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1.
J Hupert S Mobarhan T J Layden V M Papa D J Lucchesi 《Biochimie et biologie cellulaire》1991,69(8):509-514
Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation. 相似文献
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Montezinho LP B Duarte C Fonseca CP Glinka Y Layden B Mota de Freitas D Geraldes CF Castro MM 《Journal of neurochemistry》2004,90(4):920-930
In this work, we studied the effect of intracellular 3',5'-cyclic adenosine monophosphate (cAMP) on Li+ transport in SH-SY5Y cells. The cells were stimulated with forskolin, an adenylate cyclase activator, or with the cAMP analogue, dibutyryl-cAMP. It was observed that under forskolin stimulation both the Li+ influx rate constant and the Li+ accumulation in these cells were increased. Dibutyryl-cAMP also increased Li+ uptake and identical results were obtained with cortical and hippocampal neurons. The inhibitor of the Na+/Ca2+ exchanger, KB-R7943, reduced the influx of Li+ under resting conditions, and completely inhibited the effect of forskolin on the accumulation of the cation. Intracellular Ca2+ chelation, or inhibition of N-type voltage-sensitive Ca2+ channels, or inhibition of cAMP-dependent protein kinase (PKA) also abolished the effect of forskolin on Li+ uptake. The involvement of Ca2+ on forskolin-induced Li+ uptake was confirmed by intracellular free Ca2+ measurements using fluorescence spectroscopy. Exposure of SH-SY5Y cells to 1 mm Li+ for 24 h increased basal cAMP levels, but preincubation with Li+, at the same concentration, decreased cAMP production in response to forskolin. To summarize, these results demonstrate that intracellular cAMP levels regulate the uptake of Li+ in a Ca(2+)-dependent manner, and indicate that Li+ plays an important role in the homeostasis of this second messenger in neuronal cells. 相似文献
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Layden BT Abukhdeir AM Malarkey C Oriti LA Salah W Stigler C Geraldes CF Mota de Freitas D 《Biochimica et biophysica acta》2005,1741(3):339-349
Li(+) binding in subcellular fractions of human neuroblastoma SH-SY 5 Y cells was investigated using (7)Li NMR spin-lattice (T(1)) and spin-spin (T(2)) relaxation measurements, as the T(1)/T(2) ratio is a sensitive parameter of Li(+) binding. The majority of Li(+) binding occurred in the plasma membrane, microsomes, and nuclear membrane fractions as demonstrated by the Li(+) binding constants and the values of the T(1)/T(2) ratios, which were drastically larger than those observed in the cytosol, nuclei, and mitochondria. We also investigated by (31)P NMR spectroscopy the effects of chronic Li(+) treatment for 4--6 weeks on the phospholipid composition of the plasma membrane and the cell homogenate and found that the levels of phosphatidylinositol and phosphatidylserine were significantly increased and decreased, respectively, in both fractions. From these observations, we propose that Li(+) binding occurs predominantly to membrane domains, and that chronic Li(+) treatment alters the phospholipid composition at these membrane sites. These findings support those from clinical studies that have indicated that Li(+) treatment of bipolar patients results in irregularities in Li(+) binding and phospholipid metabolism. Implications of our observations on putative mechanisms of Li(+) action, including the cell membrane abnormality, the inositol depletion and the G-protein hypotheses, are discussed. 相似文献
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Jasminder Sahi Michael P. Wiggins Gil B. Gibori Thomas J. Layden Mrinalini C. Rao 《Journal of cellular physiology》1996,168(2):276-283
To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit CI− secretion, their effects on CI− transport and intracellular Ca2+ concentrations ([Ca2+]i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl− sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca2+-sensitive fluorescent probe, fura-2AM were used to assess Cl− transport and [Ca2+]i, respectively. Basal Cl− transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl− channel blocker diphenylamine-2-carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na+/K+/2Cl− cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I− > F− ≥ Cl− > Br−. DPC inhibited I− influx by ∼50%, F− influx by 80%, Cl− influx by 85%, and Br− influx by 90%. Furosemide significantly inhibited influx of Br− (84%) and Cl− (81%) but not of F− and I−. The effects of agents known to alter biological response by increasing [Ca2+]i in other epithelial systems were used to stimulate Cl− transport. Cl− influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca2+ ionophore A23187 (2.05 ± 0.40). The Cl− permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl− influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl− permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl− transport by activating the Na+/K+/2Cl− cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl− channels and the cotransporter, and their actions are cyclooxygenase-independent. © 1996 Wiley-Liss, Inc. 相似文献
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Structural model of a complex between the heterotrimeric G protein, Gsalpha, and tubulin 总被引:2,自引:0,他引:2
Layden BT Saengsawang W Donati RJ Yang S Mulhearn DC Johnson ME Rasenick MM 《Biochimica et biophysica acta》2008,1783(6):964-973
A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The alpha subunits of some heterotrimeric G proteins, including Gsalpha, have been shown to interact strongly with tubulin. Binding of Galpha to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsalpha via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsalpha was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, alpha2-beta4 (the region between switch II and switch III) and alpha3-beta5 (just distal to the switch III region) domains of Gsalpha are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gbetagamma interaction regions of Gsalpha and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Galpha and the ability of Galpha to activate tubulin GTPase. 相似文献
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THE BREEDING SYSTEM IN PRIMULA VERIS L. 总被引:4,自引:2,他引:2
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Plant resistance to many types of pathogens and pests can be achieved by the presence of disease resistance (R) genes. The nucleotide binding site-leucine rich repeat (NBS-LRR) class of R-genes is the most commonly isolated class of R-genes and makes up a super-family, which is often arranged in the genome as large multi-gene clusters. The NBS domain of these genes can be targeted by polymerase chain reaction (PCR) amplification using degenerate primers. Previous studies have used PCR derived NBS sequences to investigate both ancient R-gene evolution and recent evolution within specific plant families. However, comparative studies with the Asteraceae family have largely been ignored. In this study, we address recent evolution of NBS sequences within the Asteraceae and extend the comparison to the Arabidopsis thaliana genome. Using multiple sets of primers, NBS fragments were amplified from genomic DNA of three species from the family Asteraceae: Helianthus annuus (sunflower), Lactuca sativa (lettuce), and Cichorium intybus (chicory). Analysis suggests that Asteraceae species share distinct families of R-genes, composed of genes related to both coiled-coil (CC) and toll-interleukin-receptor homology (TIR) domain containing NBS-LRR R-genes. Between the most closely related species, (lettuce and chicory) a striking similarity of CC subfamily composition was identified, while sunflower showed less similarity in structure. These sequences were also compared to the A. thaliana genome. Asteraceae NBS gene subfamilies appear to be distinct from Arabidopsis gene clades. These data suggest that NBS families in the Asteraceae family are ancient, but also that gene duplication and gene loss events occur and change the composition of these gene subfamilies over time. 相似文献
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Minadeo N Layden B Amari LV Thomas V Radloff K Srinivasan C Hamm HE de Freitas DM 《Archives of biochemistry and biophysics》2001,388(1):7-12
Although lithium salts have been used in the treatment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmacologic action of Li+ is unknown. Based on activity studies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li+ inhibition of G-proteins may account for its pharmacologic action. We used the purified alpha subunit of the recombinant inhibitory G-protein, rGialpha1, and found that Li+ at therapeutic levels significantly inhibited the formation of the GDP.AlF4-.rGialpha1 complex. Because our studies were conducted with a purified, metal-reconstituted G-protein rather than with cell membrane suspensions, our Li+ inhibition results lend additional support to the G-protein hypothesis for Li+ action. 相似文献
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