秦岭产珠子参叶的达玛烷型皂甙研究(1)

从陕西省秦岭产珠子参(Panax japonicus C.A.Meyer var.major(Burk.)Wu etFeng)的叶中分离到十个新的达玛烷型四环三萜皂甙,经光谱测定和化学降解,其中四个的化学结构分别为珠子参甙(majoroside)F_1(1)、F_2(2)、F_3(3)和F_4(4)。同时,还分离到已知的人参甙(ginsenoside)Rd(5)、Re(6)、Rg_1(7)、Rg_2(8)和F_2(9)。     

盾叶薯蓣地上部分的三个新甾体皂甙

从盾叶薯蓣Dioscorea zingiberensis Wright地上部分分离鉴定了四个甾体皂甙,经鉴定甙A为约莫皂甙元-3-O-[α-L-鼠李吡喃糖基(1→2)]-β-D-葡萄吡喃糖甙;甙B为24α-羟基约莫皂甙元-3-O-[α-L-鼠李吡喃糖基(1→2)]β-D-葡萄吡喃糖甙;甙C为约莫皂甙元-3-O-[α-L-鼠李吡喃糖基(1→2)][β-D-葡萄吡喃糖基(1→4)]-β-D-葡萄吡喃糖基;甙D为约莫皂甙元-3-O-[α-L-鼠李吡喃糖基(1→2)][β-D-葡萄吡喃糖基(1→3)]-β-D-葡萄吡喃糖甙。前三者为新化合物,分别命名为盾叶皂甙A_1、A_2、A_3(zingiberoside A_1、A_2、A_3),其中盾叶皂甙A_2的甙元为一新甾体皂甙元,命名为盾叶皂甙元(zingiberogenin)。     

Interaction of Cibacron Blue F3G-A and Procion Red HE-3B with sheep liver 5,10-methylenetetrahydrofolate reductase

Cibacron Blue F3G-A, a probe used to monitor nucleotide binding domains in enzymes, inhibited sheep liver 5,10-methylenetetrahydrofolate reductase competitively with respect to 5-methyltetrahydrofolate and NADPH. TheK _i values obtained by kinetic methods and theK _d value for the binding of the dye to the enzyme estimated by protein fluorescence quenching were in the range 0.9–1.2 μM. Another triazine dye, Procion Red HE-3B interacted with the enzyme in an essentially similar manner to that observed with Cibacron Blue F3G-A. These results as well as the interaction of the dye with the enzyme monitored by difference spectroscopy and intrinsic protein fluorescence quenching methods indicated that the dye was probably interacting at the active site of the enzyme by binding at a hydrophobic region.     

Interaction of iodinated vinculin, metavinculin and α-actinin with cytoskeletal proteins

Iodinated vinculin, metavinculin and α-actinin were used to probe the interaction of these proteins with electrophoretically separated cytoskeletal proteins. Using the gel overlay technique, we detected strong binding of ¹²⁵I-vinculin and ¹²⁵I-metavinculin to α-actinin, 175 kDa polypeptide, talin, vinculin and metavinculin themselves, and moderate binding to actin.¹²⁵I-α-actinin was capable of interacting with vinculin and metavinculin. The specific binding of ¹²⁵-I-α-actinin to vinculin and metavinculin immobilized on a polysterene surface was also demonstrated. We suggest that the ability of vinculin and α-actinin to form a complex may be realized in microfilament-membrane linkages.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Temperature Dependence of Drug Interaction with the Platelet 5-Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox

Although [3H]imipramine is a selective radioligand for the 5-hydroxytryptamine (5-HT) transporter in human platelets, its affinity for binding to the 5-HT transporter complex at 0 degrees C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5-HT uptake at the physiological temperature of 37 degrees C (Ki = 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5-HT transporter at assay temperatures between 0 degrees C and 37 degrees C, using as radioligands [3H]imipramine (0 degrees C and 20 degrees C) and [3H]paroxetine (20 degrees C and 37 degrees C), a newly available probe for the 5-HT transporter. At 20 degrees C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p less than 0.001), validating the use of these two radioligands to study the 5-HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5-HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5-HT transporter at 0 degrees C. At 37 degrees C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5-HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5-HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5-HT transporter similar to that of imipramine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Time Course of Adaptations in Dopamine Biosynthesis, Metabolism, and Release Following Nigrostriatal Lesions: Implications for Behavioral Recovery from Brain Injury

Alterations in neostriatal dopamine metabolism, release, and biosynthesis were determined 3, 5, or 18 days following partial, unilateral destruction of the rat nigrostriatal dopamine projection. Concentrations of dopamine and each of its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) were markedly decreased in the lesioned striata at 3, 5, or 18 days postoperation. The decline in striatal high-affinity [3H]dopamine uptake closely matched the depletion of dopamine at 3 and 18 days postoperation. However, neither DOPAC, HVA, nor 3-MT concentrations were decreased to as great an extent as dopamine at any time following lesions that depleted the dopamine innervation of the striatum by greater than 80%. In these more severely lesioned animals, dopamine metabolism, estimated from the ratio of DOPAC or HVA to dopamine, was increased two- to four-fold in the injured hemisphere compared with the intact hemisphere. Dopamine release, estimated by the ratio of 3-MT to dopamine, was more increased, by five- to sixfold. Importantly, the HVA/dopamine, DOPAC/dopamine, and 3-MT/dopamine ratios did not differ between 3 and 18 days postlesioning. The rate of in vivo dopamine biosynthesis, as estimated by striatal DOPA accumulation following 3,4-dihydroxyphenylalanine (DOPA) decarboxylase inhibition with NSD 1015, was increased by 2.6- to 2.7-fold in the surviving dopamine terminals but again equally at 3 and 18 days postoperation. Thus, maximal increases in dopamine metabolism, release, and biosynthesis occur rapidly within neostriatal terminals that survive a lesion. This mobilization of dopaminergic function could contribute to the recovery from the behavioral deficits of partial denervation by increasing the availability of dopamine to neostriatal dopamine receptors. However, these presynaptic compensations are not sufficient to account for the protracted (at least 3-week) time course of sensorimotor recovery that has been observed following partial nigrostriatal lesion.     

Effects of Heavy Metal Cations and Other Sulfhydryl Reagents on Brain Dopamine D1 Receptors: Evidence for Involvement of a Thiol Group in the Conformation of the Active Site

To investigate aspects of the biochemical nature of membrane-bound dopamine D1 receptors, rat striatal homogenates were pretreated with heavy metal cations and some other chemical agents, and their effects on D1 receptors were subsequently determined using a standard [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1-N-3- benzazepine([3H]SCH 23390) binding assay. Incubation of striatal membranes with as little as 1 microM Hg2+, 10 microM Cu2+, and 10 microM Cd2+ completely prevented specific [3H]SCH 23390 binding. The effect of Cu2+, 1.5 microM, was noncompetitive in nature, whereas 3-5 microM Cu2+ afforded mixed-type inhibition. The inhibitory effect of Cu2+ was fully reversed by dithiothreitol (0.1-1 mM). Cu2+ (2 microM) did not affect the affinity of cis-flupenthixol or clozapine for remaining [3H]SCH 23390 sites. A second series of cations, Co2+ (30 microM), Ni2+ (30 microM), Mn2+ (1 mM), Ca2+ (25 mM), and Ba2+ (20 mM), inhibited specific [3H]SCH 23390 binding by 50% at the concentrations indicated. The thiol alkylating reagent N-ethylmaleimide (NEM) (0.2 mM) reduced specific binding by 70%. The effect of NEM was completely prevented by coincubation with a D1 receptor saturating concentration of SCH 23390 (20 nM) or dopamine (10 microM). The results indicated that the dopamine D1 receptor is a thiol protein and that a thiol group is essential for the ligand binding.     

Solubilization and Characterization of the Nitrendipine Receptor in Rat Brain

The nitrendipine receptor associated with the voltage-dependent calcium channel in rat brain was solubilized by detergent extraction and sonication. The detergent solution used for extraction consisted of 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.25% (wt/vol) polyoxyethylene 20 cetyl ether (Brij 58), and 0.025% (wt/vol) polyoxyethylene 17 cetyl stearyl ether (Lubrol WX) in the presence of 30% (wt/vol) glycerol as a stabilizer. The molecular weight of the receptor was estimated to be 1,800K by Sephacryl S-500 gel filtration and 800K by sucrose density gradient sedimentation. The equilibrium dissociation constant of [3H]nitrendipine to the solubilized receptors was 5.6 nM, which is approximately 10 times that of the membrane-bound receptor. The binding of nitrendipine to the receptor was inhibited noncompetitively by the structurally unrelated calcium channel inhibitors verapamil and prenylamine; their concentrations for 50% inhibition were both 1.0 X 10(-7) M, and they caused maximal inhibitions of 70 and 100%, respectively.