415 research outputs found
Synaptic tagging and capture : differential role of distinct calcium/calmodulin kinases in protein synthesis-dependent long-term potentiation
Weakly tetanized synapses in area CA1 of the hippocampus that ordinarily display long-term potentiation lasting ~3 h (called early-LTP) will maintain a longer-lasting change in efficacy (late-LTP) if the weak tetanization occurs shortly before or after strong tetanization of an independent, but convergent, set of synapses in CA1. The synaptic tagging and capture hypothesis explains this heterosynaptic influence on persistence in terms of a distinction between local mechanisms of synaptic tagging and cell-wide mechanisms responsible for the synthesis, distribution, and capture of plasticity-related proteins (PRPs). We now present evidence that distinct CaM kinase (CaMK) pathways serve a dissociable role in these mechanisms. Using a hippocampal brain-slice preparation that permits stable long-term recordings in vitro for >10 h and using hippocampal cultures to validate the differential drug effects on distinct CaMK pathways, we show that tag setting is blocked by the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) that, at low concentration, is more selective for CaMKII. In contrast, the CaMK kinase inhibitor STO-609 [7H-benzimidazo(2,1-a)benz(de)isoquinoline-7-one-3-carboxylic acid] specifically limits the synthesis and/or availability of PRPs. Analytically powerful three-pathway protocols using sequential strong and weak tetanization in varying orders and test stimulation over long periods of time after LTP induction enable a pharmacological dissociation of these distinct roles of the CaMK pathways in late-LTP and so provide a novel framework for the molecular mechanisms by which synaptic potentiation, and possibly memories, become stabilized
Higher arc nucleus-to-cytoplasm ratio during sleep in the superficial layers of the mouse cortex
The activity-regulated cytoskeleton associated protein Arc is strongly and quickly upregulated by neuronal activity, synaptic potentiation and learning. Arc entry in the synapse is followed by the endocytosis of glutamatergic AMPA receptors (AMPARs), and its nuclear accumulation has been shown in vitro to result in a small decline in the transcription of the GluA1 subunit of AMPARs. Since these effects result in a decline in synaptic strength, we asked whether a change in Arc dynamics may temporally correlate with sleep-dependent GluA1 down-regulation. We measured the ratio of nuclear to cytoplasmic Arc expression (Arc Nuc/Cyto) in the cerebral cortex of EGFP-Arc transgenic mice that were awake most of the night and then perfused immediately before lights on (W mice), or were awake most of the night and then allowed to sleep (S mice) or sleep deprived (SD mice) for the first 2 h of the light phase. In primary motor cortex (M1), neurons with high levels of nuclear Arc (High Arc cells) were present in all mice, but in these cells Arc Nuc/Cyto was higher in S mice than in W mice and, importantly, ~15% higher in S mice than in SD mice collected at the same time of day, ruling out circadian effects. Greater Arc Nuc/Cyto with sleep was observed in the superficial layers of M1, but not in the deep layers. In High Arc cells, Arc Nuc/Cyto was also ~15%–30% higher in S mice than in W and SD mice in the superficial layers of primary somatosensory cortex (S1) and cingulate cortex area 1 (Cg1). In High Arc Cells of Cg1, Arc Nuc/Cyto and cytoplasmic levels of GluA1 immunoreactivities in the soma were also negatively correlated, independent of behavioral state. Thus, Arc moves to the nucleus during both sleep and wake, but its nuclear to cytoplasmic ratio increases with sleep in the superficial layers of several cortical areas. It remains to be determined whether the relative increase in nuclear Arc contributes significantly to the overall decline in the strength of excitatory synapses that occurs during sleep. Similarly, it remains to be determined whether the entry of Arc into specific synapses is gated by sleep
Long-term Maintenance of Reduced Intraocular Pressure by Daily or Twice Daily Topical Application of Prostaglandins to Cat or Rhesus Monkey Eyes
Substantial evidence indicates that a single topical application of prostaglandins (PGs) can reduce intraocular pressure (IOP) in the eyes of several species. However, earlier literature, dealing with ocular hypertensive and inflammatory responses, shows the development of tachyphylaxis to subsequent doses of PGs. If similar tolerance developed to the ocular hypotensive effects of PGs, it would preclude the use of these agents in the treatment of chronic glaucoma. The present study shows, however, that although tachyphylaxis to the ocular hypotensive effects of PGs develops in rabbits, this is not a typical response among mammals. Significant IOP reduction was maintained in cats for up to 9 months by topical application of PGE 2 at 12-, 24-, or 48-hr intervals. The IOP reduction was jeopardized seriously only when the PG was applied every other day for several days or when, on a few occasions, 3 days were allowed to elapse between PGE 2 applications. Ocular hypotension was also maintained during the course of topical treatment of rhesus monkey eyes with PGF 2a . Short periods of pupillary constriction followed the application of each dose of PGF 2a to cat eyes, but the miotic response of rhesus monkeys to PGF 2a and cats to PGE 2 was negligible. Other apparent side effects were noted, but none of these were severe or progressive. These results clearly demonstrate that tachyphylaxis, or tolerance, is not expected to present an obstacle to the development of eicosanoids and/or their derivatives as therapeutic agents for the long-term treatment of ocular hypertension and chronic glaucoma. Invest Ophthalmol Vis Sci 24: [312][313][314][315][316][317][318][319] 1983 Early studies on the effects of prostaglandins (PGs) on the eye, primarily designed to determine the role of these autacoids in the ocular irritative response, concluded that exogenous PGs can produce increased intraocular pressure (IOP) and breakdown of the blood-aqueous barrier. 1 It has recently been shown, however, that appropriate doses of PGF 2a topically applied to the eyes of rabbits, 2 owl monkeys, 3 rhesus monkeys, and cats 4 can, in fact, reduce rather than increase IOP. In the rabbit there is only a narrow margin between the hypotensive dose of PGF 2a and a dose that causes initial hypertension. 2 Ten-to 100-fold higher PGF 2a doses than those that reduce IOP in the rabbit are required to produce the same effect in owl monkeys, rhesus monkeys, and cats, but eve
Photochemistry of the PAH pyrene in water ice: the case for ion-mediated solid-state astrochemistry
Context. Icy dust grains play an important role in the formation of complex
inter- and circumstellar molecules. Observational studies show that polycyclic
aromatic hydrocarbons (PAHs) are abundantly present in the ISM in the gas
phase. It is likely that these non-volatile species freeze out onto dust grains
as well and participate in the astrochemical solid-state network, but
experimental PAH ice studies are largely lacking. Methods. Near UV/VIS
spectroscopy is used to track the in situ VUV driven photochemistry of pyrene
containing ices at temperatures ranging from 10 to 125 K. Results. The main
photoproducts of VUV photolyzed pyrene ices are spectroscopically identified
and their band positions are listed for two host ices, \water and CO. Pyrene
ionisation is found to be most efficient in \water ices at low temperatures.
The reaction products, triplet pyrene and the 1-hydro-1-pyrenyl radical are
most efficiently formed in higher temperature water ices and in low temperature
CO ice. Formation routes and band strength information of the identified
species are discussed. Additionally, the oscillator strengths of Py, Py^+ and
PyH are derived and a quantitative kinetic analysis is performed by fitting a
chemical reaction network to the experimental data. Conclusions. Pyrene is
efficiently ionised in water ice at temperatures below 50 K. Hydrogenation
reactions dominate the chemistry in low temperature CO ice with trace amounts
of water. The results are put in an astrophysical context by determining the
importance of PAH ionisation in a molecular cloud. The photoprocessing of a
sample PAH in ice described in this manuscript indicates that PAH
photoprocessing in the solid state should also be taken into account in
astrochemical models.Comment: 11 pages, 8 figures, accepted for publication in A&
Subcellular heterogeneity of ryanodine receptor properties in ventricular myocytes with low T-tubule density
Rationale:
In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).
Objective:
To characterize RyR functional properties in relation to TT proximity, at baseline and after MI.
Methods:
Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.
Results:
In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI.
Conclusion:
TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves
Attitudes and behaviors of Japanese physicians concerning withholding and withdrawal of life-sustaining treatment for end-of-life patients: results from an Internet survey
<p>Abstract</p> <p>Background</p> <p>Evidence concerning how Japanese physicians think and behave in specific clinical situations that involve withholding or withdrawal of medical interventions for end-of-life or frail elderly patients is yet insufficient.</p> <p>Methods</p> <p>To analyze decisions and actions concerning the withholding/withdrawal of life-support care by Japanese physicians, we conducted cross-sectional web-based internet survey presenting three scenarios involving an elderly comatose patient following a severe stroke. Volunteer physicians were recruited for the survey through mailing lists and medical journals. The respondents answered questions concerning attitudes and behaviors regarding decision-making for the withholding/withdrawal of life-support care, namely, the initiation/withdrawal of tube feeding and respirator attachment.</p> <p>Results</p> <p>Of the 304 responses analyzed, a majority felt that tube feeding should be initiated in these scenarios. Only 18% felt that a respirator should be attached when the patient had severe pneumonia and respiratory failure. Over half the respondents felt that tube feeding should not be withdrawn when the coma extended beyond 6 months. Only 11% responded that they actually withdrew tube feeding. Half the respondents perceived tube feeding in such a patient as a "life-sustaining treatment," whereas the other half disagreed. Physicians seeking clinical ethics consultation supported the withdrawal of tube feeding (OR, 6.4; 95% CI, 2.5–16.3; P < 0.001).</p> <p>Conclusion</p> <p>Physicians tend to harbor greater negative attitudes toward the withdrawal of life-support care than its withholding. On the other hand, they favor withholding invasive life-sustaining treatments such as the attachment of a respirator over less invasive and long-term treatments such as tube feeding. Discrepancies were demonstrated between attitudes and actual behaviors. Physicians may need systematic support for appropriate decision-making for end-of-life care.</p
High-Density Real-Time PCR-Based in Vivo Toxicogenomic Screen to Predict Organ-Specific Toxicity
Toxicogenomics, based on the temporal effects of drugs on gene expression, is able to predict toxic effects earlier than traditional technologies by analyzing changes in genomic biomarkers that could precede subsequent protein translation and initiation of histological organ damage. In the present study our objective was to extend in vivo toxicogenomic screening from analyzing one or a few tissues to multiple organs, including heart, kidney, brain, liver and spleen. Nanocapillary quantitative real-time PCR (QRT-PCR) was used in the study, due to its higher throughput, sensitivity and reproducibility, and larger dynamic range compared to DNA microarray technologies. Based on previous data, 56 gene markers were selected coding for proteins with different functions, such as proteins for acute phase response, inflammation, oxidative stress, metabolic processes, heat-shock response, cell cycle/apoptosis regulation and enzymes which are involved in detoxification. Some of the marker genes are specific to certain organs, and some of them are general indicators of toxicity in multiple organs. Utility of the nanocapillary QRT-PCR platform was demonstrated by screening different references, as well as discovery of drug-like compounds for their gene expression profiles in different organs of treated mice in an acute experiment. For each compound, 896 QRT-PCR were done: four organs were used from each of the treated four animals to monitor the relative expression of 56 genes. Based on expression data of the discovery gene set of toxicology biomarkers the cardio- and nephrotoxicity of doxorubicin and sulfasalazin, the hepato- and nephrotoxicity of rotenone, dihydrocoumarin and aniline, and the liver toxicity of 2,4-diaminotoluene could be confirmed. The acute heart and kidney toxicity of the active metabolite SN-38 from its less toxic prodrug, irinotecan could be differentiated, and two novel gene markers for hormone replacement therapy were identified, namely fabp4 and pparg, which were down-regulated by estradiol treatment
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