Altering textural properties of fermented milk by using surface-engineered Lactococcus lactis.

PubMed

Tarazanova, Mariya; Huppertz, Thom; Kok, Jan; Bachmann, Herwig

2018-05-09

Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe-matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell-clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3-19% to 94-99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface-dependent potential of bacterial cells as structure elements in fermented foods. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Human Corneal Limbal-Epithelial Cell Response to Varying Silk Film Geometric Topography In Vitro

PubMed Central

Lawrence, Brian D.; Pan, Zhi; Liu, Aihong; Kaplan, David L.; Rosenblatt, Mark I.

2012-01-01

Silk fibroin films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding silk films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal-epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that silk film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment, and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36%–54% increase on average in initial cell attachment, which corresponded to an over 2-fold increase in FA localization when compared to other silk film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned silk film topographies. In conclusion, silk film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially in regards to FA distribution, in vitro. PMID:22705042

Avian Follicular and Interdigitating Dendritic Cells: Isolation and Morphologic, Phenotypic, and Functional Analyses

USDA-ARS?s Scientific Manuscript database

An antiserum against Eimeria tenella sporozoites was used to localize and isolate Ag-binding cells in intestinal cecal tonsils of parasite-infected chickens. Based on their tissue localization, ultrastructural features, and expression of surface markers, two subpopulations of cells were isolated, C...

Microengineering of artificial capillaries

NASA Astrophysics Data System (ADS)

Moldovan, Nicanor I.

2002-11-01

Biocompatibility and functionality of implanted inorganic medical devices is limited by the local reaction of the organism, with a recently recognized contribution of nearby microvasculature. We explored the possibility to microengineer pre-embedded microvascular networks in the surface of inorganic devices. The implants would thus function as carriers of pre-assembled microvessels, ready to expand, and contribute to local angiogenesis. Based on our own studies on the role played by local microtopography in angiogenesis (the tunneling concept), we have shown the feasibility of endothelial cells cultivation in grooves created on the surface of the materials to be implanted, either polymeric or silicon. In order to develop this new technology, we devised an in situ approach to the study of the cellular behavior on micropatterned surfaces, by use of Laser Scanning Cytometry (LSC). In this report I will present our results regarding the LSC analysis of endothelial cells cultivated in grooves made on the surface of silicon wafers, and the consequences of this treatment on endothelial physiology. When comparing the growth of endothelial cells on line patterned and non-patterned areas, in terms of several morphological parameters of cell nuclei, our data support the conclusion that lateral confinement of endothelial cells induces a quiescent state, possibly by inhibiting their ability to proliferate.

Cell surface localization of the 78 kD glucose regulated protein (GRP 78) induced by thapsigargin.

PubMed

Delpino, A; Piselli, P; Vismara, D; Vendetti, S; Colizzi, V

1998-01-01

In the present study it was found that the synthesis of the 78 kD glucose-regulated protein (GRP 78 or BIP) is vigorously induced in human rabdomiosarcoma cells (TE 671/RD) following both short-term (1 h) and prolonged (18 h) exposure to 100 nM thapsigargin (Tg). Flow cytometric analysis with a specific anti-GRP 78 polyclonal antibody showed that Tg-treated cells express the GRP 78 on the plasma membrane. Cell surface localization of the Tg-induced GRP 78 was confirmed by biotinylation of membrane-exposed proteins and subsequent isolation of the biotin-labelled proteins by streptavidin/agarose affinity chromatography. It was found that a fraction of the Tg-induced GRP 78 is present among the biotin-labelled, surface-exposed, proteins. Conversely, the GRP 78 immunoprecipitated from unfractionated lysates of Tg-treated and biotin-reacted cells was found to be biotinylated. This is the first report demonstrating surface expression of GRP 78 in cells exposed to a specific GRP 78-inducing stimulus.

Surface plasmon enhanced cell microscopy with blocked random spatial activation

NASA Astrophysics Data System (ADS)

Son, Taehwang; Oh, Youngjin; Lee, Wonju; Yang, Heejin; Kim, Donghyun

2016-03-01

We present surface plasmon enhanced fluorescence microscopy with random spatial sampling using patterned block of silver nanoislands. Rigorous coupled wave analysis was performed to confirm near-field localization on nanoislands. Random nanoislands were fabricated in silver by temperature annealing. By analyzing random near-field distribution, average size of localized fields was found to be on the order of 135 nm. Randomly localized near-fields were used to spatially sample F-actin of J774 cells (mouse macrophage cell-line). Image deconvolution algorithm based on linear imaging theory was established for stochastic estimation of fluorescent molecular distribution. The alignment between near-field distribution and raw image was performed by the patterned block. The achieved resolution is dependent upon factors including the size of localized fields and estimated to be 100-150 nm.

Expression Analysis of the Transmembrane Mucin MUC20 in Human Corneal and Conjunctival Epithelia

PubMed Central

Woodward, Ashley M.; Argüeso, Pablo

2014-01-01

Purpose. Cell surface mucins are a group of highly O-glycosylated transmembrane glycoproteins responsible for the protection of epithelial cells on mucosal surfaces. The aim of this study was to investigate the localization and regulation of mucin 20 (MUC20) at the ocular surface. Methods. Localization of MUC20 in human corneal and conjunctival epithelia was evaluated by immunofluorescence microscopy. Immortalized corneal (HCLE) and conjunctival (HCjE) cell lines were grown at different stages of differentiation and subjected to quantitative PCR and Western blot analyses. Cell surface proteins on apical cell membranes were biotinylated and isolated by neutravidin chromatography. Results. The MUC20 was detected throughout the entire human ocular surface epithelia, predominantly in cell membranes within intermediate cell layers. In conjunctiva, MUC20 also was observed in the cytoplasm of apical cells within the stratified squamous epithelium, but not in goblet cells. Quantitative PCR and immunoblotting demonstrated expression of MUC20 in HCLE and HCjE cells. Induction of differentiation with serum-containing medium resulted in upregulation of MUC20 mRNA and protein. Biotin labeling of the surface of stratified cultures revealed low levels of MUC20 protein on apical glycocalyces. Further, MUC20 was not detected in the cell culture media or in human tears, suggesting that the extracellular domain of MUC20 is not released from the ocular surface as described previously for other cell surface mucins. Conclusions. Our results indicate that MUC20 is a novel transmembrane mucin expressed by the human corneal and conjunctival epithelia, and suggest that differential expression of MUC20 during differentiation has a role in maintaining ocular surface homeostasis. PMID:25168902

The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

PubMed

Lou, Hsin-Ya; Zhao, Wenting; Zeng, Yongpeng; Cui, Bianxiao

2018-05-15

Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell-probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host-implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies allowing the visualization of membrane deformation at the cell membrane-to-substrate interface with nanometer precision and demonstrate that vertical nanostructures induce local curvatures on the plasma membrane. These local curvatures enhance the process of clathrin-mediated endocytosis and affect actin dynamics. We also present evidence that vertical nanostructures can induce significant deformation of the nuclear membrane, which can affect chromatin distribution and gene expression. Finally, we provide a brief perspective on the curvature hypothesis and the challenges and opportunities for the design of nanotopography for manipulating cell behavior.

Interconnected subsets of memory follicular helper T cells have different effector functions.

PubMed

Asrir, Assia; Aloulou, Meryem; Gador, Mylène; Pérals, Corine; Fazilleau, Nicolas

2017-10-10

Follicular helper T cells regulate high-affinity antibody production. Memory follicular helper T cells can be local in draining lymphoid organs and circulate in the blood, but the underlying mechanisms of this subdivision are unresolved. Here we show that both memory follicular helper T subsets sustain B-cell responses after reactivation. Local cells promote more plasma cell differentiation, whereas circulating cells promote more secondary germinal centers. In parallel, local memory B cells are homogeneous and programmed to become plasma cells, whereas circulating memory B cells are able to rediversify. Local memory follicular helper T cells have higher affinity T-cell receptors, which correlates with expression of peptide MHC-II at the surface of local memory B cells only. Blocking T-cell receptor-peptide MHC-II interactions induces the release of local memory follicular helper T cells in the circulating compartment. Our studies show that memory follicular helper T localization is highly intertwined with memory B cells, a finding that has important implications for vaccine design.Tfh cells can differentiate into memory cells. Here the authors describe distinct functional and phenotypic profiles of these memory Tfh cells dependent on their anatomical localization to the lymphoid organs or to the circulation.

Local delivery of molecules from a nanopipette for quantitative receptor mapping on live cells.

PubMed

Babakinejad, Babak; Jönsson, Peter; López Córdoba, Ainara; Actis, Paolo; Novak, Pavel; Takahashi, Yasufumi; Shevchuk, Andrew; Anand, Uma; Anand, Praveen; Drews, Anna; Ferrer-Montiel, Antonio; Klenerman, David; Korchev, Yuri E

2013-10-01

Using nanopipettes to locally deliver molecules to the surface of living cells could potentially open up studies of biological processes down to the level of single molecules. However, in order to achieve precise and quantitative local delivery it is essential to be able to determine the amount and distribution of the molecules being delivered. In this work, we investigate how the size of the nanopipette, the magnitude of the applied pressure or voltage, which drives the delivery, and the distance to the underlying surface influences the number and spatial distribution of the delivered molecules. Analytical expressions describing the delivery are derived and compared with the results from finite element simulations and experiments on delivery from a 100 nm nanopipette in bulk solution and to the surface of sensory neurons. We then developed a setup for rapid and quantitative delivery to multiple subcellular areas, delivering the molecule capsaicin to stimulate opening of Transient Receptor Potential Vanilloid subfamily member 1 (TRPV1) channels, membrane receptors involved in pain sensation. Overall, precise and quantitative delivery of molecules from nanopipettes has been demonstrated, opening up many applications in biology such as locally stimulating and mapping receptors on the surface of live cells.

Imaging and reconstruction of cell cortex structures near the cell surface

NASA Astrophysics Data System (ADS)

Jin, Luhong; Zhou, Xiaoxu; Xiu, Peng; Luo, Wei; Huang, Yujia; Yu, Feng; Kuang, Cuifang; Sun, Yonghong; Liu, Xu; Xu, Yingke

2017-11-01

Total internal reflection fluorescence microscopy (TIRFM) provides high optical sectioning capability and superb signal-to-noise ratio for imaging of cell cortex structures. The development of multi-angle (MA)-TIRFM permits high axial resolution imaging and reconstruction of cellular structures near the cell surface. Cytoskeleton is composed of a network of filaments, which are important for maintenance of cell function. The high-resolution imaging and quantitative analysis of filament organization would contribute to our understanding of cytoskeleton regulation in cell. Here, we used a custom-developed MA-TIRFM setup, together with stochastic photobleaching and single molecule localization method, to enhance the lateral resolution of TIRFM imaging to about 100 nm. In addition, we proposed novel methods to perform filament segmentation and 3D reconstruction from MA-TIRFM images. Furthermore, we applied these methods to study the 3D localization of cortical actin and microtubule structures in U373 cancer cells. Our results showed that cortical actins localize ∼ 27 nm closer to the plasma membrane when compared with microtubules. We found that treatment of cells with chemotherapy drugs nocodazole and cytochalasin B disassembles cytoskeletal network and induces the reorganization of filaments towards the cell periphery. In summary, this study provides feasible approaches for 3D imaging and analyzing cell surface distribution of cytoskeletal network. Our established microscopy platform and image analysis toolkits would facilitate the study of cytoskeletal network in cells.

Depth-varying density and organization of chondrocytes in immature and mature bovine articular cartilage assessed by 3d imaging and analysis.

PubMed

Jadin, Kyle D; Wong, Benjamin L; Bae, Won C; Li, Kelvin W; Williamson, Amanda K; Schumacher, Barbara L; Price, Jeffrey H; Sah, Robert L

2005-09-01

Articular cartilage is a heterogeneous tissue, with cell density and organization varying with depth from the surface. The objectives of the present study were to establish a method for localizing individual cells in three-dimensional (3D) images of cartilage and quantifying depth-associated variation in cellularity and cell organization at different stages of growth. Accuracy of nucleus localization was high, with 99% sensitivity relative to manual localization. Cellularity (million cells per cm3) decreased from 290, 310, and 150 near the articular surface in fetal, calf, and adult samples, respectively, to 120, 110, and 50 at a depth of 1.0 mm. The distance/angle to the nearest neighboring cell was 7.9 microm/31 degrees , 7.1 microm/31 degrees , and 9.1 microm/31 degrees for cells at the articular surface of fetal, calf, and adult samples, respectively, and increased/decreased to 11.6 microm/31 degrees , 12.0 microm/30 degrees , and 19.2 microm/25 degrees at a depth of 0.7 mm. The methodologies described here may be useful for analyzing the 3D cellular organization of cartilage during growth, maturation, aging, degeneration, and regeneration.

Depth-varying density and organization of chondrocytes in immature and mature bovine articular cartilage assessed by 3d imaging and analysis

NASA Technical Reports Server (NTRS)

Jadin, Kyle D.; Wong, Benjamin L.; Bae, Won C.; Li, Kelvin W.; Williamson, Amanda K.; Schumacher, Barbara L.; Price, Jeffrey H.; Sah, Robert L.

2005-01-01

Articular cartilage is a heterogeneous tissue, with cell density and organization varying with depth from the surface. The objectives of the present study were to establish a method for localizing individual cells in three-dimensional (3D) images of cartilage and quantifying depth-associated variation in cellularity and cell organization at different stages of growth. Accuracy of nucleus localization was high, with 99% sensitivity relative to manual localization. Cellularity (million cells per cm3) decreased from 290, 310, and 150 near the articular surface in fetal, calf, and adult samples, respectively, to 120, 110, and 50 at a depth of 1.0 mm. The distance/angle to the nearest neighboring cell was 7.9 microm/31 degrees , 7.1 microm/31 degrees , and 9.1 microm/31 degrees for cells at the articular surface of fetal, calf, and adult samples, respectively, and increased/decreased to 11.6 microm/31 degrees , 12.0 microm/30 degrees , and 19.2 microm/25 degrees at a depth of 0.7 mm. The methodologies described here may be useful for analyzing the 3D cellular organization of cartilage during growth, maturation, aging, degeneration, and regeneration.

Analysis of Laser Injection Condition and Electrical Properties in Local BSF for Laser Fired Contact c-Si Solar Cell Applications.

PubMed

Park, Cheolmin; Choi, Gyuho; Balaji, Nagarajan; Ju, Minkyu; Lee, Youn-Jung; Lee, Haeseok; Yi, Junsin

2018-07-01

A crystalline silicon (c-Si) local-back-contact (LBC) solar cell for which a laser-condition-optimized surface-recombination velocity (SRV), a contact resistance (Rc), and local back surface fields (LBSFs) were utilized is reported. The effect of the laser condition on the rear-side electrical properties of the laser-fired LBC solar cell was studied. The Nd:YAG-laser (1064-nm wavelength) power and frequency were varied to obtain LBSF values with a lower contact resistance. A 10-kHz laser power of 44 mW resulted in an Rc of 0.125 ohms with an LBSF thickness of 2.09 μm and a higher open-circuit voltage (VOC) of 642 mV.

Reduction and shaping of graphene-oxide by laser-printing for controlled bone tissue regeneration and bacterial killing

NASA Astrophysics Data System (ADS)

Palmieri, Valentina; Barba, Marta; Di Pietro, Lorena; Gentilini, Silvia; Chiara Braidotti, Maria; Ciancico, Carlotta; Bugli, Francesca; Ciasca, Gabriele; Larciprete, Rosanna; Lattanzi, Wanda; Sanguinetti, Maurizio; De Spirito, Marco; Conti, Claudio; Papi, Massimiliano

2018-01-01

Graphene and graphene oxide (GO) are capable of inducing stem cells differentiation into bone tissue with variable efficacy depending on reductive state of the material. Thus, modulation of osteogenic process and of bone mineral density distribution is theoretically possible by controlling the GO oxidative state. In this study, we laser-printed GO surfaces in order to obtain both a local photo-thermal GO reduction and the formation of nano-wrinkles along precise geometric pattern. Initially, after cells adhered on the surface, stem cells migrated and accumulated on the reduced and wrinkled surface. When the local density of the stem cells on the reduced stripes was high, cells started to proliferate and occupy the oxidized/flat area. The designed surfaces morphology guided stem cell orientation and the reduction accelerated differentiation. Furthermore the reduced sharp nano-wrinkles were able to enhance the GO antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), a common cause of prosthetic joints infections. This strategy can offer a revolution in present and future trends of scaffolds design for regenerative medicine.

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

PubMed

Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

2015-01-13

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. © 2014 The Authors.

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis

PubMed Central

Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

2015-01-01

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. PMID:25476450

Unexpected Role for a Serine/Threonine-Rich Domain in the Candida albicans Iff Protein Family▿†

PubMed Central

Boisramé, Anita; Cornu, Amandine; Da Costa, Grégory; Richard, Mathias L.

2011-01-01

Glycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins in Candida albicans because of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag on all members of the family. The data obtained using the complete proteins showed for Iff3 (or -9), Iff5, Iff6, and Iff8 a covalent linkage to the β-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOH-labile bonds. However, since some proteins of the Iff family were undetectable, we designed chimeric constructions using the last 60 amino acids of these proteins to test the localization signal. These constructions showed a β-1,6-glucan linkage for Iff1/Rbr3, Iff2/Hyr3, Iff4 and Iff7/Hyr4 C-terminal–Iff5 fusion proteins, and a membrane localization for the Iff10/Flo9 C terminus-Iff5 fusion protein. Immunofluorescence analyses coupled to these cell fraction data confirmed the importance of the length of the central serine/threonine-rich region for cell surface exposure. Further analysis of the Iff2/Hyr3 linkage to the cell surface showed for the first time that a serine/threonine central region of a GPI-anchored protein may be responsible for the disulfide and the NaOH bonds to the glucan and glycoproteins network and may also override the signal of the proximal ω site region. PMID:21841123

Leptin-mediated regulation of MT1-MMP localization is KIF1B dependent and enhances gastric cancer cell invasion.

PubMed

Dong, Zhaogang; Xu, Xiaofei; Du, Lutao; Yang, Yongmei; Cheng, Huanhuan; Zhang, Xin; Li, Zewu; Wang, Lili; Li, Juan; Liu, Hui; Qu, Xun; Wang, Chuanxin

2013-05-01

Leptin overexpression is closely correlated with gastric cancer (GC) invasion, but its exact effect and the underlying mechanism in tumorigenesis remain poorly understood. Membrane type 1-matrix metalloproteinase (MT1-MMP), a surface-anchored 'master switch' proteinase, is overexpressed and plays crucial roles in tumor invasion. Here, we characterized the influence of leptin on the generation and surface localization of MT1-MMP in GC and elucidated its molecular mechanisms. Our results revealed that leptin promoted GC cell invasion in vitro by upregulating MT1-MMP expression. Furthermore, cell surface biotinylation assay and flow cytometry demonstrated that the surface expression of MT1-MMP was also enhanced by leptin, and knockdown of kinesin family member 1B (KIF1B, a microtubule plus end-directed monomeric motor protein) by small interference RNA inhibited this process. Notably, coimmunoprecipitation analysis indicated that leptin enhanced the interaction of MT1-MMP with KIF1B in a time-dependent manner, which consequently contributed to GC cell invasion. Moreover, leptin increased MT1-MMP or KIF1B expression by the protein kinase B (AKT) pathway and extracellular signal-regulated kinase 1/2 partially participated in this process. However, only AKT was implicated in the leptin-mediated membrane localization of MT1-MMP. Immunohistochemistry analysis revealed that leptin, MT1-MMP and KIF1B are overexpressed in GC tissues, and they positively correlated with clinical stage and lymph node metastasis. These observations indicate that this regulatory network exists in vivo. Taken together, our findings suggest that leptin is an effective intracellular stimulator of MT1-MMP and that leptin-enhanced cell surface localization of MT1-MMP is dependent on KIF1B, which consequently plays a critical role in GC invasion.

Surface multiheme c-type cytochromes from Thermincola potens: Implications for dissimilatory metal reduction by Gram-positive bacteria

NASA Astrophysics Data System (ADS)

Carlson, H. K.; Iavarone, A. T.; Gorur, A.; Yeo, B. S.; Tran, R.; Melnyk, R. A.; Mathies, R. A.; Auer, M.; Coates, J. D.

2011-12-01

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they have been shown to be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or the humic substances analog, anthraquinone-2,6-disulfonate (AQDS). The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS and that several MHCs are localized to the cell wall or cell surface of T. potens. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results are the first direct evidence for cell-wall associated cytochromes and MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

Limitations of a localized surface plasmon resonance sensor on Salmonella detection

USDA-ARS?s Scientific Manuscript database

We have designed a localized surface plasmon resonance (LSPR) biosensor to perform the whole cell detection of Salmonella using gold nanoparticls fabricated by oblique angle deposition technique. The LSPR sensor showed a plasmon peak shift due to the Salmonella antigen and anti-Salmonella antibody r...

Regulation of AMPA receptor localization in lipid rafts

PubMed Central

Hou, Qingming; Huang, Yunfei; Amato, Stephen; Snyder, Solomon H.; Huganir, Richard L.; Man, Heng-Ye

2009-01-01

Lipid rafts are special microdomains enriched in cholesterol, sphingolipids and certain proteins, and play important roles in a variety of cellular functions including signal transduction and protein trafficking. We report that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent. Lipid rafts in mature neurons exist on the entire cell-surface and display a high degree of mobility. AMPA receptors co-localize and associate with lipid rafts in the plasma membrane. The association of AMPARs with rafts is under regulation; through the NOS–NO pathway, NMDA receptor activity increases AMPAR localization in rafts. During membrane targeting, AMPARs insert into or at close proximity of the surface raft domains. Perturbation of lipid rafts dramatically suppresses AMPA receptor exocytosis, resulting in significant reduction in AMPAR cell-surface expression. PMID:18411055

Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses

PubMed Central

Montanuy, Imma; Alejo, Ali; Alcami, Antonio

2011-01-01

Eradication of smallpox was accomplished 30 yr ago, but poxviral infections still represent a public health concern due to the potential release of variola virus or the emergence of zoonotic poxviruses, such as monkeypox virus. A critical determinant of poxvirus virulence is the inhibition of interferons (IFNs) by the virus-encoded type I IFN-binding protein (IFNα/βBP). This immunomodulatory protein is secreted and has the unique property of interacting with the cell surface in order to prevent IFN-mediated antiviral responses. However, the mechanism of its attachment to the cell surface remains unknown. Using surface plasmon resonance and cell-binding assays, we report that the IFNα/βBP from vaccinia virus, the smallpox vaccine, interacts with cell surface glycosaminoglycans (GAGs). Analysis of the contribution of different regions of the protein to cell surface binding demonstrated that clusters of basic residues in the first immunoglobulin domain mediate GAG interactions. Furthermore, mutation of the GAG-interaction motifs does not affect its IFN-binding and -blocking capacity. Functional conservation of GAG-binding sites is demonstrated for the IFNα/βBP from variola and monkeypox viruses, extending our understanding of immune modulation by the most virulent human poxviruses. These results are relevant for the design of improved vaccines and intervention strategies.—Montanuy, I., Alejo, A., Alcami, A. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses. PMID:21372110

MRCK-1 drives apical constriction in C. elegans by linking developmental patterning to force generation

PubMed Central

Marston, Daniel J.; Higgins, Christopher D.; Peters, Kimberly A.; Cupp, Timothy D.; Dickinson, Daniel J.; Pani, Ariel M.; Moore, Regan P.; Cox, Amanda H.; Kiehart, Daniel P.; Goldstein, Bob

2016-01-01

Summary Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here, we identify a myosin light chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endodermal precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, β-catenin, and cadherin become highly enriched at the apical junctions of apically-constricting cells, and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis. PMID:27451898

A Caulobacter MreB mutant with irregular cell shape exhibits compensatory widening to maintain a preferred surface area to volume ratio

PubMed Central

Harris, Leigh K.; Dye, Natalie A.; Theriot, Julie A.

2014-01-01

Summary Rod-shaped bacteria typically elongate at a uniform width. To investigate the genetic and physiological determinants involved in this process, we studied a mutation in the morphogenetic protein MreB in Caulobacter crescentus that gives rise to cells with a variable-width phenotype, where cells have regions that are both thinner and wider than wild-type. During growth, individual cells develop a balance of wide and thin regions, and mutant MreB dynamically localizes to poles and thin regions. Surprisingly, the surface area to volume ratio of these irregularly-shaped cells is, on average, very similar to wild-type. We propose that, while mutant MreB localizes to thin regions and promotes rod-like growth there, wide regions develop as a compensatory mechanism, allowing cells to maintain a wild-type-like surface area to volume ratio. To support this model, we have shown that cell widening is abrogated in growth conditions that promote higher surface area to volume ratios, and we have observed individual cells with high ratios return to wild-type levels over several hours by developing wide regions, suggesting that compensation can take place at the level of individual cells. PMID:25266768

Atomic force microscopy – looking at mechanosensors on the cell surface

PubMed Central

Heinisch, Jürgen J.; Lipke, Peter N.; Beaussart, Audrey; El Kirat Chatel, Sofiane; Dupres, Vincent; Alsteens, David; Dufrêne, Yves F.

2012-01-01

Summary Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way in which these proteins respond to mechanical stimuli and assemble into large complexes remains poorly understood at the molecular level. In the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecular resolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-molecule manipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins from yeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells. PMID:23077172

Sodium bicarbonate secretion indicated by ultrastructural cytochemical localization of HCO3(-), Cl-, and Na+ ions on rat bile duct brush cells.

PubMed

Ogata, Takuro

2005-12-01

Brush cells are widely distributed in the digestive and respiratory apparatus, but their function is still unknown. Because brush cells (BC) are found in organs secreting NaHCO3, it was hypothesized that these cells may secrete NaHCO3. To test this possibility, rat common bile duct epithelia were examined by ultrastructural cytochemical methods for localizing HCO3(-), Cl-, and Na+ ions. All three ion precipitates were few in or on BCs of rats without stimulation. Lead carbonate precipitates, which localized HCO3(-) ions by the lead nitrate-osmium method, increased markedly on the surface of the microvilli (MV) of BCs after secretin or meal stimulation, but similar precipitates were few on the luminal surface of principal cells (PCs). Silver chloride precipitates, which indicate the presence of Cl- ions by the silver-osmium method, increased in the apical cytoplasm and in MV of BCs after secretin or meal stimulation, but they were few in PCs. Sodium pyroantimonate precipitates, which localize Na+ ions by the potassium pyroantimonate-osmium method, increased on the surface of the MV, along the basolateral membrane, and in the apical cytoplasm of BCs after secretin or meal stimulation, but they were few in PCs. These results strongly suggest that BCs may be a significant source of NaHCO3 secretion.

Funnel for localizing biological cell placement and arrangement

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soscia, David; Benett, William J.; Mukerjee, Erik V.

2018-03-06

The present disclosure relates to a funnel apparatus for channeling cells onto a plurality of distinct, closely spaced regions of a seeding surface. The funnel apparatus has a body portion having an upper surface and a lower surface. The body portion forms a plurality of flow paths, at least one of which is shaped to have a decreasing cross-sectional area from the upper surface to the lower surface. The flow paths are formed at the lower surface to enable cells deposited into the flow paths at the upper surface of the funnel apparatus to be channeled into a plurality ofmore » distinct, closely spaced regions on the seeding surface positioned adjacent the lower surface.« less

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

PubMed

Hsieh, Feng-Jen; Chen, Yen-Wei; Huang, Yao-Kuan; Lee, Hsien-Ming; Lin, Chun-Hung; Chang, Huan-Cheng

2018-02-06

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

Utilizing Functionalized Nano-Paterned Surfaces as a clue to Cell Metastasis in Prostate and Breast Cancer

NASA Astrophysics Data System (ADS)

Matthews, James; Bastatas, Lyndon

2012-03-01

There is a direct relation between the survival of a patient diagnosed with prostate or breast cancer and the metastatic potential of the patient's cancer. It is therefore extremely important to prognose metastatic potentials. In this study we investigated whether the behaviors of cancer cells responding to our state of the art nano-patterns differ by the metastatic potential of the cancer cells. We have used lowly (LNCaP) and highly (CL-1) metastatic human prostate cancer cells and lowly (MCF-7) and highly (MB231) metastatic breast cancer cells. A surface functionalization study was then performed first on uniform gold and glass surfaces, then on gold nano-patterned surfaces made by nano-sphere lithography using nano-spheres in diameter of 200nm to 800nm. The gold surfaces were functionalized with fibronectin (FN) and confirmed through XPS analysis. The CL-1, MCF-7, and MB231 cells show similar proliferation on all surfaces regardless of the presence of FN, whereas LNCaP show a clear preference for FN coated surfaces. The proliferation of the LNCaP was reduced when grown on finer nano-scaffolds, but the more aggressive CL-1, MB231, and MCF-7 cells show an abnormal proliferation regardless of pattern size. The difference in adhesion is intrinsic and was verified through dual fluorescent imaging. Clear co-localization of actin-vinculin were found on CL-1, MCF-7, and MB231. However LNCaP cells showed the co-localization only on the tips of the cells. These results provide vital clues to the bio-mechanical differences between the cancer cells with different metastatic potential.

Bone cell-materials interaction on Si microchannels with bioinert coatings.

PubMed

Condie, Russell; Bose, Susmita; Bandyopadhyay, Amit

2007-07-01

Bone implant life is dependent upon integration of biomaterial surfaces with local osteoblasts. This investigation studied the effects of various microchannel parameters and surface chemistry on immortalized osteoblast precursor cell (OPC1) adhesion. Cell-materials interactions were observed within channels of varying length, width, tortuosity, convergence, divergence and chemistry. Si wafers were used to create four distinct 1cm(2) designs of varying channel dimensions. After anisotropic chemical etching to a depth of 120microm, wafers were sputter coated with gold and titanium; and on another surface SiO(2) was grown to vary the surface chemistry of these microchannels. OPC1 cells were seeded in the central cavity of each chip before incubation in tissue culture plates. On days 5, 11 and 16, samples were taken out, fixed and processed for microscopic analysis. Samples were visually characterized, qualitatively scored and analyzed. Channel walls did not contain OPC1 migration, but showed locally interrupted adhesion. Scores for channels of floor widths as narrow as 350microm were significantly reduced. No statistically significant preference was detected for gold, titanium or SiO(2) surfaces. Bands of OPC1 cells appeared to align with nearby channels, suggesting that cell morphology may be controlled by topography of the design to improve osseointegration.

Expression of Pneumocystis jirovecii Major Surface Glycoprotein in Saccharomyces cerevisiae

PubMed Central

Kutty, Geetha; England, Katherine J.; Kovacs, Joseph A.

2013-01-01

The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis organisms, recombinant Msg localized to the plasma membrane of yeast rather than to the cell wall. Msg expression was targeted to the yeast cell wall by replacing its signal peptide, serine-threonine–rich region, and glycophosphatidylinositol anchor signal region with the signal peptide of cell wall protein α-agglutinin of S. cerevisiae, the serine-threonine–rich region of epithelial adhesin (Epa1) of Candida glabrata, and the carboxyl region of the cell wall protein (Cwp2) of S. cerevisiae, respectively. Immunofluorescence analysis and treatment with β-1,3 glucanase demonstrated that the expressed Msg fusion protein localized to the yeast cell wall. Surface expression of Msg protein resulted in increased adherence of yeast to A549 alveolar epithelial cells. Heterologous expression of Msg in yeast will facilitate studies of the biologic properties of Pneumocystis Msg. PMID:23532098

Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

PubMed

Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R

2005-05-01

Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

Functional roles of cell surface peptidases in reproductive organs

PubMed Central

2004-01-01

A number of biologically active peptides have been proposed to regulate function and differentiation of reproductive organs in an autocrine and/or paracrine fashion. Regulation of the local concentrations of these peptides is one of the important factors influencing their physiological effects on target cells. Membrane‐bound cell surface peptidases can activate or inactivate biologically active peptides before peptide factors access their receptors on the cell surface. Aminopeptidase A (EC 3.4.11.7), placental leucine aminopeptidase (EC 3.4.11.3), aminopeptidase‐N/CD13 (EC 3.4.11.2), dipeptidyl peptidases IV/CD26 (EC.3.4.14.5), carboxypeptidase‐M (EC 3.4.17.12), neutral endopeptidase/CD10 (EC 3.4.24.11) and endothelin converting enzyme‐1 (EC 3.4.23) are differentially expressed on the ovary, endometrium and placenta. The inhibition of enzyme activity affects steroid hormone production by granulosa and thecal cells, decidualization of endometrium and migration of extravillous trophoblasts. These findings suggest that membrane‐bound cell surface peptidases are local regulators for cellular growth and differentiation in reproductive organs by controlling extracellular concentration of peptide factors. (Reprod Med Biol 2004; 3: 165 –176) PMID:29662383

Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria

PubMed Central

Carlson, Hans K.; Iavarone, Anthony T.; Gorur, Amita; Yeo, Boon Siang; Tran, Rosalie; Melnyk, Ryan A.; Mathies, Richard A.; Auer, Manfred; Coates, John D.

2012-01-01

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium. PMID:22307634

mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

PubMed Central

Kaltimbacher, Valérie; Bonnet, Crystel; Lecoeuvre, Gaëlle; Forster, Valérie; Sahel, José-Alain; Corral-Debrinski, Marisol

2006-01-01

As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface—the region coding for the mitochondrial targeting sequence and the 3′UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3′UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3′UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity. PMID:16751614

Formation of a physiological complex between TRPV2 and RGA protein promotes cell surface expression of TRPV2.

PubMed

Stokes, Alexander J; Wakano, Clay; Del Carmen, Kimberly A; Koblan-Huberson, Murielle; Turner, Helen

2005-03-01

The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.

Effect of intermittent shear stress on corneal epithelial cells using an in vitro flow culture model.

PubMed

Hampel, Ulrike; Garreis, Fabian; Burgemeister, Fabian; Eßel, Nicole; Paulsen, Friedrich

2018-04-27

The aim of this study was to establish and to evaluate an in vitro model for culturing human telomerase-immortalized corneal epithelial (hTCEpi) cells under adjustable medium flow mimicking the movements of the tear film on the ocular surface. Using an IBIDI pump system, cells were cultured under unidirectional, continuous or oscillating, discontinuous medium flow. Cell surface and cytoskeletal architecture were investigated by scanning electron microscopy and immunofluorescence. Gene expression of e-cadherin, occludin, tight junction protein (TJP), desmoplakin, desmocollin and mucins was investigated by real-time PCR. Protein expression of desmoplakin, TJP, occludin and e-cadherin was analyzed by western blot and localization was detected by immunofluorescence. Rose bengal staining was used to assess mucin (MUC) barrier integrity. MUC1, -4 and -16 proteins were localized by immunofluorescence. Medium flow-induced shear stress dramatically changed cellular morphology of hTCEpi. Cells subjected to discontinuous shear stress displayed the typical flattened, polygonal cell shape of the superficial layer of stratified squamous epithelia. Cell surfaces showed less bulging under shear stress and less extracellular gaps. The mRNA expression of E-cadherin, occludin and TJP were increased under oscillatory medium flow. Desmoplakin and occludin protein were upregulated under oscillatory shear stress. Stress fiber formation was not aligned to flow direction. MUC1, -4, and -16 protein were localized under all culture conditions, a regulation on mRNA expression was not detectable. Rose Bengal uptake was diminished under unidirectional conditions. Our findings suggest that shear stress as it occurs at the ocular surface during blinking exerts marked effects on corneal epithelial cells, such as changes in cellular morphology and expression of cell junctions. The described model may be useful for in vitro investigations of ocular surface epithelia as it represents a much more physiologic reproduction of the in vivo situation than the commonly applied static culture conditions. Copyright © 2018. Published by Elsevier Inc.

Molecular Chaperone BiP Interacts with Borna Disease Virus Glycoprotein at the Cell Surface▿ †

PubMed Central

Honda, Tomoyuki; Horie, Masayuki; Daito, Takuji; Ikuta, Kazuyoshi; Tomonaga, Keizo

2009-01-01

Borna disease virus (BDV) is characterized by highly neurotropic infection. BDV enters its target cells using virus surface glycoprotein (G), but the cellular molecules mediating this process remain to be elucidated. We demonstrate here that the N-terminal product of G, GP1, interacts with the 78-kDa chaperone protein BiP. BiP was found at the surface of BDV-permissive cells, and anti-BiP antibody reduced BDV infection as well as GP1 binding to the cell surface. We also reveal that BiP localizes at the synapse of neurons. These results indicate that BiP may participate in the cell surface association of BDV. PMID:19776128

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

PubMed Central

Gründel, Anne; Pfeiffer, Melanie; Jacobs, Enno

2015-01-01

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract. PMID:26667841

Generation of patterned cell co-cultures in silicone tubing using a microelectrode technique and electrostatic assembly.

PubMed

Kaji, Hirokazu; Sekine, Soichiro; Hashimoto, Masahiko; Kawashima, Takeaki; Nishizawa, Matsuhiko

2007-01-01

We report a method for producing patterned cell adhesion inside silicone tubing. A platinum needle microelectrode was inserted through the wall of the tubing and an oxidizing agent electrochemically generated at the inserted electrode. This agent caused local detachment of the anti-biofouling heparin layer from the inner surface of the tubing. The cell-adhesive protein fibronectin selectively adsorbed onto the newly exposed surface, making it possible to initiate a localized cell culture. The electrode could be readily set in place without breaking the tubular structure and, importantly, almost no culture solution leaked from the electrode insertion site after the electrode was removed. Ionic adsorption of poly-L-lysine at the tubular region retaining a heparin coating was used to switch the heparin surface from cell-repellent to cell-adhesive, thereby facilitating the adhesion of a second cell type. The combination of the electrode-based technique with electrostatic deposition enabled the formation of patterned co-cultures within the semi-closed tubular structure. The controlled co-cultures inside the elastic tubing should be of value for cell-cell interaction studies following application of chemical or mechanical stimuli and for tissue engineering-based bioreactors.

Acylation-dependent protein export in Leishmania.

PubMed

Denny, P W; Gokool, S; Russell, D G; Field, M C; Smith, D F

2000-04-14

The surface of the protozoan parasite Leishmania is unusual in that it consists predominantly of glycosylphosphatidylinositol-anchored glycoconjugates and proteins. Additionally, a family of hydrophilic acylated surface proteins (HASPs) has been localized to the extracellular face of the plasma membrane in infective parasite stages. These surface polypeptides lack a recognizable endoplasmic reticulum secretory signal sequence, transmembrane spanning domain, or glycosylphosphatidylinositol-anchor consensus sequence, indicating that novel mechanisms are involved in their transport and localization. Here, we show that the N-terminal domain of HASPB contains primary structural information that directs both N-myristoylation and palmitoylation and is essential for correct localization of the protein to the plasma membrane. Furthermore, the N-terminal 18 amino acids of HASPB, encoding the dual acylation site, are sufficient to target the heterologous Aequorea victoria green fluorescent protein to the cell surface of Leishmania. Mutagenesis of the predicted acylated residues confirms that modification by both myristate and palmitate is required for correct trafficking. These data suggest that HASPB is a representative of a novel class of proteins whose translocation onto the surface of eukaryotic cells is dependent upon a "non-classical" pathway involving N-myristoylation/palmitoylation. Significantly, HASPB is also translocated on to the extracellular face of the plasma membrane of transfected mammalian cells, indicating that the export signal for HASPB is recognized by a higher eukaryotic export mechanism.

One-step nanoimprinted hybrid micro-/nano-structure for in situ protein detection of isolated cell array via localized surface plasmon resonance

NASA Astrophysics Data System (ADS)

Ali, Riyaz Ahmad Mohamed; Villariza Espulgar, Wilfred; Aoki, Wataru; Jiang, Shu; Saito, Masato; Ueda, Mitsuyoshi; Tamiya, Eiichi

2018-03-01

Nanoplasmonic biosensors show high potentials as label-free devices for continuous monitoring in biomolecular analyses. However, most current sensors comprise multiple-dedicated layers with complicated fabrication procedures, which increases production time and manufacturing costs. In this work, we report the synergistic integration of cell-trapping microwell structures with plasmonic sensing nanopillar structures in a single-layered substrate by one-step thermal nanoimprinting. Here, microwell arrays are used for isolating cells, wherein gold-capped nanostructures sense changes in local refractive index via localized surface plasmon resonance (LSPR). Hence, proteins secreted from trapped cells can be label-freely detected as peak shifts in absorbance spectra. The fabricated device showed a detection limit of 10 ng/µL anti-IgA. In Pichia pastoris cells trial analysis, a red shift of 6.9 nm was observed over 12 h, which is likely due to the protein secretion from the cells. This approach provides an inexpensive, rapid, and reproducible alternative for mass production of biosensors for continuous biomolecular analyses.

Vector vortex beam generation with dolphin-shaped cell meta-surface.

PubMed

Yang, Zhuo; Kuang, Deng-Feng; Cheng, Fang

2017-09-18

We present a dolphin-shaped cell meta-surface, which is a combination of dolphin-shaped metallic cells and dielectric substrate, for vector vortex beam generation with the illumination of linearly polarized light. Surface plasmon polaritons are excited at the boundary of the metallic cells, then guided by the metallic structures, and finally squeezed to the tips to form highly localized strong electromagnetic fields, which generate the intensity of vector vortex beams at z component. Synchronously, the abrupt phase change produced by the meta-surface is utilized to explain the vortex phase generated by elements. The new kind of structure can be utilized for communication, bioscience, and materiality.

Surface electromagnetic waves in Fibonacci superlattices: Theoretical and experimental results

NASA Astrophysics Data System (ADS)

El Hassouani, Y.; Aynaou, H.; El Boudouti, E. H.; Djafari-Rouhani, B.; Akjouj, A.; Velasco, V. R.

2006-07-01

We study theoretically and experimentally the existence and behavior of the localized surface modes in one-dimensional (1D) quasiperiodic photonic band gap structures. These structures are made of segments and loops arranged according to a Fibonacci sequence. The experiments are carried out by using coaxial cables in the frequency region of a few tens of MHz. We consider 1D periodic structures (superlattice) where each cell is a well-defined Fibonacci generation. In these structures, we generalize a theoretical rule on the surface modes, namely when one considers two semi-infinite superlattices obtained by the cleavage of an infinite superlattice, it exists exactly one surface mode in each gap. This mode is localized on the surface either of one or the other semi-infinite superlattice. We discuss the existence of various types of surface modes and their spatial localization. The experimental observation of these modes is carried out by measuring the transmission through a guide along which a finite superlattice (i.e., constituted of a finite number of quasiperiodic cells) is grafted vertically. The surface modes appear as maxima of the transmission spectrum. These experiments are in good agreement with the theoretical model based on the formalism of the Green function.

Triangle Geometry Processing for Surface Modeling and Cartesian Grid Generation

NASA Technical Reports Server (NTRS)

Aftosmis, Michael J. (Inventor); Melton, John E. (Inventor); Berger, Marsha J. (Inventor)

2002-01-01

Cartesian mesh generation is accomplished for component based geometries, by intersecting components subject to mesh generation to extract wetted surfaces with a geometry engine using adaptive precision arithmetic in a system which automatically breaks ties with respect to geometric degeneracies. During volume mesh generation, intersected surface triangulations are received to enable mesh generation with cell division of an initially coarse grid. The hexagonal cells are resolved, preserving the ability to directionally divide cells which are locally well aligned.

Triangle geometry processing for surface modeling and cartesian grid generation

DOEpatents

Aftosmis, Michael J [San Mateo, CA; Melton, John E [Hollister, CA; Berger, Marsha J [New York, NY

2002-09-03

Cartesian mesh generation is accomplished for component based geometries, by intersecting components subject to mesh generation to extract wetted surfaces with a geometry engine using adaptive precision arithmetic in a system which automatically breaks ties with respect to geometric degeneracies. During volume mesh generation, intersected surface triangulations are received to enable mesh generation with cell division of an initially coarse grid. The hexagonal cells are resolved, preserving the ability to directionally divide cells which are locally well aligned.

Ions, metabolites, and cells: Water as a reporter of surface conditions during bacterial growth.

PubMed

Jarisz, Tasha A; Lane, Sarah; Gozdzialski, Lea; Hore, Dennis K

2018-06-14

Surface-specific nonlinear vibrational spectroscopy, combined with bulk solution measurements and imaging, is used to study the surface conditions during the growth of E. coli. As a result of the silica high surface charge density, the water structure at the silica-aqueous interface is known to be especially sensitive to pH and ionic strength, and surface concentration profiles develop that can be appreciably different from the bulk solution conditions. We illustrate that, in the presence of growing cells, a unique surface micro-environment is established as a result of metabolites accumulating on the silica surface. Even in the subsequent absence of the cells, this surface layer works to reduce the interfacial ionic strength as revealed by the enhanced signal from surface water molecules. In the presence of growing cells, an additional boost in surface water signal is attributed to a local pH that is higher than that of the bulk solution.

Ions, metabolites, and cells: Water as a reporter of surface conditions during bacterial growth

NASA Astrophysics Data System (ADS)

Jarisz, Tasha A.; Lane, Sarah; Gozdzialski, Lea; Hore, Dennis K.

2018-06-01

Surface-specific nonlinear vibrational spectroscopy, combined with bulk solution measurements and imaging, is used to study the surface conditions during the growth of E. coli. As a result of the silica high surface charge density, the water structure at the silica-aqueous interface is known to be especially sensitive to pH and ionic strength, and surface concentration profiles develop that can be appreciably different from the bulk solution conditions. We illustrate that, in the presence of growing cells, a unique surface micro-environment is established as a result of metabolites accumulating on the silica surface. Even in the subsequent absence of the cells, this surface layer works to reduce the interfacial ionic strength as revealed by the enhanced signal from surface water molecules. In the presence of growing cells, an additional boost in surface water signal is attributed to a local pH that is higher than that of the bulk solution.

Diffusion mediated localization on membrane surfaces

NASA Technical Reports Server (NTRS)

Weaver, D. L.

1982-01-01

Using the model of a cell membrane of a spherical surface in which membrane components may diffuse, the rate of localization due to trapping under diffusion control has been estimated by computing an analytical expression for the mean trapping time including the possibilities of a trapping probability less than one and/or the establishment of an equilibrium at the trap boundary.

Sorafenib suppresses TGF-β responses by inducing caveolae/lipid raft-mediated internalization/degradation of cell-surface type II TGF-β receptors: Implications in development of effective adjunctive therapy for hepatocellular carcinoma.

PubMed

Chung, Chih-Ling; Wang, Shih-Wei; Sun, Wei-Chih; Shu, Chih-Wen; Kao, Yu-Chen; Shiao, Meng-Shin; Chen, Chun-Lin

2018-04-18

Sorafenib is the only FDA approved drug for the treatment of advanced hepatocellular carcinoma (HCC) and other malignancies. Studies indicate that TGF-β signalling is associated with tumour progression in HCC. Autocrine and paracrine TGF-β promotes tumour growth and malignancy by inducing epithelial-mesenchymal transition (EMT). Sorafenib is believed to antagonize tumour progression by inhibiting TGF-β-induced EMT. It improves survival of patients but HCC later develops resistance and relapses. The underlying mechanism of resistance is unknown. Understanding of the molecular mechanism of sorafenib inhibition of TGF-β-induced signalling or responses in HCC may lead to development of adjunctive effective therapy for HCC. In this study, we demonstrate that sorafenib suppresses TGF-β responsiveness in hepatoma cells, hepatocytes, and animal liver, mainly by downregulating cell-surface type II TGF-β receptors (TβRII) localized in caveolae/lipid rafts and non-lipid raft microdomains via caveolae/lipid rafts-mediated internalization and degradation. Furthermore, sorafenib-induced downregulation and degradation of cell-surface TβRII is prevented by simultaneous treatment with a caveolae disruptor or lysosomal inhibitors. On the other hand, sorafenib only downregulates cell-surface TβRII localized in caveolae/lipid rafts but not localized in non-lipid raft microdomains in hepatic stellate cells. These results suggest that sorafenib inhibits TGF-β signalling mainly by inducing caveolae/lipid raft-mediated internalization and degradation of cell-surface TβR-II in target cells. They may also imply that treatment with agents which promote formation of caveolae/lipid rafts, TGF-β receptor kinase inhibitors (e.g., LY2157299) or TGF-β peptide antagonists (by liver-targeting delivery) may be considered as effective adjunct therapy with sorafenib for HCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Spitzenkorper, exocyst, and polarisome components in Candida albicans hyphae show different patterns of localization and have distinct dynamic properties.

PubMed

Jones, Laura A; Sudbery, Peter E

2010-10-01

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.

Immunolocalization of matrix metalloproteinase-13 on bone surface under osteoclasts in rat tibia.

PubMed

Nakamura, Hiroaki; Sato, Ginga; Hirata, Azumi; Yamamoto, Toshio

2004-01-01

Matrix metalloproteinase (MMP)-13 (an interstitial collagenase also called collagenase 3) is involved in degradation of extracellular matrix in various tissues. Using immunohistochemistry and Western blotting, we investigated localization of MMP-13 in rat tibia, to clarify the role of MMP-13 in bone resorption. MMP-13 reactivity was mainly seen on bone surfaces under osteoclasts, and in some osteocytes and their lacunae near osteoclasts. However, immunoreactivity was not seen in chondrocytes or osteoclasts. MMP-13 was also localized on cement lines in the epiphysis. In the growth plate erosion zone, perivascular cells showed MMP-13 reactivity. Immunoelectron microscopy revealed that MMP-13 was localized on the bone surfaces, under the ruffled borders and some clear zones of osteoclasts. Gold-labeled MMP-13 was closely associated with collagen fibrils. Gold labeling was also detected in Golgi apparatus of osteocytes adjacent to osteoclasts and bone lining cells. Western blotting showed that MMP-13 was mainly associated with mineralized bone matrix. These findings suggest that MMP-13 synthesized and secreted by osteoblast-lineage cells is localized under the ruffled borders of osteoclasts. MMP-13 may play an important role in degradation of type I collagen in bone matrix, acting in concert with cathepsin K and MMP-9 produced by osteoclasts. MMP-13 in perivascular cells may be involved in removal of cartilage matrix proteins such as type II collagen and aggrecan.

Suppressing the formation of lipid raft-associated Rac1/PI3K/Akt signaling complexes by curcumin inhibits SDF-1α-induced invasion of human esophageal carcinoma cells.

PubMed

Lin, Meng-Liang; Lu, Yao-Cheng; Chen, Hung-Yi; Lee, Chuan-Chun; Chung, Jing-Gung; Chen, Shih-Shun

2014-05-01

Stromal cell-derived factor-1α (SDF-1α) is a ligand for C-X-C chemokine receptor type 4 (CXCR4), which contributes to the metastasis of cancer cells by promoting cell migration. Here, we show that the SDF-1α/CXCR4 axis can significantly increase invasion of esophageal carcinoma (EC) cells. We accomplished this by examining the effects of CXCR4 knockdown as well as treatment with a CXCR4-neutralizing antibody and the CXCR4-specific inhibitor AMD3100. Curcumin suppressed SDF-1α-induced cell invasion and matrix metalloproteinase-2 (MMP-2) promoter activity, cell surface localization of CXCR4 at lipid rafts, and lipid raft-associated ras-related C3 botulinum toxin substrate 1 (Rac1)/phosphatidylinositol 3-kinase (PI3K) p85α/Akt signaling. Curcumin inhibited SDF-1α-induced cell invasion by suppressing the Rac1-PI3K signaling complex at lipid rafts but did not abrogate lipid raft formation. We further demonstrate that the attenuation of lipid raft-associated Rac1 activity by curcumin was critical for the inhibition of SDF-1α-induced PI3K/Akt/NF-κB activation, cell surface localization of CXCR4 at lipid rafts, MMP-2 promoter activity, and cell invasion. Collectively, our results indicate that curcumin inhibits SDF-1α-induced EC cell invasion by suppressing the formation of the lipid raft-associated Rac1-PI3K-Akt signaling complex, the localization of CXCR4 with lipid rafts at the cell surface, and MMP-2 promoter activity, likely through the inhibition of Rac1 activity. © 2012 Wiley Periodicals, Inc.

The finite-size effect in thin liquid crystal systems

NASA Astrophysics Data System (ADS)

Śliwa, I.

2018-05-01

Effects of surface ordering in liquid crystal systems confined between cell plates are of great theoretical and experimental interest. Liquid crystals introduced in thin cells are known to be strongly stabilized and ordered by cell plates. We introduce a new theoretical method for analyzing the effect of surfaces on local molecular ordering in thin liquid crystal systems with planar geometry of the smectic layers. Our results show that, due to the interplay between pair long-range intermolecular forces and nonlocal, relatively short-range, surface interactions, both orientational and translational orders of liquid crystal molecules across confining cells are very complex. In particular, it is demonstrated that the SmA, nematic, and isotropic phases can coexist. The phase transitions from SmA to nematic, as well as from nematic to isotropic phases, occur not simultaneously in the whole volume of the system but begin to appear locally in some regions of the LC sample. Phase transition temperatures are demonstrated to be strongly affected by the thickness of the LC system. The dependence of the corresponding shifts of phase transition temperatures on the layer number is shown to exhibit a power law character. This new type of scaling behavior is concerned with the coexistence of local phases in finite systems. The influence of a specific character of interactions of molecules with surfaces and other molecules on values of the resulting critical exponents is also analyzed.

Targeting Thromboxane A2 Receptor for Antimetastasis Therapy of Breast Cancer

DTIC Science & Technology

2012-09-01

Figure 1B, right). The predominant localization of TP in the cytosol of MDA-MB-231 cells led us to examine whether a subset of TP is expressed at cell...in the Figure 1C, MDA-MB-231 cells had positive staining at cell surface, suggesting that a subset of TP was localized at plasma membrane. We...34, ISBN 979- 953-307-183-0. 2011. Robbins GT, Nie D. PPAR gamma, bioactive lipids, and cancer progression. Front Biosci. 17:1816-34, 2012. Grants

Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium

PubMed Central

Ewald, Andrew J.; Huebner, Robert J.; Palsdottir, Hildur; Lee, Jessie K.; Perez, Melissa J.; Jorgens, Danielle M.; Tauscher, Andrew N.; Cheung, Kevin J.; Werb, Zena; Auer, Manfred

2012-01-01

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organizations. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayered epithelium. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in three-dimensional culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, whereas PAR3 displayed a cytoplasmic localization, suggesting that the apico-basal polarity was incomplete. Despite membrane localization of E-cadherin and β-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex interdigitating membrane protrusions. Single-cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes re-formed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression might reflect partial recapitulation of a normal developmental program. PMID:22344263

Ultrastructural localization of human HL-A membrane antigens by use of hybrid antibodies

PubMed Central

Neauport-Sautes, Catherine; Silvestre, Daniele; Niccolai, Marie-Gabrielle; Kourilsky, F. M.; Levy, J. P.

1972-01-01

The localization of HL-A histocompatibility antigens at the surface of human lymphocytes in electron microscopy has been studied using hybrid antibodies to bind electron-dense particles (ferritin and plant viruses) to anti-HL-A antibody. A discontinuous distribution of the markers is observed at the cell surface, which is identical with that described for H-2 antigens on mouse lymphocytes with the same technique. Double labelling experiments suggest that the areas of the cell surface where HL-A antigens are detected contain also the heterologous lymphocyte antigens detected by an anti-thymocyte serum and that HL-A antigens are not renewed at a detectable level during the period of the labelling procedure in the areas of the cell surface which are not labelled primarily with ferritin-anti-IgG-anti-HL-A complexes. The interpretation of the discontinuous labelling of HL-A antigens with direct immunoferritin techniques is discussed. ImagesFIG. 2FIG. 3FIG. 4FIG. 5 PMID:5063188

Polycystin-1 Surface Localization Is Stimulated by Polycystin-2 and Cleavage at the G Protein-coupled Receptor Proteolytic Site

PubMed Central

Chapin, Hannah C.; Rajendran, Vanathy

2010-01-01

Polycystin (PC)1 and PC2 are membrane proteins implicated in autosomal dominant polycystic kidney disease. A physiologically relevant cleavage at PC1's G protein-coupled receptor proteolytic site (GPS) occurs early in the secretory pathway. Our results suggest that PC2 increases both PC1 GPS cleavage and PC1's appearance at the plasma membrane. Mutations that prevent PC1's GPS cleavage prevent its plasma membrane localization. PC2 is a member of the trp family of cation channels and is an important PC1 binding partner. The effect of PC2 on PC1 localization is independent of PC2 channel activity, as tested using channel-inhibiting PC2 mutations. PC1 and PC2 can interact through their C-terminal tails, but removing the C-terminal tail of either protein has no effect on PC1 surface localization in human embryonic kidney 293 cells. Experiments in polarized LLC-PK cells show that apical and ciliary PC1 localization requires PC2 and that this delivery is sensitive to PC2 truncation. In sum, our work shows that PC2 expression is required for the movement of PC1 to the plasma and ciliary membranes. In fibroblast cells this localization effect is independent of PC2's channel activity or PC1 binding ability but involves a stimulation of PC1's GPS cleavage before the PC1 protein's surface delivery. PMID:20980620

Lysosomal Rerouting of Hsp70 Trafficking as a Potential Immune Activating Tool for Targeting Melanoma

PubMed Central

Juhász, Kata; Thuenauer, Roland; Spachinger, Andrea; Duda, Ernő; Horváth, Ibolya; Vígh, László; Sonnleitner, Alois; Balogi, Zsolt

2013-01-01

Tumor specific cell surface localization and release of the stress inducible heat shock protein 70 (Hsp70) stimulate the immune system against cancer cells. A key immune stimulatory function of tumor-derived Hsp70 has been exemplified with the murine melanoma cell model, B16 overexpressing exogenous Hsp70. Despite the therapeutic potential mechanism of Hsp70 transport to the surface and release remained poorly understood. We investigated principles of Hsp70 trafficking in B16 melanoma cells with low and high level of Hsp70. In cells with low level of Hsp70 apparent trafficking of Hsp70 was mediated by endosomes. Excess Hsp70 triggered a series of changes such as a switch of Hsp70 trafficking from endosomes to lysosomes and a concomitant accumulation of Hsp70 in lysosomes. Moreover, lysosomal rerouting resulted in an elevated concentration of surface Hsp70 and enabled active release of Hsp70. In fact, hyperthermia, a clinically applicable approach triggered immediate active lysosomal release of soluble Hsp70 from cells with excess Hsp70. Furthermore, excess Hsp70 enabled targeting of internalized surface Hsp70 to lysosomes, allowing in turn heat-induced secretion of surface Hsp70. Altogether, we show that excess Hsp70 expressed in B16 melanoma cells diverts Hsp70 trafficking from endosomes to lysosomes, thereby supporting its surface localization and lysosomal release. Controlled excess-induced lysosomal rerouting and secretion of Hsp70 is proposed as a promising tool to stimulate anti-tumor immunity targeting melanoma. PMID:22920897

Selective Individual Primary Cell Capture Using Locally Bio-Functionalized Micropores

PubMed Central

Liu, Jie; Bombera, Radoslaw; Leroy, Loïc; Roupioz, Yoann; Baganizi, Dieudonné R.; Marche, Patrice N.; Haguet, Vincent; Mailley, Pascal; Livache, Thierry

2013-01-01

Background Solid-state micropores have been widely employed for 6 decades to recognize and size flowing unlabeled cells. However, the resistive-pulse technique presents limitations when the cells to be differentiated have overlapping dimension ranges such as B and T lymphocytes. An alternative approach would be to specifically capture cells by solid-state micropores. Here, the inner wall of 15-µm pores made in 10 µm-thick silicon membranes was covered with antibodies specific to cell surface proteins of B or T lymphocytes. The selective trapping of individual unlabeled cells in a bio-functionalized micropore makes them recognizable just using optical microscopy. Methodology/Principal Findings We locally deposited oligodeoxynucleotide (ODN) and ODN-conjugated antibody probes on the inner wall of the micropores by forming thin films of polypyrrole-ODN copolymers using contactless electro-functionalization. The trapping capabilities of the bio-functionalized micropores were validated using optical microscopy and the resistive-pulse technique by selectively capturing polystyrene microbeads coated with complementary ODN. B or T lymphocytes from a mouse splenocyte suspension were specifically immobilized on micropore walls functionalized with complementary ODN-conjugated antibodies targeting cell surface proteins. Conclusions/Significance The results showed that locally bio-functionalized micropores can isolate target cells from a suspension during their translocation throughout the pore, including among cells of similar dimensions in complex mixtures. PMID:23469221

Mapping cell surface adhesion by rotation tracking and adhesion footprinting

NASA Astrophysics Data System (ADS)

Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

2017-03-01

Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

Epidermal growth factor-induced mobilization of a ganglioside-specific sialidase (NEU3) to membrane ruffles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamaguchi, Kazunori; Hata, Keiko; Wada, Tadashi

2006-07-28

Human ganglioside-specific sialidase, NEU3, localized at cell membranes is thought to regulate various biological processes at cell surfaces. We here explored functional subcellular localization of the sialidase by immunofluorescence and found accumulation at leading edges of cell membranes in the presence of serum in culture. In response to EGF, the sialidase redistributed rapidly to ruffling cell membranes of squamous carcinoma A431 cells and co-localized with Rac-1. NEU3 overexpression enhanced Rac-1 activation and cell migration as compared with controls in HeLa cells as well as in A431 cells. Consistent with co-localization with Rac-1 by immunofluorescence, NEU3 was found to co-precipitate withmore » activated Rac bound to GST-PAK-1 fusion protein. NEU3 silencing by siRNA, in contrast, resulted in inhibition of Rac-1 activation. These results indicate that NEU3 is able to mobilize to membrane ruffles in response to growth stimuli and activate the Rac-1 signaling by co-localization with Rac-1, leading to increased cell motility.« less

Dynamic distribution of the SecA and SecY translocase subunits and septal localization of the HtrA surface chaperone/protease during Streptococcus pneumoniae D39 cell division.

PubMed

Tsui, Ho-Ching Tiffany; Keen, Susan K; Sham, Lok-To; Wayne, Kyle J; Winkler, Malcolm E

2011-01-01

The Sec translocase pathway is the major route for protein transport across and into the cytoplasmic membrane of bacteria. Previous studies reported that the SecA translocase ATP-binding subunit and the cell surface HtrA protease/chaperone formed a single microdomain, termed "ExPortal," in some species of ellipsoidal (ovococcus) Gram-positive bacteria, including Streptococcus pyogenes. To investigate the generality of microdomain formation, we determined the distribution of SecA and SecY by immunofluorescent microscopy in Streptococcus pneumoniae (pneumococcus), which is an ovococcus species evolutionarily distant from S. pyogenes. In the majority (≥ 75%) of exponentially growing cells, S. pneumoniae SecA (SecA (Spn)) and SecY (Spn) located dynamically in cells at different stages of division. In early divisional cells, both Sec subunits concentrated at equators, which are future sites of constriction. Further along in division, SecA(Spn) and SecY(Spn) remained localized at mid-cell septa. In late divisional cells, both Sec subunits were hemispherically distributed in the regions between septa and the future equators of dividing cells. In contrast, the HtrA (Spn) homologue localized to the equators and septa of most (> 90%) dividing cells, whereas the SrtA(Spn) sortase located over the surface of cells in no discernable pattern. This dynamic pattern of Sec distribution was not perturbed by the absence of flotillin family proteins, but was largely absent in most cells in early stationary phase and in cls mutants lacking cardiolipin synthase. These results do not support the existence of an ExPortal microdomain in S. pneumoniae. Instead, the localization of the pneumococcal Sec translocase depends on the stage of cell division and anionic phospholipid content. Two patterns of Sec translocase distribution, an ExPortal microdomain in certain ovococcus-shaped species like Streptococcus pyogenes and a spiral pattern in rod-shaped species like Bacillus subtilis, have been reported for Gram-positive bacteria. This study provides evidence for a third pattern of Sec localization in the ovococcus human pathogen Streptococcus pneumoniae. The SecA motor and SecY channel subunits of the Sec translocase localize dynamically to different places in the mid-cell region during the division cycle of exponentially growing, but not stationary-phase, S. pneumoniae. Unexpectedly, the S. pneumoniae HtrA (HtrA(Spn)) protease/chaperone principally localizes to cell equators and division septa. The coincident localization of SecA(Spn), SecY (Spn), and HtrA (Spn) to regions of peptidoglycan (PG) biosynthesis in unstressed, growing cells suggests that the pneumococcal Sec translocase directs assembly of the PG biosynthesis apparatus to regions where it is needed during division and that HtrA(Spn) may play a general role in quality control of proteins exported by the Sec translocase.

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

PubMed Central

Deppert, W; Hanke, K; Henning, R

1980-01-01

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. Images PMID:6255189

Optofluidic cellular immunofunctional analysis by localized surface plasmon resonance

NASA Astrophysics Data System (ADS)

Kurabayashi, Katsuo; Oh, Bo-Ram

2014-08-01

Cytokine secretion assays provide the means to quantify intercellular-signaling proteins secreted by blood immune cells. These assays allow researchers and clinicians to obtain valuable information on the immune status of the donor. Previous studies have demonstrated that localized surface plasmon resonance (LSPR) effects enable label-free, real-time biosensing on a nanostructured metallic surface with simple optics and sensing tunability. However, limited sensitivity coupled with a lack of sample handling capability makes it challenging to implement LSPR biosensing in cellular functional immunoanalysis based on cytokine secretion assay. This paper describes our recent progress towards full development of a label-free LSPR biosensing technique to detect cell-secreted tumor necrosis factor (TNF)-α cytokines in clinical blood samples. We integrate LSPR bionanosensors in an optofluidic platform capable of handling target immune cells in a microfluidic chamber while readily permitting optical access for cytokine detection.

Method for formation of high quality back contact with screen-printed local back surface field

DOEpatents

Rohatgi, Ajeet; Meemongkolkiat, Vichai

2010-11-30

A thin silicon solar cell having a back dielectric passivation and rear contact with local back surface field is described. Specifically, the solar cell may be fabricated from a crystalline silicon wafer having a thickness from 50 to 500 micrometers. A barrier layer and a dielectric layer are applied at least to the back surface of the silicon wafer to protect the silicon wafer from deformation when the rear contact is formed. At least one opening is made to the dielectric layer. An aluminum contact that provides a back surface field is formed in the opening and on the dielectric layer. The aluminum contact may be applied by screen printing an aluminum paste having from one to 12 atomic percent silicon and then applying a heat treatment at 750 degrees Celsius.

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

PubMed

Zhang, S X; Kobayashi, T; Okada, T; García del Saz, E; Seguchi, H

1991-07-01

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.

Viscous Fingering in Deformable Systems

NASA Astrophysics Data System (ADS)

Guan, Jian Hui; MacMinn, Chris

2017-11-01

Viscous fingering is a classical hydrodynamic instability that occurs when an invading fluid is injected into a porous medium or a Hele-Shaw cell that contains a more viscous defending fluid. Recent work has shown that viscous fingering in a Hele-Shaw cell is supressed when the flow cell is deformable. However, the mechanism of suppression relies on a net volumetric expansion of the flow area. Here, we study flow in a novel Hele-Shaw cell consisting of a rigid bottom plate and a flexible top plate that deforms in a way that is volume-conserving. In other words, fluid injection into the flow cell leads to a local expansion of the flow area (outward displacement of the flexible surface) that must be coupled to non-local contraction (inward displacement of the flexible surface). We explore the impact of this volumetric confinement on steady viscous flow and on viscous fingering. We would like to thank EPSRC for the funding for this work.

Lagrangian particle drift and surface deformation in a rotating wave on a free liquid surface

NASA Astrophysics Data System (ADS)

Fontana, Paul W.; Francois, Nicolas; Xia, Hua; Punzmann, Horst; Shats, Michael

2017-11-01

A nonlinear model of a rotating wave on the free surface of a liquid is presented. The flow is assumed to be inviscid and irrotational. The wave is constructed as a superposition of two perpendicular, monochromatic standing Stokes waves and is standing-wave-like, but with ``antinodes'' or cells consisting of rotating surface gradients of alternating polarity. Lagrangian fluid particle trajectories show a rotational drift about each cell in the direction of wave rotation, corresponding to a rotating Stokes drift. Each cell therefore has a circulating flow and localized angular momentum even though the Eulerian flow is irrotational. Meanwhile, the wave sets up a static displacement of the free surface, making a trough in each cell. This static surface gradient provides a centripetal force that may account for additional rotation seen in experiments.

Quantification of surface tension and internal pressure generated by single mitotic cells

NASA Astrophysics Data System (ADS)

Fischer-Friedrich, Elisabeth; Hyman, Anthony A.; Jülicher, Frank; Müller, Daniel J.; Helenius, Jonne

2014-08-01

During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplace's law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplace's law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ~ 40 Pa and 0.2 mNm-1 during interphase to ~ 400 Pa and 1.6 mNm-1 during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*

PubMed Central

Marty, Caroline; Chaligné, Ronan; Lacout, Catherine; Constantinescu, Stefan N.; Vainchenker, William; Villeval, Jean-Luc

2009-01-01

The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPLW515L mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPLW515L within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPLW515L, MPLW515L-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPLW515L-KDEL but was not phosphorylated. We generated disulfide-bonded MPLW515L homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPLW515L-KDEL, MPLW515L-S402C-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPLW515L with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPLW515L/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling. PMID:19261614

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Comprehensive analytical model for locally contacted rear surface passivated solar cells

NASA Astrophysics Data System (ADS)

Wolf, Andreas; Biro, Daniel; Nekarda, Jan; Stumpp, Stefan; Kimmerle, Achim; Mack, Sebastian; Preu, Ralf

2010-12-01

For optimum performance of solar cells featuring a locally contacted rear surface, the metallization fraction as well as the size and distribution of the local contacts are crucial, since Ohmic and recombination losses have to be balanced. In this work we present a set of equations which enable to calculate this trade off without the need of numerical simulations. Our model combines established analytical and empirical equations to predict the energy conversion efficiency of a locally contacted device. For experimental verification, we fabricate devices from float zone silicon wafers of different resistivity using the laser fired contact technology for forming the local rear contacts. The detailed characterization of test structures enables the determination of important physical parameters, such as the surface recombination velocity at the contacted area and the spreading resistance of the contacts. Our analytical model reproduces the experimental results very well and correctly predicts the optimum contact spacing without the use of free fitting parameters. We use our model to estimate the optimum bulk resistivity for locally contacted devices fabricated from conventional Czochralski-grown silicon material. These calculations use literature values for the stable minority carrier lifetime to account for the bulk recombination caused by the formation of boron-oxygen complexes under carrier injection.

Trapping charges at grain boundaries and degradation of CH3NH3Pb(I1-x Br x )3 perovskite solar cells.

PubMed

Nguyen, Bich Phuong; Kim, Gee Yeong; Jo, William; Kim, Byeong Jo; Jung, Hyun Suk

2017-08-04

The electrical properties of CH 3 NH 3 Pb(I 1-x Br x ) 3 (x = 0.13) perovskite materials were investigated under ambient conditions. The local work function and the local current were measured using Kelvin probe force microscopy and conductive atomic force microscopy, respectively. The degradation of the perovskite layers depends on their grain size. As the material degrades, an additional peak in the surface potential appears simultaneously with a sudden increase and subsequent relaxation of the local current. The potential bending at the grain boundaries and the intragrains is the most likely reason for the change of the local current surface of the perovskite layers. The improved understanding of the degradation mechanism garnered from this study helps pave the way toward an improved photo-conversion efficiency in perovskite solar cells.

Trapping charges at grain boundaries and degradation of CH3NH3Pb(I1-x Br x )3 perovskite solar cells

NASA Astrophysics Data System (ADS)

Phuong Nguyen, Bich; Kim, Gee Yeong; Jo, William; Kim, Byeong Jo; Jung, Hyun Suk

2017-08-01

The electrical properties of CH3NH3Pb(I1-x Br x )3 (x = 0.13) perovskite materials were investigated under ambient conditions. The local work function and the local current were measured using Kelvin probe force microscopy and conductive atomic force microscopy, respectively. The degradation of the perovskite layers depends on their grain size. As the material degrades, an additional peak in the surface potential appears simultaneously with a sudden increase and subsequent relaxation of the local current. The potential bending at the grain boundaries and the intragrains is the most likely reason for the change of the local current surface of the perovskite layers. The improved understanding of the degradation mechanism garnered from this study helps pave the way toward an improved photo-conversion efficiency in perovskite solar cells.

Chitosan Nanoparticles for Nuclear Targeting: The Effect of Nanoparticle Size and Nuclear Localization Sequence Density.

PubMed

Tammam, Salma N; Azzazy, Hassan M E; Breitinger, Hans G; Lamprecht, Alf

2015-12-07

Many recently discovered therapeutic proteins exert their main function in the nucleus, thus requiring both efficient uptake and correct intracellular targeting. Chitosan nanoparticles (NPs) have attracted interest as protein delivery vehicles due to their biocompatibility and ability to escape the endosomes offering high potential for nuclear delivery. Molecular entry into the nucleus occurs through the nuclear pore complexes, the efficiency of which is dependent on NP size and the presence of nuclear localization sequence (NLS). Chitosan nanoparticles of different sizes (S-NPs ≈ 25 nm; L-NP ≈ 150 nm) were formulated, and they were modified with different densities of the octapeptide NLS CPKKKRKV (S-NPs, 0.25, 0.5, 2.0 NLS/nm(2); L-NPs, 0.6, 0.9, 2 NLS/nm(2)). Unmodified and NLS-tagged NPs were evaluated for their protein loading capacity, extent of cell association, cell uptake, cell surface binding, and finally nuclear delivery efficiency in L929 fibroblasts. To avoid errors generated with cell fractionation and nuclear isolation protocols, nuclear delivery was assessed in intact cells utilizing Förster resonance energy transfer (FRET) fluorometry and microscopy. Although L-NPs showed ≈10-fold increase in protein loading per NP when compared to S-NPs, due to higher cell association and uptake S-NPs showed superior protein delivery. NLS exerts a size and density dependent effect on nanoparticle uptake and surface binding, with a general reduction in NP cell surface binding and an increase in cell uptake with the increase in NLS density (up to 8.4-fold increase in uptake of High-NLS-L-NPs (2 NLS/nm(2)) compared to unmodified L-NPs). However, for nuclear delivery, unmodified S-NPs show higher nuclear localization rates when compared to NLS modified NPs (up to 5-fold by FRET microscopy). For L-NPs an intermediate NLS density (0.9 NLS/nm(2)) seems to provide highest nuclear localization (3.7-fold increase in nuclear delivery compared to High-NLS-L-NPs). Results indicate that a higher NLS density does not result in maximum protein nuclear localization and that a universal optimal density for NPs of different sizes does not exist.

Laser doping of boron-doped Si paste for high-efficiency silicon solar cells

NASA Astrophysics Data System (ADS)

Tomizawa, Yuka; Imamura, Tetsuya; Soeda, Masaya; Ikeda, Yoshinori; Shiro, Takashi

2015-08-01

Boron laser doping (LD) is a promising technology for high-efficiency solar cells such as p-type passivated locally diffused solar cells and n-type Si-wafer-based solar cells. We produced a printable phosphorus- or boron-doped Si paste (NanoGram® Si paste/ink) for use as a diffuser in the LD process. We used the boron LD process to fabricate high-efficiency passivated emitter and rear locally diffused (PERL) solar cells. PERL solar cells on Czochralski Si (Cz-Si) wafers yielded a maximum efficiency of 19.7%, whereas the efficiency of a reference cell was 18.5%. Fill factors above 79% and open circuit voltages above 655 mV were measured. We found that the boron-doped area effectively performs as a local boron back surface field (BSF). The characteristics of the solar cell formed using NanoGram® Si paste/ink were better than those of the reference cell.

Influence of non-adherent yeast cells on electrical characteristics of diamond-based field-effect transistors

NASA Astrophysics Data System (ADS)

Procházka, Václav; Cifra, Michal; Kulha, Pavel; Ižák, Tibor; Rezek, Bohuslav; Kromka, Alexander

2017-02-01

Diamond thin films provide unique features as substrates for cell cultures and as bio-electronic sensors. Here we employ solution-gated field effect transistors (SGFET) based on nanocrystalline diamond thin films with H-terminated surface which exhibits the sub-surface p-type conductive channel. We study an influence of yeast cells (Saccharomyces cerevisiae) on electrical characteristics of the diamond SGFETs. Two different cell culture solutions (sucrose and yeast peptone dextrose-YPD) are used, with and without the cells. We have found that transfer characteristics of the SGFETs exhibit a negative shift of the gate voltage by -26 mV and -42 mV for sucrose and YPD with cells in comparison to blank solutions without the cells. This effect is attributed to a local pH change in close vicinity of the H-terminated diamond surface due to metabolic processes of the yeast cells. The pH sensitivity of the diamond-based SGFETs, the role of cell and protein adhesion on the gate surface and the role of negative surface charge of yeast cells on the SGFETs electrical characteristics are discussed as well.

Dynamics of Agglutinin-Like Sequence (ALS) Protein Localization on the Surface of Candida Albicans

ERIC Educational Resources Information Center

Coleman, David Andrew

2009-01-01

The ALS gene family encodes large cell-surface glycoproteins associated with "C. albicans" pathogenesis. Als proteins are thought to act as adhesin molecules binding to host tissues. Wide variation in expression levels among the ALS genes exists and is related to cell morphology and environmental conditions. "ALS1," "ALS3," and "ALS4" are three of…

A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation*

PubMed Central

Graessel, Anke; Hauck, Stefanie M.; von Toerne, Christine; Kloppmann, Edda; Goldberg, Tatyana; Koppensteiner, Herwig; Schindler, Michael; Knapp, Bettina; Krause, Linda; Dietz, Katharina; Schmidt-Weber, Carsten B.; Suttner, Kathrin

2015-01-01

Naive CD4+ T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments. PMID:25991687

Down-modulation of the G-protein-coupled Estrogen Receptor, GPER, from the Cell Surface Occurs via a trans-Golgi-Proteasome Pathway*

PubMed Central

Cheng, Shi-Bin; Quinn, Jeffrey A.; Graeber, Carl T.; Filardo, Edward J.

2011-01-01

GPER is a Gs-coupled seven-transmembrane receptor that has been linked to specific estrogen binding and signaling activities that are manifested by plasma membrane-associated enzymes. However, in many cell types, GPER is predominately localized to the endoplasmic reticulum (ER), and only minor amounts of receptor are detectable at the cell surface, an observation that has caused controversy regarding its role as a plasma membrane estrogen receptor. Here, we show that GPER constitutively buds intracellularly into EEA-1+ endosomes from clathrin-coated pits. Nonvisual arrestins-2/-3 do not co-localize with GPER, and expression of arrestin-2 dominant-negative mutants lacking clathrin- or β-adaptin interaction sites fails to block GPER internalization suggesting that arrestins are not involved in GPER endocytosis. Like β1AR, which recycles to the plasma membrane, GPER co-traffics with transferrin+, Rab11+ recycling endosomes. However, endocytosed GPER does not recycle to the cell surface, but instead returns to the trans-Golgi network (TGN) and does not re-enter the ER. GPER is ubiquitinated at the cell surface, exhibits a short half-life (t½ <1 h), and is protected from degradation by the proteasome inhibitor, MG132. Disruption of the TGN by brefeldin A induces the accumulation of endocytosed GPER in Rab11+ perinuclear endosomes and prevents GPER degradation. Our results provide an explanation as to why GPER is not readily detected on the cell surface in some cell types and further suggest that TGN serves as the checkpoint for degradation of endocytosed GPER. PMID:21540189

Absorption spectra of localized surface plasmon resonance observed in an inline/picoliter spectrometer cell fabricated by a near ultraviolet femtosecond laser

NASA Astrophysics Data System (ADS)

Shiraishi, Masahiko; Nishiyama, Michiko; Watanabe, Kazuhiro; Kubodera, Shoichi

2018-03-01

Absorption spectra based on localized surface plasmon resonance (LSPR) were obtained with an inline/picoliter spectrometer cell. The spectrometer cell was fabricated into an optical glass fiber by focusing a near UV (NUV) femtosecond laser pulses at a wavelength of 400 nm with an energy of 30 μJ. The laser beam was focused from two directions opposite to each other to fabricate a through-hole spectrometer cell. A diameter of the cell was approximately 3 μm, and the length was approximately 62.5 μm, which was nearly equal to the core diameter of the optical fiber. Liquid solution of gold nanoparticles (GNPs) with a diameter of 5-10 nm was injected into the spectrometer cell with its volume of 0.4 pL. The absorption peak centered at 518 nm was observed. An increase of absorption associated with the increase of the number of nanoparticles was in agreement with the numerical calculation based on the Lambert-Beer law.

[C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein].

PubMed

Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

2017-01-01

The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.

Vertical nanopillars for highly localized fluorescence imaging

PubMed Central

Xie, Chong; Hanson, Lindsey; Cui, Yi; Cui, Bianxiao

2011-01-01

Observing individual molecules in a complex environment by fluorescence microscopy is becoming increasingly important in biological and medical research, for which critical reduction of observation volume is required. Here, we demonstrate the use of vertically aligned silicon dioxide nanopillars to achieve below-the-diffraction-limit observation volume in vitro and inside live cells. With a diameter much smaller than the wavelength of visible light, a transparent silicon dioxide nanopillar embedded in a nontransparent substrate restricts the propagation of light and affords evanescence wave excitation along its vertical surface. This effect creates highly confined illumination volume that selectively excites fluorescence molecules in the vicinity of the nanopillar. We show that this nanopillar illumination can be used for in vitro single-molecule detection at high fluorophore concentrations. In addition, we demonstrate that vertical nanopillars interface tightly with live cells and function as highly localized light sources inside the cell. Furthermore, specific chemical modification of the nanopillar surface makes it possible to locally recruit proteins of interest and simultaneously observe their behavior within the complex, crowded environment of the cell. PMID:21368157

Nanoscale imaging of photocurrent and efficiency in CdTe solar cells

DOE PAGES

Leite, Marina S.; National Inst. of Standards and Technology; Abashin, Maxim; ...

2014-10-15

The local collection characteristics of grain interiors and grain boundaries in thin film CdTe polycrystalline solar cells are investigated using scanning photocurrent microscopy. The carriers are locally generated by light injected through a small aperture (50-300 nm) of a near-field scanning optical microscope in an illumination mode. Possible influence of rough surface topography on light coupling is examined and eliminated by sculpting smooth wedges on the granular CdTe surface. By varying the wavelength of light, nanoscale spatial variations in external quantum efficiency are mapped. We find that the grain boundaries (GBs) are better current collectors than the grain interiors (GIs).more » The increased collection efficiency is caused by two distinct effects associated with the material composition of GBs. First, GBs are charged, and the corresponding built-in field facilitates the separation and the extraction of the photogenerated carriers. Second, the GB regions generate more photocurrent at long wavelength corresponding to the band edge, which can be caused by a smaller local band gap. As a result, resolving carrier collection with nanoscale resolution in solar cell materials is crucial for optimizing the polycrystalline device performance through appropriate thermal processing and passivation of defect and surfaces.« less

Highly Sensitive Detection of Target Biomolecules on Cell Surface Using Gold Nanoparticle Conjugated with Aptamer Probe

NASA Astrophysics Data System (ADS)

Kim, Hyonchol; Terazono, Hideyuki; Hayashi, Masahito; Takei, Hiroyuki; Yasuda, Kenji

2012-06-01

A method of gold nanoparticle (Au NP) labeling with backscattered electron (BE) imaging of field emission scanning electron microscopy (FE-SEM) was applied for specific detection of target biomolecules on a cell surface. A single-stranded DNA aptamer, which specifically binds to the target molecule on a human acute lymphoblastic leukemia cell, was conjugated with a 20 nm Au NP and used as a probe to label its target molecule on the cell. The Au NP probe was incubated with the cell, and the interaction was confirmed using BE imaging of FE-SEM through direct counting of the number of Au NPs attached on the target cell surface. Specific Au NP-aptamer probes were observed on a single cell surface and their spatial distributions including submicron-order localizations were also clearly visualized, whereas the nonspecific aptamer probes were not observed on it. The aptamer probe can be potentially dislodged from the cell surface with treatment of nucleases, indicating that Au NP-conjugated aptamer probes can be used as sensitive and reversible probes to label target biomolecules on cells.

Nanoscale Relationship Between CD4 and CD25 of T Cells Visualized with NSOM/QD-Based Dual-Color Imaging System

NASA Astrophysics Data System (ADS)

Fan, Jinping; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-10-01

In this study, by using of near-field scanning optical microscopy (NSOM)/immune-labeling quantum dot (QD)-based dual-color imaging system, we achieved the direct visualization of nanoscale profiles for distribution and organization of CD4 and CD25 molecules in T cells. A novel and interesting finding was that though CD25 clustering as nanodomains were observed on the surface of CD4+CD25high regulatory T cells, these CD25 nanodomains were not co-localized with CD4 nanodomains. This result presented that the formation of these CD25 nanodomains on the surface of CD4+CD25high T cells were not associated with the response of T cell receptor (TCR)/CD3-dependent signal transduction. In contrast, on the surface of CD4+CD25low T cells, CD25 molecules distributed randomly without forming nanodomains while CD4 clustering as nanodomains can be observed; on the surface of CD8+CD25+ T cells, CD25 clustering as nanodomains and co-localization with CD8 nanodomains were observed. Collectively, above these results exhibited that TCR/CD3-based microdomains were indeed required for TCR/CD3-mediated T cells activation and enhanced the immune activity of CD4+CD25low T cells or CD8+CD25+ T cells. In particular, it was found that the formation of CD25 nanodomains and their segregation from TCR/CD3 microdomains were the intrinsic capability of CD4+CD25high T cells, suggesting this specific imaging feature of CD25 should be greatly associated with the regulatory activity of CD4+CD25high T cells. Importantly, this novel NSOM/QD-based dual-color imaging system will provide a useful tool for the research of distribution-function relationship of cell-surface molecules.

Imaging of blood antigen distribution on blood cells by thermal lens microscopy

NASA Astrophysics Data System (ADS)

Kimura, Hiroko; Sekiguchi, Kazuya; Nagao, Fumiko; Mukaida, Masahiro; Kitamori, Takehiko; Sawada, Tsuguo

2000-05-01

Blood group antigens on a cell were measured by a new microscopic method, i.e. thermal lens microscopy which involves spectrometry using a laser-induced thermal-lens effect. The blood group antigen was immunologically stained using antibody labeled with colloidal gold. Human leukocyte antigens (HLA) on lymphocytes and mononuclear leukocytes were observed by the thermal lens microscope, and Lewis blood group antigens on erythrocytes and polymorphonuclear leukocytes were also observed. The antigen distribution on each cell-surface was imaged using this technique. In spite of convex surface of living cells, colloidal gold was correctly quantified by adjusting the deviation of the focal point of the probe laser by the phase of the signal. In the measurement of leukocyte antigens, antigens of HLA-A, -B, -C loci on the lymphocytes were identified and quantitated by using a single cell. The image of HLA-A, -B, -C antigen distribution on a mononuclear leukocyte was obtained. In the measurement of erythrocyte antigens, a small quantity of Lewis antigens was detected on the cord erythrocytes. Localized small quantities of membrane antigens are better quantitated without extraction or cytolysis. Our thermal lens microscope is a powerful and highly sensitive analytical tool for detecting and quantitating localized antigens in single cells and/or cell-surface-associated molecules.

Voc Degradation in TF-VLS Grown InP Solar Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Yubo; Sun, Xingshu; Johnston, Steve

2016-11-21

Here we consider two hypotheses to explain the open-circuit voltage (VOC) degradation observed in thin-film vapor-liquid-solid (TF-VLS) grown p-type InP photovoltaic cells: bandgap narrowing and local shunting. First, a bandgap (Eg) narrowing effect is hypothesized, based on the surface inhomogeneity of VLS InP captured by the photoluminescence (PL) image. The PL data was used to estimate a spatially-resolved active VOC across surface of the InP sample. Combining this data with the effective Jsc allowed an assessment of the I-V characteristics of individual unit cells. Next, an H-SPICE diode compact model was utilized to reproduce the I-V characteristics of the wholemore » sample. We find a good fit to the I-V performance of TF-VLS grown InP solar cell. Second, a local shunting effect was also considered as an alternative explanation of the VOC degradation effect. Again, PL image data was used, and small local shunt resistance was added in arbitrary elementary unit cells to represent certain dark spots seen in the PL image and dictate the VOC degradation occurred in the sample.« less

Local bone marrow renin-angiotensin system in primitive, definitive and neoplastic haematopoiesis.

PubMed

Haznedaroglu, Ibrahim C; Beyazit, Yavuz

2013-03-01

The locally active ligand peptides, mediators, receptors and signalling pathways of the haematopoietic BM (bone marrow) autocrine/paracrine RAS (renin-angiotensin system) affect the essential steps of definitive blood cell production. Haematopoiesis, erythropoiesis, myelopoiesis, formation of monocytic and lymphocytic lineages, thrombopoiesis and other stromal cellular elements are regulated by the local BM RAS. The local BM RAS is present and active even in primitive embryonic haematopoiesis. ACE (angiotensin-converting enzyme) is expressed on the surface of the first endothelial and haematopoietic cells, forming the marrow cavity in the embryo. ACE marks early haematopoietic precursor cells and long-term blood-forming CD34(+) BM cells. The local autocrine tissue BM RAS may also be active in neoplastic haematopoiesis. Critical RAS mediators such as renin, ACE, AngII (angiotensin II) and angiotensinogen have been identified in leukaemic blast cells. The local tissue RAS influences tumour growth and metastases in an autocrine and paracrine fashion via the modulation of numerous carcinogenic events, such as angiogenesis, apoptosis, cellular proliferation, immune responses, cell signalling and extracellular matrix formation. The aim of the present review is to outline the known functions of the local BM RAS within the context of primitive, definitive and neoplastic haematopoiesis. Targeting the actions of local RAS molecules could represent a valuable therapeutic option for the management of neoplastic disorders.

Direct metallization local Al-back surface field for high efficiency screen printed crystalline silicon solar cells.

PubMed

Lee, Jonghwan; Park, Cheolmin; Dao, Vinh Ai; Lee, Youn-Jung; Ryu, Kyungyul; Choi, Gyuho; Kim, Bonggi; Ju, Minkyu; Jeong, Chaehwan; Yi, Junsin

2013-11-01

In this paper, we present a detailed study on the local back contact (LBC) formation of rear-surface-passivated silicon solar cells, where both the LBC opening and metallization are realized by one-step alloying of a dot of fine pattern screen-printed aluminum paste with the silicon substrate. Based on energy dispersive spectrometer (EDS) and scanning electron microscopy (SEM) characterizations, we suggest that the aluminum distribution and the silicon concentration determine the local-back-surface-field (Al-p+) layer thickness, resistivity of the Al-p+ and hence the quality of the Al-p+ formation. The highest penetration of silicon concentration of 78.17% in aluminum resulted in the formation of a 5 microm-deep Al-p+ layer, and the minimum LBC resistivity of 0.92 x 10-6 omega cm2. The degradation of the rear-surface passivation due to high temperature of the LBC formation process can be fully recovered by forming gas annealing (FGA) at temperature and hydrogen content of 450 degrees C and 15%, respectively. The application of the optimized LBC of rear-surface-passivated by a dot of fine pattern screen(-) printed aluminum paste resulted in efficiency of up to 19.98% for the p-type czochralski (CZ) silicon wafers with 10.24 cm2 cell size at 649 mV open circuit voltage. By FGA for rear-surface passivation recovery, efficiencies up to 20.35% with a V(OC) of 662 mV, FF of 82%, and J(SC) of 37.5 mA/cm2 were demonstrated.

Atomic force microscopy recognition of protein A on Staphylococcus aureus cell surfaces by labelling with IgG-Au conjugates.

PubMed

Tatlybaeva, Elena B; Nikiyan, Hike N; Vasilchenko, Alexey S; Deryabin, Dmitri G

2013-01-01

The labelling of functional molecules on the surface of bacterial cells is one way to recognize the bacteria. In this work, we have developed a method for the selective labelling of protein A on the cell surfaces of Staphylococcus aureus by using nanosized immunogold conjugates as cell-surface markers for atomic force microscopy (AFM). The use of 30-nm size Au nanoparticles conjugated with immunoglobulin G (IgG) allowed the visualization, localization and distribution of protein A-IgG complexes on the surface of S. aureus. The selectivity of the labelling method was confirmed in mixtures of S. aureus with Bacillus licheniformis cells, which differed by size and shape and had no IgG receptors on the surface. A preferential binding of the IgG-Au conjugates to S. aureus was obtained. Thus, this novel approach allows the identification of protein A and other IgG receptor-bearing bacteria, which is useful for AFM indication of pathogenic microorganisms in poly-component associations.

Simple shearing flow of dry soap foams with TCP structure[Tetrahedrally Close-Packed

DOE Office of Scientific and Technical Information (OSTI.GOV)

REINELT,DOUGLAS A.; KRAYNIK,ANDREW M.

2000-02-16

The microrheology of dry soap foams subjected to large, quasistatic, simple shearing deformations is analyzed. Two different monodisperse foams with tetrahedrally close-packed (TCP) structure are examined: Weaire-Phelan (A15) and Friauf-Laves (C15). The elastic-plastic response is evaluated by calculating foam structures that minimize total surface area at each value of strain. The minimal surfaces are computed with the Surface Evolver program developed by Brakke. The foam geometry and macroscopic stress are piecewise continuous functions of strain. The stress scales as T/V{sup 1/3} where T is surface tension and V is cell volume. Each discontinuity corresponds to large changes in foam geometrymore » and topology that restore equilibrium to unstable configurations that violate Plateau's laws. The instabilities occur when the length of an edge on a polyhedral foam cell vanishes. The length can tend to zero smoothly or abruptly with strain. The abrupt case occurs when a small increase in strain changes the energy profile in the neighborhood of a foam structure from a local minimum to a saddle point, which can lead to symmetry-breaking bifurcations. In general, the new foam topology associated with each stable solution branch results from a cascade of local topology changes called T1 transitions. Each T1 cascade produces different cell neighbors, reduces surface energy, and provides an irreversible, film-level mechanism for plastic yield behavior. Stress-strain curves and average stresses are evaluated by examining foam orientations that admit strain-periodic behavior. For some orientations, the deformation cycle includes Kelvin cells instead of the original TCP structure; but the foam does not remain perfectly ordered. Bifurcations during subsequent T1 cascades lead to disorder and can even cause strain localization.« less

Amyloid Precursor-like Protein 2 Association with HLA Class I Molecules

PubMed Central

Tuli, Amit; Sharma, Mahak; Wang, Xiaojian; Simone, Laura C.; Capek, Haley L.; Cate, Steven; Hildebrand, William H.; Naslavsky, Naava; Caplan, Steve; Solheim, Joyce C.

2009-01-01

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2Kd and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression. PMID:19184004

Cell-material interactions revealed via material techniques of surface patterning.

PubMed

Yao, Xiang; Peng, Rong; Ding, Jiandong

2013-10-04

Cell-material interactions constitute a key fundamental topic in biomaterials study. Various cell cues and matrix cues as well as soluble factors regulate cell behaviors on materials. These factors are coupled with each other as usual, and thus it is very difficult to unambiguously elucidate the role of each regulator. The recently developed material techniques of surface patterning afford unique ways to reveal the underlying science. This paper reviews the pertinent material techniques to fabricate patterns of microscale and nanoscale resolutions, and corresponding cell studies. Some issues are emphasized, such as cell localization on patterned surfaces of chemical contrast, and effects of cell shape, cell size, cell-cell contact, and seeding density on differentiation of stem cells. Material cues to regulate cell adhesion, cell differentiation and other cell events are further summed up. Effects of some physical properties, such as surface topography and matrix stiffness, on cell behaviors are also discussed; nanoscaled features of substrate surfaces to regulate cell fate are summarized as well. The pertinent work sheds new insight into the cell-material interactions, and is stimulating for biomaterial design in regenerative medicine, tissue engineering, and high-throughput detection, diagnosis, and drug screening. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes.

PubMed

Casbon, Amy-Jo; Allen, Lee-Ann H; Dunn, Kenneth W; Dinauer, Mary C

2009-02-15

Flavocytochrome b(558), the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91(phox) (NOX2) and a smaller subunit, p22(phox). Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91(phox) and p22(phox), we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22(phox) and gp91(phox) subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes.

Pinpoint Delivery of Molecules by Using Electron Beam Addressing Virtual Cathode Display.

PubMed

Hoshino, Takayuki; Yoshioka, Moto; Wagatsuma, Akira; Miyazako, Hiroki; Mabuchi, Kunihiko

2018-03-01

Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode. The kinetic energy of the incident primary electrons to the SiN membrane was completely blocked, whereas negative charges and leaking electric current appeared on the surface of the dielectric SiN membrane within a region of 100 nm. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). Increasing fluorescence of pre-diluted PI indicated local poration and transmembrane inflow at the virtual cathode position, as well as intracellular diffusion. The transmembrane inflows depended on beam duration time and acceleration voltage. At the post-molecular delivery, a slight decrease in intracellular PI fluorescence intensity indicates membrane recovery from the poration. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.

Binding-, intracellular transport-, and biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked nephrogenic diabetes insipidus.

PubMed Central

Tsukaguchi, H; Matsubara, H; Taketani, S; Mori, Y; Seido, T; Inada, M

1995-01-01

Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunofluorescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder. Images PMID:7560098

Biofilm formation and local electrostatic force characteristics of Escherichia coli O157:H7 observed by electrostatic force microscopy

NASA Astrophysics Data System (ADS)

Oh, Y. J.; Jo, W.; Yang, Y.; Park, S.

2007-04-01

The authors report growth media dependence of electrostatic force characteristics in Escherichia coli O157:H7 biofilm through local measurement by electrostatic force microscopy (EFM). The difference values of electrostatic interaction between the bacterial surface and the abiotic surface show an exponential decay behavior during biofilm development. In the EFM data, the biofilm in the low nutrient media shows a faster decay than the biofilm in the rich media. The surface potential in the bacterial cells was changed from 957to149mV. Local characterization of extracellular materials extracted from the bacteria reveals the progress of the biofilm formation and functional complexities.

Gold nanorods as contrast agents for biological imaging: optical properties, surface conjugation, and photothermal effects†

PubMed Central

Tong, Ling; Wei, Qingshan; Wei, Alexander; Cheng, Ji-Xin

2009-01-01

Gold nanorods (NRs) have plasmon-resonant absorption and scattering in the near-infrared (NIR) region, making them attractive probes for in vitro and in vivo imaging. In the cellular environment, NRs can provide scattering contrast for darkfield microscopy, or emit a strong two-photon luminescence (TPL) due to plasmon-enhanced two-photon absorption. NRs have also been employed in biomedical imaging modalities such as optical coherence tomography (OCT) or photoacoustic tomography (PAT). Careful control over surface chemistry enhances the capacity of NRs as biological imaging agents by enabling cell-specific targeting, and by increasing their dispersion stability and circulation lifetimes. NRs can also efficiently convert optical energy into heat, and inflict localized damage to tumor cells. Laser-induced heating of NRs can disrupt cell membrane integrity and homeostasis, resulting in Ca2+ influx and the depolymerization of the intracellular actin network. The combination of plasmon-resonant optical properties, intense local photothermal effects, and robust surface chemistry render gold NRs as promising theragnostic agents. PMID:19161395

Characterization of CD44-Mediated Cancer Cell Uptake and Intracellular Distribution of Hyaluronan-Grafted Liposomes

PubMed Central

Qhattal, Hussaini Syed Sha; Liu, Xinli

2011-01-01

Hyaluronan (HA) is a biocompatible and biodegradable linear polysaccharide which is of interest for tumor targeting through cell surface CD44 receptors. HA binds with high affinity to CD44 receptors, which are overexpressed in many tumors and involved in cancer metastasis. In the present study, we investigated the impact of HA molecular weight (MW), grafting density, and CD44 receptor density on endocytosis of HA-grafted liposomes (HA-liposomes) by cancer cells. Additionally, the intracellular localization of the HA-liposomes was determined. HAs of different MWs (5-8, 10-12, 175-350, and 1600 kDa) were conjugated to liposomes with varying degrees of grafting density. HA surface density was quantified using the hexadecyltrimethylammonium bromide turbidimetric method. Cellular uptake and subcellular localization of HA-liposomes were evaluated by flow cytometry and fluorescence microscopy. Mean particle sizes of HA-liposomes ranged from 120 to 180 nm and increased with the bigger size of HA. HA-liposome uptake correlated with HA MW (5-8 < 10-12 < 175-350 kDa), grafting density, and CD44 receptor density and exceeded that obtained with unconjugated plain liposomes. HA-liposomes were taken up into cells via lipid raft-mediated endocytosis, which is both energy- and cholesterol-dependent. Once within cells, HA-liposomes localized primarily to endosomes and lysosomes. The results demonstrate that cellular targeting efficiency of HA-liposomes depends strongly upon HA MW, grafting density, and cell surface receptor CD44 density. The results support a role of HA-liposomes for targeted drug delivery. PMID:21696190

Direct interaction between surface β1,4-galactosyltransferase 1 and epidermal growth factor receptor (EGFR) inhibits EGFR activation in hepatocellular carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Wenqing; Weng, Shuqiang; Zhang, Si

2013-05-10

Highlights: •β1,4GT1 interacts with EGFR both in vitro and in vivo. •β1,4GT1 co-localizes with EGFR on the cell surface. •β1,4GT1 inhibits {sup 125}I-EGF binding to EGFR. •β1,4GT1 inhibits EGF induced EGFR dimerization and phosphorylation. -- Abstract: Our previous studies showed that cell surface β1,4-galactosyltransferase 1 (β1,4GT1) negatively regulated cell survival through inhibition and modulation of the epidermal growth factor receptor (EGFR) signaling pathway in human hepatocellular carcinoma (HCC) SMMC-7721 cells. However, the underlying mechanism remains unclear. Here we demonstrated that β1,4-galactosyltransferase 1 (β1,4GT1) interacted with EGFR in vitro by GST pull-down analysis. Furthermore, we demonstrated that β1,4GT1 bound to EGFRmore » in vivo by co-immunoprecipitation and determined the co-localization of β1,4GT1 and EGFR on the cell surface via confocal laser scanning microscopy analysis. Finally, using {sup 125}I-EGF binding experiments and Western blot analysis, we found that overexpression of β1,4GT1 inhibited {sup 125}I-EGF binding to EGFR, and consequently reduced the levels of EGFR dimerization and phosphorylation. In contrast, RNAi-mediated knockdown of β1,4GT1 increased the levels of EGFR dimerization and phosphorylation. These data suggest that cell surface β1,4GT1 interacts with EGFR and inhibits EGFR activation.« less

Microfabricated Nanotopological Surfaces for Study of Adhesion-dependent Cell mechanosensitivity**

PubMed Central

Chen, Weiqiang; Sun, Yubing

2014-01-01

Cells display high sensitivity and exhibit diverse responses to the intrinsic nanotopography of the extracellular matrix through their nanoscale cellular sensing machinery. Here, we reported a simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces using photolithography and reactive ion etching (RIE). Using RIE-generated nanorough glass surfaces, we demonstrated that local nanoroughness could provide a potent biophysical signal to regulate a diverse array of NIH/3T3 fibroblast behaviors, including cell morphology, adhesion, proliferation and migration. We further showed that cellular responses to nanotopography might be regulated by cell adhesion signaling and actin cytoskeleton remodeling. To further investigate the role of cytoskeleton contractility in nanoroughness sensing, we applied the RIE method to generate nanoroughness on the tops of an array of elastomeric poly-dimethylsiloxane (PDMS) microposts. We utilized the PDMS microposts as force sensors and demonstrated that nanoroughness could indeed regulate the cytoskeleton contractility of NIH/3T3 fibroblasts. Our results suggested that a feedback regulation and mechano-chemical integration mechanism involving adhesion signaling, actin cytoskeleton, and intracellular mechanosensory components might play an important role in regulating mechanosensitive behaviors of NIH/3T3 fibroblasts. The capability to control and further predict cellular responses to nanoroughness might suggest novel methods for developing biomaterials mimicking nanotopographic structures in vivo and suitable local cellular microenvironments for functional tissue engineering. PMID:22887768

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

PubMed Central

Tholanikunnel, Baby G.; Joseph, Kusumam; Kandasamy, Karthikeyan; Baldys, Aleksander; Raymond, John R.; Luttrell, Louis M.; McDermott, Paul J.; Fernandes, Daniel J.

2010-01-01

β2-Adrenergic receptors (β2-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β2-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β2-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β2-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β2-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β2-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals. PMID:20739277

Appropriate polarization following pharmacological rescue of V2 vasopressin receptors encoded by X-linked nephrogenic diabetes insipidus alleles involves a conformation of the receptor that also attains mature glycosylation.

PubMed

Tan, Christopher M; Nickols, Hilary Highfield; Limbird, Lee E

2003-09-12

To understand the mechanisms of G protein-coupled receptor delivery and steady state localization, we examined the trafficking itineraries of wild type (WT) and mutant V2 vasopressin receptors (V2Rs) in polarized Madin-Darby canine kidney II (MDCK II) cells and in COS M6 cells; the mutant V2Rs represent selected alleles responsible for X-linked nephrogenic diabetes insipidus. The WT V2R is localized on the plasma membrane and mediates arginine vasopressin (AVP)-stimulated cAMP accumulation, whereas the clinically relevant V2R mutants, L292P V2R, Delta V278 V2R, and R337X V2R, are retained intracellularly, are insensitive to extracellularly added AVP, and are not processed beyond initial immature glycosylation, manifest by their endoglycosidase H sensitivity. Reduced temperature and pharmacological, but not chemical, strategies rescue mutant V2Rs to the cell surface of COS M6 cells; surface rescue of L292P V2R and R337X V2R, but not of Delta V278 V2R, parallels acquisition of AVP-stimulated cAMP production. Pharmacological rescue of the L292P or R337X V2R by incubation with the membrane-permeant V2R antagonist, SR121463B, leads to a mature glycosylated form of the receptor that achieves localization on the basolateral surface of polarized MDCK II cells indistinguishable from that of the WT V2R. Surprisingly, however, the immature form of the mutant L292P V2R escapes to the apical, but not basolateral, surface of polarized MDCK II cells, even in the absence of SR121463B. These findings are consistent with the interpretation that the receptor conformation that allows appropriate processing through the N-linked glycosylation pathway is also essential for V2R targeting to the appropriate surface of polarized epithelial cells.

Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells

NASA Technical Reports Server (NTRS)

Hu, Shaohua; Chen, Jianxin; Fabry, Ben; Numaguchi, Yasushi; Gouldstone, Andrew; Ingber, Donald E.; Fredberg, Jeffrey J.; Butler, James P.; Wang, Ning

2003-01-01

We describe a novel synchronous detection approach to map the transmission of mechanical stresses within the cytoplasm of an adherent cell. Using fluorescent protein-labeled mitochondria or cytoskeletal components as fiducial markers, we measured displacements and computed stresses in the cytoskeleton of a living cell plated on extracellular matrix molecules that arise in response to a small, external localized oscillatory load applied to transmembrane receptors on the apical cell surface. Induced synchronous displacements, stresses, and phase lags were found to be concentrated at sites quite remote from the localized load and were modulated by the preexisting tensile stress (prestress) in the cytoskeleton. Stresses applied at the apical surface also resulted in displacements of focal adhesion sites at the cell base. Cytoskeletal anisotropy was revealed by differential phase lags in X vs. Y directions. Displacements and stresses in the cytoskeleton of a cell plated on poly-L-lysine decayed quickly and were not concentrated at remote sites. These data indicate that mechanical forces are transferred across discrete cytoskeletal elements over long distances through the cytoplasm in the living adherent cell.

SERS-Active Nanoinjector for Intracellular Spectroscopy

NASA Astrophysics Data System (ADS)

Vitol, Elina; Orynbayeva, Zulfiya; Bouchard, Michael; Azizkhan-Clifford, Jane; Friedman, Gary; Gogotsi, Yury

2009-03-01

We developed a multifunctional nanopipette which allows simultaneous cell injection and intacellular surface-enhanced Raman spectroscopy (SERS) analysis. SERS spectra contain the characteristic frequencies of molecular bond vibrations. This is a unique method for studying cell biochemistry and physiology on a single organelle level. Unlike the fluorescence spectroscopy, it does not require any specific staining. The principle of SERS is based on very large electromagnetic field enhancement localized around a nano-rough metallic surface. Gold colloids are widely used SERS substrates. Previously, the colloidal nanoparticles were introduced into a cell by the mechanism of endocytosis. The disadvantage of this method is the uncontrollable aggregation and distribution of gold nanoparticles inside a cell which causes a significant uncertainty in the origin of the acquired data. At the same time, the nanoparticle uptake is irreversible. We present a SERS-active nanoinjector, coated with gold nanoparticles, which enables selective signal acquisition from any point-of-interest inside a cell. The nanoinjector provides a highly localized SERS signal with sub-nanometer resolution in real time.

Cylindrical cellular geometry ensures fidelity of division site placement in fission yeast.

PubMed

Mishra, Mithilesh; Huang, Yinyi; Srivastava, Pragya; Srinivasan, Ramanujam; Sevugan, Mayalagu; Shlomovitz, Roie; Gov, Nir; Rao, Madan; Balasubramanian, Mohan

2012-08-15

Successful cytokinesis requires proper assembly of the contractile actomyosin ring, its stable positioning on the cell surface and proper constriction. Over the years, many of the key molecular components and regulators of the assembly and positioning of the actomyosin ring have been elucidated. Here we show that cell geometry and mechanics play a crucial role in the stable positioning and uniform constriction of the contractile ring. Contractile rings that assemble in locally spherical regions of cells are unstable and slip towards the poles. By contrast, actomyosin rings that assemble on locally cylindrical portions of the cell under the same conditions do not slip, but uniformly constrict the cell surface. The stability of the rings and the dynamics of ring slippage can be described by a simple mechanical model. Using fluorescence imaging, we verify some of the quantitative predictions of the model. Our study reveals an intimate interplay between geometry and actomyosin dynamics, which are likely to apply in a variety of cellular contexts.

Chemo-spectroscopic sensor for carboxyl terminus overexpressed in carcinoma cell membrane.

PubMed

Stanca, Sarmiza E; Matthäus, Christian; Neugebauer, Ute; Nietzsche, Sandor; Fritzsche, Wolfgang; Dellith, Jan; Heintzmann, Rainer; Weber, Karina; Deckert, Volker; Krafft, Christoph; Popp, Jürgen

2015-10-01

Certain carboxyl groups of the plasma membrane are involved in tumorgenesis processes. A gold core-hydroxyapatite shell (AuHA) nanocomposite is introduced as chemo-spectroscopic sensor to monitor these carboxyl groups of the cell membrane. Hydroxyapatite (HA) plays the role both of a chemical detector and of a biocompatible Raman marker. The principle of detection is based on chemical interaction between the hydroxyl groups of the HA and the carboxyl terminus of the proteins. The AuHA exhibits a surface enhanced Raman scattering (SERS) signal at 954 cm(-1) which can be used for its localization. The bio-sensing capacity of AuHA towards human skin epidermoid carcinoma (A431) and Chinese hamster ovary (CHO) cell lines is investigated using Raman microspectroscopic imaging. The localization of AuHA on cells is correlated with scanning electron microscopy, transmission electron microscopy and structured illumination fluorescence microscopy. This qualitative approach is a step towards a quantitative study of the proteins terminus. This method would enable further studies on the molecular profiling of the plasma membrane, in an attempt to provide accurate cell identification. Using a gold core-hydroxyapatite shell (AuHA) nanocomposite, the authors in this paper showed the feasibility of detecting and differentiating cell surface molecules by surface enhanced Raman scattering. Copyright © 2015 Elsevier Inc. All rights reserved.

Silk Film Topography Directs Collective Epithelial Cell Migration

PubMed Central

Rosenblatt, Mark I.

2012-01-01

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

Linear glandular trichomes of Helianthus (Asteraceae): morphology, localization, metabolite activity and occurrence

PubMed Central

Aschenbrenner, Anna-Katharina; Horakh, Silke; Spring, Otmar

2013-01-01

Capitate glandular trichomes of sunflower are well investigated, but detailed studies are lacking for the linear glandular trichomes (LGT), a second type of physiologically active plant hair present on the surface of sunflowers. Light, fluorescence and scanning electron microscopy as well as histochemical staining were used to investigate the structure and metabolite deposition of LGT. Consisting of 6–11 linearly arranged cells, LGT were found on the surface of most plant organs of Helianthus annuus. They were associated with the leaf vascular system, and also occurred along petioles, stems and the abaxial surface of chaffy bracts, ray and disc florets. The highest density was found on the abaxial surface of phyllaries. Phenotypically similar LGT were common in all species of the genus, but also occurred in most other genera of the Helianthinae so far screened. Brownish and fluorescent metabolites of an as yet unknown chemical structure, together with terpenoids, were produced and stored in apical cells of LGT. The deposition of compounds gradually progressed from the tip cell to the basal cells of older trichomes. This process was accompanied by nucleus degradation in metabolite-accumulating cells. The localization of these trichomes on prominent plant parts of the apical bud and the capitulum combined with the accumulation of terpenoids and other as yet unknown compounds suggests a chemo-ecological function of the LGT in plant–insect or plant–herbivore interaction.

Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane

PubMed Central

Paparelli, Laura; Corthout, Nikky; Wakefield, Devin L.; Sannerud, Ragna; Jovanovic-Talisman, Tijana; Annaert, Wim; Munck, Sebastian

2016-01-01

Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided. PMID:27603951

Impact of nitinol stent surface processing on in-vivo nickel release and biological response.

PubMed

Nagaraja, Srinidhi; Sullivan, Stacey J L; Stafford, Philip R; Lucas, Anne D; Malkin, Elon

2018-05-01

Although nitinol is widely used in percutaneous cardiovascular interventions, a causal relationship between nickel released from implanted cardiovascular devices and adverse systemic or local biological responses has not been established. The objective of this study was to investigate the relationship between nitinol surface processing, in-vivo nickel release, and biocompatibility. Nitinol stents manufactured using select surface treatments were implanted into the iliac arteries of minipigs for 6 months. Clinical chemistry profile, complete blood count, serum and urine nickel analyses were performed periodically during the implantation period. After explant, stented arteries were either digested and analyzed for local nickel concentration or fixed and sectioned for histopathological analysis of stenosis and inflammation within the artery. The results indicated that markers for liver and kidney function were not different than baseline values throughout 180 days of implantation regardless of surface finish. In addition, white blood cell, red blood cell, and platelet counts were similar to baseline values for all surface finishes. Systemic nickel concentrations in serum and urine were not significantly different between processing groups and comparable to baseline values during 180 days of implantation. However, stents with non-optimized surface finishing had significantly greater nickel levels in the surrounding artery compared to polished stents. These stents had increased stenosis with potential for local inflammation compared to polished stents. These findings demonstrate that proper polishing of nitinol surfaces can reduce in-vivo nickel release locally, which may aid in minimizing adverse inflammatory reactions and restenosis. Nitinol is a commonly used material in cardiovascular medical devices. However, relationships between nitinol surface finishing, in-vivo metal ion release, and adverse biological responses have yet to be established. We addressed this knowledge gap by implanting single and overlapped nitinol stents with different surface finishes to assess systemic impact on minipigs (i.e. serum and urine nickel levels, liver and kidney function, immune and blood count) over the 6 month implantation period. In addition, nickel levels and histopathology in stented arteries were analyzed on explant to determine relationships between surface processing and local adverse tissue reactions. The findings presented here highlight the importance of surface processing on in-vivo nickel release and subsequent impact on local biological response for nitinol implants. Published by Elsevier Ltd.

Regulating the migration of smooth muscle cells by a vertically distributed poly(2-hydroxyethyl methacrylate) gradient on polymer brushes covalently immobilized with RGD peptides.

PubMed

Wu, Sai; Du, Wang; Duan, Yiyuan; Zhang, Deteng; Liu, Yixiao; Wu, Bingbing; Zou, Xiaohui; Ouyang, Hongwei; Gao, Changyou

2018-05-30

The gradient localization of biological cues is of paramount importance to guide directional migration of cells. In this study, poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate)-block- poly(2-hydroxyethyl methacrylate) (P(HEMA-co-GMA)-b-PHEMA) brushes with a uniform underneath P(HEMA-co-GMA) layer and a gradient thickness of PHEMA blocks were prepared by using surface-initiated atom-transfer radical polymerization and a dynamically controlled polymerization process. The polymer chains were subsequently functionalized with the cell-adhesive arginine-glycine-aspartic acid (RGD) peptides by reaction with the glycidyl groups, and their structures and properties were characterized by X-ray photoelectron spectrometry (XPS), quartz crystal microbalance with dissipation (QCM-D) and air contact angle. Adhesion and migration processes of smooth muscle cells (SMCs) were then studied. Compared with those on the sufficiently exposed RGD surface, the cell adhesion and mobility were well maintained when the RGD peptides were localized at 18.9 nm depth, whereas the adhesion, spreading and migration rate of SMCs were significantly impaired when the RGD peptides were localized at a depth of 38.4 nm. On the RGD depth gradient surface, the SMCs exhibited preferential orientation and enhanced directional migration toward the direction of reduced thickness of the second PHEMA brushes. Half of the cells were oriented within ± 30° to the x-axis direction, and 72% of the cells moved directionally at the optimal conditions. Cell adhesion strength, arrangement of cytoskeleton, and gene and protein expression levels of adhesion-related proteins were studied to corroborate the mechanisms, demonstrating that the cell mobility is regulated by the complex and synergetic intracellular signals resulted from the difference in surface properties. Cell migration is of paramount importance for the processes of tissue repair and regeneration. So far, the gradient localization of biological cues perpendicular to the substrate, which is the usual case for the biological signaling molecules to locate in ECM in vivo, has been scarcely studied, and has not been used to guide the directional migration of cells. In this study, we prepare a depth gradient of RGD peptides along the polymer chains, which is used to guide the directional migration of SMCs after a second hydrophilic bock is prepared in a gradient manner. For the first time the directional migration of SMCs is achieved under the guidance of a depth gradient of RGD ligands. The mechanisms of different cell migration abilities are further discussed based on the results of cell adhesion, cell adhesion force, cytoskeleton alignment and expression of relative proteins and genes. This work paves a new strategy by fabricating a gradient polymer brushes with immobilized bioactive molecules to dominate the directional cell migration, and elucidates the mechanisms underlining the biased migration along RGD depth localization gradients, shedding a light for the design of novel biomaterials to control and guide cell migration and invasion. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Differential localization of lipid phosphate phosphatases 1 and 3 to cell surface subdomains in polarized MDCK cells.

PubMed

Jia, Yan-Jun; Kai, Masahiro; Wada, Ikuo; Sakane, Fumio; Kanoh, Hideo

2003-09-25

Lipid phosphate phosphatases (LPPs) are integral membrane proteins with six transmembrane domains that act as ecto-enzymes dephosphorylating a variety of extracellular lipid phosphates. Using polarized MDCK cells stably expressing human LPP1 and LPP3, we found that LPP1 was located exclusively at the apical surface whereas LPP3 was distributed mostly in the basolateral subdomain. We identified a novel apical sorting signal at the N-terminus of LPP1 composed of F(2)DKTRL(7). In the case of LPP3, a dityrosine motif present in the second cytoplasmic portion was identified as basolateral targeting signal. Our work shows that LPP1 and LPP3 are equipped with distinct sorting signals that cause them to differentially localize to the apical vs. the basolateral subdomain, respectively.

Alkali-templated surface nanopatterning of chalcogenide thin films: a novel approach toward solar cells with enhanced efficiency.

PubMed

Reinhard, Patrick; Bissig, Benjamin; Pianezzi, Fabian; Hagendorfer, Harald; Sozzi, Giovanna; Menozzi, Roberto; Gretener, Christina; Nishiwaki, Shiro; Buecheler, Stephan; Tiwari, Ayodhya N

2015-05-13

Concepts of localized contacts and junctions through surface passivation layers are already advantageously applied in Si wafer-based photovoltaic technologies. For Cu(In,Ga)Se2 thin film solar cells, such concepts are generally not applied, especially at the heterojunction, because of the lack of a simple method yielding features with the required size and distribution. Here, we show a novel, innovative surface nanopatterning approach to form homogeneously distributed nanostructures (<30 nm) on the faceted, rough surface of polycrystalline chalcogenide thin films. The method, based on selective dissolution of self-assembled and well-defined alkali condensates in water, opens up new research opportunities toward development of thin film solar cells with enhanced efficiency.

Correlation of corneal thickness, endothelial cell density and anterior chamber depth with ocular surface temperature in normal subjects.

PubMed

Pattmöller, Johanna; Wang, Jiong; Zemova, Elena; Seitz, Berthold; Eppig, Timo; Langenbucher, Achim; Szentmáry, Nóra

2015-09-01

To analyze corneal surface temperature profile in a young and healthy study population and to determine the impact of corneal thickness (CT), anterior chamber depth (ACD), and endothelial cell density (ECD) on surface temperature. In this prospective, single-center study 61 healthy right eyes of 61 subjects without tear film pathologies (mean age 24.9 ± 6.7 years) were recruited. Ocular surface temperature (OST) was measured with the Ocular Surface Thermographer TG-1000. From Pentacam HR CT and ACD, and from specular microscopy ECD and central corneal thickness (CCT) were acquired. From the raw measurement data (OST, CT and ACD) we extracted a) local OST the corneal center and 3mm away from the center at the 3, 6, and 9 o'clock positions, and b) Zernike parameters Z1, Z2 and Z3 to evaluate the general temperature profile within a 6mm circular area around the center. Overall, there was no correlation between OST and CT, ACD or ECD. Local OST did not correlate with CT at any measurement position. On average local OST was highest at measurement positions where CT was lowest, but without reaching statistical significance. Baseline OST was highest at thin corneal regions and temperature decay over time was smallest in those regions. Z1, Z2 and Z3 correlated well with CT. In healthy subjects corneal thickness, endothelial cell density and anterior chamber depth have no effect on corneal surface temperature. The general temperature profile seems to be influenced by the corneal thickness profile effecting a higher temperature and lower decay at thinner corneal regions. Copyright © 2014. Published by Elsevier GmbH.

Mechanism and control of fluid secretion.

PubMed

Oschman, J L

1977-01-01

Fluid secretion and reabsorption by a variety of plant and animal tissues appear to be accomplished by osmotic coupling between solute transport and water movement. The local osmosis model suggests that active accumulation of solutes within narrow folds at the cell surface may produce the local gradients that generate water flow. Both micropuncture techniques and electron-probe X-ray microanalysis have established that local osmotic gradients occur in absorptive epithelia, but they have not as yet been detected in secretory tissues.Hormonal control of secretion involves stimulation of solute pumps and adjustments of permeability to non-transported solutes. Since hormone receptors and pumps are often located on opposite surfaces of the cell, intracellular second messengers convey the secretory signal through cytoplasm. Much has been learned by study of insect tissues that are anatomically simple and that function for long periods in vitro. Aspects of hormone-receptor interaction have been explored, including the action of halluninogenic molecules. In insect salivary glands cyclic AMP appears to stimulate cation transport, while calcium increases anion permeability. The various second messengers probably interact with each other in complex feedback loops that stabilize the system and make it quickly responsive to hormone. Cyclic AMP may stimulate release of calcium from mitochondria. Unresolved is the way second messengers alter properties of the cell surface.

CD147-targeting siRNA inhibits cell-matrix adhesion of human malignant melanoma cells by phosphorylating focal adhesion kinase.

PubMed

Nishibaba, Rie; Higashi, Yuko; Su, Juan; Furukawa, Tatsuhiko; Kawai, Kazuhiro; Kanekura, Takuro

2012-01-01

CD147/basigin, highly expressed on the surface of malignant tumor cells including malignant melanoma (MM) cells, plays a critical role in the invasiveness and metastasis of MM. Metastasis is an orchestrated process comprised of multiple steps including adhesion and invasion. Integrin, a major adhesion molecule, co-localizes with CD147/basigin on the cell surface. Using the human MM cell line A375 that highly expresses CD147/basigin, we investigated whether CD147/basigin is involved in adhesion in association with integrin. CD147/basigin was knocked-down using siRNA targeting CD147 to elucidate the role of CD147/basigin. Cell adhesion was evaluated by adhesion assay on matrix-coated plates. The localization of integrin was inspected under a confocal microscope and the expression and phosphorylation of focal adhesion kinase (FAK), a downstream kinase of integrin, were examined by western blot analysis. Silencing of CD147/basigin in A375 cells by siRNA induced the phosphorylation of FAK at Y397. Integrin identified on the surface of parental cells was distributed in a speckled fashion in the cytoplasm of CD147 knockdown cells, resulting in morphological changes from a round to a polygonal shape with pseudopodial protrusions. Silencing of CD147/basigin in A375 cells clearly weakened their adhesiveness to collagen I and IV. Our results suggest that CD147/basigin regulates the adhesion of MM cells to extracellular matrices and of integrin β1 signaling via the phosphorylation of FAK. © 2011 Japanese Dermatological Association.

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network

PubMed Central

Swatkoski, Stephen; Matsumoto, Kazue; Campbell, Catherine B.; Petrie, Ryan J.; Dimitriadis, Emilios K.; Li, Xin; Mueller, Susette C.; Bugge, Thomas H.; Gucek, Marjan

2015-01-01

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM. PMID:25646088

The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds

PubMed Central

Sequeira, Sharon J.; Soscia, David A.; Oztan, Basak; Mosier, Aaron P.; Jean-Gilles, Riffard; Gadre, Anand; Cady, Nathaniel C.; Yener, Bülent; Castracane, James; Larsen, Melinda

2012-01-01

Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-L-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland. PMID:22285464

Energy loss from a moving vortex in superfluid helium

NASA Astrophysics Data System (ADS)

Zieve, R. J.; Frei, C. M.; Wolfson, D. L.

2012-11-01

We present measurements on both energy loss and pinning for a vortex terminating on the curved surface of a cylindrical container. We vary surface roughness, cell diameter, fluid velocity, and temperature. Although energy loss and pinning both arise from interactions between the vortex and the surface, their dependences on the experimental parameters differ, suggesting that different mechanisms govern the two effects. We propose that the energy loss stems from reconnections with a mesh of microscopic vortices that covers the cell wall, while pinning is dominated by other influences such as the local fluid velocity.

Influence of salinity on the localization of Na+/K +-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and CFTR anion channel in chloride cells of the Hawaiian goby (Stenogobius hawaiiensis)

USGS Publications Warehouse

McCormick, S.D.; Sundell, K.; Bjornsson, Bjorn Thrandur; Brown, C.L.; Hiroi, J.

2003-01-01

Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) are the three major transport proteins thought to be involved in chloride secretion in teleost fish. If this is the case, the levels of these transporters should be high in chloride cells of seawater-acclimated fish. We therefore examined the influence of salinity on immunolocalization of Na +/K+-ATPase, NKCC and CFTR in the gills of the Hawaiian goby (Stenogobius hawaiiensis). Fish were acclimated to freshwater and 20??? and 30??? seawater for 10 days. Na+/K +-ATPase and NKCC were localized specifically to chloride cells and stained throughout most of the cell except for the nucleus and the most apical region, indicating a basolateral/tubular distribution. All Na+/K +-ATPase-positive chloride cells were also positive for NKCC in all salinities. Salinity caused a slight increase in chloride cell number and size and a slight decrease in staining intensity for Na+/K +-ATPase and NKCC, but the basic pattern of localization was not altered. Gill Na+/K+-ATPase activity was also not affected by salinity. CFTR was localized to the apical surface of chloride cells, and only cells staining positive for Na+/K+-ATPase were CFTR-positive. CFTR-positive cells greatly increased in number (5-fold), area stained (53%) and intensity (29%) after seawater acclimation. In freshwater, CFTR immunoreactivity was light and occurred over a broad apical surface on chloride cells, whereas in seawater there was intense immunoreactivity around the apical pit (which was often punctate in appearance) and a light subapical staining. The results indicate that Na+/K +-ATPase, NKCC and CFTR are all present in chloride cells and support current models that all three are responsible for chloride secretion by chloride cells of teleost fish.

A broad spectrum of actin paralogs in Paramecium tetraurelia cells display differential localization and function.

PubMed

Sehring, Ivonne M; Reiner, Christoph; Mansfeld, Jörg; Plattner, Helmut; Kissmehl, Roland

2007-01-01

To localize the different actin paralogs found in Paramecium and to disclose functional implications, we used overexpression of GFP-fusion proteins and antibody labeling, as well as gene silencing. Several isoforms are associated with food vacuoles of different stages. GFP-actin either forms a tail at the lee side of the organelle, or it is vesicle bound in a homogenous or in a speckled arrangement, thus reflecting an actin-based mosaic of the phagosome surface appropriate for association and/or dissociation of other vesicles upon travel through the cell. Several paralogs occur in cilia. A set of actins is found in the cell cortex where actin outlines the regular surface pattern. Labeling of defined structures of the oral cavity is due to other types of actin, whereas yet more types are distributed in a pattern suggesting association with the numerous Golgi fields. A substantial fraction of actins is associated with cytoskeletal elements that are known to be composed of other proteins. Silencing of the respective actin genes or gene subfamilies entails inhibitory effects on organelles compatible with localization studies. Knock down of the actin found in the cleavage furrow abolishes cell division, whereas silencing of other actin genes alters vitality, cell shape and swimming behavior.

Comparative study of photothermolysis of cancer cells with nuclear-targeted or cytoplasm-targeted gold nanospheres: continuous wave or pulsed lasers

NASA Astrophysics Data System (ADS)

Huang, Xiaohua; Kang, Bin; Qian, Wei; Mackey, Megan A.; Chen, Po C.; Oyelere, Adegboyega K.; El-Sayed, Ivan H.; El-Sayed, Mostafa A.

2010-09-01

We conduct a comparative study on the efficiency and cell death pathways of continuous wave (cw) and nanosecond pulsed laser photothermal cancer therapy using gold nanospheres delivered to either the cytoplasm or nucleus of cancer cells. Cytoplasm localization is achieved using arginine-glycine-aspartate peptide modified gold nanospheres, which target integrin receptors on the cell surface and are subsequently internalized by the cells. Nuclear delivery is achieved by conjugating the gold nanospheres with nuclear localization sequence peptides originating from the simian virus. Photothermal experiments show that cell death can be induced with a single pulse of a nanosecond laser more efficiently than with a cw laser. When the cw laser is applied, gold nanospheres localized in the cytoplasm are more effective in inducing cell destruction than gold nanospheres localized at the nucleus. The opposite effect is observed when the nanosecond pulsed laser is used, suggesting that plasmonic field enhancement of the nonlinear absorption processes occurs at high localization of gold nanospheres at the nucleus. Cell death pathways are further investigated via a standard apoptosis kit to show that the cell death mechanisms depend on the type of laser used. While the cw laser induces cell death via apoptosis, the nanosecond pulsed laser leads to cell necrosis. These studies add mechanistic insight to gold nanoparticle-based photothermal therapy of cancer.

Domain decomposition by the advancing-partition method for parallel unstructured grid generation

NASA Technical Reports Server (NTRS)

Banihashemi, legal representative, Soheila (Inventor); Pirzadeh, Shahyar Z. (Inventor)

2012-01-01

In a method for domain decomposition for generating unstructured grids, a surface mesh is generated for a spatial domain. A location of a partition plane dividing the domain into two sections is determined. Triangular faces on the surface mesh that intersect the partition plane are identified. A partition grid of tetrahedral cells, dividing the domain into two sub-domains, is generated using a marching process in which a front comprises only faces of new cells which intersect the partition plane. The partition grid is generated until no active faces remain on the front. Triangular faces on each side of the partition plane are collected into two separate subsets. Each subset of triangular faces is renumbered locally and a local/global mapping is created for each sub-domain. A volume grid is generated for each sub-domain. The partition grid and volume grids are then merged using the local-global mapping.

Atomic force microscopy recognition of protein A on Staphylococcus aureus cell surfaces by labelling with IgG–Au conjugates

PubMed Central

Tatlybaeva, Elena B; Vasilchenko, Alexey S; Deryabin, Dmitri G

2013-01-01

Summary The labelling of functional molecules on the surface of bacterial cells is one way to recognize the bacteria. In this work, we have developed a method for the selective labelling of protein A on the cell surfaces of Staphylococcus aureus by using nanosized immunogold conjugates as cell-surface markers for atomic force microscopy (AFM). The use of 30-nm size Au nanoparticles conjugated with immunoglobulin G (IgG) allowed the visualization, localization and distribution of protein A–IgG complexes on the surface of S. aureus. The selectivity of the labelling method was confirmed in mixtures of S. aureus with Bacillus licheniformis cells, which differed by size and shape and had no IgG receptors on the surface. A preferential binding of the IgG–Au conjugates to S. aureus was obtained. Thus, this novel approach allows the identification of protein A and other IgG receptor-bearing bacteria, which is useful for AFM indication of pathogenic microorganisms in poly-component associations. PMID:24367742

Water at Biological Phase Boundaries: Its Role in Interfacial Activation of Enzymes and Metabolic Pathways.

PubMed

Damodaran, Srinivasan

2015-01-01

Many life-sustaining activities in living cells occur at the membrane-water interface. The pertinent questions that we need to ask are, what are the evolutionary reasons in biology for choosing the membrane-water interface as the site for performing and/or controlling crucial biological reactions, and what is the key physical principle that is very singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this chapter, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes through manipulation of the thermodynamic activity of water in the lipid-water interfacial region. The hypothesis is based on the fact that the surface pressure of a lipid monolayer is a direct measure of the thermodynamic activity of water at the lipid-water interface. Accordingly, the surface pressure-dependent activation or inactivation of interfacial enzymes is directly related to changes in the thermodynamic activity of interfacial water. Extension of this argument suggests that cells may manipulate conformations (and activities) of membrane-bound enzymes by manipulating the (re)activity of interfacial water at various locations in the membrane by localized compression or expansion of the interface. In this respect, cells may use the membrane-bound hormone receptors, lipid phase transition, and local variations in membrane lipid composition as effectors of local compression and/or expansion of membrane, and thereby local water activity. Several experimental data in the literature will be reexamined in the light of this hypothesis.

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

PubMed

MacLean, Lorna; Price, Helen; O'Toole, Peter

2016-01-01

Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

Functional subcellular distribution of β1- and β2-adrenergic receptors in rat ventricular cardiac myocytes

PubMed Central

Cros, Caroline; Brette, Fabien

2013-01-01

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells. PMID:24303124

Cisplatin loaded PMMA: mechanical properties, surface analysis and effects on Saos-2 cell culture.

PubMed

Özben, Hakan; Eralp, Levent; Baysal, Gökhan; Cort, Ayşegül; Sarkalkan, Nazli; Özben, Tomris

2013-01-01

Despite wide resection and systemic chemotherapy, bone tumors may present with local recurrences, metastases and pathological fractures. Application of bone cement containing antineoplastic drug to fill the defect after resection of metastatic lesions and to support implants has been suggested to prevent local tumor growth and implant failures. In this study, we aimed to demonstrate the effects of the addition of cisplatin which is a widely used antineoplastic drug for osteosarcoma, on the mechanical properties of bone cement, and to evaluate the cytotoxic effects of eluted cisplatin on Saos-2 cell culture. Two cement samples were prepared by mixing 100 mg and 300 mg of cisplatin powder with 40 g cement powder. The bone cement of the control group did not contain cisplatin. Mechanical analyses included 4-point bending, compression and shear testing. For cytotoxicity analysis, samples were incubated in Dulbecco's Modified Eagle's medium for 15 days. Mediums were applied to Saos-2 cell culture and cell viability was measured. Surface analyses were performed by scanning electron microscope (SEM). The addition of cisplatin did not alter the mechanical properties of bone cement. It was observed that the eluted cisplatin had cytotoxic effects on Saos-2 cells. SEM analyses demonstrated cisplatin granules on the surface of cement samples. Cisplatin maintains its cytotoxic property when released from bone cement without compromising the mechanical stability. Application of cisplatin loaded bone cement may help local control of tumor growth. We believe that our study will shed light on to these new practices for the treatment of bone cancers and will encourage future studies.

Microscale frictional strains determine chondrocyte fate in loaded cartilage.

PubMed

Bonnevie, Edward D; Delco, Michelle L; Bartell, Lena R; Jasty, Naveen; Cohen, Itai; Fortier, Lisa A; Bonassar, Lawrence J

2018-06-06

Mounting evidence suggests that altered lubricant levels within synovial fluid have acute biological consequences on chondrocyte homeostasis. While these responses have been connected to increased friction, the mechanisms behind this response remain unknown. Here, we combine a frictional bioreactor with confocal elastography and image-based cellular assays to establish the link between cartilage friction, microscale shear strain, and acute, adverse cellular responses. Our incorporation of cell-scale strain measurements reveals that elevated friction generates high shear strains localized near the tissue surface, and that these elevated strains are closely associated with mitochondrial dysfunction, apoptosis, and cell death. Collectively, our data establish two pathways by which chondrocytes negatively respond to friction: an immediate necrotic response and a longer term pathway involving mitochondrial dysfunction and apoptosis. Specifically, in the surface region, where shear strains can exceed 0.07, cells are predisposed to acute death; however, below this surface region, cells exhibit a pathway consistent with apoptosis in a manner predicted by local shear strains. These data reveal a mechanism through which cellular damage in cartilage arises from compromised lubrication and show that in addition to boundary lubricants, there are opportunities upstream of apoptosis to preserve chondrocyte health in arthritis therapy. Copyright © 2018 Elsevier Ltd. All rights reserved.

A nanodiamond-fluorescein conjugate for cell studies

NASA Astrophysics Data System (ADS)

Pedroso-Santana, Seidy; Fleitas-Salazar, Noralvis; Sarabia-Sainz, Andrei; Silva-Campa, Erika; Burgara-Estrella, Alexel; Angulo-Molina, Aracely; Melendrez, Rodrigo; Pedroza-Montero, Martin; Riera, Raul

2018-03-01

The use of nanodiamonds in studies with living systems generally involves the modification of their surfaces with functional groups. Fluorescent molecules can be attached to these groups, so that one can know the exact position of the particles in each moment of the interaction with the cells. Here we modify the surface of detonation nanodiamonds and nitrogen-vacancy center nanodiamonds using carboxylation and hydroxylation procedures. Subsequent reactions with silicates and cysteine, before addition of fluorescein allow to obtain fluorescent nano-conjugates. We used confocal microscopy to observe the position of nanodiamonds interacting with HeLa cells. At 3 h post-incubation the green fluorescence is localized in extracellular rounded like-vesicles assemblies while at 24 h the conjugates can be observed inside the cells. The measurement of the fluorescence emitted by both conjugates allowed to find an enhanced emission of fluorescein isothiocyanate (FITC) when the nitrogen-vacancy center is present. We propose the existence of a fluorescence enhancement by electron transference process. The procedure described in this work allows the functionalization of nanodiamonds with FITC and other molecules using functional surface groups and small size mediators. Also, as was proved in our work, the nanodiamond-fluorescein conjugates can be used to track nanoparticles position within the cell. Localization studies are particularly important for drug delivery applications of nanodiamonds.

Live Imaging of Kv7.2/7.3 Cell Surface Dynamics at the Axon Initial Segment: High Steady-State Stability and Calpain-Dependent Excitotoxic Downregulation Revealed.

PubMed

Benned-Jensen, Tau; Christensen, Rasmus Kordt; Denti, Federico; Perrier, Jean-Francois; Rasmussen, Hanne Borger; Olesen, Søren-Peter

2016-02-17

The voltage-gated K(+) channels Kv7.2 and Kv7.3 are located at the axon initial segment (AIS) and exert strong control over action potential generation. Therefore, changes in their localization or cell surface numbers are likely to influence neuronal signaling. However, nothing is known about the cell surface dynamics of Kv7.2/7.3 at steady state or during short-term neuronal stimulation. This is primarily attributable to their membrane topology, which hampers extracellular epitope tagging. Here we circumvent this limitation by fusing an extra phluorin-tagged helix to the N terminus of human Kv7.3. This seven transmembrane chimera, named super ecliptic phluorin (SEP)-TAC-7.3, functions and traffics as a wild-type (WT) channel. We expressed SEP-TAC-7.3 in dissociated rat hippocampal neurons to examine the lateral mobility, surface numbers, and localization of AIS Kv7.2/7.3 heteromers using live imaging. We discovered that they are extraordinarily stable and exhibit a very low surface mobility both during steady state and neuronal stimulation. In the latter case, we also found that neither localization nor cell surface numbers were changed. However, at high glutamate loads, we observed a rapid irreversible endocytosis of Kv7.2/7.3, which required the activation of NR2B-containing NMDA receptors, Ca(2+) influx, and calpain activation. This excitotoxic mechanism may be specific to ankyrin G-bound AIS proteins because Nav1.2 channels, but not AIS GABAA receptors, were also endocytosed. In conclusion, we have, for the first time, characterized the cell surface dynamics of a full-length Kv7 channel using a novel chimeric strategy. This approach is likely also applicable to other Kv channels and thus of value for the additional characterization of this ion channel subfamily. The voltage-gated K(+) channels Kv7.2 and Kv7.3 exert strong control over action potential generation, but little is known about their cell surface dynamics. Using a novel phluorin-based approach, we here show that these channels are highly stable at steady state and different types of neuronal stimulation. However, at high glutamate loads, they undergo a rapid calpain-dependent endocytosis that likely represents an early response during excitotoxic states. Copyright © 2016 the authors 0270-6474/16/362261-06$15.00/0.

Mechanisms of CDC-42 activation during contact-induced cell polarization.

PubMed

Chan, Emily; Nance, Jeremy

2013-04-01

Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure-function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts.

Gold nanorods coupled with upconverting nanophosphors for targeted thermal ablation and imaging of bladder cancer cells (Conference Presentation)

NASA Astrophysics Data System (ADS)

Cho, Suehyun K.; Su, Lih-Jen; Flaig, Thomas W.; Park, Wounjhang

2016-09-01

NaYF4:Yb3+,Er3+ upconverting nanophosphors (UCNPs) are robust and stable nanoparticles that absorb near-infrared (NIR) photons and emit green and red visible photons through energy transfer upconversion. This mechanism provides UCNPs several advantages as a bioimaging agent over traditional fluorescence imaging agent in that NIR excitation allows high-contrast imaging without autofluorescence and that they can be used for deep-tissue imaging. However, additional surface modification of UCNPs is necessary for them to be biocompatible. We use an amphiphilic polymer (poly(maleic anhydride-alt-octadecene) (PMAO) and a hetero-functional polyethylene glycol with amine and thiol ends (NH2-PEG-SH)) to make the UCNPs water-soluble. This reaction yields a carboxylic group that allows functionalization with anti-epidermal growth factor receptor (aEGFR), which provides specific binding of UCNPs to EGFR-expressing bladder cancer cells. Additionally, the thiol ends of the PEGylated UCNPs are able to bind with gold nanorods (AuNRs) to create UCNP-AuNR complexes. The localized surface plasmon of the AuNR then allow localized heating of HTB9 bladder cancer cells, enabling in situ cell killing upon detection by UCNP fluorescence. Here, we report a successful synthesis, surface modification and conjugation of aEGFR functionalized UCNP-AuNR complexes and in vitro imaging and thermal ablation studies using them. Synthesis and surface modification of UCNP-AuNR complexes are confirmed by electron microscopy. Then, a combination of brightfield, NIR confocal fluorescence, and darkfield microscopy on the UCNP-AuNR treated bladder cancer cells revealed successful cancer targeting and imaging capabilities of the complex. Finally, cell viability assay showed that NIR irradiation of UCNP-AuNR conjugated cells resulted highly selective cell killing.

Mechanisms of CDC-42 activation during contact-induced cell polarization

PubMed Central

Chan, Emily; Nance, Jeremy

2013-01-01

Summary Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure–function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts. PMID:23424200

An in vivo study of electrical charge distribution on the bacterial cell wall by atomic force microscopy in vibrating force mode

NASA Astrophysics Data System (ADS)

Marlière, Christian; Dhahri, Samia

2015-05-01

We report an in vivo electromechanical atomic force microscopy (AFM) study of charge distribution on the cell wall of Gram+ Rhodococcus wratislaviensis bacteria, naturally adherent to a glass substrate, under physiological conditions. The method presented in this paper relies on a detailed study of AFM approach/retract curves giving the variation of the interaction force versus distance between the tip and the sample. In addition to classical height and mechanical (as stiffness) data, mapping of local electrical properties, such as bacterial surface charge, was proved to be feasible at a spatial resolution better than a few tens of nanometers. This innovative method relies on the measurement of the cantilever's surface stress through its deflection far from (>10 nm) the repulsive contact zone: the variations of surface stress come from the modification of electrical surface charge of the cantilever (as in classical electrocapillary measurements) likely stemming from its charging during contact of both the tip and the sample electrical double layers. This method offers an important improvement in local electrical and electrochemical measurements at the solid/liquid interface, particularly in high-molarity electrolytes when compared to techniques focused on the direct use of electrostatic force. It thus opens a new way to directly investigate in situ biological electrical surface processes involved in numerous practical applications and fundamental problems such as bacterial adhesion, biofilm formation, microbial fuel cells, etc.We report an in vivo electromechanical atomic force microscopy (AFM) study of charge distribution on the cell wall of Gram+ Rhodococcus wratislaviensis bacteria, naturally adherent to a glass substrate, under physiological conditions. The method presented in this paper relies on a detailed study of AFM approach/retract curves giving the variation of the interaction force versus distance between the tip and the sample. In addition to classical height and mechanical (as stiffness) data, mapping of local electrical properties, such as bacterial surface charge, was proved to be feasible at a spatial resolution better than a few tens of nanometers. This innovative method relies on the measurement of the cantilever's surface stress through its deflection far from (>10 nm) the repulsive contact zone: the variations of surface stress come from the modification of electrical surface charge of the cantilever (as in classical electrocapillary measurements) likely stemming from its charging during contact of both the tip and the sample electrical double layers. This method offers an important improvement in local electrical and electrochemical measurements at the solid/liquid interface, particularly in high-molarity electrolytes when compared to techniques focused on the direct use of electrostatic force. It thus opens a new way to directly investigate in situ biological electrical surface processes involved in numerous practical applications and fundamental problems such as bacterial adhesion, biofilm formation, microbial fuel cells, etc. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr00968e

Local dynamics of glass-forming polystyrene thin films from atomistic simulation

NASA Astrophysics Data System (ADS)

Zhou, Yuxing; Milner, Scott

Despite a wide technological application ranging from protective coatings to organic solar cells, there still no consensus on the mechanism for the glass transition in polymer thin films a manifestation of the infamous glass problem under confinement. Many experimental and computational studies have observed a large deviation of nanoscale dynamical properties in thin films from the corresponding properties in bulk. In this work, we perform extensive united-atom simulations on atactic polystyrene free-standing thin films near the glass transition temperature and focus on the effect of free surface on the local dynamics. We study the segmental dynamics as a function of distance from the surface for different temperatures, from which relaxation time and thereby local Tg is obtained for each layer. We find the dynamics near free surface is not only enhanced but becomes less strongly temperature dependent as Tg is approached compared to the bulk. We find an increasing length scale associated with mobility propagation from the free surface as temperature decreases, but no correlation between local structure and enhanced relaxation rates near the surface, consistent with studies on bead-spring chains.

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

PubMed Central

Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

1996-01-01

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

Subcellular localization of Mitf in monocytic cells.

PubMed

Lu, Ssu-Yi; Wan, Hsiao-Ching; Li, Mengtao; Lin, Yi-Ling

2010-06-01

Microphthalmia-associated transcription factor (Mitf) is a transcription factor that plays an important role in regulating the development of several cell lineages. The subcellular localization of Mitf is dynamic and is associated with its transcription activity. In this study, we examined factors that affect its subcellular localization in cells derived from the monocytic lineage since Mitf is present abundantly in these cells. We identified a domain encoded by Mitf exon 1B1b to be important for Mitf to commute between the cytoplasm and the nucleus. Deletion of this domain disrupts the shuttling of Mitf to the cytoplasm and results in its retention in the nucleus. M-CSF and RANKL both induce nuclear translocation of Mitf. We showed that Mitf nuclear transport is greatly influenced by ratio of M-CSF/Mitf protein expression. In addition, cell attachment to a solid surface also is needed for the nuclear transport of Mitf.

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

PubMed

Garrigues, H Jacques; Rubinchikova, Yelena E; Rose, Timothy M

2014-03-01

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry. Copyright © 2014 Elsevier Inc. All rights reserved.

Simple shearing flow of dry soap foams with tetrahedrally close-packed structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reinelt, Douglas A.; Kraynik, Andrew M.

2000-05-01

The microrheology of dry soap foams subjected to quasistatic, simple shearing flow is analyzed. Two different monodisperse foams with tetrahedrally close-packed (TCP) structure are examined: Weaire-Phelan (A15) and Friauf-Laves (C15). The elastic-plastic response is evaluated by using the Surface Evolver to calculate foam structures that minimize total surface area at each value of strain. The foam geometry and macroscopic stress are piecewise continuous functions of strain. The stress scales as T/V{sup 1/3}, where T is surface tension and V is cell volume. Each discontinuity corresponds to large changes in foam geometry and topology that restore equilibrium to unstable configurations thatmore » violate Plateau's laws. The instabilities occur when the length of an edge on a polyhedral foam cell vanishes. The length can tend to zero smoothly or abruptly with strain. The abrupt case occurs when a small increase in strain changes the energy profile in the neighborhood of a foam structure from a local minimum to a saddle point, which can lead to symmetry-breaking bifurcations. In general, the new structure associated with each stable solution branch results from an avalanche of local topology changes called T1 transitions. Each T1 cascade produces different cell neighbors, reduces surface energy, and provides an irreversible, film-level mechanism for plastic yield behavior. Stress-strain curves and average stresses are evaluated by examining foam orientations that admit strain-periodic behavior. For some orientations, the deformation cycle includes Kelvin cells instead of the original TCP structure; but the foam does not remain perfectly ordered. Bifurcations during subsequent T1 cascades lead to disorder and can even cause strain localization. (c) 2000 Society of Rheology.« less

Flow-induced detachment of red blood cells adhering to surfaces by specific antigen-antibody bonds.

PubMed

Xia, Z; Goldsmith, H L; van de Ven, T G

1994-04-01

Fixed spherical swollen human red blood cells of blood type B adhering on a glass surface through antigen-antibody bonds to monoclonal mouse antihuman IgM, adsorbed or covalently linked on the surface, were detached by known hydrodynamic forces created in an impinging jet. The dynamic process of detachment of the specifically bound cells was recorded and analyzed. The fraction of adherent cells remaining on the surface decreased with increasing hydrodynamic force. For an IgM coverage of 0.26%, a tangential force on the order of 100 pN was able to detach almost all of the cells from the surface within 20 min. After a given time of exposure to hydrodynamic force, the fraction of adherent cells remaining increased with time, reflecting an increase in adhesion strength. The characteristic time for effective aging was approximately 4 h. Results from experiments in which the adsorbed antibody molecules were immobilized through covalent coupling and from evanescent wave light scattering of adherent cells, imply that deformation of red cells at the contact area was the principal cause for aging, rather than local clustering of the antibody through surface diffusion. Experiments with latex beads specifically bound to red blood cells suggest that, instead of breaking the antigen-antibody bonds, antigen molecules were extracted from the cell membrane during detachment.

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization

PubMed Central

Ursell, Tristan S.; Nguyen, Jeffrey; Monds, Russell D.; Colavin, Alexandre; Billings, Gabriel; Ouzounov, Nikolay; Gitai, Zemer; Shaevitz, Joshua W.; Huang, Kerwyn Casey

2014-01-01

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization. PMID:24550515

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thai,V.; Renesto, P.; Fowler, C.

Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins suchmore » as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (ie HIV-1 and SARS) or virally encoded (ie Mimivirus), are localized on viral surfaces for at least a subset of viruses.« less

Presence of multiple sites containing polar material in spherical Escherichia coli cells that lack MreB.

PubMed

Nilsen, Trine; Yan, Arthur W; Gale, Gregory; Goldberg, Marcia B

2005-09-01

In rod-shaped bacteria, certain proteins are specifically localized to the cell poles. The nature of the positional information that leads to the proper localization of these proteins is unclear. In a screen for factors required for the localization of the Shigella sp. actin assembly protein IcsA to the bacterial pole, a mutant carrying a transposon insertion in mreB displayed altered targeting of IcsA. The phenotype of cells containing a transposon insertion in mreB was indistinguishable from that of cells containing a nonpolar mutation in mreB or that of wild-type cells treated with the MreB inhibitor A22. In cells lacking MreB, a green fluorescent protein (GFP) fusion to a cytoplasmic derivative of IcsA localized to multiple sites. Secreted full-length native IcsA was present in multiple faint patches on the surfaces of these cells in a pattern similar to that seen for the cytoplasmic IcsA-GFP fusion. EpsM, the polar Vibrio cholerae inner membrane protein, also localized to multiple sites in mreB cells and colocalized with IcsA, indicating that localization to multiple sites is not unique to IcsA. Our results are consistent with the requirement, either direct or indirect, for MreB in the restriction of certain polar material to defined sites within the cell and, in the absence of MreB, with the formation of ectopic sites containing polar material.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)-Cell Interaction and the Resultant Bioeffects at the Single-cell Level.

PubMed

Li, Fenfang; Yuan, Fang; Sankin, Georgy; Yang, Chen; Zhong, Pei

2017-01-10

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

Selective cell-surface labeling of the molecular motor protein prestin

PubMed Central

McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

2011-01-01

Prestin, a multipass transmembrane protein whose N- an C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity. PMID:21651892

Influence of patterning the TCO layer on the series resistance of thin film HIT solar cells

NASA Astrophysics Data System (ADS)

Champory, Romain; Mandorlo, Fabien; Seassal, Christian; Fave, Alain

2017-01-01

Thin HIT solar cells combine efficient surface passivation and high open circuit voltage leading to high conversion efficiencies. They require a TCO layer in order to ease carriers transfer to the top surface fingers. This Transparent Conductive Oxide layer induces parasitic absorption in the low wavelength range of the solar spectrum that limits the maximum short circuit current. In case of thin film HIT solar cells, the front surface is patterned in order to increase the effective life time of photons in the active material, and the TCO layer is often deposited with a conformal way leading to additional material on the sidewalls of the patterns. In this article, we propose an alternative scheme with a local etching of both the TCO and the front a-Si:H layers in order to reduce the parasitic absorption. We study how the local resistivity of the TCO evolves as a function of the patterns, and demonstrate how the increase of the series resistance can be compensated in order to increase the conversion efficiency.

Differential localization of ErbB2 in different tissues of the rat female reproductive tract: implications for the use of specific antibodies for ErbB2 analysis.

PubMed

Idris, N; Carothers Carraway, C A; Carraway, K L

2001-11-01

ErbB2 has been implicated in numerous functions, including normal and aberrant development of a variety of tissues. Although no soluble ligand has been identified for ErbB2, we have recently shown that ASGP-2, the transmembrane subunit of the cell surface glycoprotein Muc4 (also called sialomucin complex, SMC), can act as an intramembrane ligand for ErbB2 and modulate its activity. Muc4/SMC is abundantly expressed at the apical surface of most epithelia of the rat female reproductive tract. Since Muc4/SMC can interact with ErbB2 when they are expressed in the same cell and membrane, we investigated whether these two proteins are co-expressed and co-localized in tissues of the female reproductive tract. Using an anti-ErbB2 antibody from Dako, we found moderate staining at the basolateral surface of the oviduct and also around the cell membrane of the most superficial and medial layers of the stratified epithelia of the vagina. In contrast, Neomarkers neu Ab1 antibody intensely stained the apical surface of the epithelium of the oviduct and the medial and basal layers of the stratified epithelia of the vagina, substantially overlapping the distribution of Muc4/SMC. Furthermore, Muc4/SMC and ErbB2 association in different tissues of the female reproductive tract was demonstrated by co-immunoprecipitation analysis. Interestingly, phosphorylated ErbB2 detected by anti-phospho-ErbB2 is primarily present at the apical surface of the oviduct. Thus, our results show that differentially localized forms of ErbB2 are recognized by different antibodies and raise interesting questions about the nature of the different forms of ErbB2, the mechanism for differential localization, and possible functions of ErbB2 in the female reproductive tract. They also raise a cautionary note about the use of different ErbB2 antibodies for expression and localization studies. Copyright 2001 Wiley-Liss, Inc.

Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

PubMed

Hager, Natalie A; Krasowski, Collin J; Mackie, Timothy D; Kolb, Alexander R; Needham, Patrick G; Augustine, Andrew A; Dempsey, Alison; Szent-Gyorgyi, Christopher; Bruchez, Marcel P; Bain, Daniel J; Kwiatkowski, Adam V; O'Donnell, Allyson F; Brodsky, Jeffrey L

2018-05-21

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inwardly rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorescence-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Localized Immunosuppressive Environment in the Foreign Body Response to Implanted Biomaterials

PubMed Central

Higgins, David M.; Basaraba, Randall J.; Hohnbaum, April C.; Lee, Eric J.; Grainger, David W.; Gonzalez-Juarrero, Mercedes

2009-01-01

The implantation of synthetic biomaterials initiates the foreign body response (FBR), which is characterized by macrophage infiltration, foreign body giant cell formation, and fibrotic encapsulation of the implant. The FBR is orchestrated by a complex network of immune modulators, including diverse cell types, soluble mediators, and unique cell surface interactions. The specific tissue locations, expression patterns, and spatial distribution of these immune modulators around the site of implantation are not clear. This study describes a model for studying the FBR in vivo and specifically evaluates the spatial relationship of immune modulators. We modified a biomaterials implantation in vivo model that allowed for cross-sectional in situ analysis of the FBR. Immunohistochemical techniques were used to determine the localization of soluble mediators, ie, interleukin (IL)-4, IL-13, IL-10, IL-6, transforming growth factor-β, tumor necrosis factor-α, interferon-γ, and MCP-1; specific cell types, ie, macrophages, neutrophils, fibroblasts, and lymphocytes; and cell surface markers, ie, F4/80, CD11b, CD11c, and Ly-6C, at early, middle, and late stages of the FBR in subcutaneous implant sites. The cytokines IL-4, IL-13, IL-10, and transforming growth factor-β were localized to implant-adherent cells that included macrophages and foreign body giant cells. A better understanding of the FBR in vivo will allow the development of novel strategies to enhance biomaterial implant design to achieve better performance and safety of biomedical devices at the site of implant. PMID:19528351

Localized immunosuppressive environment in the foreign body response to implanted biomaterials.

PubMed

Higgins, David M; Basaraba, Randall J; Hohnbaum, April C; Lee, Eric J; Grainger, David W; Gonzalez-Juarrero, Mercedes

2009-07-01

The implantation of synthetic biomaterials initiates the foreign body response (FBR), which is characterized by macrophage infiltration, foreign body giant cell formation, and fibrotic encapsulation of the implant. The FBR is orchestrated by a complex network of immune modulators, including diverse cell types, soluble mediators, and unique cell surface interactions. The specific tissue locations, expression patterns, and spatial distribution of these immune modulators around the site of implantation are not clear. This study describes a model for studying the FBR in vivo and specifically evaluates the spatial relationship of immune modulators. We modified a biomaterials implantation in vivo model that allowed for cross-sectional in situ analysis of the FBR. Immunohistochemical techniques were used to determine the localization of soluble mediators, ie, interleukin (IL)-4, IL-13, IL-10, IL-6, transforming growth factor-beta, tumor necrosis factor-alpha, interferon-gamma, and MCP-1; specific cell types, ie, macrophages, neutrophils, fibroblasts, and lymphocytes; and cell surface markers, ie, F4/80, CD11b, CD11c, and Ly-6C, at early, middle, and late stages of the FBR in subcutaneous implant sites. The cytokines IL-4, IL-13, IL-10, and transforming growth factor-beta were localized to implant-adherent cells that included macrophages and foreign body giant cells. A better understanding of the FBR in vivo will allow the development of novel strategies to enhance biomaterial implant design to achieve better performance and safety of biomedical devices at the site of implant.

High-resolution imaging of cellular processes across textured surfaces using an indexed-matched elastomer.

PubMed

Ravasio, Andrea; Vaishnavi, Sree; Ladoux, Benoit; Viasnoff, Virgile

2015-03-01

Understanding and controlling how cells interact with the microenvironment has emerged as a prominent field in bioengineering, stem cell research and in the development of the next generation of in vitro assays as well as organs on a chip. Changing the local rheology or the nanotextured surface of substrates has proved an efficient approach to improve cell lineage differentiation, to control cell migration properties and to understand environmental sensing processes. However, introducing substrate surface textures often alters the ability to image cells with high precision, compromising our understanding of molecular mechanisms at stake in environmental sensing. In this paper, we demonstrate how nano/microstructured surfaces can be molded from an elastomeric material with a refractive index matched to the cell culture medium. Once made biocompatible, contrast imaging (differential interference contrast, phase contrast) and high-resolution fluorescence imaging of subcellular structures can be implemented through the textured surface using an inverted microscope. Simultaneous traction force measurements by micropost deflection were also performed, demonstrating the potential of our approach to study cell-environment interactions, sensing processes and cellular force generation with unprecedented resolution. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Immunoelectrophoretic study of cell surface antigens from different Streptococcus mutans serotypes and Streptococcus sanguis.

PubMed

Ogier, J A; Klein, J P; Niddam, R; Frank, R M

1985-06-01

Antigens prepared from culture supernatants or whole cells of several cariogenic strains were examined by immunoelectrophoresis for their crossed antigenicity, with reference to Streptococcus mutans OMZ175, serotype f. Crossed immunoelectrophoresis revealed a crossreactivity between soluble extracellular and wall associated antigens of six strains of Streptococcus mutans and one strain of Streptococcus sanguis. Protease destroyed the immunoreactivity of crossreactive antigens. One of them was shown to be localized on the bacterial surface.

Temporal and mechanistic tracking of cellular uptake dynamics with novel surface fluorophore-bound nanodiamonds.

PubMed

Schrand, Amanda M; Lin, Jonathan B; Hens, Suzanne Ciftan; Hussain, Saber M

2011-02-01

Nanoparticles (NPs) offer promise for a multitude of biological applications including cellular probes at the bio-interface for targeted delivery of anticancer substances, Raman and fluorescent-based imaging and directed cell growth. Nanodiamonds (NDs), in particular, have several advantages compared to other carbon-based nanomaterials - including a rich surface chemistry useful for chemical conjugation, high biocompatibility with little reactive oxygen species (ROS) generation, physical and chemical stability that affords sterilization, high surface area to volume ratio, transparency and a high index of refraction. The visualization of ND internalization into cells is possible via photoluminescence, which is produced by direct dye conjugation or high energy irradiation that creates nitrogen vacancy centers. Here, we explore the kinetics and mechanisms involved in the intracellular uptake and localization of novel, highly-stable, fluorophore-conjugated NDs. Examination in a neuronal cell line (N2A) shows ND localization to early endosomes and lysosomes with eventual release into the cytoplasm. The addition of endocytosis and exocytosis inhibitors allows for diminished uptake and increased accumulation, respectively, which further corroborates cellular behavior in response to NDs. Ultimately, the ability of the NDs to travel throughout cellular compartments of varying pH without degradation of the surface-conjugated fluorophore or alteration of cell viability over extended periods of time is promising for their use in biomedical applications as stable, biocompatible, fluorescent probes.

Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

PubMed Central

Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

2013-01-01

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR. PMID:23922889

Dramatically reduced surface expression of NK cell receptor KIR2DS3 is attributed to multiple residues throughout the molecule.

PubMed

VandenBussche, C J; Mulrooney, T J; Frazier, W R; Dakshanamurthy, S; Hurley, C K

2009-03-01

Using flow cytometry, fluorescent microscopy and examination of receptor glycosylation status, we demonstrate that an entire killer cell immunoglobulin-like receptor (KIR) locus (KIR2DS3)--assumed earlier to be surface expressed--appears to have little appreciable surface expression in transfected cells. This phenotype was noted for receptors encoded by three allelic variants including the common KIR2DS3*001 allele. Comparing the surface expression of KIR2DS3 with that of the better-studied KIR2DS1 molecule in two different cell lines, mutational analysis identified multiple polymorphic amino-acid residues that significantly alter the proportion of molecules present on the cell surface. A simultaneous substitution of five residues localized to the leader peptide (residues -18 and -7), second domain (residues 123 and 150) and transmembrane region (residue 234) was required to restore KIR2DS3 to the expression level of KIR2DS1. Corresponding simultaneous substitutions of KIR2DS1 to the KIR2DS3 residues resulted in a dramatically decreased surface expression. Molecular modeling was used to predict how these substitutions contribute to this phenotype. Alterations in receptor surface expression are likely to affect the balance of immune cell signaling impacting the characteristics of the response to pathogens or malignancy.

Surface premelting/recrystallization governing the collapse of open-cell nanoporous Cu via thermal annealing.

PubMed

Wang, L; Zhang, X M; Deng, L; Tang, J F; Xiao, S F; Deng, H Q; Hu, W Y

2018-06-04

We systematically investigate the collapse of a set of open-cell nanoporous Cu (np-Cu) materials with the same porosity and shape but different specific surface areas, during thermal annealing, by performing large-scale molecular dynamics simulations. Two mechanisms govern the collapse of np-Cu. One is direct surface premelting, facilitating the collapse of np-Cu, when the specific surface area is less than a critical value (∼2.38 nm-1). The other is recrystallization followed by surface premelting, accelerating the sloughing of ligaments and the annihilation of voids, when the critical specific surface area is exceeded. Surface premelting results from surface reconstruction by prompting localized "disordering" and "chaos" on the surface, and the melting temperature reduces linearly with the increase of the specific surface area. Recrystallization is followed by surface premelting as the melting temperature is below the supercooling point, where a liquid is unstable and instantaneously recrystallizes.

Adhesion and proliferation of fibroblasts on RF plasma-deposited nanostructured fluorocarbon coatings: evidence of FAK activation.

PubMed

Rosso, Francesco; Marino, Gerardo; Muscariello, Livio; Cafiero, Gennaro; Favia, Pietro; D'Aloia, Erica; d'Agostino, Riccardo; Barbarisi, Alfonso

2006-06-01

We used combined plasma-deposition process to deposit smooth and nanostructured fluorocarbon coatings on polyethylenethereftalate (PET) substrates, to obtain surfaces with identical chemical composition and different roughness, and investigate the effect of surface nanostructures on adhesion and proliferation of 3T3 Swiss Albino Mouse fibroblasts. Untreated PET and polystyrene (PS) were used as controls for cell culture. We have found that the statistically significant increase of cell proliferation rate and FAK (a nonreceptor tyrosine kinase) activation detected on ROUGH fluorocarbon surfaces is due to the presence of nanostructures. Changes in cytoskeletal organization and phospho FAK (tyr 397) localization were evident after 60 min on cells adhering to ROUGH surfaces. This change was characterized by the formation of actin stress fibers along lamellar membrane protrusion instead of usual focal contacts. Also the morphology of the adhering fibroblasts (60 min) adhering on ROUGH surfaces was found quite different compared to cells adhering on smooth ones. Copyright 2006 Wiley-Liss, Inc.

Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

PubMed

Dempwolff, Felix; Reimold, Christian; Reth, Michael; Graumann, Peter L

2011-01-01

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.

Developmental localization and the role of hydroxyproline rich glycoproteins during somatic embryogenesis of banana (Musa spp. AAA)

PubMed Central

2011-01-01

Background Hydroxyproline rich glycoproteins (HRGPs) are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somatic embryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs) and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis), and by immunomodulation with the JIM11 antibody. Results Immunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs), mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs), proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM). This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages) of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP treatment showed aberrant non-compact epidermis with discontinuous ECM at the outer surface as well as much less immunolabelling with the JIM11 antibody. This treatment also decreased the plant regeneration capacity in embryogenic banana cultures. Finally, immunomodulation of surface hydroxyproline rich glycoproteins by co-culture of embryos with the JIM11 antibody resulted in a much lower germination capacity of these embryos. Conclusions These results suggest that hydroxyproline rich glycoproteins play an important developmental role, especially in the process of regeneration and germination of embryos during plant regeneration via somatic embryogenesis. Proper content and localization of hydroxyproline rich glycoproteins seem to be essential for the formation and regeneration of banana somatic embryos. PMID:21349190

Emulating Native Periosteum Cell Population and Subsequent Paracrine Factor Production To Promote Tissue Engineered Periosteum-Mediated Allograft Healing

PubMed Central

Hoffman, Michael D.

2015-01-01

Emulating autograft healing within the context of decellularized bone allografts has immediate clinical applications in the treatment of critical-sized bone defects. The periosteum, a thin, osteogenic tissue that surrounds bone, houses a heterogeneous population of stem cells and osteoprogenitors. There is evidence that periosteum-cell derived paracrine factors, specifically vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2), orchestrate autograft healing through host cell recruitment and subsequent tissue elaboration. In previous work, we demonstrated that the use of poly(ethylene glycol) (PEG) hydrogels as a tissue engineered (T.E.) periosteum to localize mesenchymal stem cells (MSCs) to the surface of decellularized bone enhances allograft healing and integration. Herein, we utilize a mixed population of 50:50 MSCs and osteoprogenitor cells to better mimic native periosteum cell population and paracrine factor production to further promote allograft healing. This mixed cell population was localized to the surface of decellularized allografts within degradable hydrogels and shown to expedite allograft healing. Specifically, bone callus formation and biomechanical graft-host integration are increased as compared to unmodified allografts. These results demonstrate the dual importance of periosteum-mediated paracrine factors orchestrating host cell recruitment as well as new bone formation while developing clinically translatable strategies for allograft healing and integration. PMID:25818449

Establishment of cell surface engineering and its development.

PubMed

Ueda, Mitsuyoshi

2016-07-01

Cell surface display of proteins/peptides has been established based on mechanisms of localizing proteins to the cell surface. In contrast to conventional intracellular and extracellular (secretion) expression systems, this method, generally called an arming technology, is particularly effective when using yeasts as a host, because the control of protein folding that is often required for the preparation of proteins can be natural. This technology can be employed for basic and applied research purposes. In this review, I describe various strategies for the construction of engineered yeasts and provide an outline of the diverse applications of this technology to industrial processes such as the production of biofuels and chemicals, as well as bioremediation and health-related processes. Furthermore, this technology is suitable for novel protein engineering and directed evolution through high-throughput screening, because proteins/peptides displayed on the cell surface can be directly analyzed using intact cells without concentration and purification. Functional proteins/peptides with improved or novel functions can be created using this beneficial, powerful, and promising technique.

Flavobacterium johnsoniae gldN and gldO Are Partially Redundant Genes Required for Gliding Motility and Surface Localization of SprB▿ †

PubMed Central

Rhodes, Ryan G.; Samarasam, Mudiarasan Napoleon; Shrivastava, Abhishek; van Baaren, Jessica M.; Pochiraju, Soumya; Bollampalli, Sreelekha; McBride, Mark J.

2010-01-01

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Mutations in gldN cause a partial defect in gliding. A novel bacteriophage selection strategy was used to aid construction of a strain with a deletion spanning gldN and the closely related gene gldO in an otherwise wild-type F. johnsoniae UW101 background. Bacteriophage transduction was used to move a gldN mutation into F. johnsoniae UW101 to allow phenotypic comparison with the gldNO deletion mutant. Cells of the gldN mutant formed nonspreading colonies on agar but retained some ability to glide in wet mounts. In contrast, cells of the gldNO deletion mutant were completely nonmotile, indicating that cells require GldN, or the GldN-like protein GldO, to glide. Recent results suggest that Porphyromonas gingivalis PorN, which is similar in sequence to GldN, has a role in protein secretion across the outer membrane. Cells of the F. johnsoniae gldNO deletion mutant were defective in localization of the motility protein SprB to the cell surface, suggesting that GldN may be involved in secretion of components of the motility machinery. Cells of the gldNO deletion mutant were also deficient in chitin utilization and were resistant to infection by bacteriophages, phenotypes that may also be related to defects in protein secretion. PMID:20038590

Vibrio cholerae use pili and flagella synergistically to effect motility switching and conditional surface attachment

NASA Astrophysics Data System (ADS)

Utada, Andrew S.; Bennett, Rachel R.; Fong, Jiunn C. N.; Gibiansky, Maxsim L.; Yildiz, Fitnat H.; Golestanian, Ramin; Wong, Gerard C. L.

2014-09-01

We show that Vibrio cholerae, the causative agent of cholera, use their flagella and mannose-sensitive hemagglutinin (MSHA) type IV pili synergistically to switch between two complementary motility states that together facilitate surface selection and attachment. Flagellar rotation counter-rotates the cell body, causing MSHA pili to have periodic mechanical contact with the surface for surface-skimming cells. Using tracking algorithms at 5 ms resolution we observe two motility behaviours: ‘roaming', characterized by meandering trajectories, and ‘orbiting’, characterized by repetitive high-curvature orbits. We develop a hydrodynamic model showing that these phenotypes result from a nonlinear relationship between trajectory shape and frictional forces between pili and the surface: strong pili-surface interactions generate orbiting motion, increasing the local bacterial loiter time. Time-lapse imaging reveals how only orbiting mode cells can attach irreversibly and form microcolonies. These observations suggest that MSHA pili are crucial for surface selection, irreversible attachment, and ultimately microcolony formation.

An ATP sensitive light addressable biosensor for extracellular monitoring of single taste receptor cell.

PubMed

Wu, Chunsheng; Du, Liping; Zou, Ling; Zhao, Luhang; Wang, Ping

2012-12-01

Adenosine triphosphate (ATP) is considered as the key neurotransmitter in taste buds for taste signal transmission and processing. Measurements of ATP secreted from single taste receptor cell (TRC) with high sensitivity and specificity are essential for investigating mechanisms underlying taste cell-to-cell communications. In this study, we presented an aptamer-based biosensor for the detection of ATP locally secreted from single TRC. ATP sensitive DNA aptamer was used as recognition element and its DNA competitor was served as signal transduction element that was covalently immobilized on the surface of light addressable potentiometric sensor (LAPS). Due to the light addressable capability of LAPS, local ATP secretion from single TRC can be detected by monitoring the working potential shifts of LAPS. The results show this biosensor can detect ATP with high sensitivity and specificity. It is demonstrated this biosensor can effectively detect the local ATP secretion from single TRC responding to tastant mixture. This biosensor could provide a promising new tool for the research of taste cell-to-cell communications as well as for the detection of local ATP secretion from other types of ATP secreting individual cells.

Circular flow patterns induced by ciliary activity in reconstituted human bronchial epithelium

NASA Astrophysics Data System (ADS)

Viallat, Annie; Khelloufi, Kamel; Gras, Delphine; Chanez, Pascal; Aix Marseille Univ., CNRS, CINaM, Marseille, France Team; Aix Marseille Univ., CNRS, Inserm, LAI, Marseille, France Team

2016-11-01

Mucociliary clearance is the transport at the surface of airways of a complex fluid layer, the mucus, moved by the beats of microscopic cilia present on epithelial ciliated cells. We explored the coupling between the spatial organisation and the activity of cilia and the transport of surface fluids on reconstituted cultures of human bronchial epithelium at air-liquid interface, obtained by human biopsies. We reveal the existence of stable local circular surface flow patterns of mucus or Newtonian fluid at the epithelium surface. We find a power law over more than 3 orders of magnitude showing that the average ciliated cell density controls the size of these flow patterns, and, therefore the distance over which mucus can be transported. We show that these circular flow patterns result from the radial linear increase of the local propelling forces (due to ciliary beats) on each flow domain. This linear increase of local forces is induced by a fine self-regulation of both cilia density and orientation of ciliary beats. Local flow domains grow and merge during ciliogenesis to provide macroscopic mucus transport. This is possible only when the viscoelastic mucus continuously exerts a shear stress on beating cilia, revealing a mechanosensitive function of cilia. M. K. Khelloufi thanks the society MedBioMed for financial support. This work was supported by the ANR MUCOCIL project, Grant ANR-13-BSV5-0015 of the French Agence Nationale de la Recherche.

Detection of microstructural defects in chalcopyrite Cu(In,Ga)Se2 solar cells by spectrally-filtered electroluminescence

NASA Astrophysics Data System (ADS)

Skvarenina, L.; Gajdos, A.; Macku, R.; Skarvada, P.

2017-12-01

The aim of this research is to detect and localize microstructural defects by using an electrically excited light emission from a forward/reverse-bias stressed pn-junction in thin-film Cu(In; Ga)Se2 solar cells with metal wrap through architecture. A different origin of the local light emission from intrinsic/extrinsic imperfections in these chalcopyrite-based solar cells can be distinguished by a spectrally-filtered electroluminescence mapping. After a light emission mapping and localization of the defects in a macro scale is performed a micro scale exploration of the solar cell surface by a scanning electron microscope which follows the particular defects obtained by an electroluminescence. In particular, these macroscopic/microscopic examinations are performed independently, then the searching of the corresponding defects in the micro scale is rather difficult due to a diffused light emission obtained from the macro scale localization. Some of the defects accompanied by a highly intense light emission very often lead to a strong local overheating. Therefore, the lock-in infrared thermography is also performed along with an electroluminescence mapping.

Characterization of hair-follicle side population cells in mouse epidermis and skin tumors

PubMed Central

Kim, Sun Hye; Sistrunk, Christopher; Miliani de Marval, Paula L.; Rodriguez-Puebla, Marcelo L.

2017-01-01

A subset of cells, termed side-population (SP), which have the ability to efflux Hoeschst 33342, have previously been demonstrated to act as a potential method to isolate stem cells. Numerous stem/progenitor cells have been localized in different regions of the mouse hair follicle (HF). The present study identified a SP in the mouse HF expressing the ABCG2 transporter and MTS24 surface marker. These cells are restricted to the upper isthmus of the HF and have previously been described as progenitor cells. Consistent with their SP characteristic, they demonstrated elevated expression of ABCG2 transporter, which participates in the dye efflux. Analysis of tumor epidermal cell lines revealed a correlation between the number of SP keratinocytes and the grade of malignancy, suggesting that the SP may play a role in malignant progression. Consistent with this idea, the present study observed an increased number of cells expressing ABCG2 and MTS24 in chemically induced skin tumors and skin tumor cell lines. This SP does not express the CD34 surface marker detected in the multipotent stem cells of the bulge region of the HF, which have been defined as tumor initiation cells. The present study concluded that a SP with properties of progenitor cells is localized in the upper isthmus of the HF and is important in mouse skin tumor progression. PMID:29181098

Cytochemical localization of phosphatases in the germ- and Sertoli cells of Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae).

PubMed

Fernandes, A P; Báo, S N

1998-08-01

Ultrastructural cytochemical techniques were used for the localization of phosphatases in spermatid and spermatozoon, as well as in Sertoli cells of Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae). Acid phosphatase was found in the acrosome. Thiamine pyrophosphatase was observed in the Golgi cisternae and in the tail spermatozoon surface. Glucose-6-phosphatase was located in the membrane complex of the acrosomal region. Already, in the Sertoli cells acid phosphatase was located in the lysosomes and glucose-6-phosphatase was observed in association with the endoplasmic reticulum and Golgi complex. These observations support the idea that various phosphatases may play some role in spermatid differentiation and in the interactions germ cells--Sertoli cells during spermiogenesis process.

Lifitegrast: First LFA-1/ICAM-1 antagonist for treatment of dry eye disease.

PubMed

Paton, D M

2016-09-01

Dry eye disease is an extremely common condition affecting millions worldwide. The underlying pathophysiological mechanism is thought to be localized inflammation of the ocular surface resulting in the localization of T cells at this surface followed by their activation and subsequent liberation of cytokines. This effect on T cells results from the binding of lymphocyte function-associated antigen-1 (LFA-1) located on T cells to intercellular adhesion molecule 1 (ICAM-1) expressed on inflamed epithelium and endothelium, and on T cells. Lifitegrast is a T-cell integrin antagonist designed to mimic ICAM-1, thus blocking the interaction of LFA-1 and ICAM-1. Lifitegrast enters the systemic circulation to a limited extent thus reducing the likelihood of unwanted systemic reactions. Clinical trials in over 2,500 subjects with dry eye disease have shown that 5.0% lifitegrast given by ocular instillation causes a significant reduction in objective and subjective signs and symptoms of the disease. These beneficial effects are associated with a relatively low incidence of unwanted effects, almost all local in nature. In light of these findings, lifitegrast was approved by the Food and Drug Administration (FDA) in 2016 for the treatment of dry eye disease, the first drug with this mechanism of action to be so approved. Copyright 2016 Prous Science, S.A.U. or its licensors. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkatagopalan, Pavithra; School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401; Microbiology Graduate Program, Arizona State University, Tempe, AZ 85287-5401

Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteinemore » tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes.« less

Identification of a Monocyte Receptor on Herpesvirus-Infected Endothelial Cells

NASA Astrophysics Data System (ADS)

Etingin, Orli R.; Silverstein, Roy L.; Hajjar, David P.

1991-08-01

The adhesion of circulating blood cells to vascular endothelium may be an initial step in atherosclerosis, inflammation, and wound healing. One mechanism for promoting cell-cell adhesion involves the expression of adhesion molecules on the surface of the target cell. Herpes simplex virus infection of endothelium induces arterial injury and has been implicated in the development of human atherosclerosis. We now demonstrate that HSV-infected endothelial cells express the adhesion molecule GMP140 and that this requires cell surface expression of HSV glycoprotein C and local thrombin generation. Monocyte adhesion to HSV-infected endothelial cells was completely inhibited by anti-GMP140 antibodies but not by antibodies to other adhesion molecules such as VCAM and ELAM-1. The induction of GMP140 expression on HSV-infected endothelium may be an important pathophysiological mechanism in virus-induced cell injury and inflammation.

A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells.

PubMed

Wu, Chunxiao; Wang, Shu

2012-01-01

Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

Enterococcus faecium biofilm formation: identification of major autolysin AtlAEfm, associated Acm surface localization, and AtlAEfm-independent extracellular DNA Release.

PubMed

Paganelli, Fernanda L; Willems, Rob J L; Jansen, Pamela; Hendrickx, Antoni; Zhang, Xinglin; Bonten, Marc J M; Leavis, Helen L

2013-04-16

Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faecium E1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faecium and renamed atlAEfm. In addition, AtlAEfm was implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. IMPORTANCE Nosocomial infections caused by Enterococcus faecium have rapidly increased, and treatment options have become more limited. This is due not only to increasing resistance to antibiotics but also to biofilm-associated infections. DNA is released in biofilm matrix via cell lysis, caused by autolysin, and acts as a matrix stabilizer. In this study, we identified and characterized the major autolysin in E. faecium, which we designated AtlAEfm. atlAEfm disruption resulted in resistance to lysis, reduced extracellular DNA (eDNA), deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and chaining. Furthermore, AtlAEfm is associated with Acm cell surface localization, resulting in less binding to collagen types I and IV in the atlAEfm mutant. We also identified AtlAEfm-independent eDNA release that contributes to cell-cell interactions in the atlAEfm mutant. These findings indicate that AtlAEfm is important in biofilm and collagen binding in E. faecium, making AtlAEfm a promising target for treatment of E. faecium infections.

Differential responses of osteoblast lineage cells to nanotopographically-modified, microroughened titanium-aluminum-vanadium alloy surfaces.

PubMed

Gittens, Rolando A; Olivares-Navarrete, Rene; McLachlan, Taylor; Cai, Ye; Hyzy, Sharon L; Schneider, Jennifer M; Schwartz, Zvi; Sandhage, Kenneth H; Boyan, Barbara D

2012-12-01

Surface structural modifications at the micrometer and nanometer scales have driven improved success rates of dental and orthopaedic implants by mimicking the hierarchical structure of bone. However, how initial osteoblast-lineage cells populating an implant surface respond to different hierarchical surface topographical cues remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible initial colonizers. Here we show that in the absence of any exogenous soluble factors, osteoblastic maturation of primary human osteoblasts (HOBs) but not osteoblastic differentiation of MSCs is strongly influenced by nanostructures superimposed onto a microrough Ti6Al4V (TiAlV) alloy. The sensitivity of osteoblasts to both surface microroughness and nanostructures led to a synergistic effect on maturation and local factor production. Osteoblastic differentiation of MSCs was sensitive to TiAlV surface microroughness with respect to production of differentiation markers, but no further enhancement was found when cultured on micro/nanostructured surfaces. Superposition of nanostructures to microroughened surfaces affected final MSC numbers and enhanced production of vascular endothelial growth factor (VEGF) but the magnitude of the response was lower than for HOB cultures. Our results suggest that the differentiation state of osteoblast-lineage cells determines the recognition of surface nanostructures and subsequent cell response, which has implications for clinical evaluation of new implant surface nanomodifications. Copyright © 2012 Elsevier Ltd. All rights reserved.

Differential Responses of Osteoblast Lineage Cells to Nanotopographically-Modified, Microroughened Titanium-Aluminum-Vanadium Alloy Surfaces

PubMed Central

Gittens, Rolando A.; Olivares-Navarrete, Rene; McLachlan, Taylor; Cai, Ye; Hyzy, Sharon L.; Schneider, Jennifer M.; Schwartz, Zvi; Sandhage, Kenneth H.; Boyan, Barbara D.

2013-01-01

Surface structural modifications at the micrometer and nanometer scales have driven improved success rates of dental and orthopaedic implants by mimicking the hierarchical structure of bone. However, how initial osteoblast-lineage cells populating an implant surface respond to different hierarchical surface topographical cues remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible initial colonizers. Here we show that in the absence of any exogenous soluble factors, osteoblastic maturation of primary human osteoblasts (HOBs) but not osteoblastic differentiation of MSCs is strongly influenced by nanostructures superimposed onto a microrough Ti6Al4V (TiAlV) alloy. The sensitivity of osteoblasts to both surface microroughness and nanostructures led to a synergistic effect on maturation and local factor production. Osteoblastic differentiation of MSCs was sensitive to TiAlV surface microroughness with respect to production of differentiation markers, but no further enhancement was found when cultured on micro/nanostructured surfaces. Superposition of nanostructures to microroughened surfaces affected final MSC numbers and enhanced production of vascular endothelial growth factor (VEGF) but the magnitude of the response was lower than for HOB cultures. Our results suggest that the differentiation state of osteoblast-lineage cells determines the recognition of surface nanostructures and subsequent cell response, which has implications for clinical evaluation of new implant surface nanomodifications. PMID:22989383

Cyclophilin A is a new M cell marker of bovine intestinal epithelium.

PubMed

Hondo, Tetsuya; Someya, Shunsuke; Nagasawa, Yuya; Terada, Shunsuke; Watanabe, Hitoshi; Chen, Xiangning; Watanabe, Kouichi; Ohwada, Shyuichi; Kitazawa, Haruki; Rose, Michael T; Nochi, Tomonori; Aso, Hisashi

2016-06-01

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches contribute to the mucosal immune response by the transcytosis of microorganisms. The mechanism by which M cells take up microorganisms, and the functional proteins by which they do this, are not clear. In order to explore one such protein, we developed a 2H5-F3 monoclonal antibody (2H5-F3 mAb) through its binding to bovine M cells, and identified the antibody reactive molecule as cyclophilin A (Cyp-A). The localization patterns of Cyp-A were very similar to the localization pattern of cytokeratin (CK) 18-positive M cells. Cyp-A was identified at the luminal surface of CK18-positive M cells in bovine jejunal and ileal FAE. The membranous localization of Cyp-A in the bovine intestinal cell line (BIE cells) increased as cells differentiated toward M cells, as determined by flow cytometry analysis. Additionally, BIE cells released Cyp-A to the extracellular space and the differentiation of BIE cells to M cells increased the secretion of Cyp-A, as determined by western blotting. Accordingly, Cyp-A may be localized in M cells in the small intestinal epithelium of cattle. The rise of the membranous localization and secretion of Cyp-A by differentiation toward M cells indicates that Cyp-A has an important role in the function of M cells. While Cyp-A of the M cell membrane may contribute to the uptake of viruses with peptidyl-prolyl cis-trans isomerase activity, in the extracellular space Cyp-A may work as a chemokine and contribute to the distribution of immuno-competent cells.

Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion.

PubMed

Holst, Mikkel Roland; Vidal-Quadras, Maite; Larsson, Elin; Song, Jie; Hubert, Madlen; Blomberg, Jeanette; Lundborg, Magnus; Landström, Maréne; Lundmark, Richard

2017-08-22

Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

ADP-glucose pyrophosphorylase is localized to both the cytoplasm and plastids in developing pericarp of tomato fruit

NASA Technical Reports Server (NTRS)

Chen, B. Y.; Wang, Y.; Janes, H. W.

1998-01-01

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

Preparation of an injectable doxorubicin surface modified cellulose nanofiber gel and evaluation of its anti-tumor and anti-metastasis activity in melanoma.

PubMed

Alizadeh, Najmeh; Akbari, Vajihe; Nurani, Maryam; Taheri, Azade

2018-03-01

Cellulose nanofibers (Cel-NFs) gel can be considered as a useful drug carrier because of its biocompatibility, high specific surface area, and high loading capacity of drugs. Injectable Cel-NFs gel could deliver doxorubicin (DOX) for localized chemotherapy of melanoma and suppress melanoma cells migration because of the physical barrier property of Cel-NFs. We prepared DOX surface modified Cel-NFs (DOX-Cel-NFs) gel by the electrostatic attachment of DOX molecules on the surface of Cel-NFs. The increase in the zeta potential of nanofibers and the changes in the FTIR spectra of DOX-Cel-NFs compared to Cel-NFs proved this attachment. DOX-Cel-NFs showed nano-fibrous structure with an average diameter of 22.32 ± 10.66 nm after analyzing using field emission scanning electron microscopy. The suitable injectability of DOX-Cel-NFs gel verified its promising application for the localized chemotherapy. DOX-Cel-NFs gel exhibited a sustained drug release manner. The cytotoxicity results showed that DOX-Cel-NFs were more cytotoxic against melanoma cancer cells than the free DOX during 48 h incubation period. Moreover, DOX-Cel-NFs gel can suppress the melanoma cancer cells migration efficiently. Thus our results emphasize the potential of DOX-Cel-NFs gel as a chemotherapeutic agent for local delivery of DOX in order to treat melanoma and prevent its metastasis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:537-545, 2018. © 2018 American Institute of Chemical Engineers.

An iridium oxide microelectrode for monitoring acute local pH changes of endothelial cells.

PubMed

Ng, Shu Rui; O'Hare, Danny

2015-06-21

pH sensors were fabricated by anodically electrodepositing iridium oxide films (AEIROFs) onto microelectrodes on chips and coated with poly(ethyleneimine) (PEI) for mechanical stability. These demonstrate super-Nernstian response to pH from pH 4.0 to 7.7 in chloride-free phosphate buffer. The surface of the chip was coated with fibronectin for the attachment of porcine aortic endothelial cells (PAECs). The working capability of the pH sensor for monitoring acute local pH changes was investigated by stimulating the PAECs with thrombin. Our results show that thrombin induced acute extracellular acidification of PAECs and dissolution of fibronectin, causing the local pH to decrease. The use of PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, reduced extracellular acidification and an increase in local pH was observed. This study shows that our pH sensors can facilitate the investigation of acute cellular responses to stimulation by monitoring the real-time, local pH changes of cells attached to the sensors.

Method for cleaning a solar cell surface opening made with a solar etch paste

DOEpatents

Rohatgi, Ajeet; Meemongkolkiat, Vichai

2010-06-22

A thin silicon solar cell having a back dielectric passivation and rear contact with local back surface field is described. Specifically, the solar cell may be fabricated from a crystalline silicon wafer having a thickness from 50 to 500 micrometers. A barrier layer and a dielectric layer are applied at least to the back surface of the silicon wafer to protect the silicon wafer from deformation when the rear contact is formed. At least one opening is made to the dielectric layer. An aluminum contact that provides a back surface field is formed in the opening and on the dielectric layer. The aluminum contact may be applied by screen printing an aluminum paste having from one to 12 atomic percent silicon and then applying a heat treatment at 750 degrees Celsius.

A comparison of caveolae and caveolin-1 to folate receptor α in retina and retinal pigment epithelium

PubMed Central

Bridges, Christy C.; El-Sherbeny, Amira; Roon, Penny; Ola, M. Shamsul; Kekuda, Ramesh; Ganapathy, Vadivel; Cameron, Richard S.; Cameron, Patricia L.

2015-01-01

Summary Caveolae are flask-shaped membrane invaginations present in most mammalian cells. They are distinguished by the presence of a striated coat composed of the protein, caveolin. Caveolae have been implicated in numerous cellular processes, including potocytosis in which caveolae are hypothesized to co-localize with folate receptor α and participate in folate uptake. Our laboratory has recently localized folate receptor α to the basolateral surface of the retinal pigment epithelium (RPE). It is present also in many other cells of the retina. In the present study, we asked whether caveolae were present in the RPE, and if so, whether their pattern of distribution was similar to folate receptor α. We also examined the distribution pattern of caveolin-1, which can be a marker of caveolae. Extensive electron microscopical analysis revealed caveolae associated with endothelial cells. However, none were detected in intact or cultured RPE. Laser scanning confocal microscopical analysis of intact RPE localized caveolin-1 to the apical and basal surfaces, a distribution unlike folate receptor α. Western analysis confirmed the presence of caveolin-1 in cultured RPE cells and laser scanning confocal microscopy localized the protein to the basal plasma membrane of the RPE, a distribution like that of folate receptor α. This distribution was confirmed by electron microscopic immunolocalization. The lack of caveolae in the RPE suggests that these structures may not be essential for folate internalization in the RPE. PMID:11508338

Surface growth kinematics via local curve evolution.

PubMed

Moulton, Derek E; Goriely, Alain

2014-01-01

A mathematical framework is developed to model the kinematics of surface growth for objects that can be generated by evolving a curve in space, such as seashells and horns. Growth is dictated by a growth velocity vector field defined at every point on a generating curve. A local orthonormal basis is attached to each point of the generating curve and the velocity field is given in terms of the local coordinate directions, leading to a fully local and elegant mathematical structure. Several examples of increasing complexity are provided, and we demonstrate how biologically relevant structures such as logarithmic shells and horns emerge as analytical solutions of the kinematics equations with a small number of parameters that can be linked to the underlying growth process. Direct access to cell tracks and local orientation enables for connections to be made to the underlying growth process.

Terrestrial Ecosystems - Land Surface Forms of the Conterminous United States

USGS Publications Warehouse

Cress, Jill J.; Sayre, Roger G.; Comer, Patrick; Warner, Harumi

2009-01-01

As part of an effort to map terrestrial ecosystems, the U.S. Geological Survey has generated land surface form classes to be used in creating maps depicting standardized, terrestrial ecosystem models for the conterminous United States, using an ecosystems classification developed by NatureServe . A biophysical stratification approach, developed for South America and now being implemented globally, was used to model the ecosystem distributions. Since land surface forms strongly influence the differentiation and distribution of terrestrial ecosystems, they are one of the key input layers in this biophysical stratification. After extensive investigation into various land surface form mapping methodologies, the decision was made to use the methodology developed by the Missouri Resource Assessment Partnership (MoRAP). MoRAP made modifications to Hammond's land surface form classification, which allowed the use of 30-meter source data and a 1-km2 window for analyzing the data cell and its surrounding cells (neighborhood analysis). While Hammond's methodology was based on three topographic variables, slope, local relief, and profile type, MoRAP's methodology uses only slope and local relief. Using the MoRAP method, slope is classified as gently sloping when more than 50 percent of the area in a 1-km2 neighborhood has slope less than 8 percent, otherwise the area is considered moderately sloping. Local relief, which is the difference between the maximum and minimum elevation in a neighborhood, is classified into five groups: 0-15 m, 16-30 m, 31-90 m, 91-150 m, and >150 m. The land surface form classes are derived by combining slope and local relief to create eight landform classes: flat plains (gently sloping and local relief = 90 m), low hills (not gently sloping and local relief = 150 m). However, in the USGS application of the MoRAP methodology, an additional local relief group was used (> 400 m) to capture additional local topographic variation. As a result, low mountains were redefined as not gently sloping and 151 m 400 m. The final application of the MoRAP methodology was implemented using the USGS 30-meter National Elevation Dataset and an existing USGS slope dataset that had been derived by calculating the slope from the NED in Universal Transverse Mercator (UTM) coordinates in each UTM zone, and then combining all of the zones into a national dataset. This map shows a smoothed image of the nine land surface form classes based on MoRAP's methodology. Additional information about this map and any data developed for the ecosystems modeling of the conterminous United States is available online at http://rmgsc.cr.usgs.gov/ecosystems/.

Magnetization of individual yeast cells by in situ formation of iron oxide on cell surfaces

NASA Astrophysics Data System (ADS)

Choi, Jinsu; Lee, Hojae; Choi, Insung S.; Yang, Sung Ho

2017-09-01

Magnetic functionalization of living cells has intensively been investigated with the aim of various bioapplications such as selective separation, targeting, and localization of the cells by using an external magnetic field. However, the magnetism has not been introduced to individual living cells through the in situ chemical reactions because of harsh conditions required for synthesis of magnetic materials. In this work, magnetic iron oxide was formed on the surface of living cells by optimizing reactions conditions to be mild sufficiently enough to sustain cell viability. Specifically, the reactive LbL strategy led to formation of magnetically responsive yeast cells with iron oxide shells. This facile and direct post-magnetization method would be a useful tool for remote manipulation of living cells with magnetic interactions, which is an important technique for the integration of cell-based circuits and the isolation of cell in microfluidic devices.

Effects of space flight on surface marker expression

NASA Astrophysics Data System (ADS)

Sonnenfeld, G.

1999-01-01

Space flight has been shown to affect expression of several cell surface markers. These markers play important roles in regulation of immune responses, including CD4 and CD8. The studies have involved flight of experimental animals and humans followed by analysis of tissue samples (blood in humans, rats and monkeys, spleen, thymus, lymph nodes and bone marrow in rodents). The degree and direction of the changes induced by space flight have been determined by the conditions of the flight. Also, there may be compartmentalization of the response of surface markers to space flight, with differences in the response of cells isolated from blood and local immune tissue. The same type of compartmentalization was also observed with cell adhesion molecules (integrins). In this case, the expression of integrins from lymph node cells differed from that of splenocytes isolated from rats immediately after space flight. Cell culture studies have indicated that there may be an inhibition in conversion of a precursor cell line to cells exhibiting mature macrophage characteristics after space flight, however, these experiments were limited as a result of technical difficulties. In general, it is clear that space flight results in alterations of cell surface markers. The biological significance of these changes remains to be established.

Modeling Local Interactions during the Motion of Cyanobacteria

PubMed Central

Galante, Amanda; Wisen, Susanne; Bhaya, Devaki; Levy, Doron

2012-01-01

Synechocystis sp., a common unicellular freshwater cyanobacterium, has been used as a model organism to study phototaxis, an ability to move in the direction of a light source. This microorganism displays a number of additional characteristics such as delayed motion, surface dependence, and a quasi-random motion, where cells move in a seemingly disordered fashion instead of in the direction of the light source, a global force on the system. These unexplained motions are thought to be modulated by local interactions between cells such as intercellular communication. In this paper, we consider only local interactions of these phototactic cells in order to mathematically model this quasi-random motion. We analyze an experimental data set to illustrate the presence of quasi-random motion and then derive a stochastic dynamic particle system modeling interacting phototactic cells. The simulations of our model are consistent with experimentally observed phototactic motion. PMID:22713858

Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.

PubMed

Lai, Zengzu; Schreiber, John R

2009-05-21

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM(197) induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor 488, followed by confocal microscopy. Labeled CRM(197) was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.

Computational modelling of the scaffold-free chondrocyte regeneration: a two-way coupling between the cell growth and local fluid flow and nutrient concentration.

PubMed

Hossain, Md Shakhawath; Bergstrom, D J; Chen, X B

2015-11-01

The in vitro chondrocyte cell culture process in a perfusion bioreactor provides enhanced nutrient supply as well as the flow-induced shear stress that may have a positive influence on the cell growth. Mathematical and computational modelling of such a culture process, by solving the coupled flow, mass transfer and cell growth equations simultaneously, can provide important insight into the biomechanical environment of a bioreactor and the related cell growth process. To do this, a two-way coupling between the local flow field and cell growth is required. Notably, most of the computational and mathematical models to date have not taken into account the influence of the cell growth on the local flow field and nutrient concentration. The present research aimed at developing a mathematical model and performing a numerical simulation using the lattice Boltzmann method to predict the chondrocyte cell growth without a scaffold on a flat plate placed inside a perfusion bioreactor. The model considers the two-way coupling between the cell growth and local flow field, and the simulation has been performed for 174 culture days. To incorporate the cell growth into the model, a control-volume-based surface growth modelling approach has been adopted. The simulation results show the variation of local fluid velocity, shear stress and concentration distribution during the culture period due to the growth of the cell phase and also illustrate that the shear stress can increase the cell volume fraction to a certain extent.

Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence.

PubMed

Rossi, Rachael M; Yum, Lauren; Agaisse, Hervé; Payne, Shelley M

2017-08-29

Cardiolipin, an anionic phospholipid that resides at the poles of the inner and outer membranes, is synthesized primarily by the putative cardiolipin synthase ClsA in Shigella flexneri An S. flexneri clsA mutant had no cardiolipin detected within its membrane, grew normally in vitro , and invaded cultured epithelial cells, but it failed to form plaques in epithelial cell monolayers, indicating that cardiolipin is required for virulence. The clsA mutant was initially motile within the host cell cytoplasm but formed filaments and lost motility during replication and failed to spread efficiently to neighboring cells. Mutation of pbgA , which encodes the transporter for cardiolipin from the inner membrane to the outer membrane, also resulted in loss of plaque formation. The S. flexneri pbgA mutant had normal levels of cardiolipin in the inner membrane, but no cardiolipin was detected in the outer membrane. The pbgA mutant invaded and replicated normally within cultured epithelial cells but failed to localize the actin polymerization protein IcsA properly on the bacterial surface and was unable to spread to neighboring cells. The clsA mutant, but not the pbgA mutant, had increased phosphatidylglycerol in the outer membrane. This appeared to compensate partially for the loss of cardiolipin in the outer membrane, allowing some IcsA localization in the outer membrane of the clsA mutant. We propose a dual function for cardiolipin in S. flexneri pathogenesis. In the inner membrane, cardiolipin is essential for proper cell division during intracellular growth. In the outer membrane, cardiolipin facilitates proper presentation of IcsA on the bacterial surface. IMPORTANCE The human pathogen Shigella flexneri causes bacterial dysentery by invading colonic epithelial cells, rapidly multiplying within their cytoplasm, and then spreading intercellularly to neighboring cells. Worldwide, Shigella spp. infect hundreds of millions of people annually, with fatality rates up to 15%. Antibiotic treatment of Shigella infections is compromised by increasing antibiotic resistance, and there is no approved vaccine to prevent future infections. This has created a growing need to understand Shigella pathogenesis and identify new targets for antimicrobial therapeutics. Here we show a previously unknown role of phospholipids in S. flexneri pathogenesis. We demonstrate that cardiolipin is required in the outer membrane for proper surface localization of IcsA and in the inner membrane for cell division during growth in the host cell cytoplasm. Copyright © 2017 Rossi et al.

Alendronate promotes bone formation by inhibiting protein prenylation in osteoblasts in rat tooth replantation model.

PubMed

Komatsu, Koichiro; Shimada, Akemi; Shibata, Tatsuya; Wada, Satoshi; Ideno, Hisashi; Nakashima, Kazuhisa; Amizuka, Norio; Noda, Masaki; Nifuji, Akira

2013-11-01

Bisphosphonates (BPs) are a major class of antiresorptive drug, and their molecular mechanisms of antiresorptive action have been extensively studied. Recent studies have suggested that BPs target bone-forming cells as well as bone-resorbing cells. We previously demonstrated that local application of a nitrogen-containing BP (N-BP), alendronate (ALN), for a short period of time increased bone tissue in a rat tooth replantation model. Here, we investigated cellular mechanisms of bone formation by ALN. Bone histomorphometry confirmed that bone formation was increased by local application of ALN. ALN increased proliferation of bone-forming cells residing on the bone surface, whereas it suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in vivo. Moreover, ALN treatment induced more alkaline phosphatase-positive and osteocalcin-positive cells on the bone surface than PBS treatment. In vitro studies revealed that pulse treatment with ALN promoted osteocalcin expression. To track the target cells of N-BPs, we applied fluorescence-labeled ALN (F-ALN) in vivo and in vitro. F-ALN was taken into bone-forming cells both in vivo and in vitro. This intracellular uptake was inhibited by endocytosis inhibitors. Furthermore, the endocytosis inhibitor dansylcadaverine (DC) suppressed ALN-stimulated osteoblastic differentiation in vitro and it suppressed the increase in alkaline phosphatase-positive bone-forming cells and subsequent bone formation in vivo. DC also blocked the inhibition of Rap1A prenylation by ALN in the osteoblastic cells. These data suggest that local application of ALN promotes bone formation by stimulating proliferation and differentiation of bone-forming cells as well as inhibiting osteoclast function. These effects may occur through endocytic incorporation of ALN and subsequent inhibition of protein prenylation.

Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation.

PubMed

Stinemetz, Emily K; Gao, Peng; Pinkston, Kenneth L; Montealegre, Maria Camila; Murray, Barbara E; Harvey, Barrett R

2017-01-01

AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation.

Processing of the major autolysin of E. faecalis, AtlA, by the zinc-metalloprotease, GelE, impacts AtlA septal localization and cell separation

PubMed Central

Pinkston, Kenneth L.; Montealegre, Maria Camila; Murray, Barbara E.

2017-01-01

AtlA is the major peptidoglycan hydrolase of Enterococcus faecalis involved in cell division and cellular autolysis. The secreted zinc metalloprotease, gelatinase (GelE), has been identified as an important regulator of cellular function through post-translational modification of protein substrates. AtlA is a known target of GelE, and their interplay has been proposed to regulate AtlA function. To study the protease-mediated post-translational modification of AtlA, monoclonal antibodies were developed as research tools. Flow cytometry and Western blot analysis suggests that in the presence of GelE, surface-bound AtlA exists primarily as a N-terminally truncated form whereas in the absence of GelE, the N-terminal domain of AtlA is retained. We identified the primary GelE cleavage site occurring near the transition between the T/E rich Domain I and catalytic region, Domain II via N-terminal sequencing. Truncation of AtlA had no effect on the peptidoglycan hydrolysis activity of AtlA. However, we observed that N-terminal cleavage was required for efficient AtlA-mediated cell division while unprocessed AtlA was unable to resolve dividing cells into individual units. Furthermore, we observed that the processed AtlA has the propensity to localize to the cell septum on wild-type cells whereas unprocessed AtlA in the ΔgelE strain were dispersed over the cell surface. Combined, these results suggest that AtlA septum localization and subsequent cell separation can be modulated by a single GelE-mediated N-terminal cleavage event, providing new insights into the post-translation modification of AtlA and the mechanisms governing chaining and cell separation. PMID:29049345

Enhancement of local surface plasmon resonance (LSPR) effect by biocompatible metal clustering based on ZnO nanorods in Raman measurements.

PubMed

Lee, Sanghwa; Lee, Seung Ho; Paulson, Bjorn; Lee, Jae-Chul; Kim, Jun Ki

2018-06-20

The development of size-selective and non-destructive detection techniques for nanosized biomarkers has many reasons, including the study of living cells and diagnostic applications. We present an approach for Raman signal enhancement on biocompatible sensing chips based on surface enhancement Raman spectroscopy (SERS). A sensing chip was fabricated by forming a ZnO-based nanorod structure so that the Raman enhancement occurred at a gap of several tens to several hundred nanometers. The effect of coffee-ring formation was eliminated by introducing the porous ZnO nanorods for the bio-liquid sample. A peculiarity of this approach is that the gold sputtered on the ZnO nanorods initially grows at their heads forming clusters, as confirmed by secondary electron microscopy. This clustering was verified by finite element analysis to be the main factor for enhancement of local surface plasmon resonance (LSPR). This clustering property and the ability to adjust the size of the nanorods enabled the signal acquisition points to be refined using confocal based Raman spectroscopy, which could be applied directly to the sensor chip based on the optimization process in this experiment. It was demonstrated by using common cancer cell lines that cell growth was high on these gold-clad ZnO nanorod-based surface-enhanced Raman substrates. The porosity of the sensing chip, the improved structure for signal enhancement, and the cell assay make these gold-coated ZnO nanorods substrates promising biosensing chips with excellent potential for detecting nanometric biomarkers secreted by cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Insights into Jumonji C-domain containing protein 6 (JMJD6): a multifactorial role in foot-and-mouth disease virus replication in cells.

PubMed

Lawrence, Paul; Rieder, Elizabeth

2017-06-01

The Jumonji C-domain containing protein 6 (JMJD6) has had a convoluted history, and recent reports indicating a multifactorial role in foot-and-mouth disease virus (FMDV) infection have further complicated the functionality of this protein. It was first identified as the phosphatidylserine receptor on the cell surface responsible for recognizing phosphatidylserine on the surface of apoptotic cells resulting in their engulfment by phagocytic cells. Subsequent study revealed a nuclear subcellular localization, where JMJD6 participated in lysine hydroxylation and arginine demethylation of histone proteins and other non-histone proteins. Interestingly, to date, JMDJ6 remains the only known arginine demethylase with a growing list of known substrate molecules. These conflicting associations rendered the subcellular localization of JMJD6 to be quite nebulous. Further muddying this area, two different groups illustrated that JMJD6 could be induced to redistribute from the cell surface to the nucleus of a cell. More recently, JMJD6 was demonstrated to be a host factor contributing to the FMDV life cycle, where it was not only exploited for its arginine demethylase activity, but also served as an alternative virus receptor. This review attempts to coalesce these divergent roles for a single protein into one cohesive account. Given the diverse functionalities already characterized for JMJD6, it is likely to continue to be a confounding protein resulting in much contention going into the near future.

Migration of the guinea pig sperm membrane protein PH-20 from one localized surface domain to another does not occur by a simple diffusion-trapping mechanism.

PubMed

Cowan, A E; Myles, D G; Koppel, D E

1991-03-01

The redistribution of membrane proteins on the surface of cells is a prevalent feature of differentiation in a variety of cells. In most cases the mechanism responsible for such redistribution is poorly understood. Two potential mechanisms for the redistribution of surface proteins are: (1) passive diffusion coupled with trapping, and (2) active translocation. We have studied the process of membrane protein redistribution for the PH-20 protein of guinea pig sperm, a surface protein required for sperm binding to the egg zona pellucida (P. Primakoff, H. Hyatt, and D. G. Myles (1985). J. Cell Biol. 101, 2239-2244). PH-20 protein is localized to the posterior head plasma menbrane of the mature sperm cell. Following the exocytotic acrosome reaction, PH-20 protein moves into the newly incorporated inner acrosomal membrane (IAM), placing it in a position favorable for a role in binding sperm to the egg zona pellucida (D. G. Myles, and P. Primakoff (1984), J. Cell Biol. 99, 1634-1641). To analyze the mechanistic basis for this protein migration, we have used fluorescence microscopy and digital image processing to characterize PH-20 protein migration in individual cells. PH-20 protein was observed to move against a concentration gradient in the posterior head plasma membrane. This result argues strongly against a model of passive diffusion followed by trapping in the IAM, and instead suggests that an active process serves to concentrate PH-20 protein toward the boundary separating the posterior head and IAM regions. A transient gradient of PH-20 concentration observed in the IAM suggests that once PH-20 protein reaches the IAM, it is freely diffusing. Additionally, we observed that migration of PH-20 protein was calcium dependent.

LAPTM5 promotes lysosomal degradation of intracellular CD3ζ but not of cell surface CD3ζ.

PubMed

Kawai, Yohei; Ouchida, Rika; Yamasaki, Sho; Dragone, Leonard; Tsubata, Takeshi; Wang, Ji-Yang

2014-07-01

The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane.

PubMed

Símová, Sárka; Klíma, Martin; Cermak, Lukas; Sourková, Vladimíra; Andera, Ladislav

2008-03-01

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

A PI4P-driven electrostatic field controls cell membrane identity and signaling in plants

PubMed Central

Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

2016-01-01

Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P). Our results further reveal that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition. PMID:27322096

A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants.

PubMed

Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

2016-06-20

Many signalling proteins permanently or transiently localize to specific organelles. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PtdIns(4)P). Our results further reveal that, contrarily to other eukaryotes, PtdIns(4)P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATOR (MAKR) family, which are involved in brassinosteroid and receptor-like kinase signalling. We anticipate that this PtdIns(4)P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition.

Glycosyltransferase-programmed stereosubstitution (GPS) to create HCELL: engineering a roadmap for cell migration.

PubMed

Sackstein, Robert

2009-07-01

During evolution of the vertebrate cardiovascular system, the vast endothelial surface area associated with branching vascular networks mandated the development of molecular processes to efficiently and specifically recruit circulating sentinel host defense cells and tissue repair cells at localized sites of inflammation/tissue injury. The forces engendered by high-velocity blood flow commensurately required the evolution of specialized cell surface molecules capable of mediating shear-resistant endothelial adhesive interactions, thus literally capturing relevant cells from the blood stream onto the target endothelial surface and permitting subsequent extravasation. The principal effectors of these shear-resistant binding interactions comprise a family of C-type lectins known as 'selectins' that bind discrete sialofucosylated glycans on their respective ligands. This review explains the 'intelligent design' of requisite reagents to convert native CD44 into the sialofucosylated glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), the most potent E-selectin counter-receptor expressed on human cells, and will describe how ex vivo glycan engineering of HCELL expression may open the 'avenues' for the efficient vascular delivery of cells for a variety of cell therapies.

MHC Class II and CD9 in human eosinophils localize to detergent-resistant membrane microdomains.

PubMed

Akuthota, Praveen; Melo, Rossana C N; Spencer, Lisa A; Weller, Peter F

2012-02-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.

MHC Class II and CD9 in Human Eosinophils Localize to Detergent-Resistant Membrane Microdomains

PubMed Central

Akuthota, Praveen; Melo, Rossana C. N.; Spencer, Lisa A.

2012-01-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor–stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR–containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4+ T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils. PMID:21885678

Regulation of MT1-MMP and MMP-2 by leptin in cardiac fibroblasts involves Rho/ROCK-dependent actin cytoskeletal reorganization and leads to enhanced cell migration.

PubMed

Schram, Kristin; Ganguly, Riya; No, Eun Kyung; Fang, Xiangping; Thong, Farah S L; Sweeney, Gary

2011-05-01

Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells. Coimmunoprecipitation analysis showed that leptin also induced the formation of a cluster of differentiation 44/MT1-MMP complex. Qualitative analysis using rhodamine-conjugated phalloidin immunofluorescence indicated that leptin stimulated actin cytoskeletal reorganization and enhanced stress fiber formation. Hence, we analyzed activation of Ras homolog gene family (Rho), member A GTPase activity and found a rapid increase in response to leptin that corresponded with increased phosphorylation of cofilin. Quantitative analysis of cytoskeleton reorganization upon separation of globular and filamentous actin by differential centrifugation confirmed the significant increase in filamentous to globular actin ratio in response to leptin, which was prevented by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated coiled-coil-forming protein kinase (ROCK) (Y-27632). Inhibition of Rho or ROCK also attenuated leptin-stimulated increases in cell surface MT1-MMP content. Pro-MMP-2 is a known MT1-MMP substrate, and we observed that enhanced cell surface MT1-MMP in response to leptin resulted in enhanced extracellular activation of pro-MMP-2 measured by gelatin zymography, which was again attenuated by inhibition of Rho or ROCK. Using wound scratch assays, we observed enhanced cell migration, but not proliferation, measured by 5-bromo2'-deoxy-uridine incorporation, in response to leptin, again via a Rho-dependent signaling mechanism. Our results suggest that leptin regulates myocardial matrix remodeling by regulating the cell surface localization of MT1-MMP in adult cardiac fibroblasts via Rho/ROCK-dependent actin cytoskeleton reorganization. Subsequent pro-MMP-2 activation then contributes to stimulation of cell migration.

Relaxation of Actinide Surfaces: An All Electron Study

NASA Astrophysics Data System (ADS)

Atta-Fynn, Raymond; Dholabhai, Pratik; Ray, Asok

2006-10-01

Fully relativistic full potential density functional calculations with a linearized augmented plane wave plus local orbitals basis (LAPW + lo) have been performed to investigate the relaxations of heavy actinide surfaces, namely the (111) surface of fcc δ-Pu and the (0001) surface of dhcp Am using WIEN2k. This code uses the LAPW + lo method with the unit cell divided into non-overlapping atom-centered spheres and an interstitial region. The APW+lo basis is used to describe all s, p, d, and f states and LAPW basis to describe all higher angular momentum states. Each surface was modeled by a three-layer periodic slab separated by 60 Bohr vacuum with four atoms per surface unit cell. In general, we have found a contraction of the interlayer separations for both Pu and Am. We will report, in detail, the electronic and geometric structures of the relaxed surfaces and comparisons with the respective non-relaxed surfaces.

Bacillus subtilis MreB Orthologs Self-Organize into Filamentous Structures underneath the Cell Membrane in a Heterologous Cell System

PubMed Central

Dempwolff, Felix; Reimold, Christian; Reth, Michael; Graumann, Peter L.

2011-01-01

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface. PMID:22069484

Slanted snaking of localized Faraday waves

NASA Astrophysics Data System (ADS)

Pradenas, Bastián; Araya, Isidora; Clerc, Marcel G.; Falcón, Claudio; Gandhi, Punit; Knobloch, Edgar

2017-06-01

We report on an experimental, theoretical, and numerical study of slanted snaking of spatially localized parametrically excited waves on the surface of a water-surfactant mixture in a Hele-Shaw cell. We demonstrate experimentally the presence of a hysteretic transition to spatially extended parametrically excited surface waves when the acceleration amplitude is varied, as well as the presence of spatially localized waves exhibiting slanted snaking. The latter extend outside the hysteresis loop. We attribute this behavior to the presence of a conserved quantity, the liquid volume trapped within the meniscus, and introduce a universal model based on symmetry arguments, which couples the wave amplitude with such a conserved quantity. The model captures both the observed slanted snaking and the presence of localized waves outside the hysteresis loop, as demonstrated by numerical integration of the model equations.

Cytosolic proteins can exploit membrane localization to trigger functional assembly

PubMed Central

2018-01-01

Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated, however, is that by localizing to membranes, proteins search in a reduced space that effectively drives up concentration. Here we derive an accurate and practical analytical theory to quantify the significance of this dimensionality reduction in regulating protein assembly on membranes. We define a simple metric, an effective equilibrium constant, that allows for quantitative comparison of protein-protein interactions with and without membrane present. To test the importance of membrane localization for driving protein assembly, we collected the protein-protein and protein-lipid affinities, protein and lipid concentrations, and volume-to-surface-area ratios for 46 interactions between 37 membrane-targeting proteins in human and yeast cells. We find that many of the protein-protein interactions between pairs of proteins involved in clathrin-mediated endocytosis in human and yeast cells can experience enormous increases in effective protein-protein affinity (10–1000 fold) due to membrane localization. Localization of binding partners thus triggers robust protein complexation, suggesting that it can play an important role in controlling the timing of endocytic protein coat formation. Our analysis shows that several other proteins involved in membrane remodeling at various organelles have similar potential to exploit localization. The theory highlights the master role of phosphoinositide lipid concentration, the volume-to-surface-area ratio, and the ratio of 3D to 2D equilibrium constants in triggering (or preventing) constitutive assembly on membranes. Our simple model provides a novel quantitative framework for interpreting or designing in vitro experiments of protein complexation influenced by membrane binding. PMID:29505559

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

PubMed

López-Alemany, Roser; Longstaff, Colin; Hawley, Stephen; Mirshahi, Massoud; Fábregas, Pere; Jardí, Merce; Merton, Elizabeth; Miles, Lindsey A; Félez, Jordi

2003-04-01

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells. Copyright 2003 Wiley-Liss, Inc.

Taxane recovery from cells of Taxus in micro- and hypergravity

NASA Technical Reports Server (NTRS)

Durzan, D. J.; Ventimiglia, F.; Havel, L.

1998-01-01

Cell suspension cultures of Taxus cuspidata produce taxanes that are released from the outer surface of cells into the culture medium as free and bound alkaloids. Paclitaxel (Taxol (TM)), is an anti-cancer drug in short supply. It has a taxane ring derived from baccatin III and a C-13 phenylisoserine side-chain. This drug is produced over a wide range of gravitational forces. Monoclonal and polyclonal antibodies to paclitaxel, baccatin III, and the C-13 phenylisoserine side chain were combined in multiple-labeling studies to localize taxanes and paclitaxel on cell surfaces or on particles released into the culture medium. Bioreactor vessel design altered the composition of taxanes recovered from cells in simulated microgravity. At 10(-2) and 2x10(-4)g, taxane recovery was reduced but biomass growth and percent paclitaxel was significantly increased. At 1 to 24g, growth was reduced with a significant recovery of total taxanes with low percent paclitaxel. Bound paclitaxel was also localized in endonuclease-rich fragmenting nuclei of individual apoptotic cells. A model is presented comprising TCH (touch) genes encoding enzymes that modify taxane-bearing xylan residues in cell walls, the calcium-sensing of gravitational forces by the cytoplasm, and the predisposition of nuclei to apoptosis. This integrates the adaptive physiological and biochemical responses of drug-producing genomes with gravitational forces.

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)-Cell Interaction and the Resultant Bioeffects at the Single-Cell Level

PubMed Central

Li, Fenfang; Yuan, Fang; Sankin, Georgy; Yang, Chen; Zhong, Pei

2017-01-01

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly-localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles. PMID:28117807

Immobilisation of a thrombopoietin peptidic mimic by self-assembled monolayers for culture of CD34+ cells.

PubMed

Lee, Eun-Ju; Be, Cheang Ly; Vinson, Andrew R; Riches, Andrew G; Fehr, Friederike; Gardiner, James; Gengenbach, Thomas R; Winkler, David A; Haylock, David

2015-01-01

Compared to soluble cytokines, surface-tethered ligands can deliver biological signalling with precise control of spatial positioning and concentration. A strategy that immobilises ligand molecules on a surface in a uniform orientation using non-cleavable linkages under physiological conditions would enhance the specific and systemic delivery of signalling in the local environment. We used mixed self-assembled monolayers (SAMs) of oxyamine- and oligo(ethylene glycol)-terminated thiols on gold to covalently install aldehyde- or ketone-functionalised ligands via oxime conjugation. Characterisation by electrochemistry and X-ray photoelectron spectroscopy showed quantitative immobilisation of the ligands on SAM surfaces. The thrombopoietin mimetic peptide, RILL, was immobilised on SAMs and the bioactivity of the substrate was demonstrated by culturing factor-dependent cells. We also optimised the immobilisation and wash conditions so that the peptide was not released into the culture medium and the immobilised RILL could be re-used for consecutive cell cultures. The surface also supported the growth of haematopoietic CD34+ cells comparable to the standard thrombopoietin-supplemented culture. Furthermore, the RILL-immobilised SAM surface was as effective in expanding uncommitted CD34+ cells as standard culture. The stimulatory effect of surface-tethered ligands in haematopoietic stem cell expansion supports the use of ligand immobilisation strategies to replicate the haematopoietic stem cell niche. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Infection rate and tissue localization of murine IL-12p40-producing monocyte-derived CD103(+) lung dendritic cells during pulmonary tuberculosis.

PubMed

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103(+) dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40(+) cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype.

Infection Rate and Tissue Localization of Murine IL-12p40-Producing Monocyte-Derived CD103+ Lung Dendritic Cells during Pulmonary Tuberculosis

PubMed Central

Leepiyasakulchai, Chaniya; Taher, Chato; Chuquimia, Olga D.; Mazurek, Jolanta; Söderberg-Naucler, Cecilia; Fernández, Carmen; Sköld, Markus

2013-01-01

Non-hematopoietic cells, including lung epithelial cells, influence host immune responses. By co-culturing primary alveolar epithelial cells and monocytes from naïve donor mice, we show that alveolar epithelial cells support monocyte survival and differentiation in vitro, suggesting a role for non-hematopoietic cells in monocyte differentiation during the steady state in vivo. CD103+ dendritic cells (αE-DC) are present at mucosal surfaces. Using a murine primary monocyte adoptive transfer model, we demonstrate that αE-DC in the lungs and pulmonary lymph nodes are monocyte-derived during pulmonary tuberculosis. The tissue localization may influence the functional potential of αE-DC that accumulate in Mycobacterium tuberculosis-infected lungs. Here, we confirm the localization of αE-DC in uninfected mice beneath the bronchial epithelial cell layer and near the vascular wall, and show that αE-DC have a similar distribution in the lungs during pulmonary tuberculosis and are detected in the bronchoalveolar lavage fluid from infected mice. Lung DC can be targeted by M. tuberculosis in vivo and play a role in bacterial dissemination to the draining lymph node. In contrast to other DC subsets, only a fraction of lung αE-DC are infected with the bacterium. We also show that virulent M. tuberculosis does not significantly alter cell surface expression levels of MHC class II on infected cells in vivo and that αE-DC contain the highest frequency of IL-12p40+ cells among the myeloid cell subsets in infected lungs. Our results support a model in which inflammatory monocytes are recruited into the M. tuberculosis-infected lung tissue and, depending on which non-hematopoietic cells they interact with, differentiate along different paths to give rise to multiple monocyte-derived cells, including DC with a distinctive αE-DC phenotype. PMID:23861965

Synthetic Spores Give Insight into the Real Thing and Reveal Functional Applications | Center for Cancer Research

Cancer.gov

Spores from bacteria, such as Bacillus subtilis, are produced to allow the bacterium’s genetic material to survive harsh environments. When the bacterium senses nutrient depletion, it divides asymmetrically into a forespore and a mother cell. The mother cell engulfs the forespore, and coat proteins synthesized by the mother cell localize to the surface of the forespore. The

Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin.

PubMed

Jenssen, Håvard; Sandvik, Kjersti; Andersen, Jeanette H; Hancock, Robert E W; Gutteberg, Tore J

2008-09-01

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.

Hybrid constructed wetlands for highly polluted river water treatment and comparison of surface- and subsurface-flow cells.

PubMed

Zheng, Yucong; Wang, Xiaochang; Xiong, Jiaqing; Liu, Yongjun; Zhao, Yaqian

2014-04-01

A series of large pilot constructed wetland (CW) systems were constructed near the confluence of an urban stream to a larger river in Xi'an, a northwestern megacity in China, for treating polluted stream water before it entered the receiving water body. Each CW system is a combination of surface-and subsurface-flow cells with local gravel, sand or slag as substrates and Phragmites australis and Typha orientalis as plants. During a one-year operation with an average surface loading of 0.053 m(3)/(m(2)·day), the overall COD, BOD, NH3-N, total nitrogen (TN) and total phosphorus (TP) removals were 72.7% ± 4.5%, 93.4% ± 2.1%, 54.0% ± 6.3%, 53.9% ± 6.0% and 69.4% ± 4.6%, respectively, which brought about an effective improvement of the river water quality. Surface-flow cells showed better NH3-N removal than their TN removal while subsurface-flow cells showed better TN removal than their NH3-N removal. Using local slag as the substrate, the organic and phosphorus removal could be much improved. Seasonal variation was also found in the removal of all the pollutants and autumn seemed to be the best season for pollutant removal due to the moderate water temperature and well grown plants in the CWs. Copyright © 2014 The Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Expression of TNF-alpha and immunohistochemical distribution of hepatic macrophage surface markers in carbon tetrachloride-induced chronic liver injury in rats.

PubMed

Orfila, C; Lepert, J C; Alric, L; Carrera, G; Beraud, M; Vinel, J P; Pipy, B

1999-10-01

In liver injury induced by carbon tetrachloride, secondary hepatic injury occurs from inflammatory processes originating from products released by activated Kupffer cells, which play a central role in hepatic inflammation. The purpose of our study was to demonstrate, in rats, the relationships between a function of the hepatic macrophages, TNF-alpha production and the state of activation of these cells, characterized by their phenotype, in the different phases of the process and development of fibrosis in a carbon tetrachloride-induced cirrhosis model. The immunohistochemical localization of proinflammatory cytokine TNF-alpha and surface surface makers (ED1 and ED2) was studied in hepatitis and cirrhosis in response to 3 and 9 weeks ingestion of carbon tetrachloride. After carbon tetrachloride ingestion, accompanying the increased necrosis, immunohistochemical analysis of liver tissue sections demonstrated the significantly increased number of cells expressing ED1, ED2 and TNF-alpha, compared to normal. The number of cells expressing the surface phenotypic markers of liver macrophages increased and this change was concomitantly associated with an increased cellular expression of TNF-alpha. Local macrophage proliferation and influx of newly recruited blood monocytes resulted in an increase of the macrophage population. The populational changes involved difference in functional activity and enhanced TNF-alpha expression. This cytokine expressed in the carbon tetrachloride-induced inflammatory process is associated with the development of fibrosis and may contribute to disease severity.

Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.

PubMed

Aplin, J D; Spanswick, C; Behzad, F; Kimber, S J; Vićovac, L

1996-07-01

Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.

A Milieu Molecule for TGF-β Required for Microglia Function in the Nervous System.

PubMed

Qin, Yan; Garrison, Brian S; Ma, Wenjiang; Wang, Rui; Jiang, Aiping; Li, Jing; Mistry, Meeta; Bronson, Roderick T; Santoro, Daria; Franco, Charlotte; Robinton, Daisy A; Stevens, Beth; Rossi, Derrick J; Lu, Chafen; Springer, Timothy A

2018-06-12

Extracellular proTGF-β is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-β is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVβ8-dependent TGF-β activation. Lrrc33 -/- mice lack CNS vascular abnormalities associated with deficiency in TGF-β-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33 -/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33 -/- microglia in the same brain suggest that there is little spreading of TGF-β activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-β provide localized and selective activation of TGF-β. Copyright © 2018 Elsevier Inc. All rights reserved.

The gene coding for the B cell surface protein CD19 is localized on human chromosome 16p11.

PubMed

Stapleton, P; Kozmik, Z; Weith, A; Busslinger, M

1995-02-01

The CD19 gene codes for one of the earliest markers of the human B cell lineage and is a target for the B lymphoid-specific transcription factor BSAP (Pax-5). The transmembrane protein CD19 has been implicated in controlling proliferation of mature B lymphocytes by modulating signal transduction through the antigen receptor. In this study, we have employed Southern blot and fluorescence in situ hybridization analyses to localize the CD19 gene to human chromosome 16p11.

Role of ER Export Signals in Controlling Surface Potassium Channel Numbers

NASA Astrophysics Data System (ADS)

Ma, Dzwokai; Zerangue, Noa; Lin, Yu-Fung; Collins, Anthony; Yu, Mei; Jan, Yuh Nung; Yeh Jan, Lily

2001-01-01

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.

Selective cell-surface labeling of the molecular motor protein prestin.

PubMed

McGuire, Ryan M; Silberg, Jonathan J; Pereira, Fred A; Raphael, Robert M

2011-06-24

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity. Copyright © 2011 Elsevier Inc. All rights reserved.

A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells

PubMed Central

Riestra, Angelica M.; Gandhi, Shiv; Sweredoski, Michael J.; Moradian, Annie; Hess, Sonja; Urban, Sinisa; Johnson, Patricia J.

2015-01-01

Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis. PMID:26684303

Dual-Function Au@Y2O3:Eu3+ Smart Film for Enhanced Power Conversion Efficiency and Long-Term Stability of Perovskite Solar Cells.

PubMed

Kim, Chang Woo; Eom, Tae Young; Yang, In Seok; Kim, Byung Su; Lee, Wan In; Kang, Yong Soo; Kang, Young Soo

2017-07-28

In the present study, a dual-functional smart film combining the effects of wavelength conversion and amplification of the converted wave by the localized surface plasmon resonance has been investigated for a perovskite solar cell. This dual-functional film, composed of Au nanoparticles coated on the surface of Y 2 O 3 :Eu 3+ phosphor (Au@Y 2 O 3 :Eu 3+ ) nanoparticle monolayer, enhances the solar energy conversion efficiency to electrical energy and long-term stability of photovoltaic cells. Coupling between the Y 2 O 3 :Eu 3+ phosphor monolayer and ultraviolet solar light induces the latter to be converted into visible light with a quantum yield above 80%. Concurrently, the Au nanoparticle monolayer on the phosphor nanoparticle monolayer amplifies the converted visible light by up to 170%. This synergy leads to an increased solar light energy conversion efficiency of perovskite solar cells. Simultaneously, the dual-function film suppresses the photodegradation of perovskite by UV light, resulting in long-term stability. Introducing the hybrid smart Au@Y 2 O 3 :Eu 3+ film in perovskite solar cells increases their overall solar-to-electrical energy conversion efficiency to 16.1% and enhances long-term stability, as compared to the value of 15.2% for standard perovskite solar cells. The synergism between the wavelength conversion effect of the phosphor nanoparticle monolayer and the wave amplification by the localized surface plasmon resonance of the Au nanoparticle monolayer in a perovskite solar cell is comparatively investigated, providing a viable strategy of broadening the solar spectrum utilization.

Jararhagin disruption of endothelial cell anchorage is enhanced in collagen enriched matrices.

PubMed

Baldo, C; Lopes, D S; Faquim-Mauro, E L; Jacysyn, J F; Niland, S; Eble, J A; Clissa, P B; Moura-da-Silva, A M

2015-12-15

Hemorrhage is one of the most striking effects of bites by viper snakes resulting in fast bleeding and ischemia in affected tissues. Snake venom metalloproteinases (SVMPs) are responsible for hemorrhagic activity, but the mechanisms involved in SVMP-induced hemorrhage are not entirely understood and the study of such mechanisms greatly depends on in vivo experiments. In vivo, hemorrhagic SVMPs accumulate on basement membrane (BM) of venules and capillary vessels allowing the hydrolysis of collagen IV with consequent weakness and rupture of capillary walls. These effects are not reproducible in vitro with conventional endothelial cell cultures. In this study we used two-dimension (2D) or three-dimension (3D) cultures of HUVECs on matrigel and observed the same characteristics as in ex vivo experiments: only the hemorrhagic toxin was able to localize on surfaces or internalize endothelial cells in 2D cultures or in the surface of tubules formed on 3D cultures. The contribution of matrigel, fibronectin and collagen matrices in jararhagin-induced endothelial cell damage was then analyzed. Collagen and matrigel substrates enhanced the endothelial cell damage induced by jararhagin allowing toxin binding to focal adhesions, disruption of stress fibers, detachment and apoptosis. The higher affinity of jararhagin to collagen than to fibronectin explains the localization of the toxin within BM. Moreover, once located in BM, interactions of jararhagin with α2β1 integrin would favor its localization on focal adhesions, as observed in our study. The accumulation of toxin in focal adhesions, observed only in cells grown in collagen matrices, would explain the enhancement of cell damage in these matrices and reflects the actual interaction among toxin, endothelial cells and BM components that occurs in vivo and results in the hemorrhagic lesions induced by viper venoms. Copyright © 2015 Elsevier Ltd. All rights reserved.

Storm observations by remote sensing and influences of gustiness on ocean waves and on generation of rogue waves

NASA Astrophysics Data System (ADS)

Pleskachevsky, Andrey L.; Lehner, Susanne; Rosenthal, Wolfgang

2012-09-01

The impact of the gustiness on surface waves under storm conditions is investigated with focus on the appearance of wave groups with extreme high amplitude and wavelength in the North Sea. During many storms characterized by extremely high individual waves measured near the German coast, especially in cold air outbreaks, the moving atmospheric open cells are observed by optical and radar satellites. According to measurements, the footprint of the cell produces a local increase in the wind field at sea surface, moving as a consistent system with a propagation speed near to swell wave-traveling speed. The optical and microwave satellite data are used to connect mesoscale atmospheric turbulences and the extreme waves measured. The parameters of open cells observed are used for numerical spectral wave modeling. The North Sea with horizontal resolution of 2.5 km and with focus on the German Bight was simulated. The wind field "storm in storm," including moving organized mesoscale eddies with increased wind speed, was generated. To take into account the rapid moving gust structure, the input wind field was updated each 5 min. The test cases idealized with one, two, and four open individual cells and, respectively, with groups of open cells, with and without preexisting sea state, as well the real storm conditions, are simulated. The model results confirm that an individual-moving open cell can cause the local significant wave height increase in order of meters within the cell area and especially in a narrow area of 1-2 km at the footprint center of a cell (the cell's diameter is 40-90 km). In a case of a traveling individual open cell with 15 m·s-1 over a sea surface with a preexisting wind sea of and swell, a local significant wave height increase of 3.5 m is produced. A group of cells for a real storm condition produces a local increase of significant wave height of more than 6 m during a short time window of 10-20 min (cell passing). The sea surface simulation from modeled wave spectra points out the appearance of wave groups including extreme individual waves with a period of about 25 s and a wavelength of more than 350 m under the cell's footprint. This corresponds well with measurement of a rogue wave group with length of about 400 m and a period of near 25 s. This has been registered at FiNO-1 research platform in the North Sea during Britta storm on November 1, 2006 at 04:00 UTC. The results can explain the appearance of rogue waves in the German Bight and can be used for ship safety and coastal protection. Presently, the considered mesoscale gustiness cannot be incorporated in present operational wave forecasting systems, since it needs an update of the wind field at spatial and temporal scales, which is still not available for such applications. However, the scenario simulations for cell structures with appropriate travel speed, observed by optical and radar satellites, can be done and applied for warning messages.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

PubMed

Bhat, Supriya V; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E S

2018-05-29

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Biodegradable Nanoneedles for Localized Delivery of Nanoparticles in Vivo: Exploring the Biointerface

PubMed Central

Chiappini, Ciro; Martinez, Jonathan O.; De Rosa, Enrica; Almeida, Carina S.

2016-01-01

Nanoneedles display potential in mediating the delivery of drugs and biologicals, as well as intracellular sensing and single cell stimulation through direct access to the cell cytoplasm. Nanoneedles enable cytosolic delivery, negotiating the cell membrane and the endolysosomal system, thus overcoming these major obstacles to the efficacy of nanotherapeutics. The low toxicity and minimal invasiveness of nanoneedles has a potential for the sustained non-immunogenic delivery of payloads in vivo, provided that the development of biocompatible nanoneedles with a simple deployment strategy is achieved. Here we present a mesoporous silicon nanoneedle array that achieves a tight interface with the cell, rapidly negotiating local biological barriers to grant temporary access to the cytosol with minimal impact on cell viability. The tightness of this interfacing enables both delivery of cell-impermeant quantum dots in vivo and live intracellular sensing of pH. Dissecting the biointerface over time elucidated the dynamics of cell association and nanoneedle biodegradation, showing rapid interfacing leading to cytosolic payload delivery within less than 30 minutes in vitro. The rapid and simple application of nanoneedles in vivo to the surface of tissues with different architectures invariably resulted in the localized delivery of quantum dots to the superficial cells and their prolonged retention. This investigation provides an understanding of the dynamics of nanoneedles’ biointerface and delivery outlining a strategy for highly local intracellular delivery of nanoparticles and cell-impermeant payloads within live tissues. PMID:25858596

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads

PubMed Central

Wang, Lin; Acosta, Miguel A.; Leach, Jennie B.; Carrier, Rebecca L.

2013-01-01

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems. PMID:23443975

Spatially monitoring oxygen level in 3D microfabricated cell culture systems using optical oxygen sensing beads.

PubMed

Wang, Lin; Acosta, Miguel A; Leach, Jennie B; Carrier, Rebecca L

2013-04-21

Capability of measuring and monitoring local oxygen concentration at the single cell level (tens of microns scale) is often desirable but difficult to achieve in cell culture. In this study, biocompatible oxygen sensing beads were prepared and tested for their potential for real-time monitoring and mapping of local oxygen concentration in 3D micro-patterned cell culture systems. Each oxygen sensing bead is composed of a silica core loaded with both an oxygen sensitive Ru(Ph2phen3)Cl2 dye and oxygen insensitive Nile blue reference dye, and a poly-dimethylsiloxane (PDMS) shell rendering biocompatibility. Human intestinal epithelial Caco-2 cells were cultivated on a series of PDMS and type I collagen based substrates patterned with micro-well arrays for 3 or 7 days, and then brought into contact with oxygen sensing beads. Using an image analysis algorithm to convert florescence intensity of beads to partial oxygen pressure in the culture system, tens of microns-size oxygen sensing beads enabled the spatial measurement of local oxygen concentration in the microfabricated system. Results generally indicated lower oxygen level inside wells than on top of wells, and local oxygen level dependence on structural features of cell culture surfaces. Interestingly, chemical composition of cell culture substrates also appeared to affect oxygen level, with type-I collagen based cell culture systems having lower oxygen concentration compared to PDMS based cell culture systems. In general, results suggest that oxygen sensing beads can be utilized to achieve real-time and local monitoring of micro-environment oxygen level in 3D microfabricated cell culture systems.

Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

PubMed Central

Gao, Junyuan; Sun, Xiurong; White, Thomas W.; Delamere, Nicholas A.; Mathias, Richard T.

2015-01-01

In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity. PMID:26536260

The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

PubMed Central

Shi, Huazhong; Kim, YongSig; Guo, Yan; Stevenson, Becky; Zhu, Jian-Kang

2003-01-01

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf. PMID:12509519

Bacteria as living patchy colloids: Phenotypic heterogeneity in surface adhesion

PubMed Central

Hermes, Michiel; Schwarz-Linek, Jana; Poon, Wilson C. K.

2018-01-01

Understanding and controlling the surface adhesion of pathogenic bacteria is of urgent biomedical importance. However, many aspects of this process remain unclear (for example, microscopic details of the initial adhesion and possible variations between individual cells). Using a new high-throughput method, we identify and follow many single cells within a clonal population of Escherichia coli near a glass surface. We find strong phenotypic heterogeneities: A fraction of the cells remain in the free (planktonic) state, whereas others adhere with an adhesion strength that itself exhibits phenotypic heterogeneity. We explain our observations using a patchy colloid model; cells bind with localized, adhesive patches, and the strength of adhesion is determined by the number of patches: Nonadherers have no patches, weak adherers bind with a single patch only, and strong adherers bind via a single or multiple patches. We discuss possible implications of our results for controlling bacterial adhesion in biomedical and other applications. PMID:29719861

PPAR-γ and Akt regulate GLUT1 and GLUT3 surface localization during Mycobacterium tuberculosis infection.

PubMed

Dasgupta, Shyamashree; Rai, Ramesh Chandra

2018-03-01

The success of Mycobacterium tuberculosis (Mtb) as a pathogen stems from its ability to manipulate the host macrophage towards increased lipid biogenesis and lipolysis inhibition. Inhibition of lipolysis requires augmented uptake of glucose into the host cell causing an upregulation of the glucose transporters GLUT1 and GLUT3 on the cell surface. Mechanism behind this upregulation of the GLUT proteins during Mtb infection is hitherto unknown and demands intensive investigation in order to understand the pathways linked with governing them. Our endeavor to investigate some of the key proteins that have been found to be affected during Mtb infection led us to investigate host molecular pathways such as Akt and PPAR-γ that remain closely associated with the survival of the bacilli by modulating the localization of glucose transporters GLUT1 and GLUT3.

Microbial diversity and impact on carbonate geochemistry across a changing geochemical gradient in a karst aquifer

PubMed Central

Gray, Cassie J; Engel, Annette S

2013-01-01

Although microbes are known to influence karst (carbonate) aquifer ecosystem-level processes, comparatively little information is available regarding the diversity of microbial activities that could influence water quality and geological modification. To assess microbial diversity in the context of aquifer geochemistry, we coupled 16S rRNA Sanger sequencing and 454 tag pyrosequencing to in situ microcosm experiments from wells that cross the transition from fresh to saline and sulfidic water in the Edwards Aquifer of central Texas, one of the largest karst aquifers in the United States. The distribution of microbial groups across the transition zone correlated with dissolved oxygen and sulfide concentration, and significant variations in community composition were explained by local carbonate geochemistry, specifically calcium concentration and alkalinity. The waters were supersaturated with respect to prevalent aquifer minerals, calcite and dolomite, but in situ microcosm experiments containing these minerals revealed significant mass loss from dissolution when colonized by microbes. Despite differences in cell density on the experimental surfaces, carbonate loss was greater from freshwater wells than saline, sulfidic wells. However, as cell density increased, which was correlated to and controlled by local geochemistry, dissolution rates decreased. Surface colonization by metabolically active cells promotes dissolution by creating local disequilibria between bulk aquifer fluids and mineral surfaces, but this also controls rates of karst aquifer modification. These results expand our understanding of microbial diversity in karst aquifers and emphasize the importance of evaluating active microbial processes that could affect carbonate weathering in the subsurface. PMID:23151637

Microbial diversity and impact on carbonate geochemistry across a changing geochemical gradient in a karst aquifer.

PubMed

Gray, Cassie J; Engel, Annette S

2013-02-01

Although microbes are known to influence karst (carbonate) aquifer ecosystem-level processes, comparatively little information is available regarding the diversity of microbial activities that could influence water quality and geological modification. To assess microbial diversity in the context of aquifer geochemistry, we coupled 16S rRNA Sanger sequencing and 454 tag pyrosequencing to in situ microcosm experiments from wells that cross the transition from fresh to saline and sulfidic water in the Edwards Aquifer of central Texas, one of the largest karst aquifers in the United States. The distribution of microbial groups across the transition zone correlated with dissolved oxygen and sulfide concentration, and significant variations in community composition were explained by local carbonate geochemistry, specifically calcium concentration and alkalinity. The waters were supersaturated with respect to prevalent aquifer minerals, calcite and dolomite, but in situ microcosm experiments containing these minerals revealed significant mass loss from dissolution when colonized by microbes. Despite differences in cell density on the experimental surfaces, carbonate loss was greater from freshwater wells than saline, sulfidic wells. However, as cell density increased, which was correlated to and controlled by local geochemistry, dissolution rates decreased. Surface colonization by metabolically active cells promotes dissolution by creating local disequilibria between bulk aquifer fluids and mineral surfaces, but this also controls rates of karst aquifer modification. These results expand our understanding of microbial diversity in karst aquifers and emphasize the importance of evaluating active microbial processes that could affect carbonate weathering in the subsurface.

Specific biomolecule corona is associated with ring-shaped organization of silver nanoparticles in cells

NASA Astrophysics Data System (ADS)

Drescher, Daniela; Guttmann, Peter; Büchner, Tina; Werner, Stephan; Laube, Gregor; Hornemann, Andrea; Tarek, Basel; Schneider, Gerd; Kneipp, Janina

2013-09-01

We correlate the localization of silver nanoparticles inside cells with respect to the cellular architecture with the molecular information in the vicinity of the particle surface by combining nanoscale 3D cryo-soft X-ray tomography (cryo-SXT) with surface-enhanced Raman scattering (SERS). The interaction of the silver nanoparticle surface with small molecules and biopolymers was monitored by SERS in vitro over time in living cells. The spectra indicate a stable, time-independent surface composition of silver nanoparticles, despite the changing environment in the endosomal structure. Cryo-SXT reveals a characteristic ring-shaped organization of the silver nanoparticles in endosomes of different cell types. The ring-like structures inside the endosomes suggest a strong association among silver particles and with membrane structures. The comparison of the data with those obtained with gold nanoparticles suggests that the interactions between the nanoparticles and with the endosomal component are influenced by the molecular composition of the corona.We correlate the localization of silver nanoparticles inside cells with respect to the cellular architecture with the molecular information in the vicinity of the particle surface by combining nanoscale 3D cryo-soft X-ray tomography (cryo-SXT) with surface-enhanced Raman scattering (SERS). The interaction of the silver nanoparticle surface with small molecules and biopolymers was monitored by SERS in vitro over time in living cells. The spectra indicate a stable, time-independent surface composition of silver nanoparticles, despite the changing environment in the endosomal structure. Cryo-SXT reveals a characteristic ring-shaped organization of the silver nanoparticles in endosomes of different cell types. The ring-like structures inside the endosomes suggest a strong association among silver particles and with membrane structures. The comparison of the data with those obtained with gold nanoparticles suggests that the interactions between the nanoparticles and with the endosomal component are influenced by the molecular composition of the corona. Electronic supplementary information (ESI) available: Description of additional experiments. Explanation of transmitted intensity and linear absorption coefficient in a cryo-XRT experiment (Fig. S1 and S2). Additional X-ray data (Fig. S3 and Movie S1). Toxicity of silver nanoparticles (Fig. S4). X-ray microscopy and SERS experiments with gold nanoparticles (Fig. S5 and S6). Size, plasmonic properties, and stability of silver and gold nanoparticles (Fig. S7-S9). Distribution of the silver nanoparticles in the cells using SERS mapping (Fig. S10). Tentative band assignments (Table S1). See DOI: 10.1039/c3nr02129g

A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

PubMed

Huberman, Lori B; Murray, Andrew W

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells.

A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

PubMed Central

Huberman, Lori B.; Murray, Andrew W.

2014-01-01

Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

Use of green fluorescent protein fusions to analyse the N- and C-terminal signal peptides of GPI-anchored cell wall proteins in Candida albicans.

PubMed

Mao, Yuxin; Zhang, Zimei; Wong, Brian

2003-12-01

Glycophosphatidylinositol (GPI)-anchored proteins account for 26-35% of the Candida albicans cell wall. To understand the signals that regulate these proteins' cell surface localization, green fluorescent protein (GFP) was fused to the N- and C-termini of the C. albicans cell wall proteins (CWPs) Hwp1p, Als3p and Rbt5p. C. albicans expressing all three fusion proteins were fluorescent at the cell surface. GFP was released from membrane fractions by PI-PLC and from cell walls by beta-glucanase, which implied that GFP was GPI-anchored to the plasma membrane and then covalently attached to cell wall glucans. Twenty and 25 amino acids, respectively, from the N- and C-termini of Hwp1p were sufficient to target GFP to the cell surface. C-terminal substitutions that are permitted by the omega rules (G613D, G613N, G613S, G613A, G615S) did not interfere with GFP localization, whereas some non-permitted substitutions (G613E, G613Q, G613R, G613T and G615Q) caused GFP to accumulate in intracellular ER-like structures and others (G615C, G613N/G615C and G613D/G615C) did not. These results imply that (i) GFP fusions can be used to analyse the N- and C-terminal signal peptides of GPI-anchored CWPs, (ii) the omega amino acid in Hwp1p is G613, and (iii) C can function at the omega+2 position in C. albicans GPI-anchored proteins.

Generation and Surface Localization of Intact M Protein in Streptococcus pyogenes Are Dependent on sagA

PubMed Central

Biswas, Indranil; Germon, Pierre; McDade, Kathleen; Scott, June R.

2001-01-01

The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019–6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor. PMID:11598078

Nuclear Localizing Peptide-Conjugated, Redox-Sensitive Polymersomes for Delivering Curcumin and Doxorubicin to Pancreatic Cancer Microtumors.

PubMed

Anajafi, Tayebeh; Yu, Junru; Sedigh, Abbas; Haldar, Manas K; Muhonen, Wallace W; Oberlander, Seth; Wasness, Heather; Froberg, Jamie; Molla, Md Shahjahan; Katti, Kalpana S; Choi, Yongki; Shabb, John B; Srivastava, D K; Mallik, Sanku

2017-06-05

Improving the therapeutic index of anticancer agents is an enormous challenge. Targeting decreases the side effects of the therapeutic agents by delivering the drugs to the intended destination. Nanocarriers containing the nuclear localizing peptide sequences (NLS) translocate to the cell nuclei. However, the nuclear localization peptides are nonselective and cannot distinguish the malignant cells from the healthy counterparts. In this study, we designed a "masked" NLS peptide which is activated only in the presence of overexpressed matrix metalloproteinase-7 (MMP-7) enzyme in the pancreatic cancer microenvironment. This peptide is conjugated to the surface of redox responsive polymersomes to deliver doxorubicin and curcumin to the pancreatic cancer cell nucleus. We have tested the formulation in both two- and three-dimensional cultures of pancreatic cancer and normal cells. Our studies revealed that the drug-encapsulated polymeric vesicles are significantly more toxic toward the cancer cells (shrinking the spheroids up to 49%) compared to the normal cells (shrinking the spheroids up to 24%). This study can lead to the development of other organelle targeted drug delivery systems for various human malignancies.

Mechanics of Fluid-Filled Interstitial Gaps. II. Gap Characteristics in Xenopus Embryonic Ectoderm.

PubMed

Barua, Debanjan; Parent, Serge E; Winklbauer, Rudolf

2017-08-22

The ectoderm of the Xenopus embryo is permeated by a network of channels that appear in histological sections as interstitial gaps. We characterized this interstitial space by measuring gap sizes, angles formed between adjacent cells, and curvatures of cell surfaces at gaps. From these parameters, and from surface-tension values measured previously, we estimated the values of critical mechanical variables that determine gap sizes and shapes in the ectoderm, using a general model of interstitial gap mechanics. We concluded that gaps of 1-4 μm side length can be formed by the insertion of extracellular matrix fluid at three-cell junctions such that cell adhesion is locally disrupted and a tension difference between cell-cell contacts and the free cell surface at gaps of 0.003 mJ/m 2 is generated. Furthermore, a cell hydrostatic pressure of 16.8 ± 1.7 Pa and an interstitial pressure of 3.9 ± 3.6 Pa, relative to the central blastocoel cavity of the embryo, was found to be consistent with the observed gap size and shape distribution. Reduction of cell adhesion by the knockdown of C-cadherin increased gap volume while leaving intracellular and interstitial pressures essentially unchanged. In both normal and adhesion-reduced ectoderm, cortical tension of the free cell surfaces at gaps does not return to the high values characteristic of the free surface of the whole tissue. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Microvillar cell surface as a natural defense system against xenobiotics: a new interpretation of multidrug resistance.

PubMed

Lange, K; Gartzke, J

2001-08-01

The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling. According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV). The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm. The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds. Because of the specific organization of epithelial Ca(2+) signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism. Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV. The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells. The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G(0)/G(1) resting phase.

New approaches for solving old problems in neuronal protein trafficking.

PubMed

Bourke, Ashley M; Bowen, Aaron B; Kennedy, Matthew J

2018-04-10

Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision. Copyright © 2018 Elsevier Inc. All rights reserved.

NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

PubMed Central

Luci, Carmelo; Bekri, Selma; Bihl, Franck; Pini, Jonathan; Bourdely, Pierre; Nouhen, Kelly; Malgogne, Angélique; Walzer, Thierry; Braud, Véronique M.; Anjuère, Fabienne

2015-01-01

Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46+ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46+ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46+ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46+ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46+ ILC in the regulation of mucosal CD8 T cell responses. PMID:26630176

Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

PubMed

Nguyen, Hoang-Minh; Mathiesen, Geir; Stelzer, Elena Maria; Pham, Mai Lan; Kuczkowska, Katarzyna; Mackenzie, Alasdair; Agger, Jane W; Eijsink, Vincent G H; Yamabhai, Montarop; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha

2016-10-04

Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach, with the enzyme of interest being displayed on the cell surface of a food-grade organism, may also be applied in production processes relevant for food industry.

The Pseudomonas aeruginosa ribbon-helix-helix DNA-binding protein AlgZ (AmrZ) controls twitching motility and biogenesis of type IV pili.

PubMed

Baynham, Patricia J; Ramsey, Deborah M; Gvozdyev, Borys V; Cordonnier, Ellen M; Wozniak, Daniel J

2006-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content, P. aeruginosa utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an algZ deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type P. aeruginosa and P. aeruginosa with the algZ deletion, the amount of PilA on the surface of the cells was drastically reduced in the algZ mutant. Analysis of algZ and algD mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that algZ (PA3385) be designated amrZ (alginate and motility regulator Z).

Characterizing Spatial Organization of Cell Surface Receptors in Human Breast Cancer with STORM

NASA Astrophysics Data System (ADS)

Lyall, Evan; Chapman, Matthew R.; Sohn, Lydia L.

2012-02-01

Regulation and control of complex biological functions are dependent upon spatial organization of biological structures at many different length scales. For instance Eph receptors and their ephrin ligands bind when opposing cells come into contact during development, resulting in spatial organizational changes on the nanometer scale that lead to changes on the macro scale, in a process known as organ morphogenesis. One technique able to probe this important spatial organization at both the nanometer and micrometer length scales, including at cell-cell junctions, is stochastic optical reconstruction microscopy (STORM). STORM is a technique that localizes individual fluorophores based on the centroids of their point spread functions and then reconstructs a composite image to produce super resolved structure. We have applied STORM to study spatial organization of the cell surface of human breast cancer cells, specifically the organization of tyrosine kinase receptors and chemokine receptors. A better characterization of spatial organization of breast cancer cell surface proteins is necessary to fully understand the tumorigenisis pathways in the most common malignancy in United States women.

Hyaluronan, CD44, and Emmprin Regulate Lactate Efflux and Membrane Localization of Monocarboxylate Transporters in Human Breast Carcinoma Cells

PubMed Central

Slomiany, Mark G.; Grass, G. Daniel; Robertson, Angela D.; Yang, Xiao Y.; Maria, Bernard L.; Beeson, Craig; Toole, Bryan P.

2013-01-01

Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin)is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells. PMID:19176383

The Cell Surface Markers Expression in Postmenopausal Women and Relation to Obesity and Bone Status.

PubMed

Horváthová, Mira; Ilavská, Silvia; Štefíková, Kornélia; Szabová, Michaela; Krivošíková, Zora; Jahnová, Eva; Tulinská, Jana; Spustová, Viera; Gajdoš, Martin

2017-07-11

The age-related changes and hormonal deprivation in postmenopausal women are associated with the immune response alteration. The excessive fat accumulation, local and systemic inflammation may lead to dysregulation in immune function and relevant health problems, including obesity and osteoporosis. We analyzed the expression of cell surface markers in the venous blood specimens, stained with fluorophores-conjugated monoclonal antibodies and analysed by multicolour flow cytometry. The significant changes of cytotoxic, naive, and memory T-lymphocytes, plasmacytoid dendritic cells (DCs) were in postmenopausal women versus fertile women. Body mass index (BMI) affected markedly the cell surface expression of CD265/RANK. Osteoporosis is linked to reduced percentage of plasmacytoid DCs, and elevated natural Treg cells ( p < 0.05). The confounding factors such as women age, BMI, bone mineral density (BMD), waist size and tissue fat affect the expression of RANK on myeloid DCs and CD40L on T-lymphocytes that might be the immunophenotypic modulators after menopause.

The Cell Surface Markers Expression in Postmenopausal Women and Relation to Obesity and Bone Status

PubMed Central

Horváthová, Mira; Ilavská, Silvia; Štefíková, Kornélia; Szabová, Michaela; Krivošíková, Zora; Jahnová, Eva; Tulinská, Jana; Spustová, Viera; Gajdoš, Martin

2017-01-01

The age-related changes and hormonal deprivation in postmenopausal women are associated with the immune response alteration. The excessive fat accumulation, local and systemic inflammation may lead to dysregulation in immune function and relevant health problems, including obesity and osteoporosis. We analyzed the expression of cell surface markers in the venous blood specimens, stained with fluorophores-conjugated monoclonal antibodies and analysed by multicolour flow cytometry. The significant changes of cytotoxic, naive, and memory T-lymphocytes, plasmacytoid dendritic cells (DCs) were in postmenopausal women versus fertile women. Body mass index (BMI) affected markedly the cell surface expression of CD265/RANK. Osteoporosis is linked to reduced percentage of plasmacytoid DCs, and elevated natural Treg cells (p < 0.05). The confounding factors such as women age, BMI, bone mineral density (BMD), waist size and tissue fat affect the expression of RANK on myeloid DCs and CD40L on T-lymphocytes that might be the immunophenotypic modulators after menopause. PMID:28696349

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release

PubMed Central

Greene, Neil G.; Narciso, Ana R.; Filipe, Sergio R.; Camilli, Andrew

2015-01-01

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens. PMID:26114646

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release.

PubMed

Greene, Neil G; Narciso, Ana R; Filipe, Sergio R; Camilli, Andrew

2015-06-01

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.

Laser-induced surface deformation microscope for the study of the dynamic viscoelasticity of plasma membrane in a living cell.

PubMed

Morisaku, Toshinori; Yui, Hiroharu

2018-05-15

A laser-induced surface deformation (LISD) microscope is developed and applied to measurement of the dynamic relaxation responses of the plasma membrane in a living cell. A laser beam is tightly focused on an optional area of cell surface and the focused light induces microscopic deformation on the surface via radiation pressure. The LISD microscope not only allows non-contact and destruction-free measurement but provides power spectra of the surface responses depending on the frequency of the intensity of the laser beam. An optical system for the LISD is equipped via a microscope, allowing us to measure the relaxation responses in sub-cellular-sized regions of the plasma membrane. In addition, the forced oscillation caused by the radiation pressure for surface deformation extends the upper limit of the frequency range in the obtained power spectra to 106 Hz, which enables us to measure relaxation responses in local regions within the plasma membrane. From differences in power-law exponents at higher frequencies, it is realized that a cancerous cell obeys a weaker single power-law than a normal fibroblast cell. Furthermore, the power spectrum of a keratinocyte cell obeys a power-law with two exponents, indicating that alternative mechanical models to a conventional soft glassy rheology model (where single power-laws explain cells' responses below about 103 Hz) are needed for the understanding over a wider frequency range. The LISD microscope would contribute to investigation of microscopic cell rheology, which is important for clarifying the mechanisms of cell migration and tissue construction.

Long-lived tissue resident HIV-1 specific memory CD8+ T cells are generated by skin immunization with live virus vectored microneedle arrays.

PubMed

Zaric, Marija; Becker, Pablo Daniel; Hervouet, Catherine; Kalcheva, Petya; Ibarzo Yus, Barbara; Cocita, Clement; O'Neill, Lauren Alexandra; Kwon, Sung-Yun; Klavinskis, Linda Sylvia

2017-12-28

The generation of tissue resident memory (T RM ) cells at the body surfaces to provide a front line defence against invading pathogens represents an important goal in vaccine development for a wide variety of pathogens. It has been widely assumed that local vaccine delivery to the mucosae is necessary to achieve that aim. Here we characterise a novel micro-needle array (MA) delivery system fabricated to deliver a live recombinant human adenovirus type 5 vaccine vector (AdHu5) encoding HIV-1 gag. We demonstrate rapid dissolution kinetics of the microneedles in skin. Moreover, a consequence of MA vaccine cargo release was the generation of long-lived antigen-specific CD8 + T cells that accumulate in mucosal tissues, including the female genital and respiratory tract. The memory CD8 + T cell population maintained in the peripheral mucosal tissues was attributable to a MA delivered AdHu5 vaccine instructing CD8 + T cell expression of CXCR3 + , CD103 +, CD49a + , CD69 + , CD127 + homing, retention and survival markers. Furthermore, memory CD8 + T cells generated by MA immunization significantly expanded upon locally administered antigenic challenge and showed a predominant poly-functional profile producing high levels of IFNγ and Granzyme B. These data demonstrate that skin vaccine delivery using microneedle technology induces mobilization of long lived, poly-functional CD8 + T cells to peripheral tissues, phenotypically displaying hallmarks of residency and yields new insights into how to design and deliver effective vaccine candidates with properties to exert local immunosurveillance at the mucosal surfaces. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models

PubMed Central

Shoji, Takuhiro; Saito, Ryuta; Chonan, Masashi; Shibahara, Ichiyo; Sato, Aya; Kanamori, Masayuki; Sonoda, Yukihiko; Kondo, Toru; Ishii, Naoto; Tominaga, Teiji

2016-01-01

Background Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. Methods CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. Results CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4+ and CD8+ T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Conclusions Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas. PMID:26917236

Cellular dosimetry calculations for Strontium-90 using Monte Carlo code PENELOPE.

PubMed

Hocine, Nora; Farlay, Delphine; Boivin, Georges; Franck, Didier; Agarande, Michelle

2014-11-01

To improve risk assessments associated with chronic exposure to Strontium-90 (Sr-90), for both the environment and human health, it is necessary to know the energy distribution in specific cells or tissue. Monte Carlo (MC) simulation codes are extremely useful tools for calculating deposition energy. The present work was focused on the validation of the MC code PENetration and Energy LOss of Positrons and Electrons (PENELOPE) and the assessment of dose distribution to bone marrow cells from punctual Sr-90 source localized within the cortical bone part. S-values (absorbed dose per unit cumulated activity) calculations using Monte Carlo simulations were performed by using PENELOPE and Monte Carlo N-Particle eXtended (MCNPX). Cytoplasm, nucleus, cell surface, mouse femur bone and Sr-90 radiation source were simulated. Cells are assumed to be spherical with the radii of the cell and cell nucleus ranging from 2-10 μm. The Sr-90 source is assumed to be uniformly distributed in cell nucleus, cytoplasm and cell surface. The comparison of S-values calculated with PENELOPE to MCNPX results and the Medical Internal Radiation Dose (MIRD) values agreed very well since the relative deviations were less than 4.5%. The dose distribution to mouse bone marrow cells showed that the cells localized near the cortical part received the maximum dose. The MC code PENELOPE may prove useful for cellular dosimetry involving radiation transport through materials other than water, or for complex distributions of radionuclides and geometries.

Simultaneous AFM topography and recognition imaging at the plasma membrane of mammalian cells.

PubMed

Chtcheglova, Lilia A; Hinterdorfer, Peter

2018-01-01

Elucidation the nano-organization of membrane proteins at/within the plasma membrane is probably the most demanding and still challenging task in cell biology since requires experimental approaches with nanoscale resolution. During last decade, atomic force microscopy (AFM)-based simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nano-maps on complex heterogeneous biosurfaces such as cells and membranes. Here we emphasize the TREC technique and explain how to unravel the nano-landscape of mammalian cells. We describe the procedures for all steps of the experiment including tip functionalization with ligand molecules, sample preparation, and localization of key molecules on the cell surface. We also discuss the current limitations and future perspectives of this technique. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Glycosylation-dependent binding of galectin-8 to activated leukocyte cell adhesion molecule (ALCAM/CD166) promotes its surface segregation on breast cancer cells.

PubMed

Fernández, Marisa M; Ferragut, Fátima; Cárdenas Delgado, Víctor M; Bracalente, Candelaria; Bravo, Alicia I; Cagnoni, Alejandro J; Nuñez, Myriam; Morosi, Luciano G; Quinta, Héctor R; Espelt, María V; Troncoso, María F; Wolfenstein-Todel, Carlota; Mariño, Karina V; Malchiodi, Emilio L; Rabinovich, Gabriel A; Elola, María T

2016-10-01

We previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell-cell interactions. Gal-8 is a "tandem-repeat"-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon. We performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization. We showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells. Our data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms. A novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Cancer cell uptake behavior of Au nanoring and its localized surface plasmon resonance induced cell inactivation

NASA Astrophysics Data System (ADS)

Chu, Che-Kuan; Tu, Yi-Chou; Chang, Yu-Wei; Chu, Chih-Ken; Chen, Shih-Yang; Chi, Ting-Ta; Kiang, Yean-Woei; Yang, Chih-Chung

2015-02-01

Au nanorings (NRIs), which have the localized surface plasmon resonance (LSPR) wavelength around 1058 nm, either with or without linked antibodies, are applied to SAS oral cancer cells for cell inactivation through the LSPR-induced photothermal effect when they are illuminated by a laser of 1065 nm in wavelength. Different incubation times of cells with Au NRIs are considered for observing the variations of cell uptake efficiency of Au NRI and the threshold laser intensity for cell inactivation. In each case of incubation time, the cell sample is washed for evaluating the total Au NRI number per cell adsorbed and internalized by the cells based on inductively coupled plasma mass spectrometry measurement. Also, the Au NRIs remaining on cell membrane are etched with KI/I2 solution to evaluate the internalized Au NRI number per cell. The threshold laser intensities for cell inactivation before washout, after washout, and after KI/I2 etching are calibrated from the circular area sizes of inactivated cells around the illuminated laser spot center with various laser power levels. By using Au NRIs with antibodies, the internalized Au NRI number per cell increases monotonically with incubation time up to 24 h. However, the number of Au NRI remaining on cell membrane reaches a maximum at 12 h in incubation time. The cell uptake behavior of an Au NRI without antibodies is similar to that with antibodies except that the uptake NRI number is significantly smaller and the incubation time for the maximum NRI number remaining on cell membrane is delayed to 20 h. By comparing the threshold laser intensities before and after KI/I2 etching, it is found that the Au NRIs remaining on cell membrane cause more effective cancer cell inactivation, when compared with the internalized Au NRIs.

The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.

PubMed

Kawada, Daiki; Kobayashi, Hiromu; Tomita, Tsuyoshi; Nakata, Eisuke; Nagano, Makoto; Siekhaus, Daria Elisabeth; Toshima, Junko Y; Toshima, Jiro

2015-01-01

Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

The development and geometry of shape change in Arabidopsis thaliana cotyledon pavement cells

PubMed Central

2011-01-01

Background The leaf epidermis is an important architectural control element that influences the growth properties of underlying tissues and the overall form of the organ. In dicots, interdigitated pavement cells are the building blocks of the tissue, and their morphogenesis includes the assembly of specialized cell walls that surround the apical, basal, and lateral (anticlinal) cell surfaces. The microtubule and actin cytoskeletons are highly polarized along the cortex of the anticlinal wall; however, the relationships between these arrays and cell morphogenesis are unclear. Results We developed new quantitative tools to compare population-level growth statistics with time-lapse imaging of cotyledon pavement cells in an intact tissue. The analysis revealed alternating waves of lobe initiation and a phase of lateral isotropic expansion that persisted for days. During lateral isotropic diffuse growth, microtubule organization varied greatly between cell surfaces. Parallel microtubule bundles were distributed unevenly along the anticlinal surface, with subsets marking stable cortical domains at cell indentations and others clearly populating the cortex within convex cell protrusions. Conclusions Pavement cell morphogenesis is discontinuous, and includes punctuated phases of lobe initiation and lateral isotropic expansion. In the epidermis, lateral isotropic growth is independent of pavement cell size and shape. Cortical microtubules along the upper cell surface and stable cortical patches of anticlinal microtubules may coordinate the growth behaviors of orthogonal cell walls. This work illustrates the importance of directly linking protein localization data to the growth behavior of leaf epidermal cells. PMID:21284861

The development and geometry of shape change in Arabidopsis thaliana cotyledon pavement cells.

PubMed

Zhang, Chunhua; Halsey, Leah E; Szymanski, Daniel B

2011-02-01

The leaf epidermis is an important architectural control element that influences the growth properties of underlying tissues and the overall form of the organ. In dicots, interdigitated pavement cells are the building blocks of the tissue, and their morphogenesis includes the assembly of specialized cell walls that surround the apical, basal, and lateral (anticlinal) cell surfaces. The microtubule and actin cytoskeletons are highly polarized along the cortex of the anticlinal wall; however, the relationships between these arrays and cell morphogenesis are unclear. We developed new quantitative tools to compare population-level growth statistics with time-lapse imaging of cotyledon pavement cells in an intact tissue. The analysis revealed alternating waves of lobe initiation and a phase of lateral isotropic expansion that persisted for days. During lateral isotropic diffuse growth, microtubule organization varied greatly between cell surfaces. Parallel microtubule bundles were distributed unevenly along the anticlinal surface, with subsets marking stable cortical domains at cell indentations and others clearly populating the cortex within convex cell protrusions. Pavement cell morphogenesis is discontinuous, and includes punctuated phases of lobe initiation and lateral isotropic expansion. In the epidermis, lateral isotropic growth is independent of pavement cell size and shape. Cortical microtubules along the upper cell surface and stable cortical patches of anticlinal microtubules may coordinate the growth behaviors of orthogonal cell walls. This work illustrates the importance of directly linking protein localization data to the growth behavior of leaf epidermal cells.

Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

PubMed

Lee, Sangyeop; Chon, Hyangah; Lee, Jiyoung; Ko, Juhui; Chung, Bong Hyun; Lim, Dong Woo; Choo, Jaebum

2014-01-15

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment. © 2013 Elsevier B.V. All rights reserved.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

PubMed

Schindelman, G; Morikami, A; Jung, J; Baskin, T I; Carpita, N C; Derbyshire, P; McCann, M C; Benfey, P N

2001-05-01

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis

PubMed Central

Schindelman, Gary; Morikami, Atsushi; Jung, Jee; Baskin, Tobias I.; Carpita, Nicholas C.; Derbyshire, Paul; McCann, Maureen C.; Benfey, Philip N.

2001-01-01

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion. PMID:11331607

GW domains of the Listeria monocytogenes invasion protein InlB are SH3-like and mediate binding to host ligands

PubMed Central

Marino, Michael; Banerjee, Manidipa; Jonquières, Renaud; Cossart, Pascale; Ghosh, Partho

2002-01-01

InlB, a surface-localized protein of Listeria monocytogenes, induces phagocytosis in non-phagocytic mammalian cells by activating Met, a receptor tyrosine kinase. InlB also binds glycosaminoglycans and the protein gC1q-R, two additional host ligands implicated in invasion. We present the structure of InlB, revealing a highly elongated molecule with leucine-rich repeats that bind Met at one end, and GW domains that dissociably bind the bacterial surface at the other. Surprisingly, the GW domains are seen to resemble SH3 domains. Despite this, GW domains are unlikely to act as functional mimics of SH3 domains since their potential proline-binding sites are blocked or destroyed. However, we do show that the GW domains, in addition to binding glycosaminoglycans, bind gC1q-R specifically, and that this binding requires release of InlB from the bacterial surface. Dissociable attachment to the bacterial surface via the GW domains may be responsible for restricting Met activation to a small, localized area of the host cell and for coupling InlB-induced host membrane dynamics with bacterial proximity during invasion. PMID:12411480

Nucleation of rotating crystals by Thiovulum majus bacteria

NASA Astrophysics Data System (ADS)

Petroff, A. P.; Libchaber, A.

2018-01-01

Thiovulum majus self-organize on glass surfaces into active two-dimensional crystals of rotating cells. Unlike classical crystals, these bacterial crystallites continuously rotate and reorganize as the power of rotating cells is dissipated by the surrounding flow. In this article, we describe the earliest stage of crystallization, the attraction of two bacteria into a hydrodynamically-bound dimer. This process occurs in three steps. First a free-swimming cell collides with the wall and becomes hydrodynamically bound to the two-dimensional surface. We present a simple model to understand how viscous forces localize cells near the chamber walls. Next, the cell diffuses over the surface for an average of 63+/- 6 s before escaping to the bulk fluid. The diffusion coefficient {D}{{eff}}=7.98 +/- 0.1 μ {{{m}}}2 {{{s}}}-1 of these 8.5 μ {{m}} diameter cells corresponds to a temperature of (4.16+/- 0.05)× {10}4 K, and thus cannot be explained by equilibrium fluctuations. Finally, two cells coalesce into a rotating dimer when the convergent flow created by each cell overwhelms their active Brownian motion. This occurs when cells diffuse to within a distance of 13.3 ± 0.2 μm of each other.

Mycobacterium tuberculosis surface protein Rv0227c contains high activity binding peptides which inhibit cell invasion.

PubMed

Rodríguez, Diana Marcela; Ocampo, Marisol; Curtidor, Hernando; Vanegas, Magnolia; Patarroyo, Manuel Elkin; Patarroyo, Manuel Alfonso

2012-12-01

Mycobacterium tuberculosis surface proteins involved in target cell invasion may be identified as a strategy for developing subunit-based, chemically-synthesized vaccines. The Rv0227c protein was thus selected to assess its role in the invasion and infection of Mycobacterium tuberculosis target cells. Results revealed Rv0227c localization on mycobacterial surface by immunoelectron microscopy and Western blot. Receptor-ligand assays using 20-mer, non-overlapping peptides covering the complete Rv0227c protein sequence revealed three high activity binding peptides for U937 phagocytic cells and seven for A549 cells. Peptide 16944 significantly inhibited mycobacterial entry to both cell lines while 16943 and 16949 only managed to inhibit entrance to U937 cells and 16951 to A549 cells. The Jnet bioinformatics tool predicted secondary structure elements for the complete protein, agreeing with elements determined for such chemically-synthesized peptides. It was thus concluded that high activity binding peptides which were able to inhibit mycobacterial entry to target cells are of great importance when selecting peptide candidates for inclusion in an anti-tuberculosis vaccine. Copyright © 2012 Elsevier Inc. All rights reserved.

Spatial and temporal control of the diazonium modification of sp2 carbon surfaces.

PubMed

Kirkman, Paul M; Güell, Aleix G; Cuharuc, Anatolii S; Unwin, Patrick R

2014-01-08

Interest in the controlled chemical functionalization of sp(2) carbon materials using diazonium compounds has been recently reignited, particularly as a means to generate a band gap in graphene. We demonstrate local diazonium modification of pristine sp(2) carbon surfaces, with high control, at the micrometer scale through the use of scanning electrochemical cell microscopy (SECCM). Electrochemically driven diazonium patterning is investigated at a range of driving forces, coupled with surface analysis using atomic force microscopy (AFM) and Raman spectroscopy. We highlight how the film density, level of sp(2)/sp(3) rehybridization and the extent of multilayer formation can be controlled, paving the way for the use of localized electrochemistry as a route to controlled diazonium modification.

Autonomous Bacterial Localization and Gene Expression Based on Nearby Cell Receptor Density

DTIC Science & Technology

2013-01-22

synthesis of novel products (Kwok, 2010). In such cases, cell populations are aligned for optimal production . While Nature’s biosynthetic toolbox is vast...less commonly examined but equally innovative strategy envisions a repro- grammed cell itself or small collections of cells as the end products of...to surfaces. In Figure 2B, we depict the level of NF binding and AI-2 production as a function of coated avidin. We used a mathematical model for E

Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages

NASA Technical Reports Server (NTRS)

Chong, H.; Vodovotz, Y.; Cox, G. W.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

1999-01-01

Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

Time-dependent grid adaptation for meshes of triangles and tetrahedra

NASA Technical Reports Server (NTRS)

Rausch, Russ D.

1993-01-01

This paper presents in viewgraph form a method of optimizing grid generation for unsteady CFD flow calculations that distributes the numerical error evenly throughout the mesh. Adaptive meshing is used to locally enrich in regions of relatively large errors and to locally coarsen in regions of relatively small errors. The enrichment/coarsening procedures are robust for isotropic cells; however, enrichment of high aspect ratio cells may fail near boundary surfaces with relatively large curvature. The enrichment indicator worked well for the cases shown, but in general requires user supervision for a more efficient solution.

Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaellquist, Linda; Rosen, Hanna; Nordenfelt, Pontus

2010-11-15

Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 withmore » clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating 'pro{sub C}'-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.« less

Chimeric antigen receptor T cells: a novel therapy for solid tumors.

PubMed

Yu, Shengnan; Li, Anping; Liu, Qian; Li, Tengfei; Yuan, Xun; Han, Xinwei; Wu, Kongming

2017-03-29

The chimeric antigen receptor T (CAR-T) cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs) expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII) was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2), and mesothelin (MSLN), as well as the challenges for CAR-T cell therapy.

Temperature-Sensitive Intracellular Traffic of α2C-Adrenergic Receptor.

PubMed

Filipeanu, Catalin M

2015-01-01

α(2C)-Adrenergic receptor (α(2C)-AR) is the least characterized adrenergic receptor subtype and still very little is known about the intracellular traffic properties and pathophysiological roles of this receptor. α(2C)-AR has an atypical subcellular localization. At 37 °C, in the vascular smooth muscle cells and in fibroblasts, the receptor is poorly localized at the plasma membrane and accumulates inside the cell. Exposure to lower temperatures stimulates α(2C)-AR transport to the cell surface. This particular intracellular trafficking of α(2C)-AR is significant in the pathology of Raynaud phenomenon. In this brief review, I will present general information on the tissue distribution and cellular localization of α(2C)-AR. Also, I will discuss the mechanisms involved in the receptor transport by focusing on the trafficking motifs and on the molecular chaperones. © 2015 Elsevier Inc. All rights reserved.

Multi-color localization microscopy of fixed cells as a promising tool to study organization of bacterial cytoskeleton

NASA Astrophysics Data System (ADS)

Vedyaykin, A. D.; Gorbunov, V. V.; Sabantsev, A. V.; Polinovskaya, V. S.; Vishnyakov, I. E.; Melnikov, A. S.; Serdobintsev, P. Yu; Khodorkovskii, M. A.

2015-11-01

Localization microscopy allows visualization of biological structures with resolution well below the diffraction limit. Localization microscopy was used to study FtsZ organization in Escherichia coli previously in combination with fluorescent protein labeling, but the fact that fluorescent chimeric protein was unable to rescue temperature-sensitive ftsZ mutants suggests that obtained images may not represent native FtsZ structures faithfully. Indirect immunolabeling of FtsZ not only overcomes this problem, but also allows the use of the powerful visualization methods arsenal available for different structures in fixed cells. In this work we simultaneously obtained super-resolution images of FtsZ structures and diffraction-limited or super-resolution images of DNA and cell surface in E. coli, which allows for the study of the spatial arrangement of FtsZ structures with respect to the nucleoid positions and septum formation.

Lectin histochemistry and alkaline phosphatase activity in the pia mater vessels of spontaneously hypertensive rats (SHR).

PubMed

Szumańska, G; Gadamski, R

1992-01-01

Some lectins were used to study the localization of sugar residues on the endothelial cell surface in the pia mater blood vessels of control (WKY) and hypertensive rats (SHR). The lectins tested recognized the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA-1), alpha-L-fucosyl (Ulex europaeus agglutinin, UEA-1), N-acetylglucosaminyl and sialyl (Wheat germ agglutinin, WGA), N-glycolyl-neuraminic acid (Limax flavus agglutinin, LFA), and N-acetyl-D-galactosaminyl (Helix pomatia agglutinin, HPA). Several differences were revealed in the presence of sugar receptors on the surface of endothelial cells between the control and the hypertensive rats. Our studies showed also differences in the localization of the tested glycoconjugates between pial capillaries, small, medium-size and large pial arteries. The histochemical evaluation of alkaline phosphatase revealed an increased activity of the enzyme in the pial vessels of SHRs as compared with control rats with a similar localization of the enzyme activity. Some differences in the distribution of lectin binding sites and alkaline phosphatase activity could be associated with the different functions of particular segments of the pial vascular network.

Effect of topography-dependent light coupling through a near-field aperture on the local photocurrent of a solar cell.

PubMed

Cao, Zhao; Ermes, Markus; Lehnen, Stephan; Carius, Reinhard; Bittkau, Karsten

2018-01-03

An aperture-type scanning near-field optical microscope (a-SNOM) is readily used for the optical and optoelectronic characterizations of a wide variety of chemical, biological and optoelectronic samples with sub-wavelength optical resolution. These samples mostly exhibit nanoscale topographic variations, which are related to local material inhomogeneity probed either by an optical contrast or by secondary effects such as photoconductivity or photoluminescence. To date, in the interpretation and evaluation of the measurement results from a-SNOM or derived methods, often only the local material inhomogeneity is taken into account. A possible influence of the optical interaction between the scanning probe and the surface topography is rarely discussed. In this paper, we present experimental and theoretical investigation of the effects of nanoscale topographic features on a-SNOM measurement results. We conduct local photocurrent measurements on a thin-film solar cell with an a-SNOM as the illumination source. A clear correlation between the photocurrent response and local topography is observed in all measurements with a signal contrast of up to ∼30%, although the sample features homogeneous permittivity and electrical properties. With the help of finite-difference time-domain (FDTD) simulations, this correlation is reproduced and local light coupling is identified as the mechanism which determines the local photocurrent response. Our results suggest that a-SNOM-based measurements of any sample with material inhomogeneity will be superimposed by the local light-coupling effect if surface topography variation exists. This effect should always be taken into consideration for an accurate interpretation of the measurement results.

Immunocytochemical localization of chymase to cytoplasmic vesicles after rat peritoneal mast cell stimulation by compound 48/80.

PubMed

Login, G R; Aoki, M; Yamakawa, M; Lunardi, L O; Digenis, E C; Tanda, N; Schwartz, L B; Dvorak, A M

1997-10-01

The subcellular events responsible for release of mediators by mast cells may help to clarify roles for mast cells in health and disease. In this study we show that the granule-associated protease chymase is also within cytoplasmic vesicles in appropriately stimulated rat peritoneal mast cells. Rat peritoneal mast cells were recovered before or 1-10 sec after exposure to the secretogogue compound 48/80 (10 micrograms/ml) and then were examined by radioimmunoassay to quantify histamine release or were processed, using routine methods for postembedding immunoelectron microscopy, to identify the subcellular localization of chymase. In comparison to unstimulated cells, compound 48/80 stimulated cells in two independent experiments showed an increase (15%, 28%) in the surface area of the cell and a decrease (12%, 6%) in the surface area of the total granule compartment before degranulation channel formation. These global cellular changes occurred in a background of transient but significant (p < 0.01) increases in the area and number of chymase-immunoreactive vesicles per microns2 cytoplasm. These changes were detectable at 5 or 7 sec after stimulation with compound 48/80 but returned to near prestimulation levels by 9 or 10 sec after addition of compound 48/80 (total cumulative histamine release was 28% by 8 sec and 47% by 14 sec). These observations suggest that vesicles participate in the early stages of regulated secretion of chymase from rat peritoneal mast cells.

GARP-TGF-β complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4+ T cells in vivo.

PubMed

Zhou, Angela X; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2013-05-15

The role of surface-bound TGF-β on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-β and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4(+) T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4(+) T cells, were also reduced in the thymus. CD4(+) T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4(+) T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-β signaling. Furthermore, inhibiting TGF-β signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4(+) T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-β and signaling, which negatively regulates GARP expression on Tregs.

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

PubMed Central

2011-01-01

Background Elevated numbers of regulatory T cells (Tregs) have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells. PMID:21615933

Fluid Mechanics, Arterial Disease, and Gene Expression.

PubMed

Tarbell, John M; Shi, Zhong-Dong; Dunn, Jessilyn; Jo, Hanjoong

2014-01-01

This review places modern research developments in vascular mechanobiology in the context of hemodynamic phenomena in the cardiovascular system and the discrete localization of vascular disease. The modern origins of this field are traced, beginning in the 1960s when associations between flow characteristics, particularly blood flow-induced wall shear stress, and the localization of atherosclerotic plaques were uncovered, and continuing to fluid shear stress effects on the vascular lining endothelial) cells (ECs), including their effects on EC morphology, biochemical production, and gene expression. The earliest single-gene studies and genome-wide analyses are considered. The final section moves from the ECs lining the vessel wall to the smooth muscle cells and fibroblasts within the wall that are fluid me chanically activated by interstitial flow that imposes shear stresses on their surfaces comparable with those of flowing blood on EC surfaces. Interstitial flow stimulates biochemical production and gene expression, much like blood flow on ECs.

Patchwork structure-function analysis of the Sendai virus matrix protein.

PubMed

Mottet-Osman, Geneviève; Miazza, Vincent; Vidalain, Pierre-Olivier; Roux, Laurent

2014-09-01

Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production. Copyright © 2014 Elsevier Inc. All rights reserved.

A compact and versatile microfluidic probe for local processing of tissue sections and biological specimens

NASA Astrophysics Data System (ADS)

Cors, J. F.; Lovchik, R. D.; Delamarche, E.; Kaigala, G. V.

2014-03-01

The microfluidic probe (MFP) is a non-contact, scanning microfluidic technology for local (bio)chemical processing of surfaces based on hydrodynamically confining nanoliter volumes of liquids over tens of micrometers. We present here a compact MFP (cMFP) that can be used on a standard inverted microscope and assist in the local processing of tissue sections and biological specimens. The cMFP has a footprint of 175 × 100 × 140 mm3 and can scan an area of 45 × 45 mm2 on a surface with an accuracy of ±15 μm. The cMFP is compatible with standard surfaces used in life science laboratories such as microscope slides and Petri dishes. For ease of use, we developed self-aligned mounted MFP heads with standardized "chip-to-world" and "chip-to-platform" interfaces. Switching the processing liquid in the flow confinement is performed within 90 s using a selector valve with a dead-volume of approximately 5 μl. We further implemented height-compensation that allows a cMFP head to follow non-planar surfaces common in tissue and cellular ensembles. This was shown by patterning different macroscopic copper-coated topographies with height differences up to 750 μm. To illustrate the applicability to tissue processing, 5 μm thick M000921 BRAF V600E+ melanoma cell blocks were stained with hematoxylin to create contours, lines, spots, gradients of the chemicals, and multiple spots over larger areas. The local staining was performed in an interactive manner using a joystick and a scripting module. The compactness, user-friendliness, and functionality of the cMFP will enable it to be adapted as a standard tool in research, development and diagnostic laboratories, particularly for the interaction with tissues and cells.

Simulation of light in-coupling through an aperture probe to investigate light propagation in a thin layer for opto-electronic application

NASA Astrophysics Data System (ADS)

Ermes, Markus; Lehnen, Stephan; Cao, Zhao; Bittkau, Karsten; Carius, Reinhard

2015-06-01

In thin optoelectronic devices, like organic light emitting diodes (OLED) or thin-film solar cells (TFSC), light propagation, which is initiated by a local point source, is of particular importance. In OLEDs, light is generated in the layer by the luminescence of single molecules, whereas in TFSCs, light is coupled into the devices by scattering at small surface features. In both applications, light propagation within the active layers has a significant impact on the optical device performance. Scanning near-field optical microscopy (SNOM) using aperture probes is a powerful tool to investigate this propagation with a high spatial resolution. Dual-probe SNOM allows simulating the local light generation by an illumination probe as well as the detection of the light propagated through the layer. In our work, we focus on the light propagation in thin silicon films as used in thin-film silicon solar cells. We investigate the light-in-coupling from an illuminating probe via rigorous solution of Maxwell's equations using a Finite-Difference Time-Domain approach, especially to gain insight into the light distribution inside a thin layer, which is not accessible in the experiment. The structures investigated include at and structured surfaces with varying illumination positions and wavelengths. From the performed simulations, we define a "spatial sensitivity" which is characteristic for the local structure and illumination position. This quantity can help to identify structures which are beneficial as well as detrimental to absorption inside the investigated layer. We find a strong dependence of the spatial sensitivity on the surface structure as well as both the absorption coefficient and the probe position. Furthermore, we investigate inhomogeneity in local light propagation resulting from different surface structures and illumination positions.

Adenovirus Entry From the Apical Surface of Polarized Epithelia Is Facilitated by the Host Innate Immune Response

PubMed Central

Kotha, Poornima L. N.; Sharma, Priyanka; Kolawole, Abimbola O.; Yan, Ran; Alghamri, Mahmoud S.; Brockman, Trisha L.; Gomez-Cambronero, Julian; Excoffon, Katherine J. D. A.

2015-01-01

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAREx8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAREx8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAREx8, to allow the initial infection the intact epithelium. In addition, CAREx8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection. PMID:25768646

Development of a reaction cell for in-situ/operando studies of surface of a catalyst under a reaction condition and during catalysis.

PubMed

Nguyen, Luan; Tao, Franklin Feng

2016-06-01

Tracking surface chemistry of a catalyst during catalysis is significant for fundamental understanding of catalytic performance of the catalyst since it allows for establishing an intrinsic correlation between surface chemistry of a catalyst at its working status and its corresponding catalytic performance. Ambient pressure X-ray photoelectron spectroscopy can be used for in-situ studies of surfaces of different materials or devices in a gas. To simulate the gaseous environment of a catalyst in a fixed-bed a flowing gaseous environment of reactants around the catalyst is necessary. Here, we report the development of a new flowing reaction cell for simulating in-situ study of a catalyst surface under a reaction condition in gas of one reactant or during catalysis in a mixture of reactants of a catalytic reaction. The homemade reaction cell is installed in a high vacuum (HV) or ultrahigh vacuum (UHV) environment of a chamber. The flowing gas in the reaction cell is separated from the HV or UHV environment through well sealings at three interfaces between the reaction cell and X-ray window, sample door and aperture of front cone of an energy analyzer. Catalyst in the cell is heated through infrared laser beam introduced through a fiber optics interfaced with the reaction cell through a homemade feedthrough. The highly localized heating on the sample holder and Au-passivated internal surface of the reaction cell effectively minimizes any unwanted reactions potentially catalyzed by the reaction cell. The incorporated laser heating allows a fast heating and a high thermal stability of the sample at a high temperature. With this cell, a catalyst at 800 °C in a flowing gas can be tracked readily.

Studies of the viral binding proteins of shrimp BP53, a receptor of white spot syndrome virus.

PubMed

Li, Chen; Gao, Xiao-Xiao; Huang, Jie; Liang, Yan

2016-02-01

The specific binding between viral attachment proteins (VAPs) of a virus and its cellular receptors on host cells mediates virus entry into host cells, which triggers subsequent viral infections. Previous studies indicate that F1 ATP synthase β subunit (named BP53), is found on the surface of shrimp cells and involved in white spot syndrome virus (WSSV) infection by functioning as a potential viral receptor. Herein, in a far-western blotting assay, three WSSV proteins with molecular weights of 28 kDa, 37 kDa, and >50 kDa were found to interact with BP53. The 28 kDa and 37 kDa proteins were identified as the envelope protein VP28 and VP37 of WSSV respectively, which could be recognized by the polyclonal antibodies. Enzyme-linked immunosorbent binding assays revealed that VP37 contributed to almost 80% of the binding capability for BP53 compared with the same amount of total WSSV protein. The relationship between BP53 and its complementary interacting protein, VP37, was visualized using a co-localization assay. Bound VP37 on the cell surface co-localized with BP53 and shared a similar subcellular location on the outer surface of shrimp cells. Pearson's correlation coefficients reached to 0.67 ± 0.05 and the Mander's overlap coefficients reached 0.70 ± 0.05, which indicated a strong relationship between the localization of BP53 and bound rVP37. This provides evidence for an interaction between BP53 and VP37 obtained at the molecular and cellular levels, supporting the hypothesis that BP53 serves as a receptor for WSSV by binding to VP37. The identification of the viral binding proteins of shrimp BP53 is helpful for better understanding the pathogenic mechanisms of WSSV to infect shrimp at the cellular level. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

PubMed Central

Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

2002-01-01

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID:11772392

Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells.

PubMed

Boensch, C; Huang, S S; Connolly, D T; Huang, J S

1999-04-09

The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.

Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin

PubMed Central

Singh, Bhuminder; Bogatcheva, Galina; Starchenko, Alina; Sinnaeve, Justine; Lapierre, Lynne A.; Williams, Janice A.; Goldenring, James R.; Coffey, Robert J.

2015-01-01

ABSTRACT Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically 156EEMETL161). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization. PMID:26272915

T cell activation is determined by the number of presented antigens.

PubMed

Deeg, Janosch; Axmann, Markus; Matic, Jovana; Liapis, Anastasia; Depoil, David; Afrose, Jehan; Curado, Silvia; Dustin, Michael L; Spatz, Joachim P

2013-01-01

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90-140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per μm(2). We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density.

T Cell Activation is Determined by the Number of Presented Antigens

PubMed Central

2013-01-01

Antigen recognition is a key event during T cell activation. Here, we introduce nanopatterned antigen arrays that mimic the antigen presenting cell surface during T cell activation. The assessment of activation related events revealed the requirement of a minimal density of 90–140 stimulating major histocompatibility complex class II proteins (pMHC) molecules per μm2. We demonstrate that these substrates induce T cell responses in a pMHC dose-dependent manner and that the number of presented pMHCs dominates over local pMHC density. PMID:24117051

Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

PubMed Central

Owen, Caroline A.

2008-01-01

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

Surface Topography Hinders Bacterial Surface Motility.

PubMed

Chang, Yow-Ren; Weeks, Eric R; Ducker, William A

2018-03-21

We demonstrate that the surface motility of the bacterium, Pseudomonas aeruginosa, is hindered by a crystalline hemispherical topography with wavelength in the range of 2-8 μm. The motility was determined by the analysis of time-lapse microscopy images of cells in a flowing growth medium maintained at 37 °C. The net displacement of bacteria over 5 min is much lower on surfaces containing 2-8 μm hemispheres than on flat topography, but displacement on the 1 μm hemispheres is not lower. That is, there is a threshold between 1 and 2 μm for response to the topography. Cells on the 4 μm hemispheres were more likely to travel parallel to the local crystal axis than in other directions. Cells on the 8 μm topography were less likely to travel across the crowns of the hemispheres and were also more likely to make 30°-50° turns than on flat surfaces. These results show that surface topography can act as a significant barrier to surface motility and may therefore hinder surface exploration by bacteria. Because surface exploration can be a part of the process whereby bacteria form colonies and seek nutrients, these results help to elucidate the mechanism by which surface topography hinders biofilm formation.

Systemic localization of seven major types of carbohydrates on cell membranes by dSTORM imaging.

PubMed

Chen, Junling; Gao, Jing; Zhang, Min; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

2016-07-25

Carbohydrates on the cell surface control intercellular interactions and play a vital role in various physiological processes. However, their systemic distribution patterns are poorly understood. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we systematically revealed that several types of representative carbohydrates are found in clustered states. Interestingly, the results from dual-color dSTORM imaging indicate that these carbohydrate clusters are prone to connect with one another and eventually form conjoined platforms where different functional glycoproteins aggregate (e.g., epidermal growth factor receptor, (EGFR) and band 3 protein). A thorough understanding of the ensemble distribution of carbohydrates on the cell surface paves the way for elucidating the structure-function relationship of cell membranes and the critical roles of carbohydrates in various physiological and pathological cell processes.

Systemic localization of seven major types of carbohydrates on cell membranes by dSTORM imaging

PubMed Central

Chen, Junling; Gao, Jing; Zhang, Min; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tian, Zhiyuan; Wang, Hongda

2016-01-01

Carbohydrates on the cell surface control intercellular interactions and play a vital role in various physiological processes. However, their systemic distribution patterns are poorly understood. Through the direct stochastic optical reconstruction microscopy (dSTORM) strategy, we systematically revealed that several types of representative carbohydrates are found in clustered states. Interestingly, the results from dual-color dSTORM imaging indicate that these carbohydrate clusters are prone to connect with one another and eventually form conjoined platforms where different functional glycoproteins aggregate (e.g., epidermal growth factor receptor, (EGFR) and band 3 protein). A thorough understanding of the ensemble distribution of carbohydrates on the cell surface paves the way for elucidating the structure-function relationship of cell membranes and the critical roles of carbohydrates in various physiological and pathological cell processes. PMID:27453176

Remote excitation fluorescence correlation spectroscopy using silver nanowires

NASA Astrophysics Data System (ADS)

Su, Liang; Yuan, Haifeng; Lu, Gang; Hofkens, Johan; Roeffaers, Maarten; Uji-i, Hiroshi

2014-11-01

Fluorescence correlation spectroscopy (FCS), a powerful tool to resolve local properties, dynamical process of molecules, rotational and translational diffusion motions, relies on the fluctuations of florescence observables in the observation volume. In the case of rare transition events or small dynamical fluctuations, FCS requires few molecules or even single molecules in the observation volume at a time to minimize the background signals. Metal nanoparticle which possess unique localized surface plasmon resonance (LSPR) have been used to reduce the observation volume down to sub-diffraction limited scale while maintain at high analyst concentration up to tens of micromolar. Nevertheless, the applications of functionalized nanoparticles in living cell are limited due to the continuous diffusion after cell uptake, which makes it difficult to target the region of interests in the cell. In this work, we demonstrate the use of silver nanowires for remote excitation FCS on fluorescent molecules in solution. By using propagation surface plasmon polaritons (SPPs) which supported by the silver nanowire to excite the fluorescence, both illumination and observation volume can be reduced simultaneously. In such a way, less perturbation is induced to the target region, and this will broaden the application scope of silver nanowire as tip in single cell endoscopy.

Psoriasin, a novel anti-Candida albicans adhesin.

PubMed

Brauner, Annelie; Alvendal, Cathrin; Chromek, Milan; Stopsack, Konrad H; Ehrström, Sophia; Schröder, Jens M; Bohm-Starke, Nina

2018-05-07

Candida albicans belongs to the normal microbial flora on epithelial surfaces of humans. However, under certain, still not fully understood conditions, it can become pathogenic and cause a spectrum of diseases, from local infections to life-threatening septicemia. We investigated a panel of antimicrobial proteins and peptides (AMPs), potentially involved in mucosal immunity against this pathogen. Out of six studied AMPs, psoriasin was most up-regulated during a mucosal infection, an acute episode of recurrent Candida vulvovaginitis, although candidacidal activity has not been demonstrated. We here show that psoriasin binds to β-glucan, a basic component of the C. albicans cell wall, and thereby inhibits adhesion of the pathogen to surfaces and increases IL-8 production by mucosal epithelial cells. In conclusion, we show a novel mechanism of action of psoriasin. By inhibiting C. albicans adhesion and by enhancing cytokine production, psoriasin contributes to the immune response against C. albicans. The antimicrobial peptide psoriasin is highly up-regulated during a local mucosal infection, Candida albicans vulvovaginitis. Psoriasin binds to β-glucan in the Candida albicans cell wall and thereby inhibits adhesion of the pathogen. Binding of psoriasin to Candida albicans induces an immune response by mucosal epithelial cells.

Localization of lead accumulated by corn plants. [Zea mays L

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malone, C.; Koeppe, D.E.; Miller, R.J.

1974-01-01

Light and electron microscopic studies of corn plants (Zea mays L.) exposed to Pb in hydroponic solution showed that the roots generally accumulated a surface Pb precipitate and slowly accumulated Pb crystals in the cell walls. The root surface precipitate formed without the apparent influence of any cell organelles. In contrast, Pb taken up by roots was concentrated in dicytosome vesicles. Dicytosome vesicles containing cell wall material fused with one another to encase the Pb deposit. This encased deposit which was surrounded by a membrane migrated toward the outside of the cell where the membrane surrounding the deposit then fusedmore » with the plasmalemma. The material surrounding the deposit then fused with the cell wall. The result of this process was a concentration of Pb deposits in the cell wall outside the plasmalemma. Similar deposits were observed in stems and leaves suggesting that Pb was transported and deposited in a similar manner.« less

Localization of Lead Accumulated by Corn Plants 1

PubMed Central

Malone, Carl; Koeppe, D. E.; Miller, Raymond J.

1974-01-01

Light and electron microscopic studies of corn plants (Zea mays L.) exposed to Pb in hydroponic solution showed that the roots generally accumulated a surface Pb precipitate and slowly accumulated Pb crystals in the cell walls. The root surface precipitate formed without the apparent influence of any cell organelles. In contrast, Pb taken up by roots was concentrated in dictyosome vesicles. Dictyosome vesicles containing cell wall material fused with one another to encase the Pb deposit. This encased deposit which was surrounded by a membrane migrated toward the outside of the cell where the membrane surrounding the deposit fused with the plasmalemma. The material surrounding the deposit then fused with the cell wall. The result of this process was a concentration of Pb deposits in the cell wall outside the plasmalemma. Similar deposits were observed in stems and leaves suggesting that Pb was transported and deposited in a similar manner. Images PMID:16658711

An uptake of cationized ferritin by alveolar type I cells in airway-instilled goat lung: distribution of anionic sites on the epithelial surface.

PubMed

Atwal, O S; Viel, L; Minhas, K J

1990-07-01

The present study has investigated ultrastructural localization of anionic sites on the luminal surface of the alveolar epithelium of goat lung by direct airway instillation of cationized ferritin (CF) in the cranial lobe of the right lung through a bronchoscope. The cationic probe decorated preferentially the luminal plasmalemmal vesicles and plasmalemma proper of alveolar type I cell. This indicated the presence of highly charged anionic microdomains at these binding sites. The ligand was internalized in the free plasmalemmal vesicles of alveolar type I cell within 2 min. Heavy decoration of vesicles at 5 min of perfusion indicated that the amount of CF internalization increased with its concentration in the alveoli. It is suggested that exposure of alveolar surface to several gases of ruminal-origin induces changes in the surface charge of luminal plasmalemma of alveolar type I cells. The significance of these anionic plasmalemmal sites is discussed in relation to the adjustment of osmotic pressure gradient across the alveolar-capillary membrane of the ruminant lung.

Mechanosensation Dynamically Coordinates Polar Growth and Cell Wall Assembly to Promote Cell Survival.

PubMed

Davì, Valeria; Tanimoto, Hirokazu; Ershov, Dmitry; Haupt, Armin; De Belly, Henry; Le Borgne, Rémi; Couturier, Etienne; Boudaoud, Arezki; Minc, Nicolas

2018-04-23

How growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning and that thickness negatively influenced growth. Thickness at growing tips exhibited a fluctuating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the CW integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall, demonstrating its pivotal role for walled cell survival. Copyright © 2018 Elsevier Inc. All rights reserved.

The way we view cellular (glyco)sphingolipids.

PubMed

Hoetzl, Sandra; Sprong, Hein; van Meer, Gerrit

2007-11-01

Mammalian cells synthesize ceramide in the endoplasmic reticulum (ER) and convert this to sphingomyelin and complex glycosphingolipids on the inner, non-cytosolic surface of Golgi cisternae. From there, these lipids travel towards the outer, non-cytosolic surface of the plasma membrane and all membranes of the endocytic system, where they are eventually degraded. At the basis of the selective, anterograde traffic out of the Golgi lies the propensity of the sphingolipids to self-aggregate with cholesterol into microdomains termed 'lipid rafts'. At the plasma membrane surface these rafts are thought to function as the scaffold for various types of (glyco) signaling domains of different protein and lipid composition that can co-exist on one and the same cell. In the past decade, various unexpected findings on the sites where sphingolipid-mediated events occur have thrown a new light on the localization and transport mechanisms of sphingolipids. These findings are largely based on biochemical experiments. Further progress in the field is hampered by a lack of morphological techniques to localize lipids with nanometer resolution. In the present paper, we critically evaluate the published data and discuss techniques and potential improvements.

Tunable plasmon-enhanced broadband light harvesting for perovskite solar cells

NASA Astrophysics Data System (ADS)

Que, Meidan; Zhu, Liangliang; Yang, Yawei; Liu, Jie; Chen, Peng; Chen, Wei; Yin, Xingtian; Que, Wenxiu

2018-04-01

In this work, we report a reliable method for synthesizing (Au, Au/Ag core)/(TiO2 shell) nanostructures with their plasmonic wavelengths covering the visible light region for perovskite solar cells. The mono- and bi-metallic core-shell nanoparticles exhibit tunable localized surface plasmon resonance wavelength and function as "light tentacle" to improve the photo-electricity conversion efficiency. Plasmonic nanoparticles with different sizes and shapes, different thicknesses of TiO2 shell and Ag interlayer are found to have a strong influence on the localized surface plasmon resonance enhancement effect. The experimental photovoltaic performance of perovskite solar cells is significantly enhanced when the plasmonic nanoparticles are embedded inmesoporous TiO2 scaffolds. A champion photo-electricity conversion efficiency of 17.85% is achieved with nanoparticles (Au/Ag, λLSPR = 650 nm), giving a 18.7% enhancement over that of the pristine device (15.04%). Finite-difference time-domain simulations show that nanorod Au in mesoporus TiO2 scaffold induces the most intense electromagnetic coupling, and provides a novel emitter for photon flux in mesoporous perovskite solar cells. These theoretical results are consistent with the corresponding experimental those. Thus, enhancing the incident light intensities around 650 nm will be most favorable to the improvement of the photo-electricity conversion efficiency of perovskite solar cells.

Distribution of glucosinolates and sulphur-rich cells in roots of field-grown canola (Brassica napus).

PubMed

McCully, Margaret E; Miller, Celia; Sprague, Susan J; Huang, Cheng X; Kirkegaard, John A

2008-01-01

To investigate the role played by the distribution pattern of glucosinolates (GSLs) in root systems in the release of biocides to the rhizosphere, GSLs have been localized, for the first time, to specific regions and cells in field-grown roots. GSL concentrations in separated tissues of canola (Brassica napus) were determined by chemical analysis, and cell-specific concentrations by extrapolation from sulphur concentrations obtained by quantitative cryo-analytical scanning electron microscopy (SEM). In roots with secondary growth, GSL concentrations in the outer secondary tissues were up to 5x those of the inner core. The highest GSL concentrations (from sulphur measurements) were in two cell layers just under the outermost periderm layer, with up to 100x published concentrations for whole roots. Primary tissues had negligible GSL. Release and renewal of the peripheral GSLs is probably a normal developmental process as secondary thickening continues and surface cells senesce, accounting for published observations that intact roots release GSLs and their biocide hydrolosates to the rhizosphere. Absence of myrosin idioblasts close to the root surface suggests that GSLs released developmentally are hydrolysed by myrosinase in the rhizosphere, ensuring a continuous localized source of biotoxic hydrolysates which can deter soil-borne pests, and influence microbial populations associated with long-lived components of the root system.

An Iron-Regulated Autolysin Remodels the Cell Wall To Facilitate Heme Acquisition in Staphylococcus lugdunensis

PubMed Central

Farrand, Allison J.; Haley, Kathryn P.; Lareau, Nichole M.; Heilbronner, Simon; McLean, John A.; Foster, Timothy

2015-01-01

Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection. PMID:26123800

Cell Surface Trafficking of TLR1 Is Differentially Regulated by the Chaperones PRAT4A and PRAT4B*

PubMed Central

Hart, Bryan E.; Tapping, Richard I.

2012-01-01

The subcellular localization of Toll-like receptors (TLRs) is critical to their ability to function as innate immune sensors of microbial infection. We previously reported that an I602S polymorphism of human TLR1 is associated with aberrant trafficking of the receptor to the cell surface, loss of responses to TLR1 agonists, and differential susceptibility to diseases caused by pathogenic mycobacteria. Through an extensive analysis of receptor deletion and point mutants we have discovered that position 602 resides within a short 6 amino acid cytoplasmic region that is required for TLR1 surface expression. This short trafficking motif, in conjunction with the adjacent transmembrane domain, is sufficient to direct TLR1 to the cell surface. A serine at position 602 interrupts this trafficking motif and prevents cell surface expression of TLR1. Additionally, we have found that ER-resident TLR chaperones, PRAT4A and PRAT4B, act as positive and negative regulators of TLR1 surface trafficking, respectively. Importantly, either over-expression of PRAT4A or knock-down of PRAT4B rescues cell surface expression of the TLR1 602S variant. We also report that IFN-γ treatment of primary human monocytes derived from homozygous 602S individuals rescues TLR1 cell surface trafficking and cellular responses to soluble agonists. This event appears to be mediated by PRAT4A whose expression is strongly induced in human monocytes by IFN-γ. Collectively, these results provide a mechanism for the differential trafficking of TLR1 I602S variants, and highlight the distinct roles for PRAT4A and PRAT4B in the regulation of TLR1 surface expression. PMID:22447933

Transposon tagging of genes for cell-cell interactions in Myxococcus xanthus.

PubMed Central

Kalos, M; Zissler, J

1990-01-01

The prokaryote Myxococcus xanthus is a model for cell interactions important in multicellular behavior. We used the transposon TnphoA to specifically identify genes for cell-surface factors involved in cell interactions. From a library of 10,700 insertions of TnphoA, we isolated 36 that produced alkaline phosphatase activity. Three TnphoA insertions tagged cell motility genes, called cgl, which control the adventurous movement of cells. The products of the tagged cgl genes could function in trans upon other cells and were localized primarily in the cell envelope and extracellular space, consistent with TnphoA tagging genes for extracellular factors controlling motility. Images PMID:2172982

The effects of non-Newtonian viscosity on the deformation of red blood cells in a shear flow

NASA Astrophysics Data System (ADS)

Sesay, Juldeh

2005-11-01

The analyses of the effects of non-Newtonian viscosity on the membrane of red blood cells (RBCs) suspended in a shear flow are presented. The specific objective is to investigate the mechanical deformation on the surfaces of an ellipsoidal particle model. The hydrodynamic stresses and other forces on the surface of the particle are used to determine the cell deformation. We extended previous works, which were based on the Newtonian fluid models, to the non-Newtonian case, and focus on imposed shear rate values between 1 and 100 per second. Two viscosity models are investigated, which respectively correspond to a normal person and a patient with cerebrovascular accident (CVA). The results are compared with those obtained assuming a Newtonian model. We observed that the orientation of the cell influences the deformation and the imposed shear rate drives the local shear rate distribution along the particle surface. The integral particle deformation for the non-Newtonian models in the given shear rate regime is higher than that for the Newtonian reference model. Finally, the deformation of the cell surface decreases as the dissipation ratio increases.

Enterococcus faecium Biofilm Formation: Identification of Major Autolysin AtlAEfm, Associated Acm Surface Localization, and AtlAEfm-Independent Extracellular DNA Release

PubMed Central

Paganelli, Fernanda L.; Willems, Rob J. L.; Jansen, Pamela; Hendrickx, Antoni; Zhang, Xinglin; Bonten, Marc J. M.; Leavis, Helen L.

2013-01-01

ABSTRACT Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faecium E1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162_2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162_2692 was considered the major autolysin in E. faecium and renamed atlAEfm. In addition, AtlAEfm was implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. PMID:23592262

Regional expression and ultrastructural localization of EphA7 in the hippocampus and cerebellum of adult rat.

PubMed

Amegandjin, Clara A; Jammow, Wafaa; Laforest, Sylvie; Riad, Mustapha; Baharnoori, Moogeh; Badeaux, Frédérique; DesGroseillers, Luc; Murai, Keith K; Pasquale, Elena B; Drolet, Guy; Doucet, Guy

2016-08-15

EphA7 is expressed in the adult central nervous system (CNS), where its roles are yet poorly defined. We mapped its distribution using in situ hybridization (ISH) and immunohistochemistry (IHC) combined with light (LM) and electron microscopy (EM) in adult rat and mouse brain. The strongest ISH signal was in the hippocampal pyramidal and granule cell layers. Moderate levels were detected in habenula, striatum, amygdala, the cingulate, piriform and entorhinal cortex, and in cerebellum, notably the Purkinje cell layer. The IHC signal distribution was consistent with ISH results, with transport of the protein to processes, as exemplified in the hippocampal neuropil layers and weakly stained pyramidal cell layers. In contrast, in the cerebellum, the Purkinje cell bodies were the most strongly immunolabeled elements. EM localized the cell surface-expression of EphA7 essentially in postsynaptic densities (PSDs) of dendritic spines and shafts, and on some astrocytic leaflets, in both hippocampus and cerebellum. Perikaryal and dendritic labeling was mostly intracellular, associated with the synthetic and trafficking machineries. Immunopositive vesicles were also observed in axons and axon terminals. Quantitative analysis in EM showed significant differences in the frequency of labeled elements between regions. Notably, labeled dendrites were ∼3-5 times less frequent in cerebellum than in hippocampus, but they were individually endowed with ∼10-40 times higher frequencies of PSDs, on their shafts and spines. The cell surface localization of EphA7, being preferentially in PSDs, and in perisynaptic astrocytic leaflets, provides morphologic evidence that EphA7 plays key roles in adult CNS synaptic maintenance, plasticity, or function. J. Comp. Neurol. 524:2462-2478, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Rescue of the colony-stimulating factor 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis.

PubMed

Ryan, G R; Dai, X M; Dominguez, M G; Tong, W; Chuan, F; Chisholm, O; Russell, R G; Pollard, J W; Stanley, E R

2001-07-01

Colony-stimulating factor 1 (CSF-1) regulates the survival, proliferation, and differentiation of mononuclear phagocytes. It is expressed as a secreted glycoprotein or proteoglycan found in the circulation or as a biologically active cell-surface glycoprotein. To investigate tissue CSF-1 regulation, CSF-1-null Csf1(op)/Csf1(op) mice expressing transgenes encoding the full-length membrane-spanning CSF-1 precursor driven by 3.13 kilobases of the mouse CSF-1 promoter and first intron were characterized. Transgene expression corrected the gross osteopetrotic, neurologic, weight, tooth, and reproductive defects of Csf1(op)/Csf1(op) mice. Detailed analysis of one transgenic line revealed that circulating CSF-1, tissue macrophage numbers, hematopoietic tissue cellularity, and hematopoietic parameters were normalized. Tissue CSF-1 levels were normal except for elevations in 4 secretory tissues. Skin fibroblasts from the transgenic mice secreted normal amounts of CSF-1 but also expressed some cell-surface CSF-1. Also, lacZ driven by the same promoter/first intron revealed beta-galactosidase expression in hematopoietic, reproductive, and other tissue locations proximal to CSF-1 cellular targets, consistent with local regulation by CSF-1 at these sites. These studies indicate that the 3.13-kilobase promoter/first intron confers essentially normal CSF-1 expression. They also pinpoint new cellular sites of CSF-1 expression, including ovarian granulosa cells, mammary ductal epithelium, testicular Leydig cells, serous acinar cells of salivary gland, Paneth cells of the small intestine, as well as local sites in several other tissues.

Emergence of complex behavior in pili-based motility in early stages of P. aeruginosa surface adaptation

NASA Astrophysics Data System (ADS)

Brill-Karniely, Yifat; Jin, Fan; Wong, Gerard C. L.; Frenkel, Daan; Dobnikar, Jure

2017-04-01

Pseudomonas aeruginosa move across surfaces by using multiple Type IV Pili (TFP), motorized appendages capable of force generation via linear extension/retraction cycles, to generate surface motions collectively known as twitching motility. Pseudomonas cells arrive at a surface with low levels of piliation and TFP activity, which both progressively increase as the cells sense the presence of a surface. At present, it is not clear how twitching motility emerges from these initial minimal conditions. Here, we build a simple model for TFP-driven surface motility without complications from viscous and solid friction on surfaces. We discover the unanticipated structural requirement that TFP motors need to have a minimal amount of effective angular rigidity in order for cells to perform the various classes of experimentally-observed motions. Moreover, a surprisingly small number of TFP are needed to recapitulate movement signatures associated with twitching: Two TFP can already produce movements reminiscent of recently observed slingshot type motion. Interestingly, jerky slingshot motions characteristic of twitching motility comprise the transition region between different types of observed crawling behavior in the dynamical phase diagram, such as self-trapped localized motion, 2-D diffusive exploration, and super-diffusive persistent motion.

Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycans Are Targeted by Bleomycin in Cancer Cells.

PubMed

Li, Xiulian; Lan, Ying; He, Yanli; Liu, Yong; Luo, Heng; Yu, Haibo; Song, Ni; Ren, Sumei; Liu, Tianwei; Hao, Cui; Guo, Yunliang; Zhang, Lijuan

2017-01-01

Bleomycin is a clinically used anti-cancer drug that produces DNA breaks once inside of cells. However, bleomycin is a positively charged molecule and cannot get inside of cells by free diffusion. We previously reported that the cell surface negatively charged glycosaminoglycans (GAGs) may be involved in the cellular uptake of bleomycin. We also observed that a class of positively charged small molecules has Golgi localization once inside of the cells. We therefore hypothesized that bleomycin might perturb Golgi-operated GAG biosynthesis. We used stable isotope labeling coupled with LC/MS analysis of GAG disaccharides simultaneously from bleomycin-treated and non-treated cancer cells. To further understand the cytotoxicity of bleomycin and its relationship to GAGs, we used sodium chlorate to inhibit GAG sulfation and commercially available GAGs to compete for cell surface GAG/bleomycin interactions in seven cell lines including CHO745 defective in both heparan sulfate and chondroitin sulfate biosynthesis. we discovered that heparan sulfate GAG was significantly undersulfated and the quantity and disaccharide compositions of GAGs were changed in bleomycin-treated cells in a concentration- and time-dependent manner. We revealed that bleomycin-induced cytotoxicity was directly related to cell surface GAGs. GAGs were targeted by bleomycin both at cell surface and at Golgi. Thus, GAGs might be the biological relevant molecules that might be related to the bleomycin-induced fibrosis in certain cancer patients, a severe side effect with largely unknown molecular mechanism. © 2017 The Author(s). Published by S. Karger AG, Basel.

Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Dustin C.; Ceresa, Brian P.

2008-11-01

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less

Upgraded metallurgical-grade silicon solar cells with efficiency above 20%

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, P.; Rougieux, F. E.; Samundsett, C.

We present solar cells fabricated with n-type Czochralski–silicon wafers grown with strongly compensated 100% upgraded metallurgical-grade feedstock, with efficiencies above 20%. The cells have a passivated boron-diffused front surface, and a rear locally phosphorus-diffused structure fabricated using an etch-back process. The local heavy phosphorus diffusion on the rear helps to maintain a high bulk lifetime in the substrates via phosphorus gettering, whilst also reducing recombination under the rear-side metal contacts. The independently measured results yield a peak efficiency of 20.9% for the best upgraded metallurgical-grade silicon cell and 21.9% for a control device made with electronic-grade float-zone silicon. The presencemore » of boron-oxygen related defects in the cells is also investigated, and we confirm that these defects can be partially deactivated permanently by annealing under illumination.« less

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

PubMed

Yamamoto, A M; Mura, C; De Lemos-Chiarandini, C; Krishnamoorthy, R; Alvarez, F

1993-06-01

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

PubMed Central

Yamamoto, A M; Mura, C; De Lemos-Chiarandini, C; Krishnamoorthy, R; Alvarez, F

1993-01-01

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:7685669

Membrane-Surface Anchoring of Charged Diacylglycerol-Lactones Correlates with Biological Activities | Center for Cancer Research

Cancer.gov

The inside cover picture shows the molecular structure of a DAG lactone derivative on top of the inner leaflet of a DMPC bilayer. The confocal microscopy image illustrates DAG-lactone-stimulated membrane localization of PKCδ-ECFP in living cells, while the space-filling model shows the surface of the C1B domain of PKCδ, the target of the lactone.

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

PubMed Central

Teissié, J; Ramos, C

1998-01-01

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion. PMID:9545050

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

PubMed

Teissié, J; Ramos, C

1998-04-01

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion.

Atomic force microscopy study of the structure function relationships of the biofilm-forming bacterium Streptococcus mutans

NASA Astrophysics Data System (ADS)

Cross, Sarah E.; Kreth, Jens; Zhu, Lin; Qi, Fengxia; Pelling, Andrew E.; Shi, Wenyuan; Gimzewski, James K.

2006-02-01

Atomic force microscopy (AFM) has garnered much interest in recent years for its ability to probe the structure, function and cellular nanomechanics inherent to specific biological cells. In particular, we have used AFM to probe the important structure-function relationships of the bacterium Streptococcus mutans. S. mutans is the primary aetiological agent in human dental caries (tooth decay), and is of medical importance due to the virulence properties of these cells in biofilm initiation and formation, leading to increased tolerance to antibiotics. We have used AFM to characterize the unique surface structures of distinct mutants of S. mutans. These mutations are located in specific genes that encode surface proteins, thus using AFM we have resolved characteristic surface features for mutant strains compared to the wild type. Ultimately, our characterization of surface morphology has shown distinct differences in the local properties displayed by various S. mutans strains on the nanoscale, which is imperative for understanding the collective properties of these cells in biofilm formation.

Rbt1 protein domains analysis in Candida albicans brings insights into hyphal surface modifications and Rbt1 potential role during adhesion and biofilm formation.

PubMed

Monniot, Céline; Boisramé, Anita; Da Costa, Grégory; Chauvel, Muriel; Sautour, Marc; Bougnoux, Marie-Elisabeth; Bellon-Fontaine, Marie-Noëlle; Dalle, Frédéric; d'Enfert, Christophe; Richard, Mathias L

2013-01-01

Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high β-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.

Convection-Diffusion Layer in an "Open Space" for Local Surface Treatment and Microfabrication using a Four-Aperture Microchemical Pen.

PubMed

Mao, Sifeng; Zhang, Yong; Zhang, Weifei; Zeng, Hulie; Nakajima, Hizuru; Lin, Jin-Ming; Uchiyama, Katsumi

2017-09-06

A four-aperture microchemical pen was used to produce a stable convection-diffusion layer in an "open space" for microreactions and microfabrication. The process represents a new method for microreactions and microfabrication in a convection-diffusion layer. To prove the concept of a convection-diffusion layer in an "open space", bovine serum albumin was labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole to confirm that the small convection-diffusion layer was effective for local surface treatment. To demonstrate the potential for microfabrication, silver patterns were fabricated on a glass surface with a convection-diffusion layer by using the silver-mirror reaction. The widths of each silver pattern could be easily controlled from 10 to 60 μm. Patterned silver lines with uniform widths or gradient widths were prepared. This is the first proof of concept study of a convection-diffusion layer in an "open space" used in local surface treatment and microfabrication on a surface. The microchemical pen represents a potential method for the region-selective microtreatment of tissues, cells, and other biological interfaces. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

L-asparaginase inhibits invasive and angiogenic activity and induces autophagy in ovarian cancer

PubMed Central

Yu, Minshu; Henning, Ryan; Walker, Amanda; Kim, Geoffrey; Perroy, Alyssa; Alessandro, Riccardo; Virador, Victoria; Kohn, Elise C

2012-01-01

Recent work identified L-asparaginase (L-ASP) as a putative therapeutic target for ovarian cancer. We suggest that L-ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L-ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion and the assessment of sialylated proteins involved in matrix-associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell-cell and cell-matrix adhesion was observed (P < 0.05–0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl LewisX (sLex) expression. No reduction in HMVEC E-selectin expression was seen consistent with the unidirectional inhibitory actions observed. L-ASP concentrations were non-toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin-1, and cleavage of LC-3, indicating cell injury did occur. These data and the known mechanism of action of L-ASP on glycosylation of nascent proteins suggest that L-ASP reduces of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLex inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behaviour, and causes cell injury initiating autophagy in tumour and vascular cells. PMID:22333033

B cell and T cell immunity in the female genital tract: potential of distinct mucosal routes of vaccination and role of tissue-associated dendritic cells and natural killer cells.

PubMed

Anjuère, F; Bekri, S; Bihl, F; Braud, V M; Cuburu, N; Czerkinsky, C; Hervouet, C; Luci, C

2012-10-01

The female genital mucosa constitutes the major port of entry of sexually transmitted infections. Most genital microbial pathogens represent an enormous challenge for developing vaccines that can induce genital immunity that will prevent their transmission. It is now established that long-lasting protective immunity at mucosal surfaces has to involve local B-cell and T-cell effectors as well as local memory cells. Mucosal immunization constitutes an attractive way to generate systemic and genital B-cell and T-cell immune responses that can control early infection by sexually transmitted pathogens. Nevertheless, no mucosal vaccines against sexually transmitted infections are approved for human use. The mucosa-associated immune system is highly compartmentalized and the selection of any particular route or combinations of routes of immunization is critical when defining vaccine strategies against genital infections. Furthermore, mucosal surfaces are complex immunocompetent tissues that comprise antigen-presenting cells and also innate immune effectors and non-immune cells that can act as 'natural adjuvants' or negative immune modulators. The functions of these cells have to be taken into account when designing tissue-specific antigen-delivery systems and adjuvants. Here, we will discuss data that compare different mucosal routes of immunization to generate B-cell and T-cell responses in the genital tract, with a special emphasis on the newly described sublingual route of immunization. We will also summarize data on the understanding of the effector and induction mechanisms of genital immunity that may influence the development of vaccine strategies against genital infections. © 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.

Nanoparticle uptake and their co-localization with cell compartments - a confocal Raman microscopy study at single cell level

NASA Astrophysics Data System (ADS)

Estrela-Lopis, I.; Romero, G.; Rojas, E.; Moya, S. E.; Donath, E.

2011-07-01

Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.

The Role of the Omental Microenvironment in Ovarian Cancer Metastatic Colonization

DTIC Science & Technology

2010-08-01

Cancer cells which have adhered to the surface of the omentum stain positively for this proliferative marker , confirming their viability under our ex...6 mice (Figure 1). Due to technical difficulties, we had to change some of the proposed immune cell markers for the FACS analysis. We are currently...for F4/80 (macrophage cell marker ) was even more dramatic. Representative data from the macrophage localization in milky spots after i.p injection of

Proliferative vitreoretinopathy in the Swine-a new model.

PubMed

Umazume, Kazuhiko; Barak, Yoreh; McDonald, Kevin; Liu, Lanhsin; Kaplan, Henry J; Tamiya, Shigeo

2012-07-24

To develop a large animal model of proliferative vitreoretinopathy (PVR) in the swine to eventually study disease pathophysiology, as well as novel therapies. PVR was induced in domestic swine by creation of a posterior vitreous detachment, creation of a retinal detachment by the injection of subretinal fluid, and intravitreal injection of green fluorescent protein-positive retinal pigment epithelial (GFP+ RPE) cells. Control eyes had the same surgical procedures without RPE cell injection. PVR was clinically graded on days 3, 7, and 14. Animals were euthanized on day 14, and enucleated eyes were analyzed by light microscopy and immunohistochemistry. Injection of GFP+ RPE cells into the vitreous cavity produced localized, traction retinal detachments by day 14 in all eyes (14 of 14); in contrast, the retina spontaneously reattached by day 3 and remained attached in all control eyes (10 of 10). Contractile epiretinal membranes on the inner retinal surface that caused the traction retinal detachments consisted predominantly of GFP+ RPE cells. These cells stained positive for cytokeratin, confirming their epithelial origin, and also expressed α-SMA and fibronectin, markers for myofibroblasts and fibrosis, respectively. We established a swine PVR model that recapitulates key clinical features found in humans and, thus, can be used to study the pathophysiology of PVR, as well as new novel therapies. GFP+ RPE cells injected into the vitreous cavity formed contractile membranes on the inner retinal surface and caused localized traction retinal detachments.

Partitioning of Electromagnetic Energy Inputs to the Thermosphere during Geomagnetic Disturbances

DTIC Science & Technology

2012-06-01

boundary of a local flux tube volume is an equipotential . Figure 4 contains maps of Poynting flux normal to a 500 km altitude surface and maps of height...as a cell quantity throughout its computational volume, we are able to generate maps of the Poynting flux, ⃗ ⃗⃗⃗⃗⃗⃗ , on altitude surfaces at...the top of the thermosphere. We used separate modules to integrate the Poynting flux over this surface to compute the total electromagnetic energy

Employing Si solar cell technology to increase efficiency of ultra-thin Cu(In,Ga)Se2 solar cells.

PubMed

Vermang, Bart; Wätjen, Jörn Timo; Fjällström, Viktor; Rostvall, Fredrik; Edoff, Marika; Kotipalli, Ratan; Henry, Frederic; Flandre, Denis

2014-10-01

Reducing absorber layer thickness below 500 nm in regular Cu(In,Ga)Se 2 (CIGS) solar cells decreases cell efficiency considerably, as both short-circuit current and open-circuit voltage are reduced because of incomplete absorption and high Mo/CIGS rear interface recombination. In this work, an innovative rear cell design is developed to avoid both effects: a highly reflective rear surface passivation layer with nano-sized local point contact openings is employed to enhance rear internal reflection and decrease the rear surface recombination velocity significantly, as compared with a standard Mo/CIGS rear interface. The formation of nano-sphere shaped precipitates in chemical bath deposition of CdS is used to generate nano-sized point contact openings. Evaporation of MgF 2 coated with a thin atomic layer deposited Al 2 O 3 layer, or direct current magnetron sputtering of Al 2 O 3 are used as rear surface passivation layers. Rear internal reflection is enhanced substantially by the increased thickness of the passivation layer, and also the rear surface recombination velocity is reduced at the Al 2 O 3 /CIGS rear interface. (MgF 2 /)Al 2 O 3 rear surface passivated ultra-thin CIGS solar cells are fabricated, showing an increase in short circuit current and open circuit voltage compared to unpassivated reference cells with equivalent CIGS thickness. Accordingly, average solar cell efficiencies of 13.5% are realized for 385 nm thick CIGS absorber layers, compared with 9.1% efficiency for the corresponding unpassivated reference cells.

Employing Si solar cell technology to increase efficiency of ultra-thin Cu(In,Ga)Se2 solar cells

PubMed Central

Vermang, Bart; Wätjen, Jörn Timo; Fjällström, Viktor; Rostvall, Fredrik; Edoff, Marika; Kotipalli, Ratan; Henry, Frederic; Flandre, Denis

2014-01-01

Reducing absorber layer thickness below 500 nm in regular Cu(In,Ga)Se2 (CIGS) solar cells decreases cell efficiency considerably, as both short-circuit current and open-circuit voltage are reduced because of incomplete absorption and high Mo/CIGS rear interface recombination. In this work, an innovative rear cell design is developed to avoid both effects: a highly reflective rear surface passivation layer with nano-sized local point contact openings is employed to enhance rear internal reflection and decrease the rear surface recombination velocity significantly, as compared with a standard Mo/CIGS rear interface. The formation of nano-sphere shaped precipitates in chemical bath deposition of CdS is used to generate nano-sized point contact openings. Evaporation of MgF2 coated with a thin atomic layer deposited Al2O3 layer, or direct current magnetron sputtering of Al2O3 are used as rear surface passivation layers. Rear internal reflection is enhanced substantially by the increased thickness of the passivation layer, and also the rear surface recombination velocity is reduced at the Al2O3/CIGS rear interface. (MgF2/)Al2O3 rear surface passivated ultra-thin CIGS solar cells are fabricated, showing an increase in short circuit current and open circuit voltage compared to unpassivated reference cells with equivalent CIGS thickness. Accordingly, average solar cell efficiencies of 13.5% are realized for 385 nm thick CIGS absorber layers, compared with 9.1% efficiency for the corresponding unpassivated reference cells. PMID:26300619

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation. PMID:22147732

Myofibroblast distribution in Dupuytren's cords: correlation with digital contracture.

PubMed

Verjee, Liaquat Suleman; Midwood, Kim; Davidson, Dominique; Essex, David; Sandison, Ann; Nanchahal, Jagdeep

2009-12-01

Dupuytren's tissue has typically been described as being composed of myofibroblast-rich palmar nodules and relatively acellular tendon-like cords. We aimed to determine myofibroblast distribution (alpha-smooth muscle actin [alpha-SMA] positive cells) within Dupuytren's tissue and to correlate histologically defined alpha-SMA-positive nodules with digital contracture and recurrent disease. One hundred and three digital Dupuytren's cords (72 fasciectomy, 31 dermofasciectomy) were stained with anti-alpha-SMA antibody. The presence of alpha-SMA-positive nodules, their surface area, and alpha-SMA-positive cells were quantified throughout excised Dupuytren's tissue. Clinical data on diathesis, flexion deformity, and previous surgeries were collected. Cords were nodular (66%) or non-nodular (34%). Nodular cords contained 1 (55%), 2 (33%), or 3 or more nodules (12%) composed of localized collections of cells. The mean total nodule surface area was 23 mm(2) (range, 1.3-105 mm(2)). Nodules contained the highest number of alpha-SMA-positive cells (mean 97%, 2374 cells/mm(2)) compared to peri-nodular areas (mean 32%, 763 cells/mm(2)), and more distant cord (mean 8%, 495 cells/mm(2)). Non-nodular cords contained 9% to 17% alpha-SMA-positive cells (mean 475-663 cells/mm(2)), with higher numbers distally. There was greater digital contracture in patients with non-nodular cords. Thirty-six of 38 proximal interphalangeal (PIP) joint-marked samples had a nodule that co-localized with the PIP joint. Nodule size did not correlate with flexion deformity or with primary or recurrent disease. We found that two thirds of digital cords were nodular. Nodules were hypercellular, the majority being alpha-SMA-positive cells. Nodules varied in size and co-localized with the PIP joint. Cord was relatively cellular throughout; a proportion of these cells were alpha-SMA-positive and cells aligned with collagen fibers. Non-nodular cords correlated with significantly greater digital flexion contracture. We propose that cells in nodular cords contract and deposit extracellular matrix components. The matrix is then remodeled in shortened configuration, and as fixed flexion deformity develops, stress shielding eventually leads to myofibroblast apoptosis, and cord becomes less cellular.

Impact of a compound droplet on a flat surface: A model for single cell epitaxy.

PubMed

Tasoglu, Savas; Kaynak, Gozde; Szeri, Andrew J; Demirci, Utkan; Muradoglu, Metin

2010-08-01

The impact and spreading of a compound viscous droplet on a flat surface are studied computationally using a front-tracking method as a model for the single cell epitaxy. This is a technology developed to create two-dimensional and three-dimensional tissue constructs cell by cell by printing cell-encapsulating droplets precisely on a substrate using an existing ink-jet printing method. The success of cell printing mainly depends on the cell viability during the printing process, which requires a deeper understanding of the impact dynamics of encapsulated cells onto a solid surface. The present study is a first step in developing a model for deposition of cell-encapsulating droplets. The inner droplet representing the cell, the encapsulating droplet, and the ambient fluid are all assumed to be Newtonian. Simulations are performed for a range of dimensionless parameters to probe the deformation and rate of deformation of the encapsulated cell, which are both hypothesized to be related to cell damage. The deformation of the inner droplet consistently increases: as the Reynolds number increases; as the diameter ratio of the encapsulating droplet to the cell decreases; as the ratio of surface tensions of the air-solution interface to the solution-cell interface increases; as the viscosity ratio of the cell to encapsulating droplet decreases; or as the equilibrium contact angle decreases. It is observed that maximum deformation for a range of Weber numbers has (at least) one local minimum at We=2. Thereafter, the effects of cell deformation on viability are estimated by employing a correlation based on the experimental data of compression of cells between parallel plates. These results provide insight into achieving optimal parameter ranges for maximal cell viability during cell printing.

Surface plasmon effects in the absorption enhancements of amorphous silicon solar cells with periodical metal nanowall and nanopillar structures.

PubMed

Lin, Hung-Yu; Kuo, Yang; Liao, Cheng-Yuan; Yang, C C; Kiang, Yean-Woei

2012-01-02

The authors numerically investigate the absorption enhancement of an amorphous Si solar cell, in which a periodical one-dimensional nanowall or two-dimensional nanopillar structure of the Ag back-reflector is fabricated such that a dome-shaped grating geometry is formed after Si deposition and indium-tin-oxide coating. In this investigation, the effects of surface plasmon (SP) interaction in such a metal nanostructure are of major concern. Absorption enhancement in most of the solar spectral range of significant amorphous Si absorption (320-800 nm) is observed in a grating solar cell. In the short-wavelength range of high amorphous Si absorption, the weakly wavelength-dependent absorption enhancement is mainly caused by the broadband anti-reflection effect, which is produced through the surface nano-grating structures. In the long-wavelength range of diminishing amorphous Si absorption, the highly wavelength-sensitive absorption enhancement is mainly caused by Fabry-Perot resonance and SP interaction. The SP interaction includes the contributions of surface plasmon polariton and localized surface plasmon.

Electromagnetically induced transparency metamaterial based on spoof localized surface plasmons at terahertz frequencies

PubMed Central

Liao, Zhen; Liu, Shuo; Ma, Hui Feng; Li, Chun; Jin, Biaobing; Cui, Tie Jun

2016-01-01

We numerically and experimentally demonstrate a plasmonic metamaterial whose unit cell is composed of an ultrathin metallic disk and four ultrathin metallic spiral arms at terahertz frequencies, which supports both spoof electric and magnetic localized surface plasmon (LSP) resonances. We show that the resonant wavelength is much larger than the size of the unit particle, and further find that the resonant wavelength is very sensitive to the particle’s geometrical dimensions and arrangements. It is clearly illustrated that the magnetic LSP resonance exhibits strong dependence to the incidence angle of terahertz wave, which enables the design of metamaterials to achieve an electromagnetically induced transparency effect in the terahertz frequencies. This work opens up the possibility to apply for the surface plasmons in functional devices in the terahertz band. PMID:27277417

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy

NASA Astrophysics Data System (ADS)

Arnal, L.; Longo, G.; Stupar, P.; Castez, M. F.; Cattelan, N.; Salvarezza, R. C.; Yantorno, O. M.; Kasas, S.; Vela, M. E.

2015-10-01

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr04644k

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noh, Hanaul; Diaz, Alfredo J.; Solares, Santiago D.

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, andmore » is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules.« less

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

DOE PAGES

Noh, Hanaul; Diaz, Alfredo J.; Solares, Santiago D.

2017-03-08

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, andmore » is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules.« less

A hexahistidine-Zn2+-dye label reveals STIM1 surface exposure

PubMed Central

Hauser, Christina T.; Tsien, Roger Y.

2007-01-01

Site-specific fluorescent labeling of proteins in vivo remains one of the most powerful techniques for imaging complex processes in live cells. Although fluorescent proteins in many colors are useful tools for tracking expression and localization of fusion proteins in cells, these relatively large tags (>220 aa) can perturb protein folding, trafficking and function. Much smaller genetically encodable domains (<15 aa) offer complementary advantages. We introduce a small fluorescent chelator whose membrane-impermeant complex with nontoxic Zn2+ ions binds tightly but reversibly to hexahistidine (His6) motifs on surface-exposed proteins. This live-cell label helps to resolve a current controversy concerning externalization of the stromal interaction molecule STIM1 upon depletion of Ca2+ from the endoplasmic reticulum. Whereas N-terminal fluorescent protein fusions interfere with surface exposure of STIM1, short His6 tags are accessible to the dye or antibodies, demonstrating externalization. PMID:17360414

Analysis and modification of defective surface aggregates on PCDTBT:PCBM solar cell blends using combined Kelvin probe, conductive and bimodal atomic force microscopy

PubMed Central

Noh, Hanaul; Diaz, Alfredo J

2017-01-01

Organic photovoltaic systems comprising donor polymers and acceptor fullerene derivatives are attractive for inexpensive energy harvesting. Extensive research on polymer solar cells has provided insight into the factors governing device-level efficiency and stability. However, the detailed investigation of nanoscale structures is still challenging. Here we demonstrate the analysis and modification of unidentified surface aggregates. The aggregates are characterized electrically by Kelvin probe force microscopy and conductive atomic force microscopy (C-AFM), whereby the correlation between local electrical potential and current confirms a defective charge transport. Bimodal AFM modification confirms that the aggregates exist on top of the solar cell structure, and is used to remove them and to reveal the underlying active layer. The systematic analysis of the surface aggregates suggests that the structure consists of PCBM molecules. PMID:28382247

Electromagnetic properties of metal-dielectric media and their applications

NASA Astrophysics Data System (ADS)

Animilli, Shravan Rakesh

The main objective of this dissertation is to investigate nano-structured random composite materials, which exhibit anomalous phenomena, such as the extraordinary enhancements of linear and non-linear optical processes due to excitation of collective electronic states, surface plasmons (SP). The main goal is to develop a time and memory efficient novel numerical method to study the properties of these random media in three dimensions (3D) by utilization of multi core processing and packages such as MPI for parallel execution. The developed numerical studies are then utilized to provide a comprehensive characterization and optimization of a surface plasmon enhanced solar cell (SPESC) and to serve as a test bed for enhanced bio and chemical sensing. In this context, this thesis work develops an efficient and exact numerical algorithm here referred to as Block Elimination Method (BE) which provides the unique capability of modeling extremely large scale composite materials (with up to 1 million strongly interacting metal or dielectric particles). This capability is crucial in order to study the electromagnetic response of large scale inhomogeneous (fractal) films and bulk composites at critical concentrations (percolation). The developed numerical method is used to accurately estimate parameters that describe the composite materials, including the effective conductivity and correlation length scaling exponents, as well as density of states and localization length exponents at the band center. This works reveals, for a first time, a unique de-localization mechanism that plays an important role in the excitation of charge-density waves, i.e. surface plasmons (SP), in metal-dielectric composites. It also shows that in 3D metal-dielectric percolation systems the local fields distribution function for frequencies close to the single particle plasmon resonance is log-normal which is a signature of a metal-dielectric phase transition manifested in the optical response of the composites. Based on the obtained numerical data a scaling theory for the higher order electric field moments is developed. A distinct evidence of singularities in the surface plasmon density of states and localization length is obtained, correlating with results previously obtained for two dimensional systems. This leads to the main finding of this work; i.e., the delocalization of surface plasmon states in percolating metal-dielectric composite materials is universally present regardless of the dimensionality of the problem. This dissertation also proposes a new approach toward developing highly efficient inorganic/organic solar cell, by presenting a method for enhancement in the optical absorption and overall cell efficiency. Specifically, the approach improves the operation characteristics of inorganic semiconductor (e.g. Si and a-Si) and organic (P3HT:PCBM) thin film solar cells by integrating a thin, inhomogeneous, metal-dielectric composite (MDC) electrode at the interface between the transparent electrode and active layer. Through numerical simulations, we show that under solar illumination, surface plasmons are excited within the fractal MDC electrode across an extremely broad range of optical frequencies, trapping the incoming light and ensuring an optimal absorption into the active layer of the solar cells. An analytical model is developed to study the I-V characteristics of the cells, providing a pathway toward achieving optimal efficiency and better understanding of the behavior of charge carriers. Using this model, it is shown that including gold MDC electrodes can lead to an enhancement in solar cell power conversion efficiency up to 33% higher compared to the benchmark device.

Sequential Reactions of Surface-Tethered Glycolytic Enzymes

PubMed Central

Mukai, Chinatsu; Bergkvist, Magnus; Nelson, Jacquelyn L.; Travis, Alexander J.

2014-01-01

SUMMARY The development of complex hybrid organic-inorganic devices faces several challenges, including how they can generate energy. Cells face similar challenges regarding local energy production. Mammalian sperm solve this problem by generating ATP down the flagellar principal piece by means of glycolytic enzymes, several of which are tethered to a cytoskeletal support via germ cell-specific targeting domains. Inspired by this design, we have produced recombinant hexokinase type 1 and glucose-6-phosphate isomerase capable of oriented immobilization on a nickel-nitrilotriacetic acid modified surface. Specific activities of enzymes tethered via this strategy were substantially higher than when randomly adsorbed. Furthermore, these enzymes showed sequential activities when tethered onto the same surface. This is the first demonstration of surface-tethered pathway components showing sequential enzymatic activities, and it provides a first step toward reconstitution of glycolysis on engineered hybrid devices. PMID:19778729

Development of viral nanoparticles for efficient intracellular delivery

NASA Astrophysics Data System (ADS)

Wu, Zhuojun; Chen, Kevin; Yildiz, Ibrahim; Dirksen, Anouk; Fischer, Rainer; Dawson, Philip E.; Steinmetz, Nicole F.

2012-05-01

Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform that can target specific cells and tissues. VNPs such as CPMV show natural affinity to cells; however, cellular uptake is inefficient. Here we show that chemical modification of the CPMV surface with a highly reactive, specific and UV-traceable hydrazone linker allows bioconjugation of polyarginine (R5) cell penetrating peptides (CPPs), which can overcome these limitations. The resulting CPMV-R5 particles were taken up into a human cervical cancer cell line (HeLa) more efficiently than native particles. Uptake efficiency was dependent on the density of R5 peptides on the surface of the VNP; particles displaying 40 R5 peptides per CPMV (denoted as CPMV-R5H) interact strongly with the plasma membrane and are taken up into the cells via an energy-dependent mechanism whereas particles displaying 10 R5 peptides per CPMV (CPMV-R5L) are only slowly taken up. The fate of CPMV-R5 versus native CPMV particles within cells was evaluated in a co-localization time course study. It was indicated that the intracellular localization of CPMV-R5 and CPMV differs; CPMV remains trapped in Lamp-1 positive endolysosomes over long time frames; in contrast, 30-50% of the CPMV-R5 particles transitioned from the endosome into other cellular vesicles or compartments. Our data provide the groundwork for the development of efficient drug delivery formulations based on CPMV-R5.Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform that can target specific cells and tissues. VNPs such as CPMV show natural affinity to cells; however, cellular uptake is inefficient. Here we show that chemical modification of the CPMV surface with a highly reactive, specific and UV-traceable hydrazone linker allows bioconjugation of polyarginine (R5) cell penetrating peptides (CPPs), which can overcome these limitations. The resulting CPMV-R5 particles were taken up into a human cervical cancer cell line (HeLa) more efficiently than native particles. Uptake efficiency was dependent on the density of R5 peptides on the surface of the VNP; particles displaying 40 R5 peptides per CPMV (denoted as CPMV-R5H) interact strongly with the plasma membrane and are taken up into the cells via an energy-dependent mechanism whereas particles displaying 10 R5 peptides per CPMV (CPMV-R5L) are only slowly taken up. The fate of CPMV-R5 versus native CPMV particles within cells was evaluated in a co-localization time course study. It was indicated that the intracellular localization of CPMV-R5 and CPMV differs; CPMV remains trapped in Lamp-1 positive endolysosomes over long time frames; in contrast, 30-50% of the CPMV-R5 particles transitioned from the endosome into other cellular vesicles or compartments. Our data provide the groundwork for the development of efficient drug delivery formulations based on CPMV-R5. Electronic supplementary information (ESI) available: Experimental details and additional supporting data. See DOI: 10.1039/c2nr30366c

Saposin C Coupled Lipid Nanovesicles Specifically Target Arthritic Mouse Joints for Optical Imaging of Disease Severity

PubMed Central

Qi, Xiaoyang; Flick, Matthew J.; Frederick, Malinda; Chu, Zhengtao; Mason, Rachel; DeLay, Monica; Thornton, Sherry

2012-01-01

Rheumatoid arthritis is a chronic inflammatory disease affecting approximately 1% of the population and is characterized by cartilage and bone destruction ultimately leading to loss of joint function. Early detection and intervention of disease provides the best hope for successful treatment and preservation of joint mobility and function. Reliable and non-invasive techniques that accurately measure arthritic disease onset and progression are lacking. We recently developed a novel agent, SapC-DOPS, which is composed of the membrane-associated lysosomal protein saposin C (SapC) incorporated into 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) lipid nanovesicles. SapC-DOPS has a high fusogenic affinity for phosphatidylserine-enriched microdomains on surfaces of target cell membranes. Incorporation of a far-red fluorophore, CellVue Maroon (CVM), into the nanovesicles allows for in vivo non-invasive visualization of the agent in targeted tissue. Given that phosphatidylserine is present only on the inner leaflet of healthy plasma membranes but is “flipped” to the outer leaflet upon cell damage, we hypothesized that SapC-DOPS would target tissue damage associated with inflammatory arthritis due to local surface-exposure of phosphatidylserine. Optical imaging with SapC-DOPS-CVM in two distinct models of arthritis, serum-transfer arthritis (e.g., K/BxN) and collagen-induced arthritis (CIA) revealed robust SapC-DOPS-CVM specific localization to arthritic paws and joints in live animals. Importantly, intensity of localized fluorescent signal correlated with macroscopic arthritic disease severity and increased with disease progression. Flow cytometry of cells extracted from arthritic joints demonstrated that SapC-DOPS-CVM localized to an average of 7–8% of total joint cells and primarily to CD11b+Gr-1+ cells. Results from the current studies strongly support the application of SapC-DOPS-CVM for advanced clinical and research applications including: detecting early arthritis onset, assessing disease progression real-time in live subjects, and providing novel information regarding cell types that may mediate arthritis progression within joints. PMID:22470501

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis*

PubMed Central

Valapala, Mallika; Maji, Sayantan; Borejdo, Julian; Vishwanatha, Jamboor K.

2014-01-01

Glutamate-induced elevation in intracellular Ca2+ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca2+-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca2+ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca2+ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. PMID:24742684

Laterally inherently thin amorphous-crystalline silicon heterojunction photovoltaic cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chowdhury, Zahidur R., E-mail: zr.chowdhury@utoronto.ca; Kherani, Nazir P., E-mail: kherani@ecf.utoronto.ca

2014-12-29

This article reports on an amorphous-crystalline silicon heterojunction photovoltaic cell concept wherein the heterojunction regions are laterally narrow and distributed amidst a backdrop of well-passivated crystalline silicon surface. The localized amorphous-crystalline silicon heterojunctions consisting of the laterally thin emitter and back-surface field regions are precisely aligned under the metal grid-lines and bus-bars while the remaining crystalline silicon surface is passivated using the recently proposed facile grown native oxide–plasma enhanced chemical vapour deposited silicon nitride passivation scheme. The proposed cell concept mitigates parasitic optical absorption losses by relegating amorphous silicon to beneath the shadowed metallized regions and by using optically transparentmore » passivation layer. A photovoltaic conversion efficiency of 13.6% is obtained for an untextured proof-of-concept cell illuminated under AM 1.5 global spectrum; the specific cell performance parameters are V{sub OC} of 666 mV, J{sub SC} of 29.5 mA-cm{sup −2}, and fill-factor of 69.3%. Reduced parasitic absorption, predominantly in the shorter wavelength range, is confirmed with external quantum efficiency measurement.« less

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

PubMed

Kim, Bong-Woo; Lee, Chang Seok; Yi, Jae-Sung; Lee, Joo-Hyung; Lee, Joong-Won; Choo, Hyo-Jung; Jung, Soon-Young; Kim, Min-Sik; Lee, Sang-Won; Lee, Myung-Shik; Yoon, Gyesoon; Ko, Young-Gyu

2010-12-01

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

Mechanotransduction across the cell surface and through the cytoskeleton

NASA Technical Reports Server (NTRS)

Wang, N.; Butler, J. P.; Ingber, D. E.

1993-01-01

Mechanical stresses were applied directly to cell surface receptors with a magnetic twisting device. The extracellular matrix receptor, integrin beta 1, induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhesion receptors did not. The cytoskeletal stiffness (ratio of stress to strain) increased in direct proportion to the applied stress and required intact microtubules and intermediate filaments as well as microfilaments. Tensegrity models that incorporate mechanically interdependent struts and strings that reorient globally in response to a localized stress mimicked this response. These results suggest that integrins act as mechanoreceptors and transmit mechanical signals to the cytoskeleton. Mechanotransduction, in turn, may be mediated simultaneously at multiple locations inside the cell through force-induced rearrangements within a tensionally integrated cytoskeleton.

Regulation of Kv2.1 K+ conductance by cell surface channel density

PubMed Central

Fox, Philip D.; Loftus, Rob J.; Tamkun, Michael M.

2013-01-01

The Kv2.1 voltage-gated K+ channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are non-conducting. Using TIRF microscopy the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared to K+ channel conductance measured by whole-cell voltage-clamp of the same cell. This approach indicated that as channel density increases non-clustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the non-conducting state with 17% conducting K+ at higher surface densities. The non-conducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immuno-fluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared to the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 days, respectively. Together these data indicate that the non-conducting state depends primarily on surface density as opposed to cluster location and that this non-conducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K+ conductance further supports a non-conducting role for Kv2.1 in excitable tissues. PMID:23325261

Hydrophobic pinning with copper nanowhiskers leads to bactericidal properties.

PubMed

Singh, Ajay Vikram; Baylan, Semanur; Park, Byung-Wook; Richter, Gunther; Sitti, Metin

2017-01-01

The considerable morbidity associated with hospitalized patients and clinics in developed countries due to biofilm formation on biomedical implants and surgical instruments is a heavy economic burden. An alternative to chemically treated surfaces for bactericidal activity started emerging from micro/nanoscale topographical cues in the last decade. Here, we demonstrate a putative antibacterial surface using copper nanowhiskers deposited by molecular beam epitaxy. Furthermore, the control of biological response is based on hydrophobic pinning of water droplets in the Wenzel regime, causing mechanical injury and cell death. Scanning electron microscopy images revealed the details of the surface morphology and non-contact mode laser scanning of the surface revealed the microtopography-associated quantitative parameters. Introducing the bacterial culture over nanowhiskers produces mechanical injury to cells, leading to a reduction in cell density over time due to local pinning of culture medium to whisker surfaces. Extended culture to 72 hours to observe biofilm formation revealed biofilm inhibition with scattered microcolonies and significantly reduced biovolume on nanowhiskers. Therefore, surfaces patterned with copper nanowhiskers can serve as potential antibiofilm surfaces. The topography-based antibacterial surfaces introduce a novel prospect in developing mechanoresponsive nanobiomaterials to reduce the risk of medical device biofilm-associated infections, contrary to chemical leaching of copper as a traditional bactericidal agent.

V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko

Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1pmore » in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V{sub o} subunit of vacuolar-type H{sup +}-ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling.« less

Cytotoxicity, intracellular localization and exocytosis of citrate capped and PEG functionalized gold nanoparticles in human hepatocyte and kidney cells.

PubMed

Tlotleng, Nonhlanhla; Vetten, Melissa A; Keter, Frankline K; Skepu, Amanda; Tshikhudo, Robert; Gulumian, Mary

2016-08-01

Surface-modified gold nanoparticles (AuNPs) are nanomaterials that hold promise in drug delivery applications. In this study, the cytotoxicity, uptake, intracellular localization, and the exocytosis of citrate-stabilized (Cit-AuNP) and polyethylene glycol (PEG)-modified gold nanoparticles with the carboxyl (COOH) terminal functional group were assessed in human embryonic kidney (HEK 293) and the human caucasian hepatocytes carcinoma (Hep G2) cell systems, representing two major accumulation sites for AuNPs. The zeta (ζ)-potential measurements confirmed the negative surface charge of the AuNPs in water and in cell growth medium. The transmission electron microscopy confirmed the size and morphology of the AuNPs. Both types of AuNPs were shown to induce cytotoxic effects in cells. The Hep G2 cells were more sensitive cell type, with the COOH-PEG-AuNPs inducing the highest toxicity at higher concentrations. Dark field microscopy and TEM images revealed that the AuNPs were internalized in cells, mostly as agglomerates. TEM micrographs further revealed that the AuNPs were confined as agglomerates inside vesicle-like compartments, likely to be endosomal and lysosomal structures as well as in the cytosol, mostly as individual particles. The AuNPs were shown to remain in cellular compartments for up to 3 weeks, but thereafter, clearance of the gold nanoparticles from the cells by exocytosis was evident. The results presented in this study may therefore give an indication on the fate of AuNPs on long-term exposure to cells and may also assist in safety evaluation of AuNPs.

Neuroendocrine cells in the gills of the bowfin Amia calva. An ultrastructural and immunocytochemical study.

PubMed

Goniakowska-Witalińska, L; Zaccone, G; Fasulo, S; Mauceri, A; Licata, A; Youson, J

1995-01-01

Neuroendocrine (NE) cells were localized by electron microscopy and immunocytochemistry in the gill epithelium of bowfin Amia calva. The NE cells are dispersed in whole epithelium of the gill as solitary cells without intraepithelial innervation. All the observed NE cells do not reach the surface of the epithelium. The NE cells are characterized by a large nucleus with patches of condensed chromatin, numerous mitochondria, a well developed Golgi apparatus and a few dense core vesicles of various size scattered in the cytoplasm. Dense core vesicles range from 100 to 560 nm in diameter, while a clear space between the electron dense core ant the limiting membrane ranges from 20 to 240 nm. Immunocytochemical observations reveal the presence of general neuroendocrine markers such as neuro-specific enolase and bioactive substances: serotonin, leu-enkephalin and met-enkephalin. we demonstrated the presence of endothelin - for the first time in fish - and suggested a local paracrine role for the NE cells. Some ultrastructural aspects and the immunocytochemical characteristics of NE cells of bowfin gills are common with those encountered in such cells of other lower vertebrate species.

Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells.

PubMed

Kerr, M; Fischer, J E; Purushotham, K R; Gao, D; Nakagawa, Y; Maeda, N; Ghanta, V; Hiramoto, R; Chegini, N; Humphreys-Beher, M G

1994-08-02

The murine transformed cell line YC-8 and beta-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was detected primarily in a perinuclear and nuclear immunostaining pattern. Western blot analysis confirmed a translocation from a cytoplasmic localization in both LSTRA and quiescent salivary cells to a membrane-associated localization in YC-8 and proliferating salivary cells.

The AWA1 Gene Is Required for the Foam-Forming Phenotype and Cell Surface Hydrophobicity of Sake Yeast

PubMed Central

Shimoi, Hitoshi; Sakamoto, Kazutoshi; Okuda, Masaki; Atthi, Ratchanee; Iwashita, Kazuhiro; Ito, Kiyoshi

2002-01-01

Sake, a traditional alcoholic beverage in Japan, is brewed with sake yeasts, which are classified as Saccharomyces cerevisiae. Almost all sake yeasts form a thick foam layer on sake mash during the fermentation process because of their cell surface hydrophobicity, which increases the cells' affinity for bubbles. To reduce the amount of foam, nonfoaming mutants were bred from foaming sake yeasts. Nonfoaming mutants have hydrophilic cell surfaces and no affinity for bubbles. We have cloned a gene from a foam-forming sake yeast that confers foaming ability to a nonfoaming mutant. This gene was named AWA1 and structures of the gene and its product were analyzed. The N- and C-terminal regions of Awa1p have the characteristic sequences of a glycosylphosphatidylinositol anchor protein. The entire protein is rich in serine and threonine residues and has a lot of repetitive sequences. These results suggest that Awa1p is localized in the cell wall. This was confirmed by immunofluorescence microscopy and Western blotting analysis using hemagglutinin-tagged Awa1p. Moreover, an awa1 disruptant of sake yeast was hydrophilic and showed a nonfoaming phenotype in sake mash. We conclude that Awa1p is a cell wall protein and is required for the foam-forming phenotype and the cell surface hydrophobicity of sake yeast. PMID:11916725

Myosin-X functions in polarized epithelial cells

PubMed Central

Liu, Katy C.; Jacobs, Damon T.; Dunn, Brian D.; Fanning, Alan S.; Cheney, Richard E.

2012-01-01

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein–Myo10 localizes to lateral membrane cell–cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis. PMID:22419816

Internal and Surface-Localized Major Surface Proteases of Leishmania spp. and Their Differential Release from Promastigotes▿

PubMed Central

Yao, Chaoqun; Donelson, John E.; Wilson, Mary E.

2007-01-01

Major surface protease (MSP), also called GP63, is a virulence factor of Leishmania spp. protozoa. There are three pools of MSP, located either internally within the parasite, anchored to the surface membrane, or released into the extracellular environment. The regulation and biological functions of these MSP pools are unknown. We investigated here the trafficking and extrusion of surface versus internal MSPs. Virulent Leishmania chagasi undergo a growth-associated lengthening in the t1/2 of surface-localized MSP, but this did not occur in the attenuated L5 strain. The release of surface-localized MSP was enhanced in a dose-dependent manner by MβCD, which chelates membrane cholesterol-ergosterol. Furthermore, incubation of promastigotes at 37°C with Matrigel matrix, a soluble basement membrane extract of Engelbreth-Holm-Swarm tumor cells, stimulated the release of internal MSP but not of surface-located MSP. Taken together, these data indicate that MSP subpopulations in distinct cellular locations are released from the parasite under different environmental conditions. We hypothesize that the internal MSP with its lengthy t1/2 does not serve as a pool for promastigote surface MSP in the sand fly vector but that it instead functions as an MSP pool ready for quick release upon inoculation of metacyclic promastigotes into mammals. We present a model in which these different MSP pools are released under distinct life cycle-specific conditions. PMID:17693594

Cytotoxicity of solid lipid nanoparticles and nanostructured lipid carriers containing the local anesthetic dibucaine designed for topical application

NASA Astrophysics Data System (ADS)

Barbosa, R. M.; da Silva, C. M. G.; Bella, T. S.; de Araújo, D. R.; Marcato, P. D.; Durán, N.; de Paula, E.

2013-04-01

Dibucaine (DBC) is powerful long-lasting local anesthetic, but it is also considered fairly toxic to the CNS. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) have attracted attention as carriers for drug delivery. The aim of this study was to develop and to evaluate the cytotoxic activity of DBC-loaded SLN and NLC against 3T3 fibroblast and HaCat keratinocyte cells. The SLN and NLC had myristyl myristate and Liponate®GC as their lipid matrices, respectively, plus a surfactant. SLN and NLC were characterized in terms in their diameter, size distribution, surface charge and DBC encapsulation efficiency. The particle size of SLN and NLC were around 234.33 and 166.62 nm, respectively. The polydispersity index was kept below 0.2 for both nanomaterials. Negative surface charges were observed for both nanoparticles, which decreased in the presence of the anesthetic. Encapsulation efficiency reached 76% and 90%, respectively, in SLN and NLC. DBC alone was found to be toxic to 3T3 and HaCat cells in culture. However, NLC and SLN loaded DBC decreased its intrinsic cytotoxic effect against 3T3 and HaCat cells. In conclusion, encapsulation of DBC in SLN and NLC decreased the in vitro toxicity of the local anesthetic, indicating the potential of these nanocarriers for clinical applications.

Sers Imaging of Nasopharyngeal Carcinoma Markers Using an Antibody-Conjugated Gold Nanoparticles Probe

NASA Astrophysics Data System (ADS)

Li, J.-H.; Du, Y.; Feng, G.-K.; Du, Y.-B.; Zhou, Y.-Q.; Zeng, M.-S.

2017-11-01

Surface-enhanced Raman scattering (SERS) nanotags as an ultrasensitive nanoprobe is becoming popular for the detection of biomarkers. Herein, antibody-conjugated gold nanoparticles (AuNPs) were used to target LMP2A in an LMP2A-infected CNE2 cell line. SERS maps showed that the LMP2A was distributed around the cell, which was consistent with the results of immunofl uorescence staining in the previous report. This location could be due to the specific binding of the bioconjugated nanotags to the receptors on the cell surface. However, the CNE2 cell line without LMP2A-infected showed no detectable signal at 1044 cm-1. The results demonstrated the potential feasibility of AuNPs nanotags as highly sensitive probes conjugated at the subcellular level for detection and localization of cancer markers in nasopharyngeal carcinoma (NPC).

SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during C. elegans gastrulation

PubMed Central

Grana, Theresa M.; Cox, Elisabeth A.; Lynch, Allison M.; Hardin, Jeff

2010-01-01

Gastrulation is the first major morphogenetic movement in development, and requires dynamic regulation of cell adhesion and the cytoskeleton. C. elegans gastrulation begins with the migration of the two endodermal precursors, Ea and Ep, from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on correct cell fate specification and polarization, apical myosin accumulation, and Wnt activated actomyosin contraction that drives apical constriction and ingression (Lee et al., 2006; Nance et al., 2005). Here, we show that Ea/Ep ingression also requires the function of either HMR-1/cadherin or SAX-7/L1CAM. Both cadherin complex components and L1CAM are localized at all sites of cell-cell contact during gastrulation. Either system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. Similar results are seen with isolated blastomeres. Ea/Ep are properly specified and appear to display correct apical-basal polarity in sax-7(eq1); hmr-1(RNAi) embryos. Significantly, in sax-7(eq1); hmr-1(RNAi) embryos Ea and Ep fail to accumulate myosin (NMY-2::GFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) embryos, apical myosin accumulation is comparable to wildtype. Thus, the cadherin and L1CAM adhesion systems are redundantly required for localized myosin accumulation, and hence for actomyosin contractility during gastrulation. We also show that sax-7 and hmr-1 function are redundantly required for Wnt-dependent spindle polarization during division of the ABar blastomere, indicating that these cell surface proteins redundantly regulate multiple developmental events in early embryos. PMID:20515680

Local convection-enhanced delivery of an anti-CD40 agonistic monoclonal antibody induces antitumor effects in mouse glioma models.

PubMed

Shoji, Takuhiro; Saito, Ryuta; Chonan, Masashi; Shibahara, Ichiyo; Sato, Aya; Kanamori, Masayuki; Sonoda, Yukihiko; Kondo, Toru; Ishii, Naoto; Tominaga, Teiji

2016-08-01

Glioblastoma is one of the most malignant brain tumors in adults and has a dismal prognosis. In a previous report, we reported that CD40, a TNF-R-related cell surface receptor, and its ligand CD40L were associated with glioma outcomes. Here we attempted to activate CD40 signaling in the tumor and determine if it exerted therapeutic efficacy. CD40 expression was examined in 3 mouse glioma cell lines (GL261, NSCL61, and bRiTs-G3) and 5 human glioma cell lines (U87, U251, U373, T98, and A172). NSCL61 and bRiTs-G3, as glioma stem cells, also expressed the glioma stem cell markers MELK and CD44. In vitro, we demonstrated direct antitumor effects of an anti-CD40 agonistic monoclonal antibody (FGK45) against the cell lines. The efficacy of FGK45 was examined by local convection-enhanced delivery of the monoclonal antibody against each glioma model. CD40 was expressed in all mouse and human cell lines tested and was found at the cell membrane of each of the 3 mouse cell lines. FGK45 administration induced significant, direct antitumor effects in vitro. The local delivery of FGK45 significantly prolonged survival compared with controls in the NSCL61 and bRiTs-G3 models, but the effect was not significant in the GL261 model. Increases in apoptosis and CD4(+) and CD8(+) T cell infiltration were observed in the bRiTs-G3 model after FGK45 treatment. Local delivery of FGK45 significantly prolonged survival in glioma stem cell models. Thus, local delivery of this monoclonal antibody is promising for immunotherapy against gliomas. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular targets and signaling pathways regulated by nuclear translocation of syndecan-1.

PubMed

Szatmári, Tünde; Mundt, Filip; Kumar-Singh, Ashish; Möbus, Lena; Ötvös, Rita; Hjerpe, Anders; Dobra, Katalin

2017-12-08

The cell-surface heparan sulfate proteoglycan syndecan-1 is important for tumor cell proliferation, migration, and cell cycle regulation in a broad spectrum of malignancies. Syndecan-1, however, also translocates to the cell nucleus, where it might regulate various molecular functions. We used a fibrosarcoma model to dissect the functions of syndecan-1 related to the nucleus and separate them from functions related to the cell-surface. Nuclear translocation of syndecan-1 hampered the proliferation of fibrosarcoma cells compared to the mutant lacking nuclear localization signal. The growth inhibitory effect of nuclear syndecan-1 was accompanied by significant accumulation of cells in the G0/G1 phase, which indicated a possible G1/S phase arrest. We implemented multiple, unsupervised global transcriptome and proteome profiling approaches and combined them with functional assays to disclose the molecular mechanisms that governed nuclear translocation and its related functions. We identified genes and pathways related to the nuclear compartment with network enrichment analysis of the transcriptome and proteome. The TGF-β pathway was activated by nuclear syndecan-1, and three genes were significantly altered with the deletion of nuclear localization signal: EGR-1 (early growth response 1), NEK11 (never-in-mitosis gene a-related kinase 11), and DOCK8 (dedicator of cytokinesis 8). These candidate genes were coupled to growth and cell-cycle regulation. Nuclear translocation of syndecan-1 influenced the activity of several other transcription factors, including E2F, NFκβ, and OCT-1. The transcripts and proteins affected by syndecan-1 showed a striking overlap in their corresponding biological processes. These processes were dominated by protein phosphorylation and post-translation modifications, indicative of alterations in intracellular signaling. In addition, we identified molecules involved in the known functions of syndecan-1, including extracellular matrix organization and transmembrane transport. Collectively, abrogation of nuclear translocation of syndecan-1 resulted in a set of changes clustering in distinct patterns, which highlighted the functional importance of nuclear syndecan-1 in hampering cell proliferation and the cell cycle. This study emphasizes the importance of the localization of syndecan-1 when considering its effects on tumor cell fate.

Surface plasmon resonance-enabled antibacterial digital versatile discs

NASA Astrophysics Data System (ADS)

Dou, Xuan; Chung, Pei-Yu; Jiang, Peng; Dai, Jianli

2012-02-01

We report the achievement of effective sterilization of exemplary bacteria including Escherichia coli and Geobacillus stearothermophilus spores on a digital versatile disc (DVD). The spiral arrangement of aluminum-covered pits generates strong surface plasmon resonance (SPR) absorption of near-infrared light, leading to high surface temperature that could even damage the DVD plastics. Localized protein denaturation and high sterilization efficiency have been demonstrated by using a fluorescence microscope and cell cultures. Numerical simulations have also been conducted to model the SPR properties and the surface temperature distribution of DVDs under laser illumination. The theoretical predictions agree reasonably well with the experimental results.

Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its function as a carcinoembryonic antigen receptor.

PubMed

Bajenova, Olga; Stolper, Eugenia; Gapon, Svetlana; Sundina, Natalia; Zimmer, Regis; Thomas, Peter

2003-11-15

Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases from colorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identified heterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76), the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1 and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of their function as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoforms are expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein has nuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleus and CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. The search of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP that may be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level of expression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternative spliced forms of CEAR is regulated in a tissue-specific manner.

Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes.

PubMed

Hirata, Tomoya; Terai, Takuya; Yamamura, Hisao; Shimonishi, Manabu; Komatsu, Toru; Hanaoka, Kenjiro; Ueno, Tasuku; Imaizumi, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-03-01

K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.

Whole cell quenched flow analysis.

PubMed

Chiang, Ya-Yu; Haeri, Sina; Gizewski, Carsten; Stewart, Joanna D; Ehrhard, Peter; Shrimpton, John; Janasek, Dirk; West, Jonathan

2013-12-03

This paper describes a microfluidic quenched flow platform for the investigation of ligand-mediated cell surface processes with unprecedented temporal resolution. A roll-slip behavior caused by cell-wall-fluid coupling was documented and acts to minimize the compression and shear stresses experienced by the cell. This feature enables high-velocity (100-400 mm/s) operation without impacting the integrity of the cell membrane. In addition, rotation generates localized convection paths. This cell-driven micromixing effect causes the cell to become rapidly enveloped with ligands to saturate the surface receptors. High-speed imaging of the transport of a Janus particle and fictitious domain numerical simulations were used to predict millisecond-scale biochemical switching times. Dispersion in the incubation channel was characterized by microparticle image velocimetry and minimized by using a horizontal Hele-Shaw velocity profile in combination with vertical hydrodynamic focusing to achieve highly reproducible incubation times (CV = 3.6%). Microfluidic quenched flow was used to investigate the pY1131 autophosphorylation transition in the type I insulin-like growth factor receptor (IGF-1R). This predimerized receptor undergoes autophosphorylation within 100 ms of stimulation. Beyond this demonstration, the extreme temporal resolution can be used to gain new insights into the mechanisms underpinning a tremendous variety of important cell surface events.

Localization and seasonal variation of blue pigment (sandercyanin) in walleye (Sander vitreus)

USGS Publications Warehouse

Schaefer, Wayne; Schmitz, Mark; Blazer, Vicki S.; Ehlinger, Tim; Berges, John

2015-01-01

Several fish species, including the walleye (Sander vitreus), have “yellow” and “blue” color morphs. In S. vitreus, one source of the blue color has been identified as a bili-binding protein pigment (sandercyanin), found in surface mucus of the fish. Little is known about the production of the pigment or about its functions. We examined the anatomical localization and seasonal variation of sandercyanin in S. vitreus from a population in McKim Lake, northwestern Ontario, Canada. Skin sections were collected from 20 fish and examined histologically. Mucus was collected from 306 fish over 6 years, and the amount of sandercyanin was quantified spectrophotometrically. Sandercyanin was found solely on dorsal surfaces of the fish and was localized to novel cells in the epidermis, similar in appearance to secretory sacciform cells. Sandercyanin concentrations were significantly higher in fish collected in summer versus other seasons. Yellow and blue morphs did not differ in amounts of sandercyanin, suggesting that the observed blue color, in fact, arises from lack of yellow pigmentation in blue morphs. The function of the sandercyanin remains unclear, but roles in photoprotection and countershading are consistent with available data.

Effects of captopril, losartan, and nifedipine on cell hypertrophy of cultured vascular smooth muscle from hypertensive Ren-2 transgenic rats

PubMed Central

Peiró, Concepción; Llergo, José L; Angulo, Javier; López-Novoa, José M; Rodríguez-López, Ana; Rodríguez-Mañas, Leocadio; Sánchez-Ferrer, Carlos F

1997-01-01

We hypothesized that tissular renin-angotensin system (RAS) induces vascular hypertrophy in hypertensive Ren-2 transgenic rats (TGR; strain name TGR(mRen2)L27). This assumption was tested in cell cultures of vascular smooth muscle (VSMC) from both hypertensive TGR and control normotensive Sprague-Dawley (SD) rats. Planar cell surface area, protein synthesis, and protein content per cell were studied, the role for locally produced angiotensin II (AII) was evaluated and the possible pharmacological interference by different drugs was analysed. By use of radioimmunoassay techniques, AII could be determined in TGR cultures (10.25±0.12 pg per 107 cells) while it could not be detected in SD ones. Under serum-free conditions, VSMC from hypertensive TGR were hypertrophic when compared to SD VSMC, as they presented a higher protein content per cell (335±18 and 288±7 pg per cell respectively; P<0.05) and increased mean planar cell surface area, as determined by image analysis (4,074±238 and 4,764±204 μm2, respectively; P<0.05). When exogenously added to cultured SD and TGR VSMC, AII (100 pM to 1 μM) promoted protein synthesis and protein content in a concentration-dependent manner without affecting DNA synthesis. Maximal effects were observed at 100 nM. At this concentration, AII effectively increased planar cell surface area in both SD and TGR cultures by ∼20%. Treatment of TGR cultures, in the absence of exogenous AII, with the angiotensin-converting enzyme inhibitor captopril or the angiotensin AT1 receptors antagonist losartan (100 nM to 10 μM) reduced planar cell surface area in a concentration-dependent manner. In addition, both captopril and losartan (10 μM), decreased protein synthesis by ∼15%. Treatment of SD VSMC, in the absence of exogenous AII, with both captopril and losartan had no effect either on planar cell surface area or protein synthesis. Treatment with the Ca2+ antagonist nifedipine (100 nM to 10 μM) reduced cell size in both SD and TGR cultures. Maximal cell reduction reached by nifedipine averaged 906±58 and 1,292±57 μm2, in SD and TGR, respectively (P<0.05). In addition, nifedipine, nitrendipine and nisoldipine (all at 10 μM) decreased protein synthesis in both cell types by 15–25%. We concluded that cultured VSMC from TGR are hypertrophic in comparison with those from SD. This cell hypertrophy can be the consequence of the expression of the transgene Ren-2 that activates a tissular RAS and locally produces AII, which acts in a paracrine, autocrine, or intracrine manner. Cell hypertrophy in TGR cultures could be selectively reduced by RAS blockade, while nifedipine decreased cell size and protein synthesis in both hypertrophic and non hypertrophic cells. PMID:9257925

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

PubMed

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a serious fungal pathogen that kills over 600,000 people annually. It converts most of its chitin, a cell wall polysaccharide, to chitosan, which is necessary for virulence. Chitin deacetylase enzymes have been identified in the cell wall, and our studies were undertaken to understand how the deacetylase is linked to the wall and where it has activity. Our results have implications for the current model of chitosan biosynthesis and further challenge the paradigm of covalent linkages between cell wall proteins and polysaccharides through a lipid modification of the protein.

Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility

PubMed Central

1993-01-01

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis. PMID:8380174

Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32).

PubMed

Chan, Derek V; Somani, Ally-Khan; Young, Andrew B; Massari, Jessica V; Ohtola, Jennifer; Sugiyama, Hideaki; Garaczi, Edina; Babineau, Denise; Cooper, Kevin D; McCormick, Thomas S

2011-05-26

Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.

Near Surface Swimming of Salmonella Typhimurium Explains Target-Site Selection and Cooperative Invasion

PubMed Central

Kreibich, Saskia; Vonaesch, Pascale; Andritschke, Daniel; Rout, Samuel; Weidner, Kerstin; Sormaz, Milos; Songhet, Pascal; Horvath, Peter; Chabria, Mamta; Vogel, Viola; Spori, Doris M.; Jenny, Patrick; Hardt, Wolf-Dietrich

2012-01-01

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria. PMID:22911370

Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8.

PubMed

Shiheido, Hirokazu; Kitagori, Koji; Sasaki, Chiyomi; Kobayashi, Shio; Aoyama, Takane; Urata, Kozue; Oku, Takuma; Hirayama, Yoshitaka; Yoshitomi, Hiroyuki; Hikida, Masaki; Yoshifuji, Hajime; Mimori, Tsuneyo; Watanabe, Takeshi; Shimizu, Jun

2014-06-01

BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3(+) T cells). BEND3(+) T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4(+) and CD8(+) T cells, and were also observed in cord blood. The stimulation of BEND3(+) T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3(+) T cells were higher than those by BEND3(-) T cells. The proportion of BEND3(+) T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3(+) T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3(+) T cells may be of importance and useful in understanding human T cell immunology.

Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8

PubMed Central

Shiheido, Hirokazu; Kitagori, Koji; Sasaki, Chiyomi; Kobayashi, Shio; Aoyama, Takane; Urata, Kozue; Oku, Takuma; Hirayama, Yoshitaka; Yoshitomi, Hiroyuki; Hikida, Masaki; Yoshifuji, Hajime; Mimori, Tsuneyo; Watanabe, Takeshi; Shimizu, Jun

2014-01-01

BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3+ T cells). BEND3+ T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4+ and CD8+ T cells, and were also observed in cord blood. The stimulation of BEND3+ T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3+ T cells were higher than those by BEND3− T cells. The proportion of BEND3+ T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3+ T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3+ T cells may be of importance and useful in understanding human T cell immunology. PMID:25400923

TMPRSS2, a novel membrane-anchored mediator in cancer pain

PubMed Central

Lam, David K.; Dang, Dongmin; Flynn, Andrea N.; Hardt, Markus; Schmidt, Brian L.

2016-01-01

More than half of all cancer patients will suffer significant pain during the course of their disease. The strategic localization of TMPRSS2, a membrane-bound serine protease, on the cancer cell surface may allow it to mediate signal transduction between the cancer cell and its extracellular environment. Here we show TMPRSS2 expression is not only dramatically increased in the primary cancers of patients but TMPRSS2-immunopositivity is also directly correlated with cancer pain severity in these patients. TMPRSS2 induced proteolytic activity, activated trigeminal neurons, and produced marked mechanical hyperalgesia when administered into the hindpaw of wild-type mice but not in PAR2-deficient mice. Co-culture of human cancer cells with murine trigeminal neurons demonstrated co-localization of TMPRSS2 with PAR2. These results point to a novel role for a cell membrane-anchored mediator in cancer pain, as well as pain in general. PMID:25734995

Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

PubMed

Luo, Yu; Russinova, Eugenia

2017-01-01

Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

A function-driven characterization of printed conductors on PV cells

NASA Astrophysics Data System (ADS)

Bellotti, Roberto; Furin, Valentina; Maras, Claire; Bartolo Picotto, Gian; Ribotta, Luigi

2018-06-01

Nowadays the development in photovoltaic (PV) cells manufacturing requires increasingly sophisticated technologies, and in order to avoid efficiency losses in PV cell, printing techniques of the front contacts have to be well controlled. To this purpose, printed linear conductors (PLCs) on a PV standard cell are characterized by morphology- and resistance-based measurements, creating a well-calibrated test structure towards the development of an application-oriented material measure. It can be noticed that morphology and texture parameters determined by stylus and optical profilers are well in agreement, and the resistance calculated from the reconstructed cross-section area matches quite well the measured resistance of fingers. Uncertainties of about 14% to 17% are estimated for local measurements of morphology-based and measured resistance of finger segments up to 5 mm length. Fingers characterized by somewhat larger roughness/waviness values (, , ) show some local irregularities, which may degrade the electrical contact of the PV front surface.

Fermi surface properties of paramagnetic NpCd11 with a large unit cell

NASA Astrophysics Data System (ADS)

Homma, Yoshiya; Aoki, Dai; Haga, Yoshinori; Settai, Rikio; Sakai, Hironori; Ikeda, Shugo; Yamamoto, Etsuji; Nakamura, Akio; Shiokawa, Yoshinobu; Takeuchi, Tetsuya; Yamagami, Hiroshi; Ōnuki, Yoshichika

2010-03-01

We succeeded in growing a high-quality single crystal of NpCd11 with the cubic BaHg11-type structure by the Cd-self flux method. The lattice parameter of a = 9.2968(2) Å and crystallographic positions of the atoms were determined by x-ray single-crystal structure analysis. From the results of the magnetic susceptibility and specific heat experiments, this compound is found to be a 5f-localized paramagnet with the singlet ground state in the crystalline electric field (CEF) scheme. Fermi surface properties were measured using the de Haas-van Alphen (dHvA) technique. Long-period oscillations were observed in the dHvA frequency range of 9.1 x 105 to 1.9 x 107 Oe, indicating small cross-sectional areas of Fermi surfaces, which is consistent with a small Brillouin zone based on a large unit cell. From the results of dHvA and magnetoresistance experiments, the Fermi surface of NpCd11 is found to consist of many kinds of closed Fermi surfaces and a multiply-connected-like Fermi surface, although the result of energy band calculations based on the 5f-localized Np3+(5f4) configuration reveals the existence of only closed Fermi surfaces. The corresponding cyclotron effective mass is small, ranging from 0.1 to 0.7 m0, which is consistent with a small electronic specific heat coefficient γ ≅ 10mJ/K2·mol, revealing no hybridization between the 5f electrons and conduction electrons.

Analysis of Microtubule Mediated Functions of Prostate Specific Membrane Antigen

DTIC Science & Technology

2006-04-01

localization of vesicles containing these markers increased to approximately 43% (38/88) when cells are incubated with tunicamycin, indicating a role...PSMA at both plasma membrane domains following nocodazole treatment. Polarity of the basolateral marker Na,K- ATPase was unaffected by nocodazole...restricted to the apical surface facing the lumen. This staining was clearly distinct from that of the endothelial cell marker CD 34 and CD31

Differential expression of the virulence-associated protein p57 and characterization of its duplicated gene rosa in virulent and attenuated strains of Renibacterium salmoninarum

USGS Publications Warehouse

O'Farrell, C. L.; Strom, M.S.

1999-01-01

Virulence mechanisms utilized by the salmonid fish pathogen Renibacterium salmoninarum are poorly understood. One potential virulence factor is p57 (also designated MSA for major soluble antigen), an abundant 57 kDa soluble protein that is predominately localized on the bacterial cell surface with significant levels released into the extracellular milieu. Previous studies of an attenuated strain, MT 239, indicated that it differs from virulent strains in the amount of surface-associated p57. In this report, we show overall expression of p57 in R. salmoninarum MT 239 is considerably reduced as compared to a virulent strain, ATCC 33209. The amount of cell-associated p57 is decreased while the level of p57 in the culture supernatant is nearly equivalent between the strains. To determine if lowered amount of cell-associated p57 was due to a sequence defect in p57, a genetic comparison was performed. Two copies of the gene encoding p57 (msa1 and msa2) were found in 33209 and MT 239, as well as in several other virulent isolates. Both copies from 33209 and MT 239 were cloned and sequenced and found to be identical to each other, and identical between the 2 strains. A comparison of msa1 and msa2 within each strain showed that their sequences diverge 40 base pairs 5, to the open reading frame, while sequences 3' to the open reading frame are essentially identical for at least 225 base pairs. Northern blot analysis showed no difference in steady state levels of rosa mRNA between the 2 strains. These data suggest that while cell-surface localization of p57 may be important for R. salmoninarum virulence, the differences in localization, and total p57 expression between 33209 anti MT 239 are not due to differences in rosa sequence or differences in steady state transcript levels.

A Conserved Role for Girdin in Basal Body Positioning and Ciliogenesis.

PubMed

Nechipurenko, Inna V; Olivier-Mason, Anique; Kazatskaya, Anna; Kennedy, Julie; McLachlan, Ian G; Heiman, Maxwell G; Blacque, Oliver E; Sengupta, Piali

2016-09-12

Primary cilia are ubiquitous sensory organelles that mediate diverse signaling pathways. Cilia position on the cell surface is determined by the location of the basal body (BB) that templates the cilium. The mechanisms that regulate BB positioning in the context of ciliogenesis are largely unknown. Here we show that the conserved signaling and scaffolding protein Girdin localizes to the proximal regions of centrioles and regulates BB positioning and ciliogenesis in Caenorhabditis elegans sensory neurons and human RPE-1 cells. Girdin depletion alters localization of the intercentriolar linker and ciliary rootlet component rootletin, and rootletin knockdown in RPE-1 cells mimics Girdin-dependent phenotypes. C. elegans Girdin also regulates localization of the apical junction component AJM-1, suggesting that in nematodes Girdin may position BBs via rootletin- and AJM-1-dependent anchoring to the cytoskeleton and plasma membrane, respectively. Together, our results describe a conserved role for Girdin in BB positioning and ciliogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Basolateral Sorting of Furin in MDCK Cells Requires a Phenylalanine-Isoleucine Motif Together with an Acidic Amino Acid Cluster

PubMed Central

Simmen, Thomas; Nobile, Massimo; Bonifacino, Juan S.; Hunziker, Walter

1999-01-01

Furin is a subtilisin-related endoprotease which processes a wide range of bioactive proteins. Furin is concentrated in the trans-Golgi network (TGN), where proteolytic activation of many precursor proteins takes place. A significant fraction of furin, however, cycles among the TGN, the plasma membrane, and endosomes, indicating that the accumulation in the TGN reflects a dynamic localization process. The cytosolic domain of furin is necessary and sufficient for TGN localization, and two signals are responsible for retrieval of furin to the TGN. A tyrosine-based (YKGL) motif mediates internalization of furin from the cell surface into endosomes. An acidic cluster that is part of two casein kinase II phosphorylation sites (SDSEEDE) is then responsible for retrieval of furin from endosomes to the TGN. In addition, the acidic EEDE sequence also mediates endocytic activity. Here, we analyzed the sorting of furin in polarized epithelial cells. We show that furin is delivered to the basolateral surface of MDCK cells, from where a significant fraction of the protein can return to the TGN. A phenylalanine-isoleucine motif together with the acidic EEDE cluster is required for basolateral sorting and constitutes a novel signal regulating intracellular traffic of furin. PMID:10082580

Glycosides derived from Hemidesmus indicus R. Br. root inhibit adherence of Salmonella typhimurium to host cells: receptor mimicry.

PubMed

Das, Sarita; Devaraj, S Niranjali

2006-09-01

For centuries, indigenous plants have been used against enteritis but their molecular targets and mode of action remain obscure. The present study was carried out to elucidate the protective and therapeutic role, if any, of glycosides from Hemidesmus indicus against S. typhimurium-induced pathogenesis. Studies were carried out in a human intestinal cell line (Int 407) and a murine macrophage cell line (P388D1) in order to evaluate its potency in local as well as systemic infections. The inhibitory role of the glycosides present in Hemidesmus indicus root extract (GHI) were tested by pre-coating the cells (both Int 407 and P388D1) with GHI prior to infection, and by neutralizing the wild-type bacteria with GHI before cell infection. In both cases, GHI protected the host cells from the cytotoxic effects of the wild S. typhimurium. This suggests that the biologically significant sugars (hexose, hexosamine, fucose and sialic acid etc) present in GHI might be mimicking host cell receptor saccharides and thereby blocking the bacterial ligands from binding to the host cells. Int 407 cells infected with wild-type bacteria had a diffused adherence pattern after 4 h incubation, but this typical character was not observed in cells infected with GHI-treated bacteria and the cells were normal in appearance at 4 h. After 18 h cells infected with wild-type bacteria were hypertrophoid with a disintegrated membrane and wrapped in a bacterial coat, whereas cells infected with treated bacteria had comparatively less morphological changes and few defective shrunken rods adhered locally. This suggests that the glycosides can change the adherence pattern of S. typhimurium from diffused to local. Treated bacteria had less adherence and invasion capability in Int 407 as well as P388D1 cells. The results show the decreased ability of adherence of GHI-treated S. typhimurium was due to a loss of surface hydrophobicity. A nonspecific binding between S. typhimurium and the glycosides was confirmed using ELISA. In summary, the glycosides of H. indicus root inhibited S. typhimurium induced pathogenesis nonspecifically, by reducing bacterial surface hydrophobicity and perhaps also by mimicking host cell receptors, thereby blocking its attachment to host cell and further pathological effects. Copyright (c) 2006 John Wiley & Sons, Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Luan; Tao, Franklin, E-mail: franklin.tao.2011@gmail.com; Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, Kansas 66045

Tracking surface chemistry of a catalyst during catalysis is significant for fundamental understanding of catalytic performance of the catalyst since it allows for establishing an intrinsic correlation between surface chemistry of a catalyst at its working status and its corresponding catalytic performance. Ambient pressure X-ray photoelectron spectroscopy can be used for in-situ studies of surfaces of different materials or devices in a gas. To simulate the gaseous environment of a catalyst in a fixed-bed a flowing gaseous environment of reactants around the catalyst is necessary. Here, we report the development of a new flowing reaction cell for simulating in-situ studymore » of a catalyst surface under a reaction condition in gas of one reactant or during catalysis in a mixture of reactants of a catalytic reaction. The homemade reaction cell is installed in a high vacuum (HV) or ultrahigh vacuum (UHV) environment of a chamber. The flowing gas in the reaction cell is separated from the HV or UHV environment through well sealings at three interfaces between the reaction cell and X-ray window, sample door and aperture of front cone of an energy analyzer. Catalyst in the cell is heated through infrared laser beam introduced through a fiber optics interfaced with the reaction cell through a homemade feedthrough. The highly localized heating on the sample holder and Au-passivated internal surface of the reaction cell effectively minimizes any unwanted reactions potentially catalyzed by the reaction cell. The incorporated laser heating allows a fast heating and a high thermal stability of the sample at a high temperature. With this cell, a catalyst at 800 °C in a flowing gas can be tracked readily.« less

The polymeric immunoglobulin receptor (secretory component) mediates transport of immune complexes across epithelial cells: a local defense function for IgA.

PubMed Central

Kaetzel, C S; Robinson, J K; Chintalacharuvu, K R; Vaerman, J P; Lamm, M E

1991-01-01

The polymeric immunoglobulin receptor (pIgR) on mucosal epithelial cells binds dimeric IgA (dIgA) on the basolateral surface and mediates transport of dIgA to the apical surface. Using Madin-Darby canine kidney epithelial cells stably transfected with pIgR cDNA, we found that soluble immune complexes (ICs) of 125I-labeled rat monoclonal antidinitrophenyl (DNP) dIgA (125I-dIgA) and DNP/biotin-bovine serum albumin were transported from the basolateral to the apical surface and then released. Monomeric IgA ICs were not transported, consistent with the specificity of pIgR for polymeric immunoglobulins. Essentially all the 125I-dIgA in apical culture supernatants was streptavidin precipitable, indicating that dIgA remained bound to antigen during transcytosis. While both dIgA and dIgA ICs bound pIgR with equal affinity (Kd approximately 8 nM), the number of high-affinity binding sites per cell was 2- to 3-fold greater for dIgA than for dIgA ICs. The extent of endocytosis of dIgA and dIgA ICs was correlated with the number of high-affinity binding sites. SDS/PAGE analysis of intracellular dIgA and dIgA ICs demonstrated that in both cases IgA remained undegraded during transport. The results suggest that the pathways of epithelial transcytosis of free dIgA and dIgA ICs are the same. Given the high population density of mucosal IgA plasma cells and the enormous surface area of pIgR-expressing mucosal epithelium, it is likely that significant local transcytosis of IgA ICs occurs in vivo. Such a process would allow direct elimination of IgA ICs at the mucosal sites where they are likely to form, thus providing an important defense function for IgA. Images PMID:1924341

Construction of a laccase chimerical gene: recombinant protein characterization and gene expression via yeast surface display.

PubMed

Bleve, G; Lezzi, C; Spagnolo, S; Rampino, P; Perrotta, C; Mita, G; Grieco, Francesco

2014-03-01

The ERY4 laccase gene from Pleurotus eryngii was expressed in Saccharomyces cerevisiae and the recombinant laccase resulted to be not biologically active. This gene was thus modified to obtain chimerical enzymes derived from the substitution of N-, C- and both N- and C-terminal regions with the corresponding regions of Ery3 laccase, another laccase isoform of P. eryngii. The chimerical isoform named 4NC3, derived from the substitution of both N- and C-terminal regions, showed the best performances in terms of enzymatic activities, affinities for different substrates and stability at a broad range of temperatures and pHs. The chimerical 4NC3 laccase isoform was displayed on the cell surface of S. cerevisiae using the N-terminal fusion with either the Pir2 or the Flo1 S. cerevisiae proteins as anchor attachment sequence. Immunofluorescence microscopy and Western blot analyses confirmed the localization of 4NC3 on the yeast cell surface. The enzyme activity on specific laccase substrates revealed that 4NC3 laccase was immobilized in active form on the cell surface. To our knowledge, this is the first example of expression of a chimerical fungal laccase by yeast cell display.

Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

PubMed Central

1995-01-01

Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

Plasma-deposited fluorocarbon polymer films on titanium for preventing cell adhesion: a surface finishing for temporarily used orthopaedic implants

NASA Astrophysics Data System (ADS)

Finke, B.; Testrich, H.; Rebl, H.; Walschus, U.; Schlosser, M.; Zietz, C.; Staehlke, S.; Nebe, J. B.; Weltmann, K. D.; Meichsner, J.; Polak, M.

2016-06-01

The design of a titanium implant surface should ideally support its later application in clinical use. Temporarily used implants have to fulfil requirements different from permanent implants: they should ensure the mechanical stabilization of the bone stock but in trauma surgery they should not be integrated into the bone because they will be removed after fracture healing. Finishing of the implant surface by a plasma-fluorocarbon-polymer (PFP) coating is a possible approach for preventing cell adhesion of osteoblasts. Two different low pressure gas-discharge plasma processes, microwave (MW 2.45 GHz) and capacitively coupled radio frequency (RF 13.56 MHz) plasma, were applied for the deposition of the PFP film using a mixture of the precursor octafluoropropane (C3F8) and hydrogen (H2). The thin films were characterized by x-ray photoelectron spectroscopy, Fourier transform infrared reflection absorption spectroscopy, and water contact angle measurements. Cell culture experiments show that cell adhesion and spreading of MG-63 osteoblasts were clearly reduced or nonexistent on these surfaces, also after 24 h of storage in the cell culture medium. In vivo data demonstrated that the local inflammatory tissue response for the PFP films deposited in MW and RF plasma were comparable to uncoated controls.

Removal forces and adhesion properties of Saccharomyces cerevisiae on glass substrates probed by optical tweezer

NASA Astrophysics Data System (ADS)

Castelain, Mickaël; Pignon, Frédéric; Piau, Jean-Michel; Magnin, Albert; Mercier-Bonin, Muriel; Schmitz, Philippe

2007-10-01

In agroindustry, the hygiene of solid surfaces is of primary importance in order to ensure that products are safe for consumers. To improve safety, one of the major ways consists in identifying and understanding the mechanisms of microbial cell adhesion to nonporous solid surfaces or filtration membranes. In this paper we investigate the adhesion of the yeast cell Saccharomyces cerevisiae (about 5μm in diameter) to a model solid surface, using well-defined hydrophilic glass substrates. An optical tweezer device developed by Piau [J. Non-Newtonian Fluid Mech. 144, 1 (2007)] was applied to yeast cells in contact with well-characterized glass surfaces. Two planes of observation were used to obtain quantitative measurements of removal forces and to characterize the corresponding mechanisms at a micrometer length scale. The results highlight various adhesion mechanisms, depending on the ionic strength, contact time, and type of yeast. The study has allowed to show a considerable increase of adhering cells with the ionic strength and has provided a quantitative measurement of the detachment forces of cultured yeast cells. Force levels are found to grow with ionic strength and differences in mobility are highlighted. The results clearly underline that a microrheological approach is essential for analyzing the adhesion mechanisms of biological systems at the relevant local scales.

Human Plasmacytoid Dendritic Cells Display and Shed B Cell Maturation Antigen upon TLR Engagement.

PubMed

Schuh, Elisabeth; Musumeci, Andrea; Thaler, Franziska S; Laurent, Sarah; Ellwart, Joachim W; Hohlfeld, Reinhard; Krug, Anne; Meinl, Edgar

2017-04-15

The BAFF-APRIL system is best known for its control of B cell homeostasis, and it is a target of therapeutic intervention in autoimmune diseases and lymphoma. By analyzing the expression of the three receptors of this system, B cell maturation Ag (BCMA), transmembrane activator and CAML interactor, and BAFF receptor, in sorted human immune cell subsets, we found that BCMA was transcribed in plasmacytoid dendritic cells (pDCs) in both blood and lymphoid tissue. Circulating human pDCs contained BCMA protein without displaying it on the cell surface. After engagement of TLR7/8 or TLR9, BCMA was detected also on the cell surface of pDCs. The display of BCMA on the surface of human pDCs was accompanied by release of soluble BCMA (sBCMA); inhibition of γ-secretase enhanced surface expression of BCMA and reduced the release of sBCMA by pDCs. In contrast with human pDCs, murine pDCs did not express BCMA, not even after TLR9 activation. In this study, we extend the spectrum of BCMA expression to human pDCs. sBCMA derived from pDCs might determine local availability of its high-affinity ligand APRIL, because sBCMA has been shown to function as an APRIL-specific decoy. Further, therapeutic trials targeting BCMA in patients with multiple myeloma should consider possible effects on pDCs. Copyright © 2017 by The American Association of Immunologists, Inc.

Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

PubMed

Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-02-28

Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of MV within the cell. Further studies demonstrated that annexin A2 interacts with the MV matrix (M) protein and mediates the localization of the M protein at the plasma membrane where MV particles are formed. The M protein lines the inner surface of the MV envelope membrane and plays a role in MV particle formation. Our results provide useful information for the understanding of the MV replication process and potential development of anti-viral agents. Copyright © 2018 American Society for Microbiology.

Ses proteins as possible targets for vaccine development against Staphylococcus epidermidis infections.

PubMed

Hofmans, Dorien; Khodaparast, Laleh; Khodaparast, Ladan; Vanstreels, Els; Shahrooei, Mohammad; Van Eldere, Johan; Van Mellaert, Lieve

2018-05-09

The opportunistic pathogen Staphylococcus epidermidis is progressively involved in device-related infections. Since these infections involve biofilm formation, antibiotics are not effective. Conversely, a vaccine can be advantageous to prevent these infections. In view of vaccine development, predicted surface proteins were evaluated on their potential as a vaccine target. Immunoglobulins directed against S. epidermidis surface proteins SesB, M, O, Q and R, were used to firstly affirm their surface location. Further, inhibitory effects of these IgGs on biofilm formation were determined in vitro on polystyrene and polyurethane surfaces and in vivo using a subcutaneous catheter mouse model. We also examined the opsonophagocytic capacity of these IgGs. Surface localization of the five Ses proteins was demonstrated both for planktonic and sessile cells, though to a variable extent. Ses-specific IgGs added to planktonic cells had a variable inhibitory effect on cell adhesion to polystyrene, while only anti-SesO IgGs decreased cell attachment to polyurethane catheters. Although phagocytic killing was only obtained after opsonisation with SesB-specific IgGs, a significant reduction of in vivo formed biofilms was observed after administration of SesB-, SesM- and SesO-specific IgGs. Regardless of their characterization or function, S. epidermidis surface proteins can be adequate targets for vaccine development aiming the prevention of device-related infections caused by invasive S. epidermidis strains. Copyright © 2018 Elsevier Ltd. All rights reserved.

Proteolysis during Tumor Cell Extravasation In Vitro: Metalloproteinase Involvement across Tumor Cell Types

PubMed Central

Voura, Evelyn B.; English, Jane L.; Yu, Hoi-Ying E.; Ho, Andrew T.; Subarsky, Patrick; Hill, Richard P.; Hojilla, Carlo V.; Khokha, Rama

2013-01-01

To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and αvβ3, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment. PMID:24194929

The nanoscale spatial organization of B-cell receptors on immunoglobulin M- and G-expressing human B-cells.

PubMed

Lee, Jinmin; Sengupta, Prabuddha; Brzostowski, Joseph; Lippincott-Schwartz, Jennifer; Pierce, Susan K

2017-02-15

B-cell activation is initiated by the binding of antigen to the B-cell receptor (BCR). Here we used dSTORM superresolution imaging to characterize the nanoscale spatial organization of immunoglobulin M (IgM) and IgG BCRs on the surfaces of resting and antigen--activated human peripheral blood B-cells. We provide insights into both the fundamental process of antigen-driven BCR clustering and differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of naive and memory B-cells to antigen. We provide evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in size and number of BCR single-molecule localizations, both resting and activated B-cells intrinsically maintain a high -frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands after antigen activation. Small, dense BCR clusters likely formed via protein-protein interactions are present on the surface of resting cells, and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein-lipid interactions. © 2017 Lee, Sengupta, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Ick Ciliary Kinase Is Essential for Planar Cell Polarity Formation in Inner Ear Hair Cells and Hearing Function.

PubMed

Okamoto, Shio; Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Kubo, Shun; Sakaguchi, Hirofumi; Furukawa, Takahisa

2017-02-22

Cellular asymmetries play crucial roles in development and organ function. The planar cell polarity (PCP) signaling pathway is involved in the establishment of cellular asymmetry within the plane of a cell sheet. Inner ear sensory hair cells (HCs), which have several rows of staircase-like stereocilia and one kinocilium located at the vertex of the stereocilia protruding from the apical surface of each HC, exhibit a typical form of PCP. Although connections between cilia and PCP signaling in vertebrate development have been reported, their precise nature is not well understood. During inner ear development, several ciliary proteins are known to play a role in PCP formation. In the current study, we investigated a functional role for intestinal cell kinase (Ick), which regulates intraflagellar transport (IFT) at the tip of cilia, in the mouse inner ear. A lack of Ick in the developing inner ear resulted in PCP defects in the cochlea, including misorientation or misshaping of stereocilia and aberrant localization of the kinocilium and basal body in the apical and middle turns, leading to auditory dysfunction. We also observed abnormal ciliary localization of Ift88 in both HCs and supporting cells. Together, our results show that Ick ciliary kinase is essential for PCP formation in inner ear HCs, suggesting that ciliary transport regulation is important for PCP signaling. SIGNIFICANCE STATEMENT The cochlea in the inner ear is the hearing organ. Planar cell polarity (PCP) in hair cells (HCs) in the cochlea is essential for mechanotransduction and refers to the asymmetric structure consisting of stereociliary bundles and the kinocilium on the apical surface of the cell body. We reported previously that a ciliary kinase, Ick, regulates intraflagellar transport (IFT). Here, we found that loss of Ick leads to abnormal localization of the IFT component in kinocilia, PCP defects in HCs, and hearing dysfunction. Our study defines the association of ciliary transport regulation with PCP formation in HCs and hearing function. Copyright © 2017 the authors 0270-6474/17/372073-13$15.00/0.

Enhancement of Cell Membrane Invaginations, Vesiculation and Uptake of Macromolecules by Protonation of the Cell Surface

PubMed Central

Ben-Dov, Nadav; Korenstein, Rafi

2012-01-01

The different pathways of endocytosis share an initial step involving local inward curvature of the cell’s lipid bilayer. It has been shown that to generate membrane curvature, proteins or lipids enforce transversal asymmetry of the plasma membrane. Thus it emerges as a general phenomenon that transversal membrane asymmetry is the common required element for the formation of membrane curvature. The present study demonstrates that elevating proton concentration at the cell surface stimulates the formation of membrane invaginations and vesiculation accompanied by efficient uptake of macromolecules (Dextran-FITC, 70 kD), relative to the constitutive one. The insensitivity of proton induced uptake to inhibiting treatments and agents of the known endocytic pathways suggests the entry of macromolecules to proceeds via a yet undefined route. This is in line with the fact that neither ATP depletion, nor the lowering of temperature, abolishes the uptake process. In addition, fusion mechanism such as associated with low pH uptake of toxins and viral proteins can be disregarded by employing the polysaccharide dextran as the uptake molecule. The proton induced uptake increases linearly in the extracellular pH range of 6.5 to 4.5, and possesses a steep increase at the range of 4> pH>3, reaching a plateau at pH≤3. The kinetics of the uptake implies that the induced vesicles release their content to the cytosol and undergo rapid recycling to the plasma membrane. We suggest that protonation of the cell’s surface induces local charge asymmetries across the cell membrane bilayer, inducing inward curvature of the cell membrane and consequent vesiculation and uptake. PMID:22558127

A high-throughput microparticle microarray platform for dendritic cell-targeting vaccines.

PubMed

Acharya, Abhinav P; Clare-Salzler, Michael J; Keselowsky, Benjamin G

2009-09-01

Immunogenomic approaches combined with advances in adjuvant immunology are guiding progress toward rational design of vaccines. Furthermore, drug delivery platforms (e.g., synthetic particles) are demonstrating promise for increasing vaccine efficacy. Currently there are scores of known antigenic epitopes and adjuvants, and numerous synthetic delivery systems accessible for formulation of vaccines for various applications. However, the lack of an efficient means to test immune cell responses to the abundant combinations available represents a significant blockade on the development of new vaccines. In order to overcome this barrier, we report fabrication of a new class of microarray consisting of antigen/adjuvant-loadable poly(D,L lactide-co-glycolide) microparticles (PLGA MPs), identified as a promising carrier for immunotherapeutics, which are co-localized with dendritic cells (DCs), key regulators of the immune system and prime targets for vaccines. The intention is to utilize this high-throughput platform to optimize particle-based vaccines designed to target DCs in vivo for immune system-related disorders, such as autoimmune diseases, cancer and infection. Fabrication of DC/MP arrays leverages the use of standard contact printing miniarraying equipment in conjunction with surface modification to achieve co-localization of particles/cells on isolated islands while providing background non-adhesive surfaces to prevent off-island cell migration. We optimized MP overspotting pin diameter, accounting for alignment error, to allow construction of large, high-fidelity arrays. Reproducible, quantitative delivery of as few as 16+/-2 MPs per spot was demonstrated and two-component MP dosing arrays were constructed, achieving MP delivery which was independent of formulation, with minimal cross-contamination. Furthermore, quantification of spotted, surface-adsorbed MP degradation was demonstrated, potentially useful for optimizing MP release properties. Finally, we demonstrate DC co-localization with PLGA MPs on isolated islands and that DCs do not migrate between islands for up to 24 h. Using this platform, we intend to analyze modulation of DC function by providing multi-parameter combinatorial cues in the form of proteins, peptides and other immuno-modulatory molecules encapsulated in or tethered on MPs. Critically, the miniaturization attained enables high-throughput investigation of rare cell populations by reducing the requirement for cells and reagents by many-fold, facilitating advances in personalized vaccines which target DCs in vivo.

Studies by immune electron microscopy of hepatitis B surface antigen in PLC/PRF/5 cells.

PubMed

Shibayama, T; Watanabe, T; Kojima, H; Yoshikawa, A; Watanabe, S; Kamimura, T; Suzuki, S; Ichida, F

1984-01-01

Electron microscopic studies of the morphology of hepatitis B surface antigen (HBsAg) produced by PLC/PRF/5 cells in vitro were carried out. Aggregates of 20-nm spherical particles in 3-day culture supernatants were observed by immune electron microscopy (IEM). Aggregates of tubular structures were found with IEM in the extracts of the cells. Tubular structures 18 to 22 nm in diameter were seen by electron microscopy (EM) in the cisternae of the endoplasmic reticulum in 2-3% of the cells. The tubular structures in the cytoplasm and extracts of PLC/PRF/5 cells resembled those observed in the hepatocytes of human carriers of hepatitis B virus (HBV). Intracellular localization of HBsAg in PLC/PRF/5 cells by direct peroxidase-conjugated antibody staining was observed on the tubular structures and the cisternal wall, which contained these structures. Rotation technique analysis indicated that the tubular structures were composed of 11 or 12 subunits.

22.7% efficient PERL silicon solar cell module with a textured front surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, J.; Wang, A.; Campbell, P.

1997-12-31

This paper describes a solar cell module efficiency of 22.7% independently measured at Sandia National Laboratories. This is the highest ever confirmed efficiency for a photovoltaic module of this size achieved by cells made from any material. This 778-cm{sup 2} module used 40 large-area double layer antireflection coated PERL (passivated emitter, rear locally-diffused) silicon cells of average efficiency of 23.1%. A textured front module surface considerably improve the module efficiency. Also reported is an independently confirmed efficiency of 23.7% for a 21.6 cm{sup 2} cell of the type used in the module. Using these PERL cells in the 1996 Worldmore » Solar Challenge solar car race from Darwin to Adelaide across Australia, Honda`s Dream and Aisin Seiki`s Aisol III were placed first and third, respectively. Honda also set a new record by reaching Adelaide in four days with an average speed of 90km/h over the 3010 km course.« less

The Microtubule-Stabilizing Protein CLASP1 Associates with the Theileria annulata Schizont Surface via Its Kinetochore-Binding Domain

PubMed Central

Huber, Sandra; Theiler, Romina; de Quervain, Daniel; Wiens, Olga; Karangenc, Tulin; Heussler, Volker; Dobbelaere, Dirk

2017-01-01

ABSTRACT Theileria is an apicomplexan parasite whose presence within the cytoplasm of a leukocyte induces cellular transformation and causes uncontrolled proliferation and clonal expansion of the infected cell. The intracellular schizont utilizes the host cell’s own mitotic machinery to ensure its distribution to both daughter cells by associating closely with microtubules (MTs) and incorporating itself within the central spindle. We show that CLASP1, an MT-stabilizing protein that plays important roles in regulating kinetochore-MT attachment and central spindle positioning, is sequestered at the Theileria annulata schizont surface. We used live-cell imaging and immunofluorescence in combination with MT depolymerization assays to demonstrate that CLASP1 binds to the schizont surface in an MT-independent manner throughout the cell cycle and that the recruitment of the related CLASP2 protein to the schizont is MT dependent. By transfecting Theileria-infected cells with a panel of truncation mutants, we found that the kinetochore-binding domain of CLASP1 is necessary and sufficient for parasite localization, revealing that CLASP1 interaction with the parasite occurs independently of EB1. We overexpressed the MT-binding domain of CLASP1 in parasitized cells. This exhibited a dominant negative effect on host MT stability and led to altered parasite size and morphology, emphasizing the importance of proper MT dynamics for Theileria partitioning during host cell division. Using coimmunoprecipitation, we demonstrate that CLASP1 interacts, directly or indirectly, with the schizont membrane protein p104, and we describe for the first time TA03615, a Theileria protein which localizes to the parasite surface, where it has the potential to participate in parasite-host interactions. IMPORTANCE T. annulata, the only eukaryote known to be capable of transforming another eukaryote, is a widespread parasite of veterinary importance that puts 250 million cattle at risk worldwide and limits livestock development for some of the poorest people in the world. Crucial to the pathology of Theileria is its ability to interact with host microtubules and the mitotic spindle of the infected cell. This study builds on our previous work in investigating the host and parasite molecules involved in mediating this interaction. Because it is not possible to genetically manipulate Theileria schizonts, identifying protein interaction partners is critical to understanding the function of parasite proteins. By identifying two Theileria surface proteins that are involved in the interaction between CLASP1 and the parasite, we provide important insights into the molecular basis of Theileria persistence within a dividing cell. PMID:28861517

Expression of nestin, mesothelin and epithelial membrane antigen (EMA) in developing and adult human meninges and meningiomas.

PubMed

Petricevic, Josko; Forempoher, Gea; Ostojic, Ljerka; Mardesic-Brakus, Snjezana; Andjelinovic, Simun; Vukojevic, Katarina; Saraga-Babic, Mirna

2011-11-01

The spatial and temporal pattern of appearance of nestin, epithelial membrane antigen (EMA) and mesothelin proteins was immunohistochemically determined in the cells of normal developing and adult human meninges and meningiomas. Human meninges developed as two mesenchymal condensations in the head region. The simple squamous epithelium on the surface of leptomeninges developed during mesenchymal to epithelial transformation. Nestin appeared for the first time in week 7, EMA in week 8, while mesothelin appeared in week 22 of development. In the late fetal period and after birth, nestin expression decreased, whereas expression of EMA and mesothelin increased. EMA appeared in all surface epithelial cells and nodules, while mesothelin was found only in some of them. In adult meninges, all three proteins were predominantly localized in the surface epithelium and meningeal nodules. In meningothelial meningiomas (WHO grade I), EMA was detected in all tumor cells except in the endothelial cells, mesothelin characterized nests of tumor cells, while nestin was found predominantly in the walls of blood vessels. The distribution pattern of those proteins in normal meningeal and tumor cells indicates that nestin might characterize immature cells, while EMA and mesothelin appeared in maturing epithelial cells. Neoplastic transformation of these specific cell lineages contributes to the cell population in meningiomas. Copyright © 2010 Elsevier GmbH. All rights reserved.

Geometrical Effects on Nonlinear Electrodiffusion in Cell Physiology

NASA Astrophysics Data System (ADS)

Cartailler, J.; Schuss, Z.; Holcman, D.

2017-12-01

We report here new electrical laws, derived from nonlinear electrodiffusion theory, about the effect of the local geometrical structure, such as curvature, on the electrical properties of a cell. We adopt the Poisson-Nernst-Planck equations for charge concentration and electric potential as a model of electrodiffusion. In the case at hand, the entire boundary is impermeable to ions and the electric field satisfies the compatibility condition of Poisson's equation. We construct an asymptotic approximation for certain singular limits to the steady-state solution in a ball with an attached cusp-shaped funnel on its surface. As the number of charge increases, they concentrate at the end of cusp-shaped funnel. These results can be used in the design of nanopipettes and help to understand the local voltage changes inside dendrites and axons with heterogeneous local geometry.

ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.

PubMed

Woodfin, Abigail; Beyrau, Martina; Voisin, Mathieu-Benoit; Ma, Bin; Whiteford, James R; Hordijk, Peter L; Hogg, Nancy; Nourshargh, Sussan

2016-02-18

Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense. © 2016 by The American Society of Hematology.

The distribution of serum albumin in the rat testis, studied by electron microscope immunocytochemistry on ultrathin frozen sections.

PubMed

Christensen, A K; Komorowski, T E; Wilson, B; Ma, S F; Stevens, R W

1985-05-01

The distribution of serum albumin is of interest in the rat testis because this protein is the principal carrier for testosterone in the plasma and interstitial fluid of this species. We have localized extravascular serum albumin in the rat testis at the electron microscope level, using gold particle immunocytochemistry on ultrathin frozen sections of tissue fixed lightly by perfusion. The same localization was obtained with three different antisera. Preabsorption and normal rabbit serum controls were negative, and Western blots of testis extracts showed major activity only at the molecular weight of albumin. Serum albumin occurred in substantial concentration throughout extracellular space in the interstitial tissue, as well as in the space between the boundary layer and the base of the seminiferous epithelium. Immunoreactivity extended between Sertoli cells, as well as around spermatogonia and early primary spermatocytes (to stage 11), but did not traverse the Sertoli-Sertoli junctions that comprise the blood-testis barrier. Macrophages in the interstitial tissue showed some endocytic activity. If perfusion fixation was carried out in a manner that flushed most of the albumin from the interstitial space, then a layer of albumin remained on the surface of Leydig cells and many macrophages but was minimal or absent on the surface of other cell types that are normally in contact with albumin, such as Sertoli cells, spermatogonia, myoid cells, lymphatic endothelium, fibroblasts, or cells of blood vessels.

Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

PubMed Central

Bell, Karen; Mitchell, Steve; Paultre, Danae; Posch, Markus; Oparka, Karl

2013-01-01

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology. PMID:23457228

Controlling Androgen receptor nuclear localization by dendrimer conjugates

NASA Astrophysics Data System (ADS)

Wang, Haoyu

Androgen Receptor (AR) antagonists, such as bicalutamide and flutamide have been used widely in the treatment of prostate cancer. Although initial treatment is effective, prostate cancer cells often acquire antiandrogen resistance with prolonged treatment. AR over-expression and AR mutations contribute to the development of antiandrogen resistant cancer. Second generation antiandrogens such as enzalutamide are more effective and show reduced AR nuclear localization. In this study, derivatives of PAN52, a small molecule antiandrogen previously developed in our lab, were conjugated to the surface of generation 4 and generation 6 PAMAM dendrimers to obtain antiandrogen PAMAM dendrimer conjugates (APDC). APDCs readily enter cells and associate with AR in the cytoplasm. Due to their large size and positive charge, they can not enter the nucleus, thus retaining AR in the cytoplasm. In addition, APDCs are effective in decreasing AR mediated transcription and cell proliferation. APDC is the first AR antagonists that inhibit DHT-induced nuclear localization of AR. By inhibiting AR nuclear localization, APDC represents a new class of antiandrogens that offer an alternative approach to addressing antiandrogen-resistant prostate cancer. Lysine post-translational modification of AR Nuclear Localization Sequence (NLS) has great impact on AR cellular localization. It is of interest to understand which modifications modulate AR translocation into the nucleus. In this study, we prepared dendrimer-based acetyltransferase mimetic (DATM), DATM is able to catalytically acetylate AR in CWR22Rv1 cells, which will be a useful tool for studying AR modification effect on AR cellular localization. Derivatives of DATM, which transfer other chemical groups to AR, can be prepared similarly, and with more dendrimer based AR modification tools prepared in future, we will be able to understand and control AR cellular localization through AR modification.

Simulation of Escherichia coli Dynamics in Biofilms and Submerged Colonies with an Individual-Based Model Including Metabolic Network Information.

PubMed

Tack, Ignace L M M; Nimmegeers, Philippe; Akkermans, Simen; Hashem, Ihab; Van Impe, Jan F M

2017-01-01

Clustered microbial communities are omnipresent in the food industry, e.g., as colonies of microbial pathogens in/on food media or as biofilms on food processing surfaces. These clustered communities are often characterized by metabolic differentiation among their constituting cells as a result of heterogeneous environmental conditions in the cellular surroundings. This paper focuses on the role of metabolic differentiation due to oxygen gradients in the development of Escherichia coli cell communities, whereby low local oxygen concentrations lead to cellular secretion of weak acid products. For this reason, a metabolic model has been developed for the facultative anaerobe E. coli covering the range of aerobic, microaerobic, and anaerobic environmental conditions. This metabolic model is expressed as a multiparametric programming problem, in which the influence of low extracellular pH values and the presence of undissociated acid cell products in the environment has been taken into account. Furthermore, the developed metabolic model is incorporated in MICRODIMS, an in-house developed individual-based modeling framework to simulate microbial colony and biofilm dynamics. Two case studies have been elaborated using the MICRODIMS simulator: (i) biofilm growth on a substratum surface and (ii) submerged colony growth in a semi-solid mixed food product. In the first case study, the acidification of the biofilm environment and the emergence of typical biofilm morphologies have been observed, such as the mushroom-shaped structure of mature biofilms and the formation of cellular chains at the exterior surface of the biofilm. The simulations show that these morphological phenomena are respectively dependent on the initial affinity of pioneer cells for the substratum surface and the cell detachment process at the outer surface of the biofilm. In the second case study, a no-growth zone emerges in the colony center due to a local decline of the environmental pH. As a result, cellular growth in the submerged colony is limited to the colony periphery, implying a linear increase of the colony radius over time. MICRODIMS has been successfully used to reproduce complex dynamics of clustered microbial communities.

Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces

PubMed Central

Venable, Alison; Mitalipova, Maisam; Lyons, Ian; Jones, Karen; Shin, Soojung; Pierce, Michael; Stice, Steven

2005-01-01

Background Pluripotent human embryonic stem cells (hESCs) have the potential to form every cell type in the body. These cells must be appropriately characterized prior to differentiation studies or when defining characteristics of the pluripotent state. Some developmentally regulated cell surface antigens identified by monoclonal antibodies in a variety of species and stem cell types have proven to be side chains of membrane glycolipids and glycoproteins. Therefore, to examine hESC surfaces for other potential pluripotent markers, we used a panel of 14 lectins, which were chosen based on their specificity for a variety of carbohydrates and carbohydrate linkages, along with stage specific embryonic antigen-4 (SSEA-4), to determine binding quantitation by flow cytometry and binding localization in adherent colonies by immunocytochemistry. Results Enriching cells for SSEA-4 expression increased the percentage of SSEA-4 positive cells to 98–99%. Using enriched high SSEA-4-expressing hESCs, we then analyzed the binding percentages of selected lectins and found a large variation in binding percentages ranging from 4% to 99% binding. Lycopersicon (tomato)esculetum lectin (TL), Ricinus communis agglutinin (RCA), and Concanavalin A (Con A) bound to SSEA-4 positive regions of hESCs and with similar binding percentages as SSEA-4. In contrast, we found Dolichos biflorus agglutinin (DBA) and Lotus tetragonolobus lectin (LTL) did not bind to hESCs while Phaseolus vulgaris leuco-agglutinin (PHA-L), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin (UEA), Phaseolus vulgaris erythro-agglutinin (PHA-E), and Maackia amurensis agglutinin (MAA) bound partially to hESCs. These binding percentages correlated well with immunocytochemistry results. Conclusion Our results provide information about types of carbohydrates and carbohydrate linkages found on pluripotent hESC surfaces. We propose that TL, RCA and Con A may be used as markers that are associated with the pluripotent state of hESCs because binding percentages and binding localization of these lectins are similar to those of SSEA-4. Non-binding lectins, DBA and LTL, may identify differentiated cell types; however, we did not find these lectins to bind to pluripotent SSEA-4 positive hESCs. This work represents a fundamental base to systematically classify pluripotent hESCs, and in future studies these lectins may be used to distinguish differentiated hESC types based on glycan presentation that accompanies differentiation. PMID:16033656

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

NASA Astrophysics Data System (ADS)

Duman, M.; Pfleger, M.; Zhu, R.; Rankl, C.; Chtcheglova, L. A.; Neundlinger, I.; Bozna, B. L.; Mayer, B.; Salio, M.; Shepherd, D.; Polzella, P.; Moertelmaier, M.; Kada, G.; Ebner, A.; Dieudonne, M.; Schütz, G. J.; Cerundolo, V.; Kienberger, F.; Hinterdorfer, P.

2010-03-01

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on α-galactosylceramide (αGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from ~ 25 to ~ 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

PubMed

Duman, M; Pfleger, M; Zhu, R; Rankl, C; Chtcheglova, L A; Neundlinger, I; Bozna, B L; Mayer, B; Salio, M; Shepherd, D; Polzella, P; Moertelmaier, M; Kada, G; Ebner, A; Dieudonne, M; Schütz, G J; Cerundolo, V; Kienberger, F; Hinterdorfer, P

2010-03-19

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Differential subcellular distribution of ion channels and the diversity of neuronal function.

PubMed

Nusser, Zoltan

2012-06-01

Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Role of Integrin Subunits in Mesenchymal Stem Cell Differentiation and Osteoblast Maturation on Graphitic Carbon-coated Microstructured Surfaces

PubMed Central

Olivares-Navarrete, Rene; Rodil, Sandra E.; Hyzy, Sharon L.; Dunn, Ginger R.; Almaguer-Flores, Argelia; Schwartz, Zvi; Boyan, Barbara D.

2015-01-01

Surface roughness, topography, chemistry, and energy promote osteoblast differentiation and increase osteogenic local factor production in vitro and bone-to-implant contact in vivo, but the mechanisms involved are not well understood. Knockdown of integrin heterodimer alpha2beta1 (α2β1) blocks the osteogenic effects of the surface, suggesting signaling by this integrin homodimer is required. The purpose of the present study was to separate effects of surface chemistry and surface structure on integrin expression by coating smooth or rough titanium (Ti) substrates with graphitic carbon, retaining surface morphology but altering surface chemistry. Ti surfaces (smooth [Ra<0.4μm], rough [Ra≥3.4μm]) were sputter-coated using a magnetron sputtering system with an ultrapure graphite target, producing a graphitic carbon thin film. Human mesenchymal stem cells and MG63 osteoblast-like cells had higher mRNA for integrin subunits α1, α2, αv, and β1 on rough surfaces in comparison to smooth, and integrin αv on graphitic-carbon-coated rough surfaces in comparison to Ti. Osteogenic differentiation was greater on rough surfaces in comparison to smooth, regardless of chemistry. Silencing integrins β1, α1, or α2 decreased osteoblast maturation on rough surfaces independent of surface chemistry. Silencing integrin αv decreased maturation only on graphitic carbon-coated surfaces, not on Ti. These results suggest a major role of the integrin β1 subunit in roughness recognition, and that integrin alpha subunits play a major role in surface chemistry recognition. PMID:25770999

Principles and biophysical applications of single particle super-localization and rotational tracking

NASA Astrophysics Data System (ADS)

Gu, Yan

While conventional Single Particle Tracking (SPT) techniques acquire 2D or 3D trajectories of particle probes, we have developed Single Particle Orientation and Rotational Tracking (SPORT) techniques to extract orientation and rotational information. Combined with DIC microscopy, the SPORT technique has been applied in biophysical studies, including membrane diffusion and intracellular transport. The rotational dynamics of nanoparticle vectors on live cell membranes was recorded and its influence on the fate of these nanoparticle vectors was elucidated. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at a temporal resolution of 5 ms under a DIC microscope. We found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charge, specific surface functional groups, and the availability of receptors on cell membranes. The study of rotational Brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances. To characterize the rotation mode of surface functionalized gold nanorods on cell membranes, the SPORT technique is combined with the correlation analysis of the bright and dark DIC intensities. The unique capabilities of visualizing and understanding rotational motions of functionalized nanoparticles on live cell membranes allow us to correlate rotational and translational dynamics in unprecedented detail and provide new insights for complex membrane processes, including electrostatic interactions, ligand-receptor binding, and lateral (confined and hopping) diffusion of membrane receptors. Surface-functionalized nanoparticles interact with the membrane in fundamentally different ways and exhibit distinct rotational modes. The early events of particle-membrane approach and attachment are directly visualized for the first time. The rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport was also visualized using high-speed SPORT with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause. To improve the localization precision of the SPT technique with DIC microscopy, a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping was introduced. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events. Finally, a novel dual-modality imaging technique has been developed to super-localize a single gold nanorod while providing its orientation and rotational information. The super-localization of the gold nanorod can be accomplished by curve fitting the modified bright-field images generated by one of the two beams laterally shifted by the first Nomarski prism in a DIC microscope. The orientation and rotational information is derived from the DIC images of gold nanorods. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment.

Principles and biophysical applications of single particle super-localization and rotational tracking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Yan

While conventional Single Particle Tracking (SPT) techniques acquire 2D or 3D trajectories of particle probes, we have developed Single Particle Orientation and Rotational Tracking (SPORT) techniques to extract orientation and rotational information. Combined with DIC microscopy, the SPORT technique has been applied in biophysical studies, including membrane diffusion and intracellular transport. The rotational dynamics of nanoparticle vectors on live cell membranes was recorded and its influence on the fate of these nanoparticle vectors was elucidated. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at a temporal resolution of 5 ms under a DIC microscope. Wemore » found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charge, specific surface functional groups, and the availability of receptors on cell membranes. The study of rotational Brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances. To characterize the rotation mode of surface functionalized gold nanorods on cell membranes, the SPORT technique is combined with the correlation analysis of the bright and dark DIC intensities. The unique capabilities of visualizing and understanding rotational motions of functionalized nanoparticles on live cell membranes allow us to correlate rotational and translational dynamics in unprecedented detail and provide new insights for complex membrane processes, including electrostatic interactions, ligand-receptor binding, and lateral (confined and hopping) diffusion of membrane receptors. Surface-functionalized nanoparticles interact with the membrane in fundamentally different ways and exhibit distinct rotational modes. The early events of particle-membrane approach and attachment are directly visualized for the first time. The rotational dynamics of cargos in both active directional transport and pausing stages of axonal transport was also visualized using high-speed SPORT with a temporal resolution of 2 ms. Both long and short pauses are imaged, and the correlations between the pause duration, the rotational behaviour of the cargo at the pause, and the moving direction after the pause are established. Furthermore, the rotational dynamics leading to switching tracks are visualized in detail. These first-time observations of cargo's rotational dynamics provide new insights on how kinesin and dynein motors take the cargo through the alternating stages of active directional transport and pause. To improve the localization precision of the SPT technique with DIC microscopy, a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping was introduced. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events. Finally, a novel dual-modality imaging technique has been developed to super-localize a single gold nanorod while providing its orientation and rotational information. The super-localization of the gold nanorod can be accomplished by curve fitting the modified bright-field images generated by one of the two beams laterally shifted by the first Nomarski prism in a DIC microscope. The orientation and rotational information is derived from the DIC images of gold nanorods. The new imaging setup has been applied to study the steric hindrance induced by relatively large cargos in the microtubule gliding assay and to track nanocargos in the crowded cellular environment.« less

Nanoscale cellular imaging with scanning angle interference microscopy.

PubMed

DuFort, Christopher; Paszek, Matthew

2014-01-01

Fluorescence microscopy is among the most widely utilized tools in cell and molecular biology due to its ability to noninvasively obtain time-resolved images of live cells with molecule-specific contrast. In this chapter, we describe a simple high-resolution technique, scanning angle interference microscopy (SAIM), for the imaging and localization of fluorescent molecules with nanometer precision along the optical axis. In SAIM, samples above a reflective surface are sequentially scanned with an excitation laser at varying angles of incidence. Interference patterns generated between the incident and reflected lights result in an emission intensity that depends on the height of a fluorophore above the silicon surface and the angle of the incident radiation. The measured fluorescence intensities are then fit to an optical model to localize the labeled molecules along the z-axis with 5-10 nm precision and diffraction-limited lateral resolution. SAIM is easily implemented on widely available commercial total internal reflection fluorescence microscopes, offering potential for widespread use in cell biology. Here, we describe the setup of SAIM and its application for imaging cellular structures near (<1 μm) the sample substrate. © 2014 Elsevier Inc. All rights reserved.

Vertical uniformity of cells and nuclei in epithelial monolayers.

PubMed

Neelam, Srujana; Hayes, Peter Robert; Zhang, Qiao; Dickinson, Richard B; Lele, Tanmay P

2016-01-22

Morphological variability in cytoskeletal organization, organelle position and cell boundaries is a common feature of cultured cells. Remarkable uniformity and reproducibility in structure can be accomplished by providing cells with defined geometric cues. Cells in tissues can also self-organize in the absence of directing extracellular cues; however the mechanical principles for such self-organization are not understood. We report that unlike horizontal shapes, the vertical shapes of the cell and nucleus in the z-dimension are uniform in cells in cultured monolayers compared to isolated cells. Apical surfaces of cells and their nuclei in monolayers were flat and heights were uniform. In contrast, isolated cells, or cells with disrupted cell-cell adhesions had nuclei with curved apical surfaces and variable heights. Isolated cells cultured within micron-sized square wells displayed flat cell and nuclear shapes similar to cells in monolayers. Local disruption of nuclear-cytoskeletal linkages resulted in spatial variation in vertical uniformity. These results suggest that competition between cell-cell pulling forces that expand and shorten the vertical cell cross-section, thereby widening and flattening the nucleus, and the resistance of the nucleus to further flattening results in uniform cell and nuclear cross-sections. Our results reveal the mechanical principles of self-organized vertical uniformity in cell monolayers.

Developmental Localization of Nephrin in Zebrafish and Medaka Pronephric Glomerulus

PubMed Central

Ichimura, Koichiro; Fukuyo, Yayoi; Nakamura, Tomomi; Powell, Rebecca; Sakai, Tatsuo; Janknecht, Ralf

2013-01-01

Slit diaphragm (SD) is a highly specialized intercellular junction between podocyte foot processes and plays a crucial role in the formation of the filtration barrier. In this study, we examined the developmental localization of Nephrin, an essential component of SD, in the pronephric glomerulus of zebrafish and medaka. In the mature glomerulus of both fish, Nephrin is localized along the glomerular basement membrane as seen in mammals, indicating that Nephrin is localized at the SD. Interestingly, Nephrin was detected already in immature podocytes before the SD and foot processes started to form in both fish. Nephrin was localized along the cell surface of immature podocytes but as different localization patterns. In zebrafish, Nephrin signal bordered the lateral membrane of podocytes, which were columnar in shape, as in rat immature podocytes. However, in medaka immature podocytes, Nephrin was localized in a punctate pattern among podocyte cell bodies. These findings suggest that Nephrin needs to be integrated to the membrane before the formation of the SD and then moves to the proper site to form the SD. Furthermore, a podocyte-specific marker, such as Nephrin, should be a useful tool for the future analysis of pronephric glomerular development in fish mutants and morphants. PMID:23324868

The effect of surface functionality on cellular trafficking of dendrimers.

PubMed

Perumal, Omathanu P; Inapagolla, Rajyalakshmi; Kannan, Sujatha; Kannan, Rangaramanujam M

2008-01-01

Dendrimers are an emerging group of nanostructured, polymeric biomaterials that have potential as non-viral vehicles for delivering drugs and genetic material to intracellular targets. They have a high charge density with tunable surface functional groups, which can alter the local environment and influence cellular interactions. This can have a significant impact on the intracellular trafficking of dendrimer-based nanodevices. With the help of flow cytometry, fluorescence microscopy, and by using specific inhibitors, the influence of surface functionality on their uptake in A549 lung epithelial cells, and subsequent intracellular distribution was investigated. In this paper, we have shown that even though all the dendrimers are taken up by fluid-phase endocytosis, significant differences in uptake mechanisms exist. Anionic dendrimers appear to be mainly taken up by caveolae mediated endocytosis in A549 lung epithelial cells, while cationic and neutral dendrimers appear to be taken in by a non-clathrin, non-caveolae mediated mechanism that may be by electrostatic interactions or other non-specific fluid-phase endocytosis. These findings open up new possibilities of targeting therapeutic agents to specific cell organelles based on surface charge.

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

PubMed

Stockis, Julie; Colau, Didier; Coulie, Pierre G; Lucas, Sophie

2009-12-01

Human Treg and Th clones secrete the latent form of TGF-beta, in which the mature TGF-beta protein is bound to the latency-associated peptide (LAP), and is thereby prevented from binding to the TGF-beta receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF-beta and bear LAP on their surface. Here, we show that latent TGF-beta, i.e. both LAP and mature TGF-beta, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF-beta mediated by binding to GARP may be necessary for the ability of Treg to activate TGF-beta upon TCR stimulation. However, it is not sufficient as lentiviral-mediated expression of GARP in human Th cells induces binding of latent TGF-beta to the cell surface, but does not result in the production of active TGF-beta upon stimulation of these Th cells.

Photovoltaic modules with cylindrical waveguides in a system for the secondary concentration of solar radiation

NASA Astrophysics Data System (ADS)

Andreev, V. M.; Davidyuk, N. Yu.; Ionova, E. A.; Rumyantsev, V. D.

2013-09-01

The parameters of the concentrating photoelectric modules with triple-junction (InGaP/GaAs/Ge) solar cells whose focusing system contains an original secondary optical element are studied. The element consists of a plane-convex lens in optical contact with the front surface of an intermediate glass plate and a cylindrical waveguide that is located on the rear side of the glass plate above the surface of the solar element. It is demonstrated that the structure of the secondary optical element provides a wide misorientation characteristic of the concentrator and the cylindrical waveguide allows a more uniform radiation density over the surface of the solar cell. The effect of chromatic aberration in the primary and secondary optical systems on the parameters of photoelectric modules is analyzed. It is demonstrated that the presence of waveguides with a length of 3-5 mm leads to effective redistribution of radiation over the surface of the solar cell whereas shorter and longer waveguides provide the local concentration of radiation at the center of the photodetecting area.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Role of Complement on Broken Surfaces After Trauma.

PubMed

Huber-Lang, Markus; Ignatius, Anita; Brenner, Rolf E

2015-01-01

Activation of both the complement and coagulation cascade after trauma and subsequent local and systemic inflammatory response represent a major scientific and clinical problem. After severe tissue injury and bone fracture, exposure of innate immunity to damaged cells and molecular debris is considered a main trigger of the posttraumatic danger response. However, the effects of cellular fragments (e.g., histones) on complement activation remain enigmatic. Furthermore, direct effects of "broken" bone and cartilage surfaces on the fluid phase response of complement and its interaction with key cells of connective tissues are still unknown. Here, we summarize data suggesting direct and indirect complement activation by extracellular and cellular danger associated molecular patterns. In addition, key complement components and the corresponding receptors (such as C3aR, C5aR) have been detected on "exposed surfaces" of the damaged regions. On a cellular level, multiple effects of complement activation products on osteoblasts, osteoclasts, chondrocytes and mesenchymal stem cells have been found.In conclusion, the complement system may be activated by trauma-altered surfaces and is crucially involved in connective tissue healing and posttraumatic systemic inflammatory response.

An N-terminal Retention Module Anchors the Giant Adhesin LapA of Pseudomonas fluorescens at the Cell Surface: A Novel Sub-family of Type I Secretion Systems.

PubMed

Smith, T Jarrod; Font, Maria E; Kelly, Carolyn M; Sondermann, Holger; O'Toole, George A

2018-02-05

LapA of Pseudomonas fluorescens Pf0-1 belongs to a diverse family of cell surface associated bacterial adhesins that are secreted via the type-1 secretion system (T1SS). We previously reported that the periplasmic protease LapG cleaves the N-terminus of LapA at a canonical dialanine motif to release the adhesin from the cell surface under conditions unfavorable to biofilm formation, thus decreasing biofilm formation. Here, we characterize LapA as the first type 1 secreted substrate that does not follow the "one-step" rule of T1SS. Rather, a novel N-terminal element, called the retention module (RM), localizes LapA at the cell surface as a secretion intermediate. Our genetic, biochemical, and molecular modeling analysis support a model wherein LapA is tethered to the cell surface through its T1SS outer membrane TolC-like pore, LapE, until LapG cleaves LapA in the periplasm. We further demonstrate this unusual retention strategy is likely conserved among LapA-like proteins, and reveals a new subclass of T1SS ABC transporters involved in transporting this group of surface-associated, LapA-like adhesins. These studies demonstrate a novel cell surface retention strategy used throughout the Proteobacteria and highlight a previously unappreciated flexibility of function for T1SS. Importance. Bacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface associated adhesins is key for initiating contact with a preferred substratum to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal Proteobacteria and provide phylogenetic evidence that these adhesins are secreted by a new subclass of T1SS ABC transporters. Copyright © 2018 American Society for Microbiology.

Antibody Against Integrin Lymphocyte Function-Associated Antigen 1 Inhibits HIV Type 1 Infection in Primary Cells Through Caspase-8-Mediated Apoptosis

PubMed Central

Walker, Tiffany N.; Cimakasky, Lisa M.; Coleman, Ebony M.; Madison, M. Nia

2013-01-01

Abstract HIV-1 infection induces formation of a virological synapse wherein CD4, chemokine receptors, and cell-adhesion molecules such as lymphocyte function-associated antigen 1 (LFA-1) form localized domains on the cell surface. Studies show that LFA-1 on the surface of HIV-1 particles retains its adhesion function and enhances virus attachment to susceptible cells by binding its counterreceptor intercellular adhesion molecule 1 (ICAM-1). This virus–cell interaction augments virus infectivity by facilitating binding and entry events. In this study, we demonstrate that inhibition of the LFA-1/ICAM-1 interaction by a monoclonal antibody leads to decreased virus production and spread in association with increased apoptosis of HIV-infected primary T cells. The data indicate that the LFA-1/ICAM-1 interaction may limit apoptosis in HIV-1-infected T cells. This phenomenon appears similar to anoikis wherein epithelial cells are protected from apoptosis conferred by ligand-bound integrins. These results have implications for further understanding HIV pathogenesis and replication in peripheral compartments and lymphoid organs. PMID:22697794

α-Actinin Promotes Surface Localization and Current Density of the Ca2+ Channel CaV1.2 by Binding to the IQ Region of the α1 Subunit.

PubMed

Tseng, Pang-Yen; Henderson, Peter B; Hergarden, Anne C; Patriarchi, Tommaso; Coleman, Andrea M; Lillya, Mark W; Montagut-Bordas, Carlota; Lee, Boram; Hell, Johannes W; Horne, Mary C

2017-07-18

The voltage-gated L-type Ca 2+ channel Ca V 1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of Ca V 1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of Ca V 1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal Ca V 1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous Ca V 1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to Ca V 1.2 supports its surface expression. Using two-hybrid screens and pull-down assays, we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α 1 1.2 subunit of Ca V 1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that Ca V 1.2 channels composed of the corresponding α-actinin-binding-deficient mutants result in a 35-40% reduction in surface expression compared to that of wild-type channels. Moreover, the mutant Ca V 1.2 channels expressed in HEK293 cells exhibit a 60-75% decrease in current density. The larger decrease in current density as compared to surface expression imparted by these α 1 1.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of Ca V 1.2 but also augments its ion conducting activity.

Grid cells on steeply sloping terrain: evidence for planar rather than volumetric encoding

PubMed Central

Hayman, Robin M. A.; Casali, Giulio; Wilson, Jonathan J.; Jeffery, Kate J.

2015-01-01

Neural encoding of navigable space involves a network of structures centered on the hippocampus, whose neurons –place cells – encode current location. Input to the place cells includes afferents from the entorhinal cortex, which contains grid cells. These are neurons expressing spatially localized activity patches, or firing fields, that are evenly spaced across the floor in a hexagonal close-packed array called a grid. It is thought that grids function to enable the calculation of distances. The question arises as to whether this odometry process operates in three dimensions, and so we queried whether grids permeate three-dimensional (3D) space – that is, form a lattice – or whether they simply follow the environment surface. If grids form a 3D lattice then this lattice would ordinarily be aligned horizontally (to explain the usual hexagonal pattern observed). A tilted floor would transect several layers of this putative lattice, resulting in interruption of the hexagonal pattern. We model this prediction with simulated grid lattices, and show that the firing of a grid cell on a 40°-tilted surface should cover proportionally less of the surface, with smaller field size, fewer fields, and reduced hexagonal symmetry. However, recording of real grid cells as animals foraged on a 40°-tilted surface found that firing of grid cells was almost indistinguishable, in pattern or rate, from that on the horizontal surface, with if anything increased coverage and field number, and preserved field size. It thus appears unlikely that the sloping surface transected a lattice. However, grid cells on the slope displayed slightly degraded firing patterns, with reduced coherence and slightly reduced symmetry. These findings collectively suggest that the grid cell component of the metric representation of space is not fixed in absolute 3D space but is influenced both by the surface the animal is on and by the relationship of this surface to the horizontal, supporting the hypothesis that the neural map of space is “multi-planar” rather than fully volumetric. PMID:26236245

Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells.

PubMed

Koh, Ai Leen; Shachaf, Catherine M; Elchuri, Sailaja; Nolan, Garry P; Sinclair, Robert

2008-12-01

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

Electron Microscopy Localization and Characterization of Functionalized Composite Organic-Inorganic SERS Nanoparticles on Leukemia Cells

PubMed Central

Koh, Ai Leen; Shachaf, Catherine M.; Elchuri, Sailaja; Nolan, Garry P.; Sinclair, Robert

2008-01-01

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet Scanning Electron Microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron detector (BSE) was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution Transmission Electron Microscope (TEM) images and Scanning Auger Electron Spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens. PMID:18995965

Rab14 and Its Exchange Factor FAM116 Link Endocytic Recycling and Adherens Junction Stability in Migrating Cells

PubMed Central

Linford, Andrea; Yoshimura, Shin-ichiro; Bastos, Ricardo Nunes; Langemeyer, Lars; Gerondopoulos, Andreas; Rigden, Daniel J.; Barr, Francis A.

2012-01-01

Summary Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions. PMID:22595670

[Ca2+]i Elevation and Oxidative Stress Induce KCNQ1 Protein Translocation from the Cytosol to the Cell Surface and Increase Slow Delayed Rectifier (IKs) in Cardiac Myocytes*

PubMed Central

Wang, Yuhong; Zankov, Dimitar P.; Jiang, Min; Zhang, Mei; Henderson, Scott C.; Tseng, Gea-Ny

2013-01-01

Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca2+]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes. PMID:24142691

Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells.

PubMed

Ackerman, G A; Wolken, K W

1981-10-01

A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites.

Surface plasmon resonance sensing: from purified biomolecules to intact cells.

PubMed

Su, Yu-Wen; Wang, Wei

2018-04-12

Surface plasmon resonance (SPR) has become a well-recognized label-free technique for measuring the binding kinetics between biomolecules since the invention of the first SPR-based immunosensor in 1980s. The most popular and traditional format for SPR analysis is to monitor the real-time optical signals when a solution containing ligand molecules is flowing over a sensor substrate functionalized with purified receptor molecules. In recent years, rapid development of several kinds of SPR imaging techniques have allowed for mapping the dynamic distribution of local mass density within single living cells with high spatial and temporal resolutions and reliable sensitivity. Such capability immediately enabled one to investigate the interaction between important biomolecules and intact cells in a label-free, quantitative, and single cell manner, leading to an exciting new trend of cell-based SPR bioanalysis. In this Trend Article, we first describe the principle and technical features of two types of SPR imaging techniques based on prism and objective, respectively. Then we survey the intact cell-based applications in both fundamental cell biology and drug discovery. We conclude the article with comments and perspectives on the future developments. Graphical abstract Recent developments in surface plasmon resonance (SPR) imaging techniques allow for label-free mapping the mass-distribution within single living cells, leading to great expansions in biomolecular interactions studies from homogeneous substrates functionalized with purified biomolecules to heterogeneous substrates containing individual living cells.

Microfabrication of proangiogenic cell-laden alginate-g-pyrrole hydrogels.

PubMed

DeVolder, Ross J; Zill, Andrew T; Jeong, Jae H; Kong, Hyunjoon

2012-11-01

Cells have been extensively studied for their uses in various therapies because of their capacities to produce therapeutic proteins and recreate new tissues. It has often been suggested that the efficacy of cell therapies can greatly be improved through the ability to localize and regulate cellular activities at a transplantation site; however, the technologies for this control are lacking. Therefore, this study reports a cell-Laden hydrogel patch engineered to support the proliferation and angiogenic growth factor expression of cells adhered to their surfaces, and to further promote neovascularization. Hydrogels consisting of alginate chemically linked with pyrrole units, termed alginate-g-pyrrole, were prepared through an oxidative cross-linking reaction between pyrrole units. Fibroblasts adhered to the alginate-g-pyrrole hydrogels, and exhibited increased proliferation and overall vascular endothelial growth factor (VEGF) expression, compared to those on pyrrole-free hydrogels. Furthermore, the alginate-g-pyrrole hydrogel surfaces were modified to present microposts, subsequently increasing the amount of pyrrole units on their surfaces. Cells adhered to the microfabricated gel surfaces exhibited increased proliferation and overall VEGF expression proportional to the density of the microposts. The resulting micropatterned alginate-g-pyrrole hydrogels exhibited increases in the size and density of mature blood vessels when implanted on chick chorioallantoic membranes (CAMs). The hydrogel system developed in this study will be broadly useful for improving the efficacy of a wide array of cell-based wound healing and tissue regenerative therapies. Copyright © 2012 Elsevier Ltd. All rights reserved.

Piezoresistance and solar cell efficiency

NASA Technical Reports Server (NTRS)

Weizer, Victor G.

1987-01-01

Diffusion-induced stresses in silicon are shown to result in large localized changes in the minority-carrier mobility which in turn can have a significant effect on cell output. Evidence is given that both compressive and tensile stresses can be generated in either the emitter or the base region. Tensile stresses in the base appear to be much more effective in altering cell performance than do compressive stresses. While most stress-related effects appear to degrade cell efficiency, this is not always the case. Evidence is presented showing that arsenic-induced stresses can result in emitter characteristics comparable to those found in the MINP cell without requiring a high degree of surface passivation.

Innate T cell responses in human gut.

PubMed

Meresse, Bertrand; Cerf-Bensussan, Nadine

2009-06-01

One arm of the gut-associated immune system is represented by a vast collection of T lymphocytes which participate in the subtle interplay between innate and adaptive immune mechanisms and maintain homeostasis at the main body external surface. Mounting data are providing exciting new insight into the innate-like mechanisms which enable intestinal T cells to rapidly sense local conditions and which broaden the spectrum of their functions and regulation at this strategic location. Herein we discuss how innate-like T cell recognition by unconventional T cell subsets and expression of innate NK receptors might modulate immune T cell responses in the human normal or diseased intestine.

The effect of internal stresses on solar cell efficiency

NASA Technical Reports Server (NTRS)

Weizer, Victor G.

1987-01-01

Diffusion induced stresses in silicon are shown to result in large localized changes in the minority carrier mobility which in turn have a significant effect on cell output. Evidence is given that both compressive and tensile stresses can be generated in either the emitter or the base region. Tensile stresses appear to be much more effective in altering cell performance. While most stress related effects appear to degrade cell efficiency, this is not always the case. Evidence is presented showing that arsenic induced stresses can result in emitter characteristics comparable to those found in the MINP cell without requiring a high degree of surface passivation.

Effects of cancer cell permeability control on the efficiency of cell damage through surface plasmon resonance of gold nanoparticle (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hsiao, Jen-Hung; Yu, Jian-He; He, Yulu; Tu, Yi-Chou; Hua, Wei-Hsiang; Low, Meng Chun; Hsieh, Cheng-Che; Kiang, Yean-Woei; Yang, Chih-Chung

2017-02-01

Cancer cell killing efficiencies based on the photothermal effect caused by the surface plasmon resonance of metal nanoparticles (NPs) and the photodynamic effect caused by the singlet oxygen generation of a photosensitizer rely on the cell uptake efficiency of metal NP and photosensitizer. Perforation and heating can increase cell membrane permeability and hence can increase the cell uptake efficiency of NPs and drugs. In this paper, we demonstrate the variations of the cell damage efficiency under the illuminations of different lasers, which can produce mainly photothermal effect, mainly photodynamic effect, and mixed effect, when a pre-perforation and a pre-heating processes are applied. Au nanorings (NRIs) with their localized surface plasmon resonance wavelength around 1064 nm are used. The perforation process is undertaken by illuminating the cell samples by a femtosecond laser at 1064 nm with the power density lower than the cell damage threshold intensity. The heating process is implemented by illuminating cells with a low power continuous laser at 1064 nm. It is found that with the pre-perforation and pre-heating processes, the photodynamic effect is enhanced because the internalized Au NRI number and hence the internalized photosensitizer (AlPcS) molecule number are increased. However, the photothermal effect can be reduced because the adsorbed Au NRIs on cell membrane are effectively internalized during the pre-perforation and pre-heating processes. The photothermal effect is more effective when Au NRIs are adsorbed on cell membrane.

Texturing Silicon Nanowires for Highly Localized Optical Modulation of Cellular Dynamics.

PubMed

Fang, Yin; Jiang, Yuanwen; Acaron Ledesma, Hector; Yi, Jaeseok; Gao, Xiang; Weiss, Dara E; Shi, Fengyuan; Tian, Bozhi

2018-06-18

Engineered silicon-based materials can display photoelectric and photothermal responses under light illumination, which may lead to further innovations at the silicon-biology interfaces. Silicon nanowires have small radial dimensions, promising as highly localized cellular modulators, however the single crystalline form typically has limited photothermal efficacy due to the poor light absorption and fast heat dissipation. In this work, we report strategies to improve the photothermal response from silicon nanowires by introducing nanoscale textures on the surface and in the bulk. We next demonstrate high-resolution extracellular modulation of calcium dynamics in a number of mammalian cells including glial cells, neurons, and cancer cells. The new materials may be broadly used in probing and modulating electrical and chemical signals at the subcellular length scale, which is currently a challenge in the field of electrophysiology or cellular engineering.

Spatial resolution versus contrast trade-off enhancement in high-resolution surface plasmon resonance imaging (SPRI) by metal surface nanostructure design.

PubMed

Banville, Frederic A; Moreau, Julien; Sarkar, Mitradeep; Besbes, Mondher; Canva, Michael; Charette, Paul G

2018-04-16

Surface plasmon resonance imaging (SPRI) is an optical near-field method used for mapping the spatial distribution of chemical/physical perturbations above a metal surface without exogenous labeling. Currently, the majority of SPRI systems are used in microarray biosensing, requiring only modest spatial resolution. There is increasing interest in applying SPRI for label-free near-field imaging of biological cells to study cell/surface interactions. However, the required resolution (sub-µm) greatly exceeds what current systems can deliver. Indeed, the attenuation length of surface plasmon polaritons (SPP) severely limits resolution along one axis, typically to tens of µm. Strategies to date for improving spatial resolution result in a commensurate deterioration in other imaging parameters. Unlike the smooth metal surfaces used in SPRI that support purely propagating surface modes, nanostructured metal surfaces support "hybrid" SPP modes that share attributes from both propagating and localized modes. We show that these hybrid modes are especially well-suited to high-resolution imaging and demonstrate how the nanostructure geometry can be designed to achieve sub-µm resolution while mitigating the imaging parameter trade-off according to an application-specific optimum.

The extracellular microenvironment explains variations in passive drug transport across different airway epithelial cell types.

PubMed

Min, Kyoung Ah; Talattof, Arjang; Tsume, Yasuhiro; Stringer, Kathleen A; Yu, Jing-Yu; Lim, Dong Hyun; Rosania, Gus R

2013-08-01

We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in the extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types.

The Extracellular Microenvironment Explains Variations in Passive Drug Transport across Different Airway Epithelial Cell Types

PubMed Central

Min, Kyoung Ah; Talattof, Arjang; Tsume, Yasuhiro; Stringer, Kathleen A.; Yu, Jing-yu; Lim, Dong Hyun; Rosania, Gus R.

2013-01-01

Purpose We sought to identify key variables in cellular architecture and physiology that might explain observed differences in the passive transport properties of small molecule drugs across different airway epithelial cell types. Methods Propranolol (PR) was selected as a weakly basic, model compound to compare the transport properties of primary (NHBE) vs. tumor-derived (Calu-3) cells. Differentiated on Transwell™ inserts, the architecture of pure vs. mixed cell co-cultures was studied with confocal microscopy followed by quantitative morphometric analysis. Cellular pharmacokinetic modeling was used to identify parameters that differentially affect PR uptake and transport across these two cell types. Results Pure Calu-3 and NHBE cells possessed different structural and functional properties. Nevertheless, mixed Calu-3 and NHBE cell co-cultures differentiated as stable cell monolayers. After measuring the total mass of PR, the fractional areas covered by Calu-3 and NHBE cells allowed deconvoluting the transport properties of each cell type. Based on the apparent thickness of the unstirred, cell surface aqueous layer, local differences in extracellular microenvironment explained the measured variations in passive PR uptake and permeation between Calu-3 and NHBE cells. Conclusion Mixed cell co-cultures can be used to compare the local effects of the extracellular microenvironment on drug uptake and transport across two epithelial cell types. PMID:23708857

A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem

PubMed Central

Burian, Agata; Uyttewaal, Magalie

2013-01-01

Cortical microtubules (CMTs) are often aligned in a particular direction in individual cells or even in groups of cells and play a central role in the definition of growth anisotropy. How the CMTs themselves are aligned is not well known, but two hypotheses have been proposed. According to the first hypothesis, CMTs align perpendicular to the maximal growth direction, and, according to the second, CMTs align parallel to the maximal stress direction. Since both hypotheses were formulated on the basis of mainly qualitative assessments, the link between CMT organization, organ geometry, and cell growth is revisited using a quantitative approach. For this purpose, CMT orientation, local curvature, and growth parameters for each cell were measured in the growing shoot apical meristem (SAM) of Arabidopsis thaliana. Using this approach, it has been shown that stable CMTs tend to be perpendicular to the direction of maximal growth in cells at the SAM periphery, but parallel in the cells at the boundary domain. When examining the local curvature of the SAM surface, no strict correlation between curvature and CMT arrangement was found, which implies that SAM geometry, and presumed geometry-derived stress distribution, is not sufficient to prescribe the CMT orientation. However, a better match between stress and CMTs was found when mechanical stress derived from differential growth was also considered. PMID:24153420

A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem.

PubMed

Burian, Agata; Ludynia, Michal; Uyttewaal, Magalie; Traas, Jan; Boudaoud, Arezki; Hamant, Olivier; Kwiatkowska, Dorota

2013-12-01

Cortical microtubules (CMTs) are often aligned in a particular direction in individual cells or even in groups of cells and play a central role in the definition of growth anisotropy. How the CMTs themselves are aligned is not well known, but two hypotheses have been proposed. According to the first hypothesis, CMTs align perpendicular to the maximal growth direction, and, according to the second, CMTs align parallel to the maximal stress direction. Since both hypotheses were formulated on the basis of mainly qualitative assessments, the link between CMT organization, organ geometry, and cell growth is revisited using a quantitative approach. For this purpose, CMT orientation, local curvature, and growth parameters for each cell were measured in the growing shoot apical meristem (SAM) of Arabidopsis thaliana. Using this approach, it has been shown that stable CMTs tend to be perpendicular to the direction of maximal growth in cells at the SAM periphery, but parallel in the cells at the boundary domain. When examining the local curvature of the SAM surface, no strict correlation between curvature and CMT arrangement was found, which implies that SAM geometry, and presumed geometry-derived stress distribution, is not sufficient to prescribe the CMT orientation. However, a better match between stress and CMTs was found when mechanical stress derived from differential growth was also considered.

Multiple internalization pathways of polyelectrolyte multilayer capsules into mammalian cells.

PubMed

Kastl, Lena; Sasse, Daniel; Wulf, Verena; Hartmann, Raimo; Mircheski, Josif; Ranke, Christiane; Carregal-Romero, Susana; Martínez-López, José Antonio; Fernández-Chacón, Rafael; Parak, Wolfgang J; Elsasser, Hans-Peter; Rivera Gil, Pilar

2013-08-27

Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (e.g., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching-off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phagolysosomes, but not with other endocytic vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to heterophagolysosomes.

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network.

PubMed

Raub, T J; Koroly, M J; Roberts, R M

1990-04-01

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle

PubMed Central

Peterson, Soren J.; Krasnow, Mark A.

2015-01-01

SUMMARY To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains including inside the cell. PMID:25557078

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle.

PubMed

Peterson, Soren J; Krasnow, Mark A

2015-01-15

To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell. Copyright © 2015 Elsevier Inc. All rights reserved.

Mucosal immunoglobulins at respiratory surfaces mark an ancient association that predates the emergence of tetrapods

PubMed Central

Xu, Zhen; Takizawa, Fumio; Parra, David; Gómez, Daniela; von Gersdorff Jørgensen, Louise; LaPatra, Scott E.; Sunyer, J. Oriol

2016-01-01

Gas-exchange structures are critical for acquiring oxygen, but they also represent portals for pathogen entry. Local mucosal immunoglobulin responses against pathogens in specialized respiratory organs have only been described in tetrapods. Since fish gills are considered a mucosal surface, we hypothesized that a dedicated mucosal immunoglobulin response would be generated within its mucosa on microbial exposure. Supporting this hypothesis, here we demonstrate that following pathogen exposure, IgT+ B cells proliferate and generate pathogen-specific IgT within the gills of fish, thus providing the first example of locally induced immunoglobulin in the mucosa of a cold-blooded species. Moreover, we demonstrate that gill microbiota is predominantly coated with IgT, thus providing previously unappreciated evidence that the microbiota present at a respiratory surface of a vertebrate is recognized by a mucosal immunoglobulin. Our findings indicate that respiratory surfaces and mucosal immunoglobulins are part of an ancient association that predates the emergence of tetrapods. PMID:26869478

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces.

PubMed

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-03-21

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces.

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

PubMed Central

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-01-01

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces. PMID:26996815

Fabrication, characterization, and biological assessment of multilayer laminin γ2 DNA coatings on titanium surfaces

NASA Astrophysics Data System (ADS)

Yang, Guoli; Zhang, Jing; Dong, Wenjing; Liu, Li; Shi, Jue; Wang, Huiming

2016-03-01

The purpose of this work was to fabricate a multilayer laminin γ2 DNA coating on a titanium surface and evaluate its biological properties. A multilayer laminin γ2 DNA coating was fabricated on titanium using a layer-by-layer assembly technique. The rate of coating degradation was evaluated by detecting the amount of cDNA remaining. Surface analysis using X-ray photoelectron spectroscopy, atomic force microscopy, and surface contact angle measurements revealed the multilayer structure to consist of cationic lipid and confirmed that a laminin γ2 DNA layer could be fabricated on titanium via the layer-by-layer assembly process. The transfection efficiency was highest for five layers in the multilayer structure. HEK293 cells cultured on the multilayer films displayed significantly higher adhesion activity than the control group. The expression of laminin γ2 and the co-localization of integrin β4 and plectin were more obvious in HN4 cells cultured on the multilayer laminin γ2 DNA coating, while weak immunoreactivities were observed in the control group. We concluded that the DNA-loaded multilayer provided a surface with good biocompatibility and that the multilayer laminin γ2 DNA coating might be effective in improving cell adhesion and the formation of hemidesmosomes on titanium surfaces.

Solid-State (13)C NMR Delineates the Architectural Design of Biopolymers in Native and Genetically Altered Tomato Fruit Cuticles.

PubMed

Chatterjee, Subhasish; Matas, Antonio J; Isaacson, Tal; Kehlet, Cindie; Rose, Jocelyn K C; Stark, Ruth E

2016-01-11

Plant cuticles on outer fruit and leaf surfaces are natural macromolecular composites of waxes and polyesters that ensure mechanical integrity and mitigate environmental challenges. They also provide renewable raw materials for cosmetics, packaging, and coatings. To delineate the structural framework and flexibility underlying the versatile functions of cutin biopolymers associated with polysaccharide-rich cell-wall matrices, solid-state NMR spectra and spin relaxation times were measured in a tomato fruit model system, including different developmental stages and surface phenotypes. The hydrophilic-hydrophobic balance of the cutin ensures compatibility with the underlying polysaccharide cell walls; the hydroxy fatty acid structures of outer epidermal cutin also support deposition of hydrophobic waxes and aromatic moieties while promoting the formation of cell-wall cross-links that rigidify and strengthen the cuticle composite during fruit development. Fruit cutin-deficient tomato mutants with compromised microbial resistance exhibit less efficient local and collective biopolymer motions, stiffening their cuticular surfaces and increasing their susceptibility to fracture.

Production of solar photovoltaic cells on the Moon

NASA Technical Reports Server (NTRS)

Criswell, David R.; Ignatiev, Alex

1991-01-01

Solar energy is directly available on the sunward lunar surface. Most, if not all, the materials are available on the Moon to make silicon based solar photovoltaic cells. A few additional types are possible. There is a small but growing literature on production of lunar derived solar cells. This literature is reviewed. Topics explored include trade-offs of local production versus import of key materials, processing options, the scale and nature of production equipment, implications of storage requirements, and the end-uses of the energy. Directions for future research and demonstrations are indicated.

Cell surface glycan engineering of neural stem cells augments neurotropism and improves recovery in a murine model of multiple sclerosis

PubMed Central

Merzaban, Jasmeen S; Imitola, Jaime; Starossom, Sarah C; Zhu, Bing; Wang, Yue; Lee, Jack; Ali, Amal J; Olah, Marta; Abuelela, Ayman F; Khoury, Samia J; Sackstein, Robert

2015-01-01

Neural stem cell (NSC)-based therapies offer potential for neural repair in central nervous system (CNS) inflammatory and degenerative disorders. Typically, these conditions present with multifocal CNS lesions making it impractical to inject NSCs locally, thus mandating optimization of vascular delivery of the cells to involved sites. Here, we analyzed NSCs for expression of molecular effectors of cell migration and found that these cells are natively devoid of E-selectin ligands. Using glycosyltransferase-programmed stereosubstitution (GPS), we glycan engineered the cell surface of NSCs (“GPS-NSCs”) with resultant enforced expression of the potent E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) and of an E-selectin-binding glycoform of neural cell adhesion molecule (“NCAM-E”). Following intravenous (i.v.) injection, short-term homing studies demonstrated that, compared with buffer-treated (control) NSCs, GPS-NSCs showed greater neurotropism. Administration of GPS-NSC significantly attenuated the clinical course of experimental autoimmune encephalomyelitis (EAE), with markedly decreased inflammation and improved oligodendroglial and axonal integrity, but without evidence of long-term stem cell engraftment. Notably, this effect of NSC is not a universal property of adult stem cells, as administration of GPS-engineered mouse hematopoietic stem/progenitor cells did not improve EAE clinical course. These findings highlight the utility of cell surface glycan engineering to boost stem cell delivery in neuroinflammatory conditions and indicate that, despite the use of a neural tissue-specific progenitor cell population, neural repair in EAE results from endogenous repair and not from direct, NSC-derived cell replacement. PMID:26153105

Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

DOE PAGES

Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; ...

2016-05-27

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed betweenmore » the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.« less

Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

PubMed Central

Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

2016-01-01

We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter.

PubMed

Dye, Natalie A; Pincus, Zachary; Fisher, Isabelle C; Shapiro, Lucy; Theriot, Julie A

2011-07-01

The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape. © 2011 Blackwell Publishing Ltd.

Mutations in the nucleotide binding pocket of MreB can alter cell curvature and polar morphology in Caulobacter

PubMed Central

Dye, Natalie A; Pincus, Zachary; Fisher, Isabelle C; Shapiro, Lucy; Theriot, Julie A

2011-01-01

Summary The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape. PMID:21564339

Developmental Localization and Methylesterification of Pectin Epitopes during Somatic Embryogenesis of Banana (Musa spp. AAA)

PubMed Central

Xu, Chunxiang; Zhao, Lu; Pan, Xiao; Šamaj, Jozef

2011-01-01

Background The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development. Methodology/Principal Findings Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment. Conclusions/Significance These data suggest that both low- and highly-methyl-esterified HG epitopes are developmentally regulated in diverse embryogenic stages during somatic embryogenesis. This study provides new information about pectin composition, HG methyl-esterification and developmental localization of pectin epitopes during somatic embryogenesis of banana. PMID:21826225

Characterization of Siglec-8 Expression on Lavage Cells after Segmental Lung Allergen Challenge.

PubMed

Johansson, Mats W; Kelly, Elizabeth A; Nguyen, Christopher L; Jarjour, Nizar N; Bochner, Bruce S

2018-06-07

Siglec-8 is present at a high level on human blood eosinophils and low level on blood basophils. Engagement of Siglec-8 on blood eosinophils causes its internalization and results in death. Siglec-8 is a potential therapeutic target in eosinophilic asthma. The aim of this study was to determine Siglec-8 levels on eosinophils and basophils recruited during lung inflammation. We analyzed surface Siglec-8 by flow cytometry on cells obtained by bronchoalveolar lavage (BAL) 48 h after segmental lung allergen challenge of human subjects with mild allergic asthma and used confocal microscopy to compare Siglec-8 distribution on BAL and blood eosinophils. Like their blood counterparts, BAL eosinophils had high unimodal surface Siglec-8, while BAL basophils had lower but detectable surface Siglec-8. BAL macrophages, monocytes, neutrophils, and plasmacytoid dendritic cells did not express surface Siglec-8. Microscopy of freshly isolated blood eosinophils demonstrated homogeneous Siglec-8 distribution over the cell surface. Upon incubation with IL-5, Siglec-8 on the surface of eosinophils became localized in patches both at the nucleopod tip and at the opposite cell pole. BAL eosinophils also had a patchy Siglec-8 distribution. We conclude that 48 h after segmental allergen challenge, overall levels of Siglec-8 expression on airway eosinophils resemble those on blood eosinophils, but with a patchier distribution, a pattern consistent with activation. Thus, therapeutic targeting of Siglec-8 has the potential to impact blood as well as lung eosinophils, which may be associated with an improved outcome in eosinophilic lung diseases. © 2018 S. Karger AG, Basel.

Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP mediated PKA-dependent Ca2+ cycling with surface membrane channels

PubMed Central

Vinogradova, Tatiana M.; Lakatta, Edward G.

2009-01-01

Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation. PMID:19573534

Research Investigation Directed Toward Extending the Useful Range of the Electromagnetic Spectrum.

DTIC Science & Technology

1983-03-31

Meyers, C. H. Liu, S. M. So, W. Hwang, and E. S. Yang, "Electrical Characterization of Electrodeposited CdTe Solar Cells," Proceedings 16th IEEE...ARO) D. J. Ehrlich and R. M. Osgood, Jr., "Laser Microchemistry, Local Nucleation Mechanisms for Photodeposition," Thin Solid Films, 90, 287 (1982...substrate Interface. Subsequently, the migrated Si atom nucleates epitaxially at the Si substrate surface through a surface reaction. It has been

Inhibition of the mobility of mouse lymphocyte surface immunoglobulins by locally bound concanavalin A.

PubMed Central

Henis, Y I; Elson, E L

1981-01-01

Fluorescence photobleaching recovery was used to study directly and quantitatively the inhibition of the lateral mobility of surface immunoglobulins (sIg) on mouse lymphocytes by localized binding of concanavalin A (Con A) coupled to platelets. Up to a threshold occupancy of about 10% of the upper cell surface by Con A-platelets, the diffusion coefficient and mobile fraction of sIg remained as in untreated cells (5.3 X 10(-10) cm2/sec and 0.65, respectively). At higher surface occupancy, these values decreased to 8 X 10(-11) cm2/sec and 0.11. The magnitude of the effect was independent of the percentage occupancy above the threshold and of the distance from the bound Con A-platelets, indicating a cooperative and propagated phenomenon. Treatment with colchicine or cytochalasin B separately induced only partial reversal of the Con A-induced modulation. Treatment with both reversal of the Con A-induced modulation. Treatment with both drugs together was synergistic and fully reversed the mobility inhibition. The modulation was unaffected by NaN3 and 2-deoxyglucose, suggesting no dependence on metabolic energy. Con A-platelets did not affect the mobility of a lipid probe. Models for the Con A-induced modulation and the relationship between the effects of Con A on sIg mobility and patch formation are discussed. PMID:6940124

[The role of endothelial cells and endothelial precursor cells in angiogenesis].

PubMed

Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

2006-01-01

Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

Plant and Animal Gravitational Biology. Part 1

NASA Technical Reports Server (NTRS)

1997-01-01

Session TA2 includes short reports covering: (1) The Interaction of Microgravity and Ethylene on Soybean Growth and Metabolism; (2) Structure and G-Sensitivity of Root Statocytes under Different Mass Acceleration; (3) Extracellular Production of Taxanes on Cell Surfaces in Simulated Microgravity and Hypergravity; (4) Current Problems of Space Cell Phytobiology; (5) Biological Consequences of Microgravity-Induced Alterations in Water Metabolism of Plant Cells; (6) Localization of Calcium Ions in Chlorella Cells Under Clinorotation; (7) Changes of Fatty Acids Content of Plant Cell Plasma Membranes under Altered Gravity; (8) Simulation of Gravity by Non-Symmetrical Vibrations and Ultrasound; and (9) Response to Simulated weightlessness of In Vitro Cultures of Differentiated Epithelial Follicular Cells from Thyroid.

Plasmonic excitation-assisted optical and electric enhancement in ultra-thin solar cells: the influence of nano-strip cross section

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sabaeian, Mohammad, E-mail: sabaiean@scu.ac.ir; Heydari, Mehdi; Ajamgard, Narges

The effects of Ag nano-strips with triangle, rectangular and trapezoid cross sections on the optical absorption, generation rate, and short-circuit current density of ultra-thin solar cells were investigated. By putting the nano-strips as a grating structure on the top of the solar cells, the waveguide, surface plasmon polariton (SPP), and localized surface plasmon (LSP) modes, which are excited with the assistance of nano-strips, were evaluated in TE and TM polarizations. The results show, firstly, the TM modes are more influential than TE modes in optical and electrical properties enhancement of solar cell, because of plasmonic excitations in TM mode. Secondly,more » the trapezoid nano-strips reveal noticeable impact on the optical absorption, generation rate, and short-circuit current density enhancement than triangle and rectangular ones. In particular, the absorption of long wavelengths which is a challenge in ultra-thin solar cells is significantly improved by using Ag trapezoid nano-strips.« less

A placebo controlled observer blind immunocytochemical and histologic study of epithelium adjacent to anogenital warts in patients treated with systemic interferon alpha in combination with cryotherapy or cryotherapy alone.

PubMed Central

Handley, J M; Maw, R D; Horner, T; Lawther, H; Walsh, M; Dinsmore, W W

1992-01-01

OBJECTIVE--To examine biopsy specimens of tissue immediately adjacent to anogenital (AG) warts which had been treated with either cryotherapy plus subcutaneous interferon (IFN) alpha 2a or cryotherapy alone, for histological features of (a) human papilloma virus (HPV) infection (b) localised cellular immune responses, to further characterise any cellular immune infiltrates with tissue immunocytochemistry, and to relate any histological, immunocytochemical findings to the treatment response of nearby AG warts. DESIGN--A randomised placebo controlled observer blind study. SETTING--Genitourinary Medicine clinic, Department of Immunopathology, Royal Victoria Hospital, Belfast, N. Ireland. SUBJECTS--Thirty patients with AG warts; 16 treated with IFN alpha 2a plus cryotherapy, and 14 treated with cryotherapy alone. OUTCOME MEASURES--(1) Light microscopic features associated with HPV infection and local cellular immune responses. (2) Indirect immunofluorescence detection of the following cell surface markers: HLA DR, alpha one antitrypsin, CD1, CD3, CD4, CD8, CD22. (3) Clinical response of AG warts to treatment. RESULTS--In pre-treatment biopsies only non specific indicators of HPV infection (acanthosis, 29/30 biopsies, and hyperkeratosis, 7/30 biopsies) were seen on light microscopy. Mononuclear cells were seen both throughout the upper dermis and centred around dermal blood vessels in 19/30 (63.3%) biopsies, and infiltrating into the epidermis in 12/30 (40%) biopsies. On indirect immunofluorescence CD3, CD8, CD4 antigen was detected on the surface of cells throughout the upper dermis in 24/29 (82.7%), 15/29 (51.7%), and 3/29 (10.3%), of biopsy specimens respectively. CD3 antigen, CD8 antigen and CD4 antigen was detected on the surface of cells infiltrating into the epidermis in 18/29 (62%), 7/29 (24.1%), and 6/29 (20.7%) of biopsy specimens respectively. CD1 antigen was seen on the surface of dendritic cells throughout the epidermis in all specimens; CD1 positive cells infiltrated into the upper dermis in 5/29 (17.2%). HLA DR was detected on the surface of dendritic cells throughout the epidermis in 22/29 (75.9%) of specimens, and on the surface of cells scattered both diffusely throughout the upper dermis and centred around dermal blood vessels in all specimens. Alpha one antitrypsin (A1AT) antigen was seen on the surface of cells in the upper dermis in 6/29 (20.7%) of biopsy specimens; no cells expressing CD22 surface antigen were seen. The nature of this local cellular immune response was not altered by treatment of nearby warts with either cryotherapy alone or cryotherapy plus systemic IFN alpha 2a, or related to the therapeutic outcome of these warts. CONCLUSIONS--(1) No convincing histological evidence of HPV infection was seen in epithelium surrounding AG warts. (2) A predominantly T cell-mediated immune response (the target of which is uncertain) was seen in this perilesional epithelium. (3) In the dosage regimens used in this study, treatment of AG warts with either systemic IFN alpha 2a plus cryotherapy or cryotherapy alone did not appear to augment localised cellular immune responses (against any presumed subclinical HPV infection) in epithelium surrounding AG warts. Images PMID:1316307

AFM probing in aqueous environment of Staphylococcus epidermidis cells naturally immobilised on glass: physico-chemistry behind the successful immobilisation.

PubMed

Méndez-Vilas, A; Gallardo-Moreno, A M; Calzado-Montero, R; González-Martín, M L

2008-05-01

AFM probing of microbial cells in liquid environments usually requires them to be physically or chemically attached to a solid surface. The fixation mechanisms may influence the nanomechanical characterization done by force curve mapping using an AFM. To study the response of a microbial cell surface to this kind of local measurement this study attempts to overcome the problem associated to the uncertainties introduced by the different fixation treatments by analysing the surface of Staphylococcus epidermidis cells naturally (non-artificially mediated) immobilised on a glass support surface. The particularities of this natural bacterial fixation process for AFM surface analysis are discussed in terms of theoretical predictions of the XDLVO model applied to the systems bacteria/support substratum and bacteria/AFM tip immersed in water. In this sense, in the first part of this study the conditions for adequate natural fixation of three S. epidermidis strains have been analyzed by taking into account the geometries of the bacterium, substrate and tip. In the second part, bacteria are probed without the risk of any possible artefacts due to the mechanical or chemical fixation procedures. Forces measured over the successfully adhered cells have (directly) shown that the untreated bacterial surface suffers from a combination of both reversible and non-reversible deformations during acquisition of force curves all taken under the same operational conditions. This is revealed directly through high-resolution tapping-mode imaging of the bacterial surface immediately following force curve mapping. The results agree with the two different types of force curves that were repeatedly obtained. Interestingly, one type of these force curves suggests that the AFM tip is breaking (rather than pushing) the cell surface during acquisition of the force curve. In this case, adhesive peaks were always observed, suggesting a mechanical origin of the measured pull-off forces. The other type of force curves shows no adhesive peaks and exhibits juxtaposing of approaching and retraction curves, reflecting elastic deformations.

Laser surface treatment of porous ceramic substrate for application in solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Mahmod, D. S. A.; Khan, A. A.; Munot, M. A.; Glandut, N.; Labbe, J. C.

2016-08-01

Laser has offered a large number of benefits for surface treatment of ceramics due to possibility of localized heating, very high heating/cooling rates and possibility of growth of structural configurations only produced under non-equilibrium high temperature conditions. The present work investigates oxidation of porous ZrB2-SiC sintered ceramic substrates through treatment by a 1072 ± 10 nm ytterbium fiber laser. A multi-layer structure is hence produced showing successively oxygen rich distinct layers. The porous bulk beneath these layers remained unaffected as this laser-formed oxide scale and protected the substrate from oxidation. A glassy SiO2 structure thus obtained on the surface of the substrate becomes subject of interest for further research, specifically for its utilization as solid protonic conductor in Solid Oxide Fuel Cells (SOFCs).