Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane¹. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex^2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein''s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (K_Na) channels by GPCRs.     

Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization

The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.     

Cell-based biosensors based on light-addressable potentiometric sensors for single cell monitoring

Cell-based biosensors incorporate cells as sensing elements that convert changes in immediate environment to signals for processing. This paper reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible cell-base biosensor that will enable us to monitor extracellular action potential of single living cell under stimulant. In order to modify chip surface and immobilize cells, we coat a layer of poly-L-ornithine and laminin on surface of LAPS chip on which rat cortical cells are grown well. When 10 microg/ml acetylcholine solution is administrated, the light pointer is focused on a single neuronal cell and the extracellular action potential of the targeted cell is recorded with cell-based biosensor based on LAPS. The results demonstrate that this kind of biosensor has potential to monitor electrophysiology of living cell non-invasive for a long term, and to evaluate drugs primarily.     

Advances in Cell‐Free Biosensors: Principle,Mechanism, and Applications

Synthetic biology has promoted the development of biosensors as tools for detecting trace substances. In the past, biosensors based on synthetic biology have been designed on living cells, but the development of cell biosensors has been greatly limited by defects such as genetically modified organism problem and the obstruction of cell membrane. However, the advent of cell‐free synthetic biology addresses these limitations. Biosensors based on the cell‐free protein synthesis system have the advantages of higher safety, higher sensitivity, and faster response time over cell biosensors, which make cell‐free biosensors have a broader application prospect. This review summarizes the workflow of various cell‐free biosensors, including the identification of analytes and signal output. The detection range of cell‐free biosensors is greatly enlarged by different recognition mechanisms and output methods. In addition, the review also discusses the applications of cell‐free biosensors in environmental monitoring and health diagnosis, as well as existing deficiencies and aspects that should be improved. In the future, through continuous improvement and optimization, the potential of cell‐free biosensors will be stimulated, and their application fields will be expanded.     

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.     

Antifungal activity in triterpene glycosides from the sea cucumber Actinopyga lecanora

Bioassay-guided fractionation of methanol extract of sea cucumber Actinopyga lecanora led to the isolation of a new triterpene glycoside (1), along with two known glycosides holothurin B (3) and holothurin A (4). The structure has been elucidated on the basis of extensive 2D NMR spectroscopic analysis. The saponin (3) showed in vitro antifungal activity against all the twenty fungal test isolates including ATCC strain and was found to be most effective against Trychophyton mentagrophytes and Sporothrix schenckii, MIC range of 1.56 microg/ml.     

Isolation and functional analysis of a 24-residue linear alpha-helical antimicrobial peptide from Korean blackish cicada, Cryptotympana dubia (Homoptera)

A new antimicrobial peptide, cryptonin, was isolated and characterized from the adult Korean blackish cicada, Cryptotympana dubia. It consists of 24 amino acid residues and has a molecular weight of 2,704 Da on mass spectroscopy. The predicted alpha-helical structure analysis and increased helix percent in 40% trifloroethanol of cryptonin suggests that it belongs to the typical linear alpha-helix forming peptide. Binding of the biotin-labeled cryptonin at the surface of E. coli cells and increased influx of propidium iodide in E. coli after cryptonin treatment indicates that it kills microbial cells by binding bacterial cell surfaces and disrupting the cell permeability. Cryptonin showed strong antibacterial (MIC 1.56-25 microg/ml) and antifungal (MIC 3.12-50 microg/ml) activities against tested bacteria and fungi including two antibiotic-resistant bacterial strains; methicilin-resistant S. aureus and vancomycin-resistant Enterococci (MIC 25 microg/ml, each).     

Bioactive oleanolic acid saponins and other constituents from the roots of Viguiera decurrens

The bisdesmoside oleanolic acid saponin, 3-0-(methyl-beta-D-glucuronopyranosiduronoate)-28-0-beta-D-glucopyranosyl-oleanolate along with nine known compounds (two diterpenic acids, one chromene, three triterpenes, one steroidal glycoside, and two monodesmoside oleanolic acid saponins), were obtained from Viguiera decurrens roots. The chemical structure of the bisdesmoside oleanolic saponin was determined by chemical and NMR spectral evidence. A mixture of monodesmoside saponins displayed cytotoxic activity against P388 and COLON cell lines (ED50= 2.3 and 3.6 microg/ml, respectively). Two of the known compounds showed insecticidal activity against the Mexican bean beetle larvae (Epilachna varivestis).     

The effect of cytochalasin J on kinetochore structure in PtK1 cells is mitotic cycle dependent

Mitotic PtK1 cells were arrested in mitosis with nocodazole to determine the effect of cytochalasin J (CJ) on kinetochore structure in arrested and nocodazole-released cells. In previous studies it was shown that CJ had a more pronounced effect on alteration of kinetochore structure and spindle microtubule (MT) architecture when applied during prophase or prometaphase. In this study, mitotic cells were treated at preanaphase for 10 min with 1 microg/ml nocodazole, or in 1 microg/ml nocodazole and 10 microg/ml CJ to allow for the advancement of the 'mitotic clock'. Thus it can be determined if either changes in the timing of mitosis, the maturation of the kinetochore, and/or the lack of MT connection to the kinetochore affects the ability of CJ to detach or alter the attachment of chromosomes to the developing spindle. Preanaphase cells treated with 1 microg/ml nocodazole for 10 min and released into 10 microg/ml CJ showed significant changes in MT organization and kinetochore structure. MTs nucleated at the centrosome are fragmented and kinetochore structure was significantly altered showing only two laminae with few MTs inserted into this structure. Preanaphase cells treated with 1 microg/ml nocodazole and 10 microg/ml CJ for 10 min and released into 10 microg/ml CJ showed similar, but more pronounced, effects on kinetochore structure and spindle MT organization. We interpret these results to suggest that CJ treatment has a greater effect on MT attachment and kinetochore structure in nocodazole pre-treated cells, where the kinetochore structure is mature and the mitotic cycle has been advanced.     

压电生物传感器研究进展

作为一项新兴综合型科学技术,生物传感器是近年来生物化学、分子生物学、传感器技术等领域的研究热点之一。本介绍了压电生物传感器的基本原理、组成、分类,并对近年来国内外的研究进展、生物识别元件的固定化技术以及压电生物传感器的发展趋势作综合评述。     

Alterations in human red blood cell membrane properties induced by the lipopolysaccharide from Proteus mirabilis S1959

The effect of lipopolysaccharide (LPS, endotoxin), isolated from Proteus mirabilis S1959 strain, on red blood cell (RBC) membranes in whole cells as well as on isolated membranes was studied. Lipid membrane fluidity, conformational state of membrane proteins and the osmotic fragility of RBCs were examined using electron paramagnetic resonance spectroscopy and spectrophotometric method. Lipid membrane fluidity was determined using three spin-labeled fatty acids: 5-, 12- and 16-doxylstearic acid (5-, 12- and 16-DS). The addition of LPS S1959 to RBC suspension resulted in an increase in membrane fluidity, as indicated by 12-DS. At the concentrations of 0.5 and 1 mg/ml, LPS treatment led to a significant (P<0.05) increase in lipid membrane fluidity in the deeper region of lipid bilayer (determined by 12-DS). The conformational changes in membrane proteins were determined using two covalently bound spin labels, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl and 4-iodoacetamido-2,2,6,6-tetramethylpiperidine-1-oxyl (ISL). The highest concentration of endotoxin significantly (P<0.05) decreased the relative rotational correlation time of ISL and significantly (P<0.05) increased the osmotic fragility of RBCs. The effect of endotoxin was much more profound in isolated membranes than in intact cells treated with LPS. At the concentrations 0.5 and 1 mg/ml, LPS led to a significant increase in h(w)/h(s) ratio. These results indicated increased membrane protein mobility, mainly in the spectrin-actin complex in membrane cytoskeleton. These data suggest that LPS-induced alterations in membrane lipids and cytoskeleton proteins of RBCs lead to loss of membrane integrity.     

Purification and cloning of lectin that induce cell apoptosis from Allium chinense

A 8.7 kDa lectin with high agglutin activity was isolated by affinity chromatography and cloned from Allium chinense in this study. For the MTT assay, approximately 60 µg/ml A. chinense lectin (ACL) inhibited 50% of the human hepatoma Hep-3B cells grown after 48 h. In addition, no antiproliferative effect was observed on normal human umbilical vein endothelial cells (HUVEC) even at 100 µg/ml concentration. After treatments with ACL on Hep-3B cells, morphologic changes in the nucleus and cytoskeleton were observed under laser scanning confocal microscopy with 4′,6-diamidino-2-phenylindole and tubulin Alexa Fluor 488 staining; whereas, the mitochondrial membrane potential was observed through Mito Tracker Red CMXRos staining. The results showed that ACL led to cell morphology and structure change (e.g., round cell shrinkage). Moreover, ACL resulted in significant change in the shape of the nucleus, damaged the cytoskeleton when tubulin was degraded, and reduced the mitochondrial transmembrane potential. By contrast, no changes were observed on HUVEC cells under the same treatment conditions. DNA fragmentation analysis was used to detect DNA damage. Western blot showed that ACL upregulated caspase-3 and Bax expression during apoptosis and cloned the structural gene of ACL with an open reading frame of 456 bp encoding 151 amino acid residues. The results showed that ACL is a potential anticancer drug.     

Cytoskeletal changes in mouse embryonic fibroblasts exposed to colcemid. Electron microscopic research on platinum replicas

Cytoskeletons of colcemid-treated mouse embryo fibroblasts were studied using platinum replica technique. In the control cells, cytoskeletal components were oriented along direction of cell polarization. Structure of the control cytoskeleton changed regularly from the cell active edge to its centre forming several zones. Distribution of microtubules by colcemid led to significant changes in the organization of actin cytoskeleton. Both orientation and zonal differentiation of cytoskeleton disappeared in colcemid-treated fibroblasts. Changes in the fine structure of microfilament sheath were most prominent. Control sheath was composed of stretched tightly packed microfilaments. Colcemid treatment transformed it into fine microfilament meshwork, normally characteristic only for ruffle zone. Alterations of the fine structure of focal contacts and ruffles were also observed in treated cells. The role of microtubules in the organization of intracellular tensions and in the distribution of sites of actin polymerization is discussed.     

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

Saponin 1 is a triterpeniod saponin extracted from Anemone taipaiensis, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, in vivo study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant in vitro and in vivo anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.     

Role of apoptosis in photodynamic sensitivity of human tumour cell lines

Photodynamic therapy (PDT) using a photosensitizer, such as haematoporphyrin derivative (HpD), in conjunction with visible light is a promising new modality to treat localized cancer. Cell death caused by PDT (through the generation of reactive oxygen species) can occur either by apoptosis (interphase death or as a secondary event following mitosis) and/or necrosis depending on the cell type, concentration and intracellular localization of the sensitizer, and the light dose. Since, apoptosis induced by PDT treatment plays an important role in determining the photodynamic efficacy, in the present work we have investigated the role of apoptotic cell death in relation to the observed differences in sensitivity to HpD-PDT between a human glioma cell line (BMG-1) carrying wild-type tumour suppressor gene p53 and a human squamous carcinoma cell line (4451) with mutated p53. HpD (photosan-3; PS-3) -PDT induced apoptosis was studied by: [A] flow-cytometric analysis of DNA content (sub G0/G1 population); [B] phosphatidylserine externalization (Annexin-V +ve cells); [C] cell size and cytoskeleton reorganization (light-scatter analysis); and [D] fluorescence microscopy (morphological features). PS-3-PDT induced a significantly higher level of apoptosis in BMG-1 cells as compared to 4451 cells. This was dependent on the concentration of PS-3 as well as post-irradiation time in both the cell lines. At 2.5 microg/ml of PS-3 the fraction of BMG-1 cells undergoing apoptosis (60%) was nearly 6 folds higher than 4451 cells (10%). In BMG-1 cells the induction of apoptosis increased with PS-3 concentration up to 5 microg/ml (>80%). However, a decrease was observed at a concentration of 10 microg/ml, possibly due to a shift in the mode of cell death from apoptosis to necrosis. In 4451 cells, on the other hand, the increase in apoptosis could be observed even up to 10 microg/ml of PS-3 (60%). Present results show that the higher sensitivity to PS-3-PDT in glioma cells arise on account of a higher level of apoptosis and suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in certain cell types.     

Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.     

黄蝉和姜花生殖细胞内肌动蛋白微丝的定位

用荧光标记的鬼笔碱染色,对离体的黄蝉和姜花的生殖细胞内肌动蛋白微丝的分布进行了研究,结果证明两种植物的生殖细胞内部都存在一个微丝网络,黄蝉生殖细胞的比姜花的简单,微丝束较粗。但姜花生殖细胞的网络微丝束比黄蝉的更紧密地环绕着核。用免疫荧光技术在黄蝉生殖细胞的分裂前期和中期,可以观察到一些微丝束的存在,但在分裂后期和末期细胞内的肌动蛋白则变为颗粒状。     

Cdc42: an important regulator of neuronal morphology

Regulation of neuronal morphology and activity-dependent synaptic modifications involves reorganization of the actin cytoskeleton. Dynamic changes of the actin cytoskeleton in many cell types are controlled by small GTPases of the Rho family, such as RhoA, Rac1 and Cdc42. As key regulators of both actin and microtubule cytoskeleton, Rho GTPases have also emerged as important regulators of dendrite and spine structural plasticity. Multiple studies suggest that Rac1 and Cdc42 are positive regulators promoting neurite outgrowth and growth cone protrusion, while the activation of RhoA induces stress fiber formation, leading to growth cone collapse and neurite retraction. This review focuses on recent advances in our understanding of the molecular mechanisms underlying physiological and pathological functions of Cdc42 in the nervous system. We also discuss application of different FRET-based biosensors as a powerful approach to examine the dynamics of Cdc42 activity in living cells.     

Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity

Intestinal absorptive cells may modulate both the structure and function of occluding junctions by a cytoskeleton dependent mechanism (Madara, J. L., 1983, J. Cell Biol., 97:125-136). To further examine the putative relationship between absorptive cell occluding junctions and the cytoskeleton, we assessed the effects of cytochalasin D (CD) on occluding junction function and structure in guinea pig ileum using ultrastructural and Ussing chamber techniques. Maximal decrements in transepithelial resistance and junctional charge selectivity were obtained with 10 micrograms/ml CD and the dose-response curves for these two functional parameters were highly similar. Analysis of simultaneous flux studies of sodium and the nonabsorbable extracellular tracer mannitol suggested that CD opened a transjunctional shunt and that this shunt could fully account for the increase in sodium permeability and thus the decrease in resistance. Structural studies including electron microscopy of detergent-extracted cytoskeletal preparations revealed that 10 micrograms/ml CD produced condensation of filamentous elements of the peri-junctional contractile ring and that this was associated with brush border contraction as assessed by scanning electron microscopy. Quantitative freeze-fracture studies revealed marked aberrations in absorptive cell occluding junction structure including diminished strand number, reduced strand-strand cross-linking, and failure of strands to impede the movement of intramembrane particles across them. In aggregate these studies show that CD-induced perturbation of the absorptive cell cytoskeleton results in production of a transepithelial shunt which is fully explained by a defect in the transjunctional pathway. Furthermore, substantial structural abnormalities in occluding junction structure accompany this response. Lastly, the abnormalities in occluding junction structure and function coincide with structural changes in and contraction of the peri-junctional actin-myosin ring. These data suggest that a functionally relevant association may exist between the cytoskeleton and the occluding junction of absorptive cells. We speculate that such an association may serve as a mechanism by which absorptive cells regulate paracellular transport.