Establishment of the model of white blood cell membrane chromatography and screening of antagonizing TLR4 receptor component from Atractylodes macrocephala Koidz

A model of white blood cell membrane chromatography (WB-CMC) was established to screen active component from Atractylodes macrocephala Koidz. The component can antagonize Toll-like receptor 4 (TLR₄) and inhibit inflammatory reaction. In the model of WB-CMC, cell membrane stationary phase (CMSP) was prepared by immobilizing the rabbit white blood cell membrane (WBCM) onto the surface of silica carrier and taxinol was used as a model molecule. The active component which can act on WBCM and its receptor (such as TLR₄) as an effective target in A. macrocephala was determined by using a replacement experiment. The anti-inflammatory effects of the active component were tested by using pharmacological methods in vivo. The results indicated that the retention characteristics of atractylenolide I as active component was similar to that of taxinol in the model of WB-CMC. And so, atractylenolide I acted on the WBCM and TLR₄ and its anti-inflammatory activity was related with antagonizing TLR₄. Therefore, the interaction between the active component and WBCM and its receptor can be simulated by the model of WB-CMC in vitro. This model can be used to screen active components and to study effective characteristics for acting on definite targets.     

Establishment of the model of white blood cell membrane chromatography and screening of antagonizing TLR_4 receptor component from Atractylodes macrocephala Koidz

Inflammation is a common and fundamental patho- physiological response. The inflammatory reaction is characterized by activation of white blood cell (WBC) with an accompanying vascular reaction. Recently, it has been found that the inflammatory reaction d…     

Evidence of Phytoalexins in Rhizome of Atractylodis Maceocephalae Koidz Infected with Sclerotium rolfsii sacc Following Treatment with the Polysaccharides of Chrysanthemum indicum

Atractylenolides compounds (atractylenolide‐I, atractylenolide‐II and atractylenolide‐III) extracted from Atractylenolides macrocephala were analysed for their differential presence in the rhizome of both the infected and healthy plants and its fungitoxicity on prophylactic treatment with polysaccharides of Chrysanthemum indicum against Sclerotium rolfsii. It was evident that the plant under stress by pathogen has instigated the significant synthesis and accumulation of atractylenolide‐II and atractylenolide‐III in the rhizome of plants as a result of elicitation with polysaccharides of C. indicum. Our results also showed that the absence of atractylenolide‐II in the rhizome made the plant susceptible to S. rolfsii. Furthermore, on in vitro antifungal studies, exogenous application of atractylenolide‐II with the concentration of 200 μg/ml showed a significant inhibitory effect on mycelial growth by achieving a 77.23% antifungal activity rate, and a minimal inhibitory effect at a concentration of 12.5 μg/ml with atractylenolide‐II. To our knowledge, this novel study on phytoalexins in A. macrocephala suggested that A. macrocephala plants produce elevated levels of atractylenolide‐II and atractylenolide‐III in a pattern typical of phytoalexins, in response to an eliciting treatment after infection.     

Neuroprotection of Atractylenolide III from Atractylodis macrocephalae Against Glutamate-Induced Neuronal Apoptosis Via Inhibiting Caspase Signaling Pathway

Glutamate-induced excitotoxicity appears to play a crucial role in neurological disorders. Neuroprotection against glutamate-induced excitotoxicity has been proposed as a therapeutic strategy for preventing and/or treating these excitotoxicity-mediated diseases. In the present study, atractylenolide III, which exhibited significantly neuroprotective effect against glutamate-induced neuronal apoptosis, was isolated from Atractylodes macrocephala by means of bioactivity-guided fractionation. The inhibitory effect of atractylenolide III on glutamate-induced neuronal apoptosis was in a concentration-dependent manner. The anti-apoptotic property of atractylenolide III might be mediated, in part, via inhibiting caspase signaling pathway. Atractylenolide III may have therapeutic potential in excitotoxicity-mediated neurological diseases.     

Species Differentiation and Quality Evaluation for Atractylodes Medicinal Plants by GC/MS Coupled with Chemometric Analysis

The utilization of rhizomes from the genus Atractylodes has been challenging due to their closely related origins. In this study, we developed an analytical strategy to differentiate Atractylodes lancea (A. lancea), Atractylodes chinensis (A. chinensis), Atractylodes japonica (A. japonica), and Atractylodes macrocephala (A. macrocephala), and compared their volatile compositions. Gas chromatography-mass spectrometry (GC/MS) was used to analyze the volatile profiles of essential oils extracted from 59 batches of samples. Chemometric methods enabled a better understanding of the differences in volatile oils between the four species and identified significant components affecting their classification and quality. A total of 50 volatile components were identified from the essential oils by GC/MS. Unsupervised and supervised chemometric analyses accurately distinguished A. lancea, A. chinensis, A. japonica, and A. macrocephala. Furthermore, five characteristic chemical markers, namely hinesol, β-eudesmol, atractylon, atractylodin and atractylenolide I, were obtained, and their respective percentage contents in individual species and samples were determined. This study provides a valuable reference for the quality evaluation of medicinal plants with essential oils and holds significance for species differentiation and the rational clinical application of Atractylodes herbs.     

Sevoflurane Inhibits Nuclear Factor-κB Activation in Lipopolysaccharide-Induced Acute Inflammatory Lung Injury via Toll-Like Receptor 4 Signaling

Background

Infection is a common cause of acute lung injury (ALI). This study was aimed to explore whether Toll-like receptors 4 (TLR₄) of airway smooth muscle cells (ASMCs) play a role in lipopolysaccharide (LPS)-induced airway hyperresponsiveness and potential mechanisms.

Methods

In vivo: A sensitizing dose of LPS (50 µg) was administered i.p. to female mice before anesthesia with either 3% sevoflurane or phenobarbital i.p. After stabilization, the mice were challenged with 5 µg of intratracheal LPS to mimic inflammatory attack. The effects of sevoflurane were assessed by measurement of airway responsiveness to methacholine, histological examination, and IL-1, IL-6, TNF-α levels in bronchoalveolar lavage fluid (BALF). Protein and gene expression of TLR₄ and NF-κB were also assessed. In vitro: After pre-sensitization of ASMCs and ASM segments for 24h, levels of TLR₄ and NF-κB proteins in cultured ASMCs were measured after continuous LPS exposure for 1, 3, 5, 12 and 24h in presence or absence of sevoflurane. Constrictor and relaxant responsiveness of ASM was measured 24 h afterwards.

Results

The mRNA and protein levels of NF-κB and TLR₄ in ASM were increased and maintained at high level after LPS challenge throughout 24h observation period, both in vivo and in vitro. Sevoflurane reduced LPS-induced airway hyperresponsiveness, lung inflammatory cell infiltration and proinflammatory cytokines release in BALF as well as maximal isometric contractile force of ASM segments to acetylcholine, but it increased maximal relaxation response to isoproterenol. Treatment with specific NF-κB inhibitor produced similar protections as sevoflurane, including decreased expressions of TLR₄ and NF-κB in cultured ASMCs and improved pharmacodynamic responsiveness of ASM to ACh and isoproterenol.

Conclusions

This study demonstrates the crucial role of TLR₄ activation in ASMCs during ALI in response to LPS. Sevoflurane exerts direct relaxant and anti-inflammatory effects in vivo and in vitro via inhibition of TLR₄/NF-κB pathway.     

不同土壤硒水平对白术幼苗生长及部分生理特性的影响

硒(Se)为植物非必需营养元素,但适量的硒对植物生长发育具有促进作用,因此研究植物生长对硒的响应及其生理机制具有重要意义。该研究采用盆栽试验,设置6种不同的土壤硒处理(0、5、10、25、50、100 mg·kg^-1),观察不同硒处理对白术幼苗生理生长的影响及其综合效应。结果表明：(1)相比于对照处理,5 mg·kg^-1的硒处理可以提高白术幼苗鲜重、叶片叶绿素相对含量(SPAD值)以及可溶性糖(SS)含量,增幅分别为8.8%、8.4%和60.3%;显著降低白术幼苗叶片的过氧化物酶(POD)和过氧化氢酶(CAT)活性;同时减少叶片可溶性蛋白(SP)和丙二醛(MDA)含量,降幅分别为32.1%和18.9%。(2)25 mg·kg^-1以上硒处理则不利于白术幼苗的生长,会导致MDA和超氧阴离子自由基(·O₂^-)含量显著上升,使白术幼苗的抗氧化系统遭受胁迫。(3)主成分分析表明,鲜重、株高、叶片SP含量、CAT和POD活性等可作为反映白术幼苗生理生长的重要特征指标。综上表明,土施适量硒可...     

Genetic drift outweighs natural selection at toll‐like receptor (TLR) immunity loci in a re‐introduced population of a threatened species

During population establishment, genetic drift can be the key driver of changes in genetic diversity, particularly while the population is small. However, natural selection can also play a role in shaping diversity at functionally important loci. We used a well‐studied, re‐introduced population of the threatened Stewart Island robin (N = 722 pedigreed individuals) to determine whether selection shaped genetic diversity at innate immunity toll‐like receptor (TLR) genes, over a 9‐year period of population growth following establishment with 12 genetic founders. We found no evidence for selection operating with respect to TLR diversity on first‐year overwinter survival for the majority of loci, genotypes and alleles studied. However, survival of individuals with TLR4_BE genotype was significantly improved: these birds were less than half as likely to die prior to maturity compared with all other TLR4 genotypes. Furthermore, the population frequency of this genotype, at a two‐fold excess over Hardy–Weinberg expectation, was increased by nonrandom mating. Near‐complete sampling and full pedigree and reproductive data enabled us to eliminate other potential causes of these patterns including inbreeding, year effects, density dependence, selection on animals at earlier life history stages or genome‐level association of the TLR4_E allele with ‘good genes’. However, comparison of observed levels of gene diversity to predictions under simulated genetic drift revealed results consistent with neutral expectations for all loci, including TLR4. Although selection favoured TLR4_BE heterozygotes in this population, these effects were insufficient to outweigh genetic drift. This is the first empirical study to show that genetic drift can overwhelm natural selection in a wild population immediately following establishment.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

猫须草抗炎活性成分研究

为了研究猫须草(Clerodendranthus spicatus)抗炎活性成分,该研究采用活性追踪的方法,利用硅胶、MCI柱色谱以及HPLC等分离技术,对猫须草抗炎活性部位进行分离纯化,通过波谱数据分析和文献比对鉴定化合物结构,并利用LPS诱导的RAW 264.7巨噬细胞炎症模型进行抗炎活性评价。结果表明:(1)从猫须草抗炎活性部位分离得到10个化合物,分别鉴定为泡桐素(1)、鼠尾草素(2)、对苯二甲酸二辛酯(3)、N-(N-苯甲酰基-L-苯丙酰基)-L-苯基丙醇(4)、fragransin B₁(5)、6,7,8,4''-四甲氧基黄酮(6)、N-反式-阿魏酰酪胺(7)、N-顺式-阿魏酰酪胺(8)、trans-N-cinnamoyltyramine(9)、新海胆灵 A(10),其中化合物1、4、8-10为首次从该植物中分离得到。(2)抗炎结果显示,猫须草抗炎活性成分主要存在于中低极性部位,从中分离得到的大部分化合物显示出一定的NO生成抑制活性,其中酰胺类成分(7-9)均具有较好的抗炎活性,表明该类成分是猫须草抗炎作用的主要成分之一。该研究丰富了猫须草抗炎物质基础,为其开发利用提供科学依据。     

Role of membrane gangliosides in the binding and action of bacterial toxins

Summary Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside G_M1. Toxin-resistant cells which lack G_M1 can be sensitized to cholera toxin by treating them with G_M1. Cholera toxin specifically protects G_M1 from cell surface labeling procedures and only G_M1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that G_M1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to G_M1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. G_M1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin ofE. coli is very similar to cholera toxin in both form and function and can also use G_M1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides G_D1b and G_T1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established G_M1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.     

Endosomal location of dopamine receptors in neuronal cell cytoplasm

Five subtypes of dopamine receptor exist in two subfamilies: two D₁-like (D₁ and D₅) and three D₂-like (D₂, D₃ and D₄). We produced novel monoclonal antibodies against all three D₂-like receptors and used them to localize receptors in Ntera-2 (NT-2) cells, the human neuronal precursor cell line. Most of the immunostaining for all three receptors colocalized with mannose-6-phosphate receptor, a marker for late endosomes formed by internalization of the plasma membrane. This result was obtained with antibodies against three different epitopes on the D₃ receptor, to rule out the possibility of cross-reaction with another protein, and controls without primary antibody or in the presence of competitor antigen were completely negative. In rat cerebral cortex and hippocampus, some of the dopamine receptor staining was found in similar structures in neuronal cell cytoplasm. Only some of the neurons were positive for dopamine receptors and the pattern was consistent with previously-reported patterns of innervation by dopamine-producing neurons. Endosomal dopamine receptors may provide a useful method for identifying cell bodies of dopamine-responsive neurons to complement methods that detect only active receptors in the neuronal cell membrane.     

The effect of staurosporine, a protein kinase inhibitor, on asialoglycoprotein receptor endocytosis

Receptor-mediated endocytosis via coated pits is modulated by the activity of protein kinases and protein phosphorylation. We examined the effects of the potent protein kinase inhibitor staurosporine (SSP) on endocytosis of the asialoglycoprotein (ASGP) receptor in HepG2 cells. Staurosporine caused a rapid (<2 min) inhibition of ligand internalization from the cell surface. In contrast the rate of receptor exocytosis from intracellular compartments to the cell surface was not altered (t_1/2 = 8 min). This resulted in increased ASGP receptors at the plasma membrane (140% of control) while the total number of receptors per cell was unchanged. Receptor up-regulation was half-maximal at 30 nM SSP. At this concentration staurosporine also inhibited the internalization of iodinated transferrin by HepG2 cells and SK Hep-1 cells, another human hepatoma-derived cell line. Staurosporine was without effect on the non-receptor-mediated uptake of Lucifer yellow by pinocytosis. We investigated the possible involvement of protein kinase C in the inhibitory effects of staurosporine on receptor endocytosis. The active protein kinase C inhibitor H7 did not inhibit ASGP receptor internalization. Furthermore depletion of cellular protein kinase C by overnight incubation with 1 μM phorbol myristate acetate did not abrogate the SSP effect. Together these data suggest that the mechanism of SSP action is independent of the inhibition of protein kinase C. In conclusion staurosporine is a potent and rapid inhibitor of receptor trafficking which is specific for receptor internalization from the plasma membrane.     

Insecticidal and Repellant Activities of Polyacetylenes and Lactones Derived from Atractylodes lancea Rhizomes

During a screening program for new agrochemicals from Chinese medicinal herbs and local wild plants, the petroleum ether (PE) extract of Atractylodes lancea (Thunb. ) rhizomes was found to possess repellent and contact activities against Tribolium castaneum adults. Bioactivity‐directed chromatographic separation of PE extract on repeated silica‐gel columns led to the isolation of two polyacetylenes, atractylodin and atractylodinol ( 1 and 2 , resp.), and two lactones, atractylenolides II and III ( 3 and 4 , resp.). The structures of the compounds were elucidated based on NMR spectra. The four isolated compounds were evaluated for their insecticidal and repellent activities against T. castaneum. Atractylodin exhibited strong contact activity against T. castaneum adults with a LD₅₀ value of 1.83 μg/adult. Atractylodin and atractylenolide II also possessed strong repellenct activities against T. castaneum adults. After 4‐h exposure, >90% repellency was achieved with atractylodin at a low concentration of 0.63 μg/cm². The results indicated that atractylodin ( 1 ) and atractylenolide II ( 3 ) have a good potential as a source for natural repellents, and 1 has the potential to be developed as natural insecticide.     

A combination of pharmacophore modeling,atom-based 3D-QSAR,molecular docking and molecular dynamics simulation studies on PDE4 enzyme inhibitors

Phosphodiesterases 4 enzyme is an attractive target for the design of anti-inflammatory and bronchodilator agents. In the present study, pharmacophore and atom-based 3D-QSAR studies were carried out for pyrazolopyridine and quinoline derivatives using Schrödinger suite 2014-3. A four-point pharmacophore model was developed using 74 molecules having pIC₅₀ ranging from 10.1 to 4.5. The best four feature model consists of one hydrogen bond acceptor, two aromatic rings, and one hydrophobic group. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a high correlation coefficient (R^2?=?.9949), cross validation coefficient (Q^2?=?.7291), and Pearson-r (.9107) at six component partial least square factor. The external validation indicated that our QSAR model possessed high predictive power with R² value of .88. The generated model was further validated by enrichment studies using the decoy test. Molecular docking, free energy calculation, and molecular dynamics (MD) simulation studies have been performed to explore the putative binding modes of these ligands. A 10-ns MD simulation confirmed the docking results of both stability of the 1XMU–ligand complex and the presumed active conformation. Outcomes of the present study provide insight in designing novel molecules with better PDE4 inhibitory activity.     

Using genetic diversity information to establish core collections of Stylosanthes capitata and Stylosanthes macrocephala

Stylosanthes species are important forage legumes in tropical and subtropical areas. S. macrocephala and S. capitata germplasm collections that consist of 134 and 192 accessions, respectively, are maintained at the Brazilian Agricultural Research Corporation Cerrados (Embrapa-Cerrados). Polymorphic microsatellite markers were used to assess genetic diversity and population structure with the aim to assemble a core collection. The mean values of H_O and H_E for S. macrocephala were 0.08 and 0.36, respectively, whereas the means for S. capitata were 0.48 and 0.50, respectively. Roger’s genetic distance varied from 0 to 0.83 for S. macrocephala and from 0 to 0.85 for S. capitata. Analysis with STRUCTURE software distinguished five groups among the S. macrocephala accessions and four groups among those of S. capitata. Nei’s genetic diversity was 27% in S. macrocephala and 11% in S. capitata. Core collections were assembled for both species. For S. macrocephala, all of the allelic diversity was represented by 23 accessions, whereas only 13 accessions were necessary to represent all allelic diversity for S. capitata. The data presented herein evidence the population structure present in the Embrapa-Cerrados germplasm collections of S. macrocephala and S. capitata, which may be useful for breeding programs and germplasm conservation.     

Isolation and characterization of 11 microsatellite loci from Carex macrocephala (Cyperaceae)

Carex macrocephala (Cyperaceae) is a species of great conservation concern along the northwest coast of North America. Using a biotinylated-streptavidin bead protocol, we successfully isolated and characterized 11 microsatellite loci from a C. macrocephala genomic library. Although these loci are polymorphic, the scored populations exhibit a significant deficiency in heterozygosity. Carex macrocephala is a self-pollinating rhizomatously spreading sedge, consequently this may contribute to the observed patterns.     

Electrophysiological and Theoretical Analysis of Depolarization-Dependent Outward Currents in the Dendritic Membrane of an Identified Nonspiking Interneuron in Crayfish

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 ± 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I _s) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I _t1) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I _t2) was blocked by 100 M 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at –21.0 mV in I _t1, and –38.0 mV in I _t2. I _s showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at –36.0 mV in I _s, –31.0 mV in I _t1 and –40.0 mV in I _t2. The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.     

DIDS increases K+ secretion through an I sKchannel in apical membrane of vestibular dark cell epithelium of gerbil

Vestibular dark cell epithelium secretes K⁺ via I _sKchannels in the apical membrane. The previous observation that disulfonic stilbenes increased the equivalent short circuit current (I _sc) suggested that these agents might be useful investigative tools in this tissue. The present experiments were conducted to determine if the increase in I _scwas associated with an increase in K⁺ flux and if the effect was directly on the I _sKchannel or indirectly via a cytosolic intermediary. Measurements of transepithelial K⁺ flux with the K⁺-selective vibrating probe and of changes in net cellular solute flux by measurements of epithelial cell height showed that 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) increased K⁺ flux by a factor of 1.96±0.71 and caused net solute efflux. The apical membrane was partitioned with a macropatch pipette and DIDS was applied either to the membrane outside the pipette, inside the pipette or to the entire apical membrane. DIDS inside the pipette increased the current across the patch, the membrane conductance, the slowly-inactivating (I _sK) component of the membrane current and shifted the reversal voltage toward the equilibrium potential for K⁺. DIDS outside the patch decreased the patch current and conductance, consistent with shunting of current away from the membrane patch. These findings strongly support the notion that DIDS increases K⁺ secretion through I _sKchannels in the apical membrane of vestibular dark cell epithelium by acting directly on the channels or on a tightly colocalized membrane component.We thank Dr. Peter J.S. Smith and Alan Shipley of the National Vibrating Probe Facility at the Marine Biological Laboratory at Woods Hole, MA for their support and assistance in the measurements of K⁺ flux. This work was supported by National Institutes of Health grants R01-DC00212, R29-DC1098 and P41-RR01395.