and high UHF power. Superposed epoch analysis shows that for State 1, the K-line intensity presents 3.5 min chromospheric oscillations with maxima occurring 21 s after G-band intensity maxima implying a 150-210 km effective height difference. For State 2, the G-band and K-line intensity maxima are simultaneous, suggesting that in the highly magnetized environment sites of G-band and K-line emission may be spatially close together. Analysis of observations obtained with Hinode/SOT confirm a scaling range in the G-band spectrum up to 53 mHz also consistent with turbulent motions as well as the identification of two distinct states in terms of the H-line intensity and G-band power as functions of G-band intensity.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (β-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected β-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead β-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and β-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Novel role of androgens in mitochondrial fission and apoptosis

Androgen and androgen receptors (AR) play critical roles in the proliferation of prostate cancer through transcriptional regulation of target genes. Here, we found that androgens upregulated the expression of dynamin-related protein 1 (Drp1), which is involved in the induction of mitochondrial fission, a common event in mitosis and apoptosis. Clinical tissue samples and various prostate cancer cell lines revealed a positive correlation between Drp1 and AR levels. Treatment of androgen-sensitive cells with an AR agonist, R1881, and antagonist, bicalutamide, showed that Drp1 is transcriptionally regulated by androgens, as confirmed by an AR ChIP-seq assay. Live imaging experiments using pAcGFP1-Mito stably transfected LNCaP (mito-green) cells revealed that androgen did not induce significant mitochondrial fission by itself, although Drp1 was upregulated. However, when treated with CGP37157 (CGP), an inhibitor of mitochondrial Ca��⁺ efflux, these cells exhibited mitochondrial fission, which was further enhanced by pretreatment with R1881, suggesting that androgen-induced Drp1 expression facilitated CGP-induced mitochondrial fission. This enhanced mitochondrial fission was correlated with increased apoptosis. Transfection with dominant-negative (DN-Drp1, K38A) rescued cells from increased apoptosis, confirming the role of androgen-induced Drp1 in the observed apoptosis with combination treatment. Furthermore, we found that CGP reduced the expression of Mfn1, a protein that promotes mitochondrial fusion, a process which opposes fission. We suggest that androgen-increased Drp1 enhanced mitochondrial fission leading to apoptosis. The present study shows a novel role for androgens in the regulation of mitochondrial morphology that could potentially be utilized in prostate cancer therapy.