一种真菌对人参皂苷Rg3的转化

[目的]筛选长白山人参土壤中的活性微生物,转化人参总皂苷及单体人参皂苷产生稀有抗肿瘤成份.[方法]从长白山人参根际土壤中分离各类菌株,对人参总皂苷及单体人参皂苷进行微生物转化,并通过硅胶柱层析等方法对转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定;结合菌落形态、产孢结构、孢子形态特征以及菌株ITS rDNA核酸序列分析,对活性菌株进行鉴定.[结果]从长白山人参根际土壤中分离各类真菌菌株68株,有12株菌株对人参总皂苷有转化活性,其中菌株SYP2353对二醇组人参皂苷Rg3具有较强的转化活性.[结论]阳性菌株SYP2353被鉴定为疣孢漆斑菌(Myrothecium verrucaria),能将人参皂苷Rg3转化为稀有人参皂苷Rh2及二醇组人参皂苷苷元PPD,为稀有人参皂苷Rh2的制备提供了新的方法.     

Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition

The epithelial Na(+) channel (ENaC) is typically formed by three homologous subunits (alpha, beta, and gamma) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na(+) self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na(+). All 16 Cys residues within alpha and gamma ECLs and selected beta ECL Cys residues were individually mutated to alanine or serine residues. The Na(+) self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight alpha (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one beta (Cys-7), and nine gamma (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na(+) self-inhibition. Na(+) self-inhibition was eliminated by simultaneous mutations of either the last three alpha ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both alpha and gamma ECLs. By analyzing the Na(+) self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the alphaECL (alphaCys-1/alphaCys-6, alphaCys-4/alphaCys-5, alphaCys-7/alphaCys-16, alphaCys-10/alphaCys-13, and alphaCys-11/alphaCys-12) and one pair within the gammaECL (gammaCys-7/gammaCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the alpha and gamma ENaC subunits are required to establish the tertiary structure that ensures a proper Na(+) self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.     

Essential role for NHERF in cAMP-mediated inhibition of the Na+-HCO3- co-transporter in BSC-1 cells

Prior studies have indicated a requirement for the PDZ domain-containing protein, Na(+)/H(+) Exchanger Regulatory Factor (NHERF), for protein kinase A (PKA)-mediated inhibition of the renal basolateral Na(+)-HCO(3)(-) co-transporter (NBC). The present studies explore the potential mechanisms by which NHERF transduces cAMP signals to inhibit NBC. In BSC-1 cells, cells that express NBC but lack NHERF, 8-bromo-cAMP (100 microm for 15 min) failed to inhibit transport until wild-type mNHERF-(1-355) was expressed. mNHERF-(116-355) containing PDZ II and C-terminal ezrin-binding sequences or a mutant unphosphorylated form of rabbit NHERF effectively transduced the cAMP signals that inhibited NBC. By contrast, mNHERF-(1-126) encompassing N-terminal PDZ I and mNHERF-(1-325), which lacks ezrin-binding, failed to support cAMP inhibition of NBC activity. NBC and NHERF did not associate with each other in yeast two-hybrid or co-immunoprecipitation assays, and confocal microscopy indicated distinct subcellular localization of the two proteins. NBC was phosphorylated in BSC-1 cells, but its phosphorylation was not increased by cAMP nor was immunoprecipitated NBC phosphorylated by PKA in vitro. Acute exposure of mNHERF-(1-355)-expressing BSC-1 cells to cAMP did not change cell surface expression of NBC. Although these results established an essential role for NHERF in cAMP-mediated inhibition of NBC in BSC-1 cells, they also suggest a novel mechanism for NHERF-mediated signal transduction distinct from that previously characterized from studies of other NHERF targets.     

A new subunit of the epithelial Na+ channel identifies regions involved in Na+ self-inhibition

The epithelial Na+ channel (ENaC) is the apical entry pathway for Na+ in many Na+-reabsorbing epithelia. ENaC is a heterotetrameric protein composed of homologous alpha, beta, and gamma subunits. Mutations in ENaC cause severe hypertension or salt wasting in humans; and consequently, ENaC activity is tightly controlled. According to the concept of Na+ self-inhibition, the extracellular Na+ ion itself can reduce ENaC activity. The molecular basis for Na+ self-inhibition is unknown. Here, we describe cloning of a new ENaC subunit from Xenopus laevis (epsilonxENaC). epsilonxENaC can replace alphaxENaC and formed functional, highly selective, amiloride-sensitive Na+ channels when coexpressed with betaxENaC and gammaxENaC. Channels containing epsilonxENaC showed strong inhibition by extracellular Na+. This Na+ self-inhibition was significantly slower than for alphaxENaC-containing channels. Using site-directed mutagenesis, we show that the proximal part of the large extracellular domain controls the speed of self-inhibition. This suggests that this region is involved in conformational changes during Na+ self-inhibition.     

SRC family kinases mediate epithelial Na+ channel inhibition by endothelin

The epithelial Na(+) channel (ENaC) is implicated in the pathogenesis of salt-sensitive hypertension. Recent evidence from animal models suggests that the vasoactive peptide, endothelin (ET-1), may be an important negative regulator of ENaC in vivo. We investigated the signaling pathway involved in endothelin-mediated ENaC inhibition. Experiments were performed in NIH 3T3 cells stably expressing genes for the three (alpha, beta, and gamma) ENaC subunits. In whole cell patch clamp experiments, we found that ET-1 treatment induced a dose-dependent decrease in amiloride-sensitive currents. Using receptor-specific antagonists, we determined that the effects of ET-1 were attributed to activation of the ET(B) receptor. Moreover, the inhibitory effect of ET-1 on ENaC could be completely blocked when cells were pretreated with the selective Src family kinase inhibitor, PP2. Further studies revealed that basal Src family kinase activity strongly regulates ENaC whole cell currents and single channel gating. These results suggest that Src family kinases lie in a signaling pathway activated by ET-1 and are components of a novel negative regulatory cascade resulting in ENaC inhibition.     

Regulation of the epithelial Na+ channel by intracellular Na+

The hypothesis that the intracellularNa⁺ concentration([Na⁺]_i)is a regulator of the epithelialNa⁺ channel (ENaC) was tested withthe Xenopus oocyte expression systemby utilizing a dual-electrode voltage clamp.[Na⁺]_iaveraged 48.1 ± 2.2 meq (n = 27)and was estimated from the amiloride-sensitive reversal potential.[Na⁺]_iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 MNa₂SO₄.Within minutes of injection,[Na⁺]_istabilized and remained elevated at 97.8 ± 6.5 meq(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after theinitial injection of 0.5 and 0.25 MNa₂SO₄,respectively. This increase of[Na⁺]_icaused a biphasic inhibition of ENaC currents. In oocytes injected with0.5 MNa₂SO₄(n = 9), a rapid decrease of inwardamiloride-sensitive slope conductance(g_Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slowerdecrease to 0.304 ± 0.043 of control at 30 min were observed.Similar but smaller inhibitions were also observed with the injectionof 0.25 MNa₂SO₄.Injection of isotonicK₂SO₄(70 mM) or isotonicK₂SO₄made hypertonic with sucrose (70 mMK₂SO₄-1.2M sucrose) was without effect. Injection of a 0.5 M concentration ofeitherK₂SO₄,N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thusincreases of[Na⁺]_ihave multiple specific inhibitory effects on ENaC that can betemporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

     

Protective effect of ginsenoside Rg3 on lung injury in diabetic rats

Ginsenoside has been used to treat diabetes, while ginsenoside Rg3 is the main active ingredient component of ginseng and is used to study its effects on lung tissue damage in diabetic rats. In this paper, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry were applied to detect the proliferation and apoptosis of BEAS-2B cells treated with different concentrations of Rg3. The inflammatory response and pathological change in the lung tissue of diabetic rats treated with Rg3 were evaluated by enzyme-linked immunosorbent assay, quantative real-time polymerase chain reaction, and hematoxylin and eosin staining immunohistochemistry. Meanwhile, PI3K and MAPK signaling pathway proteins in lung tissue were determined by Western blot analysis. The results showed that ginsenoside Rg3 had no significant influence on the proliferation and apoptosis of BEAS-2B cells. Ginsenoside Rg3 can inhibit inflammatory response and promote the activation of PI3K and MAPK signaling pathways to prevent damages of lung tissues induced by hyperglycemia. The protective effect provided by ginsenoside Rg3 indicates that ginsenoside Rg3 is a potential drug for the treatment of diabetes.     

In vivo rat metabolism and pharmacokinetic studies of ginsenoside Rg3

Metabolism of an anti-tumor active component of Panax ginseng, ginsenoside (20R)-Rg(3), was studied for better understanding its pharmacokinetics in rat. LC-MS was used to determine Rg(3) and its metabolites in rat plasma, urine and feces samples. An average half-life of 18.5 min was obtained after the ginsenoside was intravenously dosed at 5 mg/kg. However, Rg(3) was not detected in rat plasma collected after oral administration at 100 mg/kg. Only 0.97-1.15% Rg(3) of the dosed amount was determined in feces. Hydrolysis and oxygenated metabolites were detected and identified in feces collected after oral administration by using LC-MS and MS-MS.     

Effects of ginsenoside Rg2 on the 5-HT3A receptor-mediated ion current in Xenopus oocytes

Treatment with ginsenosides, major active ingredients of Panax ginseng, produces a variety of pharmacological or physiological responses with effects on the central and peripheral nervous systems. Recent reports showed that ginsenoside Rg2 inhibits nicotinic acetylcholine receptor-mediated Na+ influx and channel activity. In the present study, we investigated the effect of ginsenoside Rg2 on human 5-hydroxytryptamine3A (5-HT3A) receptor channel activity, which is also one of the ligand-gated ion channel families. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured using the two-electrode voltage clamp technique. The ginsenoside Rg2 itself had no effect on the oocytes that were injected with H2O as well as on the oocytes that were injected with the 5-HT3A receptor cRNA. In the oocytes that were injected with the 5-HT3A receptor cRNA, the pretreatment of ginsenoside Rg2 inhibited the 5-HT-induced inward peak current (I5-HT) The inhibitory effect of ginsenoside Rg2 on I5-HT was dose dependent and reversible. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 was 22.3 +/- 4.6 microM. The inhibition of I5-HT by ginsenoside Rg2 was non-competitive and voltage-independent. These results indicate that ginsenoside Rg2 might regulate the 5-HT3A receptors that are expressed in Xenopus oocytes. Further, this regulation on the ligand-gated ion channel activity by ginsenosides might be one of the pharmacological actions of Panax ginseng.     

Estimation of Na+ dwell time in the gramicidin A channel. Na+ ions as blockers of H+ currents

The permeation of Na+ through gramicidin A channels shows a simple saturation with increasing Na+ concentration that can be described by two different models. The first model assumes that one Na+ binds to the channel with high affinity (approximately 30 M-1) and that conduction occurs by a 'knock-on' mechanism requiring double occupancy of the channel; the other model assumes that Na+ binding is of low affinity (less than 1 M-1), and that double occupancy of the channel is rare. NMR measurements have shown tight Na+ binding, favoring the first model, but measurements of flux ratios and water transport support the second model. We present here a relatively model-independent measurement of the dwell time of Na+ inside the channel, in which we characterize the fluctuations in H+ current through the channel induced by 'block' from the more slowly permeating Na+ ions. The mean Na+ dwell time inside the channel is estimated to be approximately 10 ns at a membrane potential of 200 mV. This result is inconsistent with tight Na+ binding, thus favoring the second model.     

Proteasomal degradation of Kir6.2 channel protein and its inhibition by a Na+ channel blocker aprindine

ATP-sensitive K+ channels (K(ATP):SUR2A+Kir6.2) play a pivotal role in cardiac protection against ischemia and reperfusion injury. When expressed in COS cells, Kir6.2 was short-lived with a half-life time of 1.9 h. The half-life time of Kir6.2 was prolonged by proteasome inhibitors MG132, ALLN, proteasome inhibitor 1, and lactacystine, but not at all by a lysosomal inhibitor chloroquine. MG132 also increased the level of ubiquitinated Kir6.2 without affecting its localization in the endoplasmic reticulum and Golgi apparatus. In electrophysiological recordings, MG132 augmented nicorandil-activated K(ATP) currents in COS cells expressing SUR2A and Kir6.2 as well as the same currents in neonatal rat cardiomyocytes. Like MG132, a Na+ channel blocker aprindine prolonged the half-life time of Kir6.2 and augmented K(ATP). Finally, both aprindine and MG132 inhibited the 20S proteasome activity in vitro. These results suggest a novel activity of aprindine to enhance K(ATP) currents by inhibiting proteasomal degradation of Kir 6.2 channels, which may be beneficial in the setting of cardiac ischemia.     

功能化外泌体载人参皂苷Rg1治疗缺血性脑卒中

本研究旨在探讨c(RGDyK)肽修饰的骨髓间充质干细胞外泌体作为一种多功能载体材料负载人参皂苷Rg1 (ginsenoside Rg1, G-Rg1)对缺血性脑卒中的治疗效果和潜在作用机制。以线栓法制备SD大鼠短暂性大脑中动脉缺血性模型(transient middle cerebral occlusion model, tMCAO),并将造模成功的SD大鼠随机分为tMCAO组、Exo组、游离G-Rg1组、Exo-Rg1组和cRGD-Exo-Rg1组,并以sham组作为对照。采用2,3,5-三苯基四氯化铵(2,3,5-triphenyltetrachloride, TTC)染色法检测脑梗死体积;免疫荧光法观察神经元、血管内皮细胞数量变化;Western blotting实验检测相关蛋白的表达。结果表明cRGD-Exo-Rg1可通过激活PI3K/AKT通路上调血管内皮生长因子(vascular endothelial growth factor, VEGF)和缺氧诱导因子(hypoxia-inducible factors, HIF-1α)的表达,促进血管生成和神经生成,有效减少脑梗死体...     

Molecular identification of the Na+-activated K+ channel

Progress in understanding sodium-activated potassium channels (K(Na)), suggested to function in excitable cells both during physiological conditions and protectively during hypoxia, has been limited by their unknown molecular identity. In this issue of Neuron, Salkoff and coworkers now show that members of the Slo gene family, Slo2.1 and Slo2.2, encode functional K(Na) channels.     

Ras couples phosphoinositide 3-OH kinase to the epithelial Na+ channel

Aldosterone induces the expression of the small G protein K-Ras. Both K-Ras and its 1st effector phosphoinositide 3-OH kinase (PI3-K) are necessary and sufficient for the activation of ENaC increasing channel open probability. The cell signaling mechanism by which K-Ras enhances ENaC activity, however, is uncertain. We demonstrate here that K-Ras significantly activates human ENaC reconstituted in Chinese hamster ovary cells approximately 3-fold. Activation in response to K-Ras was sensitive to the irreversible PI3-K inhibitor wortmannin but not the competitive LY294002 inhibitor of this phospholipid kinase. Similarly, a PI3-K 1st effector-specific Ras mutant (G12:C40) enhanced ENaC activity in a wortmannin but not LY294002 sensitive manner. Constitutively active PI3-K also enhanced ENaC activity but in a wortmannin and LY294002 sensitive manner with the effects of PI3-K and K-Ras not being additive. The activation of ENaC by PI3-K was also sensitive to intracellular GDPbetaS. Constitutively active PI3-K that is incapable of interacting with K-Ras (K227E p110alpha) acted as dominant negative with respect to the regulation of ENaC even in the presence of K-Ras. K-Ras is known to directly interact with PI3-K with aldosterone promoting this interaction. Here we demonstrate that K-Ras also interacts with ENaC through an, as yet, undetermined mechanism. We conclude that K-Ras enhances ENaC activity by localizing PI3-K near the channel and stimulating of PI3-K activity.     

Combined blockade of the Na+ channel and the Na+/H+ exchanger virtually prevents ischemic Na+ overload in rat hearts

Blocking either the Na⁺ channel or the Na⁺/H⁺ exchanger (NHE) has been shown to reduce Na⁺ and Ca²⁺ overload during myocardial ischemia and reperfusion, respectively, and to improve post-ischemic contractile recovery. The effect of combined blockade of both Na⁺ influx routes on ionic homeostasis is unknown and was tested in this study. [Na⁺]_i, pH_i and energy-related phosphates were measured using simultaneous ²³Na- and ³¹P-NMR spectroscopy in isolated rat hearts. Eniporide (3 μM) and/or lidocaine (200 μM) were administered during 5 min prior to 40 min of global ischemia and 40 min of drug free reperfusion to block the NHE and the Na⁺ channel, respectively. Lidocaine reduced the rise in [Na⁺]_i during the first 10 min of ischemia, followed by a rise with a rate similar to the one found in untreated hearts. Eniporide reduced the ischemic Na⁺ influx during the entire ischemic period. Administration of both drugs resulted in a summation of the effects found in the lidocaine and eniporide groups. Contractile recovery and infarct size were significantly improved in hearts treated with both drugs, although not significantly different from hearts treated with either one of them.     

Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate

It has been recognized that ginsenoside Rg3 is not naturally produced in ginseng although this ginsenoside can accumulate in red ginseng as the result of a thermal process. In order to determine whether or not Rg3 is synthesized in ginseng, hairy roots were treated with methyl jasmonate (MJ). From HPLC analysis, no peak for Rg3 was observed in the controls. However, Rg3 did accumulate in hairy roots that were MJ-treated for 7?days. Rg3 content was 0.42?mg/g (dry weight). To gain more insight into the effects of MJ on UDP-glucosyltransferase (UGT) activity, we attempted to evaluate ginsenoside Rg3 biosynthesis by UGT. A new peak for putative Rg3 was observed, which was confirmed by LC-MS/MS analysis. Our findings indicate that the proteins extracted from our hairy root lines can catalyze Rg3 from Rh2. This suggests that our ginseng hairy root lines possess Rg3 biosynthesis capacity.