Structures, chemotaxonomic significance, cytotoxic and Na(+),K(+)-ATPase inhibitory activities of new cardenolides from Asclepias curassavica.

Five new cardenolide lactates (1­5) and one new dioxane double linked cardenolide glycoside (17) along with 15 known compounds (6­16 and 18­21) were isolated from the ornamental milkweed Asclepias curassavica. Their structures were elucidated by extensive spectroscopic methods (IR, UV, MS, 1D- and 2D-NMR). The molecular structures and absolute configurations of 1­3 and 17 were further confirmed by single-crystal X-ray diffraction analysis. Simultaneous isolation of dioxane double linked cardenolide glycosides (17­21) and cardenolide lactates (1­5) provided unique chemotaxonomic markers for this genus. Compounds 1­21 were evaluated for the inhibitory activities against DU145 prostate cancer cells. The dioxane double linked cardenolide glycosides showed the most potent cytotoxic effect followed by normal cardenolides and cardenolide lactates, while the C21 steroids were non-cytotoxic. Enzymatic assay established a correlation between the cytotoxic effects in DU145 cancer cells and the Ki for the inhibition of Na(+),K(+)-ATPase. Molecular docking analysis revealed relatively strong H-bond interactions between the bottom of the binding cavity and compounds 18 or 20, and explained why the dioxane double linked cardenolide glycosides possessed higher inhibitory potency on Na(+),K(+)-ATPase than the cardenolide lactate.

Detecting metabolites of different transition metal-lithospermate B complexes after intravenous injection in rats.

AIM: Lithospermate B (LSB) isolated from the traditional Chinese medicine danshen (Salvia miltiorrhiza) is an effective Na(+)/K(+)-ATPase inhibitor and used to treat congestive heart failure. The inhibition of LSB on Na(+)/K(+)-ATPase is potentiated by forming complexes with transition metal ions. Here we investigated the safety and metabolites of different transition metal-LSB complexes in rats. METHODS: LSB complexed with six different transition metal ions (Mg(2+), Zn(2+), Cr(3+), Co(2+), Ni(2+) and Mn(2+)) were prepared. Adult male SD rats were injected with the different metal-LSB complexes (50 mg/kg, iv), and their bile and blood samples were collected. The metabolites of the metal-LSB complexes in the samples were analyzed using mass spectroscopy. RESULTS: In rats injected with LSB complexed with Mg(2+), Zn(2+), Cr(3+), Ni(2+) or Mn(2+), LSB and its four putative metabolites were equivalently detected in their bile samples. Mn(2+)-LSB exhibited distinct metabolite profiles compared with the other four metal-LSB complexes. The four putative metabolites were identified as 3-monomethyl-LSB, 3,3''-dimethyl-LSB, 3,3'''-dimethyl-LSB and 3,3'',3'''-trimethyl-LSB. The tracking of successive bile samples of rats injected with Mg(2+)-LSB, Zn(2+)-LSB and Mn(2+)-LSB concurrently demonstrated that LSB was firstly methylated at position 3, then at position 3'', and, finally, the 3''' hydroxyl group. All rats injected with Co(2+)-LSB died. CONCLUSION: Zn(2+)-LSB, Cr(3+)-LSB, Ni(2+)-LSB or Mn(2+)-LSB produces identical four methylated metabolites of LSB in rats, and seemed to be as safe as LSB or Mg(2+)-LSB.

Enhancing the potency of lithospermate B for inhibiting Na+/K+-ATPase activity by forming transition metal ion complexes.

AIM: To determine whether replacing Mg(2+) in magnesium lithospermate B (Mg-LSB) isolated from danshen (Salvia miltiorrhiza) with other metal ions could affect its potency in inhibition of Na(+)/K(+)-ATPase activity. METHODS: Eight metal ions (Na(+), K(+), Mg(2+), Cr(3+), Mn(2+), Co(2+), Ni(2+), and Zn(2+)) were used to form complexes with LSB. The activity of Na(+)/K(+)-ATPase was determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP. Human adrenergic neuroblastoma cell line SH-SY5Y was used to assess the intracellular Ca(2+) level fluctuation and cell viability. The metal binding site on LSB and the binding mode of the metal-LSB complexes were detected by NMR and visible spectroscopy, respectively. RESULTS: The potencies of LSB complexed with Cr(3+), Mn(2+), Co(2+), or Ni(2+) increased by approximately 5 times compared to the naturally occurring LSB and Mg-LSB. The IC50 values of Cr-LSB, Mn-LSB, Co-LSB, Ni-LSB, LSB, and Mg-LSB in inhibition of Na(+)/K(+)-ATPase activity were 23, 17, 26, 25, 101, and 128 µmol/L, respectively. After treatment of SH-SY5Y cells with the transition metal-LSB complexes (25 µmol/L), the intracellular Ca(2+) level was substantially elevated, and the cells were viable for one day. The transition metals, as exemplified by Co(2+), appeared to be coordinated by two carboxylate groups and one carbonyl group of LSB. Titration of LSB against Co(2+) demonstrated that the Co-LSB complex was formed with a Co(2+):LSB molar ratio of 1:2 or 1:1, when [Co(2+)] was less than half of the [LSB] or higher than the [LSB], respectively. CONCLUSION: LSB complexed with Cr(3+), Mn(2+), Co(2+), or Ni(2+) are stable, non-toxic and more potent in inhibition of Na(+)/K(+)-ATPase. The transition metal-LSB complexes have the potential to be superior substitutes for cardiac glycosides in the treatment of congestive heart failure.

Active ingredients in Chinese medicines promoting blood circulation as Na+/K+ -ATPase inhibitors.

The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na(+)/K(+)-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na(+)/K(+)-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na(+)/K(+)-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na(+)/K(+)-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na(+)/K(+)-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na(+)/K(+)-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na(+)/K(+)-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.

Docking study of the precursor peptide of mastoparan onto its putative processing enzyme, dipeptidyl peptidase IV: a revisit to molecular ticketing.

Stepwise-cleavage process of promastoparans to reach maturity was investigated theoretically by combining ab initio folding and unbounded docking. The comparison between the structures of the promastoparans both before and after docking were examined along with the hydrogen bonding interaction pattern between the dipetidyl peptidase IV (DPPIV) and promastoparans to reveal how the endpoint of this stepwise cleavage is recognized among these promastoparans with highly resemble amino acid sequences. The current approach of folding and docking study provides structural insight on the stepwise cleavage process.

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevates intracellular Ca(2+) level in SH-SY5Y cells.

AIM: To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na(+)/K(+)-ATPase, leads to the elevation of intracellular Ca(2+) level as observed in cells treated with cardiac glycosides. METHODS: Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca(2+) levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na(+)/Ca(2+) exchanger inhibitor) and 2-APB (IP(3) receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na(+)/K(+)-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope. RESULTS: severe toxicity was observed in cells treated with ouabain of concentration higher than 1 micromol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca(2+) levels were substantially elevated by MLB (1 micromol/L) and ouabain (1 micromol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 micromol/L) or 2-APB (100 micromol/L). Equivalent interaction with the binding cavity of Na(+)/K(+)-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 micromol/L), but not MLB (1 mumol/L), induced dendritic shrink of SH-SY5Y cells. CONCLUSION: Comparable to ouabain, MLB leads to the elevation of intracellular Ca(2+) level presumably via the same mechanism by inhibiting Na(+)/K(+)-ATPase. The elevated Ca(2+) levels seem to be supplied by Ca(2+) influx through the reversed mode of the Na(+)/Ca(2+) exchanger and intracellular release from endoplasmic reticulum.

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na+/K+ -ATPase.

AIM: To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain. METHODS: The inhibitory potency of ouabain and the identified steroid-like compounds on Na(+)/K(+)-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na(+)/K(+)-ATPase. RESULTS: All the examined steroid-like compounds displayed more or less inhibition on Na(+)/K(+)-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na(+)/K(+)-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K(+) binding sites of Na(+)/K(+)-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na(+)/K(+)-ATPase. CONCLUSION: Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na(+)/K(+)-ATPase.

Effect of sugar positions in ginsenosides and their inhibitory potency on Na+/K+-ATPase activity.

AIM: To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin. METHODS: The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginsenosides and ouabain on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na+/K+-ATPase. RESULTS: Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na+/K+-ATPase activity. However, their inhibitory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 position of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these positions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na+/K+-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure. CONCLUSION: The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhibition of Na+/K+-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure.

Synergic effect of curcumin and its structural analogue (Monoacetylcurcumin) on anti-influenza virus infection.

Curcumin (Cur), a polyphenolic compound extracted from spice and common food colourant turmeric, contains versatile bio-activities. Monoacetylcurcumin (MAC), a structural analogue of Cur, differs from Cur by acetyl modification, but retains enone groups. Comparative analysis revealed MAC effectively inhibited influenza virus infection (IAV) to a similar extent as, if not superior to, curcumin. Both compounds mildly reduced viral NA activity. Surprisingly, unlike Cur, the MAC inhibition of IAV did not occur through the blocking of HA activity. However, MAC strongly dampened Akt phosphorylation, the prerequisite signalling for efficient IAV propagation. A much stronger inhibition effect on IAV infection was observed when MAC treatment was in combination with Cur. Collectively, MAC demonstrated clear antiviral activity, and likely inhibited IAV via multiple mechanisms that were not identical to Cur. Importantly, Cur and MAC in combination synergistically inhibited IAV infection.

Detection of lithospermate B in rat plasma at the nanogram level by LC/MS in multi reaction monitoring mode.

Low bioavailability and high binding affinity to plasma proteins led to the difficulty for the quantitative detection of lithospermate B (LSB) in plasma. This study aimed to develop a protocol for detecting LSB in plasma. A method was employed to quantitatively detect LSB of 5-500 ng/mL by LC/MS spectrometry in multi reaction monitoring mode via monitoring two major fragments with m/z values of 519 and 321 in the MS2 spectrum. To set up an adequate extraction solution to release LSB captured by plasma proteins, recovery yields of LSB extracted from rat plasma acidified by formic acid or HCl in the presence or absence of EDTA and caffeic acid were detected and compared using the above quantitative method. High recovery yield (∼90%) was achieved when LSB (5-500 ng/mL) mixed in rat plasma was acidified by HCl (5 M) in the presence of EDTA (0.5 M) and caffeic acid (400 µg/mL). The lower limit of detection and the lower limit of quantification for LSB in the spiked plasma were calculated to be 1.8 and 5.4 ng/mL, respectively. Good accuracy (within ±10%) and precision (less than 10%) of intra- and inter-day quality controlled samples were observed. Oral bioavailability of LSB in rat model was detected via this optimized extraction method, and the maximum plasma concentration (Cmax) was found to be 1034.3 ± 510.5 µg/L at tmax around 10 min, and the area under the plasma concentration-time curve (AUC) was 1414.1 ± 851.2 µg·h/L.

In vitro assay to estimate tea astringency via observing flotation of artificial oil bodies sheltered by caleosin fused with histatin 3.

Astringency, a sensory characteristic of food and beverages rich in polyphenols, mainly results from the formation of complexes between polyphenols and salivary proteins, causing a reduction of the lubricating properties of saliva. To develop an in vitro assay to estimate the astringency of oolong tea infusion, artificial oil bodies were constituted with sesame oil sheltered by a modified caleosin fused with histatin 3, one of the human salivary small peptides. Aggregation of artificial oil bodies was induced when they were mixed with oolong tea infusion or its major polyphenolic compound, (-)-epigallocatechin gallate (EGCG) of 100µM as observed in light microscopy. The aggregated artificial oil bodies gradually floated on top of the solution and formed a visible milky layer whose thickness was in proportion to the concentrations of tea infusion. This assay system was applied to test four different oolong tea infusions with sensory astringency corresponding to their EGCG contents. The result showed that relative astringency of the four tea infusions was correlated to the thickness of floated artificial oil bodies, and could be estimated according to the standard curve generated by simultaneously observing a serial dilution of the tea infusion with the highest astringency.

Identification of steroleosin in oil bodies of pine megagametophytes.

Three classes of integral proteins termed oleosin, caleosin and steroleosin have been identified in seed oil bodies of diverse angiosperm species. Recently, two oleosin isoforms and one caleosin were identified in megagametophyte oil bodies of pine (Pinus massoniana), a representative gymnosperm species. In this study, a putative steroleosin of approximately 41 kDa was observed in isolated oil bodies of pine megagametophytes, and its corresponding cDNA fragment was obtained by PCR cloning and further confirmed by mass spectrometric analysis. Phylogenetic tree analysis showed that pine steroleosin was evolutionarily more closely-related to steroleosin-B than steroleosin-A found in angiosperm seed oil bodies. As expected, artificial oil bodies constituted with recombinant steroleosin over-expressed in Escherichia coli were less stable and larger than native pine oil bodies. Filipin staining of artificial oil bodies sheltered by recombinant steroleosin with or without its sterol binding domain showed that the sterol binding domain was responsible for the sterol binding capability of steroleosin. Sterol-coupling dehydrogenase activity was demonstrated in artificial oil bodies constituted with recombinant steroleosin as well as in purified pine oil bodies.

Ginkgoghrelins, unique acylated flavonoid diglycosides in Folium Ginkgo, stimulate growth hormone secretion via activation of the ghrelin receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Folium Ginkgo, the dried leaf of Ginkgo biloba L. is a traditional Chinese medicine listed in the Pharmacopoeia of the People's Republic of China with several therapeutic effects, including prevention of aging. It is used as herbal medicine for the treatment of several aging-related diseases. The therapeutic effects of Folium Ginkgo on aging-related diseases are suspected to be similar to the anti-aging effects of growth hormone release induced by ghrelin. MATERIALS AND METHODS: Candidate components responsible for the anti-aging effects via the ghrelin receptor-activated pathway were searched from the known compounds found in Folium Ginkgo. Two acylated flavonoid diglycosides, tentatively named ginkgoghrelins in this study, were selected and isolated from the methanol extract of Folium Ginkgo, and their chemical structures were confirmed by spectroscopic analysis. These two compounds were examined for their capability of stimulating growth hormone release of rat primary anterior pituitary cells via activation of the ghrelin receptor. The major metabolites of ginkgoghrelins in rat bile were detected after intravenous injection and structurally analyzed by mass spectroscopy. Molecular modeling of ginkgoghrelins docking to the ghrelin receptor was exhibited to explore the possible interaction within the binding pocket. RESULTS: Similar to growth hormone-releasing hormone-6 (GHRP-6), a synthetic analog of ghrelin, ginkgoghrelins were demonstrated to stimulate growth hormone secretion of rat primary anterior pituitary cells in a dose dependent manner, and the stimulation was inhibited by [d-Arg1, d-Phe5, d-Trp7,9, Leu11]-substance P, an inverse agonist of the ghrelin receptor. Putative metabolites of ginkgoghrelins via glucuronidation and methylation were detected in bile of rats after intravenous injection. Molecular modeling and docking showed that ginkgoghrelins as well as GHRP-6 could fit in and adequately interact with the binding pocket of the ghrelin receptor. CONCLUSION: The results suggest that ginkgoghrelins are putative components partly accounting for the anti-aging effects of Folium Ginkgo possibly via activation of the ghrelin receptor, and possess great potential to be developed as non-peptidyl analogs of ghrelin.

Novel stereoselective bufadienolides reveal new insights into the requirements for Na(+), K(+)-ATPase inhibition by cardiotonic steroids.

Cardiotonic steroids (CTS) are clinically important drugs for the treatment of heart failure owing to their potent inhibition of cardiac Na(+), K(+)-ATPase (NKA). Bufadienolides constitute one of the two major classes of CTS, but little is known about how they interact with NKA. We report a remarkable stereoselectivity of NKA inhibition by native 3ß-hydroxy bufalin over the 3α-isomer, yet replacing the 3ß-hydroxy group with larger polar groups in the same configuration enhances inhibitory potency. Binding of the two (13)C-labelled glycosyl diastereomers to NKA were studied by solid-state NMR (SSNMR), which revealed interactions of the glucose group of the 3ß- derivative with the inhibitory site, but much weaker interactions of the 3α- derivative with the enzyme. Molecular docking simulations suggest that the polar 3ß-groups are closer to the hydrophilic amino acid residues in the entrance of the ligand-binding pocket than those with α-configuration. These first insights into the stereoselective inhibition of NKA by bufadienolides highlight the important role of the hydrophilic moieties at C3 for binding, and may explain why only 3ß-hydroxylated bufadienolides are present as a toxic chemical defence in toad venom.

Identification of biosynthetic intermediates of teaghrelins and teaghrelin-like compounds in oolong teas, and their molecular docking to the ghrelin receptor.

Teaghrelins are unique acylated flavonoid tetraglycosides found in Chin-shin oolong tea, and have been demonstrated to be promising oral ghrelin analogues. The biosynthetic pathway of teaghrelins from quercetin-3-O-rutinoside (rutin) or kaempferol-3-O-rutinoside (nicotiflorin) was proposed to comprise three enzymatic steps according to the identification of putative intermediates in Chin-shin oolong tea. In addition to the two known teaghrelins in Chin-shin oolong tea, four teaghrelin-like compounds with different attachments of glycosides were identified in various oolong teas. Molecular modeling and docking were used to evaluate theoretically whether the putative biosynthetic intermediates of teaghrelins and the four teaghrelin-like compounds could be potential candidates of ghrelin analogues. The results showed that the attachment of a coumaroyl group was crucial for these tea compounds to bind to the ghrelin receptor. However, the additional attachment of a rhamnosyl glycoside to the flavonoid backbone of teaghrelin-like compounds at C-7 significantly reduced their binding affinity with the ghrelin receptor.

Analysis of lipophilic compounds of tea coated on the surface of clay teapots.

The surface of a clay teapot tends to be coated with a waterproof film after constant use for tea preparation. The waterproof films of two kinds of teapots (zisha and zhuni) used for preparing oolong tea and old oolong tea were extracted and subjected to gas chromatography-mass spectrometry analysis. The results showed that comparable constituents were detected in these films; they were primarily fatty acids and linear hydrocarbons that were particularly rich in palmitic acid and stearic acid. To explore the source of these two abundant fatty acids, the fatty acid compositions of fresh tea leaves, granules, infusion, and vapor of infusion were analyzed by gas chromatography. Fresh tea leaves were rich in palmitic acid (C-16:0), unsaturated linolenic acid (C-18:3), linoleic acid (C-18:2), and oleic acid (C-18:1), which were presumably from the phospholipid membrane. During the process of manufacturing oolong tea, the three unsaturated fatty acids may be substantially degraded or oxidized to stearic acid (C-18:0), which was enriched with palmitic acid in the tea granules and in the infusion. The vapor of the tea infusion is primarily composed of palmitic acid and stearic acid. Thus, the coated films of teapots mostly originated from the lipophilic compounds of the tea infusions.

Emoghrelin, a unique emodin derivative in Heshouwu, stimulates growth hormone secretion via activation of the ghrelin receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Heshouwu, the root of Polygonum multiflorum, is an anti-aging Chinese traditional medicine. Fresh (raw) Heshouwu is commonly converted to processed Heshouwu by specialized heating to alleviate its side effects of diarrhea presumably caused by anthraquinones. However, raw Heshouwu has been noted to be better than processed Heshouwu regarding anti-aging effects. The therapeutic effects of raw Heshouwu on aging-related diseases were somehow similar to the anti-aging effects of growth hormone release induced by ghrelin MATERIALS AND METHODS: Major ingredients in the methanol extract from raw Heshouwu were separated and identified. Emodin-8-O-(6'-O-malonyl)-glucoside, a unique anthraquinone glycoside known to be completely eliminated in the conversion process of Heshouwu was isolated. This emodin derivative, tentatively named emoghrelin, was examined for its cytotoxicity and capability of stimulating growth hormone release of rat primary anterior pituitary cells via activation of the ghrelin receptor. Moreover, molecular modeling of emoghrelin docking to the ghrelin receptor was exhibited to explore the possible interaction within the binding pocket. RESULTS: No apparent cytotoxicity was observed for emoghrelin of 10(-7)-10(-4)M. Similar to growth hormone-releasing hormone-6 (GHRP-6), a synthetic analog of ghrelin, emoghrelin was demonstrated to stimulate growth hormone secretion of rat primary anterior pituitary cells in a dose dependent manner, and the stimulation was inhibited by [d-Arg(1), d-Phe(5), d-Trp(7,9), Leu(11)]-substance P, an antagonist of the ghrelin receptor. Molecular modeling and docking showed that emoghrelin as well as GHRP-6 could fit in and adequately interact with the binding pocket of the ghrelin receptor. CONCLUSION: The results suggest that emoghrelin is a key ingredient accounting for the anti-aging effects of Heshouwu, and possesses great potential to be a promising non-peptidyl analog of ghrelin.

Identification of caleosin and two oleosin isoforms in oil bodies of pine megagametophytes.

Numerous oil bodies of 0.2-2 µm occupied approximately 80% of intracellular space in mature pine (Pinus massoniana) megagametophytes. They were stably isolated and found to comprise mostly triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Fatty acids released from the triacylglycerols of pine oil bodies were mainly unsaturated, including linoleic acid (60%), adrenic acid (12.3%) and vaccenic acid (9.7%). Proteins extracted from pine oil bodies were subjected to immunological cross-recognition, and the results showed that three proteins of 28, 16 and 14 kDa were detected by antibodies against sesame seed caleosin, sesame oleosin-L and lily pollen oleosin-P, respectively. Complete cDNA fragments encoding these three pine oil-body proteins, tentatively named caleosin, oleosin-L and oleosin-G, were obtained by PCR cloning and further confirmed by mass spectrometric analysis. Consistently, phylogenetic tree analyses showed that pine caleosin was closely-related to the caleosin of cycad megagametophyte among known caleosin sequences. While pine oleosin-L was found clustered with seed oleosin isoforms of angiosperm species, oleosin-G was distinctively grouped with the oleosin-P of lily pollen. The oleosin-G identified in pine megagametophytes seems to represent a new class of seed oleosin isoform evolutionarily close to the pollen oleosin-P.

Binding orientation and specificity of calmodulin to rat olfactory cyclic nucleotide-gated ion channel.

Calmodulin (CaM), the primary intracellular Ca(2+) receptor, regulates a large number of key enzymes and controls a wide spectrum of important biological responses. Recognition between CaM and its target sequence in rat olfactory cyclic nucleotide-gated ion channel (OLFp) was investigated by circular dichroism (CD), fluorescence, and NMR spectroscopy. Fluorescence data showed the OLFp tightly bound to CaM with a dissociation constant of 12 nM in a 1:1 stoichiometry. Far-UV CD data showed that approximately 60% of OLFp residues formed α-helical structures when associated with CaM. NMR data showed that most of the (15)N-(1)H HSQC cross-peaks of the (15)N-labeled CaM not only shifted but also split into two sets of peaks upon association with the OLFp. Our data indicated that the two distinct CaM/OLFp complexes existed simultaneously with stable structures that were not interexchangeable within the NMR time scale. In light of the palindromic sequence of OLFp (FQRIVRLVGVIRDW) for CaM targeting, we proposed that the helical OLFp with C2 symmetry may bind to CaM in two orientations. This hypothesis is supported by the observation that only one set of (15)N-(1)H HSQC cross-peaks of the (15)N-labeled CaM was detected upon association with OLFp-M13 chimeric peptide (OLFMp), a mutated OLFp lacking the palindromic feature. The binding specificity of OLFMp to CaM was restored when the palindromic feature was destroyed. Binding modes of CaM/OLFp and CaM/OLFMp simulated by molecular docking were in accord with their distinct patterns observed in HSQC spectra. Our studies suggest that the palindromic residues in OLFp are crucial for the orientation-specific recognition by CaM.

Inhibition of Na(+)/K(+) -ATPase by antcins, unique steroid-like compounds in Antrodia camphorate.

The inhibition of Na(+)/K(+) -ATPase by versatile steroid-like compounds contributes to the putative therapeutic effects of many Chinese medicinal cardiac products via the same molecular mechanism triggered by cardiac glycosides. Five major steroid-like compounds, antcin A, B, C, H, and K were isolated from Niuchangchih (Antrodia camphorata), a unique Taiwan mushroom, and all inhibited Na(+)/K(+) -ATPase. Antcin A exhibited significantly higher inhibitory potency than the other four antcins, though weaker than ginsenoside Rh2 . In contrast, cortisone (an analogous steroid with anti-inflammatory effects stronger than antcin A) showed no detectable inhibitory potency. Molecular modeling has shown that antcins bind to Na(+)/K(+) -ATPase with the steroidal skeleton structurally upside-down in comparison with ginsenoside Rh2 . The inhibitory potency of antcin A is attributed to steroidal hydrophobic interaction within the binding pocket and the formation of three hydrogen bonds between its carboxyl group and two cationic residues around the cavity entrance of Na(+)/K(+) -ATPase. The presence of an additional carbonyl or hydroxyl group at C7 of the other four antcins leads to severe repulsion in the hydrophobic pocket, and thus significantly reduces inhibitory potency. It is proposed that antcin A is a bi-functional compound that exerts anti-inflammatory effects and that enhances blood circulation via two different molecular mechanisms.