Photolytic Release Research Articles

Gait Abnormalities and Aberrant D2 Receptor Expression and Signaling in Mice Carrying the Human Pathogenic Mutation DRD2I212F.

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. That allelic variant D2-I212F is a constitutively active and G protein-biased receptor. We now describe mice engineered using CRISPR-Cas9-mediated gene editing technology to carry the D2-I212F variant. Drd2I212F mice exhibited gait abnormalities resembling those in other mouse models of chorea and/or dystonia and had striatal D2 receptor expression that was decreased approximately 30% per Drd2I212F allele. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons and striatum from Drd2I212F mice, caused by G protein activation of potassium channels, exhibited slow kinetics (e.g., approximately four- to sixfold slower decay) compared with Drd2 +/+ mice. Current decay initiated by photolytic release of the D2 antagonist sulpiride from CyHQ-sulpiride was also ∼fourfold slower in midbrain slices from Drd2I212F mice than Drd2 +/+ mice. Furthermore, in contrast to Drd2 +/+ mice, in which dopamine is several-fold more potent at neurons in the nucleus accumbens than in the dorsal striatum, reflecting activation of Gα o versus Gα i, dopamine had similar potencies in those two brain regions of Drd2I212F mice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of Drd2 +/+ mice, had no effect on dopamine potency in Drd2 I212F mice. The results demonstrate the pathogenicity of the D2-I212F mutation and the utility of this mouse model for investigating the role of pathogenic DRD2 variants in early-onset hyperkinetic movement disorders. SIGNIFICANCE STATEMENT: The first dopamine receptor mutation to cause a movement disorder, D2-I212F, was recently identified. The mutation makes receptor activation of G protein-mediated signaling more efficient. To confirm the pathogenesis of D2-I212F, this study reports that mice carrying this mutation have gait abnormalities consistent with the clinical phenotype. The mutation also profoundly alters D2 receptor expression and function in vivo. This mouse model will be useful for further characterization of the mutant receptor and for evaluation of potential therapeutic drugs.

Constructing ultra-stable photothermal plastics assisted by carbon dots with photocaged reactivity

Constructing ultra-stable photothermal plastics assisted by carbon dots with photocaged reactivity

Inactivation of Competitive Decay Channels Leads to Enhanced Coumarin Photochemistry.

In the development of photolabile protecting groups, it is of high interest to selectively modify photochemical properties with structural changes as simple as possible. In this work, knowledge of fluorophore optimization was adopted and used to design new coumarin‐ based photocages. Photolysis efficiency was selectively modulated by inactivating competitive decay channels, such as twisted intramolecular charge transfer (TICT) or hydrogen‐bonding, and the photolytic release of the neurotransmitter serotonin was demonstrated. Structural modifications inspired by the fluorophore ATTO 390 led to a significant increase in the uncaging cross section that can be further improved by the simple addition of a double bond. Ultrafast transient absorption spectroscopy gave insights into the underlying solvent‐dependent photophysical dynamics. The chromophores presented here are excellently suited as new photocages in the visible wavelength range due to their simple synthesis and their superior photochemical properties.

A novel variant of the human dopamine D2 receptor, associated with a hyperkinetic movement disorder, mediates enhanced activation of G proteins

A novel DRD2 variant with a mutation in exon 5 of DRD2(c.634A>T; p.I212F)has been identified in a Dutch family. Ile212 is at the N-terminus of the D2 receptor (D2R) 3rd cytoplasmic loop. D2-I212F is associated with a novel hyperkinetic movement disorder characterized by progressive chorea and cervical dystonia. We hypothesized that determining the effect of the mutation on D2R function might explain how D2-I212F causes the movement disorder. Molecular dynamics (MD) simulations of human D2R models suggested that the presence of Phe212 in D2R separates the side chains of Arg132, at the cytoplasmic end of the 3rd transmembrane domain (TM3), and Glu368, at the cytoplasmic end of TM6, spontaneously breaking the ionic lock in the inactive D2-I212F. This ionic lock contributes to maintaining unliganded receptors in an inactive conformation, and it is broken in agonist-activated receptors. Conversely, preventing formation of the lock frequently creates a constitutively active receptor. In vitro and ex vivo studies supported the hypothesis that D2-I212F is a constitutively active receptor: We expressed D2L/S-I212F in HEK293 cells and assayed activation of Gαi1 and GαoA by bioluminescence resonance energy transfer. We observed increased quinpirole potency to activate Gαi1 by D2L/S-I212F (2.8 or 3.5 nM for D2L or D2S, respectively) compared to D2L/S-WT (19 or 27 nM) whereas quinpirole potency for activating GαoA was not substantially changed by the I212F mutation. On the other hand, D2L/S-I212F exhibited increased basal activation of both Gαi/o proteins compared to D2L/S-WT (set as 0%); basal GαoA activation by D2L/S-I212F (43 or 57% of maximal stimulation, respectively) was significantly higher than D2L/S-I212F-mediated basal Gαi1 activation (25% and 35%, respectively). We then used AAV to express Cre recombinase-regulated D2S-WT or D2S-I212F in dopamine neurons of D2 autoreceptor knockout mice and characterized D2R activation of G protein-regulated inward-rectifying potassium channels (GIRKs). When CyHQ-sulpiride (5 μM) was circulated over a midbrain slice, photolytic release of sulpiride produced a small inhibition of a tonic GIRK current in cells expressing D2-WT (-9 pA), and a much larger inhibition in cells expressing D2-I212F (-62 pA). The enhanced inhibition of tonic current by the mutant receptor could reflect constitutive activation of G proteins or a heightened sensitivity of the mutant to dopamine. To distinguish between these possibilities, midbrain slices were treated with reserpine to deplete endogenous dopamine. This treatment abolished the response to sulpiride in cells expressing D2-WT and greatly decreased the response in cells expressing D2-I212F, indicating that most of the tonic current was due to endogenous dopamine to which D2-I212F may be more sensitive, but that D2-I212F also displayed some constitutive activity in the presumed absence of dopamine. Thus, our in silico, in vitro, and ex vivo studies all indicate that the mutation enhances constitutive and agonist-induced activation of G proteins by D2R

Photolytic Release of a Caged Inhibitor of an Endogenous Transcription Factor Enables Optochemical Control of CREB-Mediated Gene Expression

A direct optochemical method for regulating gene function has been developed based on uncaging of an inactive caged precursor that fragments to produce a CREB (cAMP-response element binding protein) inhibitor that binds to an endogenous transcription factor responsible for regulating CREB-mediated gene expression levels.

Probing Single Synapses via the Photolytic Release of Neurotransmitters.

The development of two-photon microscopy has revolutionized our understanding of how synapses are formed and how they transform synaptic inputs in dendritic spines—tiny protrusions that cover the dendrites of pyramidal neurons that receive most excitatory synaptic information in the brain. These discoveries have led us to better comprehend the neuronal computations that take place at the level of dendritic spines as well as within neuronal circuits with unprecedented resolution. Here, we describe a method that uses a two-photon (2P) microscope and 2P uncaging of caged neurotransmitters for the activation of single and multiple spines in the dendrites of cortical pyramidal neurons. In addition, we propose a cost-effective description of the components necessary for the construction of a one laser source-2P microscope capable of nearly simultaneous 2P uncaging of neurotransmitters and 2P calcium imaging of the activated spines and nearby dendrites. We provide a brief overview on how the use of these techniques have helped researchers in the last 15 years unravel the function of spines in: (a) information processing; (b) storage; and (c) integration of excitatory synaptic inputs.

Controlled light-induced gas phase nitric oxide release from S-nitrosothiol-doped silicone rubber films

The light induced nitric oxide (NO) release properties of S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) NO donors doped within polydimethylsiloxane (PDMS) films (PDMS-SNAP and PDMS-GSNO respectively) for potential inhaled NO (iNO) applications is examined. To achieve photolytic release of gas phase NO from the PDMS-SNAP and PDMS-GSNO films, narrow-band LED light sources are employed and the NO concentration in a N2 sweep gas above the film is monitored with an electrochemical NO sensor. The NO release kinetics using LED sources with different nominal wavelengths and optical power densities are reported. The effect of the NO donor loading within the PDMS films is also examined. The NO release levels can be controlled by the LED triggered release from the NO donor-doped silicone rubber films in order to generate therapeutic levels in a sweep gas for suitable durations potentially useful for iNO therapy. Hence this work may lay the groundwork for future development of a highly portable iNO system for treatment of patients with pulmonary hypertension, hypoxemia, and cystic fibrosis.

Photolytic release of bioactive carboxylic acids from fused pyran conjugates

New ester cages bearing the coumarin (2H-benzopyran-2-one) skeleton with extended π-systems as phototriggers, for glycine and β-alanine, as models of carboxylic acid bifunctional molecules with biological relevance, were evaluated under photolysis conditions at 254, 300, 350 and 419 nm of irradiation in a RPR-100 photochemical reactor. The processes were followed by HPLC-UV detection and 1H NMR with collection of kinetic data. The results showed a correlation between the photolysis efficiency and the increasing extension of the conjugation for both glycine and β-alanine, showing that the 7-aminocoumarin afforded the best results at all wavelengths tested.From a study of the time-resolved fluorescence behaviour, these compounds were also found to exhibit more complex fluorescence decay kinetics. This was attributed to the presence of conjugated and non-conjugated coumarin species.

Solvent-Controlled Chemoselectivity in the Photolytic Release of Hydroxamic Acids and Carboxamides from Solid Support.

The synthetic utility and theoretical basis of a photolabile hydroxylamine-linker are presented. The developed protocols enable the efficient synthesis and chemoselective photolytic release of either hydroxamates or carboxamides from solid support. The bidetachable mode of the linker unit is uniquely dependent on the solvent. Hydroxamic acids are obtained by performing photolysis in protic solvents, whereas photolysis in aprotic solvents enables the selective release of carboxamides.

A spectroscopic study of the excited state proton transfer processes of (8-bromo-7-hydroxyquinolin-2-yl)methyl-protected phenol in aqueous solutions.

A combination of spectroscopic methods and density functional theory (DFT) computations was used to study the excited state proton transfer (ESPT) processes of (8-bromo-7-hydroxyquinolin-2-yl)methyl-protected phenol (BHQ-OPh). Characterization of the prototropic forms of BHQ-OPh in different solvent environments revealed that the neutral form predominates in acetonitrile and in 1 : 1 acetonitrile/water (pH 5.0), whereas the anionic form predominates in 1 : 1 acetonitrile/PBS (pH 7.4). Both the neutral and anionic forms were significantly populated in 1 : 1 acetonitrile/water. Upon irradiation in acetonitrile the triplet neutral form was observed, whereas the triplet anionic form was detected in 1 : 1 acetonitrile/PBS (pH 7.4). The existence of the triplet tautomeric form of BHQ-OPh in both 1 : 1 acetonitrile/water and 1 : 1 acetonitrile/water (pH 5.0), and the ESPT processes from the neutral to the anionic to the tautomeric forms in the excited state were observed using time-resolved spectroscopy. A reaction mechanism in 1 : 1 acetonitrile/water and 1 : 1 acetonitrile/water (pH 5.0) was proposed based on the spectroscopic and DFT computational results. A comparison of the results for BHQ-OPh with those of BHQ-OAc reveals that the initial prototropic states and photochemical processes are similar. The understanding gained of the initial photo-induced processes of BHQ-based photoremovable protecting groups (PPGs) is useful for the design of new quinolinyl-based PPGs for specialized applications.

Optogenetic Regulation of Tunable Gene Expression in Yeast Using Photo-Labile Caged Methionine.

Light-mediated gene expression enables the noninvasive regulation of cellular functions. Apart from their classical application of regulating single cells with high spatiotemporal resolution, we highlight the potential of light-mediated gene expression for biotechnological issues. Here, we demonstrate the first light-mediated gene regulation in Saccharomyces cerevisiae using the repressible pMET17 promoter and the photolabile NVOC methionine that releases methionine upon irradiation with UVA light. In this system, the expression can be repressed upon irradiation and is reactivated due to consumption of methionine. The photolytic release allows precise control over the methionine concentration and therefore over the repression duration. Using this light regulation mechanism, we were able to apply an in-house constructed 48-well cultivation system which allows parallelized and automated irradiation programs as well as online detection of fluorescence and growth. This system enables screening of multiple combinations of several repression/derepression intervals to realize complex expression programs (e.g., a stepwise increase of temporally constant expression levels, linear expression rates with variable slopes, and accurate control over the expression induction, although we used a repressible promoter.) Thus, we were able to control all general parameters of a gene expression experiment precisely, namely start, pause, and stop at desired time points, as well as the ongoing expression rate. Furthermore, we gained detailed insights into single-cell expression dynamics with spatiotemporal resolution by applying microfluidics cultivation technology combined with fluorescence time-lapse microscopy.

Functional Indicators of Glutamate Transport in Single Striatal Astrocytes and the Influence of Kir4.1 in Normal and Huntington Mice.

This study evaluates single-cell indicators of glutamate transport in sulforhodamine 101-positive astrocytes of Q175 mice, a knock-in model of Huntington's disease (HD). Transport-related fluorescent ratio signals obtained with sodium-binding benzofuran isophtalate (SBFI) AM from unperturbed or voltage-clamped astrocytes and respective glutamate transporter currents (GTCs) were induced by photolytic or synaptic glutamate release and isolated pharmacologically. The HD-induced deficit ranged from -27% (GTC maximum at -100 mV in Ba(2+)) to -41% (sodium transients in astrocytes after loading SBFI-AM). Our specific aim was to clarify the mechanism(s) by which Kir4.1 channels can influence glutamate transport, as determined by either Na(+) imaging or transport-associated electrical signals. A decrease of Kir4.1 conductance was mimicked with Ba(2+) (200 μm), and an increase of Kir4.1 expression was obtained by intravenous administration of AAV9-gfaABC1D-Kir4.1-EGFP. The decrease of Kir4.1 conductance reduced the sodium transients but increased the amplitudes of somatic GTCs. Accordingly, after genetic upregulation of Kir4.1, somatic GTCs were found to be decreased. In individual cells, there was a negative correlation between Kir4.1 currents and GTCs. The relative effect of the Kir4.1 conductance was higher in the astrocyte periphery. These and other results suggest that the Kir4.1 conductance affects glutamate transporter activity in a dual manner: (1) by providing the driving force (voltage dependency of the transport itself) and (2) by limiting the lateral charge transfer (thereby reducing the interference with other electrogenic transporter functions). This leads to the testable prediction that restoring the high conductance state of passive astrocytes will not only normalize glutamate uptake but also restore other astrocytic transporter activities afflicted with HD. Insufficiency of astrocytic glutamate uptake is a major element in the pathophysiology of neurodegenerative diseases. Considering the heterogeneity of astrocytes and their differential susceptibility to therapeutic interventions, it becomes necessary to evaluate the determinants of transport activity in individual astroglial cells. We have examined intracellular Na(+) transients and glutamate transporter currents as the most telling indicators of glutamate clearance after synaptic or photolytic release of glutamate in striatal slices. The results show that, in Huntington's disease, glutamate uptake activity critically depends on Kir4.1. These channels enable the high conductance state of the astrocytic plasma membrane, which ensures the driving force for glutamate transport and dumps the transport-associated depolarization along the astrocyte processes. This has significant implications for developing therapeutic targets.

Synthesis and Characterization of the Novel Nitric Oxide (NO) Donating Compound, S-nitroso-N-acetyl-D-penicillamine Derivatized Cyclam (SNAP-Cyclam).

Nitric oxide (NO) has been heavily studied over the past two decades due to its multitude of physiological functions and its potential therapeutic promise. Of major interest is the desire to fabricate or coat implanted devices with an NO releasing material that will impart the appropriate dose and duration of NO release to positively mediate the biological response to the medical device, thereby improving its safety and efficacy. To date, this goal has not yet been achieved, despite very promising early research. Herein, we describe the synthesis and NO release properties of a novel NO donor which covalently links the S-nitrosothiol, S-nitroso-N-acetyl-D-penicillamine (SNAP), to the macrocycle, cyclam (SNAP-cyclam). This compound can then be blended into a wide variety of polymer matrices, imparting NO release to the polymer system. This release can be initiated and controlled by transition metal catalysis, thermal degradation or photolytic release of NO from the composite NO-releasing material. SNAP-cyclam is capable of releasing physiologically relevant levels of NO for up to 3 months in vitro when blended into poly(l-lactic acid) thin films.

No evidence for altered intracellular calcium-handling in airway smooth muscle cells from human subjects with asthma.

BackgroundAsthma is characterized by airway hyper-responsiveness and variable airflow obstruction, in part as a consequence of hyper-contractile airway smooth muscle, which persists in primary cell culture. One potential mechanism for this hyper-contractility is abnormal intracellular Ca2+ handling.MethodsWe sought to compare intracellular Ca2+ handling in airway smooth muscle cells from subjects with asthma compared to non-asthmatic controls by measuring: i) bradykinin-stimulated changes in inositol 1,4,5-trisphosphate (IP3) accumulation and intracellular Ca2+ concentration, ii) sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression, iii) mechanisms of cytoplasmic Ca2+ clearance assessed following instantaneous flash photolytic release of Ca2+ into the cytoplasm.ResultsWe found no differences in airway smooth muscle cell basal intracellular Ca2+ concentrations, bradykinin-stimulated IP3 accumulation or intracellular Ca2+ responses. Quantification of SERCA2 mRNA or protein expression levels revealed no differences in ASM cells obtained from subjects with asthma compared to non-asthmatic controls. We did not identify differences in intracellular calcium kinetics assessed by flash photolysis and calcium uncaging independent of agonist-activation with or without SERCA inhibition. However, we did observe some correlations in subjects with asthma between lung function and the different cellular measurements of intracellular Ca2+ handling, with poorer lung function related to increased rate of recovery following flash photolytic elevation of cytoplasmic Ca2+ concentration.ConclusionsTaken together, the experimental results reported in this study do not demonstrate major fundamental differences in Ca2+ handling between airway smooth muscle cells from non-asthmatic and asthmatic subjects. Therefore, increased contraction of airway smooth muscle cells derived from asthmatic subjects cannot be fully explained by altered Ca2+ homeostasis.

Evidence of calcium-permeable AMPA receptors in dendritic spines of CA1 pyramidal neurons.

GluA2-lacking, calcium-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) have unique properties, but their presence at excitatory synapses in pyramidal cells is controversial. We have tested certain predictions of the model that such receptors are present in CA1 cells and show here that the polyamine spermine, but not philanthotoxin, causes use-dependent inhibition of synaptically evoked excitatory responses in stratum radiatum, but not s. oriens, in cultured and acute hippocampal slices. Stimulation of single dendritic spines by photolytic release of caged glutamate induced an N-methyl-d-aspartate receptor-independent, use- and spermine-sensitive calcium influx only at apical spines in cultured slices. Bath application of glutamate also triggered a spermine-sensitive influx of cobalt into CA1 cell dendrites in s. radiatum. Responses of single apical, but not basal, spines to photostimulation displayed prominent paired-pulse facilitation (PPF) consistent with use-dependent relief of cytoplasmic polyamine block. Responses at apical dendrites were diminished, and PPF was increased, by spermine. Intracellular application of pep2m, which inhibits recycling of GluA2-containing AMPARs, reduced apical spine responses and increased PPF. We conclude that some calcium-permeable, polyamine-sensitive AMPARs, perhaps lacking GluA2 subunits, are present at synapses on apical dendrites of CA1 pyramidal cells, which may allow distinct forms of synaptic plasticity and computation at different sets of excitatory inputs.

Ip3 Receptor Mediated Ca Release in Atrial Cells from Normal and Failing Hearts

We studied excitation-contraction coupling (ECC) and inositol-1,4,5-triphosphate (IP3)-dependent Ca release in atrial cells from normal and heart failure (HF) rabbit hearts. Elementary Ca release events (Ca puffs) from IP3 receptors (IP3Rs) were detected in permeabilized myocytes after inhibition of ryanodine-receptor mediated Ca spark activity with tetracaine. Puff activity was induced by IP3 exposure and blocked by 2-APB, identifying their origin from IP3Rs. In normal myocytes puff activity was low and restricted to the cell periphery, whereas in HF myocytes their overall frequency was increased and puffs were more prominent in the cell center. Compared to normal cells, in HF atrial myocytes diastolic [Ca]i was increased, action potential (AP)-induced Ca transients (CaTs) were larger in amplitude (primarily due to enhanced Ca release from central non-junctional sarcoplasmic reticulum, SR), propagation of activation from cell periphery to the center was accelerated, and fractional SR Ca release was increased, whereas no difference in SR Ca load was observed. These differences were largely abolished by exposure to 2-APB, suggesting an increased basal IP3 activity in HF. Angiotensin II (AngII) increased diastolic [Ca]i in normal and HF myocytes whereas the effect was more pronounced in HF. The amplitude of AP-dependent CaTs was increased in the presence of AngII or after photolytical release of caged IP3 in normal cells, whereas the opposite effect was observed in HF. The frequency of spontaneous Ca waves was increased in HF, and returned to levels comparable to normal myocytes after 2-APB exposure. In conclusion, HF atrial myocytes revealed enhanced Ca release during ecc and more frequent spontaneous Ca waves, presumably resulting from increased basal IP3R-mediated Ca release. However, additional neurohumoral stimulation with AngII, as it might occur in-vivo in cardiac disease, negatively affected atrial Ca signaling during ECC.

Use of Creatine Kinase To Induce Multistep Reactions in Infrared Spectroscopic Experiments

An extension of current approaches to trigger enzymatic reactions in reaction-induced infrared difference spectroscopy experiments is described. A common procedure is to add a compound that induces a reaction in the protein of interest. To be able to induce multistep reactions, we explored here the use of creatine kinase (CK) for the study of phosphate transfer mechanisms. The enzymatic reaction of CK could be followed using bands at 1614 and 979 cm(-1) for creatine phosphate consumption, at 944 cm(-1) for ADP consumption, and at 1243, 992, and 917 cm(-1) for ATP formation. The potential of CK to induce multistep reactions in infrared spectroscopic experiments was demonstrated using the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) as the protein of interest. ADP binding to the ATPase was triggered by photolytic release of ADP from P(3)-1-(2-nitro)phenylethyl ADP (caged ADP). CK added in small amounts converted the released ADP to ATP on the time scale of minutes. This phosphorylated the ATPase and led to the formation of the first phosphoenzyme intermediate Ca2E1P. Thus a difference spectrum could be obtained that reflected the reaction from the ADP ATPase complex to the first phosphoenzyme intermediate. Comparison with a phosphorylation spectrum obtained when the initial state was the ATP ATPase complex revealed the contribution of ATP's γ-phosphate to the conformational change of the ATPase upon nucleotide binding: γ-phosphate binding modifies the structure of a β-sheet, likely in the phosphorylation domain, and shifts its spectral position from ~1640 to ~1630 cm(-1). Upon phosphorylation of the ATPase, the β-sheet relaxes back to a structure that is intermediate between that adopted in the ADP bound state and that in the ATP bound state.

‘Eventless’ InsP3‐dependent SR‐Ca2+ release affecting atrial Ca2+ sparks

Augmented inositol 1,4,5-trisphosphate receptor (InsP3R) function has been linked to a variety of cardiac pathologies, including cardiac arrhythmia. The contribution of inositol 1,4,5-trisphosphate-induced Ca(2+) release (IP3ICR) in excitation-contraction coupling (ECC) under physiological conditions, as well as under cellular remodelling, remains controversial. Here we test the hypothesis that local IP3ICR directly affects ryanodine receptor (RyR) function and subsequent Ca(2+)-induced Ca(2+) release in atrial myocytes. IP3ICR was evoked by UV-flash photolysis of caged InsP3 under whole-cell configuration of the voltage-clamp technique in atrial myocytes isolated from C57/BL6 mice. Photolytic release of InsP3 was accompanied by a significant increase in the Ca(2+) release event frequency (4.14 ± 0.72 vs. 6.20 ± 0.76 events (100 μm)(-1) s(-1)). These individual photolytically triggered Ca(2+) release events were identified as Ca(2+) sparks, which originated from RyR openings. This was verified by Ca(2+) spark analysis and pharmacological separation between RyR and InsP3R-dependent sarcoplasmic reticulum (SR)-Ca(2+) release (2-aminoethoxydiphenyl borate, xestospongin C, tetracaine). Significant SR-Ca(2+) flux but eventless SR-Ca(2+) release through InsP3R were characterized using SR-Ca(2+) leak/SR-Ca(2+) load measurements. These results strongly support the idea that IP3ICR can effectively modulate RyR openings and Ca(2+) spark probability. We conclude that eventless and highly efficient InsP3-dependent SR-Ca(2+) flux is the main mechanism of functional cross-talk between InsP3Rs and RyRs, which may be an important factor in the modulation of ECC sensitivity.

Acidocalcisomes of Trypanosoma brucei have an inositol 1,4,5-trisphosphate receptor that is required for growth and infectivity

Acidocalcisomes are acidic calcium stores rich in polyphosphate and found in a diverse range of organisms. The mechanism of Ca(2+) release from these organelles was unknown. Here we present evidence that Trypanosoma brucei acidocalcisomes possess an inositol 1,4,5-trisphosphate receptor (TbIP(3)R) for Ca(2+) release. Localization studies in cell lines expressing TbIP(3)R in its endogenous locus fused to an epitope tag revealed its partial colocalization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes. IP(3) was able to stimulate Ca(2+) release from a chicken B-lymphocyte cell line in which the genes for all three vertebrate IP(3)Rs have been stably ablated (DT40-3KO) and that were stably expressing TbIP(3)R, providing evidence of its function. IP(3) was also able to release Ca(2+) from permeabilized trypanosomes or isolated acidocalcisomes and photolytic release of IP(3) in intact trypanosomes loaded with Fluo-4 elicited a transient Ca(2+) increase in their cytosol. Ablation of TbIP(3)R by RNA interference caused a significant reduction of IP(3)-mediated Ca(2+) release in trypanosomes and resulted in defects in growth in culture and infectivity in mice. Taken together, the data provide evidence of the presence of a functional IP(3)R as a Ca(2+) release channel in acidocalcisomes of trypanosomes and suggest that a Ca(2+) signaling pathway that involves acidocalcisomes is required for growth and establishment of infection.

Kinetics of thermal and photolytic degradation of decabromodiphenyl ether (BDE 209) in backcoated textile samples

Kinetics of the thermal and photolytic degradation of decabromodiphenyl ether (DBE 209) was studied using HPLC. Samples lost an amount of ∼8.4% (w/w), 24% (w/w), 39.4% (w/w) and 28.5% of the amount of DBE 209 originally present in the samples due to ageing at 25, 60, 90°C and UV exposure, respectively. The thermal and photolytic release was found to follow the first order kinetics with rate constants estimated to be 3.6×10−3, 1.03×10−2, 3.6×10−2 and 3.94×10−2day−1, respectively. Ageing of the textile samples enhanced the release of the DBE 209 from the backcoated textile. Photodegradation of BDE 209 into lower congeners of brominated flame retardants was also observed for the UV-aged samples.Migration of DBE 209 from the backcoated textile into biological fluids was studied using Head-over-Heels and contact-Blotting test for unaged, thermally and UV aged samples. The presence of biological fluids (sweat, saliva and juice) was found to enhance the migration of DBE 209 compared to water. Migration of BDE 209 into artificial biological fluids is significantly increased for samples previously exposed to UV radiation or thermally aged. An increase from 0.6% (w/w) to a maximum of 2% (w/w) of the amount of BDE 209 migrated into artificial biological fluids due to ageing conditions in the presence of biological fluid was recorded.