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Terms modified by Same Cell Selected AbstractsModulation of calcium signalling by intracellular organelles seen with targeted aequorinsACTA PHYSIOLOGICA, Issue 1 2009M. T. Alonso Abstract The cytosolic Ca2+ signals that trigger cell responses occur either as localized domains of high Ca2+ concentration or as propagating Ca2+ waves. Cytoplasmic organelles, taking up or releasing Ca2+ to the cytosol, shape the cytosolic signals. On the other hand, Ca2+ concentration inside organelles is also important in physiology and pathophysiology. Comprehensive study of these matters requires to measure [Ca2+] inside organelles and at the relevant cytosolic domains. Aequorins, the best-known chemiluminescent Ca2+ probes, are excellent for this end as they do not require stressing illumination, have a large dynamic range and a sharp Ca2+ -dependence, can be targeted to the appropriate location and engineered to have the proper Ca2+ affinity. Using this methodology, we have evidenced the existence in chromaffin cells of functional units composed by three closely interrelated elements: (1) plasma membrane Ca2+ channels, (2) subplasmalemmal endoplasmic reticulum and (3) mitochondria. These Ca2+ -signalling triads optimize Ca2+ microdomains for secretion and prevent propagation of the Ca2+ wave towards the cell core. Oscillatory cytosolic Ca2+ signals originate also oscillations of mitochondrial Ca2+ in several cell types. The nuclear envelope slows down the propagation of the Ca2+ wave to the nucleus and filters high frequencies. On the other hand, inositol-trisphosphate may produce direct release of Ca2+ to the nucleoplasm in GH3 pituitary cells, thus providing mechanisms for selective nuclear signalling. Aequorins emitting at different wavelengths, prepared by fusion either with green or red fluorescent protein, permit simultaneous and independent monitorization of the Ca2+ signals in different subcellular domains within the same cell. [source] Electrical penetration graphs of the nymphal stage of Bemisia argentifoliiENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 2 2003Y.X. Jiang Abstract Electrical penetration graph (EPG, DC system) waveforms were recorded from first, second, and third instar Bemisia argentifolii nymphs. Waveforms recorded were similar among the three instars. Four waveforms were recorded and were named C, J, L, and H. Waveform J is new, whereas waveforms C, L, and H of B. argentifolii nymphs were similar to those published previously from greenhouse whitefly nymphs. As in the previous study on greenhouse whitefly nymphs, there was variation in each of waveforms C, L, and H. Waveform C was recorded at an extracellular voltage level, and represents a pathway phase where the stylets penetrate the plant tissue in an intercellular pathway. At the end of waveform C, the voltage dropped to an intracellular level, indicating penetration of a living cell, and the stylet tips then remained in that cell for the rest of the EPG recording, which was sometimes as long as 16 h. Three waveforms (J, L, and H) were recorded during this intracellular phase, beginning with J, a brief (average = 31 s), low amplitude, irregular waveform. J appeared only at the beginning of the intracellular phase, and was followed by either L (five out of eight times) or H (three out of eight times). Waveforms L and H then alternated with one another for the remainder of the intracellular phase. The most conspicuous difference between L and H was the frequency of their voltage fluctuations; L had a lower frequency and H a higher frequency. Usually the shape of waveform L was dominated by voltage peaks in a positive direction, while waveform H was characterized by strong voltage peaks in a negative direction; although some variants of both L and H had distinct voltage peaks in both directions. The electrical origin of both the positive and negative voltage peaks was electromotive force (emf) fluctuation rather than resistance fluctuation. During waveform H, copious amounts of honeydew were produced, indicating that the penetrated cell was a sieve element. We conclude, therefore, that H represents phloem sap ingestion; and because J and L are produced in the same cell as H, then phloem phase is represented by waveforms J, L, and H. The biological correlations for J and L are not yet known. [source] MULTIPLE HIV-1 INFECTION OF CELLS AND THE EVOLUTIONARY DYNAMICS OF CYTOTOXIC T LYMPHOCYTE ESCAPE MUTANTSEVOLUTION, Issue 9 2009Dominik Wodarz Cytotoxic T lymphocytes (CTL) are an important branch of the immune system, killing virus-infected cells. Many viruses can mutate so that infected cells are not killed by CTL anymore. This escape can contribute to virus persistence and disease. A prominent example is HIV-1. The evolutionary dynamics of CTL escape mutants in vivo have been studied experimentally and mathematically, assuming that a cell can only be infected with one HIV particle at a time. However, according to data, multiple virus particles frequently infect the same cell, a process called coinfection. Here, we study the evolutionary dynamics of CTL escape mutants in the context of coinfection. A mathematical model suggests that an intermediate strength of the CTL response against the wild-type is most detrimental for an escape mutant, minimizing overall virus load and even leading to its extinction. A weaker or, paradoxically, stronger CTL response against the wild-type both lead to the persistence of the escape mutant and higher virus load. It is hypothesized that an intermediate strength of the CTL response, and thus the suboptimal virus suppression observed in HIV-1 infection, might be adaptive to minimize the impact of existing CTL escape mutants on overall virus load. [source] DEFINED ORDER OF EVOLUTIONARY ADAPTATIONS: EXPERIMENTAL EVIDENCEEVOLUTION, Issue 7 2008Erez Oxman Organisms often adapt to new conditions by means of beneficial mutations that become fixed in the population. Often, full adaptation requires several different mutations in the same cell, each of which may affect a different aspect of the behavior. Can one predict order in which these mutations become fixed? To address this, we experimentally studied evolution of Escherichia coli in a growth medium in which the effects of different adaptations can be easily classified as affecting growth rate or the lag-phase duration. We find that adaptations are fixed in a defined and reproducible order: first reduction of lag phase, and then an increase of the exponential growth rate. A population genetics theory explains this order, and suggests growth conditions in which the order of adaptations is reversed. We experimentally find this order reversal under the predicted conditions. This study supports a view in which the evolutionary path to adaptation in a new environment can be captured by theory and experiment. [source] Green fluorescent protein , a bright idea for the study of bacterial protein localizationFEMS MICROBIOLOGY LETTERS, Issue 1 2001Gregory J Phillips Abstract Use of the green fluorescent protein (GFP) of Aequorea victoria as a reporter for protein and DNA localization has provided sensitive, new approaches for studying the organization of the bacterial cell, leading to new insights into diverse cellular processes. GFP has many characteristics that make it useful for localization studies in bacteria, primarily its ability to fluoresce when fused to target polypeptides without the addition of exogenously added substrates. As an alternative to immunofluorescence microscopy, the expression of gfp gene fusions has been used to probe the function of cellular components fundamental for DNA replication, translation, protein export, and signal transduction, that heretofore have been difficult to study in living cells. Moreover, protein and DNA localization can now be monitored in real time, revealing that several proteins important for cell division, development and sporulation are dynamically localized throughout the cell cycle. The use of additional GFP variants that permit the labeling of multiple components within the same cell, and the use of GFP for genetic screens, should continue to make this a valuable tool for addressing complex questions about the bacterial cell. [source] Anode Interfacial Tuning via Electron-Blocking/Hole-Transport Layers and Indium Tin Oxide Surface Treatment in Bulk-Heterojunction Organic Photovoltaic CellsADVANCED FUNCTIONAL MATERIALS, Issue 4 2010Alexander W. Hains Abstract The effects of anode/active layer interface modification in bulk-heterojunction organic photovoltaic (OPV) cells is investigated using poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) and/or a hole-transporting/electron-blocking blend of 4,4,-bis[(p -trichlorosilylpropylphenyl)-phenylamino]biphenyl (TPDSi2) and poly[9,9-dioctylfluorene- co - N -[4-(3-methylpropyl)]-diphenylamine] (TFB) as interfacial layers (IFLs). Current,voltage data in the dark and AM1.5G light show that the TPDSi2:TFB IFL yields MDMO-PPV:PCBM OPVs with substantially increased open-circuit voltage (Voc), power conversion efficiency, and thermal stability versus devices having no IFL or PEDOT:PSS. Using PEDOT:PSS and TPDSi2:TFB together in the same cell greatly reduces dark current and produces the highest Voc (0.91,V) by combining the electron-blocking effects of both layers. ITO anode pre-treatment was investigated by X-ray photoelectron spectroscopy to understand why oxygen plasma, UV ozone, and solvent cleaning markedly affect cell response in combination with each IFL. O2 plasma and UV ozone treatment most effectively clean the ITO surface and are found most effective in preparing the surface for PEDOT:PSS deposition; UV ozone produces optimum solar cells with the TPDSi2:TFB IFL. Solvent cleaning leaves significant residual carbon contamination on the ITO and is best followed by O2 plasma or UV ozone treatment. [source] Transcriptionally active nuclei are selective in mature multinucleated osteoclastsGENES TO CELLS, Issue 10 2010Min-Young Youn Multinucleation is indispensable for the bone-resorbing activity of mature osteoclasts. Although multinucleation is evident in mature osteoclasts and certain other cell types, putative regulatory networks among nuclei remain poorly characterized. To address this issue, transcriptional activity of each nucleus in a multinucleated osteoclast was assessed by detecting the distributions of nuclear proteins by immunocytochemistry and primary transcripts by RNA FISH. Patterns of epigenetic histone markers governing transcription as well as localization of tested nuclear receptor proteins appeared indistinguishable among nuclei in differentiated Raw264 cells and mouse mature osteoclasts. However, RNAPII-Ser5P/2P and NFATc1 proteins were selectively distributed in certain nuclei in the same cell. Similarly, the distributions of primary transcripts for osteoclast-specific genes (Nfatc1, Ctsk and Acp5) as well as a housekeeping gene (beta-tubulin) were limited in certain nuclei within individual cells. By fusing two Raw264 cell lines that stably expressed ZsGreen-NLS and DsRed-NLS proteins, transmission of nuclear proteins across all of the nuclei in a cell could be observed, presumably through the shared cytoplasm. Taken together, we conclude that although nuclear proteins are diffusible among nuclei, only certain nuclei within a multinucleated osteoclast are transcriptionally active. [source] Novel role of nectin: implication in the co-localization of JAM-A and claudin-1 at the same cell,cell adhesion membrane domainGENES TO CELLS, Issue 8 2008Kaori Kuramitsu Tight junctions (TJs) are formed at the apical side of adherens junctions (AJs) in epithelial cells. Major cell adhesion molecules (CAMs) at TJs are JAM and claudin, whereas major CAMs at AJs are nectin and cadherin. We previously showed that nectin initially forms cell,cell adhesion and then recruits cadherin to the nectin-based cell,cell adhesion sites to form AJs, followed by the recruitment of JAM and claudin to the apical side of AJs to form TJs. We investigated the roles of nectin in the formation of TJs by expressing various combinations of CAMs in L fibroblasts with no TJs or AJs. Co-expression of one of the AJ CAMs and one of the TJ CAMs formed two separate cell,cell adhesion membrane domains (CAMDs). Co-expression of nectin-3 and E-cadherin formed the same CAMD, but co-expression of JAM-A and claudin-1 did not form the same CAMD. Co-expression of JAM-A and claudin-1 with nectin-3, but not E-cadherin, made them form the same CAMD, which was separated from the nectin-based CAMD. Nectin-3 required afadin, a nectin- and F-actin-binding protein, for this ability. In conclusion, nectin plays a novel role in the co-localization of JAM and claudin at the same CAMD. [source] Hepatitis B and C virus coinfection: A novel model system reveals the absence of direct viral interference,HEPATOLOGY, Issue 1 2009Pantxika Bellecave Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been associated with severe liver disease and frequent progression to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests reciprocal replicative suppression of the two viruses, or viral interference. However, interactions between HBV and HCV have been difficult to study due to the lack of appropriate model systems. We have established a novel model system to investigate interactions between HBV and HCV. Stable Huh-7 cell lines inducibly replicating HBV were transfected with selectable HCV replicons or infected with cell culture,derived HCV. In this system, both viruses were found to replicate in the same cell without overt interference. Specific inhibition of one virus did not affect the replication and gene expression of the other. Furthermore, cells harboring replicating HBV could be infected with cell culture,derived HCV, arguing against superinfection exclusion. Finally, cells harboring replicating HBV supported efficient production of infectious HCV. Conclusion: HBV and HCV can replicate in the same cell without evidence for direct interference in vitro. Therefore, the viral interference observed in coinfected patients is probably due to indirect mechanisms mediated by innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV,HCV coinfection and may contribute to its clinical management in the future. (HEPATOLOGY 2009.) [source] Mechanism of antigen presentation after hypertonic loading of soluble antigensIMMUNOLOGY, Issue 4 2002Georg A. Enders Summary Hypertonic loading of proteins into cells has been used to introduce soluble proteins into the major histocompatibility complex class I pathway of antigen presentation followed by cytotoxic T-lymphocyte (CTL) induction. The precise mechanism for this pathway is not completely understood. The antigen is either processed and presented by/on the same cell or by professional antigen-presenting cells (APC) after taking up the antigen from damaged or apoptotic cells. After loading labelled ovalbumin (OVA), it could be co-precipitated with the proteasome complex, supporting the role of this pathway for antigen processing. The processing speed however, appeared to be slow since intact OVA could be detected inside the cells even after 18 hr. This corresponded well with the processing of OVA by isolated proteasomes. On the other hand, enough peptides for recognition of target cells by CTLs were generated in this reaction. One reason for the low level of processing might be that hypertonic loading may damage the cells and inhibit direct processing. In fact, at least 50% of the cells became positive for Annexin V binding after hypertonic loading which indicates severe membrane alterations usually associated with the progress of apoptosis. Annexin V binds to phosphatidylserine residues which also serve as ligand for CD36 expressed on monocytes and some immature dendritic cells. This may direct the phagocytic pathway to hypertonically loaded cells and thus enable professional APCs to present OVA-peptides. Therefore, in addition to the direct processing of OVA, CTLs can be primed by professional APC after uptake of apoptotic, OVA-loaded cells. [source] Epidermal growth factor receptor and cancer: control of oncogenic signalling by endocytosisJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 5a 2008Michael Vibo Grandal ,,Introduction ,,Endocytosis of EGFR -,Kinase activity -,Clathrin-coated pits -,Ubiquitination -,Effects of EGFR-ErbB2 heterodimerization on EGFR internalization ,,Cellular and molecular requirements for lysosomal degradation of EGFR -,Intracellular EGFR degradation depends on luminal sorting at multivesicular bodies -,Molecular requirements for EGFR sorting in multivesicular endosomes Abstract The epidermal growth factor receptor (EGFR) and other members of the EGFR/ErbB receptor family of receptor tyrosine kinases (RTKs) are important regulators of proliferation, angiogenesis, migration, tumorigenesis and metastasis. Overexpression, mutations, deletions and production of autocrine ligands contribute to aberrant activation of the ErbB proteins. The signalling output from EGFR is complicated given that other ErbB proteins are often additionally expressed and activated in the same cell, resulting in formation of homo-and/or heterodimers. In particular, association of EGFR with ErbB2 prevents its down-regulation, underscoring the importance of the cellular background for EGFR effects. Signalling from ErbB proteins can either be terminated by dissociation of ligand resulting in dephosphorylation, or blunted by degradation of the receptors. Although proteasomal targeting of ErbB proteins has been described, lysosomal degradation upon ligand-induced endocytosis seems to play the major role in EGFR down-regulation. Preclinical and clinical data have demonstrated that EGFR is a central player in cancer, especially in carcinomas, some brain tumours and in non-small cell lung cancer. Such studies have further validated EGFR as an important molecular target in cancer treatment. This review focuses on mechanisms involved in ligand-induced EGFR activation and endocytic down-regulation. A better understanding of EGFR biology should allow development of more tumour-selective therapeutic approaches targeting EGFR-induced signalling. [source] Non-muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2010Patrick G. Flynn Abstract Non-muscle myosin II (NM II) helps mediate survival and apoptosis in response to TNF-alpha (TNF), however, NM II's mechanism of action in these processes is not fully understood. NM II isoforms are involved in a variety of cellular processes and differences in their enzyme kinetics, localization, and activation allow NM II isoforms to have distinct functions within the same cell. The present study focused on isoform specific functions of NM IIA and IIB in mediating TNF induced apoptosis. Results show that siRNA knockdown of NM IIB, but not NM IIA, impaired caspase cleavage and nuclear condensation in response to TNF. NM II's function in promoting cell death signaling appears to be independent of actomyosin contractility (AMC) since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF induced caspase cleavage. Immunoprecipitation studies revealed associations of NM IIB with clathrin, FADD, and caspase 8 in response to TNF suggesting a role for NM IIB in TNFR1 endocytosis and the formation of the death inducing signaling complex (DISC). These findings suggest that NM IIB promotes TNF cell death signaling in a manner independent of its force generating property. J. Cell. Biochem. 9999: 1365,1375, 2010. © 2010 Wiley-Liss, Inc. [source] Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2004Meenal Mehrotra Abstract Osteoblastic migration and proliferation in response to growth factors are essential for skeletal development, bone remodeling, and fracture repair, as well as pathologic processes, such as metastasis. We studied migration in response to platelet-derived growth factor (PDGF, 10 ng/ml) in a wounding model. PDGF stimulated a twofold increase in migration of osteoblastic MC3T3-E1 cells and murine calvarial osteoblasts over 24,48 h. PDGF also stimulated a tenfold increase in 3H-thymidine (3H-TdR) incorporation in MC3T3-E1 cells. Migration and DNA replication, as measured by BrdU incorporation, could be stimulated in the same cell. Blocking DNA replication with aphidicolin did not reduce the distance migrated. To examine the role of mitogen-activated protein (MAP) kinases in migration and proliferation, we used specific inhibitors of p38 MAP kinase, extracellular signal regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). For these signaling studies, proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) using flow cytometry. Inhibition of the p38 MAP kinase pathway by SB203580 and SB202190 blocked PDGF-stimulated migration but had no effect on proliferation. Inhibition of the ERK pathway by PD98059 and U0126 inhibited proliferation but did not inhibit migration. Inhibition of JNK activity by SP600125 inhibited both migration and proliferation. Hence, the stimulation of migration and proliferation by PDGF occurred by both overlapping and independent pathways. The JNK pathway was involved in both migration and proliferation, whereas the p38 pathway was predominantly involved in migration and the ERK pathway predominantly involved in proliferation. © 2004 Wiley-Liss, Inc. [source] Evaluation of rapid volume changes of substrate-adherent cells by conventional microscopy 3D imagingJOURNAL OF MICROSCOPY, Issue 3 2004F. BOUDREAULT Summary Precise measurement of rapid volume changes of substrate-adherent cells is essential to understand many aspects of cell physiology, yet techniques to evaluate volume changes with sufficient precision and high temporal resolution are limited. Here, we describe a novel imaging method that surveys the rapid morphology modifications of living, substrate-adherent cells based on phase-contrast, digital video microscopy. Cells grown on a glass substrate are mounted in a custom-designed, side-viewing chamber and subjected to hypotonic swelling. Side-view images of the rapidly swelling cell, and at the end of the assay, an image of the same cell viewed from a perpendicular direction through the substrate, are acquired. Based on these images, off-line reconstruction of 3D cell morphology is performed, which precisely measures cell volume, height and surface at different points during cell volume changes. Volume evaluations are comparable to those obtained by confocal laser scanning microscopy (,Volume , 14%), but our method has superior temporal resolution limited only by the time of single-image acquisition, typically ,100 ms. The advantages of using standard phase-contrast microscopy without the need for cell staining or intense illumination to monitor cell volume make this system a promising new tool to investigate the fundamentals of cell volume physiology. [source] Antiapoptotic and antiautophagic effects of glial cell line-derived neurotrophic factor and hepatocyte growth factor after transient middle cerebral artery occlusion in ratsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 10 2010Jingwei Shang Abstract Glial cell line-derived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF) are strong neurotrophic factors, which function as antiapoptotic factors. However, the neuroprotective effect of GDNF and HGF in ameliorating ischemic brain injury via an antiautophagic effect has not been examined. Therefore, we investigated GDNF and HGF for changes of infarct size and antiapoptotic and antiautophagic effects after transient middle cerebral artery occlusion (tMCAO) in rats. For the estimation of ischemic brain injury, the infarct size was calculated at 24 hr after tMCAO by HE staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) was performed for evaluating the antiapoptotic effect. Western blot analysis of microtubule-associated protein 1 light chain 3 (LC3) and immunofluorescence analysis of LC3 and phosphorylated mTOR/Ser2448 (p-mTOR) were performed for evaluating the antiautophagic effect. GDNF and HGF significantly reduced infarct size after cerebral ischemia. The amounts of LC3-I plus LC3-II (relative to ,-tubulin) were significantly increased after tMCAO, and GDNF and HGF significantly decreased them. GDNF and HGF significantly increased p-mTOR-positive cells. GDNF and HGF significantly decreased the numbers of TUNEL-, LC3-, and LC3/TUNEL double-positive cells. LC3/TUNEL double-positive cells accounted for about 34.3% of LC3 plus TUNEL-positive cells. This study suggests that the protective effects of GDNF and HGF were greatly associated with not only the antiapoptotic but also the antiautophagic effects; maybe two types of cell death can occur in the same cell at the same time, and GDNF and HGF are capable of ameliorating these two pathways. © 2010 Wiley-Liss, Inc. [source] Two different Pseudomonas aeruginosa chemosensory signal transduction complexes localize to cell poles and form and remould in stationary phaseMOLECULAR MICROBIOLOGY, Issue 1 2006Zehra Tüzün Güvener Summary Pseudomonas aeruginosa has sets of sensory genes designated che and che2. The che genes are required for flagella-mediated chemotaxis. The che2 genes are expressed in the stationary phase of growth and are probably also involved in flagella,mediated behavioural responses. P. aeruginosa also has 26 chemoreceptor genes, six of which are preferentially expressed in stationary phase. Subcellular localization experiments indicated that Che proteins form signal transduction complexes at cell poles throughout growth. Cyan fluorescent protein (CFP)-tagged McpA, a stationary phase-expressed chemoreceptor, appeared and colocalized with yellow fluorescent protein (YFP)-tagged CheA when cells entered stationary phase. This indicates that P. aeruginosa chemotaxis protein complexes are subject to remoulding by chemoreceptor proteins that are expressed when cells stop growing. CheA-CFP and CheY2-YFP tagged proteins that were coexpressed in the same cell had separate subcellular locations, indicating that Che2 proteins do not enter into direct physical interactions with Che proteins. Che2 protein complex formation required McpB, another stationary phase induced chemoreceptor that is predicted to be soluble. This implies that Che2 complexes have a function that depends on just one chemoreceptor. Our results suggest that motile P. aeruginosa cells have signal transduction systems that are adapted to allow non-growing cells to sense and respond to their environment differently from actively growing cells. [source] Does the lipid membrane composition of arsonoliposomes affect their anticancer activity?MOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 5 2009A cell culture study Abstract Sonicated arsonoliposomes were prepared using arsonolipid with palmitic acid acyl chain (C16), mixed with phosphatidylcholine (PC)-based or 1,2-distearoyl- sn -glycero-3-phosphocholine (DSPC)-based, and cholesterol (Chol) with C16/DSPC/Chol 8:12:10 molar ratio. PEG-lipid (1,2-distearoyl- sn -glycero-3-phosphoethanolamine conjugated to polyethylenoglycol 2000) containing vesicles (PEGylated-arsonoliposomes; PC-based and DSPC-based) were also prepared. The cytotoxicity of these arsonoliposomes towards different cancer cells (human promyelocytic leukaemia NB4, Prostatic cancer PC3, human breast adenocarcinoma MDA-MB-468, human T-lymphocyte (MT-4) and also towards human umbilical vein endothelial cells (HUVECs) was evaluated by calculating the arsonoliposome-induced growth inhibition of the cells by the MTT assay. IC-50 values were interpolated from cell number/arsonoliposome concentration curves. The results reveal that all types of arsonoliposomes evaluated significantly inhibit the growth of most of the cancer cells studied (PC3, NB4, MT4) with the exception of the MDA-MB-468 breast cancer cells which were minimally affected by arsonoliposomes; in some cases even less than HUVEC. Nevertheless, for the same cell type the differences between the different types of arsonoliposomes were significant but not proportional to their stability, indicating that the formation of arsonoliposomes with very stable membranes is not a problem for their anticancer activity. Thereby it is concluded that arsonoliposome composition should be adjusted in accordance to their in vivo kinetics and the desired, for each specific application, biodistribution of As and/or encapsulated drug. [source] Simultaneous imaging of membrane antigen and the corresponding chromosomal locus in pathology archivesPATHOLOGY INTERNATIONAL, Issue 12 2005Hisaki Igarashi A new procedure for the simultaneous staining of membranous antigens, such as tyrosine kinase-type cell surface receptor HER2 (c-erbB2), and the corresponding chromosome (chromosome 17 for c-erbB2) in the same cell for use in examining pathology archives is presented. A multistep procedure involving microwave-assisted fluorescence in situ hybridization and immunofluorescence yielded cell images having c-erbB2 on the membrane and genomic signals from the chromosome 17 centromere and the c-erbB2 locus. Furthermore, a combination of microwave-assisted chromogenic in situ hybridization and immunohistochemistry found colorized signals from both chromosome 17 centromere in the nuclei and c-erbB2 on the membranes of individual cells. Quantitative image analysis further confirmed the presence of a significantly stronger c-erbB2 immunoreactivity on cells containing three or more signals from chromosome 17 than from those with less than three signals. It was possible to extend the constellation of cell surface markers and corresponding chromosomes or locus-specific makers to several other genes including CDH1. In this case, the disappearances of CDH1 expression, a CDH1 locus signal, and a centromere enumeration probe (CEP) 16 signal were simultaneously demonstrated in the less-adhesive tumor cells. Thus, it is believed that this procedure might pave the way for exploiting pathology archives for the genotype,phenotype analysis of individual cells. [source] Coexistence of gastric- and intestinal-type endocrine cells in gastric and intestinal mixed intestinal metaplasia of the human stomachPATHOLOGY INTERNATIONAL, Issue 4 2005Takafumi Otsuka Intestinal metaplasia (IM) in the human stomach has previously been classified into a gastric and intestinal mixed (GI-IM) and a solely intestinal phenotype (I-IM). The phenotypes of mucous and endocrine cells were evaluated in 3034 glandular ducts associated with chronic gastritis. In the pyloric region, the relative expression of gastric endocrine cell markers, such as gastrin and somatostatin, decreased gradually from glandular ducts with only gastric mucous cell phenotype (G type) to GI-IM toward I-IM, while that of the intestinal endocrine cell markers, glicentin, gastric inhibitory polypeptide (GIP), and glucagon-like peptide-1 (GLP-1) was inversely correlated. In the fundic region, gastrin-positive, cells, emerged, in, the, pseudo-pyloric, and, GI-IM glands, whereas I-IM glands did not possess any gastrin-positive cells, suggesting the presence of a distinct pathway of intestinalization. Double staining revealed coexistence of gastrin- and GLP-1-positive cells in the same gland and occasionally in the same cell in GI-IM glands. These results suggest that the phenotypes of endocrine cells are in line with those for mucous counterparts and support the concept that all of the different types of mucous and endocrine cells in normal and IM glands might be derived from a single progenitor cell in each gland. [source] Food uptake in the mixotrophic Dinophysis acuminataTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005LUCIE MARANDA Evidence of food uptake in the photosynthetic genus Dinophysis comes solely from the presence of food vacuoles, as no photosynthetic cells have ever been observed in the act of feeding. We examined the feeding ecology of D. acuminata in natural populations and under laboratory conditions. Using depth-integrated sampling of the water column, we determined the frequency of food vacuolated cells at 2-h intervals over a 24-h period in a shallow marine embayment. Food vacuoles in preserved cells were enumerated using Nomarski differential interference contrast microscopy; ultrastructural characters were recorded by transmission electron microscopy. A peak in the feeding activity was observed toward dusk for an abundant June population, with 26% of cells with at least one food vacuole. Mechanisms of concurrent carbon acquisition were evident from the presence of chloroplasts with starch grains and food vacuoles within the same cell. Vacuole content could not be identified. In a preliminary 2-wk long simulated grazing experiment, a mixture of two hypothesized preys, Rhodomonas salina and Dunaliella tertiolecta, was offered to D. acuminata; the Dinophysis populations decreased steadily and at the same rate, whether food was present or not. The evaluation of the food vacuole frequency will be repeated in the coming season to verify the observed pattern, while grazing experiments will include a variety of food items and incubation conditions. Our current inability to successfully culture any photosynthetic Dinophysis limits ecophysiological approaches, either at the population or cellular level, to manipulation of field samples. Supported by National Institutes of Health Grant GM62126-01A1. [source] Conditional length distributions induced by the coverage of two points by a Poisson Voronoï tessellation: application to a telecommunication modelAPPLIED STOCHASTIC MODELS IN BUSINESS AND INDUSTRY, Issue 4 2006Catherine Gloaguen Abstract The end points of a fixed segment in the Euclidian plane covered by a Poisson Voronoï tessellation belong to the same cell or to two distinct cells. This marks off one or two points of the underlying Poisson process that are the nucleus(i) of the cell(s). Our interest lies in the geometrical relationship between these nuclei and the segment end points as well as between the nuclei. We investigate their probability distribution functions conditioning on the number of nuclei, taking into account the length of the segment. The aim of the study is to establish some tools to be used for the analysis of a telecommunication problem related to the pricing of leased lines. We motivate and give accurate approximations of the probability of common coverage and of the length distributions that can be included in spreadsheet codes as an element of simple cost functions. Copyright © 2006 John Wiley & Sons, Ltd. [source] Therapeutic control of B cell activation via recruitment of Fc, receptor IIb (CD32B) inhibitory function with a novel bispecific antibody scaffold,ARTHRITIS & RHEUMATISM, Issue 7 2010Maria-Concetta Veri Objective To exploit the physiologic Fc, receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications. Methods DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the ,-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. Results DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. Conclusion This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans. [source] Glutamate levels and activity of the T cell voltage-gated potassium Kv1.3 channel in patients with systemic lupus erythematosusARTHRITIS & RHEUMATISM, Issue 5 2008C. Poulopoulou Objective Alterations in glutamate homeostasis and Kv1.3 voltage-gated potassium channel function have been independently associated with T cell dysfunction, whereas selective blockade of Kv1.3 channels inhibits T cell activation and improves T cell,mediated manifestations in animal models of autoimmunity. Because low extracellular glutamate concentrations enhance the activity of this channel in normal T cells ex vivo, we undertook this study to examine serum glutamate concentrations and Kv1.3 channel activity in patients with systemic lupus erythematosus (SLE). Methods We used high-performance liquid chromatography for glutamate measurements, and we used the whole-cell patch-clamp technique for electrophysiologic studies performed in freshly isolated, noncultured peripheral T cells. Results Mean ± SD serum concentrations of glutamate were lower in patients with either clinically quiescent SLE (77 ± 27 ,M [n = 18]) or active SLE (61 ± 36 ,M [n = 16]) than in healthy controls (166 ± 64 ,M [n = 24]) (both P < 0.0001). The intrinsic gating properties of the Kv1.3 channels in lupus T cells were found to be comparable with those in healthy control,derived T cells. Notably, electrophysiologic data from SLE patient,derived T cells exposed to extracellular glutamate concentrations similar to their respective serum levels (50 ,M) demonstrated Kv1.3 current responses enhanced by almost 20% (P < 0.01) compared with those subsequently obtained from the same cell in the presence of glutamate concentrations within control serum levels (200 ,M). Conclusion Based on the key role of Kv1.3 channel activity in lymphocyte physiology, an enhancing in vivo effect of low serum glutamate concentrations on the functional activity of this channel may contribute to lupus T cell hyperactivity. Studies to further elucidate Kv1.3 responses in SLE, as well as the possible pathogenetic role of this unsuspected metabolic abnormality, may have therapeutic implications for SLE patients. [source] Multiple P2X receptors on guinea-pig pelvic ganglion neurons exhibit novel pharmacological propertiesBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2001Yu Zhong Application of ATP and ,,,-methylene ATP (,,meATP) to voltage-clamped guinea-pig pelvic neurons produced three types of inward currents. A fast-desensitizing response was present in 5% (25/660) of neurons, 70% gave slowly-desensitizing currents, and the remainder had biphasic responses. Slowly-desensitizing responses were characterized pharmacologically. The response to ,,meATP 100 ,M was 46±27% (range 0 , 100%) of that evoked by ATP 100 ,M in the same cell. Cross-desensitization indicated the presence of ,,meATP-sensitive and -insensitive receptors. The concentration-response curve for ,,meATP had an EC50 of 55 ,M, and a Hill coefficient of 0.99, while at the ,,meATP-insensitive receptor, ATP had an EC50 of 73 ,M, with a Hill coefficient of 1.78. The response to ,,meATP was blocked by pyridoxalphosphate-6-azophenyl-2,,4,-disulphonic acid (PPADS), suramin and Cibacron blue. However, the ,,meATP-insensitive receptor was inhibited by PPADS, but not by the other two antagonists. 2,- (or 3,-) O -trinitrophenyl-ATP was 10 times more potent in inhibiting responses to ,,meATP than to ATP (at the ,,meATP-insensitive receptor). Lowering extracellular pH potentiated responses to ,,meATP and ATP, while raising pH attenuated them. Co-application of Zn2+ (3 , 300 ,M) inhibited the responses to ,,meATP and ATP, with IC50 values of 286 and 60 ,M, respectively. In conclusion, unlike rat and mouse pelvic ganglion neurons, which only express P2X2 homomers, at least three distinct P2X receptors are present in guinea-pig pelvic neurons, probably homomeric P2X2, P2X3 and heteromeric P2X2/3 receptors. However, some of the novel pharmacological properties observed suggest that the guinea-pig P2X receptor subtypes may differ from their rat orthologues. British Journal of Pharmacology (2001) 132, 221,233; doi:10.1038/sj.bjp.0703778 [source] Riluzole inhibits the persistent sodium current in mammalian CNS neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000Andrea Urbani Abstract The effects of 0.1,100 ,m riluzole, a neuroprotective agent with anticonvulsant properties, were studied on neurons from rat brain cortex. Patch-clamp whole-cell recordings in voltage-clamp mode were performed on thin slices to examine the effects of the drug on a noninactivating (persistent) Na+ current (INa,p). INa,p was selected because it enhances neuronal excitability near firing threshold, which makes it a potential target for anticonvulsant drugs. When added to the external solution, riluzole dose-dependently inhibited INa,p up to a complete blocking of the current (EC50 2 ,m), showing a significant effect at therapeutic drug concentrations. A comparative dose-effect study was carried out in the same cells for the other main known action of riluzole, the inhibitory effect on the fast transient sodium current. This effect was confirmed in our experiments, but we found that it was achieved at levels much higher than putative therapeutic concentrations. Only the effect on INa,p, and not that on fast sodium current, can account for the reduction in neuronal excitability observed in cortical neurons following riluzole treatment at therapeutic concentrations, and this might represent a novel mechanism accounting for the anticonvulsant and neuroprotective properties of riluzole. [source] Efficient killing of SW480 colon carcinoma cells by a signal transducer and activator of transcription (STAT) 3 hairpin decoy oligodeoxynucleotide , interference with interferon-,-STAT1-mediated killingFEBS JOURNAL, Issue 9 2009Ali Tadlaoui Hbibi The signal transducers and activators of transcription (STATs) convey signals from the membrane to the nucleus in response to cytokines or growth factors. STAT3 is activated in response to cytokines involved mostly in cell proliferation; STAT1 is activated by cytokines, including interferon-,, involved in defence against pathogens and the inhibition of cell proliferation. STAT3, which is frequently activated in tumour cells, is a valuable target with respect to achieving inhibition of tumour cell proliferation. Indeed, its inhibition results in cell death. We previously observed that inhibition of the transcription factor nuclear factor-,B, a key regulator of cell proliferation, with decoy oligodeoxynucleotides results in cell death. We used a similar approach for STAT3. A hairpin STAT3 oligodeoxynucleotide was added to a colon carcinoma cell line in which it induced cell death as efficiently as the STAT3 inhibitor stattic. The hairpin STAT3 oligodeoxynucleotide co-localized with STAT3 within the cytoplasm, prevented STAT3 localization to the nucleus, blocked a cyclin D1 reporter promoter and associated with STAT3 in pull-down assays. However, the same cells were efficiently killed by interferon-,. This effect was counteracted by the STAT3 oligodeoxynucleotide, which was found to efficiently inhibit STAT1. Thus, although it can inhibit STAT3, the hairpin STAT3 oligodeoxynucleotide appears also to inhibit STAT1-mediated interferon-, cell killing, highlighting the need to optimize STAT3-targeting oligodeoxynucleotides. [source] Accumulation of multiple forms of lamin A with down-regulation of FACE-1 suppresses growth in senescent human cellsGENES TO CELLS, Issue 3 2007Ryo Ukekawa 5-Bromodeoxyuridine (BrdU) clearly induces a senescence-like phenomenon in every cell type. Proteome analysis revealed that lamin A and C were most highly increased in the nuclei of HeLa cells upon addition of BrdU. Immunoblot analysis also revealed marked accumulation of nuclear prelamin A. Consistently, farnesylated-proteins converting enzyme 1 (FACE-1) was markedly down-regulated in the same cells. Similar phenomena were also observed in normal human fibroblasts undergoing replicative senescence. Immunochemical analysis confirmed the above results. Lamin A is a major component of lamina and responsible for several genetic diseases. Thus, we ectopically expressed a wild-type, a mature type and a premature type of lamin in HeLa cells. All of these forms similarly inhibited colony formation and delayed cell cycle progression mainly through G2 phase. These results suggest that a change in the amount of lamin A, rather than appearance of its truncated form, is responsible for growth retardation in affected cells. [source] Lesions of the mammillary body region alter hippocampal movement signals and theta frequency: Implications for path integration modelsHIPPOCAMPUS, Issue 9 2008Patricia E. Sharp Abstract Cells throughout the hippocampal formation are involved in processing spatial information. These same cells also show an influence of locomotor activity, and these movement signals are thought to be critical for the path integration abilities of these cells. Nuclei in the mammillary region provide ascending influences to the hippocampal formation and have been implicated in influencing both hippocampal spatial and theta signals. Here, we report the effects of mammillary lesions on movement-related signals in several hippocampal subregions. We find first, as predicted by earlier work, these lesions cause an approximately 1 Hz reduction in the frequency of theta modulation of cell firing. According to recent theoretical work, this might, in turn, be expected to influence the size of hippocampal place fields. Our data do not confirm this prediction for any of the hippocampal regions examined. Second, we report lesion effects on the relationship between firing rate and running speed for the hippocampal cells. These lesions caused a reduction in both the slope and intercept of rate-by-speed functions for cells in the hippocampus and postsubiculum. Surprisingly, cells in subiculum showed an opposite effect, so that the excitatory influence of locomotion was enhanced. Path integration theories predict that the speed at which path integration occurs is related to the strength of this movement signal. In remarkable accordance with this prediction, we report that the timing of the place cell signals is slowed following mammillary lesions for hippocampal and postsubicular cells, but, in contrast, is speeded up for subicular cells. In fact, the timing for place signals across lesion condition and brain region is predicted by a single linear function which relates timing to the strength of the running speed signal. Thus, these data provide remarkable support for some aspects of current path integration theory, while posing a challenge for other aspects of these same theories. © 2008 Wiley-Liss, Inc. [source] Encapsulated-Dye All-Organic Charged Colored Ink Nanoparticles for Electrophoretic Image DisplayADVANCED MATERIALS, Issue 48 2009Sun Wha Oh Electrophoretic ink nanoparticles with high mobility are successfully fabricated by dispersion polymerization. The color of test cells can be changed by applying a bias voltage, as shown in the figure: the lower row shows the same cells as the upper row but with an applied voltage. These all-organic, encapsulated-dye, electrophoretic ink particles are expected to reduce the fabrication cost of e-ink in electrophoretic image display cells. [source] Signalling responses linked to betulinic acid-induced apoptosis are antagonized by MEK inhibitor U0126 in adherent or 3D spheroid melanoma irrespective of p53 statusINTERNATIONAL JOURNAL OF CANCER, Issue 5 2006Manuel Rieber Abstract MEK1/2 inhibitors like U0126 can potentiate or antagonize the antitumor activity of cytotoxic agents such as cisplatin, paclitaxel or vinblastine, depending on the drug or the target cells. We now investigated whether U0126, differentially regulates melanoma signaling in response to UV radiation or betulinic acid, a drug lethal against melanoma. This report shows that U0126 inhibits early response (ERK) kinase activation and cyclin A expression in wt p53 C8161 melanoma exposed to either UV radiation or betulinic acid. However, U0126 does not protect from UV damage, but counteracts betulinic acid-mediated apoptosis in the same cells. Protection from the latter drug by joint treatment with U0126 was also evident in wt p53 MelJuso melanoma and mutant p53 WM164 melanoma. The latter cells were the most responsive to betulinic acid, showing a selective decline in the cdk4 protein, without a comparable change in other key cell cycle proteins like cdc2, cdk2, cdk7 or cyclin A, prior to apoptosis-associated PARP fragmentation. Laser scanning cytometry also showed that betulinic acid induced a significant increase in chromatin condensation in WM164 melanoma irrespective of whether they were in adherent form or as multicellular spheroids. All these betulinic acid-induced changes were counteracted by U0126. Our data show for the first time that (a) cdk4 protein is an early target of betulinic acid-induced apoptosis and (b) unrestricted ERK signaling favours betulinic acid-induced apoptosis, but this is counteracted by U0126, partly through counteracting chromatin condensation and restoring Akt activation decreased by betulinic acid treatment. © 2005 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||