补骨脂中呋喃香豆素类物质的积累规律及组织定位

应用高效液相色谱技术,分析了补骨脂(Psoralea corylifolia)不同器官、不同发育时期补骨脂素和异补骨脂素的含量变化规律。2种呋喃香豆素类物质在补骨脂的根、茎、叶、花、果实和种子中均有积累,其含量在果实中最高,根中最低,幼嫩茎叶高于成熟茎叶,成熟期果实高于幼嫩期果实。通过组织化学和荧光显微技术对呋喃香豆素类物质的定位研究,同时结合分泌腔的系数与2种呋喃香豆素的含量相关性分析,证明补骨脂中分泌腔是2种呋喃香豆素积累的主要场所。     

On the action and mechanism of withaferin-A from Withania somnifera,a novel and potent melanin dispersing agent in frog melanophores

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.     

Biotic elicitor enhanced production of psoralen in suspension cultures of Psoralea corylifolia L.

Cell cultures of Psoralea corylifolia L. were established from the leaf disk derived callus. The effect of different biotic elicitors prepared from the fungal extract (Aspergillus niger and Penicillium notatum), yeast extract and chitosan with different concentrations was studied. The increased synthesis of psoralen in 16-day old cell cultures under 16 h of light and 8 h of dark period was studied. Elicitation of psoralen in A. niger elicitor treated cells was found 9-fold higher over control cells. Treating the cells with P. notatum, yeast extract and chitosan elicitors lead to four to seven-fold higher psoralen accumulation over control cells. The extract of A. niger at 1.0% v/v increased the significant accumulation of psoralen (9850 μg/g DCW) in the cultured cells. Our study clearly shows that all the elicitors had the potential to increase the accumulation of psoralen but the A. niger elicitor at 1.0% v/v induced maximum accumulation.     

补骨脂中异戊烯基黄酮类化合物及其药理作用研究进展

综述补骨脂(Psoralea corylifolia)中的异戊烯基黄酮类化合物及其药理作用的研究进展.     

补骨脂叶中补骨脂素和异补骨脂素研究

通过研究补骨脂叶中是否含有补骨脂素和异补骨脂素,寻找含此类活性成分的新药源。分别采用四种不同的浸提方法对补骨脂叶甲醇溶液进行高效液相色谱分析,结合紫外光谱比对,发现补骨脂叶中含有补骨脂素类成分,且不同的处理方法使化学成分发生了显著的量变。新鲜补骨脂叶中补骨脂素和异补骨脂素以及另外两种化学成分的含量均明显高于烘干、晾干、研磨的叶子,补骨脂叶在干制过程中此四种化学成分的含量显著降低。     

Somatic embryogenesis and plant regeneration from root segments of Psoralea corylifolia L., an endangered medicinally important plant

Summary An efficient plant regeneration protocol has been developed from root explants of Psoralea corylifolia L., an endangered medicinally important herbaceous plant species belonging to the family Fabaceae. Nodular embryogenic callus was initiated from young root segments cultured on Murashige and Skoog (MS) medium (1962) supplemented with α-naphthaleneacetic acid (NAA; 2.68–13.42 μM) or 2,4-dichlorophenoxyacetic acid (2.4-D; 2.25–11.25 μM) in combination with 6-benzylaminopurine (BA: 2.2. μM). thiamine HCl (2.9 μM), L-glutamine (342.23 μM) and sucrose (3.0% w/v). The highest frequency (95.2%) of embryogenic calluses was obtained on MS medium supplemented with the growth regulators NAA (10.74 μM) and BA (2.2 μM). Development and maturation of somatic embryos was achieved after transfer of embryogenic calluses to MS medium supplemented with 1.34 μM NAA or 1.12 μM 2,4-D and 4.4–13.2 μM BA. The maximum number (13.8±1.34) of cotyledonary stage somatic embryos was obtained on MS medium containing 1.34 μM NAA and 13.2 μM BA. Germination of somatic embryos occurred on MS medium without any growth regulators and also on MS medium enriched with BA (1.1–8.8 μM), although the maximum germination frequency (76.1%) was obtained on 4.4 μM BA plus 1.45 μM gibberellic acid (GA₃). Plant regeneration without complete somatic embryo maturation was also achieved by transferring clumps of nodular embryogenic calluses onto MSO medium or MS medium supplemented with NAA (1.34 μM) and BA (2.2–8.8 μM). The highest frequency of plant regeneration (93.3%) and mean number of plantlets (15.4±0.88) were obtained on MS medium containing 1.34 μM NAA and 4.4 μM BA. Regenerated plants with well-developed root systems were transferred to pots where they grew vigorously, attained maturity and produced fertile seeds.     

Mediation of cholino-piperine like receptors by extracts of Piper nigrum induces melanin dispersion in Rana tigerina tadpole melanophores

Aim: The present study was carried out to determine the effects of lyophilized dried fruit extracts of Piper nigrum and pure piperine on the tadpole melanophores of frog Rana tigerina which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The nature of specific cellular receptors present on the neuro-melanophore junction and their involvement in pigmentary responses has been explored.

Material: Effects of lyophilized extracts of P. nigrum and pure piperine were studied on the isolated tail melanophores of tadpoles of the frog R. tigerina as per the modified method.

Results: The extract of P. nigrum and its active ingredient piperine caused significant melanin dispersal responses leading to darkening of the tail melanophores, which were completely antagonized by atropine and hyoscine. These per se melanin dispersal effects were also found to be markedly potentiated by neostigmine an anticholinesterase agent.

Conclusion: It appears that the melanin dispersal effects of the extracts of P. nigrum and pure piperine leading to skin darkening are mediated by cholinergic muscarinic or piperine-like receptors having similar properties.     

Enhanced production of phytoestrogenic isoflavones from hairy root cultures of Psoralea corylifolia L. Using elicitation and precursor feeding

The effect of biotic elicitors (yeast extract, chitosan), signaling molecule (salicylic acid), and polyamines (putrescine and spermidine) was studied with respect to isoflavones accumulation in hairy root cultures of Psoralea corylifolia L. Untreated hairy roots (control) accumulated 1.55% dry wt of daidzein and 0.19% dry wt of genistein. In precursor feeding experiment, phenylalanine at 2 mM concentration led to 1.3 fold higher production of daidzein (1.91% dry wt) and genistein (0.27% dry wt). In biotic elicitors, chitosan (2 mg/L) was found to be the most efficient elicitor to induce daidzein (2.78% dry wt) and genistein (0.279% dry wt) levels in hairy roots. Salicylic acid at 1 mM concentration stimulated the maximum accumulation of daidzein (2.2% dry wt) and genistein (0.228% dry wt) 2 days after elicitation. In case of polyamines, putrescine (50 mM) resulted in highest accumulation of daidzein (3.01% dry wt) and genistein (0.227% dry wt) after 5 days of addition. Present results indicated the effectiveness of elicitation and precursor feeding on isoflavones accumulation in hairy roots of P. corylifolia. This is the first report of elicitation on isoflavones production by hairy roots of P. corylifolia.     

Analyzing the responses of saccharin in context with melatonin receptors on the melanophores of the fish Labeo rohita (Ham.)

The hormone melatonin regulates the biological clock and assist in various other physiologies of vertebrates. Present work is intended to check the affinity of saccharin towards the melatonin receptors and the possible role of saccharin interference in the melatonin physiology. The present in vitro study is based on the working model of isolated scale melanophores in the dorso-lateral region of Labeo rohita. The pigment cells were incubated in the agonist and the antagonists within a limited time frame and subsequently their Melanophore Size Index (MSI) were calculated. The inferences were drafted through the observed signal transduction upshots in pigment translocations within the melanophores. Saccharin, in a wide dose range, has consistently induced a concentration-related aggregation similar to the aggregatory effect as shown by melatonin on the melanophores. Binding of saccharin with the receptors and eliciting its aggregatory effect is partially dependent on the release of neurotransmitters. The aggregatory effects were found to be significantly blocked by luzindole, K185, and prazosin, which are the potent melatonin receptor blockers, at the higher concentrations of saccharin. Hence, all the three subtypes of melatonin receptors viz. MT₁, MT₂, and MT₃ are participating in saccharin-mediated aggregations. Blocking by neomycin shows that Ca²⁺ ions are very crucial in dispensing the aggregatory effect of the sweetener. This research demands that an intensive and careful thorough study should be made about saccharin, specifically its effects upon melatonin physiology, before its unwarranted use as the food ingredients for human use.     

Muscarinic agonists acting through M2 acetylcholine receptors stimulate the migration of an NO-sensitive guanylyl cyclase to the plasma membrane of bovine tracheal smooth muscle

Muscarinic agonists acting on bovine tracheal smooth muscle (BTSM) induce two separate cGMP signals, one at 20?sec associated with NO-sensitive-soluble-guanylyl-cyclase (NO-sGC) and another at 60?sec, linked to natriuretic-peptide-GC. The 20-sec-cGMP novel cascade starts with mAChRs, via unknown components, activates an NO-sGC. To unravel this cascade, in crude membranes isolated from intact BTSM strips exposed to muscarinic agonists, we detected GC activities increments at 20?sec and 60?sec. The 20-sec-GC is a NO-sensitive-GC, identified as α₁β₁-heterodimer. In reconstitution experiments with purified plasma membranes and cytosol, muscarinic agonists induced an NO-sGC migration in a dose-dependent manner, being inhibited by muscarinic antagonists displaying an M₂AChR profile and blocked by PTX, suggesting the involvement of G_o/G_i proteins. The NO-sGC related to migration was isolated and identified as an α₁β₁-heterodimer. This work shows that muscarinic agonists in BTSM induce a massive and selective α₁β₁-NO-sGC migration from cytoplasm to plasma membranes being responsible for the 20-sec-cGMP signal.     

Peculiarities of phospholipase C-dependent release of CA2+ from intracellular stores upon activation of choline and purine receptors in myocytes of the guinea-pig small intestine

Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 μM carbachol, CCh, as well as 10.0 and 100.0 μM ATP) induced a rise in the [Ca²⁺]_i. Carbachol evoked an increase in the [Ca²⁺]_i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise in the [Ca²⁺]_i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca²⁺]_i in myocytes. The absence of the slow component in the [Ca²⁺]_i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is not related to activation of the entry of Ca²⁺ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca²⁺]_i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 μM), completely blocked rises in the [Ca²⁺]_i induced by both CCh and ATP; therefore, the release of Ca²⁺ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the difference in the release of Ca²⁺ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional effects of the above-mentioned neurotransmitters on smooth muscles. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 3–10, January–February, 2006.     

Response of sensory receptors of the cat's hindlimb to a transient,step-function DC electric field

There are two possible mechanisms of effects of large electric fields on animals, one caused by the electric field at the body surface and the other caused by the electric current induced inside the body. The purpose of the present experiments was to investigate the former possibility by recording action potentials from afferent fibers innervating various sensory receptors in the cat's hindlimb. Cat hairs were attracted to the upper electrode when exposed to DC electric fields of 180 kV/m or greater, and action potentials were evoked in the afferent fibers innervating G₁, G₂, and down hair receptors. No action potentials were evoked in afferent fibers innervating type I, type II, field receptors, muscle spindles, or joint receptors. These results indicate that a strong DC electric field induced movement of the hairs, eventually evoked excitation of the hair receptors, but that other receptors located under the skin were not influenced by electric field exposure.     

Evidence for the presence of A1 and A2 adenosine receptors in the ventral aorta of the dogfish shark,Squalus acanthias

Isolated, endothelium-free rings of vascular smooth muscle (VSM) from the ventral aorta of the dogfish shark, Squalus acanthias, were used to examine the vasoactive effects of various adenosine agonists. Cumulative addition of 2-chloroadenosine (2 Cl-ADO) over the concentration range 10 nM-1 mM resulted in a biphasic response, with a significant increase in tension at 1 microM and a more significant decline in tension at 100 microM and 1 mM, suggesting that this tissue may possess both A1 and A2 adenosine receptors. N6-Cyclopentyladenosine (N-6 CPA) and N6-(2-phenylisopropyl)adenosine, R(-)isomer (R-PIA), generally considered to be more A1 specific, also produced slight, but significant increases in tension, but only at relatively high concentrations. The more specific A1 agonist, N6-(25)-[2-endo-norbonyl] adenosine [(S)-ENBA] produced a significant increase in tension at 1 pM, reaching 28% above control at 10 nM. The response to (S)-ENBA was also biphasic, with a fall in tension at 10 microM. The relatively non-specific agonist 5'-N-ethylcarboxamidoadenosine (NECA) produced a small, but significant, increase in tension at 1 microM, with no subsequent decline in tension at higher concentrations. These results allow us to assign a tentative structure-activity relationship (SAR) for an increase in tension of (S)-ENBA much much greater than R-PIA greater than or equal to 2-Cl ADO = N-6 CPA = NECA; for the decrease, the SAR is (S)-ENBA greater than 2-Cl ADO greater than R-PIA greater than N-6 CPA = NECA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic administration of the antidepressants phenelzine,despiramine, clomipramine,or maprotiline decreases binding to 5-hydroxytryptamine2A receptors without affecting benzodiazepine binding sites in rat brain

Summary 1. The effects of chronic administration of five antidepressant drugs on the benzodiazepine and 5-HT_2A binding sites in the same rat brain were assessed.2. Clomipramine, desipramine, maprotiline, fluoxetine, and phenelzine (all 10 mg/kg/day) were administered s.c. for 21 days by Alzet osmotic minipumps.3. Results showed that none of the drugs changed the density or affinity of benzodiazepine binding sites, yet at the same dose all the drugs with the exception of fluoxetine decreased binding to 5-HT_2A receptors in the same animals.     

Identification, synthesis and properties of a consensus peptide recognized by a monoclonal antibody directed to various type I cytokine receptors

Previous works demonstrated that the monoclonal antibody (MAb) called R7B4 is directed to an epitope shared by receptors for lactogenic and somatogenic hormones as well as interleukins 2 and 6 (IL-2 and IL-6). The MAb inhibited the biological effects of those hormones and cytokines by impairing their binding to receptors. It is known that the receptors for growth hormones (GH), prolactins (PRL), IL-2, and IL-6 pertain to the type I cytokine receptor family, sharing the common motif WSXWS or the homologous F(Y)GEFS. Thus, a set of 34 decapeptides corresponding to diverse receptors containing those sequences were synthesized by the PEPSCAN method and their reactions with MAb R7B4 were measured by ELISA. The MAb significantly recognized 21 peptides, allowing us to establish the consensus sequence HGYWSEWSPE as a portion of the R7B4 epitope. The consensus peptide was synthesized and purified by conventional methods, and its capacity to bind to MAb R7B4 paratope confirmed. Moreover, polyclonal Ab to the peptide elicited in mice were able to inhibit the hGH binding to lactogenic, somatogenic and human specific liver receptors. This fact suggests that the consensus peptide could be used as an immunogen to produce anti-hGH receptor Ab behaving as hormone or cytokine antagonists in certain pathological conditions.     

Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, fungi, and eubacteria.

We demonstrate that three integral membrane receptors of mammals--the ecotropic retroviral leukemia receptor (ERR), the human retroviral receptor (HRR), and the T-cell early activator (Tea)--are homologous to a family of transporters specific for amino acids, polyamines, and choline (APC), which catalyze solute uniport, solute:cation symport, or solute:solute antiport in yeast, fungi, and eubacteria. Interestingly, the ERR membrane protein was recently shown to function as a cation:amino acid cotransporter. A binary sequence similarity matrix and an evolutionary tree of the 14 members of this family, illustrating their sequence similarities and divergences, were constructed. Other proteins, including the developmentally controlled GerAII spore germination protein of Bacillus subtilis and the acetylcholine receptor of Drosophila melanogaster gave sequence comparison scores of a sufficiently large magnitude to suggest (but not to establish) a common evolutionary origin with members of the APC family. We report an extended and corrected Tea cDNA sequence and show that the mammalian Tea and ERR encoding genes are differentially expressed in tissues and cell lines. Furthermore, the two mammalian cDNA sequences hybridize with other vertebrate and yeast genomic DNAs under stringent conditions. These observations support the notion that cell surface receptor proteins in mammals are transport proteins that share a common origin with transport proteins of single-celled organisms. Thus, permeases of essential metabolites may function pathologically as viral receptors.     

Contribution of conserved polar glutamine, asparagine and threonine residues and glycosylation to agonist action at human P2X1 receptors for ATP

The role of conserved polar glutamine, asparagine and threonine residues in the large extracellular loop, and glycosylation, to agonist action at human P2X1 receptors was tested by generating alanine substitution mutants. For the majority of mutants (Q56A, Q95A, T104A, T109A, Q112A, Q114A, T146A, N153A, T158A, N184A, N191A, N242A, N300A) alanine substitution had no effect on ATP potency. The mutants Q95A, Q112A, Q114A and T158A showed changes in efficacy for the partial agonists BzATP and Ap5A, suggesting that these polar residues may contribute to the gating of the channel. The mutants T186A, N204A and N290A had six-, three- and 60-fold decreases in ATP potency, respectively. For T186A and N290A, the partial agonists BzATP and Ap5A were no longer agonists but still bind to the receptor as shown by the ability to modulate the response to co-applied ATP. N153, N184 and N242 are glycosylated in the endoplasmic reticulum and N300 acquires complex glycosylation in the golgi. These results aid in refining a model for ATP binding at the P2X1 receptor where the residues F185T186, and the conserved triplet N290F291R292, are likely to play a role in ATP action at the receptor.     

Tricyclic antidepressants, but not the selective serotonin reuptake inhibitor fluoxetine, bind to the S1S2 domain of AMPA receptors

The hypothesis that depression is caused solely by a decrease in synaptic availability of monoaminergic neurotransmitters has been questioned over the past two decades. Based on accumulating data, it seems more plausible that cross-talk exists between neurotransmitters in the CNS, including the glutamatergic system. Glutamate, the major fast excitatory neurotransmitter in the CNS, is the natural agonist for the ionotropic glutamate receptors, a family of ligand-gated ion channels including NMDA (N-methyl-D-aspartate), AMPA (amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), and kainate receptors. In this work, we show that five tricyclic antidepressants bind to the S1S2 domain of the GluR2 subunit of the AMPA receptor. A combination of fluorescence quenching, Stern-Volmer analyses, and protease protection assays differentiate the binding of each antidepressant. These analyses provide no evidence for the binding of the selective serotonin reuptake inhibitor, fluoxetine, to this domain. The data presented provides further support for a role of the glutamatergic system in antidepressant activity.