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Loss of the RNA-binding protein FMRP (Fragile X Mental Retardation Protein) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na+-activated K+ channels (KNa), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were co-localized at the periphery of isolated BC neurons and the two proteins could be reciprocally co-immunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly-activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to the inside-out patches containing native Aplysia Slack channels increased channel opening and, in current clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that in BC neurons Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel-FMRP interactions may link changes in neuronal firing to changes in protein translation.
The FMR1 gene encodes the Fragile X Mental Retardation protein (FMRP), an RNA-binding protein (Willemsen et al., 2003) that is expressed in many tissues and at high levels in neurons (Devys et al., 1993; Fahling et al., 2009). FMRP binds a subset of mRNAs and regulates their translation at synapses in response to activation of Group1 metabotropic glutamate receptors and other stimuli (Bassell and Warren, 2008). Loss of FMRP leads to altered synaptic function and loss of protein synthesis-dependent plasticity (Bassell and Warren, 2008; Li et al., 2009). In humans, absence of FMRP causes Fragile X Syndrome (FXS) the most common form of inherited intellectual disability (Willemsen et al., 2003; Bear et al., 2004; Bagni and Greenough, 2005). FMRP interacts with a range of mRNA targets and a variety of intracellular protein partners. Recent studies suggest that one of these FMRP-interacting proteins is the Na+-activated K+ channel (KNa) Slack (Brown et al., 2010). Biochemical experiments indicate that FMRP interacts with the cytoplasmic C-terminal domain of Slack. Direct application of FMRP to the cytoplasmic face of excised patches containing Slack channels enhances channel activity. Whether the protein-protein interactions of FMRP with ion channels directly influence the excitability of neurons is, however, unknown.
A model system for investigation of changes in neuronal excitability is that of the BC (bag cell) neurons of the marine mollusc, Aplysia californica. These neurons control a series of reproductive behavior culminating in egg laying. Although these peptidergic neurons display no spontaneous firing, they undergo an ~30 minute afterdischarge of actions potentials in response to brief synaptic stimulation, triggering the release of several neuroactive peptides (Conn and Kaczmarek, 1989). The electrical characteristics of BC neurons, including their resting potential, input resistance and action potential height and width have been shown to be potently modulated by several signaling pathways including the cyclic-AMP-dependent protein kinase (Kaczmarek et al., 1980), protein kinase C (White et al., 1998) and tyrosine kinases/phosphatases(Wilson and Kaczmarek, 1993)
KNa channels are widely expressed and have been recorded in neurons of a variety of species where they contribute to the afterhyperpolarization that follows one or more evoked action potentials (Bader et al., 1985; Wallen et al., 2007; Nanou et al., 2008). KNa channels generally have a large unitary conductance with various degrees of voltage dependency and are gated by increases in Na+ and Cl− (Joiner et al., 1998; Bhattacharjee et al., 2003; Yuan et al., 2003; Bhattacharjee and Kaczmarek, 2005; Santi et al., 2006; Chen et al., 2009). In this study, we have characterized the native KNa channels in Aplysia BC neurons and provide evidence that they are encoded by the Slack gene. We have also demonstrated that Slack exists in a protein-protein complex with FMRP in these neurons, and that, as predicted by mammalian studies, intracellular injection of FMRP acutely increases KNa current and alters their firing response to depolarizing current pulses. Furthermore, we show that Slack channels are required for a prolonged change in the excitability of BC neurons that requires new protein synthesis.
Adult A. californica weighing 1507#x2013;200 g were obtained from Marine Specimens Unlimited (San Francisco, CA) or Marinus Inc. (Long Beach, CA). Primary cultures of isolated BC neurons were prepared as described previously (White et al., 1998; Magoski et al., 2002), and were maintained in normal artificial sea water (nASW) containing (in mM): 460 NaCl, 10.4 KCl, 11 CaCl2, 55 MgCl2, and 15 HEPES; plus 100 U ml−1 penicillin and 0.1 mg−1 streptomycin; pH 7.8.
We identified an Aplysia homolog of Slack based on a tblastn search of the Aplysia genome trace archive and amplified partial Slack cDNA by PCR from an Aplysia BC neuron cDNA library. PCR products were purified and cloned into the T/A cloning vector pCR2.1 (Invitrogen). The products were sequenced by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale.
Pre-designed silencer select siRNAs (Aplysia Slack, Shab and scrambled siRNA) were purchased from Ambion. For each gene siRNA, we targeted two different sites, and then mixed the two different siRNA products for treatment of neurons. The target sequences were chosen from Aplysia Slack sequence between the start codon and the S6 transmembrane domain: sense, GGUCUAGUCUCAACCUACATT and GGUCAGAUAUCAACUGGUATT; for Aplysia Shab, the target sequences were: sense, GGAUAGUGUUUAUCAAGCATT and GGAUUUUAAUGCGAAGUUUTT. Commercially available scrambled siRNA (Ambion), which has previously been reported as a negative control in Aplysia neurons (Paganoni and Ferreira, 2005; Jordan et al., 2007) was used as the non-targeting sequence. All siRNAs were obtained in annealed and desalted form and were dissolved in siRNA buffer (Ambion). Isolated neurons were microinjected using pipettes containing 1 μM scrambled siRNA, Slack siRNA, Shab siRNA in 200 mM KCl. Intact clusters of neurons were treated with 20 μM Slack siRNA or scrambled siRNA in nASW. Immunocytochemistry, western blotting, single cell electrophysiology and discharge experiments were carried out 4–6 days after onset of siRNA treatment.
Staining of 1–2 day cultured BC neurons was performed on coverslips coated with 1 μg/ml poly(D) lysine, and after fixation with 4% paraformaldehyde in 400 mM sucrose/nASW. Coverslips were washed twice with PBS and blocked with 5% goat serum/PBS before incubation with primary antibodies. The anti-Slack antibody we used for immunostaining (N3/26 NeuroMab produced by UC Davis/NIH NeuroMab Facility; Davis, CA) was raised against an antigen sequence that has 30% identity and 62% similarity compared with Aplysia Slack sequence. Rabbit anti-FMRP antibody was obtained from Abcam (MA). The coverslips were inverted on 100 μl of primary antibody solution (anti-Slack antibody, 1:1000 dilution, anti- FMRP antibody, 5 μg/ml in 5% goat serum/PBS,) and placed in a humidified chamber at 4 °C overnight, washed extensively with PBS, and then incubated for 2 h at room temperature with CY3-conjugated goat anti-mouse IgG and fluoresence-conjugated anti-rabbit IgG secondary antibodies. The coverslips were washed and mounted on glass slides using mounting medium. The stained slides were viewed and photographed using Axiovision software (Zeiss).
In order to test whether anti-FMRP antibodies raised in rabbits recognize Aplysia FMRP, we expressed Aplysia Fmr1 in CHO cells. We subcloned Aplysia Fmr1 (Genbank#AAQ18136) from pNEX3 into the pcDNA3 vector, and then transfected it into CHO cells using lipofectamine. The transiently-transfected cells were grown on glass coverslips to 60–70% confluence and fixed in PBS containing 4% paraformaldehyde for 10 min. After washing with PBS, cells were permeabilized in PBS containing 1% bovine serum albumin and 0.2% Triton X-100 for 10 min. Primary rabbit-anti-FMRP antibody (Abcam, MA; 5 μg/ml in 5% goat serum/PBS) was added to the coverslips and incubated for 1 hour, washed 3 times and then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:500) for 30 mins, washed again, and mounted on glass slides with Citifluor mounting medium (Ted Pella, Inc., Redding, CA) for fluorescence microscopy.
BC neuron clusters were dissected from 150–200 g animals. The clusters were homogenized in 100 μl of homogenization buffer (Zhang et al., 2008b).The samples were then centrifuged at 5,000 × g for 20 min. The supernatants were transferred to 1.5 ml Eppendorf tubes. Tubes were incubated at 4° C with rotation overnight after addition of 1 μg of purified mouse anti-FMRP antibody or rabbit anti-Slack antibody. After adding 30 μl protein A-Sepharose beads (50% (v/v) in Triton X-100 buffer; Sigma), the samples were rotated at 4°C for 2 h. The immunoprecipitates were collected by centrifugation at 3000 × g for 1 min and washed five times in 800 μl of Triton X-100 buffer. After washes, the beads were incubated in sample buffer at room temperature for 30 mins. The samples were applied to SDS-acrylamide gels. For immunoblotting, the SDS gel-electrophoresed proteins were transferred to an immunoblot polyvinylidene difluoride membrane (Bio-Rad). Nonspecific binding was blocked by incubating the blot in Tris-buffered saline Tween 20 (TBST) buffer with 5% milk for 1 h at room temperature. The blots were incubated overnight at 4 °C with one of the following primary antibodies: rabbit anti-Slack antibody (1:500, Biosynthesis Inc., TX); or rabbit anti-Slack antibody preincubated with excess antigen obtained from rat Slack purified protein; or mouse anti-FMRP antibody (1 μg/ml, EMD Millipore, MA); or mouse anti-FMRP antibody preincubated with excess recombinant FMRP antigen (Novus Biologicals, Littleton, CO). Next, the blots were washed four times for 30 min each, followed by application of secondary horseradish peroxidase-linked anti-rabbit or anti mouse antibodies at 1:10,000 dilution for 1 h at room temperature. After three washes of 30 min each in TBST, immunoreactive proteins were visualized with detection reagent (Amersham Biosciences). In parallel experiments, we used anti-rabbit IgG or anti-mouse IgG (The Jackson Laboratory, Bar Harbor, ME) as controls for immunoprecipitation.
Current clamp and voltage clamp recordings were made from BC neurons using an AxonClamp 2B (Axon Instruments, Union City, CA). For current clamp recordings, the single sharp electrode, bridge balance method was used. For voltage clamp analysis, the discontinuous single electrode approach was used, in which the membrane potential is changed using a high frequency train of current pulses and measurements of membrane potential are made immediately prior to each current pulse. The discontinuous single electrode voltage clamp amplifier was run at a switching frequency of 1–2 kHz. To ensure adequate voltage control, the individual pulses were monitored on an oscilloscope and adjusted so that the period of voltage sampling corresponded to at least nine electrode time constants. Microelectrodes were pulled from borosilicate glass capillaries (1.2-mm ID, TW 120F-4; World Precision Instruments, Sarasota, FL) and had tip diameters of < 1 μm with resistances of ~2 MΩ when filled with 3 M KCl. Using a Picospritzer (General Valve, Fairfield, NJ) 100 ms in duration and an injection pressure of 14 psi. Recordings were filtered at 3 kHz using the Axonclamp built-in Bessel filter and sampled at 2 kHz using a Digidata 1200 A/D converter (Axon Instruments) using Clampex software (version 8.2; Axon Instruments).
Recombinant FMRP(1–298) was obtained from Novus Biologicals (Littleton, CO). For intracellular injections of FMRP, the pipette solution contained 530 mM K+ acetate 11 mM Tris-HCl, pH 7.2,, and 100 nM recombinant FMRP(1–298) and the external solution contained (in mM) 460 NaCl, 10.4 KCl, 11 CaCl2, 55 MgCl2, and 15 Hepes; pH 7.8, with modifications as described in the text.
BC neurons were cultured in nASW. Electrodes had a resistance of 5–10 MΩ for single channel recordings. Excised inside-out patch recordings were performed using symmetrical K solutions: 470 mM KCl, 66 mM MgCl2, 15 mM HEPES, 2 mM EGTA. The cytoplasmic face of the membrane was perfused with bath solution supplemented with Na+ to 0, 120, or 200 mM free Na+. Data were acquired with an EPC-7 amplifier (HEKA Electronics, Mahone Bay, Nova Scotia, Canada), a Digidata 1200 analog-to-digital (A/D) converter (Axon Instruments, Foster City, CA), and the clampex acquisition program of pClamp (version 8.2; Axon Instruments). Current was sampled at 10 kHz and filtered at 1 kHz with a Bessel filter (Frequency Devices, Haverhill, MS). Data were gathered in 1–3 min intervals while holding the patch at +30 mV.
Abdominal ganglia were placed in a recording chamber at 14°C. A wide-bore, fire-polished glass suction electrode placed at the distal end of one connective, and a recording suction electrode was placed at the rostral end of the corresponding bag cell neuron cluster (Kaczmarek et al., 1978). Current pulse trains (20 V pulses of 2.5 msec duration at 6 Hz for 10–20 sec) were delivered with a Grass S88 stimulator and isolation unit, and voltage was recorded using a Warner DP-301 differential amplifier. Within 15 min of the end of a first afterdischarge, a similar pulse train was applied one or two times to ensure that the cells had entered the refractory period. To test the effect of the protein synthesis inhibitor anisomycin on the afterdischarge, 3 μM anisomycin was added to clusters at least 30 min before stimulation.
We identified an Aplysia ortholog of Slack based on a tblastn search of the Aplysia genome trace archive and amplified Slack cDNA by PCR from an Aplysia BC neuron cDNA library. Several Slack isoforms, differing at their 3'-ends, are found in BC neurons. The longest full sequence has a 3.531kb coding region corresponding to six putative membrane-spanning regions (S1–S6) and a long, presumably cytoplasmic, carboxy terminal (Genbank access number is HG413690). This Aplysia Slack shares 46% identity and 72% similarity with human SLACK (Needleman-Wunch method. BLOSUM62). A region of sequence between S6 and the first RCK domain is also highly conserved between rat and human Slack.
Like Slack, FMRP has been conserved throughout evolution. The Aplysia nervous system expresses a single Fmr1 gene (Moroz et al., 2006) that is 74.4% identical to that of the mouse Fmr1 at the genomic level (AlignX, Vector NTI software). FMRP contains two heterogeneous nuclear ribonucleoprotein (hnRNAP) K-homology (KH) domains and one arginine-glycine domain (RGG box) that can mediate protein/RNA interactions (Ramos et al., 2006; Bassell and Warren, 2008; Bechara et al., 2009). The major functional mRNA-binding domains of FMRP are the KH1, KH2, and RGG domains, which are 69, 83, and 83% identical between rat and Aplysia. Moreover, the sequence of the N-terminus of Aplysia FMRP is 58% identical and 76% similar to that of mouse FMRP and 55% identical, 75% similar to that of human FMRP. Antibodies against this conserved N-terminus of Human FMRP specifically recognize a single band on western blots of BC neuron homogenates that is of the expected size for a close ortholog (75kDa, Fig. 1B). Recent work has shown that, in mammalian cells, Slack can form a protein complex with FMRP (Brown et al., 2010). We therefore carried out co-immunoprecipitation experiments to test whether Slack interacts with FMRP in Aplysia neurons. Using extracts from the BC neurons, we found that immunoprecipitates obtained using the mouse anti-FMRP antibody contained a Slack-immunoreactive band with a molecular weight of 90 kDa, a size comparable to that reported in Lamprey (Nanou et al., 2008) (Fig. 1A). The rabbit anti-Slack antibody was generated against the C-terminus of rat Slack (Bhattacharjee et al., 2002) and the Slack-immunoreactive band in Aplysia neurons was completely eliminated when the anti-Slack antibodies were pre-adsorbed with purified rat Slack protein (Fig. 1A). Conversely, a single FMRP-immunoreactive band was detected in immunoprecipitates obtained using the anti-Slack antibodies (Fig. 1B). Again, the FMRP immunoreactive band was eliminated when the anti-FMRP antibodies were pre-adsorbed with human FMRP(1–298). No bands were detected when control IgG was used for immunoprecipitation or when secondary antibody alone was used for immunoblotting (Fig. 1A, B).
We also carried out double immunocytochemistry to examine the subcellular localization of Slack and FMRP in isolated cultured BC neurons (Fig. 1C). In order to test the specificity of the rabbit anti-FMRP antibody we used for immunocytochemistry, we transiently transfected CHO cells with Aplysia Fmr1. On subsequent immunostaining of the transfected cells, strong staining of the transfected cells was seen using the anti-FMRP antibody, but no staining was detected in untransfected CHO cells (Data not shown). In BC neurons, immunoreactivity for FMRP was diffusely distributed throughout the cells including the somata and neurites, whereas Slack immunoreactivity was highly enriched at the periphery of the neurons, consistent with localization to the plasma membrane. Merged images demonstrate that, at the light level, Slack and FMRP immunoreactivity is co-localized at the periphery of the cells and within the neurites. Overlap appeared strongest at the plasma membrane of the somata, perhaps because the ratio of FMRP to Slack is reduced in the neurites. No staining was detected when secondary antibodies alone were used (Fig. 1C).
To test whether KNa channels are present in the plasma membrane of BC neurons, we first recorded outward currents from isolated cultured neurons using the sharp single electrode voltage-clamp technique. Intracellular injections of Na+ were made using pulses of pressure (14 psi, 1–2 pulses, 100 msec duration) applied to the intracellular electrodes which contained either 0.5M Na+ acetate, NaCl or Na2SO4. We found that intracellular injections of Na+ produced a significant increase in the outward current (Fig. 2A, B, C, n = 6, p< 0.05). This increased current does not depend on the co-injection anion. In contrast, intracellular injections of KCl, using the same injection parameters, produced no significant change in outward currents (Fig. 2A, B, n = 3).
Further evidence for the presence of Na+-activated K+ channels was obtained using excised inside-out patches from cultured BC neurons, using symmetrical 470 mM KCl recording solutions with no Ca2+ ions at the cytoplasmic face. Under these conditions, it was possible to readily identify these channels based on their large unitary conductance, voltage dependence of opening (an increase in P0 with depolarization) and sensitivity to Na+ ions. Little channel activity was detected at + 30 mV in the absence of Na+ (Fig. 2D, E). When the Na+ concentration at the cytoplasmic face of the patch was raised to 120 mM, channel activity increased markedly, and further elevation of the Na+ concentration to 200 mM produced a further increase in open probability from 0.05 ± 0.01 (0 Na+), to 0.37 ± 0.02 (120 mM Na+), to 0.52 ± 0.02 (200 mM Na+). Subconductance states could sometimes also be observed in the single channel recordings, and, in amplitude histograms produced a slight asymmetry in peaks for the closed state (Fig. 2E). When the amplitude of the KNa channels was plotted as a function of voltage, the unitary conductance was found to be ~167 pS (Fig. 2F, n = 3).
To confirm the correspondence between Slack channels and the KNa currents in BC neurons, we tested the effects of decreasing the synthesis of native KNa channels by microinjecting double-strand siRNA against Aplysia Slack into isolated cultured BC neurons. This method has been shown to inhibit gene expression specifically in various organisms including Aplysia (Lee et al., 2001). By comparing the level of immunostaining for Slack in isolated neurons injected with the Slack siRNA with that in control neurons from the same animal, we confirmed that levels of the Slack channel in BC neurons is decreased after Slack expression is knocked down using this approach (Fig. 3A). The diffuse localization of FMRP throughout somata and neurites did not appear to be altered by Slack siRNA (Fig. 3A). Intact ganglia containing clusters of BC neurons were treated with Slack siRNA or scrambled siRNA (Korneev et al., 2002). After 5–6 days, Slack protein was greatly reduced in Slack siRNA-treated cells compared to scrambled siRNA-treated cells, as determined by Western blotting (Fig. 3B, C).
We then tested the effects of the siRNA on the electrophysiological characteristics of BC neurons. As a positive control, we used siRNA against the Aplysia voltage-dependent Shab (Kv2.1) K+ channel, which is also expressed in BC neurons but which has very different characteristics from those of Slack (Quattrocki et al., 1994; Zhang et al., 2008a). As a negative control, we used a scrambled siRNA. To test the effects of each of these siRNA reagents, BC neurons isolated from the same animal were divided into two groups. In the first group, 1 μM siRNA was injected into each neuron, while the second control group was not injected. Three days after injection of the siRNA, outward currents were recorded from both groups of cells. After normalization for capacitance, the averaged current of all the siRNA-injected cells was subtracted from the mean current in control cells. This subtracted trace corresponds to the mean change in outward currents produced by siRNA injection.
The subtracted trace for treatment with Slack siRNA revealed a biphasic outward current, with a rapid (50–100 msec) transient component that was followed by a more slowly activating component (Fig. 3D, E, n = 8). Injections of control scrambled siRNA produced no significant difference current in the subtracted traces (Fig. 3F, n = 4). Treatment with Shab siRNA resulted in an outward difference current that activated within ~50 msec and then partially inactivated over the remainder of a 600 msec command pulse (Fig. 3G, n = 3). This slowly inactivating current very closely matches that reported for the Shab current in expression systems and in native BC neurons (Quattrocki et al., 1994).
Unlike the subtracted Slack siRNA trace, Slack currents in heterologous expression systems activate slowly with no transient component at the onset of a depolarizing pulse (Yang et al., 2006). To test the possibility that this transient component in native BC neurons reflects a transient enhanced activation of Slack channels by Na+ entry through voltage-dependent Na+ channels at the onset of the depolarizing commands, we repeated the experiments using Slack siRNA, but recorded all currents in the presence of tetrodotoxin (TTX) at a concentration (100 μM) known to block voltage-dependent Na+ channels in this species (Knox et al., 1996; Hung and Magoski, 2007). Under these conditions the transient component was suppressed but the slowly activating component was still present (Fig. 3H, n = 6, siRNA + TTX treated; n = 3, control).While we cannot be confident that voltage-control was adequate to clamp transient Na+ currents themselves, our findings are consisted with the hypothesis that the slower transient component of K+ current reflects activation of KNa channels by Na+ entry at the onset of voltage commands.
We also tested the effects of Slack siRNA treatment on the mean resting potential of BC neurons recorded in the current clamp mode. Reduction of Slack expression was found to produce a depolarization (mean resting potential of Slack siRNA-treated neurons = −38.2 ± 1.8 mV (n = 9) vs −44.7 ± 0.8 mV (n = 14) in controls, p < 0.002), suggesting that KNa currents contribute to the resting conductance.
We tested the effects of recombinant FMRP that contains amino acids 1–298 (FMRP(1–298)) on macroscopic currents and on the electrical properties of isolated BC neurons. This truncated FMRP contains the N-terminal domain (NDF; aa1–134) of FMRP that has been shown to serve as a platform for the majority of known protein-protein interactions of FMRP with other cellular signaling components, but lacks the C-terminal RNA-binding region (Jin et al., 2004; Darnell et al., 2005; Ramos et al., 2006). We found that injecting FMRP(1–298) into isolated BC neurons under voltage-clamp produces an increase in net outward current. The increase in current began 1–5 minutes after injection and reached a maximum at about 20 mins. Like the subtracted Slack siRNA current, the difference currents obtained by subtracting the pre-injection currents from those in the same cell after injection of FMRP(1–298) were biphasic with an early transient component lasting 50–100 msec followed by a more slowly activating component (Fig. 4A, G, n = 4). The FMRP(1–298)-induced currents progressively increased with test potentials up to ~ +30 mV after which they declined with further depolarization (Yuan et al., 2003; Yang et al., 2006) (Fig. 4F). Injection of heat-inactivated FMRP(1–298) into the neurons maintained in a normal Na+-containing external medium had no effect on outward currents (Fig. 4B, G, n = 4)
The effects of FMRP(1–298) on outward current were dependent on Na+ ions. As with the Slack-component of current in the siRNA treated cells, the early transient component of current induced by FMRP(1–298) was eliminated if the cells were pretreated with sodium channel blocker TTX (100 μM) (Fig. 4C). The effects of FMRP(1–298) on the slow component of outward current were also dependent on Na+. When external Na+ was replaced by N-Methyl-D-Glucamine (NMDG), the total outward current was reduced (Fig. 4D). Moreover, injection of FMRP(1–298) into neurons in this Na+-free external medium was found to have no effect on either the amplitude or kinetic behavior of outward currents (Fig. 4E, G, n = 4). Overall, these findings are consistent with an acute enhancement of native Slack KNa currents by injection of FMRP(1–298).
We then tested whether, as has been demonstrated in Xenopus oocytes expressing rat Slack channels, the FMRP protein acutely enhances the activity of native KNa channels in neurons. We excised inside-out patches containing KNa channels from isolated BC neurons and applied recombinant FMRP(1–298) (100 nM) to the cytoplasmic face of the patches. This produced a marked increase in channel opening, raising the channel open probability from 0.19 ± 0.09 to 0.30 ± 0.11 (Fig. 5A, B, C, n = 4). As has also been described for Slack channels in heterologous expression systems (Brown et al., 2010), application of FMRP suppressed the occurrence of subconductance states (Fig. 5B).
To test the acute effects of FMRP on intrinsic electrical properties, isolated neurons were recorded in the current-clamp mode using single microelectrodes. Intracellular microinjection of FMRP(1–298) was found to decrease the input resistance and hyperpolarize the resting membrane potential (Fig. 5D, E, n = 4). In addition, the width of action potentials evoked by depolarizing currents was reduced within ~2 minutes of injection. Because a change in resting potential can in itself produce changes in action potential width, we introduced a bias current to adjust the membrane potential of each neuron to its pre-injection value. Under these conditions the mean reduction in width measured at 50% spike amplitude was 4 ± 1 ms (n = 4). As with the voltage-clamp experiments, injection of heat-inactivated FMRP(1–298) had no effect on the neurons in the current-clamp mode (Fig. 5D, E, n = 4).
In response to brief stimulation of their synaptic input, BC neurons in the abdominal ganglion of the mollusk Aplysia generate a ~30 minute discharge of action potentials, which is followed by a prolonged inhibited period during which further stimulation of the input either fails to trigger an afterdischarge or triggers discharges of reduced duration (Kupfermann and Kandel, 1970; Kauer and Kaczmarek, 1985). In vivo, this sequence of changes in excitability serves to trigger a series of reproductive behaviors culminating in egg-laying and to limit their frequency (Kupfermann and Kandel, 1970). Recovery from the inhibited period occurs gradually over ~18 hrs, such that stimulation one day after evoking a first discharge (Day 2) triggers discharges that are of the same duration as those on Day 1(Fig 6A, C, n = 14).
We tested the effects of suppression of Slack expression on stimulus-evoked discharges in BC neurons and on the recovery from the prolonged inhibited period. Treatment of bag cell clusters in abdominal ganglia for 5 days with Slack siRNA had no effect on the duration of a first discharge or on the subsequent entry of the neurons into the prolonged inhibited period (Fig. 6B, C, n = 6). Slack siRNA-treated BC neurons failed, however, to recover from the inhibited period (Fig. 6B, C, n = 6). In contrast, BC clusters treated with the scrambled siRNA for 6 days generated full length discharge both on day 5 and on day 6, indicating full recovery from the inhibited state induced on day 5 (Fig. 6C, n = 6).
Previous work has shown that stimulation of BC neurons triggers an increase in synthesis of some proteins, and that this is dependent on the presence of extracellular Na+ ions (Wayne et al., 2004). We therefore tested whether recovery from the inhibited period requires new protein synthesis. As described previously (Montarolo et al., 1986), we found that pre-treatment of clusters of BC neurons with the protein synthesis inhibitor anisomycin (3 μM) inhibited incorporation of S35-methionine into new proteins by 95.4 ± 0.04 % (n = 4). Anisomycin had no effect on the duration of a first discharge (Fig. 6D, n = 15) or on the subsequent inhibited state. One day later, however, stimulation could only trigger action potentials and short discharges that are identical to those in the inhibited state. This was not a non-specific effect of protein synthesis inhibition because pre-incubation of BC neurons for 24 hrs in anisomycin without stimulation had no effect on the ability of stimulation to trigger discharges (Fig. 6D, n = 10).
Our results indicate that the interaction between Slack KNa channels and the mRNA-binding protein FMRP, previously documented in the nervous system of mice (Brown et al., 2010) is an evolutionarily conserved one. Like their mammalian counterparts, Slack channels in Aplysia have a large unitary conductance, and, upon depolarization, are activated in a biphasic manner. Rapid activation by Na+ entry through voltage-dependent Na+ channels is followed by a slower phase of activation as the depolarization is maintained. It is likely that both persistent Na+ currents and other cation channels contribute to this sustained phase (Lu et al., 2010; Hage and Salkoff, 2012). It is also possible that some activation of KNa channels by voltage steps occurs even at resting levels of internal Na+ ions, which would change only slowly on removal of external Na+.
The KNa current in BC neurons is enhanced by introduction of FMRP both in excised patches and in intact cells, leading to a hyperpolarization of the resting potential and a narrowing of action potentials. In mammalian cells, application of FMRP to KNa channels in patches from Slack-transfected cells enhances channel opening in part by suppressing the occurrence of subconductance states (Brown et al., 2010). We also observed the same effect for KNa channels in BC neurons. Nevertheless, subconductance states appeared less prominent than in transfected cells, suggesting that some binding of endogenous FMRP may occur in the patches excised from BC neurons. Regulation of the amplitude of KNa currents by FMRP is likely to be a dynamic one, subject to changes in factors such as the phosphorylation state of Slack or FMRP, which may change rapidly with neuronal stimulation.
It seems unlikely that the interaction between Slack and FMRP serves purely as a mechanism to regulate KNa current amplitude. A variety of recent studies have suggested that the gating of ion channels, triggered by depolarization or neuronal activity, may instigate cytoplasmic signaling cascades that could alter gene transcription or other biochemical events (An et al., 2000; Gomez-Ospina et al., 2006; Hegle et al., 2006; Kaczmarek, 2006). Thus, it is possible that activation of Slack channels by Na+ elevations during neuronal firing alters the ability of FMRP to participate in one or more of its functions, such as regulation of translation of its cargo mRNAs, although there is at present no direct evidence for this. FMRP is known to be required for activity-dependent increases in protein translation, most likely by suppressing translation of its cargo mRNAs until neurons become active (Li et al., 2009; Schutt et al., 2009).
A "non-conducting" function of this sort for Slack channels is consistent with the finding that suppression of Slack channels prevents recovery of BC neurons from a prolonged inhibited state that normally follow the characteristic ~30 minute discharge of these cells and that this recovery requires the synthesis of new proteins. Thus Slack channels could comprise part of a pathway that links neuronal firing to activity-driven protein synthesis. The strongest activation of KNa channels occurs during periods of repetitive neuronal activity, during which time intracellular Na+ levels are elevated (Yang et al., 2007; Nanou et al., 2008). Moreover is has been found that activity- dependent synthesis of a neuropeptide precursor during the discharge of BC neurons is abolished by transient removal of external Na+ ions at the onset of the discharge (Wayne et al., 2004).
While the hypothesis that Slack channel activity modulates the ability of FMRP to liberate mRNAs for translation is an attractive one, our present findings do not eliminate a variety of alternative hypotheses. For example, although suppression of Slack channels does not prevent discharges or entry into the inhibited period, changes in action potentials or resting membrane potential produced by decreased Slack levels could indirectly alter intracellular events such as calcium transients. These could, in turn, alter cell metabolism or rates of protein synthesis even if Slack channels function solely to regulate the intrinsic electrical properties of neurons.
Our findings have demonstrated that the interaction of Slack with FMRP is one that is conserved across species. In addition, they raise the possibility that it serves to link changes in neuronal firing to changes in the translation of FMRP-associated mRNAs. In addition to the recovery from the inhibited period of BC neurons, a variety of other long-lasting changes in the excitability of neurons and in their ability to release neurotransmitter require new protein synthesis (Wang et al., 2009; Jin et al., 2011). Many such changes underlie learning, memory and alterations in animal behavior. Definitive tests of the role of Slack channels in such events will require identification of the proteins whose synthesis is required for these prolonged changes in excitability.
This work was supported by the National Institutes of Health grants: NS18492 and HD067517 (L.K.K.)
No conflict of interest
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's Canada Institute for Scientific and Technical Information in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
This project was supported by the Deutsche Forschungsgemeinschaft (DFG) and the Fonds der Chemischen Industrie (FCI). We thank Olaf Ackermann for assistance with HPLC. We thank Prof. Thorsten Bach and his group for helpful discussions and generous support.
Catalyzed Tandem Reaction of 3-Silyloxy-1,5-enynes Consisting of Cyclization and Pinacol Rearrangement†
Article first published online: 20 FEB 2007
DOI: 10.1002/anie.200604544
Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Angewandte Chemie International Edition
Volume 46, Issue 13, pages 2310–2313, March 19, 2007
Additional Information
How to Cite
Kirsch, Stefan F., Binder, Jörg T., Crone, B., Duschek, A., Haug, Timm T., Liébert, C. and Menz, H. (2007), Catalyzed Tandem Reaction of 3-Silyloxy-1,5-enynes Consisting of Cyclization and Pinacol Rearrangement. Angew. Chem. Int. Ed., 46: 2310–2313. doi: 10.1002/anie.200604544
- †
Publication History
- Issue published online: 9 MAR 2007
- Article first published online: 20 FEB 2007
- Manuscript Received: 6 NOV 2006
Funded by
- Deutsche Forschungsgemeinschaft (DFG)
- Fonds der Chemischen Industrie (FCI)
- cyclization;
- domino reactions;
- gold;
- homogeneous catalysis;
- rearrangement
Au-spicious! Under silver-free conditions, simple 3-silyloxy-1,5-enynes were converted into complex cyclopentenes by a gold(I)-catalyzed sequence that likely proceeds through a carbocyclization followed by a pinacol rearrangement. For the final demetalation step, isopropyl alcohol and N-iodosuccinimide are effectively utilized, and the resulting products are set up for a wealth of further reactions.

subset: commoncrawl


text:
706 Tasting Notes
So I still have a couple of these and had brought one to work at one point in case of pu-erh emergency. I decided to make this in my perfect tea mug with a western gongfu hybrid. After my attempt to brew the Chenpi Ripe Tangering pu-erh Western style the other day, I thought I'd better try a rinse. I got the water to a boil then did a ten second rinse. It smelled very earthy and dark (and a little fishy) after this so I did another ten second rinse. Still smells earthy, dark and strong but not offensive. The tuo pretty much collapsed at this point and my infuser is fall of very finely ground leaves, like coffee grinds (I see I had that experience with this the first time as well).
After all this, I gave it a 30 second steep which yields an aroma of earth, barn and mushrooms. Maybe damp hay. Not offensive, just strong. Like a farm, but not unpleasant. I swear! It's not that it smells like manure, but it does smell like a wooden structure that's lived in by animals. I think people with a rural sensibility might understand that. Or I'm crazy. :)
So indtead of writing the rest of this note, I accidentally talked with a co-worker for a while. Whoops. Impressions from the past are: it wasn't very strong in flavour, despite the depth of colour and aroma. I was probably a little skimpy on time given the two rinses BUT leaving it like this kept it fairly fresh and allowed me to sip along mindlessly which was actually perfect in the circumstance. No strong notes really came to the fore, so my assessment of this now (with more pu under my belt) is that it is good for a casual puerh when I want something rich but am not in the mood for black tea. It's a beginner puerh, which makes sense given that it's a mini tuocha with tiny leaves. I'm gonna go make more now…
Haha I'm a city person mostly, but I totally get the barn smell. It's like clean animals and hay and fresh straw and outdoors and all those different scents that combine to make that barn smell.
How many tuochas did you use for your mug? A twelve ounce mug really needs 2 tuochas. You might get better results.
Allan, I've had particular tea before with a stronger taste and it was not so pleasant. Surprisingly, this followed the recommendations of Teavivre (at least from two years ago when I first received it). I personally wouldn't want two.
Wooooow. Just looked at Teavivre's new recommendations for this tea Western style:
Teapot 8oz Tea:2p 5-8 min 100ºC/212ºF
That would be beyond undrinkable for me. I might try it as an experiment someday as I have been surprised before. But wow.
Sample 4 outof 5 from Teavivre. Thanks! I am spending the day at home catching up on life. Between my two jobs, I have had only a few days off this month and I am rarely home to enjoy them. Evenings are equally busy with visiting family and so on. Today is the last official day of summer and after a massive torrential rain overnight I am enjoying some sun and warm temperatures. It's just after noon and I've already made two loaves of bread and a batch of english muffins which will be gratefully enjoyed throughout the week when I have no time to make dough.
So, here I sit with a cup of this. I was pleasantly surprised to see massive chunks of tangerine peel in with the puerh. I used half the sample in my perfect tea mug for a few minutes with water that (apparently) cooled to 96 before pouring. The result is a deep brown like a stout or dark ale. The aroma is musty and barnlike, but not in the usual pleasant way. It's overpowering. Shoot. I haven't made puerh Western style in a while…shoulda done some sort of a rinse first. I'm going try drinking this but if it's too strong, I can always pour it out and try again with a re-steep. D'oh.
Huh. This has a totally different taste profile than the aroma suggests. I get smoke. No barn or hay. Maybe something musty if that ties into the smoke. It's strong with a sense of bitterness like a coffee edge but not unpleasant. This is a very potent mug. VERY strong. I am tempted to add a bit of milk to dilute it because I'm not super interesting in drinking it as is. Which sucks, because this is my fault and this is my first tea of the day. I will not rate this currently because I need to try again under better circumstances.
All I can say for sure right now is that it is very bold and strong with a smoky edge. No sweetness. Whew.
Flavors: Musty, Smoke, Wood
Third of five recent Teavivre samples, and I am saving the unusual ones for last. The beau and I recently drank the regular Silver Needle and I am interested to see if I get nuch difference in the organic. I am going Western again and following the instructions with 75 degree water and the full pouch in the teapot. Around four minutes steeping a light yellow liquor with some serious melon and cucumber aromas.
First sips come in light, a bit melony and sweet. I recently ate some seasoned crackers and the remains of those are overpowering the tea. This is a very light tea, not one that holds up well to this particular pairing. I think this should be enjoyed on its own so you can get the full sense of light sweet hay and honeydew/cucumber flavours. No bitterness or astringency at all, I like this more than the regular Silver Needle from last week. This stands out more and has a bit of a floral note that I appreciate. I think I still preferred the white peony but this hits the spot right now.
I can't rule out steeping parameters as a factor in my results but this stand on its own much better than it's non-organic counterpart. A very nice light white that is easy to sip exactly as is.
EDIT: second steep at boiling for a minute is really nice. Similar flavours but even more bold. Nice to sip while I sit in the sun before going to work at the library. We were up later than usual checking out the Northern lights. Amazing!
Flavors: Cucumber, Floral, Hay, Honeydew, Melon
This is quite possibly my first "Random Steepings" entry. Tada! The beau and I had Chinese food "in town" tonight and I was curious about the tea. The waitress said there was a loose leaf oolong and we asked for a pot of that. It came loose but was brewing up really dark so we investigated. Turned out, this is definitely shu puerh. She had no more info about it but we told her a bit about puerh and how she might like to brew it if she wants to try it again. It was actually really nice and held up to Western steeping really well. This encouraged me to try more of our pu Western style. Yay!
Also excuse the (many?) Typos. I'm on my phone and Steepster won't let me see what I am typing so I am just hoping it turns out legible…now it's letting me. Grr. That was pretty bad. I think I got most of them.
First thoughts: Wow, those are big! The leaves are uniformly whole, about 1.5 inches long.
I got some of this in my recent package of samples from Teavivre and now that Steepster seems less broken, I'm finally reviewing it. I don't often drink white tea but when I do I tend to enjoy it. I steeped this for about two minutes in 75 degree water per the instructions on the sample. I used the whole packet to make a pot for the beau and I since he is a big fan of white teas.
At two minutes western style, this is light tea. Very light. Maybe too light. I poured the tea back into the pot for another minute steeping – and made a mess. Whoops. Then I forgot it, and it went two more minutes. So, a four minute steep it is. The water was only 75 when poured though, so I'm not worried about over-steeping.
Now, we have a lot more flavour. It is still a light-tea but full-bodied and sweet with some notes of hay and melon or cucumber. No bitterness, astringency or dryness. Light but yummy. Sort of unassuming. Another tea that I like when I drink but don't always think to go for. The beau proclaims it "really good." He gets a bit of jasmine (which is likely floral in general). He had a sip of scotch before trying it though, so his impressions are now confused.
I don't think we will have any problem enjoying these samples and I am interested to compare it to the Organic Silver Needle we also received. I think I will try a second steep of this later at higher temps as well to see what we get.
It's been a little while since I've had the white peony, but so far I would say I liked that a little better. It had a little more oomph for me and stood out a bit more. I think it also had different brewing parameters though which can really change the outcome.
Flavors: Cucumber, Melon, Sweet
This is a bit of a revelation. I still have half a pound of this from my hasty mega-purchase a few years ago. I constantly give it away, but it reproduces like gremlins.
Knowing its tendency to become a bit bitter and dry, I steeped it quick (about 2.5 minutes) and added a small splash of milk and a bit of honey. This brings it back to sweet, creamy, vanilla. Not my favourite and I definitely regret buying so much but this will make it really easy to drink some more at work. No re-steeping, dear heaven.
Flavors: Cream, Vanilla
I find it makes a difference. Sometimes you need a bit more leaf to make up for the loss in time. Another option is to use water that has cooled for a minute or so. I think it is the decaf tea that lends the unpleasant bitter/dry aspect and these tricks help defeat it.
Sooo, I am finally trying this – the first of my Mandala sampler. This is my first Mandala experience, but not my first puerh. I already know I prefer Shu, so this is a great opportunity. Dry, the leaf smells sweet and reminds me (as usual) of sweet hay, damp earth, wood and sweet clean animals.
I followed the instructions provided with the teas which suggested the whole serving for one session or half that for a lighter brew (or in my case for a small quantity of liquid). I gave a 15 second rinse with boiling water, then 30 second steep with boiling again. The result is not bitter or astringent, though it isn't as sweet as expected. It has a bit of dryness and some general earthiness but not a lot else. It's definitely earthy and mushroom-y. The beau gets a fishy smell but I don't. He says "it's really strong. It's not bad though."
2: 45 seconds, boiling. The leaves are still pretty clumped up, and the liquor is quite dark with an earthy, mushroom aroma. The flavour matches the first, but maybe a bit less dry. Strong, rich, earthy.
3: 1 minutes, boiling. Might as well mention that due to my lack of a sharing pitcher, I am brewing in my small gaiwan, and pouring into the larger one then pouring into our cups with a dribble at the end for my teapet. It's a messy but equitable system. This steep smells very strong. Like an earthy greenhouse or barn. As always, this isn't negative. Still no bitterness, but a bit dry. No sweetness, which I had in other shu. I think this is maybe the result of the small leaf?It's bold, fo shu! (See what I did there?) The beau says the fish is gone (I never got that!) and that it is more earthy. "Best steep so far."
4: 1 minute 15 seconds, boiling. I can't imagine how strong this would be with the full sample. I definitely made the right choice for my brewing setup to split this in two. I was looking at other notes and saw some comparisons to coffee which seem accurate but it isn't unpleasant and bitter like coffee. More an explanation of the depth and richness. This steep gets some sweetness finally. I like it. Much the same, but better. The beau says "now this tastes like a typical pu-erh, the strong taste is gone." We agree on that for once.
5: 1 minutes 30 seconds, boiling. Whoa sweetness. Something fruity. Appley? This is different. I like it. Best so far. Reading other notes again – man, did my experience contradict EVERYONE else? Oh well!
6: 1 minute 45 seconds, boiling. The problem with this steeping method is that the gaiwan burns my fingers with boiling water, but the water is cool enough to drink by the time it is poured in my cup. I could go through a lot of steeps very quickly if it weren't for the need to get water boiling again regularly. Taste is similar to 5. Sweet, lighter, a sense of apple or maybe apple peel. I don't know why I say that, it isn't strong enough to really explain, but it comes to mind anyway.
From here on, I'm going to just steep and enjoy without reporting. This can get exhausting. I need a bigger sample so I can try it gongfu, and Western-ish as well. For now, this was nice and different from other shu I have tried. I don't think I need a cake of it, as it doesn't hit my hot spots (currently). Really nice to experience though. Great education!
Flavors: Apple Skins, Earth
I have this tea and have brewed it. I think you would do better with shorter steeps, no more than 15 sec initially.
Well, I haven't had this in years and I expected my opinion to be different as my tastes have changed so much, but I actually still like it. My youngest brother got it for my while in Toronto, – he got me a couple teas he wanted to try. Smart cookie. I forgot I had had this one and my initial impression was favourable. I love jasmine, the lemon scent was strong and the oolong seemed subtle. All good for me.
Steeped, those impressions held true. I didn't notice the oolong underneath the lemon, but it was definitely there since it tasted a bit creamier and smoother than lemon myrtle on its own. The jasmine was present underneath the lemon, but very pleasant. Youngest brother tried it hot and said it just tasted like water. At this point, he decided he wanted it cold and stronger. I put the tea in a travel mug and told him to refrigerate overnight and try today. I don't know if that helped him out but I do remember that people new to tea tend to like it much stronger than I do. I thought this was a great balance, where he found it plain. Just goes to show how subjective tastes are!
Flavors: Jasmine, Lemon
Ah this is one of my all time favourites. I find everything about it pretty much perfectly balanced. It's so interesting to me, when people love something I find really mediocre, or dislike something I really love, not just because we like different things, but because the experience of that tea is so different that if you didn't know better you'd never think we were drinking the same thing.
My husband also drinks tea and I enjoy comparing notes with him because we rarely have the same impressions even when sharing a pot – or a cup. It's really interesting and has me questioning how unique our sense experiences are. I often wondered if everyone else saw colours the same as I do, this is just an extension of that, I guess!
I used to kind of just assume that people experienced sensory stuff in the same way I do. I never really thought about it, but figured it was all pretty similar.
Which is particularly funny, because I experience synsthesia, so there's some sensory stuff that I experience in a way that doesn't even have shared context with other people – but I kind of obliviously didn't realize this for years, until I clued into the fact that people looked at me weird when I described tasting colours.
As a child, my eyes went poorly very quickly. It never occurred to me that I couldn't see, I just assumed that it was like that for everyone and was copying school notes off friends rather than the board, etc. After nearly a year of this, someone caught on and I was sent for an eye test. The results were so bad that I was sent for another one because the adults didn't believe me. Luckily it didn't end in anything more than a strong prescription for glasses and eyes that continually (but now gradually) deteriorate BUT I think it set me further on the path of realizing how unique our experiences are and how difficult it is to ever know that people see/hear/feel/smell/experience things the same way. It led to me especially wondering about sight but I eventually started questioning everything. I was also a huge reader and quite imaginative, but I think the dramatic sight change had an impact.
Synesthesia has long fascinated me and I am sometimes disappointed that I don't have any synesthetic associations, that I know of. I used to associate colours with numbers but I am fairly convinced that was a result of paint-by-numbers always using the same code! After reading more about it, I am a little relieved that my brain isn't firing that much that often, but it's still fascinating.
It's really different for every person who experiences it.
Mine is pretty random and I don't experience it all the time or with all things. There are certain things that taste like colours (colours don't have a taste, colours are a flavour), certain sounds or music produce colours, flashes of light or patterns, and certain smells have a temperature separate from their usual association. I also have other crossed wires which I tend to forget about till I'm experiencing them.
So some examples, parsnips and lime jello taste green, but spinach or green tea doesn't. Dutch licorice rooibos tastes a very specific shade of grey-blue, kind of cornflowerish, but other licorice or blueberries don't.
Part of ATB's 9pm is a glowing deep blue. ATB's Don't Stop is a bright, sunny yellow. Foster the People's Pumped up Kicks is pale blue on top and brown on the bottom with a kind of wavy dividing line, like an abstract landscape. Some of the Swans' music is a pretty wild experience with a lot of visuals going on. Most music is just music.
Cucumbers smell cool, but some artificial cucumber fragrances that are otherwise dead on smell hot. Cinnamon, mint, pepper etc have no temperature.
It's all pretty innocuous and generally pretty neat. I like experiencing it.
Some of my friends have a totally different synesthetic experience. One of them experiences colours as tastes in her mouth, which can be great or disgusting.
You know what Steepster? This tea is going on four years old and I still have 50 grams left. You know what else? It's still really good. I get grapes, I get raisin, I get a bit of natural sweetness and a hint of white tea. I don't get bitterness or astringency. I don't get bubblegum. I don't get anything overly artificial. This was a really well balanced white tea blend that Davids put out for Christmas nearly four years ago and I really liked it. I still like it. I wish they would return to these less flavoured blends that hold up to a bit of abuse. I am so happy with this right now. Trying out whites and green oolongs lately has inspired me to broaden my tea horizons, on occasion at least. Usually the beau takes care of everything that isn't a black tea in our house but I've been on a sampling role lately and I will be keeping this one at work with me for the times I want a flavoured or light tea.
By the way, the grapiness of this is like purple grapes I find. No tart, not aggressive, not fake or candy-like. Really nice. A white base was the way to go. Way better than the newer grape tea they did a while ago.
Flavors: Grapes, Raisins, Sweet
I've opted to do a follow-up note on this tea as I am steeping it Western style and am curious how it will hold up. I am also happy to report that this samples doesn't have any of the crushed leaves or powder that I encountered in my earlier sample. I feel badly for suggesting that it is a broken leaf tea when I apparently experienced an anomaly.
As per Teavivres Western instructions, I am steeping the whole pouch in my Perfect Tea mug for one minute in boiling water. I've got to admit that despite my innate specticism about some of these steeping parameters, Teavivre is REALLY good about making them accurate to the specific tea. I tend to brew per my own general guidelines but following the exact directions for each tea yields a different (and sometimes better) result for the Teavivre teas. The dedication to optimal brewing per tea and per batch is remarkable, and much appreciated – even from a brewing sinner.
After this minute in the boiling bath, I have a boldly aromatic tea with hints of cinnamon, spice, hay. I can smell it from across my desk, it has a note that suggests a potential bitterness, but is generally appealing. It also maybe smells a bit roasty, and perhaps a bit floral.
First sips reflect the flavour quite well. Some sweet baking spice flavour, some roastiness, no bitterness or astringency. It reminds me of the Taiwan Oriental Beauty I sampled the other day. Very nice!
EDIT: Second steep: As usual, I got distracted with work and this went 2 or possibly closer to 3 minutes with boiling water. This results in some astringency, a very dry tongue with the start of bitterness. I don't like that, but I know it is my fault so I shan't try a third. If I can get past the self-inflicted astringency, it tastes much like the first. Bakey and roasty with a bit of sweet and spice. Like snickerdoodles or cinnamon buns.


text:
Despicable Me 2010 PG
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Gibbon intro guitar pro by This Town Needs Guns, added: December 14th, 2012
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+ - Calif. scientists push plan to 'evaporate' asteroids->
Submitted by netbuzz
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You have a headache again. What are you going to do? Probably the same as always, head for the medicine cabinet, take a pain pill, it only partially works. Okay then let's turn out the lights and lie down.
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3. Apples
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Everyone loves apples, so this is a pleasant way to relieve those bothersome headaches.
4. Tea
It does not matter what kind of tea you like. Whether you like herbal, Chai, white, green, or black they all are helpful. To get the most benefit from your tea make it hot and add a lemon slice or two.
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2012 CBA & Re-alignment: Lockout in Effect. Thanks Gary/Donald! PART II
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I think Mr. Burgundy is right. If the owners and Fehr truly pushed this into next season you'd be looking at a wholly different situation than at the end of the last lockout.
For starters, fans are already more pissed and able to communicate as a group about boycotts and things of that nature than the last time, because of social media. That is going to compound over time. There will be more and more people spouting negative publicity about the NHL and parties involved as we go, and it will start to catch on as people like Puck Daddy and others give them free advertising / links to a big audience. Especially if it's well into the summer and there's no end in sight. Fans are going to lose hope..
Second, if you can kill a whole season and show so little respect for fans that you move it into a consecutive season... you risk in a big way, permanently alienating a huge swath of the US fan base (to the tune of 30-40% of ticket buying and subscription buying fans IMO), and a significant portion of Canadian fans will stick to watching other leagues as well. Effectively you will kill hockey in the United States except at the lower levels. It would be a fan-base disaster IMO. MANY fans would say "**** off" and mean it. Just leave with an attitude of indifference. Because at that point you're truly being played for a fool.
I mean what self-respecting person keeps hanging on in a situation like that when there's so many other options for entertainment or useful things to do when passing time? I can guarantee 100% as it is now, I will not pay for tickets this year if they come back and will only pay for Center Ice if there's a "we're sorry fans" discount applied (say 40 or 50% off remaining games). If they went into next year, I would never buy another ticket, never subscribe again, never buy another piece of merchandise. I would watch what I could on free TV, do some fantasy hockey maybe and that's it. And there would be probably hundreds of thousands of fans just like me doing the same thing. And then another group that wouldn't even watch on free tv, driving ad revenues down and making it even less viable a sport.
To tell you the truth, if they go into next year and the NHL ends up folding because of financial collapse (losing money even after they come back), I wouldn't shed a tear that's for sure. F off. Let the players go where they want and if you start a new league it better be in real hockey markets only or I won't bother because we'll end up in the same place again.
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The formation of phenolic diterpenes in Rosmarinus officinalis L. under Mediterranean climate
- S. Munné-Bosch,
- L. Alegre,
- K. Schwarz
- … show all 3 hide
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The formation of phenolic diterpenes in the leaves of rosemary plants grown under Mediterranean climate was investigated. Low precipitation coincided with high solar radiation during the summer, resulting in water and light stress of the plants. The highest concentrations of the major diterpenes carnosic acid and carnosol were found during the winter and the lowest concentrations during the summer, which is likely to be the result of their enzymatic conversion in the presence of singlet oxygen. All other diterpenes followed different trends during the season. The concentration of carnosic acid increased with the relative water content of the leaf but decreased with increasing solar radiation and temperature. 11,12-di-O-Methylisorosmanol was found for the first time in rosemary leaves.
- Title
- The formation of phenolic diterpenes in Rosmarinus officinalis L. under Mediterranean climate
- Journal
European Food Research and Technology
Volume 210, Issue 4 , pp 263-267
- Cover Date
- 2000-02
- 10.1007/s002179900108
- Print ISSN
- 1438-2377
- Online ISSN
- 1438-2385
- Publisher
- Springer-Verlag
- Additional Links
- Keywords
- Key words Rosmarinus officinalis
- Phenolic diterpenes
- Carnosic acid
- Carnosol
- 11,12-Di-O-methylisorosmanol
- Industry Sectors
- Authors
- S. Munné-Bosch (A1)
- L. Alegre (A1)
- K. Schwarz (A2)
- Author Affiliations
- A1. Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Avenida Diagonal 645, E-08028 Barcelona, Spain, ES
- A2. Institut für Lebensmittelwissenschaft, Universität Hannover, Wunstorfer Strasse 14, D-30453 Hannover, Germany e-mail: email@example.com, DE