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Despite recent advances, the link between the evolution of atmospheric CO2 and climate during the Eocene greenhouse remains uncertain. In particular, modelling studies suggest that in order to achieve the global warmth that characterised the early Eocene, warmer climates must be more sensitive to CO2 forcing than colder climates. Here, we test this assertion in the geological record by combining a new high-resolution boron isotope-based CO2 record with novel estimates of Global Mean Temperature. We find that Equilibrium Climate Sensitivity (ECS) was indeed higher during the warmest intervals of the Eocene, agreeing well with recent model simulations, and declined through the Eocene as global climate cooled. These observations indicate that the canonical IPCC range of ECS (1.5 to 4.5 °C per doubling) is unlikely to be appropriate for high-CO2 warm climates of the past, and the state dependency of ECS may play an increasingly important role in determining the state of future climate as the Earth continues to warm.

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The Miocene Climatic Optimum (MCO, 14-17 Ma) was ~3-4 °C warmer than present, similar to estimates for 2100. Coincident with the MCO is the Monterey positive carbon isotope (δ13C) excursion, with oceans more depleted in 12C relative to 13C than any time in the past 50 Myrs. The long-standing Monterey Hypothesis uses this excursion to invoke massive marine organic carbon burial and draw-down of atmospheric CO2 as a cause for the subsequent Miocene Climate Transition and Antarctic glaciation. However, this hypothesis cannot explain the multi-Myr lag between the δ13C excursion and global cooling. We use planktic foraminiferal B/Ca, δ11B, δ13C, and Mg/Ca to reconstruct surface ocean carbonate chemistry and temperature. We propose that the MCO was associated with elevated oceanic dissolved inorganic carbon caused by volcanic degassing, global warming, and sea-level rise. A key negative feedback of this warm climate was the organic carbon burial on drowned continental shelves.

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Ile-177 and Ser-180 are conserved residues in the first transmembrane segment (S1) of the Shaker, Shab, Shaw, and Shal subfamilies of voltage-gated K+ channels. Here we report that the mutation of these residues in Kv1.1 to leucine, proline, or arginine abolished the expression of outward potassium currents in Xenopus oocytes. Co-injection of these mutant cRNAs and wild type Kv1.1 cRNA into Xenopus oocytes exerted a potent dominant negative effect resulting in the suppression of Kv1.1-encoded currents. Transient transfection experiments of COS-7 cells revealed that the S1 mutants directed the synthesis of Kv1.1 polypeptides. Quantitative co-immunoprecipitation assays revealed that most of the S1 mutants co-assembled and formed both homo- and heteromultimeric complexes. Furthermore, the mutated polypeptides could reach the plasma membranes of transfected Sol8 cells. We conclude that mutations of Ile-177 and Ser-180 do not interfere with either the assembly of multimeric channel complexes or the targeting of these complexes to the plasma membrane. It is likely that these residues are involved in helix-helix interactions that are critical to the proper functioning of voltage-gated potassium channels.

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Two prominent proteins (30 and 33 kD) in a purified preparation of the sheep pineal gland were studied. Amino acid analysis of tryptic peptides indicated that the 33-kD protein was the epsilon isoform of the 14-3-3 family of proteins, and that the 30-kD protein was the zeta isoform. The sheep pineal gland was found to have six other 14-3-3 isoforms in addition to the epsilon and zeta, suggesting that copurification of the epsilon and zeta forms may reflect the existence of homo- or heterodimers comprised of these isoforms. To characterize 14-3-3 proteins further in the pineal gland, the full sequence of the epsilon isoform and a partial sequence of the zeta isoform were cloned from a rat pineal cDNA library and are reported here. Tissue distribution studies using Western blot analysis revealed that rat pineal and retina have levels of 14-3-3 protein similar to those found in brain, and that relatively low levels occur in other tissues. This investigation also revealed the epsilon isoform was present at high levels in the rat pineal gland early in development and decreased steadily thereafter and that 30-kD isoforms exhibited the inverse developmental pattern.

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Three different experimental approaches were used to establish that the first transmembrane segment (S1) is important for K+ channel assembly. First, hydrodynamic analyses of in vitro translated Kv1.1 N-terminal domain containing the S1 segment coassembles to form homotetrameric complexes, whereas deletion of the S1 segment abolishes coassembly. Second, coimmunoprecipitation experiments of cotranslated Kv1.1 and Kv1.5 truncated polypeptides show that the S1 segment is essential for coimmunoprecipitation. Third, dominant negative experiments in Xenopus oocytes confirm that over-expression of either the S1 segment or the N-terminal domain is sufficient for abolishing the expression of functional Kv1.1 and Kv1.5 K+ channels. These data indicate that S1 segment plays an important role in the coassembly of homo- and heterotetrameric K+ channels.

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Cholera toxin (CT) treatment (50 micrograms/ml) was used to down regulate the alpha subunit of the stimulatory guanine nucleotide binding protein (Gs alpha) in pineal glands in organ culture, as has been seen in non-neural tissue. A 15 h treatment reduces Gs alpha by approximately 75% as measured using semi-quantitative Western blot technology. In contrast, this treatment does not alter the abundance of G beta, Gi alpha or Go alpha. This effect on Gs alpha was still apparent following a 36-h washout period. The 48-h CT treatment increased cyclic AMP accumulation 10- to 17-fold but blocked the norepinephrine (NE)-induced increase in cyclic AMP accumulation, presumably reflecting the loss of Gs alpha. This treatment did not, however, inhibit protein synthesis or stimulation of arylalkylamine N-acetyltransferase (NAT) activity produced by treatment with either DB-cyclic AMP (N6,2'-O-dibutyryl adenosine 3',5' monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP derivatives. This indicates that a 48-h CT treatment was not generally toxic. In contrast, this treatment blocked subsequent CT stimulation of NAT. The effects of CT treatment on the adrenergic stimulation of NAT was examined using treatments which selectively produced alpha- or beta-adrenergic stimulation. alpha 1-Adrenergic activation of the pineal gland elevates [Ca2+]i, which potentiates effects of cyclic AMP; in these studies the response to alpha-adrenergic activation was markedly increased in 48 h CT-treated glands, reflecting Ca2+ potentiation of the effects of elevated levels of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

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Denervation and other forms of stimulus deprivation cause an increase in the magnitude of subsequent responses, a phenomenon commonly referred to as denervation supersensitivity. This has been well demonstrated with the cyclic AMP response to norepinephrine in the pineal gland. In the present report, we address the question of whether stimulus deprivation alters alpha and beta subunits of the GTP binding regulatory protein that stimulates adenylyl cyclase activity (Gs). Stimulus deprivation of the pineal gland was produced by denervation (superior cervical ganglionectomy), decentralization of the superior cervical ganglia, or by exposure of the animal to continuous lighting. All increased both the alpha and beta subunits of Gs (Gs alpha and G beta) by up to fourfold, as estimated using semiquantitative western blot technology. These effects were detectable after 1 day of stimulus deprivation and were sustained for 2 weeks. The stimulatory effects of constant light-induced stimulus deprivation were also apparent by measuring cholera toxin-dependent ADP-ribosylation of Gs alpha, which revealed a four-fold increase in the amount of labeled substrate. The results of in vivo studies were confirmed with in vitro studies, which demonstrated a spontaneous increase in both Gs alpha and G beta during 72 h of organ culture. The constant light-induced increases in both Gs alpha and G beta were prevented by continuous administration of isoproterenol (0.3 mg/kg/day), supporting the suggestion that adrenergic stimulation controls the levels of Gs alpha and G beta. These studies indicate that stimulus deprivation increases both Gs alpha and G beta.(ABSTRACT TRUNCATED AT 250 WORDS)

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Pinealocytes and retinal photoreceptor cells contain an unusual cytoplasmic complex composed of the G beta gamma dimer of GTP-binding regulatory proteins (G-proteins) tightly bound to an acidic 33 kDa phosphoprotein termed MEKA or phosducin; MEKA is a substrate of cyclic AMP-dependent protein kinase. This study characterized the developmental appearance of these and two related proteins, G gamma and S-antigen, in pineal and retinal tissue. MEKA was absent in the pineal gland prior to birth, at a time when it was possible to detect G beta in pineal cytoplasm, indicating that the appearance of G beta in the cytoplasm precedes that of MEKA and does not appear to require the presence of MEKA. The absence of MEKA at this time indicates that the cyclic AMP stimulation of pineal serotonin N-acetyltransferase activity is not mediated by MEKA, which has been considered as a possible role of MEKA. After postnatal day 7, pineal MEKA and cytoplasmic G beta increased in a parallel manner, with peak values occurring at about postnatal day 21. Thereafter, both proteins in the pineal gland decreased in a parallel fashion to 10 and 35% of their peak values, respectively; in contrast, the cytoplasmic protein S-antigen and membrane associated G beta remained at maximal levels after this time. Whereas both MEKA and G beta decreased late in development in the pineal gland, these proteins either increased or remained constant in the retina. These tissue-specific patterns were found to differ from those of another cytosolic protein found exclusively in the pineal gland and retina, S-antigen, which remained constant after day 21 in the pineal gland but decreased in the retina late in life.

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The adult rat pineal gland contains relatively high concentrations of Gsa, low amounts of both Gia and Goa, and undetectable levels of GTa. During development the amounts of 45 kDa Gsa and of Gia remain constant. In contrast, 42 kDa Gsa and Goa are nearly absent at birth and increase in abundance markedly thereafter. GTa is undetectable at any age. It would appear that multiple mechanisms regulate the expression of G-proteins in the pineal gland.

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Digitalis-like compounds (DLC) were shown to be a normal constituent of the skin and plasma of toads. In order to assess the possible physiological role of these compounds in the toad, their levels were determined in the brain, plasma and skin following acclimation in different NaCl solutions. We demonstrate that an increase in salt concentrations in the animal medium from 0 to 1.2% decreased the levels of DLC in the brain by 50% without altering significantly its levels in the plasma and skin. An increase in medium salt concentration to 1.5% resulted in a 50% increase of DLC levels in the skin without changing its levels in the plasma or brain. These results suggest that skin and brain DLC may participate in the long-term salt and water homeostasis in the toad, while the plasma compound either participates in the short-term regulations of salt and water homeostasis or have some other, unknown, function.

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Pinched-off presynaptic nerve terminals (synaptosomes) possess significant regulatory volume increase (RVI) and regulatory volume decrease (RVD) capabilities. Following a swelling induced by a hypotonic challenge, the synaptosomes regulate their volume and adjust it, in 2 min, to within 5% of its initial value (RVD) at an initial rate of -0.77 +/- 0.10%/s (mean +/- SEM). Following a shrinking induced by a hypertonic challenge, the synaptosomes also regulate their volume at an initial rate of 0.18 +/- 0.02%/s (RVI), resulting in a new steady state, reached within 5-10 min, with a synaptosomal volume below the original volume. The omission of Na+ or K+ ions from the extrasynaptosomal medium reduces the initial rate of RVI by 72.5 and 66.5%, respectively. The "loop diuretics" bumetanide and furosemide significantly inhibited the RVI of the synaptosomes. In contrast, ouabain, amiloride, or 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid did not have any significant effect on RVI parameters. Furthermore, bumetanide-sensitive 86Rb uptake by rat brain synaptosomes was stimulated threefold by a hypertonic perturbation of 30%. Thus we conclude that the RVI of synaptosomes is mainly due to a stimulation of the Na+, K+, Cl- co-transport system induced by the synaptosomal shrinking following the hypertonic challenge.

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The binding of [3H]-bumetanide to rat brain synaptosomes revealed the existence of two binding sites. The high affinity site (R1 = 46.6 fmoles/mg protein) binds bumetanide and furosemide with Kd1 of 13 nM and 1.5 microM respectively, while the low affinity site (R2 = 1.37 nmoles/mg protein) is characterized by Kd2 of 200 microM and 680 microM for bumetanide and furosemide, respectively. Bumetanide sensitive 86Rb uptake was 34 +/- 14.5, 38.3 +/- 1.4, 18.6 +/- 1.3 and 29.0 +/- 6.1% of total 86Rb uptake in synaptic plasma membrane vesicles, rat brain synaptosomes, Neuroblastoma N1E115 cell line and chick chest muscle cells, respectively. Furosemide and bumetanide inhibited 86Rb uptake to rat brain SPM- vesicles in a dose dependent fashion. Half maximal inhibition (IC50) was observed at 20 nM and 4 microM for bumetanide and furosemide, respectively. Bumetanide-sensitive transport was dependent on extravesicular sodium and chloride concentrations with a Km of 21 and 25 mM for the two ions, respectively. These results demonstrate the existence of a "loop diuretic" sensitive carrier-mediated K+ transport system in brain and other excitable cells.

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This study was undertaken in order to assess the functional role of acetylcholinesterase (AChE) in cultures of chick skeletal muscle cells. Cultures of skeletal myotubes were prepared by mechanical dissociation of limb muscle removed from 11-day-old chick embryos and plating at a concentration of 0.8 X 10(6) cells/ml. Cultures incubated for 4-10 days were used for electrophysiological studies with intracellular microelectrodes. Individual myotubes differed with respect to the time course of repolarization following depolarization by acetylcholine (ACh), some cells repolarizing within 2-3 min and others only after 8-10 min. Physostigmine (10(-8)-10(-6) M) prolonged or sometimes completely prevented repolarization following ACh-induced depolarization. These results demonstrate that hydrolysis of ACh by AChE in cultured chick skeletal myotubes plays an important role in the repolarization of these cells following ACh-induced depolarization.

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