The Endogenous GRP78 Interactome in Human Head and Neck Cancers: A Deterministic Role of Cell Surface GRP78 in Cancer Stemness.

PubMed

Chen, Hsin-Ying; Chang, Joseph Tung-Chieh; Chien, Kun-Yi; Lee, Yun-Shien; You, Guo-Rung; Cheng, Ann-Joy

2018-01-11

Cell surface glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, was suggested to be a cancer stem cell marker, but the influence of this molecule on cancer stemness is poorly characterized. In this study, we developed a mass spectrometry platform to detect the endogenous interactome of GRP78 and investigated its role in cancer stemness. The interactome results showed that cell surface GRP78 associates with multiple molecules. The influence of cell population heterogeneity of head and neck cancer cell lines (OECM1, FaDu, and BM2) according to the cell surface expression levels of GRP78 and the GRP78 interactome protein, Progranulin, was investigated. The four sorted cell groups exhibited distinct cell cycle distributions, asymmetric/symmetric cell divisions, and different relative expression levels of stemness markers. Our results demonstrate that cell surface GRP78 promotes cancer stemness, whereas drives cells toward a non-stemlike phenotype when it chaperones Progranulin. We conclude that cell surface GRP78 is a chaperone exerting a deterministic influence on cancer stemness.

Soluble fragments of e-cadherin cell-adhesion molecule increase in urinary-excretion of cancer-patients, potentially indicating its shedding from epithelial tumor-cells.

PubMed

Katayama, M; Hirai, S; Yasumoto, M; Nishikawa, K; Nagata, S; Otsuka, M; Kamihagi, K; Kato, I

1994-11-01

E-cadherin (Ecad) is well known to be a calcium-ion-dependent cell-cell adhesion molecule expressed mostly in epithelial tissues. Previous immunohistochemical studies suggested that this cell adhesion molecule acts as an invasion suppressor and is negligibly detected in cancer metastatic regions. Soluble Ecad fragments derived from the proteolysed membrane-associated form were detected in culture supernatants of two cell lines, COLO 205 and A-431, with normal distribution of cell surface Ecad. Soluble Ecad levels released into culture of COLO 205 exhibiting reduced cell-cell adhesion were apparently elevated above those of A-431 with tight cell-cell adhesion. Furthermore, human circulation and urine continuously contain soluble Ecad which consists mainly of homogeneous 75-85 kDa extracellular domains. Soluble Ecad urinary level per urinary creatinine level was found to be significantly elevated in 53% of patients suffering from various types of cancers including lung, liver, stomach, colon and rectal cancers, as compared with those in the age-matched healthy subjects. These results suggest that dysfunction of cell surface Ecad is responsible for its enhanced proteolytic shedding in tumorigenesis, which may lead to the decrease of cell surface Ecads. Furthermore, excretion of high levels of soluble Ecad fragments potentially indicates the progression of epithelial tumors excessively degrading cell surface Ecad in clinical subjects.

Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine.

PubMed

Zaitseva, Elena; Zaitsev, Eugene; Melikov, Kamran; Arakelyan, Anush; Marin, Mariana; Villasmil, Rafael; Margolis, Leonid B; Melikyan, Gregory B; Chernomordik, Leonid V

2017-07-12

HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling, but this remains controversial. Here we report that formation of the pre-fusion Env-CD4-coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS). HIV-1-induced PS redistribution depends on Ca 2+ signaling triggered by Env-coreceptor interactions and involves the lipid scramblase TMEM16F. Externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Blocking externalized PS or suppressing TMEM16F inhibited Env-mediated fusion. Exogenously added PS promoted fusion, with fusion dependence on PS being especially strong for cells with low surface density of coreceptors. These findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion complexes and likely focuses the infection on cells conducive to PS signaling. Published by Elsevier Inc.

The fine structure of the stomach mucosa of the Llama (Llama guanacoe). II. The fundic region of the hind stomach.

PubMed

Luciano, L; Reale, E; von Engelhardt, W

1980-01-01

The epithelium of the fundic region mucosa of the hind stomach in the Llama guanacoe has been studied using morphological and histochemical methods. Morphology suggests that solute and water absorption may occur in the epithelium of the surface and of the foveolae, although this absorption can not be estimated because of the extensive secretion of the gastric glands. The same cells of the surface and foveolar epithelium show numerous secretory granules. The glands reveal neck cells, chief cells, a large number of oxyntic cells, four types of endocrine cells (A-like, ECL, D and EC), brush cells and wandering cells. PAS and Alcian blue reactions for light microscopy suggest a secretion of neutral and acidic mucosubstances in the surface and foveolar epithelium, of neutral mucosubstances only in the neck cells. Periodic acid-thiocarbohydrazide silver proteinate (PA-TCH-SP) reaction for electron microscopy confirms the presence of neutral mucosubstances within the secretory granules of the surface, foveolar and neck epithelial cells. In all these cells, the reaction product is also evident within sacculi and vesicles of the maturing surface of the Golgi apparatus. A positive PA-TCH-SP reaction also occurs on the membrane (and not on the contents) of the Golgi apparatus (maturing surface) and of the secretory granules of the chief cells as well as on the membrane of the Golgi apparatus and of apical vesicles and tubules of the oxyntic cells. In addition, silver granules slightly enhance the electron desity of the contents of the secretory granules in the endocrine cells. Morphological and histochemical findings are discussed and compared with results described by others for monogastric mammals.

Interactions between cells and ionized dendritic biomaterials: Flow cytometry and fluorescence spectroscopic studies

NASA Astrophysics Data System (ADS)

Kannan, R. M.; Kolhe, Parag; Khandare, Jayant; Kannan, Sujatha; Lieh-Lai, Mary

2004-03-01

Dendrimers and hyperbranched polymers are a new class of macromolecules characterized by large density of "tunable" peripheral functional groups. Therefore dendrimers can serve as a model macromolecular system to study the influence of molecular geometry and charge density on transport across biological barriers, especially cellular interfaces. The effect of size, end-functionality, surface charge (pH), and the nature of the cell surface are expected to play an important role in transport, and are investigated using flow cytometry, fluorescene microscopy and UV/Vis spectroscopy. Our results suggest that at physiological pH, cationic polyamidoamine (PAMAM) dendrimers can enter the A549 cancer lung epithelial cells within 5 minutes, perhaps due to the favorable interaction between anionic surface receptors of cells and cationic PAMAM dendrimer, through adsorptive endocytosis. On the other hand, hyperbranched polyol, which is a neutral polymer at physiological pH, enters cells at a much slower rate. The entry of hyperbranched polyol may be because of fluid-phase pinocytosis. Our results also indicate that the dendritic polymers enter the cell surface much more rapidly than linear polymers, and some small drugs, suggesting that the high density of functional groups plays a key role in the interaction with the cell surface, and the subsequent transport inside.

Plasma Membrane Sterol Distribution Resembles the Surface Topography of Living Cells

PubMed Central

2007-01-01

Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8–2.5 μm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1—a putative raft marker—did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane. PMID:17065557

Direct association of thioredoxin-1 (TRX) with macrophage migration inhibitory factor (MIF): regulatory role of TRX on MIF internalization and signaling.

PubMed

Son, Aoi; Kato, Noriko; Horibe, Tomohisa; Matsuo, Yoshiyuki; Mochizuki, Michika; Mitsui, Akira; Kawakami, Koji; Nakamura, Hajime; Yodoi, Junji

2009-10-01

Thioredoxin-1 (TRX) is a small (14 kDa) multifunctional protein with the redox-active site Cys-Gly-Pro-Cys. Macrophage migration inhibitory factor (MIF) is a 12 kDa cytokine belonging to the TRX family. Historically, when we purified TRX from the supernatant of ATL-2 cells, a 12 kDa protein was identified along with TRX, which was later proved to be MIF. Here, we show that TRX and MIF form a complex in the cell and the culture supernatant of ATL-2 cells. Using a BIAcore assay, we confirmed that TRX has a specific affinity with MIF. We also found that extracellular MIF was more effectively internalized into the ATL-2 cells expressing TRX on the cell surface, than the Jurkat T cells which do not express surface TRX. Moreover, anti-TRX antibody blocked the MIF internalization, suggesting that the cell surface TRX is involved in MIF internalization into the cells. Furthermore, anti-TRX antibody inhibited MIF-mediated enhancement of TNF-alpha production from macrophage RAW264.7 cells. These results suggest that the cell surface TRX serves as one of the MIF binding molecules or MIF receptor component and inhibits MIF-mediated inflammatory signals.

Evolution of Cell Size Homeostasis and Growth Rate Diversity during Initial Surface Colonization of Shewanella oneidensis.

PubMed

Lee, Calvin K; Kim, Alexander J; Santos, Giancarlo S; Lai, Peter Y; Lee, Stella Y; Qiao, David F; Anda, Jaime De; Young, Thomas D; Chen, Yujie; Rowe, Annette R; Nealson, Kenneth H; Weiss, Paul S; Wong, Gerard C L

2016-09-06

Cell size control and homeostasis are fundamental features of bacterial metabolism. Recent work suggests that cells add a constant size between birth and division ("adder" model). However, it is not known how cell size homeostasis is influenced by the existence of heterogeneous microenvironments, such as those during biofilm formation. Shewanella oneidensis MR-1 can use diverse energy sources on a range of surfaces via extracellular electron transport (EET), which can impact growth, metabolism, and size diversity. Here, we track bacterial surface communities at single-cell resolution to show that not only do bacterial motility appendages influence the transition from two- to three-dimensional biofilm growth and control postdivisional cell fates, they strongly impact cell size homeostasis. For every generation, we find that the average growth rate for cells that stay on the surface and continue to divide (nondetaching population) and that for cells that detach before their next division (detaching population) are roughly constant. However, the growth rate distribution is narrow for the nondetaching population, but broad for the detaching population in each generation. Interestingly, the appendage deletion mutants (ΔpilA, ΔmshA-D, Δflg) have significantly broader growth rate distributions than that of the wild type for both detaching and nondetaching populations, which suggests that Shewanella appendages are important for sensing and integrating environmental inputs that contribute to size homeostasis. Moreover, our results suggest multiplexing of appendages for sensing and motility functions contributes to cell size dysregulation. These results can potentially provide a framework for generating metabolic diversity in S. oneidensis populations to optimize EET in heterogeneous environments.

Comparative evaluation of the three different surface treatments - conventional, laser and Nano technology methods in enhancing the surface characteristics of commercially pure titanium discs and their effects on cell adhesion: An in vitro study.

PubMed

Vignesh; Nayar, Sanjna; Bhuminathan; Mahadevan; Santhosh, S

2015-04-01

The surface area of the titanium dental implant materials can be increased by surface treatments without altering their shape and form, thereby increasing the biologic properties of the biomaterial. A good biomaterial helps in early cell adhesion and cell signaling. In this study, the commercially pure titanium surfaces were prepared to enable machined surfaces to form a control material and to be compared with sandblasted and acid-etched surfaces, laser treated surfaces and titanium dioxide (20 nm) Nano-particle coated surfaces. The surface elements were characterized. The biocompatibility was evaluated by cell culture in vitro using L929 fibroblasts. The results suggested that the titanium dioxide Nano-particle coated surfaces had good osteoconductivity and can be used as a potential method for coating the biomaterial.

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

Calcium and initial surface binding phase of pinocytosis in Amoeba proteus.

PubMed

Prusch, R D

1986-08-01

The uptake of membrane-bound solute and external medium by bulk-phase pinocytosis in Amoeba proteus is influenced by the level of Ca2+ in the external medium. Increasing external Ca2+ to approximately 10(-4) M increases pinocytotic intensity, while increases in Ca2+ above this level decrease the intensity of pinocytosis. The initial interaction of pinocytotic inducers and Ca2+ at the surface of Amoeba proteus was therefore examined. Alcian blue and Na+, both inducers of pinocytosis, differ in the manner with which they associate with the amoeba surface, suggesting the possibility of different pinocytosis-inducing sites on the amoeba surface. Low levels of external Ca2+ in the range of 3 X 10(-5) to 1.5 X 10(-4) M increase the amount of cationic inducer associated with the cell surface while, at the same time, decreasing anion association with the cell surface. It is suggested that Ca2+ influences ion association with the cell surface by controlling the availability of negative surface sites, which in turn influences pinocytotic intensity.

Effect of respiratory syncytial virus (RSV) infection on the adherence of pathogenic bacteria to human epithelial cells

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

Faden, H.; Hong, J.J.; Ogra, P.L.

1986-03-01

The effect of RSV infection on the adherence of Streptococcus pneumoniae (SP), Haemophilus influenzae (HI) and Staphylococcus aureus (SA) to human epithelial cells was determined. RSV-infected Hep-2 cell cultures at different stages of expression of surface viral antigens and bacteria labeled with /sup 3/H-thymidine were employed to examine the kinetics of bacterial adherence to virus-infected cells. RSV infection did not alter the magnitude of adherence of HI or SA to HEp-2 cells. However, adherence of SP to HEp-2 cells was significantly (P < 0.01) enhanced by prior RSV infection. The degree of adherence was directly related to the amount ofmore » viral antigen expressed on the cell surface. The adherence was temperature dependent, with maximal adherence observed at 37/sup 0/C. Heat-inactivation of SP did not alter adherence characteristics. These data suggest that RSV infection increases adherence of SP to the surface of epithelial cells in vitro. Since attachment of bacteria to mucosal surfaces is the first step in many infections, it is suggested that viral infections of epithelial cells render them more susceptible to bacterial adherence. Thus, RSV infection in vivo may predispose children to SP infections, such as in otitis media, by increasing colonization with SP.« less

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

PubMed

Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki

2014-02-01

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.

Infection of Polarized MDCK Cells with Herpes Simplex Virus 1: Two Asymmetrically Distributed Cell Receptors Interact with Different Viral Proteins

NASA Astrophysics Data System (ADS)

Sears, Amy E.; McGwire, Bradford S.; Roizman, Bernard

1991-06-01

Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable.

Do cell surface trafficking impairments account for variable cell surface sodium iodide symporter levels in breast cancer?

PubMed Central

Beyer, S.J.; Jimenez, R.E.; Shapiro, C.L.; Cho, J.Y.; Jhiang, S.M.

2009-01-01

The Na+/I- symporter (NIS) is a transmembrane glycoprotein that mediates iodide uptake into thyroid follicular cells and serves as the molecular basis of radioiodine imaging and therapy for thyroid cancer patients. The finding that NIS protein is present in 80-90% of breast tumors suggests that breast cancer patients may also benefit from NIS-mediated radionuclide imaging and targeted therapy. However, only 17-25% of NIS-positive breast tumors have detectable radionuclide uptake activity. The discrepancy between NIS expression and radionuclide uptake activity is most likely contributed by variable cell surface NIS protein levels. Apart from the prevalent view that NIS cell surface trafficking impairments account for the variability, our current study proposes that differential levels of NIS expression may also account for variable cell surface NIS levels among breast tumors. We address the need to confirm the identity of intracellular NIS staining to reveal the mechanisms underlying variable cell surface NIS levels. In addition, we warrant a quantitative correlation between cell surface NIS levels and radionuclide uptake activity in patients such that the cell surface NIS levels required for radionuclide imaging can be defined and the defects impairing NIS activity can be recognized. PMID:18500672

Surface glycosaminoglycans mediate adherence between HeLa cells and Lactobacillus salivarius Lv72.

PubMed

Martín, Rebeca; Martín, Carla; Escobedo, Susana; Suárez, Juan E; Quirós, Luis M

2013-09-17

The adhesion of lactobacilli to the vaginal surface is of paramount importance to develop their probiotic functions. For this reason, the role of HeLa cell surface proteoglycans in the attachment of Lactobacillus salivarius Lv72, a mutualistic strain of vaginal origin, was investigated. Incubation of cultures with a variety of glycosaminoglycans (chondroitin sulfate A and C, heparin and heparan sulfate) resulted in marked binding interference. However, no single glycosaminoglycan was able to completely abolish cell binding, the sum of all having an additive effect that suggests cooperation between them and recognition of specific adhesins on the bacterial surface. In contrast, chondroitin sulfate B enhanced cell to cell attachment, showing the relevance of the stereochemistry of the uronic acid and the sulfation pattern on binding. Elimination of the HeLa surface glycosaminoglycans with lyases also resulted in severe adherence impairment. Advantage was taken of the Lactobacillus-glycosaminoglycans interaction to identify an adhesin from the bacterial surface. This protein, identify as a soluble binding protein of an ABC transporter system (OppA) by MALDI-TOF/(MS), was overproduced in Escherichia coli, purified and shown to interfere with L. salivarius Lv72 adhesion to HeLa cells. These data suggest that glycosaminoglycans play a fundamental role in attachment of mutualistic bacteria to the epithelium that lines the cavities where the normal microbiota thrives, OppA being a bacterial adhesin involved in the process.

Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells.

PubMed

Sala-Rabanal, Monica; Yurtsever, Zeynep; Nichols, Colin G; Brett, Tom J

2015-03-17

Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl(-) channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers.

A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

PubMed

Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

2017-05-01

Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Identification of a Supramolecular Functional Architecture of Streptococcus mutans Adhesin P1 on the Bacterial Cell Surface*

PubMed Central

Heim, Kyle P.; Sullan, Ruby May A.; Crowley, Paula J.; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F.; Brady, L. Jeannine

2015-01-01

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. PMID:25666624

Identification of a supramolecular functional architecture of Streptococcus mutans adhesin P1 on the bacterial cell surface.

PubMed

Heim, Kyle P; Sullan, Ruby May A; Crowley, Paula J; El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Tang, Wenxing; Besingi, Richard; Dufrene, Yves F; Brady, L Jeannine

2015-04-03

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Glycosylation controls cooperative PECAM-VEGFR2-β3 integrin functions at the endothelial surface for tumor angiogenesis.

PubMed

Imamaki, Rie; Ogawa, Kazuko; Kizuka, Yasuhiko; Komi, Yusuke; Kojima, Soichi; Kotani, Norihiro; Honke, Koichi; Honda, Takashi; Taniguchi, Naoyuki; Kitazume, Shinobu

2018-05-02

Most of the angiogenesis inhibitors clinically used in cancer treatment target the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway. However, the current strategies for treating angiogenesis have limited efficacy. The issue of how to treat angiogenesis and endothelial dysfunction in cancer remains a matter of substantial debate. Here we demonstrate a glycosylation-dependent regulatory mechanism for tumor angiogenesis. St6gal1 -/- mice, lacking the α2,6-sialylation enzyme, were shown to exhibit impaired tumor angiogenesis through enhanced endothelial apoptosis. In a previous study, St6gal1 -/- endothelial cells exhibited a reduction in the cell surface residency of platelet endothelial cell adhesion molecule (PECAM). In this study, we found that cooperative functionality of PECAM-VEGFR2-integrin β3 was disturbed in St6gal1 -/- mice. First, cell surface PECAM-VEGFR2 complexes were lost, and both VEGFR2 internalization and the VEGFR-dependent signaling pathway were enhanced. Second, enhanced anoikis was observed, suggesting that the absence of α2,6-sialic acid leads to dysregulated integrin signaling. Notably, ectopic expression of PECAM increased cell surface integrin-β3, indicating that the reduction of cell surface integrin-β3 involves loss-of-endothelial PECAM. The results suggest that the cell surface stability of these glycoproteins is significantly reduced by the lack of α2,6-sialic acid, leading to abnormal signal transduction. The present findings highlight that α2,6-sialylation is critically involved in endothelial survival by controlling the cell surface stability and signal transduction of angiogenic molecules, and could be a novel target for anti-angiogenesis therapy.

Early detection of disease program: Evaluation of the cellular immune response

NASA Technical Reports Server (NTRS)

Criswell, B. S.; Knight, V.; Martin, R. R.; Kasel, J. A.

1975-01-01

Surfaces of normal, cultured, and mitogen-stimulated mouse lymphoid cells were examined by scanning electron microscopy (SEM). Lymphocytes with smooth, highly villous and intermediate surfaces were observed in cell suspensions from both spleens and thymuses of normal mice and from spleens of congenitally athymic (nude) mice. Several strain-specific surface features were noted, including the spine-like appearance of microvilli on C57B1/6 lymphocytes. Although thymus cell suspensions contained somewhat more smooth cells than did spleen cell preparations, lymphocyte derivation could not be inferred from SEM examination. Studies of cells stimulated with mitogenic agents for thymus-derived lymphocytes (concanavalin A) or for bone marrow-derived lymphocytes (lipopolysaccharide) suggested that, in the mouse, development of a complex villous surface is a general concomitant of lymphocyte activation and transformation.

Morphological Observations of Mesenchymal Stem Cell Adhesion to a Nanoperiodic-Structured Titanium Surface Patterned Using Femtosecond Laser Processing

NASA Astrophysics Data System (ADS)

Oya, Kei; Aoki, Shun; Shimomura, Kazunori; Sugita, Norihiko; Suzuki, Kenji; Nakamura, Norimasa; Fujie, Hiromichi

2012-12-01

It is known that the adhesive and anisotropic properties of cell-derived biomaterials are affected by micro- or nanoscale structures processed on culture surfaces. In the present study, the femtosecond laser processing technique was used to scan a laser beam at an intensity of approximately the ablation threshold level on a titanium surface for nanoscale processing. Microscopy observation revealed that the processed titanium exhibited a periodic-patterned groove structure at the surface; the width and depth of the groove were 292 ±50 and 99 ±31 nm, respectively, and the periodic pitch of the groove was 501 ±100 nm. Human synovium-derived mesenchymal stem cells were cultured on the surface at a cell density of 3.0×103 cells/cm2 after 4 cell passages. For comparison, the cells were also cultured on a nonprocessed titanium surface under the condition identical to that of the processed surface. Results revealed that the duration for cell attachment to the surface was markedly reduced on the processed titanium as compared with the nonprocessed titanium. Moreover, on the processed titanium, cell extension area significantly increased while cell orientation was aligned along the direction of the periodic grooves. These results suggest that the femtosecond laser processing improves the adhesive and anisotropic properties of cells by producing the nanoperiodic structure on titanium culture surfaces.

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

Tailorable Surface Morphology of 3D Scaffolds by Combining Additive Manufacturing with Thermally Induced Phase Separation.

PubMed

Di Luca, Andrea; de Wijn, Joost R; van Blitterswijk, Clemens A; Camarero-Espinosa, Sandra; Moroni, Lorenzo

2017-08-01

The functionalization of biomaterials substrates used for cell culture is gearing towards an increasing control over cell activity. Although a number of biomaterials have been successfully modified by different strategies to display tailored physical and chemical surface properties, it is still challenging to step from 2D substrates to 3D scaffolds with instructive surface properties for cell culture and tissue regeneration. In this study, additive manufacturing and thermally induced phase separation are combined to create 3D scaffolds with tunable surface morphology from polymer gels. Surface features vary depending on the gel concentration, the exchanging temperature, and the nonsolvent used. When preosteoblasts (MC-3T3 cells) are cultured on these scaffolds, a significant increase in alkaline phosphatase activity is measured for submicron surface topography, suggesting a potential role on early cell differentiation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface-Selective Preferential Production of Reactive Oxygen Species on Piezoelectric Ceramics for Bacterial Killing.

PubMed

Tan, Guoxin; Wang, Shuangying; Zhu, Ye; Zhou, Lei; Yu, Peng; Wang, Xiaolan; He, Tianrui; Chen, Junqi; Mao, Chuanbin; Ning, Chengyun

2016-09-21

Reactive oxygen species (ROS) can be used to kill bacterial cells, and thus the selective generation of ROS from material surfaces is an emerging direction in antibacterial material discovery. We found the polarization of piezoelectric ceramic causes the two sides of the disk to become positively and negatively charged, which translate into cathode and anode surfaces in an aqueous solution. Because of the microelectrolysis of water, ROS are preferentially formed on the cathode surface. Consequently, the bacteria are selectively killed on the cathode surface. However, the cell experiment suggested that the level of ROS is safe for normal mammalian cells.

Galactosyltransferase and Concanavalin A Agglutination of Cells

PubMed Central

Podolsky, Daniel K.; Weiser, Milton M.; Mont, J. Thomas La; Isselbacher, Kurt J.

1974-01-01

A correlation has been observed between concanavalin A agglutination of various cell types and the presence of surface membrane galactosyltransferase (1-O-α-D-Galactosyl-myo-inositol:raffinose galactosyltransferase, EC 2.4.1.67) activity. Moreover, a reduction to less than 50% of cell surface galactosyltransferase activity occurred after treatment with concanavalin A; other cell surface glycosyltransferase enzyme activities examined were unaffected by concanavalin A treatment. To confirm the participation of cell surface galactosyltransferase in concanavalin A-induced cell agglutination, the enzyme from rabbit erythrocytes was solubilized by sonication and purified by preparative polyacrylamide gel electrophoresis. It was possible to achieve a purified preparation of rabbit erythrocyte galactosyltransferase by separation on concanavalin A-Sepharose. The purified enzyme showed visible immunoprecipitation (Ouchterlony) with concanavalin A. Furthermore, human erythrocytes, which are not normally agglutinated by concanavalin A, became agglutinable by the lectin when the erythrocytes were preincubated with purified galactosyltransferase. These experiments suggest a direct and possible specific role of cell surface galactosyltransferase enzyme in the mechanism of concanavalin A agglutination of cells. Images PMID:4522801

Stem Cell Therapy for Healing Wounded Skin and Soft Tissues

DTIC Science & Technology

2012-07-01

changes of ASC surface markers due to repetitive in vitro sub-culturing. ASCs were harvested, washed in PBS to remove cell culture medium, and resuspended...Our in vitro and in vivo studies suggest that ASC and BM-MSC are not identical, though they have similar surface markers . We found that topically...ofpolybrene. Transduced cells were selected by treating 10 J.!g/rnl ofblasticidin. GFP expressing cells were further selected by flow cytometry using

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

NASA Astrophysics Data System (ADS)

Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second aim, the response of surface-bound integrins to flow-related shear stress was examined. Based on fluorescent analysis, total alphavbeta 3, alpha1beta1, and alpha5beta 1 appeared to increase on stainless steel after 90-minute low shear stress exposure, whereas only alpha5beta1 appeared to increase when exposed to high shear. 65/35 poly(D, L-lactide-co-glycolide) exhibited increased total binding of alpha5beta1 and alphaMbeta2, when exposed to either shear stress level. Exposure to either shear stress regimen appeared to increase binding of all integrins on the combinational surface. These responses to shear stress suggest differential integrin binding affinity compared to stainless steel. Using antibodies specific to the integrin subunits, the apparent increase in surface-bound integrins was found to be related to a surface disassociation of alpha and beta subunits. The third aim evaluated human aortic endothelial cells and acute monocytic leukemia cells (THP-1) cell binding to the tested biomaterial surfaces under both static and flow conditions. Both stainless steel and the combinational surface had increased endothelial cell binding compared to monocyte attachment. Pre-incubation of the surface with the specific integrins significantly inhibited human aortic endothelial cell binding. Aim four was designed to investigate the influence of surface bound integrins on human aortic endothelial cell migration under shear stress. If biomaterial surface integrin binding patterns are specific, then pre-bound surface integrins should competitively inhibit binding of cellular integrins to the surface. Cell migration distance on to alphavbeta3, alpha 1beta1, and alpha5beta1 pre-incubated stainless steel was decreased ten-fold, and decreased by three-fold on both 65/35 poly(D, L-lactide-coglycolide) and combinational surfaces compared to the respective bare surfaces. In contrast, migration distance on to alphaMbeta2 pre-coated stainless steel and combinational surface was decreased by only sixty percent and only fifty percent on alphaMbeta2 precoated 65/35 poly(D, L -lactide-co-glycolide). These results suggested that surface binding sites are selective and critical in governing endothelial cell migration. In conclusion, these results support the hypothesis that a surface that encourages specific integrin binding would promote differential cell binding. The novel integrin binding model used in this investigation may be a methodology that can be employed to evaluate potential vascular biomaterials.

TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

PubMed

Oida, Takatoku; Weiner, Howard L

2010-11-24

It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs. We generated anti-mouse LAP mAbs by immunizing TGF-β(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells. Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

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

Takahashi, Tadanobu; Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST, JST, and COE Program in the 21st Century, Shizuoka 422-8526; Moriyama, Yusuke

Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism.

Calreticulin is expressed on the cell surface of activated human peripheral blood T lymphocytes in association with major histocompatibility complex class I molecules.

PubMed

Arosa, F A; de Jesus, O; Porto, G; Carmo, A M; de Sousa, M

1999-06-11

Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of beta2-free MHC class I molecules.

Activation of the novel estrogen receptor G protein-coupled receptor 30 (GPR30) at the plasma membrane.

PubMed

Filardo, E; Quinn, J; Pang, Y; Graeber, C; Shaw, S; Dong, J; Thomas, P

2007-07-01

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17beta-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17beta-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application.

PubMed

Vabbilisetty, Pratima; Boron, Mallorie; Nie, Huan; Ozhegov, Evgeny; Sun, Xue-Long

2018-02-28

Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell's functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine-poly(ethylene glycol)-dibenzocyclooctyne (DSPE-PEG 2000 -DBCO) and cholesterol-PEG-dibenzocyclooctyne (CHOL-PEG 2000 -DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids.

Surface Structure Characterization of Aspergillus fumigatus Conidia Mutated in the Melanin Synthesis Pathway and Their Human Cellular Immune Response

PubMed Central

Bayry, Jagadeesh; Beaussart, Audrey; Dufrêne, Yves F.; Sharma, Meenu; Bansal, Kushagra; Kniemeyer, Olaf; Aimanianda, Vishukumar; Brakhage, Axel A.; Kaveri, Srini V.; Kwon-Chung, Kyung J.

2014-01-01

In Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface. PMID:24818666

Bacterial cell surface properties: role of loosely bound extracellular polymeric substances (LB-EPS).

PubMed

Zhao, Wenqiang; Yang, Shanshan; Huang, Qiaoyun; Cai, Peng

2015-04-01

This study investigated the effect of loosely bound extracellular polymeric substances (LB-EPS) on the comprehensive surface properties of four bacteria (Bacillus subtilis, Streptococcus suis, Escherichia coli and Pseudomonas putida). The removal of LB-EPS from bacterial surfaces by high-speed centrifugation (12,000×g) was confirmed by SEM images. Viability tests showed that the percentages of viable cells ranged from 95.9% to 98.0%, and no significant difference was found after treatment (P>0.05). FTIR spectra revealed the presence of phosphodiester, carboxylic, phosphate, and amino functional groups on bacteria surfaces, and the removal of LB-EPS did not alter the types of cell surface functional groups. Potentiometric titration results suggested the total site concentrations on the intact bacteria were higher than those on LB-EPS free bacteria. Most of the acidity constants (pKa) were almost identical, except the increased pKa values of phosphodiester groups on LB-EPS free S. suis and E. coli surfaces. The electrophoretic mobilities and hydrodynamic diameters of the intact and LB-EPS free bacteria were statistically unchanged (P>0.05), indicating LB-EPS had no influence on the net surface charges and size distribution of bacteria. However, LB-ESP could enhance cell aggregation processes. The four LB-EPS free bacteria all exhibited fewer hydrophobicity values (26.1-65.0%) as compared to the intact cells (47.4-69.3%), suggesting the removal of uncharged nonpolar compounds (e.g., carbohydrates) in LB-EPS. These findings improve our understanding of the changes in cell surface characterizations induced by LB-EPS, and have important implications for assessing the role of LB-EPS in bacterial adhesion and transport behaviors. Copyright © 2015 Elsevier B.V. All rights reserved.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

PubMed

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

CHANGES IN THE MORPHOLOGY AND POLYSACCHARIDE CONTENT OF MICROCYSTIS AERUGINOSA (CYANOBACTERIA) DURING FLAGELLATE GRAZING(1).

PubMed

Yang, Zhou; Kong, Fanxiang; Shi, Xiaoli; Zhang, Min; Xing, Peng; Cao, Huansheng

2008-06-01

To investigate the changes in the morphology and polysaccharide content of Microcystis aeruginosa (Kütz.) Kütz. during flagellate grazing, cultures of M. aeruginosa were exposed to grazing Ochromonas sp. for a period of 9 d under controlled laboratory conditions. M. aeruginosa responded actively to flagellate grazing and formed colonies, most of which were made up of several or dozens of cells, suggesting that flagellate grazing may be one of the biotic factors responsible for colony formation in M. aeruginosa. When colonies were formed, the cell surface ultrastructure changed, and the polysaccharide layer on the surface of the cell wall became thicker. This change indicated that synthesis and secretion of extracellular polysaccharide (EPS) of M. aeruginosa cells increased under flagellate grazing pressure. The contents of soluble extracellular polysaccharide (sEPS), bound extracellular polysaccharide (bEPS), and total polysaccharide (TPS) in colonial cells of M. aeruginosa increased significantly compared with those in single cells. This finding suggested that the increased amount of EPS on the cell surface may play a role in keeping M. aeruginosa cells together to form colonies. © 2008 Phycological Society of America.

The importance of surface recombination and energy-bandgap narrowing in p-n-junction silicon solar cells

NASA Technical Reports Server (NTRS)

Fossum, J. G.; Lindholm, F. A.; Shibib, M. A.

1979-01-01

Experimental data demonstrating the sensitivity of open-circuit voltage to front-surface conditions are presented for a variety of p-n-junction silicon solar cells. Analytical models accounting for the data are defined and supported by additional experiments. The models and the data imply that a) surface recombination significantly limits the open-circuit voltage (and the short-circuit current) of typical silicon cells, and b) energy-bandgap narrowing is important in the manifestation of these limitations. The models suggest modifications in both the structural design and the fabrication processing of the cells that would result in substantial improvements in cell performance. The benefits of one such modification - the addition of a thin thermal silicon-dioxide layer on the front surface - are indicated experimentally.

Osteogenic potential of in situ TiO2 nanowire surfaces formed by thermal oxidation of titanium alloy substrate

NASA Astrophysics Data System (ADS)

Tan, A. W.; Ismail, R.; Chua, K. H.; Ahmad, R.; Akbar, S. A.; Pingguan-Murphy, B.

2014-11-01

Titanium dioxide (TiO2) nanowire surface structures were fabricated in situ by a thermal oxidation process, and their ability to enhance the osteogenic potential of primary osteoblasts was investigated. Human osteoblasts were isolated from nasal bone and cultured on a TiO2 nanowires coated substrate to assess its in vitro cellular interaction. Bare featureless Ti-6Al-4V substrate was used as a control surface. Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression were examined on the TiO2 nanowire surfaces as compared to the control surfaces after 2 weeks of culturing. Cell adhesion and cell proliferation were assayed by field emission scanning electron microscope (FESEM) and Alamar Blue reduction assay, respectively. The nanowire surfaces promoted better cell adhesion and spreading than the control surface, as well as leading to higher cell proliferation. Our results showed that osteoblasts grown onto the TiO2 nanowire surfaces displayed significantly higher production levels of alkaline phosphatase (ALP), extracellular (ECM) mineralization and genes expression of runt-related transcription factor (Runx2), bone sialoprotein (BSP), ostoepontin (OPN) and osteocalcin (OCN) compared to the control surfaces. This suggests the potential use of such surface modification on Ti-6Al-4V substrates as a promising means to improve the osteointegration of titanium based implants.

A conserved WW domain-like motif regulates invariant chain-dependent cell-surface transport of the NKG2D ligand ULBP2.

PubMed

Uhlenbrock, Franziska; van Andel, Esther; Andresen, Lars; Skov, Søren

2015-08-01

Malignant cells expressing NKG2D ligands on their cell surface can be directly sensed and killed by NKG2D-bearing lymphocytes. To ensure this immune recognition, accumulating evidence suggests that NKG2D ligands are trafficed via alternative pathways to the cell surface. We have previously shown that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA/B. Substitution of Trp to alanine resulted in cell-surface inhibition of ULBP2 in different cancer cell lines. Moreover, the mutated ULBP2 constructs were retained and not degraded inside the cell, indicating a crucial role of this conserved Trp-motif in trafficking. Finally, overexpression of Ii increased surface expression of wt ULBP2 while Trp-mutants could not be expressed, proposing that this Trp-motif is required for an Ii-dependent cell-surface transport of ULBP2. Aberrant soluble ULBP2 is immunosuppressive. Thus, targeting a distinct protein module on the ULBP2 sequence could counteract this abnormal expression of ULBP2. Copyright © 2015 Elsevier Ltd. All rights reserved.

Calcium and initial surface binding phase of pinocytosis in Amoeba proteus

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

Prusch, R.D.

1986-08-01

The uptake of membrane-bound solute and external medium by bulk-phase pinocytosis in Amoeba proteus is influenced by the level of Ca/sup 2 +/ in the external medium. Increasing external Ca/sup 2 +/ to approx.10/sup -4/ M increases pinocytotic intensity, while increases in Ca/sup 2 +/ above this level decrease the intensity of pinocytosis. The initial interaction of pinocytotic inducers and Ca/sup +2/ at the surface of A moeba proteus was therefore examined. Alcain blue and Na/sup +/, both inducers of pinocytosis, differ in the manner with which they associate with the amoeba surface, suggesting the possibility of different pinocytosis-inducing sitesmore » on the amoeba surface. Low levels of external Ca/sup 2 +/ in the range of 3 x 10/sup -5/ to 4.5 x 10/sup -4/ M increase the amount of cationic inducer associated with the cell surface while, at the same time, decreasing anion association with the cell surface. It is suggested that Ca/sup 2 +/ influences ion association with the cell surface by controlling the availability of negative surface sites, which in turn influences pinocytotic intensity. Surface binding of Na/sup +/, Ca/sup 2 +/ and Cl/sup -/ was determined by adding /sup 22/Na, /sup 45/Ca or /sup 36/Cl.« less

Antiadhesive Character of Mucin O-glycans at the Apical Surface of Corneal Epithelial Cells

PubMed Central

Sumiyoshi, Mika; Ricciuto, Jessica; Tisdale, Ann; Gipson, Ilene K.; Mantelli, Flavio; Argüeso, Pablo

2008-01-01

Purpose Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal–limbal epithelial (HCLE) cells. Methods Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-α-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell–cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber. Results Benzyl-α-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-α-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface. Conclusions These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface–associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye). PMID:18172093

EMMPRIN Regulates Cytoskeleton Reorganization and Cell Adhesion in Prostate Cancer

PubMed Central

Zhu, Haining; Zhao, Jun; Zhu, Beibei; Collazo, Joanne; Gal, Jozsef; Shi, Ping; Liu, Li; Ström, Anna-Lena; Lu, Xiaoning; McCann, Richard O.; Toborek, Michal; Kyprianou, Natasha

2011-01-01

Background Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment. Methods In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) vs. malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization. Results EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins. Conclusions Our results suggest that EMMPRIN regulates cell adhesion, invasion and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis. PMID:21563192

EMMPRIN regulates cytoskeleton reorganization and cell adhesion in prostate cancer.

PubMed

Zhu, Haining; Zhao, Jun; Zhu, Beibei; Collazo, Joanne; Gal, Jozsef; Shi, Ping; Liu, Li; Ström, Anna-Lena; Lu, Xiaoning; McCann, Richard O; Toborek, Michal; Kyprianou, Natasha

2012-01-01

Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment. In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) versus malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization. EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression, or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins. Our results suggest that EMMPRIN regulates cell adhesion, invasion, and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis. Copyright © 2011 Wiley Periodicals, Inc.

[Quantitative characterization of nuclear polymorphism of the epithelial cells in fibroadenomatosis and cancer of the breast].

PubMed

Ganina, K P; Petunin, Iu I; Timoshenko, Ia G

1989-01-01

A method for quantitative analysis of epithelial cell nuclear polymorphism was suggested, viz. identification of general statistical population using Petunin's criterion. This criterion was employed to assess heterogeneity of visible surface of interphase epithelial cell nuclei and to assay nuclear DNA level in fibroadenomatous hyperplasia and cancer of the breast. Heterogeneity index (h), alongside with other parameters, appeared useful for quantitative assessment of the disease: heterogeneity index values ranging 0.1-0.4 point to pronounced heterogeneity of epithelial cell nucleus surface and DNA level, and are suggestive of malignant transformation of tissue, whereas benign proliferation of the epithelium is usually characterized by 0.4 less than h less than or equal to 0.9.

Coaction of intercellular adhesion and cortical tension specifies tissue surface tension

PubMed Central

Manning, M. Lisa; Foty, Ramsey A.; Steinberg, Malcolm S.; Schoetz, Eva-Maria

2010-01-01

In the course of animal morphogenesis, large-scale cell movements occur, which involve the rearrangement, mutual spreading, and compartmentalization of cell populations in specific configurations. Morphogenetic cell rearrangements such as cell sorting and mutual tissue spreading have been compared with the behaviors of immiscible liquids, which they closely resemble. Based on this similarity, it has been proposed that tissues behave as liquids and possess a characteristic surface tension, which arises as a collective, macroscopic property of groups of mobile, cohering cells. But how are tissue surface tensions generated? Different theories have been proposed to explain how mesoscopic cell properties such as cell–cell adhesion and contractility of cell interfaces may underlie tissue surface tensions. Although recent work suggests that both may be contributors, an explicit model for the dependence of tissue surface tension on these mesoscopic parameters has been missing. Here we show explicitly that the ratio of adhesion to cortical tension determines tissue surface tension. Our minimal model successfully explains the available experimental data and makes predictions, based on the feedback between mechanical energy and geometry, about the shapes of aggregate surface cells, which we verify experimentally. This model indicates that there is a crossover from adhesion dominated to cortical-tension dominated behavior as a function of the ratio between these two quantities. PMID:20616053

Biodegradation and surfactant-mediated biodegradation of diesel fuel by 218 microbial consortia are not correlated to cell surface hydrophobicity.

PubMed

Owsianiak, Mikołaj; Szulc, Alicja; Chrzanowski, Łukasz; Cyplik, Paweł; Bogacki, Mariusz; Olejnik-Schmidt, Agnieszka K; Heipieper, Hermann J

2009-09-01

In this study, we elucidated the role of cell surface hydrophobicity (microbial adhesion to hydrocarbons method, MATH) and the effect of anionic rhamnolipids and nonionic Triton X-100 surfactants on biodegradation of diesel fuel employing 218 microbial consortia isolated from petroleum-contaminated soils. Applied enrichment procedure with floating diesel fuel as a sole carbon source in liquid cultures resulted in consortia of varying biodegradation potential and diametrically different cell surface properties, suggesting that cell surface hydrophobicity is a conserved parameter. Surprisingly, no correlations between cell surface hydrophobicity and biodegradation of diesel fuel were found. Nevertheless, both surfactants altered cell surface hydrophobicity of the consortia in similar manner: increased for the hydrophilic and decreased for the hydrophobic cultures. In addition to this, the surfactants exhibited similar influence on diesel fuel biodegradation: Increase was observed for initially slow-degrading cultures and the opposite for fast degraders. This indicates that in the surfactant-mediated biodegradation, effectiveness of surfactants depends on the specification of microorganisms and not on the type of surfactant. In contrary to what was previously reported for pure strains, cell surface hydrophobicity, as determined by MATH, is not a good descriptor of biodegrading potential for mixed cultures.

Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

PubMed

Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

2015-10-01

Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value to the covalent grafting immobilization methods. However no differences in exposure of the cell binding domains were observed (ELISA results) before SDS rinsing, suggesting that pFN protein grafting to the surface is initially kinetically driven be a stochastic random adsorption phenomenon. Covalent grafting acts in the final stage as a process that simply tethers and stabilizes (or freezes) the initial conformation/orientation of the adsorbed protein on the surface. In addition covalent linkage via the SSMPB approach is likely favored by surface-induce exposure of one of the normally hidden free thiol group pair, thus optimizing covalent linkage to the surface. However after SDS rinsing, this "tethering"/"freezing" effect was significantly more prominent for the GA grafting approach (due to greater number of potential covalent links between the protein and the surface) compared to that for the SSMPB approach. This hypothesis was buttressed by the improved resistance to denaturation (smaller conformational lability) for the GA compared to the SMPB approach and improved exposure of the cell binding domain for the former (>50%) even after SDS rinsing. These results are promising in that they suggest covalent tethering of fibronectin to PS substrate in a monolayer range, with significantly improved irreversible protein surface bonding via both approaches (compared to that for mere adsorption). The latter are likely applicable to a wide range of proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

High cell surface death receptor expression determines type I versus type II signaling.

PubMed

Meng, Xue Wei; Peterson, Kevin L; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D; Gores, Gregory J; Kaufmann, Scott H

2011-10-14

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Zinc Binding and Dimerization of Streptococcus pyogenes Pyrogenic Exotoxin C Are Not Essential for T-Cell Stimulation

DTIC Science & Technology

2003-03-14

streptococcal superantigen binding to MHCII on the surface of cells (7–9), suggesting an essential role in both MHCII molecular recognition and TCR-mediated...extent, mutations of side chains found in a second conserved MHCII alpha-chain-binding site consisting of a hydrophobic surface loop decreased T-cell...fraction of dimer is present at T-cell stimulatory concentrations of Spe-C following mutation of the unpaired side chain of cys- teine at residue 27 to

Sustained neurotensin exposure promotes cell surface recruitment of NTS2 receptors

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

Perron, Amelie; Sharif, Nadder; Gendron, Louis

2006-05-12

In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [{sup 125}I]-NT binding. We found that NTS2-expressing cells retained the same surface receptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores,more » as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.« less

Microtubule array reorientation in response to hormones does not involve changes in microtubule nucleation modes at the periclinal cell surface

PubMed Central

Atkinson, Samantha; Kirik, Angela; Kirik, Viktor

2014-01-01

Aligned microtubule arrays spatially organize cell division, trafficking, and determine the direction of cell expansion in plant cells. In response to changes in environmental and developmental signals, cells reorganize their microtubule arrays into new configurations. Here, we tested the role of microtubule nucleation during hormone-induced microtubule array reorientation. We have found that in the process of microtubule array reorientation the ratios between branching, parallel, and de-novo nucleations remained constant, suggesting that the microtubule reorientation mechanism does not involve changes in nucleation modes. In the ton2/fass mutant, which has reduced microtubule branching nucleation frequency and decreased nucleation activity of the γ-tubulin complexes, microtubule arrays were able to reorient. Presented data suggest that reorientation of microtubules into transverse arrays in response to hormones does not involve changes in microtubule nucleation at the periclinal cell surface PMID:25135522

Fine tuning cellular recognition: The function of the leucine rich repeat (LRR) trans-membrane protein, LRT, in muscle targeting to tendon cells.

PubMed

Gilsohn, Eli; Volk, Talila

2010-01-01

The formation of complex tissues during embryonic development is often accompanied by directed cellular migration towards a target tissue. Specific mutual recognition between the migrating cell and its target tissue leads to the arrest of the cell migratory behavior and subsequent contact formation between the two interacting cell types. Recent studies implicated a novel family of surface proteins containing a trans-membrane domain and single leucine-rich repeat (LRR) domain in inter-cellular recognition and the arrest of cell migration. Here, we describe the involvement of a novel LRR surface protein, LRT, in targeting migrating muscles towards their corresponding tendon cells in the Drosophila embryo. LRT is specifically expressed by the target tendon cells and is essential for arresting the migratory behavior of the muscle cells. Additional studies in Drosophila S2 cultured cells suggest that LRT forms a protein complex with the Roundabout (Robo) receptor, essential for guiding muscles towards their tendon partners. Genetic analysis supports a model in which LRT performs its activity non-autonomously through its interaction with the Robo receptors expressed on the muscle surfaces. These results suggest a novel mechanism of intercellular recognition through interactions between LRR family members and Robo receptors.

Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface

PubMed Central

DAS, R.; PLOW, E. F.

2013-01-01

Summary Background Plasminogen (Plg) binding to cell surface Plg receptors (Plg-Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg-R and its cell surface expression is upregulated when monocytes are differentiated to macrophages via a pathway dependent on L-type Ca2+ channels and intracellular Ca2+. Objectives We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on themacrophage surface. Methods THP-1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outermembrane exposure of phosphatidylserine (PS). Results H2B interacted directly with PS via an electrostatic interaction. Anti-PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L-type Ca2+ channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. Conclusions These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L-type Ca2+ channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to. PMID:21040449

Synthesis and biological evaluation of sialyl-oligonucleotide conjugates targeting leukocyte B trans-membranal receptor CD22 as delivery agents for nucleic acid drugs.

PubMed

St-Pierre, Gabrielle; Pal, Sudip; Østergaard, Michael E; Zhou, Tianyuan; Yu, Jinghua; Tanowitz, Michael; Seth, Punit P; Hanessian, Stephen

2016-06-01

Antisense oligonucleotides (ASOs) modified with ligands which target cell surface receptors have the potential to significantly improve potency in the target tissue. This has recently been demonstrated using triantennary N-acetyl d-galactosamine conjugated ASOs. CD22 is a cell surface receptor expressed exclusively on B cells thus presenting an attractive target for B cell specific delivery of drugs. Herein, we reported the synthesis of monovalent and trivalent ASO conjugates with biphenylcarbonyl (BPC) modified sialic acids and their study as ASO delivery agents into B cells. CD22 positive cells exhibited reduced potency when treated with ligand modified ASOs and mechanistic examination suggested reduced uptake into cells potentially as a result of sequestration of ASO by other cell-surface proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Presence of closely spaced protein thiols on the surface of mammalian cells.

PubMed Central

Donoghue, N.; Yam, P. T.; Jiang, X. M.; Hogg, P. J.

2000-01-01

It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the gamma-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active. PMID:11206065

Capture and release of cells using a temperature-responsive surface that immobilizes an antibody through DNA duplex formation.

PubMed

Kimura, Tsuyoshi; Nakamura, Naoko; Umeda, Kanji; Hashimoto, Yoshihide; Kishida, Akio

We synthesized a temperature-responsive surface that immobilized an antibody via DNA duplex formation for selective capture and release of target cells. Polyethylene films were modified by grafting poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), which were prepared at various ratios of NIPAAm/AAc. The increased hydrophilicity of P(NIPAAm-co-PAA) film with decreased temperature was confirmed by water contact angle measurement. Single strand DNA (20mer) was chemically immobilized on the surface and then antibody (anti-mouse CD45, mCD45) modified with the complementary single strand DNA was immobilized on the surface through DNA duplex formation. The mCD45 antibody immobilization was confirmed by immunostaining. HeLa cells (mCD45 negative) and mouse bone marrow (BM) cells (mCD45 positive) were adhered on the surfaces at 37 °C. Although HeLa cells were detached by 4 °C incubation, BM cells were still adhered on the surface and then the adhered cells were released by DNase treatment. From these results, it was suggested that cells could be selectively captured and collected by using a film having surface that immobilizes an antibody via DNA duplex formation.

Upregulation of cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on magnesium alloy surface coating with tricalcium phosphate.

PubMed

Jiang, Tianlong; Guo, Lei; Ni, Shenghui; Zhao, Yuyan

2015-04-01

Magnesium (Mg) alloys have been demonstrated to be viable orthopedic implants because of mechanical and biocompatible properties similar to natural bone. In order to improve its osteogenic properties, a porous β-tricalcium phosphate (β-TCP) was coated on the Mg-3AI-1Zn alloy by alkali-heat treatment technique. The human bone-derived cells (SaOS-2) were cultured on (β-TCP)-Mg-3AI-1Zn in vitro, and the osteoblast response, the morphology and the elements on this alloy surface were investigated. Also, the regulation of key intracellular signalling proteins was investigated in the SaOS-2 cells cultured on alloy surface. The results from scanning electron microscope and immunofluorescence staining demonstrated that (β-TCP)-Mg-3AI-1Zn induced significant osteogenesis. SaOS-2 cell proliferation was improved by β-TCP coating. Moreover, the (β-TCP)-Mg-3AI-1Zn surface induced activation of key intracellular signalling proteins in SaOS-2 cells. We observed an enhanced activation of Src homology and collagen (Shc), a common point of integration between bone morphogenetic protein 2, and the Ras/mitogen-activated protein kinase (MAPK) pathway. ERK1/2 MAP kinase activation was also upregulated, suggesting a role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the (β-TCP)-Mg-3AI-1Zn. These results suggest that β-TCP coating may contribute to successful osteoblast function on Mg alloy surface. (β-TCP)-Mg-3AI-1Zn may upregulate cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on Mg alloy surface.

Megakaryocyte Polyploidization and Proplatelet Formation in Low-Attachment Conditions.

PubMed

Schlinker, Alaina C; Duncan, Mark T; DeLuca, Teresa A; Whitehead, David C; Miller, William M

2016-07-15

In vitro -derived platelets (PLTs), which could provide an alternative source of PLTs for patient transfusions, are formed from polyploid megakaryocytes (MKs) that extend long cytoplasmic projections, termed proplatelets (proPLTs). In this study, we compared polyploidization and proPLT formation (PPF) of MKs cultured on surfaces that either promote or inhibit protein adsorption and subsequent cell adhesion. A megakaryoblastic cell line exhibited increased polyploidization and arrested PPF on a low-attachment surface. Primary human MKs also showed low levels of PPF on the same surface, but no difference in ploidy. Importantly, both cell types exhibited accelerated PPF after transfer to a surface that supports attachment, suggesting that pre-culture on a non-adhesive surface may facilitate synchronization of PPF and PLT generation in culture.

Surface structure characterization of Aspergillus fumigatus conidia mutated in the melanin synthesis pathway and their human cellular immune response.

PubMed

Bayry, Jagadeesh; Beaussart, Audrey; Dufrêne, Yves F; Sharma, Meenu; Bansal, Kushagra; Kniemeyer, Olaf; Aimanianda, Vishukumar; Brakhage, Axel A; Kaveri, Srini V; Kwon-Chung, Kyung J; Latgé, Jean-Paul; Beauvais, Anne

2014-08-01

In Aspergillus fumigatus, the conidial surface contains dihydroxynaphthalene (DHN)-melanin. Six-clustered gene products have been identified that mediate sequential catalysis of DHN-melanin biosynthesis. Melanin thus produced is known to be a virulence factor, protecting the fungus from the host defense mechanisms. In the present study, individual deletion of the genes involved in the initial three steps of melanin biosynthesis resulted in an altered conidial surface with masked surface rodlet layer, leaky cell wall allowing the deposition of proteins on the cell surface and exposing the otherwise-masked cell wall polysaccharides at the surface. Melanin as such was immunologically inert; however, deletion mutant conidia with modified surfaces could activate human dendritic cells and the subsequent cytokine production in contrast to the wild-type conidia. Cell surface defects were rectified in the conidia mutated in downstream melanin biosynthetic pathway, and maximum immune inertness was observed upon synthesis of vermelone onward. These observations suggest that although melanin as such is an immunologically inert material, it confers virulence by facilitating proper formation of the A. fumigatus conidial surface. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The chemotaxis-like Che1 pathway has an indirect role in adhesive cell properties of Azospirillum brasilense.

PubMed

Siuti, Piro; Green, Calvin; Edwards, Amanda Nicole; Doktycz, Mitchel J; Alexandre, Gladys

2011-10-01

The Azospirillum brasilense chemotaxis-like Che1 signal transduction pathway was recently shown to modulate changes in adhesive cell surface properties that, in turn, affect cell-to-cell aggregation and flocculation behaviors rather than flagellar-mediated chemotaxis. Attachment to surfaces and root colonization may be functions related to flocculation. Here, the conditions under which A. brasilense wild-type Sp7 and che1 mutant strains attach to abiotic and biotic surfaces were examined using in vitro attachment and biofilm assays combined with atomic force microscopy and confocal microscopy. The nitrogen source available for growth is found to be a major modulator of surface attachment by A. brasilense and could be promoted in vitro by lectins, suggesting that it depends on interaction with surface-exposed residues within the extracellular matrix of cells. However, Che1-dependent signaling is shown to contribute indirectly to surface attachment, indicating that distinct mechanisms are likely underlying flocculation and attachment to surfaces in A. brasilense. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Transport of IRW, an ovotransferrin-derived antihypertensive peptide, in human intestinal epithelial Caco-2 cells.

PubMed

Bejjani, Satyanarayana; Wu, Jianping

2013-02-20

IRW is an egg ovotransferrin-derived ACE inhibitory peptide. The purpose of this study was to evaluate the stability and transcellular transport of IRW in Caco-2 cell monolayers. The stability of IRW was monitored on the apical (AP) surface while its transport was studied from AP to basal (BL) and from BL to AP surfaces. The results revealed that IRW is resistant against intestinal peptidase up to 60 min. Transport of IRW was not affected by addition of wortamanin, a transcytosis inhibitor. However, in the presence of cytochalasin D, a gap junction disruptor, transport of IRW was significantly increased, suggesting a possible passive transport from AP to BL surface. A higher transport of IRW from AP to BL surface than that from BL to AP surface suggests a passive-mediated transport. Moreover, in the presence of glycyl-sarcosine, a substrate for peptide transporter PepT 1, transport of IRW was reduced from AP to BL surface. The above observations showed atypical transport of IRW in Caco-2 cell monolayers. Thus, IRW may possibly be absorbed intact into the site of action for controlling hypertension.

Antimicrobial design of titanium surface that kill sessile bacteria but support stem cells adhesion

NASA Astrophysics Data System (ADS)

Zhu, Chen; Bao, Ni-Rong; Chen, Shuo; Zhao, Jian-Ning

2016-12-01

Implant-related bacterial infection is one of the most severe postoperative complications in orthopedic or dental surgery. In this context, from the perspective of surface modification, increasing efforts have been made to enhance the antibacterial capability of titanium surface. In this work, a hierarchical hybrid surface architecture was firstly constructed on titanium surface by two-step strategy of acid etching and H2O2 aging. Then silver nanoparticles were firmly immobilized on the hierarchical surface by ion implantation, showing no detectable release of silver ions from surface. The designed titanium surface showed good bioactivity. More importantly, this elaborately designed titanium surface can effectively inactivate the adherent S. aureus on surface by virtue of a contact-killing mode. Meanwhile, the designed titanium surface can significantly facilitate the initial adhesion and spreading behaviors of bone marrow mesenchymal stem cells (MSCs) on titanium. The results suggested that, the elaborately designed titanium surface might own a cell-favoring ability that can help mammalian cells win the initial adhesion race against bacteria. We hope the present study can provide a new insight for the better understanding and designing of antimicrobial titanium surface, and pave the way to satisfying clinical requirements.

Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application

PubMed Central

2018-01-01

Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell’s functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine–poly(ethylene glycol)–dibenzocyclooctyne (DSPE–PEG2000–DBCO) and cholesterol–PEG–dibenzocyclooctyne (CHOL–PEG2000–DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids. PMID:29503972

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

Oligosaccharide ligands for NKR-P1 protein activate NK cells and cytotoxicity

NASA Astrophysics Data System (ADS)

Bezouška, Karel; Yuen, Chun-Ting; O'Brien, Jacqui; Childs, Robert A.; Chai, Wengang; Lawson, Alexander M.; Drbal, Karel; Fišerová, Anna; Posíšil, Miloslav; Feizi, Ten

1994-11-01

A diversity of high-affinity Oligosaccharide ligands are identified for NKR-P1, a membrane protein on natural killer (NK) cells which contains an extracellular Ca2+-dependent lectin domain. Interactions of such oligosaccharides on the target cell surface with NKR-P1 on the killer cell surface are crucial both for target cell recognition and for delivery of stimulatory or inhibitory signals linked to the NK cytolytic machinery. NK-resistant tumour cells are rendered susceptible by preincubation with liposomes expressing NKR-P1 ligands, suggesting that purging of tumour or virally infected cells in vivo may be a therapeutic possibility.

Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

PubMed Central

Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

2012-01-01

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

Various fates of neuronal progenitor cells observed on several different chemical functional groups

NASA Astrophysics Data System (ADS)

Liu, Xi; Wang, Ying; He, Jin; Wang, Xiu-Mei; Cui, Fu-Zhai; Xu, Quan-Yuan

2011-12-01

Neuronal progenitor cells cultured on gold-coated glass surfaces modified by different chemical functional groups, including hydroxyl (-OH), carboxyl (-COOH), amino (-NH2), bromo (-Br), mercapto (-SH), - Phenyl and methyl (-CH3), were studied here to investigate the influence of surface chemistry on the cells' adhesion, morphology, proliferation and functional gene expression. Focal adhesion staining indicated in the initial culture stage cells exhibited morphological changes in response to different chemical functional groups. Cells cultured on -NH2 grafted surface displayed focal adhesion plaque and flattened morphology and had the largest contact area. However, their counter parts on -CH3 grafted surface displayed no focal adhesion and rounded morphology and had the smallest contact area. After 6 days culture, the proliferation trend was as follows: -NH2 > -SH> -COOH> - Phenyl > - Br > -OH> -CH3. To determine the neural functional properties of the cells affected by surface chemistry, the expression of glutamate decarboxylase (GAD67), nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF) were characterized. An increase of GAD67 expression was observed on -NH2, -COOH and -SH grafted surfaces, while no increase in NGF and BDNF expression was observed on any chemical surfaces. These results highlight the importance of surface chemistry in the fate determination of neuronal progenitor cells, and suggest that surface chemistry must be considered in the design of biomaterials for neural tissue engineering.

Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.

PubMed Central

Iino, R; Koyama, I; Kusumi, A

2001-01-01

Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions). PMID:11371443

Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis.

PubMed

San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

2015-10-01

This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis

PubMed Central

San, Kaungmyat; Long, Janet; Michels, Corinne A; Gadura, Nidhi

2015-01-01

This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain. PMID:26185055

Exopolysaccharide-Independent Social Motility of Myxococcus xanthus

PubMed Central

Hu, Wei; Hossain, Muhaiminu; Lux, Renate; Wang, Jing; Yang, Zhe; Li, Yuezhong; Shi, Wenyuan

2011-01-01

Social motility (S motility), the coordinated movement of large cell groups on agar surfaces, of Myxococcus xanthus requires type IV pili (TFP) and exopolysaccharides (EPS). Previous models proposed that this behavior, which only occurred within cell groups, requires cycles of TFP extension and retraction triggered by the close interaction of TFP with EPS. However, the curious observation that M. xanthus can perform TFP-dependent motility at a single-cell level when placed onto polystyrene surfaces in a highly viscous medium containing 1% methylcellulose indicated that “S motility” is not limited to group movements. In an apparent further challenge of the previous findings for S motility, mutants defective in EPS production were found to perform TFP-dependent motility on polystyrene surface in methylcellulose-containing medium. By exploring the interactions between pilin and surface materials, we found that the binding of TFP onto polystyrene surfaces eliminated the requirement for EPS in EPS- cells and thus enabled TFP-dependent motility on a single cell level. However, the presence of a general anchoring surface in a viscous environment could not substitute for the role of cell surface EPS in group movement. Furthermore, EPS was found to serve as a self-produced anchoring substrate that can be shed onto surfaces to enable cells to conduct TFP-dependent motility regardless of surface properties. These results suggested that in certain environments, such as in methylcellulose solution, the cells could bypass the need for EPS to anchor their TPF and conduct single-cell S motility to promote exploratory movement of colonies over new specific surfaces. PMID:21245931

Cell surface acid-base properties of the cyanobacterium Synechococcus: Influences of nitrogen source, growth phase and N:P ratios

NASA Astrophysics Data System (ADS)

Liu, Yuxia; Alessi, D. S.; Owttrim, G. W.; Kenney, J. P. L.; Zhou, Qixing; Lalonde, S. V.; Konhauser, K. O.

2016-08-01

The distribution of many trace metals in the oceans is controlled by biological uptake. Recently, Liu et al. (2015) demonstrated the propensity for a marine cyanobacterium to adsorb cadmium from seawater, suggesting that cell surface reactivity might also play an important role in the cycling of metals in the oceans. However, it remains unclear how variations in cyanobacterial growth rates and nutrient supply might affect the chemical properties of their cellular surfaces. In this study we used potentiometric titrations and Fourier Transform Infrared (FT-IR) spectrometry to profile the key metabolic changes and surface chemical responses of a Synechococcus strain, PCC 7002, during different growth regimes. This included testing various nitrogen (N) to phosphorous (P) ratios (both nitrogen and phosphorous dependent), nitrogen sources (nitrate, ammonium and urea) and growth stages (exponential, stationary, and death phase). FT-IR spectroscopy showed that varying the growth substrates on which Synechococcus cells were cultured resulted in differences in either the type or abundance of cellular exudates produced or a change in the cell wall components. Potentiometric titration data were modeled using three distinct proton binding sites, with resulting pKa values for cells of the various growth conditions in the ranges of 4.96-5.51 (pKa1), 6.67-7.42 (pKa2) and 8.13-9.95 (pKa3). According to previous spectroscopic studies, these pKa ranges are consistent with carboxyl, phosphoryl, and amine groups, respectively. Comparisons between the titration data (for the cell surface) and FT-IR spectra (for the average cellular changes) generally indicate (1) that the nitrogen source is a greater determinant of ligand concentration than growth phase, and (2) that phosphorus limitation has a greater impact on Synechococcus cellular and extracellular properties than does nitrogen limitation. Taken together, these techniques indicate that nutritional quality during cell growth can noticeably influence the expression of cell surface ligands and their measurable densities. Given that cell surface charge ultimately affects metal adsorption, our results suggest that the cycling of metals by Synechococcus cells in the oceans may vary regionally.

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

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

PubMed Central

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel

2016-01-01

Summary Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN. PMID:27826507

How Escherichia coli lands and forms cell clusters on a surface: a new role of surface topography

PubMed Central

Gu, Huan; Chen, Aaron; Song, Xinran; Brasch, Megan E.; Henderson, James H.; Ren, Dacheng

2016-01-01

Bacterial response to surface topography during biofilm formation was studied using 5 μm tall line patterns of poly (dimethylsiloxane) (PDMS). Escherichia coli cells attached on top of protruding line patterns were found to align more perpendicularly to the orientation of line patterns when the pattern narrowed. Consistently, cell cluster formation per unit area on 5 μm wide line patterns was reduced by 14-fold compared to flat PDMS. Contrasting the reduced colony formation, cells attached on narrow patterns were longer and had higher transcriptional activities, suggesting that such unfavorable topography may present a stress to attached cells. Results of mutant studies indicate that flagellar motility is involved in the observed preference in cell orientation on narrow patterns, which was corroborated by the changes in cell rotation pattern before settling on different surface topographies. These findings led to a set of new design principles for creating antifouling topographies, which was validated using 10 μm tall hexagonal patterns. PMID:27412365

Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells

PubMed Central

Sänger, Christian; Mühlberger, Elke; Ryabchikova, Elena; Kolesnikova, Larissa; Klenk, Hans-Dieter; Becker, Stephan

2001-01-01

Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein. PMID:11152500

Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

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

Chang, Eun-Ju; Kim, Hong-Hee; Huh, Jung-Eun

2005-02-01

The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growthmore » factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue.« less

Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates

PubMed Central

1977-01-01

A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D- galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies. PMID:833547

Effect of sialic acid loss on dendritic cell maturation

PubMed Central

Crespo, Hélio J; Guadalupe Cabral, M; Teixeira, Alexandra V; Lau, Joseph T Y; Trindade, Hélder; Videira, Paula A

2009-01-01

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I−/− and ST6Gal.I−/− mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I−/− and ST6Gal.I−/− strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading. PMID:19740323

CD40 expression in Wehi-164 cell line

PubMed Central

Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

2010-01-01

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body’s defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system. PMID:20496113

CD40 expression in Wehi-164 cell line.

PubMed

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

2010-07-01

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body's defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system.

Specialized cell surface structures in cellulolytic bacteria.

PubMed Central

Lamed, R; Naimark, J; Morgenstern, E; Bayer, E A

1987-01-01

The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose. Images PMID:3301817

SYNTHESIS, INTRACELLULAR DISTRIBUTION, AND SECRETION OF IMMUNOGLOBULIN AND H-2 ANTIGEN IN MURINE SPLENOCYTES

PubMed Central

Wernet, Dorothee; Vitetta, Ellen S.; Uhr, Jonathan W.; Boyse, Edward A.

1973-01-01

A/J spleen cells were labeled with [3H]leucine and at intervals thereafter were homogenized and separated into microsomes and cell sap. Ig and H-2 antigens were assayed in the cell fractions and cell supernatants using immunoprecipitation. In addition, cells labeled by enzymatic radioiodination were incubated to determine the rates of release of Ig and H-2 antigens from the surface. The results indicate that the majority of Ig and H-2 antigens remain membrane bound throughout their intracellular life. In contrast to Ig, H-2 antigens are neither secreted nor shed from the cell surface. It is suggested that Ig is a peripheral protein of the cell membrane, whereas H-2 antigens are integral ones. The release of Ig on a fragment of plasma membrane could occur at fixed cell surface areas that contain no H-2 antigens or from which they have migrated before release. PMID:4200648

Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

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

Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

1995-04-10

Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, althoughmore » its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.« less

Emergence of fractal geometry on the surface of human cervical epithelial cells during progression towards cancer

NASA Astrophysics Data System (ADS)

Dokukin, M. E.; Guz, N. V.; Woodworth, C. D.; Sokolov, I.

2015-03-01

Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation.

Emerging of fractal geometry on surface of human cervical epithelial cells during progression towards cancer

PubMed Central

Dokukin, M. E.; Guz, N. V.; Woodworth, C.D.; Sokolov, I.

2015-01-01

Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation. PMID:25844044

The influence of surface carbohydrates during in vitro infection of mammalian cells by the dermatophyte Trichophyton rubrum.

PubMed

Esquenazi, Daniele; Alviano, Celuta S; de Souza, Wanderley; Rozental, Sonia

2004-04-01

In order to better understand the role played by surface glycoconjugates during host cell adhesion and endocytosis of Trichophyton rubrum, we looked for the presence of carbohydrate-binding adhesins on the microconidia surface and their role on cellular interaction with epithelial and macrophages cells. The interaction of T. rubrum with chinese hamster ovary epithelial cells and their glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, that express mannose and galactose, respectively. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates to the interaction medium, pretreatment with lectins and with sodium periodate decreased the adhesion and endocytic index for all mutants. The ability of the fungus to penetrate into mammalian cells was confirmed in experiments using macrophages treated with cytochalasin D. Flow cytometric analysis showed that this fungus recognizes mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 than at 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature for the microconidia adhesins. The presence of lectin-like molecules in fungus cell could be observed by scanning electron microscopy of the fungus incubated with colloidal-gold labeled neoglycoproteins. Our results suggest that T. rubrum has the ability to invade mammalian cells and expresses carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. These adhesins may play an important role on the adhesion and invasion of the fungus during the infectious process of dermatophytosis.

Borrelia burgdorferi upregulates the adhesion molecules E-selectin, P-selectin, ICAM-1 and VCAM-1 on mouse endothelioma cells in vitro.

PubMed

Böggemeyer, E; Stehle, T; Schaible, U E; Hahne, M; Vestweber, D; Simon, M M

1994-06-01

In order to obtain more information on processes leading to Borrelia burgdorferi-induced inflammation in the host, we have developed an in vitro model to study the upregulation of cell surface expression of adhesion molecules on endothelial cells by spirochetes. A mouse endothelioma cell line, derived from brain capillaries, bEnd3, was used as indicator population. bEnd3 cells were incubated with preparations of viable, inactivated or sonicated spirochetes and the expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 was monitored by immunocytochemistry and quantified by cell surface ELISA. We show that all three spirochetal preparations are able to upregulate cell surface expression of E-selectin, P-selectin, ICAM-1 and VCAM-1 on bEnd 3 cells in a dose-dependent manner. The kinetics of cell surface expression of the individual adhesion molecules in the presence of Borrelia burgdorferi showed maxima at about 50 h of incubation or later; this was distinct from results obtained with sonicated-preparations of Escherichia coli bacteria or with enterobacterial LPS where peak expression was observed between 4 h and 16 h. The fact that Borrelia burgdorferi does not contain conventional LPS suggests that the mode of induction of adhesion molecules on endothelial cells is influenced by the phenotype of bacteria. At the peak of spirochete-induced cell surface expression of adhesion molecules (approximately 50 h), bEnd3 cells were found to bind cells of a VLA-4+ B lymphoma line (L1-2) much more efficiently than untreated control cells. The binding of L1-2 cells to presensitized bEnd3 cells was significantly inhibited (more than 75%) in the presence of monoclonal antibodies to both VLA-4 and its endothelial counterreceptor VCAM-1. These findings demonstrate that Borrelia burgdorferi organisms are able to induce functionally active adhesion molecules on endothelial cells in vitro and suggest that E-selectin, P-selectin, ICAM-1 and VCAM-1 play an important role in the pathogenesis of spirochetal infection.

The behavior of MC3T3-E1 cells on chitosan/poly-L-lysine composite films: effect of nanotopography, surface chemistry, and wettability.

PubMed

Zheng, Zhenhuan; Zhang, Ling; Kong, Lijun; Wang, Aijun; Gong, Yandao; Zhang, Xiufang

2009-05-01

In the present work, a series of composite films were produced from chitosan/poly-L-lysine blend solutions. The surface topography, chemistry, and wettability of composite films were characterized by atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and contact angle assay, respectively. For all composite films, blending with poly-L-lysine induced changes in surface chemistry and wettability. Interestingly, it was also found that increasing poly-L-lysine weight fraction in blend solutions could result in different nanoscaled surface topographic features, which displayed particle-, granule-, or fiber-dominant morphologies. MC3T3-E1 osteoblast-like cells were cultured on all composite films to evaluate the effects of surface nanotopography, chemistry, and wettability on cell behavior. The observations indicated that MC3T3-E1 cell behavior was affected by surface topography, chemistry, and wettability simultaneously and that cells showed strong responses to surface topography. On fiber-dominant surface, cells fully spread with obvious cytoskeleton organization and exhibited significantly higher level of adhesion and proliferation compared with particle- or granule-dominant surfaces. Furthermore, fiber-dominant surface also induced greater expression of mature osteogenic marker osteocalcin and higher mineralization based on RT-PCR and von Kossa staining. The results suggest that topographic modification of chitosan substratum at the nanoscale may be exploited in regulating cell behavior for its applications in tissue engineering.

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.

Cell behavior on gallium nitride surfaces: peptide affinity attachment versus covalent functionalization.

PubMed

Foster, Corey M; Collazo, Ramon; Sitar, Zlatko; Ivanisevic, Albena

2013-07-02

Gallium nitride is a wide band gap semiconductor that demonstrates a unique set of optical and electrical properties as well as aqueous stability and biocompatibility. This combination of properties makes gallium nitride a strong candidate for use in chemical and biological applications such as sensors and neural interfaces. Molecular modification can be used to enhance the functionality and properties of the gallium nitride surface. Here, gallium nitride surfaces were functionalized with a PC12 cell adhesion promoting peptide using covalent and affinity driven attachment methods. The covalent scheme proceeded by Grignard reaction and olefin metathesis while the affinity driven scheme utilized the recognition peptide isolated through phage display. This study shows that the method of attaching the adhesion peptide influences PC12 cell adhesion and differentiation as measured by cell density and morphological analysis. Covalent attachment promoted monolayer and dispersed cell adhesion while affinity driven attachment promoted multilayer cell agglomeration. Higher cell density was observed on surfaces modified using the recognition peptide. The results suggest that the covalent and affinity driven attachment methods are both suitable for promoting PC12 cell adhesion to the gallium nitride surface, though each method may be preferentially suited for distinct applications.

Megakaryocyte Polyploidization and Proplatelet Formation in Low-Attachment Conditions

PubMed Central

Schlinker, Alaina C.; Whitehead, David C.; Miller, William M.

2016-01-01

In vitro-derived platelets (PLTs), which could provide an alternative source of PLTs for patient transfusions, are formed from polyploid megakaryocytes (MKs) that extend long cytoplasmic projections, termed proplatelets (proPLTs). In this study, we compared polyploidization and proPLT formation (PPF) of MKs cultured on surfaces that either promote or inhibit protein adsorption and subsequent cell adhesion. A megakaryoblastic cell line exhibited increased polyploidization and arrested PPF on a low-attachment surface. Primary human MKs also showed low levels of PPF on the same surface, but no difference in ploidy. Importantly, both cell types exhibited accelerated PPF after transfer to a surface that supports attachment, suggesting that pre-culture on a non-adhesive surface may facilitate synchronization of PPF and PLT generation in culture. PMID:27087780

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.

Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

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

Makarova, Olga V.; Adams, Daniel L.; Divan, Ralu

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and withoutmore » an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. Copyright 2016 The Authors. Published by Elsevier B.V. All rights reserved.« less

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.

The RSV F and G glycoproteins interact to form a complex on the surface of infected cells

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

Low, Kit-Wei; Tan, Timothy; Ng, Ken

2008-02-08

In this study, the interaction between the respiratory syncytial virus (RSV) fusion (F) protein, attachment (G) protein, and small hydrophobic (SH) proteins was examined. Immunoprecipitation analysis suggested that the F and G proteins exist as a protein complex on the surface of RSV-infected cells, and this conclusion was supported by ultracentrifugation analysis that demonstrated co-migration of surface-expressed F and G proteins. Although our analysis provided evidence for an interaction between the G and SH proteins, no evidence was obtained for a single protein complex involving all three of the virus proteins. These data suggest the existence of multiple virus glycoproteinmore » complexes within the RSV envelope. Although the stimulus that drives RSV-mediated membrane fusion is unknown, the association between the G and F proteins suggest an indirect role for the G protein in this process.« less

Rare TREM2 variants associated with Alzheimer's disease display reduced cell surface expression.

PubMed

Sirkis, Daniel W; Bonham, Luke W; Aparicio, Renan E; Geier, Ethan G; Ramos, Eliana Marisa; Wang, Qing; Karydas, Anna; Miller, Zachary A; Miller, Bruce L; Coppola, Giovanni; Yokoyama, Jennifer S

2016-09-02

Rare variation in TREM2 has been associated with greater risk for Alzheimer's disease (AD). TREM2 encodes a cell surface receptor expressed on microglia and related cells, and the R47H variant associated with AD appears to affect the ability of TREM2 to bind extracellular ligands. In addition, other rare TREM2 mutations causing early-onset neurodegeneration are thought to impair cell surface expression. Using a sequence kernel association (SKAT) analysis in two independent AD cohorts, we found significant enrichment of rare TREM2 variants not previously characterized at the protein level. Heterologous expression of the identified variants showed that novel variants S31F and R47C displayed significantly reduced cell surface expression. In addition, we identified rare variant R136Q in a patient with language-predominant AD that also showed impaired surface expression. The results suggest rare TREM2 variants enriched in AD may be associated with altered TREM2 function and that AD risk may be conferred, in part, from altered TREM2 surface expression.

Surface Mechanoengineering of a Zr-based Bulk Metallic Glass via Ar-Nanobubble Doping to Probe Cell Sensitivity to Rigid Materials

DOE PAGES

Huang, Lu; Tian, Mengkun; Wu, Dong; ...

2017-11-24

In this paper, a new materials platform, utilizing the amorphous microstructure of bulk metallic glasses (BMGs) and the versatility of ion implantation, was developed for the fundamental investigation of cell responses to substrate-rigidity variations in the gigapascal modulus range, which was previously unattainable with polymeric materials. The surface rigidity of a Zr-Al- Ni-Cu-Y BMG was modulated with low-energy Ar-ion implantation owing to the impartment of Ar nanobubbles into the amorphous matrix. Surface softening was achieved due to the formation of nanobubble-doped transitional zones in the Zrbased BMG substrate. Bone-forming cell studies on this newly designed platform demonstrated that mechanical cues,more » accompanied with the potential effects of other surface properties (i.e. roughness, morphology, and chemistry), contributed to modulating cell behaviors. Cell adhesion and actin filaments were found to be less established on less stiff surfaces, especially on the surface with an elastic modulus of 51 GPa. Cell growth appeared to be affected by surface mechanical properties. A lower stiffness was generally related to a higher growth rate. Findings in this study broadened our fundamental understanding concerning the mechanosensing of bone cells on stiff substrates. It also suggests that surface mechano-engineering of metallic materials could be a potential strategy to promote osseointegration of such materials for bone-implant applications. Further investigations are proposed to fine tune the ion implantation variables in order to further distinguish the surface-mechanical effect on bone-forming cell activities from the contributions of other surface properties.« less

Surface Mechanoengineering of a Zr-based Bulk Metallic Glass via Ar-Nanobubble Doping to Probe Cell Sensitivity to Rigid Materials

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

Huang, Lu; Tian, Mengkun; Wu, Dong

In this paper, a new materials platform, utilizing the amorphous microstructure of bulk metallic glasses (BMGs) and the versatility of ion implantation, was developed for the fundamental investigation of cell responses to substrate-rigidity variations in the gigapascal modulus range, which was previously unattainable with polymeric materials. The surface rigidity of a Zr-Al- Ni-Cu-Y BMG was modulated with low-energy Ar-ion implantation owing to the impartment of Ar nanobubbles into the amorphous matrix. Surface softening was achieved due to the formation of nanobubble-doped transitional zones in the Zrbased BMG substrate. Bone-forming cell studies on this newly designed platform demonstrated that mechanical cues,more » accompanied with the potential effects of other surface properties (i.e. roughness, morphology, and chemistry), contributed to modulating cell behaviors. Cell adhesion and actin filaments were found to be less established on less stiff surfaces, especially on the surface with an elastic modulus of 51 GPa. Cell growth appeared to be affected by surface mechanical properties. A lower stiffness was generally related to a higher growth rate. Findings in this study broadened our fundamental understanding concerning the mechanosensing of bone cells on stiff substrates. It also suggests that surface mechano-engineering of metallic materials could be a potential strategy to promote osseointegration of such materials for bone-implant applications. Further investigations are proposed to fine tune the ion implantation variables in order to further distinguish the surface-mechanical effect on bone-forming cell activities from the contributions of other surface properties.« less

Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier*

PubMed Central

Argüeso, Pablo; Guzman-Aranguez, Ana; Mantelli, Flavio; Cao, Zhiyi; Ricciuto, Jessica; Panjwani, Noorjahan

2009-01-01

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins. PMID:19556244

Influence of Substrates on the Surface Characteristics and Membrane Proteome of Fibrobacter succinogenes S85

PubMed Central

Raut, Mahendra P.; Karunakaran, Esther; Mukherjee, Joy; Biggs, Catherine A.; Wright, Phillip C.

2015-01-01

Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane. PMID:26492413

Clostridium perfringens Iota Toxin: Binding Studies and Characterization of Cell Surface Receptor by Fluorescence-Activated Cytometry

PubMed Central

Stiles, Bradley G.; Hale, Martha L.; Marvaud, Jean-Christophe; Popoff, Michel R.

2000-01-01

The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37°C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37°C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane. PMID:10816501

Clostridium perfringens iota toxin: binding studies and characterization of cell surface receptor by fluorescence-activated cytometry.

PubMed

Stiles, B G; Hale, M L; Marvaud, J C; Popoff, M R

2000-06-01

The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37 degrees C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37 degrees C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.

Flavobacterium johnsoniae sprB Is Part of an Operon Spanning the Additional Gliding Motility Genes sprC, sprD, and sprF ▿ †

PubMed Central

Rhodes, Ryan G.; Nelson, Shawn S.; Pochiraju, Soumya; McBride, Mark J.

2011-01-01

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the gliding motor that propels cell surface adhesins, such as the 669-kDa SprB. A novel protein secretion apparatus called the Por secretion system (PorSS) is required for assembly of SprB on the cell surface. Genetic and molecular analyses revealed that sprB is part of a seven-gene operon spanning 29.3 kbp of DNA. In addition to sprB, three other genes of this operon (sprC, sprD, and sprF) are involved in gliding. Mutations in sprB, sprC, sprD, and sprF resulted in cells that failed to form spreading colonies on agar but that exhibited some motility on glass in wet mounts. SprF exhibits some similarity to Porphyromonas gingivalis PorP, which is required for secretion of gingipain protease virulence factors via the P. gingivalis PorSS. F. johnsoniae sprF mutants produced SprB protein but were defective in localization of SprB to the cell surface, suggesting a role for SprF in secretion of SprB. The F. johnsoniae PorSS is involved in secretion of extracellular chitinase in addition to its role in secretion of SprB. SprF was not needed for chitinase secretion and may be specifically required for SprB secretion by the PorSS. Cells with nonpolar mutations in sprC or sprD produced and secreted SprB and propelled it rapidly along the cell surface. Multiple paralogs of sprB, sprC, sprD, and sprF are present in the genome, which may explain why mutations in sprB, sprC, sprD, and sprF do not result in complete loss of motility and suggests the possibility that semiredundant SprB-like adhesins may allow movement of cells over different surfaces. PMID:21131497

Formation of bone-like mineralized matrix by periodontal ligament cells in vivo: a morphological study in rats.

PubMed

Hiraga, Toru; Ninomiya, Tadashi; Hosoya, Akihiro; Takahashi, Masafumi; Nakamura, Hiroaki

2009-01-01

Periodontal ligament (PDL) is a unique connective tissue that not only connects cementum and alveolar bone to support teeth, but also plays an important role in reconstructing periodontal tissues. Previous studies have suggested that PDL cells have osteogenic potential; however, they lack precise histological examinations. Here, we studied bone-like matrix formation by PDL cells in rats using morphological techniques. Rat and human PDL cells exhibited substantial alkaline phosphatase activity and induced mineralization in vitro. RT-PCR analyses showed that PDL cells expressed the osteoblast markers, Runx2, osterix, and osteocalcin. These results suggest that PDL cells share similar phenotypes with osteoblasts. To examine the bone-like matrix formation in vivo, PDL cells isolated from green fluorescent protein (GFP)-transgenic rats were inoculated with hydroxyapatite (HA) disks into wild-type rats. Five weeks after the implantation, the pores in HA disks were occupied by GFP-positive cells. Mineralized matrix formation was also found on the surface of HA pores. At 12 weeks, some of the pores were filled with bone-like mineralized matrices (BLMM), which were positive for the bone matrix proteins, osteopontin, bone sialoprotein, and osteocalcin. Immunohistochemical examination revealed that most of the osteoblast- and osteocyte-like cells on or in the BLMM were GFP-positive, suggesting that the BLMM were directly formed by the inoculated PDL cells. On the pore surfaces, Sharpey's fiber-like structures embedded in cementum-like mineralized layers were also observed. These results collectively suggest that PDL cells have the ability to form periodontal tissues and could be a useful source for regenerative therapies of periodontal diseases.

Immobilization of concanavalin A receptors during differentiation of neuroblastoma cells.

PubMed

Fishman, M C; Dragsten, P R; Spector, I

1981-04-30

Neuroblastoma cells serve as a useful model of neuronal development because compounds such as dimethyl sulphoxide (DMSO) and dibutyryl cyclic AMP cause them to undergo a process of controlled differentiation in tissue culture, during which they can extend long processes, develop characteristic excitability mechanisms, synthesize neurotransmitters and form synapses. We have used the technique of fluorescence photobleaching recovery to study the lateral mobility of cell-surface constituents during the differentiation of neuroblastoma clone N1E-115 cells. The concanavalin A (Con A) binding sites appear as discrete patches distributed over the entire cell surface and exhibit lateral mobility in undifferentiated cells comparable with that of surface glycoproteins of other cells. After induction of differentiation, however, the vast majority of Con A binding sites become immobilized, and we present data which suggest that the mechanism of this immobilization may involve linkage to the internal actin network.

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.

SRC-like adaptor protein regulates B cell development and function.

PubMed

Dragone, Leonard L; Myers, Margaret D; White, Carmen; Sosinowski, Tomasz; Weiss, Arthur

2006-01-01

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

Anti-LRP/LR Specific Antibody IgG1-iS18 Impedes Adhesion and Invasion of Liver Cancer Cells

PubMed Central

Chetty, Carryn; Khumalo, Thandokuhle; Da Costa Dias, Bianca; Reusch, Uwe; Knackmuss, Stefan; Little, Melvyn; Weiss, Stefan F. T.

2014-01-01

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson's correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction. PMID:24798101

A Cell-surface Phylome for African Trypanosomes

PubMed Central

Jackson, Andrew P.; Allison, Harriet C.; Barry, J. David; Field, Mark C.; Hertz-Fowler, Christiane; Berriman, Matthew

2013-01-01

The cell surface of Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at: http://www.genedb.org/Page/trypanosoma_surface_phylome. PMID:23556014

Characterization of cell surface and extracellular matrix remodeling of Azospirillum brasilense chemotaxis-like 1 signal transduction pathway mutants by atomic force microscopy.

PubMed

Edwards, Amanda Nicole; Siuti, Piro; Bible, Amber N; Alexandre, Gladys; Retterer, Scott T; Doktycz, Mitchel J; Morrell-Falvey, Jennifer L

2011-01-01

To compete in complex microbial communities, bacteria must sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the regulation of multiple behaviors in response to changes in the environment, including motility patterns, exopolysaccharide production, and cell-to-cell interactions. In Azospirillum brasilense, cell surface properties, including exopolysaccharide production, are thought to play a direct role in promoting flocculation. Recently, the Che1 chemotaxis-like pathway from A. brasilense was shown to modulate flocculation, suggesting an associated modulation of cell surface properties. Using atomic force microscopy, distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains were detected. Whereas the wild-type strain produces a smooth mucosal extracellular matrix after 24 h, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition, lectin-binding assays, and comparison of lipopolysaccharides profiles suggest that the extracellular matrix differs between the cheA1 and the cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that disruption of the Che1 pathway is correlated with distinctive changes in the extracellular matrix, which likely result from changes in surface polysaccharides structure and/or composition. FEMS Microbiology Letters © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

Amyloid-β peptide on sialyl-Lewis(X)-selectin-mediated membrane tether mechanics at the cerebral endothelial cell surface.

PubMed

Askarova, Sholpan; Sun, Zhe; Sun, Grace Y; Meininger, Gerald A; Lee, James C-M

2013-01-01

Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewis(x) (sLe(x)) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLe(x) and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf ), and produced a bimodal population of Fmtf , suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf . In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB.

Amyloid-β Peptide on Sialyl-LewisX-Selectin-Mediated Membrane Tether Mechanics at the Cerebral Endothelial Cell Surface

PubMed Central

Askarova, Sholpan; Sun, Zhe; Sun, Grace Y.; Meininger, Gerald A.; Lee, James C-M.

2013-01-01

Increased deposition of amyloid-β peptide (Aβ) at the cerebral endothelial cell (CEC) surface has been implicated in enhancement of transmigration of monocytes across the brain blood barrier (BBB) in Alzheimer's disease (AD). In this study, quantitative immunofluorescence microscopy (QIM) and atomic force microscopy (AFM) with cantilevers biofunctionalized by sialyl-Lewisx (sLex) were employed to investigate Aβ-altered mechanics of membrane tethers formed by bonding between sLex and p-selectin at the CEC surface, the initial mechanical step governing the transmigration of monocytes. QIM results indicated the ability for Aβ to increase p-selectin expression at the cell surface and promote actin polymerization in both bEND3 cells (immortalized mouse CECs) and human primary CECs. AFM data also showed the ability for Aβ to increase cell stiffness and adhesion probability in bEND3 cells. On the contrary, Aβ lowered the overall force of membrane tether formation (Fmtf), and produced a bimodal population of Fmtf, suggesting subcellular mechanical alterations in membrane tethering. The lower Fmtf population was similar to the results obtained from cells treated with an F-actin-disrupting drug, latrunculin A. Indeed, AFM results also showed that both Aβ and latrunculin A decreased membrane stiffness, suggesting a lower membrane-cytoskeleton adhesion, a factor resulting in lower Fmtf. In addition, these cerebral endothelial alterations induced by Aβ were abrogated by lovastatin, consistent with its anti-inflammatory effects. In sum, these results demonstrated the ability for Aβ to enhance p-selectin expression at the CEC surface and induce cytoskeleton reorganization, which in turn, resulted in changes in membrane-cytoskeleton adhesion and membrane tethering, mechanical factors important in transmigration of monocytes through the BBB. PMID:23593361

The role of surface carbohydrates on the interaction of microconidia of Trichophyton mentagrophytes with epithelial cells.

PubMed

Esquenazi, Daniele; de Souza, Wanderley; Alviano, Celuta Sales; Rozental, Sonia

2003-03-20

The presence of carbohydrate-binding adhesins on the microconidia of Trichophyton mentagrophytes surface and their role on cellular interactions were investigated. Flow cytometry showed that this fungus recognizes the sugars mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 degrees C than 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature of the microconidia adhesin. The interaction of the fungus to Chinese hamster ovary epithelial cells and its glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, which express mannose and galactose, respectively, as the terminal carbohydrate on the cell surface. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside to the interaction medium, pretreatment of Lec1 and Lec2 cells with lectins Concanavalina A and Arachis hypogaea and pretreatment with sodium periodate decreased the adhesion and the endocytic index. Examination of thin section by transmission electron microscopy showed that after fungal ingestion by Lec2 cells the fungi are enclosed in a 'loose'-type vacuole while the other cells are found within a 'tight'-type membrane-bound cytoplasmic vacuole. Our results suggest the occurrence of carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. This may have a role in the adhesion process during the infectious process of dermatophytosis.

Effect of Stratification on Surface Properties of Corneal Epithelial Cells

PubMed Central

Yáñez-Soto, Bernardo; Leonard, Brian C.; Raghunathan, Vijay Krishna; Abbott, Nicholas L.; Murphy, Christopher J.

2015-01-01

Purpose The purpose of this study was to determine the influence of mucin expression in an immortalized human corneal epithelial cell line (hTCEpi) on the surface properties of cells, such as wettability, contact angle, and surface heterogeneity. Methods hTCEpi cells were cultured to confluence in serum-free medium. The medium was then replaced by stratification medium to induce mucin biosynthesis. The mucin expression profile was analyzed using quantitative PCR and Western blotting. Contact angles were measured using a two-immiscible liquid method, and contact angle hysteresis was evaluated by tilting the apparatus and recording advancing and receding contact angles. The spatial distribution of mucins was evaluated with fluorescently labeled lectin. Results hTCEpi cells expressed the three main ocular mucins (MUC1, MUC4, and MUC16) with a maximum between days 1 and 3 of the stratification process. Upon stratification, cells caused a very significant increase in contact angle hysteresis, suggesting the development of spatially discrete and heterogeneously distributed surface features, defined by topography and/or chemical functionality. Although atomic force microscopy measurements showed no formation of appreciable topographic features on the surface of the cells, we observed a significant increase in surface chemical heterogeneity. Conclusions The surface chemical heterogeneity of the corneal epithelium may influence the dynamic behavior of tear film by “pinning” the contact line between the cellular surface and aqueous tear film. Engineering the surface properties of corneal epithelium could potentially lead to novel treatments in dry eye disease. PMID:26747762

Contact Killing of Bacteria on Copper Is Suppressed if Bacterial-Metal Contact Is Prevented and Is Induced on Iron by Copper Ions

PubMed Central

Mathews, Salima; Hans, Michael

2013-01-01

Bacteria are rapidly killed on copper surfaces, and copper ions released from the surface have been proposed to play a major role in the killing process. However, it has remained unclear whether contact of the bacteria with the copper surface is also an important factor. Using laser interference lithography, we engineered copper surfaces which were covered with a grid of an inert polymer which prevented contact of the bacteria with the surface. Using Enterococcus hirae as a model organism, we showed that the release of ionic copper from these modified surfaces was not significantly reduced. In contrast, killing of bacteria was strongly attenuated. When E. hirae cells were exposed to a solid iron surface, the loss of cell viability was the same as on glass. However, exposing cells to iron in the presence of 4 mM CuSO4 led to complete killing in 100 min. These experiments suggest that contact killing proceeds by a mechanism whereby the metal-bacterial contact damages the cell envelope, which, in turn, makes the cells susceptible to further damage by copper ions. PMID:23396344

Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding.

PubMed

Fayadat, L; Niccoli-Sire, P; Lanet, J; Franc, J L

1999-04-09

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.

Nano-hydroxyapatite-coated metal-ceramic composite of iron-tricalcium phosphate: Improving the surface wettability, adhesion and proliferation of mesenchymal stem cells in vitro.

PubMed

Surmeneva, Maria A; Kleinhans, Claudia; Vacun, Gabriele; Kluger, Petra Juliane; Schönhaar, Veronika; Müller, Michaela; Hein, Sebastian Boris; Wittmar, Alexandra; Ulbricht, Mathias; Prymak, Oleg; Oehr, Christian; Surmenev, Roman A

2015-11-01

Thin radio-frequency magnetron sputter deposited nano-hydroxyapatite (HA) films were prepared on the surface of a Fe-tricalcium phosphate (Fe-TCP) bioceramic composite, which was obtained using a conventional powder injection moulding technique. The obtained nano-hydroxyapatite coated Fe-TCP biocomposites (nano-HA-Fe-TCP) were studied with respect to their chemical and phase composition, surface morphology, water contact angle, surface free energy and hysteresis. The deposition process resulted in a homogeneous, single-phase HA coating. The ability of the surface to support adhesion and the proliferation of human mesenchymal stem cells (hMSCs) was studied using biological short-term tests in vitro. The surface of the uncoated Fe-TCP bioceramic composite showed an initial cell attachment after 24h of seeding, but adhesion, proliferation and growth did not persist during 14 days of culture. However, the HA-Fe-TCP surfaces allowed cell adhesion, and proliferation during 14 days. The deposition of the nano-HA films on the Fe-TCP surface resulted in higher surface energy, improved hydrophilicity and biocompatibility compared with the surface of the uncoated Fe-TCP. Furthermore, it is suggested that an increase in the polar component of the surface energy was responsible for the enhanced cell adhesion and proliferation in the case of the nano-HA-Fe-TCP biocomposites. Copyright © 2015 Elsevier B.V. All rights reserved.

ABI domain containing proteins contribute to surface protein display and cell division in Staphylococcus aureus

PubMed Central

Frankel, Matthew B.; Wojcik, Brandon; DeDent, Andrea C.; Missiakas, Dominique M.; Schneewind, Olaf

2012-01-01

Summary The human pathogen Staphyloccocus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harbored transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross walls and in the relative abundance of staphylococci with cross walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. PMID:20923422

Swimming Motility Reduces Deposition to Silica Surfaces

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

Lu, Nanxi; Massoudieh, Arash; Liang, Xiaomeng

The role of swimming motility on bacterial transport and fate in porous media was evaluated. We present microscopic evidence showing that strong swimming motility reduces attachment of Azotobacter vinelandii cells to silica surfaces. Applying global and cluster statistical analyses to microscopic videos taken under non-flow conditions, wild type, flagellated A. vinelandii strain DJ showed strong swimming ability with an average speed of 13.1 μm/s, DJ77 showed impaired swimming averaged at 8.7 μm/s, and both the non-flagellated JZ52 and chemically treated DJ cells were non-motile. Quantitative analyses of trajectories observed at different distances above the collector of a radial stagnation pointmore » flow cell (RSPF) revealed that both swimming and non-swimming cells moved with the flow when at a distance of at least 20 μm from the collector surface. Near the surface, DJ cells showed both horizontal and vertical movement diverging them from reaching surfaces, while chemically treated DJ cells moved with the flow to reach surfaces, suggesting that strong swimming reduced attachment. In agreement with the RSPF results, the deposition rates obtained for two-dimensional multiple-collector micromodels were also lowest for DJ, while DJ77 and JZ52 showed similar values. Strong swimming specifically reduced deposition on the upstream surfaces of the micromodel collectors.« less

ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus.

PubMed

Frankel, Matthew B; Wojcik, Brandon M; DeDent, Andrea C; Missiakas, Dominique M; Schneewind, Olaf

2010-10-01

The human pathogen Staphylococcus aureus requires cell wall anchored surface proteins to cause disease. During cell division, surface proteins with YSIRK signal peptides are secreted into the cross-wall, a layer of newly synthesized peptidoglycan between separating daughter cells. The molecular determinants for the trafficking of surface proteins are, however, still unknown. We screened mutants with non-redundant transposon insertions by fluorescence-activated cell sorting for reduced deposition of protein A (SpA) into the staphylococcal envelope. Three mutants, each of which harboured transposon insertions in genes for transmembrane proteins, displayed greatly reduced envelope abundance of SpA and surface proteins with YSIRK signal peptides. Characterization of the corresponding mutations identified three transmembrane proteins with abortive infectivity (ABI) domains, elements first described in lactococci for their role in phage exclusion. Mutations in genes for ABI domain proteins, designated spdA, spdB and spdC (surface protein display), diminish the expression of surface proteins with YSIRK signal peptides, but not of precursor proteins with conventional signal peptides. spdA, spdB and spdC mutants display an increase in the thickness of cross-walls and in the relative abundance of staphylococci with cross-walls, suggesting that spd mutations may represent a possible link between staphylococcal cell division and protein secretion. © 2010 Blackwell Publishing Ltd.

Quantitative analysis of osteoblast behavior on microgrooved hydroxyapatite and titanium substrata.

PubMed

Lu, Xiong; Leng, Yang

2003-09-01

The effects of implant surface topography and chemistry on osteoblast behavior have been a research focus because of their potential importance in orthopedic and dental applications. This work focused on the topographic effects of hydroxyapatite (HA) and titanium (Ti) surface that had identical micropatterns to determine whether there was synergistic interaction between surface chemistry and surface topography. Surface microgrooves with six different groove widths (4, 8, 16, 24, 30, and 38 microm) and three different groove depths (2, 4, and 10 microm) were made on single crystalline silicon wafers using microfabrication techniques. Ti and HA thin films were coated on the microgrooves by radio-frequency magnetron sputtering. After that, human osteoblast-like cells were seeded and cultured on the microgrooved surfaces for up to 7 days. The cells' behavior was examined using scanning electron microscopy after cells were fixed and dehydrated. Statistical analysis was based on quantitative data of orientation angle, evaluating the contact guidance, and form index, describing cell shape or cell morphology changes. The contact guidance and cell shape changes were observed on the HA and Ti microgrooves. No difference in orientation angle between HA and Ti microgrooves was found. This might suggest that surface chemistry was not a significant influence on cell guidance. However, the form index analysis indicated an interaction between topographic effects and surface chemistry. Thus, conclusions about surface topographic effects on cell behavior drawn from one type of material cannot simply be applied to another type of material. Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 66A: 677-687, 2003

The effect of charging rate on the graphite electrode of commercial lithium-ion cells: A post-mortem study

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

Somerville, L.; Bareno, J.; Trask, S.

Increased charging rates negatively affect the lifetime of lithium-ion cells by increasing cell resistance and reducing capacity. This work is a post-mortem study of 18650 cells subjected to charge rates of 0.7-, 2-, 4-, and 6-C. For cells charged at 0.7-C to 4-C, this performance degradation is primarily related to surface film thickness with no observable change in surface film chemical composition. However, at charge rates of 6-C, the chemical composition of the surface film changes significantly, suggesting that this change is the reason for the sharper increase in cell resistance compared to the lower charge rates. In addition, wemore » found that surface film formation was not uniform across the electrode. Surface film was thicker and chemically different along the central band of the electrode “jelly roll”. This result is most likely attributable to an increase in temperature that results from non-uniform electrode wetting during manufacture. As a result, this non-uniform change further resulted in active material delamination from the current collector owing to chemical changes to the binder for the cell charged at 6-C.« less

The effect of charging rate on the graphite electrode of commercial lithium-ion cells: A post-mortem study

DOE PAGES

Somerville, L.; Bareno, J.; Trask, S.; ...

2016-10-22

Increased charging rates negatively affect the lifetime of lithium-ion cells by increasing cell resistance and reducing capacity. This work is a post-mortem study of 18650 cells subjected to charge rates of 0.7-, 2-, 4-, and 6-C. For cells charged at 0.7-C to 4-C, this performance degradation is primarily related to surface film thickness with no observable change in surface film chemical composition. However, at charge rates of 6-C, the chemical composition of the surface film changes significantly, suggesting that this change is the reason for the sharper increase in cell resistance compared to the lower charge rates. In addition, wemore » found that surface film formation was not uniform across the electrode. Surface film was thicker and chemically different along the central band of the electrode “jelly roll”. This result is most likely attributable to an increase in temperature that results from non-uniform electrode wetting during manufacture. As a result, this non-uniform change further resulted in active material delamination from the current collector owing to chemical changes to the binder for the cell charged at 6-C.« less

Fabrication and surface modification of macroporous poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds for human skin fibroblast cell culture.

PubMed

Yang, Jian; Shi, Guixin; Bei, Jianzhong; Wang, Shenguo; Cao, Yilin; Shang, Qingxin; Yang, Guanghui; Wang, Wenjing

2002-12-05

The fabrication and surface modification of a porous cell scaffold are very important in tissue engineering. Of most concern are high-density cell seeding, nutrient and oxygen supply, and cell affinity. In the present study, poly(L-lactic acid) and poly(L-lactic-co-glycolic acid) (70/30) cell scaffolds with different pore structures were fabricated. An improved method based on Archimedes' Principle for measuring the porosity of scaffolds, using a density bottle, was developed. Anhydrous ammonia plasma treatment was used to modify surface properties to improve the cell affinity of the scaffolds. The results show that hydrophilicity and surface energy were improved. The polar N-containing groups and positive charged groups also were incorporated into the sample surface. A low-temperature treatment was used to maintain the plasma-modified surface properties effectively. It would do help to the further application of plasma treatment technique. Cell culture results showed that pores smaller than 160 microm are suitable for human skin fibroblast cell growth. Cell seeding efficiency was maintained at above 99%, which is better than the efficiency achieved with the common method of prewetting by ethanol. The plasma-treatment method also helped to resolve the problem of cell loss during cell seeding, and the negative effects of the ethanol trace on cell culture were avoided. The results suggest that anhydrous ammonia plasma treatment enhances the cell affinity of porous scaffolds. Mass transport issues also have been considered. Copyright 2002 Wiley Periodicals, Inc.

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.

Combinatorial growth of oxide nanoscaffolds and its influence in osteoblast cell adhesion

NASA Astrophysics Data System (ADS)

Acevedo-Morantes, Claudia Y.; Irizarry-Ortiz, Roberto A.; Caceres-Valencia, Pablo G.; Singh, Surinder P.; Ramirez-Vick, Jaime E.

2012-05-01

We report a novel method for high-throughput investigations on cell-material interactions based on metal oxide nanoscaffolds. These scaffolds possess a continuous gradient of various titanium alloys allowing the compositional and morphological variation that could substantially improve the formation of an osseointegrative interface with bone. The model nanoscaffold has been fabricated on commercially pure titanium (cp-Ti) substrate with a compositional gradients of tin (Sn), chromium (Cr), and niobium (Nb) deposited using a combinatorial approach followed by annealing to create native oxide surface. As an invitro test system, the human fetal osteoblastic cell line (hFOB 1.19) has been used. Cell-adhesion of hFOB 1.19 cells and the suitability of these alloys have been evaluated for cell-morphology, cell-number, and protein adsorption. Although, cell-morphology was not affected by surface composition, cell-proliferation rates varied significantly with surface metal oxide composition; with the Sn- and Nb-rich regions showing the highest proliferation rate and the Cr-rich regions presenting the lowest. The results suggest that Sn and Nb rich regions on surface seems to promote hFOB 1.19 cell proliferation and may therefore be considered as implant material candidates that deserve further analysis.

Effects of scaffold surface morphology on cell adhesion and survival rate in vitreous cryopreservation of tenocyte-scaffold constructs

NASA Astrophysics Data System (ADS)

Wang, Zhi; Qing, Quan; Chen, Xi; Liu, Cheng-Jun; Luo, Jing-Cong; Hu, Jin-Lian; Qin, Ting-Wu

2016-12-01

The purpose of this study was to investigate the effects of scaffold surface morphology on cell adhesion and survival rate in vitreous cryopreservation of tenocyte-scaffold constructs. Tenocytes were obtained from tail tendons of rats. Polydimethylsiloxane (PDMS) was used to fabricate three types of scaffolds with varying surface morphological characteristics, i.e., smooth, micro-grooved, and porous surfaces, respectively. The tenocytes were seeded on the surfaces of the scaffolds to form tenocyte-scaffold constructs. The constructs were cryopreserved in a vitreous cryoprotectant (CPA) with a multi-step protocol. The cell adhesion to scaffolds was observed with electronic scanning microscopy (SEM). The elongation index of the living tenocytes and ratio of live/dead cell number were examined based on a live/dead dual fluorescent staining technique, and the survival rate of tenocytes was studied with flow cytometry (FC). The results showed the shapes of tenocytes varied between the different groups: flat or polygonal (on smooth surface), spindle (on micro-grooved surface), and spindle or ellipse (on porous surface). After thawing, the porous surface got the most living tenocytes and a higher survival rate, suggesting its potential application for vitreous cryopreservation of engineered tendon constructs.

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.

Novel INTeraction of MUC4 and galectin: potential pathobiological implications for metastasis in lethal pancreatic cancer.

PubMed

Senapati, Shantibhusan; Chaturvedi, Pallavi; Chaney, William G; Chakraborty, Subhankar; Gnanapragassam, Vinayaga S; Sasson, Aaron R; Batra, Surinder K

2011-01-15

Several studies have reported aberrant expression of MUC4 in pancreatic cancer (PC), which is associated with tumorigenicity and metastasis. Mechanisms through which MUC4 promote metastasis of PC cells to distant organs are poorly defined. Identification of MUC4-galectin-3 interaction and its effect on the adhesion of cancer cells to endothelial cells were done by immunoprecipitation and cell-cell adhesion assays, respectively. Serum galectin-3 level for normal and PC patients were evaluated through ELISA. In the present study, we have provided clinical evidence that the level of galectin-3 is significantly elevated in the sera of PC patients with metastatic disease compared with patients without metastasis (P = 0.04) and healthy controls (P = 0.00001). Importantly, for the first time, we demonstrate that MUC4 present on the surface of circulating PC cells plays a significant role in the transient and reversible attachment (docking) of circulating tumor cells to the surface of endothelial cells. Further, exogenous galectin-3 at concentrations similar to that found in the sera of PC patients interacts with MUC4 via surface glycans such as T antigens, which results in the clustering of MUC4 on the cell surface and a stronger attachment (locking) of circulating tumor cells to the endothelium. Altogether, these findings suggest that PC cell-associated MUC4 helps in the docking of tumor cells on the endothelial surface. During cancer progression, MUC4-galectin-3 interaction-mediated clustering of MUC4 may expose the surface adhesion molecules, which in turn promotes a stronger attachment (locking) of tumor cells to the endothelial surface. ©2010 AACR.

DAP12 impacts trafficking and surface stability of killer immunoglobulin-like receptors on natural killer cells

PubMed Central

Mulrooney, Tiernan J.; Posch, Phillip E.; Hurley, Carolyn Katovich

2013-01-01

KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules. PMID:23715743

Superparamagnetic poly(methyl methacrylate) nanoparticles surface modified with folic acid presenting cell uptake mediated by endocytosis

NASA Astrophysics Data System (ADS)

Feuser, Paulo Emilio; Jacques, Amanda Virtuoso; Arévalo, Juan Marcelo Carpio; Rocha, Maria Eliane Merlin; dos Santos-Silva, Maria Claudia; Sayer, Claudia; de Araújo, Pedro H. Hermes

2016-04-01

The encapsulation of superparamagnetic nanoparticles (MNPs) in polymeric nanoparticles (NPs) with modified surfaces can improve targeted delivery and induce cell death by hyperthermia. The goals of this study were to synthesize and characterize surface modified superparamagnetic poly(methyl methacrylate) with folic acid (FA) prepared by miniemulsion polymerization (MNPsPMMA-FA) and to evaluate their in vitro cytotoxicity and cellular uptake in non-tumor cells, murine fibroblast (L929) cells and tumor cells that overexpressed folate receptor (FR) β, and chronic myeloid leukemia cells in blast crisis (K562). Lastly, hemolysis assays were performed on human red blood cells. MNPsPMMA-FA presented an average mean diameter of 135 nm and a saturation magnetization (Ms) value of 37 emu/g of iron oxide, as well as superparamagnetic behavior. The MNPsPMMA-FA did not present cytotoxicity in L929 and K562 cells. Cellular uptake assays showed a higher uptake of MNPsPMMA-FA than MNPsPMMA in K562 cells when incubated at 37 °C. On the other hand, MNPsPMMA-FA showed a low uptake when endocytosis mechanisms were blocked at low temperature (4 °C), suggesting that the MNPsPMMA-FA uptake was mediated by endocytosis. High concentrations of MNPsPMMA-FA showed hemocompatibility when incubated for 24 h in human red blood cells. Therefore, our results suggest that these carrier systems can be an excellent alternative in targeted drug delivery via FR.

Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15-34), antimicrobial peptides from rattlesnake venom.

PubMed

Pérez-Peinado, Clara; Dias, Susana Almeida; Domingues, Marco M; Benfield, Aurélie H; Freire, João Miguel; Rádis-Baptista, Gandhi; Gaspar, Diana; Castanho, Miguel A R B; Craik, David J; Henriques, Sónia Troeira; Veiga, Ana Salomé; Andreu, David

2018-02-02

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Biological and Mechanical Effects of Micro-Nanostructured Titanium Surface on an Osteoblastic Cell Line In vitro and Osteointegration In vivo.

PubMed

Hao, Jingzu; Li, Ying; Li, Baoe; Wang, Xiaolin; Li, Haipeng; Liu, Shimin; Liang, Chunyong; Wang, Hongshui

2017-09-01

Hybrid micro-nanostructure implant surface was produced on titanium (Ti) surface by acid etching and anodic oxidation to improve the biological and mechanical properties. The biological properties of the micro-nanostructure were investigated by simulated body fluid (SBF) soaking test and MC3T3-E1 cell co-culture experiment. The cell proliferation, spreading, and bone sialoprotein (BSP) gene expression were examined by MTT, SEM, and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the mechanical properties were evaluated by instrumented nanoindentation test and friction-wear test. Furthermore, the effect of the micro-nanostructure surface on implant osteointegration was examined by in vivo experiment. The results showed that the formation of bone-like apatite was accelerated on the micro-nanostructured Ti surface after immersion in simulated body fluid, and the proliferation, spreading, and BSP gene expression of the MC3T3-E1 cells were also upregulated on the modified surface. The micro-nanostructured Ti surface displayed decreased friction coefficient, stiffness value, and Young's modulus which were much closer to those of the cortical bone, compared to the polished Ti surface. This suggested much better mechanical match to the surrounding bone tissue of the micro-nanostructured Ti surface. Furthermore, the in vivo animal experiment showed that after implantation in the rat femora, the micro-nanostructure surface displayed higher bonding strength between bone tissues and implant; hematoxylin and eosin (H&E) staining suggested that much compact osteoid tissue was observed at the interface of Micro-nano-Ti-bone than polished Ti-bone interface after implantation. Based on these results mentioned above, it was concluded that the improved biological and mechanical properties of the micro-nanostructure endowed Ti surface with good biocompatibility and better osteointegration, implying the enlarged application of the micro-nanostructure surface Ti implants in future.

Neurogenic differentiation of human umbilical cord mesenchymal stem cells on aligned electrospun polypyrrole/polylactide composite nanofibers with electrical stimulation

NASA Astrophysics Data System (ADS)

Zhou, Junfeng; Cheng, Liang; Sun, Xiaodan; Wang, Xiumei; Jin, Shouhong; Li, Junxiang; Wu, Qiong

2016-09-01

Adult central nervous system (CNS) tissue has a limited capacity to recover after trauma or disease. Recent medical cell therapy using polymeric biomaterialloaded stem cells with the capability of differentiation to specific neural population has directed focuses toward the recovery of CNS. Fibers that can provide topographical, biochemical and electrical cues would be attractive for directing the differentiation of stem cells into electro-responsive cells such as neuronal cells. Here we report on the fabrication of an electrospun polypyrrole/polylactide composite nanofiber film that direct or determine the fate of mesenchymal stem cells (MSCs), via combination of aligned surface topography, and electrical stimulation (ES). The surface morphology, mechanical properties and electric properties of the film were characterized. Comparing with that on random surface film, expression of neurofilament-lowest and nestin of human umbilical cord mesenchymal stemcells (huMSCs) cultured on film with aligned surface topography and ES were obviously enhanced. These results suggest that aligned topography combining with ES facilitates the neurogenic differentiation of huMSCs and the aligned conductive film can act as a potential nerve scaffold.

Cellular internalization mechanism and intracellular trafficking of filamentous M13 phages displaying a cell-penetrating transbody and TAT peptide.

PubMed

Kim, Aeyung; Shin, Tae-Hwan; Shin, Seung-Min; Pham, Chuong D; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

2012-01-01

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005 ≈ 0.01%) than that of TAT-M13 (0.001 ≈ 0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs.

Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

PubMed Central

Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

2012-01-01

Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

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

Graphene oxide assisted synthesis of GaN nanostructures for reducing cell adhesion.

PubMed

Yang, Rong; Zhang, Ying; Li, Jingying; Han, Qiusen; Zhang, Wei; Lu, Chao; Yang, Yanlian; Dong, Hongwei; Wang, Chen

2013-11-21

We report a general approach for the synthesis of large-scale gallium nitride (GaN) nanostructures by the graphene oxide (GO) assisted chemical vapor deposition (CVD) method. A modulation effect of GaN nanostructures on cell adhesion has been observed. The morphology of the GaN surface can be controlled by GO concentrations. This approach, which is based on the predictable choice of the ratio of GO to catalysts, can be readily extended to the synthesis of other materials with controllable nanostructures. Cell studies show that GaN nanostructures reduced cell adhesion significantly compared to GaN flat surfaces. The cell-repelling property is related to the nanostructure and surface wettability. These observations of the modulation effect on cell behaviors suggest new opportunities for novel GaN nanomaterial-based biomedical devices. We believe that potential applications will emerge in the biomedical and biotechnological fields.

Endothelial cell regulation of leukocyte infiltration in inflammatory tissues

PubMed Central

Mantovani, A.; Introna, M.; Dejana, E.

1995-01-01

Endothelial cells play an important, active role in the onset and regulation of inflammatory and immune reactions. Through the production of chemokines they attract leukocytes and activate their adhesive receptors. This leads to the anchorage of leukocytes to the adhesive molecules expressed on the endothelial surface. Leukocyte adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. Adhesive molecules (such as PECAM) on the endothelial cell surface might also ‘direct’ leukocytes through the intercellular junction by haptotaxis. The information available on the molecular structure and functional properties of endothelial chemokines, adhesive molecules or junction organization is still fragmentary. Further work is needed to clarify how they interplay in regulating leukocyte infiltration into tissues. PMID:18475659

Immobilization of heparin/poly-(L)-lysine nanoparticles on dopamine-coated surface to create a heparin density gradient for selective direction of platelet and vascular cells behavior.

PubMed

Liu, Tao; Liu, Yang; Chen, Yuan; Liu, Shihui; Maitz, Manfred F; Wang, Xue; Zhang, Kun; Wang, Jian; Wang, Yuan; Chen, Junying; Huang, Nan

2014-05-01

Restenosis, thrombosis formation and delayed endothelium regeneration continue to be problematic for coronary artery stent therapy. To improve the hemocompatibility of the cardiovascular implants and selectively direct vascular cell behavior, a novel kind of heparin/poly-l-lysine (Hep/PLL) nanoparticle was developed and immobilized on a dopamine-coated surface. The stability and structural characteristics of the nanoparticles changed with the Hep:PLL concentration ratio. A Hep density gradient was created on a surface by immobilizing nanoparticles with various Hep:PLL ratios on a dopamine-coated surface. Antithrombin III binding quantity was significantly enhanced, and in plasma the APTT and TT times as coagulation tests were prolonged, depending on the Hep density. A low Hep density is sufficient to prevent platelet adhesion and activation. The sensitivity of vascular cells to the Hep density is very different: high Hep density inhibits the growth of all vascular cells, while low Hep density could selectively inhibit smooth muscle cell hyperplasia but promote endothelial progenitor cells and endothelial cell proliferation. These observations provide important guidance for modification of surface heparinization. We suggest that this method will provide a potential means to construct a suitable platform on a stent surface for selective direction of vascular cell behavior with low side effects. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

An incomplete trafficking defect to the cell-surface leads to paradoxical thrombocytosis for human and murine MPL P106L.

PubMed

Favale, Fabrizia; Messaoudi, Kahia; Varghese, Leila N; Boukour, Siham; Pecquet, Christian; Gryshkova, Vitalina; Defour, Jean Philippe; Albu, Roxana-Irina; Bluteau, Olivier; Ballerini, Paola; Leverger, Guy; Plo, Isabelle; Debili, Najet; Raslova, Hana; Favier, Remi; Constantinescu, Stefan N; Vainchenker, William

2016-12-29

The mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34 + cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl -/- mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [ 125 I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells. © 2016 by The American Society of Hematology.

Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression.

PubMed

Cowden Dahl, Karen D; Robertson, Sarah E; Weaver, Valerie M; Simon, M Celeste

2005-04-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hifalpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alphavbeta3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O(2)). Culturing B16F0 melanoma cells at 1.5% O(2) resulted in increased alphavbeta3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O(2) tension influence placental invasion and tumor migration by increasing cell surface expression of alphavbeta3 integrin.

Hypoxia-inducible Factor Regulates αvβ3 Integrin Cell Surface Expression

PubMed Central

Cowden Dahl, Karen D.; Robertson, Sarah E.; Weaver, Valerie M.; Simon, M. Celeste

2005-01-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and β aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvβ3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvβ3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvβ3 integrin. PMID:15689487

Neuroglian-positive plasmatocytes of Manduca sexta and the initiation of hemocyte attachment to foreign surfaces.

PubMed

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

2006-01-01

Observations of hemocyte aggregation on abiotic surfaces suggested that certain plasmatocytes from larvae of Manduca sexta act as foci for hemocyte aggregation. To establish how these particular plasmatocytes form initial attachments to foreign surfaces, they were cultured separately from other selected populations of hemocytes. While all circulating plasmatocytes immunolabel with anti-beta-integrin monoclonal antibody (MAb), only these larger plasmatocytes immunolabel with a MAb to the adhesion protein neuroglian. Neuroglian-negative plasmatocytes and granular cells that have been magnetically segregated from the majority of granular cells adhere to each other but fail to adhere to foreign substrata; by contrast, neuroglian-positive plasmatocytes that segregate with most granular cells adhere firmly to a substratum. Hemocytes form stable aggregates around the large, neuroglian-positive plasmatocytes. However, if neuroglian-positive plasmatocytes are separated from most granular cells, attachment of these plasmatocytes to foreign surfaces is suppressed.

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

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

Takabe, Piia, E-mail: piia.takabe@uef.fi; Bart, Geneviève; Ropponen, Antti

2015-09-10

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanomamore » cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.« less

The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.

PubMed Central

Ringwald, M; Schuh, R; Vestweber, D; Eistetter, H; Lottspeich, F; Engel, J; Dölz, R; Jähnig, F; Epplen, J; Mayer, S

1987-01-01

We have determined the amino acid sequence of the Ca2+-dependent cell adhesion molecule uvomorulin as it appears on the cell surface. The extracellular part of the molecule exhibits three internally repeated domains of 112 residues which are most likely generated by gene duplication. Each of the repeated domains contains two highly conserved units which could represent putative Ca2+-binding sites. Secondary structure predictions suggest that the putative Ca2+-binding units are located in external loops at the surface of the protein. The protein sequence exhibits a single membrane-spanning region and a cytoplasmic domain. Sequence comparison reveals extensive homology to the chicken L-CAM. Both uvomorulin and L-CAM are identical in 65% of their entire amino acid sequence suggesting a common origin for both CAMs. Images Fig. 1. Fig. 4. Fig. 7. PMID:3501370

Single-Cell Imaging and Spectroscopic Analyses of Cr(VI) Reduction on the Surface of Bacterial Cells

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

Wang, Yuanmin; Sevinc, Papatya C.; Belchik, Sara M.

2013-01-22

We investigate single-cell reduction of toxic Cr(VI) by the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 (MR-1), an important bioremediation process, using Raman spectroscopy and scanning electron microscopy (SEM) combined with energy-dispersive X-ray spectroscopy (EDX). Our experiments indicate that the toxic and highly soluble Cr(VI) can be efficiently reduced to the less toxic and non-soluble Cr2O3 nanoparticles by MR-1. Cr2O3 is observed to emerge as nanoparticles adsorbed on the cell surface and its chemical nature is identified by EDX imaging and Raman spectroscopy. Co-localization of Cr2O3 and cytochromes by EDX imaging and Raman spectroscopy suggests a terminal reductase role for MR-1more » surface-exposed cytochromes MtrC and OmcA. Our experiments revealed that the cooperation of surface proteins OmcA and MtrC makes the reduction reaction most efficient, and the sequence of the reducing reactivity of the MR-1 is: wild type > single mutant @mtrC or mutant @omcA > double mutant (@omcA-@mtrC). Moreover, our results also suggest that the direct microbial Cr(VI) reduction and Fe(II) (hematite)-mediated Cr(VI) reduction mechanisms may co-exist in the reduction processes.« less

GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface.

PubMed

Hiscox, Stephen; Hallett, Maurice B; Morgan, B Paul; van den Berg, Carmen W

2002-05-03

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent. Copyright 2002 Elsevier Science (USA).

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

PubMed Central

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté

2017-01-01

ABSTRACT Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation. PMID:28089989

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells.

PubMed

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

2017-01-15

Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation. © 2017. Published by The Company of Biologists Ltd.

Revealing the cell-material interface with nanometer resolution by FIB-SEM

PubMed Central

Santoro, Francesca; Zhao, Wenting; Joubert, Lydia-Marie; Duan, Liting; Schnitker, Jan; van de Burgt, Yoeri; Lou, Hsin-Ya; Liu, Bofei; Salleo, Alberto; Cui, Lifeng; Cui, Yi; Cui, Bianxiao

2018-01-01

The interface between cells and non-biological surfaces regulates cell attachment, chronic tissue responses, and ultimately the success of medical implants or biosensors. Clinical and laboratory studies show that topological features of the surface profoundly influences cellular responses, e.g. titanium surfaces with nano- and microtopographical structures enhance osteoblast attachment and host-implant integration as compare to smooth surface. To understand how cells and tissues respond to different topographical features, it is of critical importance to directly visualize the cell-materials interface at the relevant nanometer length scale. Here, we present a new method for in situ examination of the cell-to-material interface at any desired location, based on focused-ion beam milling and scanning electron microscopy imaging (FIB-SEM) to resolve the cell membrane-to-material interface with 10 nm resolution. By examining how cell membranes interact with topographical features such as nanoscale protrusions or invaginations, we discovered that the cell membrane readily deforms inward and wraps around protruding structures, but hardly deforms outward to contour invaginating structures. This asymmetric membrane response (inward vs. outward deformation) causes the cleft width between the cell membrane and the nanostructure surface to vary for more than an order of magnitude. Our results suggest that surface topology is a crucial consideration for the development of medical implants or biosensors whose performances are strongly influenced by the cell-to-material interface. We anticipate that the method can be used to explore the direct interaction of cells/tissue with medical devices such as metal implants in the future. PMID:28682058

Cicada Wing Surface Topography: An Investigation into the Bactericidal Properties of Nanostructural Features.

PubMed

Kelleher, S M; Habimana, O; Lawler, J; O' Reilly, B; Daniels, S; Casey, E; Cowley, A

2016-06-22

Recently, the surface of the wings of the Psaltoda claripennis cicada species has been shown to possess bactericidal properties and it has been suggested that the nanostructure present on the wings was responsible for the bacterial death. We have studied the surface-based nanostructure and bactericidal activity of the wings of three different cicadas (Megapomponia intermedia, Ayuthia spectabile and Cryptotympana aguila) in order to correlate the relationship between the observed surface topographical features and their bactericidal properties. Atomic force microscopy and scanning electron microscopy performed in this study revealed that the tested wing species contained a highly uniform, nanopillar structure on the surface. The bactericidal properties of the cicada wings were investigated by assessing the viability of autofluorescent Pseudomonas fluorescens cells following static adhesion assays and targeted dead/live fluorescence staining through direct microscopic counting methods. These experiments revealed a 20-25% bacterial surface coverage on all tested wing species; however, significant bactericidal properties were observed in the M. intermedia and C. aguila species as revealed by the high dead:live cell ratio on their surfaces. The combined results suggest a strong correlation between the bactericidal properties of the wings and the scale of the nanotopography present on the different wing surfaces.

Inactivation of Clostridium perfringens spores adhered onto stainless steel surface by agents used in a clean-in-place procedure.

PubMed

Alzubeidi, Yasmeen S; Udompijitkul, Pathima; Talukdar, Prabhat K; Sarker, Mahfuzur R

2018-07-20

Enterotoxigenic Clostridium perfringens, a leading foodborne pathogen can be cross-contaminated from food processing stainless steel (SS) surfaces to the finished food products. This is mostly due to the high resistance of C. perfringens spores adhered onto SS surfaces to various disinfectants commonly used in food industries. In this study, we aimed to investigate the survivability and adherence of C. perfringens spores onto SS surfaces and then validate the effectiveness of a simulated Clean-in-Place (CIP) regime on inactivation of spores adhered onto SS surfaces. Our results demonstrated that, 1) C. perfringens spores adhered firmly onto SS surfaces and survived for at-least 48 h, unlike their vegetative cells who died within 30 min, after aerobic incubation at refrigerated and ambient temperatures; 2) Spores exhibited higher levels of hydrophobicity than vegetative cells, suggesting a correlation between cell surface hydrophobicity and adhesion to solid surfaces; 3) Intact spores were more hydrophobic than the decoated spores, suggesting a positive role of spore coat components on spores' hydrophobicity and thus adhesion onto SS surfaces; and finally 4) The CIP regime (NaOH + HNO 3 ) successfully inactivated C. perfringens spores adhered onto SS surfaces, and most of the effect of CIP regime appeared to be due to the NaOH. Collectively, our current findings may well contribute towards developing a strategy to control cross-contamination of C. perfringens spores into food products, which should help reducing the risk of C. perfringens-associated food poisoning outbreaks. Copyright © 2018 Elsevier B.V. All rights reserved.

In vitro effects of cisplatin-functionalized silica nanoparticles on chondrocytes

NASA Astrophysics Data System (ADS)

Bhowmick, Tridib Kumar; Yoon, Diana; Patel, Minal; Fisher, John; Ehrman, Sheryl

2010-10-01

In this study, we evaluated the combined effect of a known toxic molecule, cisplatin, in combination with relatively nontoxic nanoparticles, amorphous fumed silica, on chondrocyte cells. Cisplatin was attached to silica nanoparticles using aminopropyltriethoxy silane as a linker molecule, and characterized in terms of size, shape, specific surface area, as well as the dissolution of cisplatin from the silica surface. The primary particle diameter of the as-received silica nanoparticles ranged from 7.1 to 61 nm, estimated from measurements of specific surface area, and the primary particles were aggregated. The effects of cisplatin-functionalized silica particles with different specific surface areas (41, 85, 202, 237, and 297 m2/g) were compared in vitro on chondrocytes, the parenchymal cell of hyaline cartilage. The results show that adverse effects on cell function, as evidenced by reduced metabolic activity measured by the MTT assay and increased membrane permeability observed using the Live/Dead stain, can be correlated with specific surface area of the silica. Cisplatin-functionalized silica nanoparticles with the highest specific surface area incited the greatest response, which was almost equivalent to that induced by free cisplatin. This result suggests the importance of particle specific surface area in interactions between cells and surface-functionalized nanomaterials.

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.

Cell surface control of the multiubiquitination and deubiquitination of high-affinity immunoglobulin E receptors.

PubMed Central

Paolini, R; Kinet, J P

1993-01-01

Multiubiquitination of proteins is a critical step leading to selective degradation for many polypeptides. Therefore, activation-induced multiubiquitination of cell surface receptors, such as the platelet-derived growth factor (PDGF) receptor and the T cell antigen (TCR) receptor, may correspond to a degradation pathway for ligand-receptor complexes. Here we show that the antigen-induced engagement of high-affinity immunoglobulin E receptors (Fc epsilon RI) results in the immediate multiubiquitination of Fc epsilon RI beta and gamma chains. This ubiquitination is independent of receptor phosphorylation and is restricted to activated receptors. Surprisingly, receptor multiubiquitination is immediately reversible when receptors are disengaged. Therefore, multiubiquitination and deubiquitination of Fc epsilon RI receptors is controlled at the cell surface by receptor engagement and disengagement. The rapidity, specificity and, most importantly, the reversibility of the activation-induced receptor multiubiquitination suggest that this process may turn on/off a cell surface receptor signaling function thus far unsuspected. Images PMID:8382611

Surface photovoltage spectroscopy applied to gallium arsenide surfaces

NASA Technical Reports Server (NTRS)

Bynik, C. E.

1975-01-01

The experimental and theoretical basis for surface photovoltage spectroscopy is outlined. Results of this technique applied to gallium arsenide surfaces, are reviewed and discussed. The results suggest that in gallium arsenide the surface voltage may be due to deep bulk impurity acceptor states that are pinned at the Fermi level at the surface. Establishment of the validity of this model will indicate the direction to proceed to increase the efficiency of gallium arsenide solar cells.

Patterns of surface burrow plugging in a colony of black-tailed prairie dogs occupied by black-footed ferrets

USGS Publications Warehouse

Eads, David E.; Biggins, Dean E.

2012-01-01

Black-tailed prairie dogs (Cynomys ludovicianus) can surface-plug openings to a burrow occupied by a black-footed ferret (Mustela nigripes). At a coarse scale, surface plugs are more common in colonies of prairie dogs occupied by ferrets than in colonies without ferrets. However, little is known about spatial and temporal patterns of surface plugging in a colony occupied by ferrets. In a 452-ha colony of black-tailed prairie dogs in South Dakota, we sampled burrow openings for surface plugs and related those data to locations of ferrets observed during spotlight surveys. Of 67,574 burrow openings in the colony between June and September 2007, 3.7% were plugged. In a colony-wide grid of 80 m × 80 m cells, the occurrence of surface plugging (≥1 opening plugged) was greater in cells used by ferrets (93.3% of cells) than in cells not observably used by ferrets (70.6%). Rates of surface plugging (percentages of openings plugged) were significantly higher in cells used by ferrets (median = 3.7%) than in cells without known ferret use (median = 3.2%). Also, numbers of ferret locations in cells correlated positively with numbers of mapped surface plugs in the cells. To investigate surface plugging at finer temporal and spatial scales, we compared rates of surface plugging in 20-m-radius circle-plots centered on ferret locations and in random plots 1–4 days after observing a ferret (Jun–Oct 2007 and 2008). Rates of surface plugging were greater in ferret-plots (median = 12.0%) than in random plots (median = 0%). For prairie dogs and their associates, the implications of surface plugging could be numerous. For instance, ferrets must dig to exit or enter plugged burrows (suggesting energetic costs), and surface plugs might influence microclimates in burrows and consequently influence species that cannot excavate soil (e.g., fleas that transmit the plague bacterium Yersinia pestis).

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.

Adsorption of Amorphous Silica Nanoparticles onto Hydroxyapatite Surfaces Differentially Alters Surfaces Properties and Adhesion of Human Osteoblast Cells

PubMed Central

Kalia, Priya; Brooks, Roger A.; Kinrade, Stephen D.; Morgan, David J.; Brown, Andrew P.; Rushton, Neil; Jugdaohsingh, Ravin

2016-01-01

Silicon (Si) is suggested to be an important/essential nutrient for bone and connective tissue health. Silicon-substituted hydroxyapatite (Si-HA) has silicate ions incorporated into its lattice structure and was developed to improve attachment to bone and increase new bone formation. Here we investigated the direct adsorption of silicate species onto an HA coated surface as a cost effective method of incorporating silicon on to HA surfaces for improved implant osseointegration, and determined changes in surface characteristics and osteoblast cell adhesion. Plasma-sprayed HA-coated stainless steel discs were incubated in silica dispersions of different concentrations (0–42 mM Si), at neutral pH for 12 h. Adsorbed Si was confirmed by XPS analysis and quantified by ICP-OES analysis following release from the HA surface. Changes in surface characteristics were determined by AFM and measurement of surface wettability. Osteoblast cell adhesion was determined by vinculin plaque staining. Maximum Si adsorption to the HA coated disc occurred after incubation in the 6 mM silica dispersion and decreased progressively with higher silica concentrations, while no adsorption was observed with dispersions below 6 mM Si. Comparison of the Si dispersions that produced the highest and lowest Si adsorption to the HA surface, by TEM-based analysis, revealed an abundance of small amorphous nanosilica species (NSP) of ~1.5 nm in diameter in the 6 mM Si dispersion, with much fewer and larger NSP in the 42 mM Si dispersions. 29Si-NMR confirmed that the NSPs in the 6 mM silica dispersion were polymeric and similar in composition to the larger NSPs in the 42 mM Si dispersion, suggesting that the latter were aggregates of the former. Amorphous NSP adsorbed from the 6 mM dispersion on to a HA-coated disc surface increased the surface’s water contact angle by 53°, whereas that adsorbed from the 42 mM dispersion decreased the contact angle by 18°, indicating increased and decreased hydrophobicity, respectively. AFM showed an increase in surface roughness of the 6 mM Si treated surface, which correlated well with an increase in number of vinculin plaques. These findings suggest that NSP of the right size (relative to charge) adsorb readily to the HA surface, changing the surface characteristics and, thus, improving osteoblast cell adhesion. This treatment provides a simple way to modify plasma-coated HA surfaces that may enable improved osseointegration of bone implants. PMID:26863624

Effects of titanium surface topography on morphology and in vitro activity of human gingival fibroblasts.

PubMed

Ramaglia, L; Capece, G; Di Spigna, G; Bruno, M P; Buonocore, N; Postiglione, L

2013-01-01

The aim of the present study was to evaluate in vitro the biological behavior of human gingival fibroblasts cultured on two different titanium surfaces. Titanium test disks were prepared with a machined, relatively smooth (S) surface or a rough surface (O) obtained by a double acid etching procedure. Primary cultures of human gingival fibroblasts were plated on the experimental titanium disks and cultured up to 14 days. Titanium disk surfaces were analysed by scanning electron microscopy (SEM). Cell proliferation and a quantitative analysis by ELISA in situ of ECM components as CoI, FN and TN were performed. Results have shown different effects of titanium surface microtopography on cell expression and differentiation. At 96 hours of culture on experimental surfaces human gingival fibroblasts displayed a favourable cell attachment and proliferation on both surfaces although showing some differences. Both the relatively smooth and the etched surfaces interacted actively with in vitro cultures of human gingival fibroblasts, promoting cell proliferation and differentiation. Results suggested that the microtopography of a double acid-etched rough surface may induce a greater Co I and FN production, thus conditioning in vivo the biological behaviour of human gingival fibroblasts during the process of peri-implant soft tissue healing.

The Role of Titanium Surface Microtopography on Adhesion, Proliferation, Transformation, and Matrix Deposition of Corneal Cells.

PubMed

Zhou, Chengxin; Lei, Fengyang; Chodosh, James; Paschalis, Eleftherios I

2016-04-01

Titanium (Ti) is an excellent implantable biomaterial that can be further enhanced by surface topography optimization. Despite numerous data from orthopedics and dentistry, the effect of Ti surface topography on ocular cells is still poorly understood. In light of the recent adaptation of Ti in the Boston Keratoprosthesis artificial cornea, we attempted to perform an extended evaluation of the effect of Ti surface topography on corneal cell adhesion, proliferation, cytotoxicity, transformation, and matrix deposition. Different surface topographies were generated on medical grade Ti-6Al-4V-ELI (extra-low interstitial), with linearly increased roughness (polished to grit blasted). Biological response was evaluated in vitro using human corneal limbal epithelial (HCLE) cells, stromal fibroblasts (HCF), and endothelial cells (HCEnC). None of the Ti surface topographies caused cytotoxicity to any of the three corneal cell types. However, rough Ti surface inhibited HCLE and HCF cell adhesion and proliferation, while HCEnC proliferation was unaffected. Long-term experiments with HCF revealed that rough Ti surface with R(a) (the arithmetic average of the profile height from the mean line) ≥ 1.15 μm suppressed HCF focal adhesion kinase phosphorylation, changed fibroblast morphology, and caused less aligned and reduced deposition of collagen matrix as compared to smooth Ti (R(a) ≤ 0.08 μm). In the presence of transforming growth factor β1 (TGFβ1) stimulation, rough Ti inhibited alpha-smooth muscle actin (α-SMA) expression and collagen deposition, leading to decreased myofibroblast transformation and disorganization of the collagen fibrils as compared to smooth Ti. This study suggests that Ti surface topography regulates corneal cell behavior in a tissue-dependent manner that varies across the corneal strata. Contrary to the accepted paradigm, smooth surface topography can enhance cell adhesion and proliferation and increase matrix deposition by corneal cells.

Inhibition and enhancement of microbial surface colonization: the role of silicate composition

USGS Publications Warehouse

Roberts, Jennifer A.

2004-01-01

Classical treatment of cell attachment by models of filtration or coulombic attraction assumes that attachment of cells to mineral surfaces would be controlled by factors such as response to predation, collision efficiency, or coulombic attraction between the charged groups at the mineral and cell surfaces. In the study reported here, the passive model of attachment was investigated using a native microbial consortium and a variety of Al- and Fe-bearing silicates and oxides to determine if other controls, such as mineral composition, also influence the interaction between cells and surfaces. Results from in situ colonization studies in an anaerobic groundwater at pH 6.8 combined with most probable number analyses (MPN) of surface-adherent cells demonstrate that electrostatic effects dominate microbial colonization on positively charged oxide surfaces regardless of mineral composition. In contrast, on negatively charged silicate minerals and glasses, the solid phase composition is a factor in determining the extent of microbial colonization, as well as the diversity of the attached community. In particular, silicates containing more than 1.2% Al exhibit less biomass than Al-poor silicates and MPN suggests a shift in community diversity, possibly indicating Al toxicity on these surfaces. When Fe is present in the silicate, however, this trend is reversed and abundant colonization of the surface is observed. Here, microorganisms preferentially colonize those silicate surfaces that offer beneficial nutrients and avoid those that contain potentially toxic elements.

Adsorption and transport of charged vs. neutral hydrophobic molecules at the membrane of murine erythroleukemia (MEL) cells.

PubMed

Zeng, Jia; Eckenrode, Heather M; Dai, Hai-Lung; Wilhelm, Michael J

2015-03-01

The adsorption and transport of hydrophobic molecules at the membrane surface of pre- and post-DMSO induced differentiated murine erythroleukemia (MEL) cells were examined by time- and wavelength-resolved second harmonic light scattering. Two medium (<600 Da) hydrophobic molecules, cationic malachite green (MG) and neutral bromocresol purple (BCP), were investigated. While it was observed that the MG cation adsorbs onto the surface of the MEL cell, neutral BCP does not. It is suggested that an electrostatic interaction between the opposite charges of the cation and the MEL cell surface is the primary driving force for adsorption. Comparisons of adsorption density and free energy, measured at different pH and cell morphology, indicate that the interaction is predominantly through sialic acid carboxyl groups. MG cation adsorption densities have been determined as (0.6±0.3)×10(6) μm(-2) on the surface of undifferentiated MEL cells, and (1.8±0.5)×10(7) μm(-2) on differentiated MEL cells, while the deduced adsorption free energies are effectively identical (ca. -10.9±0.1 and -10.8±0.1 kcal mol(-1), respectively). The measured MG densities indicate that the total number of surface carboxyl groups is largely conserved following differentiation, and therefore the density of carboxylic groups is much larger on the differentiated cell surface than the undifferentiated one. Finally, in contrast to synthetic liposomes and bacterial membranes, surface adsorbed MG cations are unable to traverse the MEL cell membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

The responses of immune cells to iron oxide nanoparticles.

PubMed

Xu, Yaolin; Sherwood, Jennifer A; Lackey, Kimberly H; Qin, Ying; Bao, Yuping

2016-04-01

Immune cells play an important role in recognizing and removing foreign objects, such as nanoparticles. Among various parameters, surface coatings of nanoparticles are the first contact with biological system, which critically affect nanoparticle interactions. Here, surface coating effects on nanoparticle cellular uptake, toxicity and ability to trigger immune response were evaluated on a human monocyte cell line using iron oxide nanoparticles. The cells were treated with nanoparticles of three types of coatings (negatively charged polyacrylic acid, positively charged polyethylenimine and neutral polyethylene glycol). The cells were treated at various nanoparticle concentrations (5, 10, 20, 30, 50 μg ml(-1) or 2, 4, 8, 12, 20 μg cm(-2)) with 6 h incubation or treated at a nanoparticle concentration of 50 μg ml(-1) (20 μg cm(-2)) at different incubation times (6, 12, 24, 48 or 72 h). Cell viability over 80% was observed for all nanoparticle treatment experiments, regardless of surface coatings, nanoparticle concentrations and incubation times. The much lower cell viability for cells treated with free ligands (e.g. ~10% for polyethylenimine) suggested that the surface coatings were tightly attached to the nanoparticle surfaces. The immune responses of cells to nanoparticles were evaluated by quantifying the expression of toll-like receptor 2 and tumor necrosis factor-α. The expression of tumor necrosis factor-α and toll-like receptor 2 were not significant in any case of the surface coatings, nanoparticle concentrations and incubation times. These results provide useful information to select nanoparticle surface coatings for biological and biomedical applications. Copyright © 2016 John Wiley & Sons, Ltd.

Role of geometrical cues in bone marrow-derived mesenchymal stem cell survival, growth and osteogenic differentiation.

PubMed

Gupta, Dhanak; Grant, David M; Zakir Hossain, Kazi M; Ahmed, Ifty; Sottile, Virginie

2018-02-01

Mesenchymal stem cells play a vital role in bone formation process by differentiating into osteoblasts, in a tissue that offers not a flat but a discontinuous three-dimensional (3D) topography in vivo. In order to understand how geometry may be affecting mesenchymal stem cells, this study explored the influence of 3D geometry on mesenchymal stem cell-fate by comparing cell growth, viability and osteogenic potential using monolayer (two-dimensional, 2D) with microsphere (3D) culture systems normalised to surface area. The results suggested lower cell viability and reduced cell growth in 3D. Alkaline phosphatase activity was higher in 3D; however, both collagen and mineral deposition appeared significantly lower in 3D, even after osteogenic supplementation. Also, there were signs of patchy mineralisation in 3D with or without osteogenic supplementation as early as day 7. These results suggest that the convex surfaces on microspheres and inter-particulate porosity may have led to variable cell morphology and fate within the 3D culture. This study provides deeper insights into geometrical regulation of mesenchymal stem cell responses applicable for bone tissue engineering.

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.

Engineered porous silicon counter electrodes for high efficiency dye-sensitized solar cells.

PubMed

Erwin, William R; Oakes, Landon; Chatterjee, Shahana; Zarick, Holly F; Pint, Cary L; Bardhan, Rizia

2014-06-25

In this work, we demonstrate for the first time, the use of porous silicon (P-Si) as counter electrodes in dye-sensitized solar cells (DSSCs) with efficiencies (5.38%) comparable to that achieved with platinum counter electrodes (5.80%). To activate the P-Si for triiodide reduction, few layer carbon passivation is utilized to enable electrochemical stability of the silicon surface. Our results suggest porous silicon as a promising sustainable and manufacturable alternative to rare metals for electrochemical solar cells, following appropriate surface modification.

Kupffer cell/tumor cell interactions and hepatic metastasis in colorectal cancer.

PubMed

Meterissian, S H; Toth, C A; Steele, G; Thomas, P

1994-06-15

The degree of interaction with Kupffer cells of two moderately well differentiated cell lines, CX-1 and CCl-188 of high metastatic potential (61%) were compared to two poorly differentiated cell lines, MIP-101 and Clone A of low metastatic potential (6%) in the intrasplenic injection model for liver metastasis. MIP-101 and Clone A bound significantly better to mouse Kupffer cells in vitro than either CX-1 or CCL-188. We also identified specific cell surface proteins mediating attachment of colorectal carcinoma cells to murine Kupffer cells. Kupffer cells were radiolabelled and their surface proteins incubated with MIP-101 and CX-1. Two radiolabelled proteins from murine Kupffer cells of 14 and 34 kDa were identified consistently binding to the tumor cells. Binding of both proteins was inhibited by asialofetuin but not by fetuin. This suggests that the major binding proteins between Kupffer cells and colorectal cancer cells are galactose binding lectins.

Proventricular adenocarcinoma in a Humboldt penguin (Spheniscus humboldti) and a great horned owl (Bubo virginianus); identification of origin by mucin histochemistry.

PubMed

Yonemaru, Kayoko; Sakai, Hiroki; Asaoka, Yoshiji; Yanai, Tokuma; Fukushi, Hideto; Watanabe, Ken; Hirai, Katsuya; Masegi, Toshiaki

2004-02-01

Cases of proventricular neoplasm in a Humboldt penguin (Spheniscus humboldti) and a great horned owl (Bubo virginianus) were observed. Microscopically, the neoplastic cells formed branching tubules or acini in both cases. Galactose oxidase-Schiff (GOS) staining revealed that the cytoplasm of the normal surface epithelium and surface mucosubstances of the proventriculus adjacent to the neoplasm were positive in both cases. The neoplastic cells in both cases were also classified as GOS-positive. Therefore, the two proventricular neoplasms in this report were diagnosed as proventricular adenocarcinoma that arose from the proventricular surface epithelium. This study suggests that the mucosubstances, which the neoplastic cells produced, were a useful index for identifying the origin of the neoplastic cells in the birds.

Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

PubMed Central

1993-01-01

Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to record single L-type Ca2+ channel activity produce a positive shift in the voltage dependence of activation (approximately 32 mV in 100 mM Ba2+). PMID:7687645

Biophysical Effects of a Polymeric Biosurfactant in Candida krusei and Candida albicans Cells.

PubMed

Ferreira, Gabriella Freitas; Dos Santos Pinto, Bruna Lorrana; Souza, Eliene Batista; Viana, José Lima; Zagmignan, Adrielle; Dos Santos, Julliana Ribeiro Alves; Santos, Áquila Rodrigues Costa; Tavares, Priscila Batista; Denadai, Ângelo Márcio Leite; Monteiro, Andrea Souza

2016-12-01

This study evaluated the effects of a polymeric biosurfactant produced by Trichosporon montevideense CLOA72 in the adhesion of Candida albicans and Candida krusei cells to human buccal epithelial cells and its interference in biofilm formation by these strains. The biofilm inhibition by biosurfactant (25 mg/mL) in C. krusei and C. albicans in polystyrene was reduced up to 79.5 and 85 %, respectively. In addition, the zeta potential and hydrodynamic diameter of the yeasts altered as a function of the biosurfactant concentration added to the cell suspension. The changes in the cell surface characteristics and the interface modification can contribute to the inhibition of the initial adherence of yeasts cells to the surface. In addition, the analyses of the biofilm matrix and planktonic cell surfaces demonstrated differences in carbohydrate and protein concentrations for the two studied strains, which may contribute to the modulation of cell adhesion or consolidation of biofilms, especially in C. krusei. This study suggests a possible application of the of CLOA72 biosurfactant in inhibiting the adhesion and formation of biofilms on biological surfaces by yeasts of the Candida genus.

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

Induction of non-apoptotic cell death by morphinone in human promyelocytic leukemia HL-60 cells.

PubMed

Takeuchi, Risa; Hoshijima, Hiroshi; Nagasaka, Hiroshi; Chowdhury, Shahead Ali; Kikuchi, Hirotaka; Kanda, Yumiko; Kunii, Shiro; Kawase, Masami; Sakagami, Hiroshi

2006-01-01

As previously suggested, codeinone (oxidation product of codeine) induces non-apoptotic cell death, characterized by marginal caspase activation and the lack of DNA fragmentation in HL-60 human promyelocytic leukemia cells, which was inhibited by N-acetyl-L-cysteine. Whether, morphinone, an oxidative metabolite of morphine, also induced a similar type of cell death in HL-60 cells was investigated. Morphinone showed slightly higher cytotoxic activity against human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, HSC-4, NA, Ca9-22, promyelocytic leukemia HL-60, cervical carcinoma HeLa) than against normal oral human cells (gingival fibroblast HGF, pulp cells HPC, periodontal ligament fibroblast HPLF). Morphinone also induced an almost undetectable level of internucleosomal DNA fragmentation in the HL-60 cells. Morphinone did not activate caspase-8 or -9 in these cells. Morphinone dose-dependently activated caspase-3 in both HL-60 and HSC-2 cell lines, but to a much lesser extent than actinomycin D. Electron microscopy demonstrated that morphinone induced mitochondrial shrinkage, vacuolization and production of autophagosome and the loss of cell surface microvilli, without destruction of cell surface and nuclear membranes in the HL-60 cells. The autophagy inhibitor 3-methyladenine (0.3-10 mM) slightly inhibited the morphinone-induced cytotoxicity, when corrected for its own cytotoxicity. These data suggest that morphinone induces non-apoptotic cell death in HL-60 cells.

An in vitro investigation of bacteria-osteoblast competition on oxygen plasma-modified PEEK.

PubMed

Rochford, Edward T J; Subbiahdoss, Guruprakash; Moriarty, T Fintan; Poulsson, Alexandra H C; van der Mei, Henny C; Busscher, Henk J; Richards, R Geoff

2014-12-01

Polyetheretherketone (PEEK) films were oxygen plasma treated to increase surface free energy and characterized by X-ray photoelectron microscopy, atomic force microscopy, and water contact angles. A parallel plate flow chamber was used to measure Staphylococcus epidermidis, Staphylococcus aureus, and U-2 OS osteosarcomal cell-line adhesion to the PEEK films in separate monocultures. In addition, bacteria and U-2 OS cells were cocultured to model competition between osteoblasts and contaminating bacteria for the test surfaces. Plasma treatment of the surfaces increased surface oxygen content and decreased the hydrophobicity of the materials, but did not lead to a significant difference in bacterial or U-2 OS cell adhesion in the monocultures. In the S. epidermidis coculture experiments, the U-2 OS cells adhered in greater numbers on the treated surfaces compared to the untreated PEEK and spread to a similar extent. However, in the presence of S. aureus, cell death of the U-2 OS occurred within 10 h on all surfaces. The results of this study suggest that oxygen plasma treatment of PEEK may maintain the ability of osteoblast-like cells to adhere and spread, even in the presence of S. epidermidis contamination, without increasing the risk of preoperative bacterial adhesion. Therefore, oxygen plasma-treated PEEK remains a promising method to improve implant surface free energy for osseointegration. © 2014 Wiley Periodicals, Inc.

Heat shock 70-kDa protein 8 isoform 1 is expressed on the surface of human embryonic stem cells and downregulated upon differentiation.

PubMed

Son, Yeon Sung; Park, Jae Hyun; Kang, Young Kook; Park, Jin-Sung; Choi, Hong Seo; Lim, Ji Young; Lee, Jeoung Eun; Lee, Jung Bok; Ko, Myoung Seok; Kim, Yong-Sam; Ko, Jeong-Heon; Yoon, Hyun Soo; Lee, Kwang-Woong; Seong, Rho Hyun; Moon, Shin Yong; Ryu, Chun Jeih; Hong, Hyo Jeong

2005-01-01

The cell-surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC-specific cell-surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz-hES1, and selected 26 MAbs that were able to bind to Miz-hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz-hES1 cells, either. Of these, MAb 20-202S (IgG1, kappa) immunoprecipitated a cell-surface protein of 72-kDa from the lysate of biotin-labeled Miz-hES1 cells, which was identified to be heat shock 70-kDa protein 8 isoform 1 (HSPA8) by quadrupole time-of-flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz-hES4, Miz-hES6, and HSF6. Two-color flow cytometric analysis of Miz-hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage-specific embryonic antigen 3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-81. Flow cytometric and Western blot analyses using various cells showed that MAb 20-202S specifically bound to the HSPA8 protein on the surface of Miz-hES1, contrary to other anti-HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz-hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20-202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell-surface marker for undifferentiated hESCs.

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

PubMed

Feriotto, G; Calza, R; Bergamini, C M; Griffin, M; Wang, Z; Beninati, S; Ferretti, V; Marzola, E; Guerrini, R; Pagnoni, A; Cavazzini, A; Casciano, F; Mischiati, C

2017-03-01

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Disruption of SorCS2 reveals differences in the regulation of stereociliary bundle formation between hair cell types in the inner ear

PubMed Central

Taylor, Ruth R.; Lovett, Michael; Jagger, Daniel J.

2017-01-01

Behavioural anomalies suggesting an inner ear disorder were observed in a colony of transgenic mice. Affected animals were profoundly deaf. Severe hair bundle defects were identified in all outer and inner hair cells (OHC, IHC) in the cochlea and in hair cells of vestibular macular organs, but hair cells in cristae were essentially unaffected. Evidence suggested the disorder was likely due to gene disruption by a randomly inserted transgene construct. Whole-genome sequencing identified interruption of the SorCS2 (Sortilin-related VPS-10 domain containing protein) locus. Real-time-qPCR demonstrated disrupted expression of SorCS2 RNA in cochlear tissue from affected mice and this was confirmed by SorCS2 immuno-labelling. In all affected hair cells, stereocilia were shorter than normal, but abnormalities of bundle morphology and organisation differed between hair cell types. Bundles on OHC were grossly misshapen with significantly fewer stereocilia than normal. However, stereocilia were organised in rows of increasing height. Bundles on IHC contained significantly more stereocilia than normal with some longer stereocilia towards the centre, or with minimal height differentials. In early postnatal mice, kinocilia (primary cilia) of IHC and of OHC were initially located towards the lateral edge of the hair cell surface but often became surrounded by stereocilia as bundle shape and apical surface contour changed. In macular organs the kinocilium was positioned in the centre of the cell surface throughout maturation. There was disruption of the signalling pathway controlling intrinsic hair cell apical asymmetry. LGN and Gαi3 were largely absent, and atypical Protein Kinase C (aPKC) lost its asymmetric distribution. The results suggest that SorCS2 plays a role upstream of the intrinsic polarity pathway and that there are differences between hair cell types in the deployment of the machinery that generates a precisely organised hair bundle. PMID:28346477

Living Toroids - Cells on Toroidal Surfaces

NASA Astrophysics Data System (ADS)

Chang, Ya-Wen; Angelini, Thomas; Marquez, Samantha; Kim, Harold; Fernandez-Nieves, Alberto

2014-03-01

Cellular environment influences a multitude of cellular functions by providing chemical and physical signals that modulate cell behavior, dynamics, development, and eventually survival. Substrate mechanics has been recognized as one of the important physical cues that governs cell behavior at single cell level as well as in collective cell motion. Past research has suggested several contact-guided behaviors to be the result of surface curvature. However, studies on the effect of curvature are relatively scarce likely due to the difficulty in generating substrates with well-defined curvature. Here we describe the generation of toroidal droplets, which unlike spherical droplets, have regions of both positive and negative Gaussian curvature. Additionally, the range of curvatures can be controlled by varying the size and aspect ratio of the torus. Cells are either encapsulated inside toroidal droplets or located on toroidal hydrogel surfaces. Preliminary studies use B. Subtilis to study the organization of bacteria biofilms. When confined in droplets surrounded by yield-stress fluid, bacteria self-organize into heterogeneous biofilm at fluid- substrate interface. It is found that the surface curvature in the sub-millimeter scale has little effect on biofilm architecture.

House dust mite induces expression of intercellular adhesion molecule-1 in EoL-1 human eosinophilic leukemic cells.

PubMed

Kwon, Byoung Chul; Sohn, Myung Hyun; Kim, Kyung Won; Kim, Eun Soo; Kim, Kyu-Earn; Shin, Myeong Heon

2007-10-01

The house dust mite (HDM) is considered to be the most common indoor allergen associated with bronchial asthma. In this study, we investigated whether crude extract of the HDM Dermatophagoides farinae could activate human eosinophilic leukemic cells (EoL-1) to induce upregulation of cell-surface adhesion molecules. When EoL-1 cells were incubated with D. farinae extract, expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased on the cell surfaces compared to cells incubated with medium alone. In contrast, surface expression of CD11b and CD49d in EoL-1 cells was not affected by D. farinae extract. In addition, pretreatment of cells with NF-kappaB inhibitor (MG-132) or JNK inhibitor (SP600125) significantly inhibited ICAM-1 expression promoted by HDM extract. However, neither p38 MAP kinase inhibitor nor MEK inhibitor prevented HDM-induced ICAM-1 expression in EoL-1 cells. These results suggest that crude extract of D. farinae induces ICAM-1 expression in EoL-1 cells through signaling pathways involving both NF-kappaB and JNK.

House Dust Mite Induces Expression of Intercellular Adhesion Molecule-1 in EoL-1 Human Eosinophilic Leukemic Cells

PubMed Central

Kwon, Byoung Chul; Sohn, Myung Hyun; Kim, Kyung Won; Kim, Eun Soo; Kim, Kyu-Earn

2007-01-01

The house dust mite (HDM) is considered to be the most common indoor allergen associated with bronchial asthma. In this study, we investigated whether crude extract of the HDM Dermatophagoides farinae could activate human eosinophilic leukemic cells (EoL-1) to induce upregulation of cell-surface adhesion molecules. When EoL-1 cells were incubated with D. farinae extract, expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased on the cell surfaces compared to cells incubated with medium alone. In contrast, surface expression of CD11b and CD49d in EoL-1 cells was not affected by D. farinae extract. In addition, pretreatment of cells with NF-κB inhibitor (MG-132) or JNK inhibitor (SP600125) significantly inhibited ICAM-1 expression promoted by HDM extract. However, neither p38 MAP kinase inhibitor nor MEK inhibitor prevented HDM-induced ICAM-1 expression in EoL-1 cells. These results suggest that crude extract of D. farinae induces ICAM-1 expression in EoL-1 cells through signaling pathways involving both NF-κB and JNK. PMID:17982228

Effects of a hybrid micro/nanorod topography-modified titanium implant on adhesion and osteogenic differentiation in rat bone marrow mesenchymal stem cells.

PubMed

Zhang, Wenjie; Li, Zihui; Huang, Qingfeng; Xu, Ling; Li, Jinhua; Jin, Yuqin; Wang, Guifang; Liu, Xuanyong; Jiang, Xinquan

2013-01-01

Various methods have been used to modify titanium implant surfaces with the aim of achieving better osseointegration. In this study, we fabricated a clustered nanorod structure on an acid-etched, microstructured titanium plate surface using hydrogen peroxide. We also evaluated biofunctionalization of the hybrid micro/nanorod topography on rat bone marrow mesenchymal stem cells. Scanning electron microscopy and x-ray diffraction were used to investigate the surface topography and phase composition of the modified titanium plate. Rat bone marrow mesenchymal stem cells were cultured and seeded on the plate. The adhesion ability of the cells was then assayed by cell counting at one, 4, and 24 hours after cell seeding, and expression of adhesion-related protein integrin β1 was detected by immunofluorescence. In addition, a polymerase chain reaction assay, alkaline phosphatase and Alizarin Red S staining assays, and osteopontin and osteocalcin immunofluorescence analyses were used to evaluate the osteogenic differentiation behavior of the cells. The hybrid micro/nanoscale texture formed on the titanium surface enhanced the initial adhesion activity of the rat bone marrow mesenchymal stem cells. Importantly, the hierarchical structure promoted osteogenic differentiation of these cells. This study suggests that a hybrid micro/nanorod topography on a titanium surface fabricated by treatment with hydrogen peroxide followed by acid etching might facilitate osseointegration of a titanium implant in vivo.

Tuning Surface Chemistry of Polyetheretherketone by Gold Coating and Plasma Treatment

NASA Astrophysics Data System (ADS)

Novotná, Zdeňka; Rimpelová, Silvie; Juřík, Petr; Veselý, Martin; Kolská, Zdeňka; Hubáček, Tomáš; Borovec, Jakub; Švorčík, Václav

2017-06-01

Polyetheretherketone (PEEK) has good chemical and biomechanical properties that are excellent for biomedical applications. However, PEEK exhibits hydrophobic and other surface characteristics which cause limited cell adhesion. We have investigated the potential of Ar plasma treatment for the formation of a nanostructured PEEK surface in order to enhance cell adhesion. The specific aim of this study was to reveal the effect of the interface of plasma-treated and gold-coated PEEK matrices on adhesion and spreading of mouse embryonic fibroblasts. The surface characteristics (polarity, surface chemistry, and structure) before and after treatment were evaluated by various experimental techniques (gravimetry, goniometry, X-ray photoelectron spectroscopy (XPS), and electrokinetic analysis). Further, atomic force microscopy (AFM) was employed to examine PEEK surface morphology and roughness. The biological response of cells towards nanostructured PEEK was evaluated in terms of cell adhesion, spreading, and proliferation. Detailed cell morphology was evaluated by scanning electron microscopy (SEM). Compared to plasma treatment, gold coating improved PEEK wettability. The XPS method showed a decrease in the carbon concentration with increasing time of plasma treatment. Cell adhesion determined on the interface between plasma-treated and gold-coated PEEK matrices was directly proportional to the thickness of a gold layer on a sample. Our results suggest that plasma treatment in a combination with gold coating could be used in biomedical applications requiring enhanced cell adhesion.

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

Immunity to Intracellular Salmonella Depends on Surface-associated Antigens

PubMed Central

Claudi, Beatrice; Mazé, Alain; Schemmer, Anne K.; Kirchhoff, Dennis; Schmidt, Alexander; Burton, Neil; Bumann, Dirk

2012-01-01

Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens. PMID:23093937

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins.

PubMed

García-Bayona, Leonor; Guo, Monica S; Laub, Michael T

2017-03-21

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

PubMed

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Inhibition of experimental ascending urinary tract infection by an epithelial cell-surface receptor analogue

NASA Astrophysics Data System (ADS)

Edén, C. Svanborg; Freter, R.; Hagberg, L.; Hull, R.; Hull, S.; Leffler, H.; Schoolnik, G.

1982-08-01

It has been shown that the establishment of urinary tract infection by Escherichia coli is dependent on attachment of the bacteria to epithelial cells1-4. The attachment involves specific epithelial cell receptors, which have been characterized as glycolipids5-10. Reversible binding to cell-surface mannosides may also be important4,11-13. This suggests an approach to the treatment of infections-that of blocking bacterial attachment with cell membrane receptor analogues. Using E. coli mutants lacking one or other of the two binding specificities (glycolipid and mannose), we show here that glycolipid analogues can block in vitro adhesion and in vivo urinary tract infection.

Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B

PubMed Central

Wang, Pan; He, Jie; Sun, Yufei; Reynolds, Matthew; Zhang, Li; Han, Shuangyan; Liang, Shuli; Sui, Haixin; Lin, Ying

2016-01-01

To modify the Pichia pastoris cell surface, two classes of hydrophobins, SC3 from Schizophyllum commune and HFBI from Trichoderma reesei, were separately displayed on the cell wall. There was an observable increase in the hydrophobicity of recombinant strains. Candida antarctica lipase B (CALB) was then co-displayed on the modified cells, generating strains GS115/SC3-61/CALB-51 and GS115/HFBI-61/CALB-51. Interestingly, the hydrolytic and synthetic activities of strain GS115/HFBI-61/CALB-51 increased by 37% and 109%, respectively, but decreased by 26% and 43%, respectively, in strain GS115/SC3-61/CALB-51 compared with the hydrophobin-minus recombinant strain GS115/CALB-GCW51. The amount of glycerol by-product from the transesterification reaction adsorbed on the cell surface was significantly decreased following hydrophobin modification, removing the glycerol barrier and allowing substrates to access the active sites of lipases. Electron micrographs indicated that the cell wall structures of both recombinant strains appeared altered, including changes to the inner glucan layer and outer mannan layer. These results suggest that the display of hydrophobins can change the surface structure and hydrophobic properties of P. pastoris, and affect the catalytic activities of CALB displayed on the surface of P. pastoris cells. PMID:26969039

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.

Constrained Adherable Area of Nanotopographic Surfaces Promotes Cell Migration through the Regulation of Focal Adhesion via Focal Adhesion Kinase/Rac1 Activation.

PubMed

Lim, Jiwon; Choi, Andrew; Kim, Hyung Woo; Yoon, Hyungjun; Park, Sang Min; Tsai, Chia-Hung Dylan; Kaneko, Makoto; Kim, Dong Sung

2018-05-02

Cell migration is crucial in physiological and pathological processes such as embryonic development and wound healing; such migration is strongly guided by the surrounding nanostructured extracellular matrix. Previous studies have extensively studied the cell migration on anisotropic nanotopographic surfaces; however, only a few studies have reported cell migration on isotropic nanotopographic surfaces. We herein, for the first time, propose a novel concept of adherable area on cell migration using isotropic nanopore surfaces with sufficient nanopore depth by adopting a high aspect ratio. As the pore size of the nanopore surface was controlled to 200, 300, and 400 nm in a fixed center-to-center distance of 480 nm, it produced 86, 68, and 36% of adherable area, respectively, on the fabricated surface. A meticulous investigation of the cell migration in response to changes in the constrained adherable area of the nanotopographic surface showed 1.4-, 1.5-, and 1.6-fold increase in migration speeds and a 1.4-, 2-, and 2.5-fold decrease in the number of focal adhesions as the adherable area was decreased to 86, 68, and 36%, respectively. Furthermore, a strong activation of FAK/Rac1 signaling was observed to be involved in the promoted cell migration. These results suggest that the reduced adherable area promotes cell migration through decreasing the FA formation, which in turn upregulates FAK/Rac1 activation. The findings in this study can be utilized to control the cell migration behaviors, which is a powerful tool in the research fields involving cell migration such as promoting wound healing and tissue repair.

Distribution of serotonergic and dopaminergic nerve fibers in the salivary gland complex of the cockroach Periplaneta americana

PubMed Central

Baumann, Otto; Dames, Petra; Kühnel, Dana; Walz, Bernd

2002-01-01

Background The cockroach salivary gland consists of secretory acini with peripheral ion-transporting cells and central protein-producing cells, an extensive duct system, and a pair of reservoirs. Salivation is controled by serotonergic and dopaminergic innervation. Serotonin stimulates the secretion of a protein-rich saliva, dopamine causes the production of a saliva without proteins. These findings suggest a model in which serotonin acts on the central cells and possibly other cell types, and dopamine acts selectively on the ion-transporting cells. To examine this model, we have analyzed the spatial relationship of dopaminergic and serotonergic nerve fibers to the various cell types. Results The acinar tissue is entangled in a meshwork of serotonergic and dopaminergic varicose fibers. Dopaminergic fibers reside only at the surface of the acini next to the peripheral cells. Serotonergic fibers invade the acini and form a dense network between central cells. Salivary duct segments close to the acini are locally associated with dopaminergic and serotonergic fibers, whereas duct segments further downstream have only dopaminergic fibers on their surface and within the epithelium. In addition, the reservoirs have both a dopaminergic and a serotonergic innervation. Conclusion Our results suggest that dopamine is released on the acinar surface, close to peripheral cells, and along the entire duct system. Serotonin is probably released close to peripheral and central cells, and at initial segments of the duct system. Moreover, the presence of serotonergic and dopaminergic fiber terminals on the reservoir indicates that the functions of this structure are also regulated by dopamine and serotonin. PMID:12095424

Distribution of serotonergic and dopaminergic nerve fibers in the salivary gland complex of the cockroach Periplaneta americana.

PubMed

Baumann, Otto; Dames, Petra; Kühnel, Dana; Walz, Bernd

2002-06-24

The cockroach salivary gland consists of secretory acini with peripheral ion-transporting cells and central protein-producing cells, an extensive duct system, and a pair of reservoirs. Salivation is controlled by serotonergic and dopaminergic innervation. Serotonin stimulates the secretion of a protein-rich saliva, dopamine causes the production of a saliva without proteins. These findings suggest a model in which serotonin acts on the central cells and possibly other cell types, and dopamine acts selectively on the ion-transporting cells. To examine this model, we have analyzed the spatial relationship of dopaminergic and serotonergic nerve fibers to the various cell types. The acinar tissue is entangled in a meshwork of serotonergic and dopaminergic varicose fibers. Dopaminergic fibers reside only at the surface of the acini next to the peripheral cells. Serotonergic fibers invade the acini and form a dense network between central cells. Salivary duct segments close to the acini are locally associated with dopaminergic and serotonergic fibers, whereas duct segments further downstream have only dopaminergic fibers on their surface and within the epithelium. In addition, the reservoirs have both a dopaminergic and a serotonergic innervation. Our results suggest that dopamine is released on the acinar surface, close to peripheral cells, and along the entire duct system. Serotonin is probably released close to peripheral and central cells, and at initial segments of the duct system. Moreover, the presence of serotonergic and dopaminergic fiber terminals on the reservoir indicates that the functions of this structure are also regulated by dopamine and serotonin.

Biofunctionalization of a titanium surface with a nano-sawtooth structure regulates the behavior of rat bone marrow mesenchymal stem cells

PubMed Central

Zhang, Wenjie; Li, Zihui; Liu, Yan; Ye, Dongxia; Li, Jinhua; Xu, Lianyi; Wei, Bin; Zhang, Xiuli; Liu, Xuanyong; Jiang, Xinquan

2012-01-01

Background: The topography of an implant surface can serve as a powerful signaling cue for attached cells and can enhance the quality of osseointegration. A series of improved implant surfaces functionalized with nanoscale structures have been fabricated using various methods. Methods: In this study, using an H2O2 process, we fabricated two size-controllable sawtooth-like nanostructures with different dimensions on a titanium surface. The effects of the two nano-sawtooth structures on rat bone marrow mesenchymal stem cells (BMMSCs) were evaluated without the addition of osteoinductive chemical factors. Results: These new surface modifications did not adversely affect cell viability, and rat BMMSCs demonstrated a greater increase in proliferation ability on the surfaces of the nano-sawtooth structures than on a control plate. Furthermore, upregulated expression of osteogenic-related genes and proteins indicated that the nano-sawtooth structures promote osteoblastic differentiation of rat BMMSCs. Importantly, the large nano-sawtooth structure resulted in the greatest cell responses, including increased adhesion, proliferation, and differentiation. Conclusion: The enhanced adhesion, proliferation, and osteogenic differentiation abilities of rat BMMSCs on the nano-sawtooth structures suggest the potential to induce improvements in bone-titanium integration in vivo. Our study reveals the key role played by the nano-sawtooth structures on a titanium surface for the fate of rat BMMSCs and provides insights into the study of stem cell-nanostructure relationships and the related design of improved biomedical implant surfaces. PMID:22927760

Fine Tuning of Tissues' Viscosity and Surface Tension through Contractility Suggests a New Role for α-Catenin

PubMed Central

Stirbat, Tomita Vasilica; Mgharbel, Abbas; Bodennec, Selena; Ferri, Karine; Mertani, Hichem C.; Rieu, Jean-Paul; Delanoë-Ayari, Hélène

2013-01-01

What governs tissue organization and movement? If molecular and genetic approaches are able to give some answers on these issues, more and more works are now giving a real importance to mechanics as a key component eventually triggering further signaling events. We chose embryonic cell aggregates as model systems for tissue organization and movement in order to investigate the origin of some mechanical constraints arising from cells organization. Steinberg et al. proposed a long time ago an analogy between liquids and tissues and showed that indeed tissues possess a measurable tissue surface tension and viscosity. We question here the molecular origin of these parameters and give a quantitative measurement of adhesion versus contractility in the framework of the differential interfacial tension hypothesis. Accompanying surface tension measurements by angle measurements (at vertexes of cell-cell contacts) at the cell/medium interface, we are able to extract the full parameters of this model: cortical tensions and adhesion energy. We show that a tunable surface tension and viscosity can be achieved easily through the control of cell-cell contractility compared to cell-medium one. Moreover we show that -catenin is crucial for this regulation to occur: these molecules appear as a catalyser for the remodeling of the actin cytoskeleton underneath cell-cell contact, enabling a differential contractility between the cell-medium and cell-cell interface to take place. PMID:23390488

Characterization of adhesive properties of red blood cells using surface acoustic wave induced flows for rapid diagnostics

NASA Astrophysics Data System (ADS)

Sivanantha, Ninnuja; Ma, Charles; Collins, David J.; Sesen, Muhsincan; Brenker, Jason; Coppel, Ross L.; Neild, Adrian; Alan, Tuncay

2014-09-01

This letter presents a method which employs surface acoustic wave induced acoustic streaming to differentially peel treated red blood cells (RBCs) off a substrate based on their adhesive properties and separate populations of pathological cells from normal ones. We demonstrate the principle of operation by comparing the applied power and time required to overcome the adhesion displayed by healthy, glutaraldehyde-treated or malaria-infected human RBCs. Our experiments indicate that the method can be used to differentiate between various cell populations contained in a 9 μl droplet within 30 s, suggesting potential for rapid diagnostics.

Glycan Engagement Dictates Hydrocephalus Induction by Serotype 1 Reovirus

PubMed Central

Stencel-Baerenwald, Jennifer; Reiss, Kerstin; Blaum, Bärbel S.; Colvin, Daniel; Li, Xiao-Nan; Abel, Ty; Boyd, Kelli; Stehle, Thilo

2015-01-01

ABSTRACT Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease. PMID:25736887

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains.

PubMed

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.

A model study of factors involved in adhesion of Pseudomonas fluorescens to meat.

PubMed Central

Piette, J P; Idziak, E S

1992-01-01

A study was undertaken to investigate the factors involved in the adhesion of Pseudomonas fluorescens to model meat surfaces (tendon slices). Adhesion was fast (less than 2.5 min) and was not suppressed by killing the cells with UV, gamma rays, or heat, indicating that physiological activity was not required. In various salt solutions (NaCl, KCl, CaCl2, MgCl2), adhesion increased with increasing ionic strength up to 10 to 100 mM, suggesting that, at low ionic strengths, electrostatic interactions were involved in the adhesion process. At higher ionic strengths (greater than 10 to 100 mM) or in the presence of Al3+ ions, adhesion was sharply reduced. Selectively blocking of carboxyl or amino groups at the cell surface by chemical means did not affect adhesion. These groups are therefore not directly involved in an adhesive bond with tendon. Given a sufficient cell concentration (10(10) CFU.ml-1) in the adhesion medium, the surface of tendon was almost entirely covered with adherent bacteria. This suggests that if the adhesion is specific, the attachment sites on the tendon surface must be located within collagen or proteoglycan molecules. Images PMID:1444387

A scanning electron microscopic study of 34 cases of acute granulocytic, myelomonocytic, monoblastic and histiocytic leukemia.

PubMed

Polliack, A; McKenzie, S; Gee, T; Lampen, N; de Harven, E; Clarkson, B D

1975-09-01

This report describes the surface architecture of leukemic cells, as seen by scanning electron microscopy in 34 patients with acute nonlymphoblastic leukemia. Six patients with myeloblastic, 4 with promyelocytic, 10 with myelomonocytic, 8 with monocytic, 4 with histiocytic and 2 with undifferentiated leukemia were studied. Under the scanning electron microscope most leukemia histiocytes and monocytes appeared similar and were characterized by the presence of large, well developed broad-based ruffled membranes or prominent raised ridge-like profiles, resembling ithis respect normal monocytes. Most cells from patients with acute promyelocytic or myeloblastic leukemia exhibited narrower ridge-like profiles whereas some showed ruffles or microvilli. Patients with myelomonocytic leukemia showed mixed populations of cells with ridge-like profiles and ruffled membranes whereas cells from two patients with undifferentiated leukemia had smooth surfaces, similar to those encountered in cells from patients with acute lymphoblastic leukemia. It appears that nonlymphoblastic and lymphoblastic leukemia cells (particularly histiocytes and monocytes) can frequently be distinquished on the basis of their surface architecture. The surface features of leukemic histiocytes and monocytes are similar, suggesting that they may belong to the same cell series. The monocytes seem to have characteristic surface features recognizable with the scanning electron microscope and differ from most cells from patients with acute granulocytic leukemia. Although overlap of surface features and misidentification can occur, scanning electron microscopy is a useful adjunct to other modes of microscopy in the study and diagnosis of acute leukemia.

Effect of flagella expression on adhesion of Achromobacter piechaudii to chalk surfaces.

PubMed

Nejidat, A; Saadi, I; Ronen, Z

2008-12-01

To examine flagella role and cell motility in adhesion of Achromobacter piechaudii to chalk. Transmission electron microscopy revealed that stationary cells have thicker and longer flagella than logarithmic cells. SDS-PAGE analysis showed that flagellin was more abundant in stationary cells than logarithmic ones. Sonication or inhibition of flagellin synthesis caused a 30% reduction in adhesion to chalk. Preincubation of chalk with flagella extracts reduced adhesion, by 50%. Three motility mutants were isolated. Mutants 94 and 153 were nonmotile, expressed normal levels of flagellin, have regular flagella and exhibited reduced adhesion. Mutant 208 expressed low levels of flagellin, no flagella and a spherical cell shape but with normal adhesion capacity. Multiple cell surface factors affect the adhesion efficiency to chalk. Flagella per se through physical interaction and through cell motility contribute to the adhesion process. The adhesion behaviour of mutant 208 suggests that cell shape can compensate for flagellar removal and motility. Physiological status affects bacterial cell surface properties and hence adhesion efficiency to chalk. This interaction is essential to sustain biodegradation activities and thus, remediation of contaminated chalk aquifers.

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

PubMed

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

Micropattern array with gradient size (µPAGS) plastic surfaces fabricated by PDMS (polydimethylsiloxane) mold-based hot embossing technique for investigation of cell-surface interaction.

PubMed

Choi, Min Jin; Park, Ju Young; Cha, Kyoung Je; Rhie, Jong-Won; Cho, Dong-Woo; Kim, Dong Sung

2012-12-01

Recently, it was found that the variations of physical environment significantly affect cell behaviors including cell proliferation, migration and differentiation. Through a plastic surface with controlled mechanical properties such as stiffness, one can change the orientation and migration of cells in a particular direction, thereby determining cell behaviors. In this study, we demonstrate a polydimethylsiloxane (PDMS) mold-based hot embossing technique for rapid, simple and low-cost replication of polystyrene (PS) surfaces having micropatterns. The PDMS mold was fabricated by UV-photolithography followed by PDMS casting; the elastomeric properties of PDMS enabled us to obtain conformal contact of the PDMS mold to a PS surface and to create high transcription quality of micropatterns on the PS surface. Two different types of circular micropillar and microwell arrays were successfully replicated on the PS surfaces based on the suggested technique. The micropatterns were designed to have various diameters (2-150 µm), spacings (2-160 µm) and heights (1.4, 2.4, 8.2 and 14.9 µm), so as to generate the gradient of physical properties on the surface. Experimental parametric studies indicated that (1) the embossing temperature became a critical processing parameter as the aspect ratio of micropattern increased and (2) the PDMS mold-based hot embossing could successfully replicate micropatterns, even having an aspect ratio of 2.7 for micropattern diameter of 6 µm, with an optimal processing condition (embossing pressure and temperature of 0.4 MPa and 130 °C, respectively) in this study. We carried out cell experiments with adipose-derived stem cells on the replicated PS surface with the height of 1.4 µm to investigate cellular behaviors in response to the micropattern array with gradient size. Cellular experiment results showed that the micropillar-arrayed surface improved cell proliferation as compared with the microwell-arrayed surface. We could also estimate the ranges of pattern sizes having the desired effects on the cellular behaviors.

Effects of cadmium on intercellular junctions in a renal epithelial cell line grown on permeable membrane supports

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

Prozialeck, W.C.; Niewenhuis, R.J.

1991-03-11

Recent findings from the authors laboratories have shown that Cd{sup 2+} has relatively specific damaging effects on adhering and occluding junctions in the established porcine renal epithelial cell line, LLC-PK{sub 1}. The present studies were undertaken in order to further characterize the junction-perturbing effects of Cd{sup 2+} in polarized monolayers of LLC-PK{sub 1} cells, and to begin to identify the mechanisms underlying these effects. LLC-PK{sub 1} cells were grown to confluency on Millicell HA chambers and exposed to Cd{sup 2+} in polarized monolayers of LLC-PK{sub 1} cells, and to begin to identify the mechanisms underlying these effects. LLC-PK{sub 1} cellsmore » were grown to confluency on Millicell HA chambers an exposed to Cd{sup 2+} by adding CdCl{sub 2} to the solutions on either side of the cell monolayer. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that exposure to Cd{sup 2+} caused a pronounced decrease in transepithelial resistance without causing the cells to detach from the Millicell membrane. This decrease in resistance occurred more quickly and was much more pronounced when Cd{sup 2+} was added to the basolateral surface rather than the apical surface. Furthermore, the effects of Cd{sup 2+} were greatly reduced when excess Ca{sup 2+} was present in the medium. These results suggest that Cd{sup 2+} was present in the medium. These results suggest that Cd{sup 2+} may disrupt cell-cell junctions by interacting with Ca{sup 2+} binding sites or Ca{sup 2+} channels that are oriented toward the basolateral cell surface.« less

Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin.

PubMed Central

Su, H; Watkins, N G; Zhang, Y X; Caldwell, H D

1990-01-01

The major outer membrane protein (MOMP) of Chlamydia trachomatis is characterized by four symmetrically spaced variable domains (VDs I to IV) whose sequences vary among serotypes. The surface-exposed portions of these VDs contain contiguous sequences that are both serotyping determinants and in vivo target sites for neutralizing antibodies. Previous studies using surface proteolysis of C. trachomatis B implicated VDs II and IV of the MOMP of this serotype in the attachment of chlamydiae to host cells. In this study, we used monoclonal antibodies (MAbs) specific to antigenic determinants located in VDs II and IV of the MOMP of serotype B to further investigate the role of the MOMP in the attachment of chlamydiae to host cells. MABs specific to serotype- and subspecies-specific epitopes located in exposed VDs II and IV, respectively, neutralized chlamydial infectivity for hamster kidney cells by blocking chlamydial attachment. We radioiodinated these MAbs and used them to determine the number and topology of the surface-exposed VDs II and IV epitopes on chlamydial elementary bodies. VDs II and IV each comprised approximately 2.86 x 10(4) negatively charged sites and were in proximity on the chlamydial cell surface. These studies suggest that the MAbs blocked chlamydial attachment by inhibiting electrostatic interactions with host cells. We examined the effects of thermal inactivation on both chlamydial attachment and conformation of the MOMP. Heat-inactivated chlamydiae failed to attach to host cells and exhibited a conformational change in an inaccessible invariant hydrophobic nonapeptide sequence located within VD IV of the MOMPs of C. trachomatis serotypes. These findings suggest that in addition to electrostatic interactions, a common hydrophobic component of the MOMP also contributes to the binding of chlamydiae to host cells. Thus, we propose that the MOMP functions as a chlamydial adhesin by promoting nonspecific (electrostatic and hydrophobic) interactions with host cells. Surface-accessible negatively charged VDs appear to be important in electrostatic binding, while the invariant region of VD IV may provide a subsurface hydrophobic depression which further promotes binding of chlamydiae to host cells through hydrophobic interactions. Images PMID:2318528

Downregulation of cell surface molecules during noncytopathic infection of T cells with human immunodeficiency virus.

PubMed Central

Stevenson, M; Zhang, X H; Volsky, D J

1987-01-01

Noncytopathic infection of human T-lymphoid cell line CR-10 with human immunodeficiency virus (HIV) (CEM-N1T isolate) resulted in a gradual loss of cell surface receptors for OKT4/OKT4A (HIV receptor), OKT8, OKT3, and OKT11 but not for OKT9 (transferrin receptor) within 10 days after infection. Surface receptor decline was accompanied by a rapid increase in HIV antigens and mRNA expression. Multireceptor downregulation was also observed in three T-lymphoid cell lines (MT-4, CEM, and HBD-1) cytopathically infected with the HIV/N1T virus and in HUT-78 cells infected with the HIV/SF-2 isolate. HIV-infected and uninfected CR-10 cells contained similar levels of mRNAs coding for T3, T8, T9, T11, HLA-A2, and HLA-B7 proteins. By densitometry, fully infected CR-10 cells showed approximately 75% reduction in T4 and tubulin (beta chain) mRNA levels when compared with uninfected CR-10 cells. No such reduction was detected in HIV-infected MT-4 and HBD-1 cells. A T-cell receptor gene (beta chain) rearrangement study revealed that no distinct CR-10 subpopulation was selected out upon infection with HIV. Our results suggest that the reduction in cell surface receptors observed between 1 and 2 weeks postinfection cannot be directly attributed to similar reductions in mRNA levels coding for these receptor proteins. We conclude that HIV infection induces posttranscriptional downregulation of several T-cell surface receptors. Images PMID:3500327

Characterization of novel biomarkers in selecting for subtype specific medulloblastoma phenotypes.

PubMed

Liang, Lisa; Aiken, Christopher; McClelland, Robyn; Morrison, Ludivine Coudière; Tatari, Nazanin; Remke, Marc; Ramaswamy, Vijay; Issaivanan, Magimairajan; Ryken, Timothy; Del Bigio, Marc R; Taylor, Michael D; Werbowetski-Ogilvie, Tamra E

2015-11-17

Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Medulloblastoma is the most common primary malignant pediatric brain cancer and consists of 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. Given the heterogeneity within and between medulloblastoma variants, surface marker profiles may be subtype-specific. Here, we employed a high throughput flow cytometry screen to identify differentially expressed cell surface markers in self-renewing vs. non-self-renewing SHH medulloblastoma cells. The top 25 markers were reduced to 4, CD271/p75NTR/NGFR, CD106/VCAM1, EGFR and CD171/NCAM-L1, by evaluating transcript levels in SHH tumors relative to samples representing the other variants. However, only CD271/p75NTR/NGFR and CD171/NCAM-L1 maintain differential expression between variants at the protein level. Functional characterization of CD271, a low affinity neurotrophin receptor, in cell lines and primary cultures suggested that CD271 selects for lower self-renewing progenitors or stem cells. Moreover, CD271 levels were negatively correlated with expression of SHH pathway genes. Our study reveals a novel role for CD271 in SHH medulloblastoma and suggests that targeting CD271 pathways could lead to the design of more selective therapies that lessen the broad impact of current treatments on developing nervous systems.

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

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

Surfactant protein D delays Fas- and TRAIL-mediated extrinsic pathway of apoptosis in T cells.

PubMed

Djiadeu, Pascal; Kotra, Lakshmi P; Sweezey, Neil; Palaniyar, Nades

2017-05-01

Only a few extracellular soluble proteins are known to modulate apoptosis. We considered that surfactant-associated protein D (SP-D), an innate immune collectin present on many mucosal surfaces, could regulate apoptosis. Although SP-D is known to be important for immune cell homeostasis, whether SP-D affects apoptosis is unknown. In this study we aimed to determine the effects of SP-D on Jurkat T cells and human T cells dying by apoptosis. Here we show that SP-D binds to Jurkat T cells and delays the progression of Fas (CD95)-Fas ligand and TRAIL-TRAIL receptor induced, but not TNF-TNF receptor-mediated apoptosis. SP-D exerts its effects by reducing the activation of initiator caspase-8 and executioner caspase-3. SP-D also delays the surface exposure of phosphatidylserine. The effect of SP-D was ablated by the presence of caspase-8 inhibitor, but not by intrinsic pathway inhibitors. The binding ability of SP-D to dying cells decreases during the early stages of apoptosis, suggesting the release of apoptotic cell surface targets during apoptosis. SP-D also delays FasL-induced death of primary human T cells. SP-D delaying the progression of the extrinsic pathway of apoptosis could have important implications in regulating immune cell homeostasis at mucosal surfaces.

EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK- anaplastic large-cell lymphoma.

PubMed

Schmidt, Janine; Bonzheim, Irina; Steinhilber, Julia; Montes-Mojarro, Ivonne A; Ortiz-Hidalgo, Carlos; Klapper, Wolfram; Fend, Falko; Quintanilla-Martínez, Leticia

2017-09-01

Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL.

Differences in the Mechanical Properties of the Developing Cerebral Cortical Proliferative Zone between Mice and Ferrets at both the Tissue and Single-Cell Levels.

PubMed

Nagasaka, Arata; Shinoda, Tomoyasu; Kawaue, Takumi; Suzuki, Makoto; Nagayama, Kazuaki; Matsumoto, Takeo; Ueno, Naoto; Kawaguchi, Ayano; Miyata, Takaki

2016-01-01

Cell-producing events in developing tissues are mechanically dynamic throughout the cell cycle. In many epithelial systems, cells are apicobasally tall, with nuclei and somata that adopt different apicobasal positions because nuclei and somata move in a cell cycle-dependent manner. This movement is apical during G2 phase and basal during G1 phase, whereas mitosis occurs at the apical surface. These movements are collectively referred to as interkinetic nuclear migration, and such epithelia are called "pseudostratified." The embryonic mammalian cerebral cortical neuroepithelium is a good model for highly pseudostratified epithelia, and we previously found differences between mice and ferrets in both horizontal cellular density (greater in ferrets) and nuclear/somal movements (slower during G2 and faster during G1 in ferrets). These differences suggest that neuroepithelial cells alter their nucleokinetic behavior in response to physical factors that they encounter, which may form the basis for evolutionary transitions toward more abundant brain-cell production from mice to ferrets and primates. To address how mouse and ferret neuroepithelia may differ physically in a quantitative manner, we used atomic force microscopy to determine that the vertical stiffness of their apical surface is greater in ferrets (Young's modulus = 1700 Pa) than in mice (1400 Pa). We systematically analyzed factors underlying the apical-surface stiffness through experiments to pharmacologically inhibit actomyosin or microtubules and to examine recoiling behaviors of the apical surface upon laser ablation and also through electron microscopy to observe adherens junction. We found that although both actomyosin and microtubules are partly responsible for the apical-surface stiffness, the mouse

Differences in the Mechanical Properties of the Developing Cerebral Cortical Proliferative Zone between Mice and Ferrets at both the Tissue and Single-Cell Levels

PubMed Central

Nagasaka, Arata; Shinoda, Tomoyasu; Kawaue, Takumi; Suzuki, Makoto; Nagayama, Kazuaki; Matsumoto, Takeo; Ueno, Naoto; Kawaguchi, Ayano; Miyata, Takaki

2016-01-01

Cell-producing events in developing tissues are mechanically dynamic throughout the cell cycle. In many epithelial systems, cells are apicobasally tall, with nuclei and somata that adopt different apicobasal positions because nuclei and somata move in a cell cycle–dependent manner. This movement is apical during G2 phase and basal during G1 phase, whereas mitosis occurs at the apical surface. These movements are collectively referred to as interkinetic nuclear migration, and such epithelia are called “pseudostratified.” The embryonic mammalian cerebral cortical neuroepithelium is a good model for highly pseudostratified epithelia, and we previously found differences between mice and ferrets in both horizontal cellular density (greater in ferrets) and nuclear/somal movements (slower during G2 and faster during G1 in ferrets). These differences suggest that neuroepithelial cells alter their nucleokinetic behavior in response to physical factors that they encounter, which may form the basis for evolutionary transitions toward more abundant brain-cell production from mice to ferrets and primates. To address how mouse and ferret neuroepithelia may differ physically in a quantitative manner, we used atomic force microscopy to determine that the vertical stiffness of their apical surface is greater in ferrets (Young's modulus = 1700 Pa) than in mice (1400 Pa). We systematically analyzed factors underlying the apical-surface stiffness through experiments to pharmacologically inhibit actomyosin or microtubules and to examine recoiling behaviors of the apical surface upon laser ablation and also through electron microscopy to observe adherens junction. We found that although both actomyosin and microtubules are partly responsible for the apical-surface stiffness, the mouse

Specificity of marine microbial surface interactions.

PubMed Central

Imam, S H; Bard, R F; Tosteson, T R

1984-01-01

The macromolecular surface components involved in intraspecific cell surface interactions of the green microalga Chlorella vulgaris and closely associated bacteria were investigated. The specific surface attachment between this alga and its associated bacteria is mediated by lectin-like macromolecules associated with the surfaces of these cells. The binding activity of these surface polymers was inhibited by specific simple sugars; this suggests the involvement of specific receptor-ligand binding sites on the interactive surfaces. Epifluorescent microscopic evaluation of bacteria-alga interactions in the presence and absence of the macromolecules that mediate these interactions showed that the glycoproteins active in these processes were specific to the microbial sources from which they were obtained. The demonstration and definition of the specificity of these interactions in mixed microbial populations may play an important role in our understanding of the dynamics of marine microbial populations in the sea. PMID:6508293

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

PubMed

Gordon, Sheldon R; Wood, Meredith

2009-03-01

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

Analysis of surface properties of fixed and live cells using derivatized agarose beads.

PubMed

Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

2002-01-01

A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

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

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

Kononchik, Joseph P.; Vancini, Ricardo; Brown, Dennis T., E-mail: dennis_brown@ncsu.edu

Sindbis Virus (SV), the prototype alphavirus in the family togaviridae, infects both mammalian and insect cells. The ability of SV to infect cells possessing significantly different biochemical environments suggests that there may be a common mode of entry into each cell type. Previous studies show that up to 4 h post infection cells are permeable to small ions and alpha sarcin suggesting that the plasma membrane is compromised as infection takes place. Thin-section electron microscopy has also shown SV to bind to the plasma membrane and lose its electron dense core through a pore like structure developed upon interaction ofmore » the virus with the cell surface. Using freeze-fracture replicas, thin-sections and antibody labeling the data presented herein show virus associated with intramembrane particles on mosquito cells. These data suggest that the intramembrane particles associated with SV may be part of the pore structure consisting of virus proteins and cell receptor.« less

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed Central

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-01-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells. PMID:3855866

Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

PubMed

Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

1985-02-01

The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells.

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.

Multistage adsorption of diffusing macromolecules and viruses

NASA Astrophysics Data System (ADS)

Chou, Tom; D'Orsogna, Maria R.

2007-09-01

We derive the equations that describe adsorption of diffusing particles onto a surface followed by additional surface kinetic steps before being transported across the interface. Multistage surface kinetics occurs during membrane protein insertion, cell signaling, and the infection of cells by virus particles. For example, viral entry into healthy cells is possible only after a series of receptor and coreceptor binding events occurs at the cellular surface. We couple the diffusion of particles in the bulk phase with the multistage surface kinetics and derive an effective, integrodifferential boundary condition that contains a memory kernel embodying the delay induced by the surface reactions. This boundary condition takes the form of a singular perturbation problem in the limit where particle-surface interactions are short ranged. Moreover, depending on the surface kinetics, the delay kernel induces a nonmonotonic, transient replenishment of the bulk particle concentration near the interface. The approach generalizes that of Ward and Tordai [J. Chem. Phys. 14, 453 (1946)] and Diamant and Andelman [Colloids Surf. A 183-185, 259 (2001)] to include surface kinetics, giving rise to qualitatively new behaviors. Our analysis also suggests a simple scheme by which stochastic surface reactions may be coupled to deterministic bulk diffusion.

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

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

Hiyoshi, Masateru; Hashimoto, Michihiro; Yukihara, Mamiko

Highlights: •Many mutations were identified in Fms as a putative genetic cause of HDLS. •All of the mutations tested severely impair the kinase activity. •Most of the mutations also impair the trafficking to the cell surface. •These defects further suggest that HDLS is caused by a loss of Fms function. -- Abstract: The tyrosine kinase Fms, the cell surface receptor for M-CSF and IL-34, is critical for microglial proliferation and differentiation in the brain. Recently, a number of mutations have been identified in Fms as a putative genetic cause of hereditary diffuse leukoencephalopathy with spheroids (HDLS), implying an important rolemore » of microglial dysfunction in HDLS pathogenesis. In this study, we initially confirmed that 11 mutations, which reside within the ATP-binding or major tyrosine kinase domain, caused a severe impairment of ligand-induced Fms auto-phosphorylation. Intriguingly, we found that 10 of the 11 mutants also showed a weak cell surface expression, which was associated with a concomitant increase in the low molecular weight hypo-N-glycosylated immature gp130Fms-like species. Indeed, the mutant proteins heavily accumulated to the Golgi-like perinuclear regions. These results indicate that all of the Fms mutations tested severely impair the kinase activity and most of the mutations also impair the trafficking to the cell surface, further suggesting that HDLS is caused by the loss of Fms function.« less

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

Rebamipide suppresses PolyI:C-stimulated cytokine production in human conjunctival epithelial cells.

PubMed

Ueta, Mayumi; Sotozono, Chie; Yokoi, Norihiko; Kinoshita, Shigeru

2013-09-01

We previously documented that ocular surface epithelial cells could regulate ocular surface inflammation and suggested that, while Toll-like receptor 3 upregulates, EP3, one of the prostaglandin E2 receptors, downregulates ocular surface inflammation. Others reported that rebamipide, a gastroprotective drug, could not only increase the gastric mucus production, but also suppressed gastric mucosal inflammation and that it was dominantly distributed in mucosal tissues. The eyedrop form of rebamipide, approved in Japan for use in the treatment of dry eye diseases, upregulates mucin secretion and production, thereby suppressing superficial punctate keratopathy on the ocular surface of patients with this disease. In the current study, we investigated whether rebamipide has anti- inflammatory effects on the ocular surface. To examine the effects of rebamipide on polyI:C-induced cytokine expression by primary human conjunctival epithelial cells, we used enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction assay. We studied the effects of rebamipide on ocular surface inflammation in our murine experimental allergic conjunctivitis (EAC) model. Rebamipide could suppress polyI:C-induced cytokine production and the expression of mRNAs for CXCL10, CXCL11, RANTES, MCP-1, and IL-6 in human conjunctival epithelial cells. In our EAC model, the topical administration of rebamipide suppressed conjunctival allergic eosinophil infiltration. The topical application of rebamipide on the ocular surface might suppress ocular surface inflammation by suppressing the production of cytokines by ocular surface epithelial cells.

Multiscale Model of Swarming Bacteria

NASA Astrophysics Data System (ADS)

Alber, Mark

2011-03-01

Many bacteria can rapidly traverse surfaces from which they are extracting nutrient for growth. They generate flat, spreading colonies, called swarms because they resemble swarms of insects. In the beginning of the talk, swarms of the M. xanthus will be described in detail. Individual M. xanthus cells are elongated; they always move in the direction of their long axis; and they are in constant motion, repeatedly touching each other. As a cell glides, the slime capsule of a cell interacts with the bare agar surface, non-oriented slime which arises from the surface contact with the slime capsule, or oriented slime trails. Remarkably, cells regularly reverse their gliding directions. In this talk a detailed cell- and behavior-based computational model of M. xanthus swarming will be used to demonstrate that reversals of gliding direction and cell bending are essential for swarming and that specific reversal frequencies result in optimal swarming rate of the whole population. This suggests that the circuit regulating reversals evolved to its current sensitivity under selection for growth achieved by swarming.

Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

PubMed Central

Chiu, Yu-Hsin; Alvarez-Baron, Claudia; Kim, Eun Young

2010-01-01

Large-conductance Ca2+-activated K+ (BKCa) channels regulate the physiology of many cell types. A single vertebrate gene variously known as Slo1, KCa1.1, or KCNMA1 encodes the pore-forming subunits of BKCa channel but is expressed in a potentially very large number of alternative splice variants. Two splice variants of Slo1, Slo1VEDEC and Slo1QEERL, which differ at the extreme COOH terminus, show markedly different steady-state expression levels on the cell surface. Here we show that Slo1VEDEC and Slo1QEERL can reciprocally coimmunoprecipitate, indicating that they form heteromeric complexes. Moreover, coexpression of even small amounts of Slo1VEDEC markedly reduces surface expression of Slo1QEERL and total Slo1 as indicated by cell-surface biotinylation assays. The effects of Slo1VEDEC on steady-state surface expression can be attributed primarily to the last five residues of the protein based on surface expression of motif-swapped constructs of Slo1 in human embryonic kidney (HEK) 293T cells. In addition, the presence of the VEDEC motif at the COOH terminus of Slo1 channels is sufficient to confer a dominant-negative effect on cell surface expression of itself or other types of Slo1 subunits. Treating cells with short peptides containing the VEDEC motif increased surface expression of Slo1VEDEC channels transiently expressed in HEK293T cells and increased current through endogenous BKCa channels in mouse podocytes. Slo1VEDEC and Slo1QEERL channels are removed from the HEK293T cell surface with similar kinetics and to a similar extent, which suggests that the inhibitory effect of the VEDEC motif is exerted primarily on forward trafficking into the plasma membrane. PMID:20051533

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

Pinus Monophylla (Single Needled Pinyon Pine) show morphological changes in needle cell size and stomata over the past 100 years of rising CO2 in Western Arid Ecosystems.

NASA Astrophysics Data System (ADS)

Van De Water, P. K.

2016-12-01

The size, frequency, and morphology of leaf surface stomata is used to reconstruct past levels of atmospheric carbon dioxide over geologic time. This technique relies on measuring cell and cell-clusters to correlate with changes of known carbon dioxide levels in the atmosphere. Unfortunately, not all plants are suitable because the occurrence and placement of stomatal cell-complexes differ significantly between plant families. Monocot and dicot angiosperms exhibit different types of stomata and stomatal complexes that lack order and thus are unsuitable. But, in gymnosperms, the number and distribution of stomata and pavement cells is formalized and can be used to reconstruct past atmospheric carbon dioxide levels. However, characteristic of each plant species must still be considered. For example, conifers are useful but are divided into two-needle to five-needle pines, or have irregular surface morphology (Pseudotsuga sp. and Tsuga sp. needles). This study uses Pinus monophylla an undivided needle morphology, that being a cylinder has no interior surface cells. Pinus monophylla (single needle pinyon) needles were collected along Geiger Grade (Nevada State Highway 341, Reno) in 2005 and 2013 from 1500m to 2195m. Herbarium samples were also collected from 13 historic collections made between 1911 and 1994. The study determined changes with elevation and/or over time using in these populations. Using Pinus monophylla, insured needles represented a single surface with stomata, stomatal complex cells, and co-occurring pavement cell types. Results show decreased stomatal densities (stomata/area), stomatal index (stomata/stomata + epidermal cells) and stable stomata per row (stomata/row) . Epidermal cell density (Epidermal Cells /Area), and Pavement cell density (Pavement cell/area) track stomatal density similarly. Data comparison, using elevation in the 2005 and 2013 collections showed no-significant trends. Individual stomatal complexes show no differences in the size and shape over time or with elevation. Stomata morphology and the stomatal pores appear conservative. However some complex cells show a morphology suggesting they are not fully formed and functional. These characteristics appear often in the modern material suggesting some stomata never fully develop.

Antimicrobial metallic copper surfaces kill Staphylococcus haemolyticus via membrane damage.

PubMed

Santo, Christophe Espírito; Quaranta, Davide; Grass, Gregor

2012-03-01

Recently, copper (Cu) in its metallic form has regained interest for its antimicrobial properties. Use of metallic Cu surfaces in worldwide hospital trials resulted in remarkable reductions in surface contaminations. Yet, our understanding of why microbes are killed upon contact to the metal is still limited and different modes of action have been proposed. This knowledge, however, is crucial for sustained use of such surfaces in hospitals and other hygiene-sensitive areas. Here, we report on the molecular mechanisms by which the Gram-positive Staphylococcus haemolyticus is inactivated by metallic Cu. Staphylococcus haemolyticus was killed within minutes on Cu but not on stainless steel demonstrating the antimicrobial efficacy of metallic Cu. Inductively coupled plasma mass spectroscopy (ICP-MS) analysis and in vivo staining with Coppersensor-1 indicated that cells accumulated large amounts of Cu ions from metallic Cu surfaces contributing to lethal damage. Mutation rates of Cu- or steel-exposed cells were similarly low. Instead, live/dead staining indicated cell membrane damage in Cu- but not steel-exposed cells. These findings support a model of the cellular targets of metallic Cu toxicity in bacteria, which suggests that metallic Cu is not genotoxic and does not kill via DNA damage. In contrast, membranes constitute the likely Achilles' heel of Cu surface-exposed cells.

Antimicrobial metallic copper surfaces kill Staphylococcus haemolyticus via membrane damage

PubMed Central

Santo, Christophe Espírito; Quaranta, Davide; Grass, Gregor

2012-01-01

Recently, copper (Cu) in its metallic form has regained interest for its antimicrobial properties. Use of metallic Cu surfaces in worldwide hospital trials resulted in remarkable reductions in surface contaminations. Yet, our understanding of why microbes are killed upon contact to the metal is still limited and different modes of action have been proposed. This knowledge, however, is crucial for sustained use of such surfaces in hospitals and other hygiene-sensitive areas. Here, we report on the molecular mechanisms by which the Gram-positive Staphylococcus haemolyticus is inactivated by metallic Cu. Staphylococcus haemolyticus was killed within minutes on Cu but not on stainless steel demonstrating the antimicrobial efficacy of metallic Cu. Inductively coupled plasma mass spectroscopy (ICP-MS) analysis and in vivo staining with Coppersensor-1 indicated that cells accumulated large amounts of Cu ions from metallic Cu surfaces contributing to lethal damage. Mutation rates of Cu- or steel-exposed cells were similarly low. Instead, live/dead staining indicated cell membrane damage in Cu- but not steel-exposed cells. These findings support a model of the cellular targets of metallic Cu toxicity in bacteria, which suggests that metallic Cu is not genotoxic and does not kill via DNA damage. In contrast, membranes constitute the likely Achilles’ heel of Cu surface-exposed cells. PMID:22950011

CD45RO enriches for activated, highly mutated human germinal center B cells

PubMed Central

Jackson, Stephen M.; Harp, Natessa; Patel, Darshna; Zhang, Jeffrey; Willson, Savannah; Kim, Yoon J.; Clanton, Christian

2007-01-01

To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker. PMID:17644737

Sonication reduces the attachment of Salmonella Typhimurium ATCC 14028 cells to bacterial cellulose-based plant cell wall models and cut plant material.

PubMed

Tan, Michelle S F; Rahman, Sadequr; Dykes, Gary A

2017-04-01

This study investigated the removal of bacterial surface structures, particularly flagella, using sonication, and examined its effect on the attachment of Salmonella Typhimurium ATCC 14028 cells to plant cell walls. S. Typhimurium ATCC 14028 cells were subjected to sonication at 20 kHz to remove surface structures without affecting cell viability. Effective removal of flagella was determined by staining flagella of sonicated cells with Ryu's stain and enumerating the flagella remaining by direct microscopic counting. The attachment of sonicated S. Typhimurium cells to bacterial cellulose-based plant cell wall models and cut plant material (potato, apple, lettuce) was then evaluated. Varying concentrations of pectin and/or xyloglucan were used to produce a range of bacterial cellulose-based plant cell wall models. As compared to the non-sonicated controls, sonicated S. Typhimurium cells attached in significantly lower numbers (between 0.5 and 1.0 log CFU/cm 2 ) to all surfaces except to the bacterial cellulose-only composite without pectin and xyloglucan. Since attachment of S. Typhimurium to the bacterial cellulose-only composite was not affected by sonication, this suggests that bacterial surface structures, particularly flagella, could have specific interactions with pectin and xyloglucan. This study indicates that sonication may have potential applications for reducing Salmonella attachment during the processing of fresh produce. Copyright © 2016 Elsevier Ltd. All rights reserved.

Surface display of metal binding domain derived from PbrR on Escherichia coli specifically increases lead(II) adsorption.

PubMed

Hui, Chang-Ye; Guo, Yan; Yang, Xue-Qin; Zhang, Wen; Huang, Xian-Qing

2018-05-01

To improve the Pb 2+ biosorption capacity of the potential E. coli biosorbent, a putative Pb 2+ binding domain (PbBD) derived from PbrR was efficiently displayed on to the E. coli cell surface. The PbBD was obtained by truncating the N-terminal DNA-binding domain and C-terminal redundant amino acid residues of the Pb 2+ -sensing transcriptional factor PbrR. Whole-cell sorbents were constructed with the full-length PbrR and PbBD of PbrR genetically engineered onto the surface of E. coli cells using Lpp-OmpA as the anchor. Followed by a 1.71-fold higher display of PbBD than PbrR, the presence of PbBD on the surface of E. coli cells enabled a 1.92-fold higher Pb 2+ biosorption than that found in PbrR-displayed cells. Specific Pb 2+ binding via PbBD was the same as Pb 2+ binding via the full-length PbrR, with no observable decline even in the presence of Zn 2+ and Cd 2+ . Since surface-engineered E. coli cells with PbBD increased the Pb 2+ binding capacity and did not affect the adsorption selectivity, this suggests that surface display of the metal binding domain derived from MerR-like proteins may be used for the bioremediation of specific toxic heavy metals.

Migration of lymphocytes on fibronectin-coated surfaces: temporal evolution of migratory parameters

NASA Technical Reports Server (NTRS)

Bergman, A. J.; Zygourakis, K.; McIntire, L. V. (Principal Investigator)

1999-01-01

Lymphocytes typically interact with implanted biomaterials through adsorbed exogenous proteins. To provide a more complete characterization of these interactions, analysis of lymphocyte migration on adsorbed extracellular matrix proteins must accompany the commonly performed adhesion studies. We report here a comparison of the migratory and adhesion behavior of Jurkat cells (a T lymphoblastoid cell line) on tissue culture treated and untreated polystyrene surfaces coated with various concentrations of fibronectin. The average speed of cell locomotion showed a biphasic response to substrate adhesiveness for cells migrating on untreated polystyrene and a monotonic decrease for cells migrating on tissue culture-treated polystyrene. A modified approach to the persistent random walk model was implemented to determine the time dependence of cell migration parameters. The random motility coefficient showed significant increases with time when cells migrated on tissue culture-treated polystyrene surfaces, while it remained relatively constant for experiments with untreated polystyrene plates. Finally, a cell migration computer model was developed to verify our modified persistent random walk analysis. Simulation results suggest that our experimental data were consistent with temporally increasing random motility coefficients.

Surface expression and CEA binding of hnRNP M4 protein in HT29 colon cancer cells.

PubMed

Laguinge, Luciana; Bajenova, Olga; Bowden, Emma; Sayyah, Jacqueline; Thomas, Peter; Juhl, Hartmut

2005-01-01

Carcinoembryonic antigen (CEA) has been shown to participate in the progression and metastatic growth of colorectal cancer. However, its biological function remains elusive. Recently, we found that CEA protects colon cancer cells from undergoing apoptosis, suggesting a complex role that includes signal transduction activity. Additionally, it was reported that CEA binds to Kupffer cells and macrophages to a membrane-anchored homolog of heterogeneous nuclear protein M4 (hnRNP M4), which subsequently was named CEA-receptor (CEAR). Cytoplasmatic and membranous expression of CEAR in CEA-positive colon cancer tissues prompted us to analyze the CEA-CEAR interaction in HT29 colon cancer cells. Both, CEA and CEAR were found on the cell surface of HT29 cells, as demonstrated by confocal microscopy. Imaging analysis suggested co-localization and, thus, interaction of both molecules. To confirm this observation, immunoprecipitation experiments and Western blot analysis were performed and indicated binding of CEA and CEAR. Immunoprecipitation of CEA resulted in a pull down of CEAR. The pull down of CEAR correlated with the amount of CEA as demonstrated by ribozyme targeting of CEA. Finally, external treatment of HT29 cells with soluble CEA induced tyrosine phosphorylation of CEAR, suggesting a CEA-dependent role of CEAR in signal transduction. Future experiments will elucidate whether the CEA-CEAR interaction is involved in CEA's antiapoptotic role and mediates the prometastatic properties of CEA in colon cancer cells.

One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior.

PubMed

Marinkovic, Milos; Block, Travis J; Rakian, Rubie; Li, Qihong; Wang, Exing; Reilly, Matthew A; Dean, David D; Chen, Xiao-Dong

2016-01-01

For more than 100years, cells and tissues have been studied in vitro using glass and plastic surfaces. Over the last 10-20years, a great body of research has shown that cells are acutely sensitive to their local environment (extracellular matrix, ECM) which contains both chemical and physical cues that influence cell behavior. These observations suggest that modern cell culture systems, using tissue culture polystyrene (TCP) surfaces, may fail to reproduce authentic cell behavior in vitro, resulting in "artificial outcomes." In the current study, we use bone marrow (BM)- and adipose (AD)-derived stromal cells to prepare BM-ECM and AD-ECM, which are decellularized after synthesis by the cells, to mimic the cellular niche for each of these tissues. Each ECM was characterized for its ability to affect BM- and AD-mesenchymal stem cell (MSC) proliferation, as well as proliferation of three cancer cell lines (HeLa, MCF-7, and MDA-MB-231), modulate cell spreading, and direct differentiation relative to standard TCP surfaces. We found that both ECMs promoted the proliferation of MSCs, but that this effect was enhanced when the tissue-origin of the cells matched that of the ECM (i.e. BM-ECM promoted the proliferation of BM-MSCs over AD-MSCs, and vice versa). Moreover, BM- and AD-ECM were shown to preferentially direct MSC differentiation towards either osteogenic or adipogenic lineage, respectively, suggesting that the effects of the ECM were tissue-specific. Further, each ECM influenced cell morphology (i.e. circularity), irrespective of the origin of the MSCs, lending more support to the idea that effects were tissue specific. Interestingly, unlike MSCs, these ECMs did not promote the proliferation of the cancer cells. In an effort to further understand how these three culture substrates influence cell behavior, we evaluated the chemical (protein composition) and physical properties (architecture and mechanical) of the two ECMs. While many structural proteins (e.g. collagen and fibronectin) were found at equivalent levels in both BM- and AD-ECM, the architecture (i.e. fiber orientation; surface roughness) and physical properties (storage modulus, surface energy) of each were unique. These results, demonstrating differences in cell behavior when cultured on the three different substrates (BM- and AD-ECM and TCP) with differences in chemical and physical properties, provide evidence that the two ECMs may recapitulate specific elements of the native stem cell niche for bone marrow and adipose tissues. More broadly, it could be argued that ECMs, elaborated by cells ex vivo, serve as an ideal starting point for developing tissue-specific culture environments. In contrast to TCP, which relies on the "one size fits all" paradigm, native tissue-specific ECM may be a more rational model to approach engineering 3D tissue-specific culture systems to replicate the in vivo niche. We suggest that this approach will provide more meaningful information for basic research studies of cell behavior as well as cell-based therapeutics. Published by Elsevier B.V.

Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae

PubMed Central

Tacchi, Jessica L.; Raymond, Benjamin B. A.; Haynes, Paul A.; Berry, Iain J.; Widjaja, Michael; Bogema, Daniel R.; Woolley, Lauren K.; Jenkins, Cheryl; Minion, F. Chris; Padula, Matthew P.; Djordjevic, Steven P.

2016-01-01

Mycoplasma hyopneumoniae is a genome-reduced, cell wall-less, bacterial pathogen with a predicted coding capacity of less than 700 proteins and is one of the smallest self-replicating pathogens. The cell surface of M. hyopneumoniae is extensively modified by processing events that target the P97 and P102 adhesin families. Here, we present analyses of the proteome of M. hyopneumoniae-type strain J using protein-centric approaches (one- and two-dimensional GeLC–MS/MS) that enabled us to focus on global processing events in this species. While these approaches only identified 52% of the predicted proteome (347 proteins), our analyses identified 35 surface-associated proteins with widely divergent functions that were targets of unusual endoproteolytic processing events, including cell adhesins, lipoproteins and proteins with canonical functions in the cytosol that moonlight on the cell surface. Affinity chromatography assays that separately used heparin, fibronectin, actin and host epithelial cell surface proteins as bait recovered cleavage products derived from these processed proteins, suggesting these fragments interact directly with the bait proteins and display previously unrecognized adhesive functions. We hypothesize that protein processing is underestimated as a post-translational modification in genome-reduced bacteria and prokaryotes more broadly, and represents an important mechanism for creating cell surface protein diversity. PMID:26865024

Influence of local anesthetics upon human polymorphonuclear leukocyte function in vitro. Reduction of lysosomal enzyme release and superoxide anion production

PubMed Central

1977-01-01

Cationic local anesthetics have been reported to influence cellular responses to surface stimuli by interfering with the function of microtubules and microfilaments. Since unimpaired microtubule and microfilament functions are required by human polymorphonuclear leukocytes in order to respond normally to surface stimulation, we have studied effects of the local anesthetic, tetracaine on the function and morphology of these cells in vitro. Tetracaine (0.25--1.0 mM) significantly reduced extracellular release of the lysosomal enzymes, beta-glucuronidase and lysozyme from polymorphonuclear leukocytes exposed to serum-treated zymosan (a particulate stimulus), zymosan- treated serum (a soluble stimulus), and to the surface-active lectin, concanavalin A. Tetracaine also significantly reduced superoixde anion production (superoxide dismutase-inhibitable cytochrome c reduction) by these cells. Tetrancaine was not cytotoxic and its effects could be reversed completely by washing cells once with buffer. Electron microscope examination of tetracaine-treated cells revealed marked alterations of surface membranes. Microtubules and microfilaments appeared normal in "resting" polymorphonuclear leukocytes, but the increase in microtubules normally observed in stimulated cells was not seen after tetracaine treatment. These results suggest that tetracaine interferes with those interactions between immune reactants and the polymorphonuclear leukocyte cell surface which provoke exocytosis and increased oxidative metabolism. PMID:195003

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.

Nanoporous metals for biodegradable implants: Initial bone mesenchymal stem cell adhesion and degradation behavior.

PubMed

Heiden, Michael; Huang, Sabrina; Nauman, Eric; Johnson, David; Stanciu, Lia

2016-07-01

Nanostructured Fe-Mn and Fe-Mn-Zn metal scaffolds were generated through a well-controlled selective leaching process in order to fulfill the growing demand for adjustable degradation rates and improved cellular response of resorbable materials. Mouse bone marrow mesenchymal stem cells (D1 ORL UVA) were seeded onto eleven, carefully chosen nanoporous surfaces for 24 h in vitro. Using a combination of fluorescence microscopy, scanning electron microscopy (SEM), and an MTS assay, it was discovered that scaffolds with nanoscale roughened surfaces had increased cell attachment by up to 123% compared to polished smooth Fe-Mn surfaces. Significant cell spreading and construction of cell multilayers were also apparent after 24 h, suggesting better adhesion. Additionally, static electrochemical polarization experiments revealed an improvement of up to 26% in the actual rate of biodegradation for Fe-Mn surface-modified materials. However, any residual concentration of zinc after leaching was shown to slightly increase corrosion resistance. The results demonstrate that selectively leached, nanostructured Fe-Mn surfaces have the potential of being tailored to a diverse set of transient implant scenarios, while also effectively boosting overall biocompatibility, initial cell attachment, and degradation rate. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1747-1758, 2016. © 2016 Wiley Periodicals, Inc.

Realization of 13.6% Efficiency on 20 μm Thick Si/Organic Hybrid Heterojunction Solar Cells via Advanced Nanotexturing and Surface Recombination Suppression.

PubMed

He, Jian; Gao, Pingqi; Liao, Mingdun; Yang, Xi; Ying, Zhiqin; Zhou, Suqiong; Ye, Jichun; Cui, Yi

2015-06-23

Hybrid silicon/polymer solar cells promise to be an economically feasible alternative energy solution for various applications if ultrathin flexible crystalline silicon (c-Si) substrates are used. However, utilization of ultrathin c-Si encounters problems in light harvesting and electronic losses at surfaces, which severely degrade the performance of solar cells. Here, we developed a metal-assisted chemical etching method to deliver front-side surface texturing of hierarchically bowl-like nanopores on 20 μm c-Si, enabling an omnidirectional light harvesting over the entire solar spectrum as well as an enlarged contact area with the polymer. In addition, a back surface field was introduced on the back side of the thin c-Si to minimize the series resistance losses as well as to suppress the surface recombination by the built high-low junction. Through these improvements, a power conversion efficiency (PCE) up to 13.6% was achieved under an air mass 1.5 G irradiation for silicon/organic hybrid solar cells with the c-Si thickness of only about 20 μm. This PCE is as high as the record currently reported in hybrid solar cells constructed from bulk c-Si, suggesting a design rule for efficient silicon/organic solar cells with thinner absorbers.

Matrigel immobilization on the shish-kebab structured poly(ɛ-caprolactone) nanofibers for skin tissue engineering

NASA Astrophysics Data System (ADS)

Jing, Xin; Mi, Hao-Yang; Peng, Xiang-Fang; Turng, Lih-Sheng

2016-03-01

Surface properties of tissue engineering scaffolds such as topography, hydrophilicity, and functional groups play a vital role in cell adhesion, migration, proliferation, and apoptosis. First, poly(ɛ-caprolactone) (PCL) shish-kebab scaffolds (PCL-SK), which feature a three-dimensional structure comprised of electrospun PCL nanofibers covered by periodic, self-induced PCL crystal lamellae on the surface, was created to mimic the nanotopography of native collagen fibrils in the extracellular matrix (ECM). Second, matrigel was covalently immobilized on the surface of alkaline hydrolyzed PCL-SK scaffolds to enhance their hydrophilicity. This combined approach not only mimics the nanotopography of native collagen fibrils, but also simulates the surface features of collagen fibrils for cell growth. To investigate the viability of such scaffolds, HEF1 fibroblast cell assays were conducted and the results revealed that the nanotopography of the PCL-SK scaffolds facilitated cell adhesion and proliferation. The matrigel functionalization on PCL-SK scaffolds further enhanced cellular response, which suggested elevated biocompatibility and greater potential for skin tissue engineering applications.

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.

The levels and kinetics of oxygen tension detectable at the surface of human dermal fibroblast cultures.

PubMed

Tokuda, Y; Crane, S; Yamaguchi, Y; Zhou, L; Falanga, V

2000-03-01

Low oxygen tension has recently been shown to stimulate cell growth and clonal expansion, as well as synthesis and transcription of certain growth factors and extracellular matrix components. These results have been obtained by exposing cell cultures to a hypoxic environment. Using an oxygen probe, we have now studied how experimental conditions affect the oxygen tension detectable at the cell surface. Dissolved oxygen tension was directly related to the height of the medium above the cell surface (r = 0.8793, P = 0.021), but was constant when no cells were present in the flask (r = -0. 9732, P = 0.001). In both human dermal fibroblasts and NIH/3T3 cultures, oxygen tension decreased linearly as cell density increased (r = -0.835, P < 0.0001; r = -0.916, P < 0.0001, respectively). When human dermal fibroblasts were exposed to 2% O(2), maximum hypoxic levels (0 mmHg) were achieved within approximately 15 min, and the recovery time was within a similar time frame. The addition of rotenone, an inhibitor of cellular respiration, blocked this decrease in oxygen tension at the cell surface, suggesting that cellular consumption of oxygen is responsible for the decline. Finally, we examined the cell-surface oxygen tension in control and acutely wounded human skin equivalents (HSE), consisting of a keratinocyte layer over a type I collagen matrix containing fibroblasts. We found that oxygen tension dropped significantly (P < 0.0001) in acutely wounded areas of HSE as compared to unwounded areas of HSE and that this drop was prevented by the addition of mitomycin C. These results indicate that cell-surface oxygen tension is indirectly related to cell density, and that the amount of detectable oxygen at the cell surface is a function of cell density, the oxygen tension in the incubator, and increased cellular activity, as occurs after injury. Copyright 2000 Wiley-Liss, Inc.

Application of nanostructured biochips for efficient cell transfection microarrays

NASA Astrophysics Data System (ADS)

Akkamsetty, Yamini; Hook, Andrew L.; Thissen, Helmut; Hayes, Jason P.; Voelcker, Nicolas H.

2007-01-01

Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

Inactivation of the srtA Gene Affects Localization of Surface Proteins and Decreases Adhesion of Streptococcus pneumoniae to Human Pharyngeal Cells In Vitro

PubMed Central

Kharat, Arun S.; Tomasz, Alexander

2003-01-01

Inactivation of sortase gene srtA in Streptococcus pneumoniae strain R6 caused the release of β-galactosidase and neuraminidase A (NanA) from the cell wall into the surrounding medium. Both of these surface proteins contain the LPXTG motif in the C-terminal domain. Complementation with plasmid-borne srtA reversed protein release. Deletion of murM, a gene involved in the branching of pneumococcal peptidoglycan, also caused partial release of β-galactosidase, suggesting preferential attachment of the protein to branched muropeptides in the cell wall. Inactivation of srtA caused decreased adherence to human pharyngeal cells in vitro but had no effect on the virulence of a capsular type III strain of S. pneumoniae in the mouse intraperitoneal model. The observations suggest that—as in other gram-positive bacteria—sortase-dependent display of proteins occurs in S. pneumoniae and that some of these proteins may be involved in colonization of the human host. PMID:12704150

The formation mechanism for printed silver-contacts for silicon solar cells.

PubMed

Fields, Jeremy D; Ahmad, Md Imteyaz; Pool, Vanessa L; Yu, Jiafan; Van Campen, Douglas G; Parilla, Philip A; Toney, Michael F; van Hest, Maikel F A M

2016-04-01

Screen-printing provides an economically attractive means for making Ag electrical contacts to Si solar cells, but the use of Ag substantiates a significant manufacturing cost, and the glass frit used in the paste to enable contact formation contains Pb. To achieve optimal electrical performance and to develop pastes with alternative, abundant and non-toxic materials, a better understanding the contact formation process during firing is required. Here, we use in situ X-ray diffraction during firing to reveal the reaction sequence. The findings suggest that between 500 and 650 °C PbO in the frit etches the SiNx antireflective-coating on the solar cell, exposing the Si surface. Then, above 650 °C, Ag(+) dissolves into the molten glass frit - key for enabling deposition of metallic Ag on the emitter surface and precipitation of Ag nanocrystals within the glass. Ultimately, this work clarifies contact formation mechanisms and suggests approaches for development of inexpensive, nontoxic solar cell contacting pastes.

Hydroxamate anchors for improved photoconversion in dye-sensitized solar cells.

PubMed

Brewster, Timothy P; Konezny, Steven J; Sheehan, Stafford W; Martini, Lauren A; Schmuttenmaer, Charles A; Batista, Victor S; Crabtree, Robert H

2013-06-03

We present the first analysis of performance of hydroxamate linkers as compared to carboxylate and phosphonate groups when anchoring ruthenium-polypyridyl dyes to TiO2 surfaces in dye-sensitized solar cells (DSSCs). The study provides fundamental insight into structure/function relationships that are critical for cell performance. Our DSSCs have been produced by using newly synthesized dye molecules and characterized by combining measurements and simulations of experimental current density-voltage (J-V) characteristic curves. We show that the choice of anchoring group has a direct effect on the overall sunlight-to-electricity conversion efficiency (η), with hydroxamate anchors showing the best performance. Solar cells based on the pyridyl-hydroxamate complex exhibit higher efficiency since they suppress electron transfer from the photoanode to the electrolyte and have superior photoinjection characteristics. These findings suggest that hydroxamate anchoring groups should be particularly valuable in DSSCs and photocatalytic applications based on molecular adsorbates covalently bound to semiconductor surfaces. In contrast, analogous acetylacetonate anchors might undergo decomposition under similar conditions suggesting limited potential in future applications.

The formation mechanism for printed silver-contacts for silicon solar cells

DOE PAGES

Fields, Jeremy D.; Ahmad, Md. Imteyaz; Pool, Vanessa L.; ...

2016-04-01

Screen-printing provides an economically attractive means for making Ag electrical contacts to Si solar cells, but the use of Ag substantiates a significant manufacturing cost, and the glass frit used in the paste to enable contact formation contains Pb. To achieve optimal electrical performance and to develop pastes with alternative, abundant, and non-toxic materials requires understanding the contact formation process during firing. Here, we use in-situ X-ray diffraction during firing to reveal the reaction sequence. The findings suggest that between 500 degrees C and 650 degrees C PbO in the frit etches the SiNx antireflective-coating on the solar cell, exposingmore » the Si surface. Then, above 650 degrees C, Ag+ dissolves into the molten glass frit -- key for enabling deposition of metallic Ag on the emitter surface and precipitation of Ag nanocrystals within the glass. Ultimately, this work clarifies contact formation mechanisms and suggests approaches for development of inexpensive, nontoxic solar cell contacting pastes.« less

The role of a cell surface inhibitor in early signal transduction associated with the regulation of cell division and differentiation

NASA Technical Reports Server (NTRS)

Johnson, T. C.; Enebo, D. J.; Moos, P. J.; Fattaey, H. K.; Spooner, B. S. (Principal Investigator)

1992-01-01

Serum stimulation of quiescent human fibroblast cultures resulted in a hyperphosphorylation of the nuclear retinoblastoma gene susceptibility product (RB). However, serum stimulation in the presence of 9 x 10(-8) M of a purified bovine sialoglycopeptide (SGP) cell surface inhibitor abrogated the hyperphosphorylation of the RB protein and the subsequent progression of cells through the mitotic cycle. The experimental results suggest that the SGP mediated its cell cycle arrest at a site in the cell cycle that was at the time of RB phosphorylation or somewhat upstream of the modification of this regulatory protein of cell division. Both cells serum-deprived and serum stimulated in the presence of the SGP displayed only a hypophosphorylated RB protein, consistent with the SGP-mediated cell cycle arrest point being near the G1/S interface.

Methylammonium lead mixed halide films processed with a new composition for planar perovskite solar cells

NASA Astrophysics Data System (ADS)

Park, Ban-Suk; Lee, Seojun; Yoon, Saemon; Ha, Tae-Jun; Kang, Dong-Won

2018-01-01

In this work, we propose a new mixed halide precursor composition for MAPbI3-xClx organic/inorganic perovskite (PRV) solar cells. PRV films made with a new precursor composition of (MAI: PbCl2: PbI2 = 2 : 1 : 1) could be crystallized at lower temperature (70 °C) and shorter annealing duration (60 min), whereas previous standard composition (MAI: PbCl2 = 3 : 1) requires multi-step and high temperature (from 75 °C to 130 °C) annealing for longer durations (∼100 min). By adopting the suggested composition, much uniform surface morphology of PRV light harvester was obtained even though non-polar solvent washing was not introduced yet. Also, when the suitable toluene washing treatment was introduced, PRV surfaces of highly compact and large crystallites with regular distribution were achieved without any pinhole, which offered significant improvements in fill factor (41 → 65%) and power conversion efficiency (5.85 → 9.39%) of PRV cells. The suggested new precursor composition contributing for surface topography can be widely utilized for inverted planar PRV devices with low-temperature and simple processing.

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.

Ganglioside inhibition of sup 125 I-plasmin binding to colorectal carcinoma cells

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

Liepkalns, V.A.; Burtin, M.C.; Correc, P.

1990-01-01

The pre-incubation of human colorectal carcinoma cells SW 1116 with 25 to 100 uM purified gangliosides resulted in 35-60% inhibition of specific {sup 125}I-plasmin binding to the cell surface. After 5 to 6 days in culture, tumor cells were pre-incubated at 4 degrees for 1 to 4 h followed by post-incubation with {sup 125}I-plasmin by techniques previously described. At 25 uM the capacity for inhibition of plasmin binding was GT1b greater than GQ1b greater than or equal to GD1a greater than GM1 less than or equal to GgOse 4Cer. Thus a terminal sialyl moiety appears to be necessary (p lessmore » than 0.05) although exogenous N-acetyl neuraminic acid was ineffective (p greater than 0.05), indicating a role for the lipid portion of the ganglioside. Other (glyco)lipids such as sphingosine, fucolipid H-1 and sulfatide were without significant effect. The inhibition could not be reversed by the presence of 10 mM Ca+2, EDTA, pre-treatment of the cell with carboxypeptidase or pretreatment of plasmin with neuraminidases. The inhibition was however reversed by post-incubation in control medium without exogenous ganglioside. Cell counts determined prior to, and after ganglioside incubation showed that the effect was not due to cell death or detachment from the culture surface. The dissociation constant for {sup 125}I-plasmin binding was 5.6 x 10(-8) M (700,000 sites/cell), but in the presence of trisialoganglioside (GT1b), Scatchard plots suggested diversification of binding sites with 280,000 sites/cell at Kd 2.6 x 10(-8) M and 820,000 sites/cell at Kd 2.1 x 10(-7) M. Another interpretation of the Scatchard plot in the presence of ganglioside was that the glycolipid imposed negative cooperativity on plasmin binding to the cell surface. These results suggest that certain gangliosides can affect tumor cell invasiveness by altering protease binding to the cell surface.« less

Sparging and agitation-induced injury of cultured animals cells: Do cell-to-bubble interactions in the bulk liquid injure cells?

PubMed

Michaels, J D; Mallik, A K; Papoutsakis, E T

1996-08-20

It has been established that the forces resulting from bubbles rupturing at the free air (gas)/liquid surface injure animal cells in agitated and/or sparged bioreactors. Although it has been suggested that bubble coalescence and breakup within agitated and sparged bioreactors (i.e., away from the free liquid surface) can be a source of cell injury as well, the evidence has been indirect. We have carried out experiments to examine this issue. The free air/liquid surface in a sparged and agitated bioractor was eliminated by completely filling the 2-L reactor and allowing sparged bubbles to escape through an outlet tube. Two identical bioreactors were run in parallel to make comparisons between cultures that were oxygenated via direct air sparging and the control culture in which silicone tubing was used for bubble-free oxygenation. Thus, cell damage from cell-to-bubble interactions due to processes (bubble coalescence and breakup) occurring in the bulk liquid could be isolated by eliminating damage due to bubbles rupturing at the free air/liquid surface of the bioreactor. We found that Chinese hamster ovary (CHO) cells grown in medium that does not contain shear-protecting additives can be agitated at rates up to 600 rpm without being damaged extensively by cell-to bubble interactions in the bulk of the bioreactor. We verified this using both batch and high-density perfusion cultures. We tested two impeller designs (pitched blade and Rushton) and found them not to affect cell damage under similar operational conditions. Sparger location (above vs. below the impeller) had no effect on cell damage at higher agitation rates but may affect the injury process at lower agitation intensities (here, below 250 rpm). In the absence of a headspace, we found less cell damage at higher agitation intensities (400 and 600 rpm), and we suggest that this nonintuitive finding derives from the important effect of bubble size and foam stability on the cell damage process. (c) 1996 John Wiley & Sons, Inc.

Applied electric field enhances DRG neurite growth: influence of stimulation media, surface coating and growth supplements

NASA Astrophysics Data System (ADS)

Wood, Matthew D.; Willits, Rebecca Kuntz

2009-08-01

Electrical therapies have been found to aid repair of nerve injuries and have been shown to increase and direct neurite outgrowth during stimulation. This enhanced neural growth existed even after the electric field (EF) or stimulation was removed, but the factors that may influence the enhanced growth, such as stimulation media or surface coating, have not been fully investigated. This study characterized neurite outgrowth and branching under various conditions: EF magnitude and application time, ECM surface coating, medium during EF application and growth supplements. A uniform, low-magnitude EF (24 or 44 V m-1) was applied to dissociated chick embryo dorsal root ganglia seeded on collagen or laminin-coated surfaces. During the growth period, cells were either exposed to NGF or N2, and during stimulation cells were exposed to either unsupplemented media (Ca2+) or PBS (no Ca2+). Parallel controls for each experiment included cells exposed to the chamber with no stimulation and cells remaining outside the chamber. After brief electrical stimulation (10 min), neurite length significantly increased 24 h after application for all conditions studied. Of particular interest, increased stimulation time (10-100 min) further enhanced neurite length on laminin but not on collagen surfaces. Neurite branching was not affected by stimulation on any surface, and no preferential growth of neurites was noted after stimulation. Overall, the results of this report suggest that short-duration electric stimulation is sufficient to enhance neurite length under a variety of conditions. While further data are needed to fully elucidate a mechanism for this increased growth, these data suggest that one focus of those investigations should be the interaction between the growth cone and the substrata.

Surface expression of squamous cell carcinoma antigen (SCCA) can be increased by the preS1(21-47) sequence of hepatitis B virus.

PubMed

Ruvoletto, M G; Tono, N; Carollo, D; Vilei, T; Trentin, L; Muraca, M; Marino, M; Gatta, A; Fassina, G; Pontisso, P

2004-03-01

A variant of the serpin squamous cell carcinoma antigen (SCCA) has been identified as a hepatitis B virus binding protein and high expression of SCCA has recently been found in hepatocarcinoma. Since HBV is involved in liver carcinogenesis, experiments were carried out to examine the effect of HBV preS1 envelope protein on SCCA expression. Surface and intracellular staining for SCCA was assessed by FACS analysis. Preincubation of HepG2 cells and primary human hepatocytes with preS1 protein or with preS1(21-47) tetrameric peptide significantly increased the surface expression of SCCA, without modification of its overall cellular burden, suggesting a surface redistribution of the serpin. An increase in HBV binding and internalization was observed after pre-incubation of the cells with preS1 preparations, compared to cells preincubated with medium alone. Pretreatment of cells with DMSO, while not influencing SCCA basal expression, was responsible for an increase in the efficiency of HBV internalization and this effect was additive to that obtained after incubation with preS1 preparations. In conclusion, the HBV preS1(21-47) sequence is able to induce overexpression of SCCA at the cell surface facilitating virus internalization, while the increased efficiency of HBV entry following DMSO addition is not mediated by SCCA.

Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination.

PubMed

Fonseca, Nuno A; Rodrigues, Ana S; Rodrigues-Santos, Paulo; Alves, Vera; Gregório, Ana C; Valério-Fernandes, Ângela; Gomes-da-Silva, Lígia C; Rosa, Manuel Santos; Moura, Vera; Ramalho-Santos, João; Simões, Sérgio; Moreira, João Nuno

2015-11-01

Breast cancer stem cells (CSC) are thought responsible for tumor growth and relapse, metastization and active evasion to standard chemotherapy. The recognition that CSC may originate from non-stem cancer cells (non-SCC) through plastic epithelial-to-mesenchymal transition turned these into relevant cell targets. Of crucial importance for successful therapeutic intervention is the identification of surface receptors overexpressed in both CSC and non-SCC. Cell surface nucleolin has been described as overexpressed in cancer cells as well as a tumor angiogenic marker. Herein we have addressed the questions on whether nucleolin was a common receptor among breast CSC and non-SCC and whether it could be exploited for targeting purposes. Liposomes functionalized with the nucleolin-binding F3 peptide, targeted simultaneously, nucleolin-overexpressing putative breast CSC and non-SCC, which was paralleled by OCT4 and NANOG mRNA levels in cells from triple negative breast cancer (TNBC) origin. In murine embryonic stem cells, both nucleolin mRNA levels and F3 peptide-targeted liposomes cellular association were dependent on the stemness status. An in vivo tumorigenic assay suggested that surface nucleolin overexpression per se, could be associated with the identification of highly tumorigenic TNBC cells. This proposed link between nucleolin expression and the stem-like phenotype in TNBC, enabled 100% cell death mediated by F3 peptide-targeted synergistic drug combination, suggesting the potential to abrogate the plasticity and adaptability associated with CSC and non-SCC. Ultimately, nucleolin-specific therapeutic tools capable of simultaneous debulk multiple cellular compartments of the tumor microenvironment may pave the way towards a specific treatment for TNBC patient care. Copyright © 2015 Elsevier Ltd. All rights reserved.

Conversion of Stationary to Invasive Tumor Initiating Cells (TICs): Role of Hypoxia in Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) Trafficking

PubMed Central

Li, Jian; Zucker, Stanley; Pulkoski-Gross, Ashleigh; Kuscu, Cem; Karaayvaz, Mihriban; Ju, Jingfang; Yao, Herui; Song, Erwei; Cao, Jian

2012-01-01

Emerging evidence has implicated the role of tumor initiating cells (TICs) in the process of cancer metastasis. The mechanism underlying the conversion of TICs from stationary to invasive remains to be characterized. In this report, we employed less invasive breast cancer TICs, SK-3rd, that displays CD44high/CD24low with high mammosphere-forming and tumorigenic capacities, to investigate the mechanism by which stationary TICs are converted to invasive TICs. Invasive ability of SK-3rd TICs was markedly enhanced when the cells were cultured under hypoxic conditions. Given the role of membrane type 1-matrix metalloproteinase (MT1-MMP) in cancer invasion/metastasis, we explored a possible involvement of MT1-MMP in hypoxia-induced TIC invasion. Silencing of MT1-MMP by a shRNA approach resulted in diminution of hypoxia-induced cell invasion in vitro and metastasis in vivo. Under hypoxic conditions, MT1-MMP redistributed from cytoplasmic storage pools to the cell surface of TICs, which coincides with the increased cell invasion. In addition, CD44, a cancer stem-like cell marker, inversely correlated with increased cell surface MT1-MMP. Interestingly, cell surface MT1-MMP gradually disappeared when the hypoxia-treated cells were switched to normoxia, suggesting the plasticity of TICs in response to oxygen content. Furthermore, we dissected the pathways leading to upregulated MT1-MMP in cytoplasmic storage pools under normoxic conditions, by demonstrating a cascade involving Twist1-miR10b-HoxD10 leading to enhanced MT1-MMP expression in SK-3rd TICs. These observations suggest that MT1-MMP is a key molecule capable of executing conversion of stationary TICs to invasive TICs under hypoxic conditions and thereby controlling metastasis. PMID:22679501

Age-related impairment of endothelial progenitor cell migration correlates with structural alterations of heparan sulfate proteoglycans.

PubMed

Williamson, Kate A; Hamilton, Andrew; Reynolds, John A; Sipos, Peter; Crocker, Ian; Stringer, Sally E; Alexander, Yvonne M

2013-02-01

Aging poses one of the largest risk factors for the development of cardiovascular disease. The increased propensity toward vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells to contribute to vascular repair and regeneration. Although there is evidence to suggest that colony forming unit-Hill cells and circulating angiogenic cells are subject to age-associated changes that impair their function, the impact of aging on human outgrowth endothelial cell (OEC) function has been less studied. We demonstrate that OECs isolated from cord blood or peripheral blood samples from young and old individuals exhibit different characteristics in terms of their migratory capacity. In addition, age-related structural changes were discovered in OEC heparan sulfate (HS), a glycocalyx component that is essential in many signalling pathways. An age-associated decline in the migratory response of OECs toward a gradient of VEGF significantly correlated with a reduction in the relative percentage of the trisulfated disaccharide, 2-O-sulfated-uronic acid, N, 6-O-sulfated-glucosamine (UA[2S]-GlcNS[6S]), within OEC cell surface HS polysaccharide chains. Furthermore, disruption of cell surface HS reduced the migratory response of peripheral blood-derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Together these findings suggest that aging is associated with alterations in the fine structure of HS on the cell surface of OECs. Such changes may modulate the migration, homing, and engraftment capacity of these repair cells, thereby contributing to the progression of endothelial dysfunction and age-related vascular pathologies. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Notch Signaling Modulates MUC16 Biosynthesis in an In Vitro Model of Human Corneal and Conjunctival Epithelial Cell Differentiation

PubMed Central

Xiong, Linjie; Woodward, Ashley M.

2011-01-01

Purpose. Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and differentiation in wet-surfaced epithelia. We sought to determine whether Notch signaling contributes to maintaining mucosal homeostasis by modulating the biosynthesis of cell surface-associated mucins in an in vitro model of human corneal (HCLE) and conjunctival (HCjE) epithelial cell differentiation. Methods. HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction. Results. Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. Conclusions. These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface. PMID:21508102

Cellular basis for neonatally induced T-suppressor activity. Primary B cell maturation is blocked by suppressor-helper interactions restricted by loci on chromosome 12

PubMed Central

1985-01-01

The cellular mechanism and genetic restriction of neonatally induced HA- specific suppressor T (Ts) cells have been examined. The in vivo effect of these Ts cells on antibody production, primary B cell proliferation, B cell surface marker changes, and helper T (Th) cell priming during primary responses to HA have been determined. The results indicate that, although antigen-induced B cell proliferative responses and surface marker changes occur in the presence of Ts cells, differentiation to Ig secretion, and long-lived memory B cell production are prevented. Further, antigen-specific Th cell priming is completely ablated by Ts cells, suggesting that Ts act by preventing the delivery of Th signals required for both the later stages of primary B cell maturation, and the formation of memory B cell populations. Finally, in vivo cell mixing experiments using congenic mice indicate that this Ts-Th interaction is restricted by loci on mouse chromosome 12. PMID:2580040

Monoclonal Antibody and an Antibody-Toxin Conjugate to a Cell Surface Proteoglycan of Melanoma Cells Suppress in vivo Tumor Growth

NASA Astrophysics Data System (ADS)

Bumol, T. F.; Wang, Q. C.; Reisfeld, R. A.; Kaplan, N. O.

1983-01-01

A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

[The epithelial junctional zones of the anal canal and cervix uterus: the ultrastructure of tumors of these zones].

PubMed

Chernyĭ, A P; Iakovleva, N I

1990-01-01

Relationships between squamous and columnar epithelia in the anal canal and cervix uteri of postnatal period and fetus were studied. The transitional stratified epithelial lining, which is called junctional epithelium, is interposed between the mentioned epithelia. The junctional epithelium has variable numbers of layers of epidermoid cells, which differ from cells of atypical squamous epithelium by some ultrastructural features of the cytoskeleton and cell surface and by a low content of glycogen. The hypothesis on the physiological significance of this epithelium is proposed. Ultrastructural features of the cytoskeleton and cell surface suggest that anal basaloid carcinomas and some cervical squamous carcinomas may develop from so-called junctional epithelium.

Increased affinity of endothelial cells to NiTi using ultraviolet irradiation: An in vitro study.

PubMed

Tateshima, Satoshi; Kaneko, Naoki; Yamada, Masahiro; Duckwiler, Gary; Vinuela, Fernando; Ogawa, Takahiro

2018-04-01

Nickel-titanium alloy (NiTi) is one of the most popular materials used endovascularly because of its shape memory and superelasticity. The NiTi device needs to be covered by endothelial cells after being placed in the blood vessel to reduce ischemic complications. The objective of this study was to examine the impact of ultraviolet (UV) irradiation on the biocompatibility of NiTi surfaces with endothelial cells. NiTi sheets were treated with UV irradiation for 48 h and human aorta derived endothelial cells were used in this study. UV irradiation converted the NiTi surface to hydrophilic state and increased albumin adsorption. The number of endothelial cell migration, attachment, proliferation as well as their metabolic activity were significantly increased on UV treated NiTi. This study provides the first evidence of the photoactivation of NiTi surfaces by UV irradiation and demonstrates improved biocompatibility of UV-treated NiTi surfaces with vascular endothelial cells. These results suggest that UV irradiation may promote endothelialization of NiTi devices in blood vessels. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1034-1038, 2018. © 2017 Wiley Periodicals, Inc.

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

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

Curbo, Sophie; Gaudin, Raphael; Carlsten, Mattias

2009-12-25

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4R{alpha} receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown tomore » be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.« less

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

Evidence of adaptation, niche separation and microevolution within the genus Polaromonas on Arctic and Antarctic glacial surfaces.

PubMed

Gawor, Jan; Grzesiak, Jakub; Sasin-Kurowska, Joanna; Borsuk, Piotr; Gromadka, Robert; Górniak, Dorota; Świątecki, Aleksander; Aleksandrzak-Piekarczyk, Tamara; Zdanowski, Marek K

2016-07-01

Polaromonas is one of the most abundant genera found on glacier surfaces, yet its ecology remains poorly described. Investigations made to date point towards a uniform distribution of Polaromonas phylotypes across the globe. We compared 43 Polaromonas isolates obtained from surfaces of Arctic and Antarctic glaciers to address this issue. 16S rRNA gene sequences, intergenic transcribed spacers (ITS) and metabolic fingerprinting showed great differences between hemispheres but also between neighboring glaciers. Phylogenetic distance between Arctic and Antarctic isolates indicated separate species. The Arctic group clustered similarly, when constructing dendrograms based on 16S rRNA gene and ITS sequences, as well as metabolic traits. The Antarctic strains, although almost identical considering 16S rRNA genes, diverged into 2 groups based on the ITS sequences and metabolic traits, suggesting recent niche separation. Certain phenotypic traits pointed towards cell adaptation to specific conditions on a particular glacier, like varying pH levels. Collected data suggest, that seeding of glacial surfaces with Polaromonas cells transported by various means, is of greater efficiency on local than global scales. Selection mechanisms present of glacial surfaces reduce the deposited Polaromonas diversity, causing subsequent adaptation to prevailing environmental conditions. Furthermore, interactions with other supraglacial microbiota, like algae cells may drive postselectional niche separation and microevolution within the Polaromonas genus.

A Phosphoinositide 3-Kinase (PI3K)-serum- and glucocorticoid-inducible Kinase 1 (SGK1) Pathway Promotes Kv7.1 Channel Surface Expression by Inhibiting Nedd4-2 Protein*

PubMed Central

Andersen, Martin Nybo; Krzystanek, Katarzyna; Petersen, Frederic; Bomholtz, Sofia Hammami; Olesen, Søren-Peter; Abriel, Hugues; Jespersen, Thomas; Rasmussen, Hanne Borger

2013-01-01

Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells. PMID:24214981

A phosphoinositide 3-kinase (PI3K)-serum- and glucocorticoid-inducible kinase 1 (SGK1) pathway promotes Kv7.1 channel surface expression by inhibiting Nedd4-2 protein.

PubMed

Andersen, Martin Nybo; Krzystanek, Katarzyna; Petersen, Frederic; Bomholtz, Sofia Hammami; Olesen, Søren-Peter; Abriel, Hugues; Jespersen, Thomas; Rasmussen, Hanne Borger

2013-12-27

Epithelial cell polarization involves several kinase signaling cascades that eventually divide the surface membrane into an apical and a basolateral part. One kinase, which is activated during the polarization process, is phosphoinositide 3-kinase (PI3K). In MDCK cells, the basolateral potassium channel Kv7.1 requires PI3K activity for surface-expression during the polarization process. Here, we demonstrate that Kv7.1 surface expression requires tonic PI3K activity as PI3K inhibition triggers endocytosis of these channels in polarized MDCK. Pharmacological inhibition of SGK1 gave similar results as PI3K inhibition, whereas overexpression of constitutively active SGK1 overruled it, suggesting that SGK1 is the primary downstream target of PI3K in this process. Furthermore, knockdown of the ubiquitin ligase Nedd4-2 overruled PI3K inhibition, whereas a Nedd4-2 interaction-deficient Kv7.1 mutant was resistant to both PI3K and SGK1 inhibition. Altogether, these data suggest that a PI3K-SGK1 pathway stabilizes Kv7.1 surface expression by inhibiting Nedd4-2-dependent endocytosis and thereby demonstrates that Nedd4-2 is a key regulator of Kv7.1 localization and turnover in epithelial cells.

Efficient biological conversion of carbon monoxide (CO) to carbon dioxide (CO2) and for utilization in bioplastic production by Ralstonia eutropha through the display of an enzyme complex on the cell surface.

PubMed

Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

2015-06-25

An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of the CO2-utilizing Ralstonia eutropha and successfully resulted in a 3.3-fold increase in conversion efficiency. These results suggest that this complexed system may be a promising strategy for CO2 utilization as a biological tool for the production of bioplastics.

Enhanced cell-surface display of a heterologous protein using SED1 anchoring system in SED1-disrupted Saccharomyces cerevisiae strain.

PubMed

Bamba, Takahiro; Inokuma, Kentaro; Hasunuma, Tomohisa; Kondo, Akihiko

2018-03-01

Yeast displaying enzymes on the cell surface are used for developing whole-cell biocatalysts. High enzyme activity on the cell surface is required in certain applications such as direct ethanol production from lignocellulosic materials. However, the cell surface enzyme activity is limited by several factors, one of which is the protein amount of the yeast cell wall. In this study, we attempted to improve the incorporation capacity of a displayed heterologous enzyme by disrupting a native cell-wall protein. β-Glucosidase (BGL1) from Aspergillus aculeatus was fused with Saccharomyces cerevisiae Sed1 and displayed on the cell surface of S. cerevisiae BY4741 strain and its SED1 disruptant. Sed1 is one of the most abundant stationary phase yeast cell wall protein. A time course analysis revealed that BGL1 activity of the control strain reached saturation after 48 h of cultivation. In contrast, the BGL1 activity of the SED1 disruptant increased until 72 h of cultivation and was 22% higher than that of the control strain. We also performed relative quantification of cell wall proteins of these strains by nanoscale ultra pressure liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nano-UPLC-MS E ). The amount of the cell wall-associated BGL1 per unit dry cell-weight of the SED1 disruptant was 19% higher than that of the control strain. These results suggested that the incorporation capacity of the cell wall for BGL1 was increased by disruption of SED1. Disruption of SED1 would be a promising approach for improving display efficiency of heterologous protein fused with Sed1. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The effects of fine-scale substratum roughness on diatom community structure in estuarine biofilms.

PubMed

Sweat, L Holly; Johnson, Kevin B

2013-09-01

Benthic diatoms are a major component of biofilms that form on surfaces submerged in marine environments. Roughness of the underlying substratum affects the settlement of both diatoms and subsequent macrofouling colonizers. This study reports the effects of roughness on estuarine diatom communities established in situ in the Indian River Lagoon, FL, USA. Natural communities were established on acrylic panels with a range of surface roughnesses. Smoother substrata exhibited higher cell density, species richness, and diversity. Twenty-three of 58 species were found either exclusively or more abundantly on the smooth surfaces compared to one or both roughened treatments. The results suggest a greater ability of benthic diatoms to recruit and colonize smooth surfaces, which is probably explained by a higher degree of contact between the cells and the surface.

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.

Biomechanical properties of jaw periosteum-derived mineralized culture on different titanium topography.

PubMed

Att, Wael; Kubo, Katsutoshi; Yamada, Masahiro; Maeda, Hatsuhiko; Ogawa, Takahiro

2009-01-01

This study evaluated the biomechanical properties of periosteum-derived mineralized culture on different surface topographies of titanium. Titanium surfaces modified by machining or by acid etching were analyzed using scanning electron microscopy (SEM). Rat mandibular periosteum-derived cells were cultured on either of the titanium surfaces. Cell proliferation was evaluated by cell counts, and gene expression was analyzed using a reverse-transcriptase polymerase chain reaction. Alkaline phosphatase (ALP) stain assay was employed to evaluate osteoblastic activity. Matrix mineralization was examined via von Kossa stain assay, total calcium deposition, and SEM. The hardness and elastic modulus of mineralized cultures were measured using a nano-indenter. The machined surface demonstrated a flat topographic configuration, while the acid-etched surface revealed a uniform micron-scale roughness. Both cell density and ALP activity were significantly higher on the machined surface than on the acid-etched surface. The expression of bone-related genes was up-regulated or enhanced on the acid-etched surface compared to the machined surface. Von Kossa stain showed significantly greater positive areas for the machined surface compared to the acid-etched surface, while total calcium deposition was statistically similar. Mineralized culture on the acid-etched surface was characterized by denser calcium deposition, more mature collagen deposition on the superficial layer, and larger and denser globular matrices inside the matrix than the culture on the machined surface. The mineralized matrix on the acid-etched surface was two times harder than on the machined surface, whereas the elastic modulus was comparable between the two surfaces. The design of this study can be used as a model to evaluate the effect of implant surface topography on the biomechanical properties of periosteum-derived mineralized culture. The results suggest that mandibular periosteal cells respond to different titanium surface topographies differently enough to produce mineralized matrices with different biomechanical qualities.

Pancreatic cancer cells express CD44 variant 9 and multidrug resistance protein 1 during mitosis.

PubMed

Kiuchi, Shizuka; Ikeshita, Shunji; Miyatake, Yukiko; Kasahara, Masanori

2015-02-01

Pancreatic cancer is one of the most lethal cancers with high metastatic potential and strong chemoresistance. Its intractable natures are attributed to high robustness in tumor cells for their survival. We demonstrate here that pancreatic cancer cells (PCCs) with an epithelial phenotype upregulate cell surface expression of CD44 variant 9 (CD44v9), an important cancer stem cell marker, during the mitotic phases of the cell cycle. Of five human CD44(+) PCC lines examined, three cell lines, PCI-24, PCI-43 and PCI-55, expressed E-cadherin and CD44 variants, suggesting that they have an epithelial phenotype. By contrast, PANC-1 and MIA PaCa-2 cells expressed vimentin and ZEB1, suggesting that they have a mesenchymal phenotype. PCCs with an epithelial phenotype upregulated cell surface expression of CD44v9 in prophase, metaphase, anaphase and telophase and downregulated CD44v9 expression in late-telophase, cytokinesis and interphase. Sorted CD44v9-negative PCI-55 cells resumed CD44v9 expression when they re-entered the mitotic stage. Interestingly, CD44v9(bright) mitotic cells expressed multidrug resistance protein 1 (MDR1) intracellularly. Upregulated expression of CD44v9 and MDR1 might contribute to the intractable nature of PCCs with high proliferative activity. Copyright © 2014 Elsevier Inc. All rights reserved.

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins

PubMed Central

García-Bayona, Leonor; Guo, Monica S; Laub, Michael T

2017-01-01

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common. DOI: http://dx.doi.org/10.7554/eLife.24869.001 PMID:28323618

Cell-polymer interactions of fluorescent polystyrene latex particles coated with thermosensitive poly(N-isopropylacrylamide) and poly(N-vinylcaprolactam) or grafted with poly(ethylene oxide)-macromonomer.

PubMed

Vihola, Henna; Marttila, Anna-Kaisa; Pakkanen, Jukka S; Andersson, Mirja; Laukkanen, Antti; Kaukonen, Ann Marie; Tenhu, Heikki; Hirvonen, Jouni

2007-10-01

Cell-polymer interactions of thermosensitive poly(N-isopropylacrylamide) (PNIPAM) or poly(N-vinylcaprolactam) (PVCL) coated particles with RAW264.7 macrophages and intestinal Caco-2 cells were evaluated. Nanosized particles were prepared by modifying the surface of fluorescent polystyrene (FPS) particles with the thermosensitive polymer gels or with poly(ethylene oxide) (PEO)-macromonomer grafts. The particles were characterized by IR-spectroscopy for functional groups, light scattering for size distribution and zeta-potential for surface charge. Effects of temperature and polymer coating/grafting on the cellular interactions were evaluated by cell association/uptake and visualized by confocal scanning microscope. PEO and PNIPAM inhibited the polymer-cell contact by steric repulsion, evidenced by weak attachment of the particles. PVCL-coated FPS was adsorbed on the cells more strongly, especially at 37 degrees C, because of more hydrophobic nature at higher temperatures. The results suggest feasibility of the PNIPAM and PVCL for biotechnological/pharmaceutical applications, as the cell-particle interactions may be modified by size, surface charge, hydrophobicity, steric repulsion and temperature.

Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

PubMed Central

Collier, Ivan E.; Legant, Wesley; Marmer, Barry; Lubman, Olga; Saffarian, Saveez; Wakatsuki, Tetsuro; Elson, Elliot; Goldberg, Gregory I.

2011-01-01

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions. PMID:21912660

Leukocyte adhesion: High-speed cells with ABS.

PubMed

van der Merwe, P A

1999-06-03

In order to decide where to exit blood vessels and enter tissues, leukocytes roll along endothelial surfaces. Recent studies suggest that an 'automatic braking system' (ABS), involving selectin cell-adhesion molecules, enables leukocytes to roll at a fairly constant velocity despite large variations in blood flow rate.

Post-adsorption process of Yb phosphate nano-particle formation by Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Jiang, MingYu; Ohnuki, Toshihiko; Tanaka, Kazuya; Kozai, Naofumi; Kamiishi, Eigo; Utsunomiya, Satoshi

2012-09-01

In this study, we have investigated the post-adsorption process of ytterbium (Yb) phosphate nano-particle formation by Saccharomyces cerevisiae (yeast). The yeast grown in P-rich medium were exposed to 1.44 × 10-4 mol/L Yb(III) solution for 2-120 h, and 2 months at 25 ± 1 °C at an initial pH of 3, 4, or 5, respectively. Ytterbium concentrations in solutions decreased as a function of exposure time. Field-emission scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy (FESEM), transmission electron microscopy (TEM), and synchrotron-based extended X-ray absorption fine structure (EXAFS) analyses revealed that nano-sized blocky Yb phosphate with an amorphous phase formed on the yeast cells surfaces in the solutions with Yb. These nano-sized precipitates that formed on the cell surfaces remained stable even after 2 months of exposure at 25 ± 1 °C around neutral pHs. The EXAFS data revealed that the chemical state of the accumulated Yb on the cell surfaces changed from the adsorption on both phosphate and carboxyl sites at 30 min to Yb phosphate precipitates at 5 days, indicating the Yb-phosphate precipitation as a major post-adsorption process. In addition, the precipitation of Yb phosphate occurred on cell surfaces during 7 days of exposure in Yb-free solution after 2 h of exposure (short-term Yb adsorption) in Yb solution. These results suggest that the released P from the inside of yeast cells reacted with adsorbed Yb on cell surfaces, resulting in the formation of Yb precipitates, even though no P was added to the exposure solution. In an abiotic system, the EXAFS data showed that the speciation of sorbed Yb on the reference materials, carboxymethyl cellulose and Ln resin, did not change even when the Yb was exposed to P solution, without forming Yb phosphate precipitates. This result strongly suggests that the cell surface of the yeast plays an important role in the Yb-phosphate precipitation process, not only as a carrier of the functional groups but also as a substrate inducing the nucleation of phosphate nanoparticles. Stable nano-sized Yb phosphate precipitates formed on yeast cell surfaces in the present study, which implies that this post-adsorption nano-particle formation process caused by microbial cells should be one of the important processes governing the long-term migration of heavy rare earth elements and presumably trivalent actinides in geological repository.

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

PubMed

Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

2018-04-17

Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

A fish eye out of water: epithelial surface projections on aerial and aquatic corneas of the 'four-eyed fish' Anableps anableps.

PubMed

Simmich, Joshua; Temple, Shelby E; Collin, Shaun P

2012-03-01

Vertebrate corneas feature a variety of microprojections, to which a tear film adheres. These microprojections are formed by folds in epithelial cell membranes, which increase surface area, stabilise the tear film and enhance movement of nutritional and waste products across cell membranes. Differences in corneal microprojections among vertebrates have been correlated with habitat and differ markedly between terrestrial and aquatic species. This study investigated epithelial microprojections of both the aerial (dorsal) and aquatic (ventral) corneal surfaces of the 'four-eyed fish' Anableps anableps using scanning electron microscopy. The central region of the dorsal cornea, which projects above the water, had a density of 16,387 ± 3,995 cells per mm(2) , while the central region of the ventral cornea (underwater) had a density of 22,428 ± 6,387 cells per mm(2), a difference that suggests an environmental adaptation along the two visual axes. Both corneal surfaces were found to possess microridges rather than microvilli or microplicae characteristic of terrestrial/aerial vertebrates. Microridges were 142 ± 9 nm wide and did not differ (p = 0.757) between dorsal and ventral corneas. Microridges were consistently separated by a distance of 369 ± 9 nm across both corneas. Dorsal-ventral differences in corneal epithelial cell density in Anableps anableps suggest a difference in osmotic pressure of the two corneas. The modest differences in the microprojections indicate that the need to secure the tear film underlying each optical axis is of prime importance, due to the likelihood that a persistent layer of water normally covers both dorsal and ventral corneal surfaces and that maintaining a transparent optical pathway for vision is critical for a species prone to predation from both above and below the water's surface. © 2012 The Authors. Clinical and Experimental Optometry © 2012 Optometrists Association Australia.

Adhesion of glucosyltransferase phase variants to Streptococcus gordonii bacterium-glucan substrata may involve lipoteichoic acid.

PubMed

Vickerman, M M; Jones, G W

1992-10-01

Growing Streptococcus gordonii Spp+ phase variants, which have normal levels of glucosyltransferase (GTF) activity, use sucrose to promote their accumulation on surfaces by forming a cohesive bacterium-insoluble glucan polymer mass (BPM). Spp- phase variants, which have lower levels of GTF activity, do not form BPMs and do not remain in BPMs formed by Spp+ cells when grown in mixed cultures. To test the hypothesis that segregation of attached Spp+ and unattached Spp- cells was due to differences in adhesiveness, adhesion between washed, [3H]thymidine-labeled cells and preformed BPM substrata was measured. Unexpectedly, the results showed that cells of both phenotypes, as well as GTF-negative cells, attached equally well to preformed BPMs, indicating that attachment to BPMs was independent of cell surface GTF activity. Initial characterization of this binding interaction suggested that a protease-sensitive component on the washed cells may be binding to lipoteichoic acids sequestered in the BPM, since exogenous lipoteichoic acid inhibited adhesion. Surprisingly, the adhesion of both Spp+ and Spp- cells was markedly inhibited in the presence of sucrose, which also released lipoteichoic acid from the BPM. These in vitro findings suggest that, in vivo, sucrose and lipoteichoic acid may modify dental plaque development by enhancing or inhibiting the attachment of additional bacteria.

Adhesion of glucosyltransferase phase variants to Streptococcus gordonii bacterium-glucan substrata may involve lipoteichoic acid.

PubMed Central

Vickerman, M M; Jones, G W

1992-01-01

Growing Streptococcus gordonii Spp+ phase variants, which have normal levels of glucosyltransferase (GTF) activity, use sucrose to promote their accumulation on surfaces by forming a cohesive bacterium-insoluble glucan polymer mass (BPM). Spp- phase variants, which have lower levels of GTF activity, do not form BPMs and do not remain in BPMs formed by Spp+ cells when grown in mixed cultures. To test the hypothesis that segregation of attached Spp+ and unattached Spp- cells was due to differences in adhesiveness, adhesion between washed, [3H]thymidine-labeled cells and preformed BPM substrata was measured. Unexpectedly, the results showed that cells of both phenotypes, as well as GTF-negative cells, attached equally well to preformed BPMs, indicating that attachment to BPMs was independent of cell surface GTF activity. Initial characterization of this binding interaction suggested that a protease-sensitive component on the washed cells may be binding to lipoteichoic acids sequestered in the BPM, since exogenous lipoteichoic acid inhibited adhesion. Surprisingly, the adhesion of both Spp+ and Spp- cells was markedly inhibited in the presence of sucrose, which also released lipoteichoic acid from the BPM. These in vitro findings suggest that, in vivo, sucrose and lipoteichoic acid may modify dental plaque development by enhancing or inhibiting the attachment of additional bacteria. PMID:1398940

Na+/H+ exchange regulatory factor 1 is required for ROMK1 K+ channel expression in the surface membrane of cultured M-1 cortical collecting duct cells.

PubMed

Suzuki, Takashi; Nakamura, Kazuyoshi; Mayanagi, Taira; Sobue, Kenji; Kubokawa, Manabu

2017-07-22

The ROMK1 K + channel, a member of the ROMK channel family, is the major candidate for the K + secretion pathway in the renal cortical collecting duct (CCD). ROMK1 possesses a PDZ domain-binding motif at its C-terminus that is considered a modulator of ROMK1 expression via interaction with Na + /H + exchange regulatory factor (NHERF) 1 and NHERF2 scaffold protein. Although NHERF1 is a potential binding partner of the ROMK1 K + channel, the interaction between NHERF1 and K + channel activity remains unclear. Therefore, in this study, we knocked down NHERF1 in cultured M-1 cells derived from mouse CCD and investigated the surface expression and K + channel current in these cells after exogenous transfection with EGFP-ROMK1. NHERF1 knockdown resulted in reduced surface expression of ROMK1 as indicated by a cell biotinylation assay. Using the patch-clamp technique, we further found that the number of active channels per patched membrane and the Ba 2+ -sensitive whole-cell K + current were decreased in the knockdown cells, suggesting that reduced K + current was accompanied by decreased surface expression of ROMK1 in the NHERF1 knockdown cells. Our results provide evidence that NHERF1 mediates K + current activity through acceleration of the surface expression of ROMK1 K + channels in M-1 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

PubMed

Persson, Maria; Lorite, Gabriela S; Kokkonen, Hanna E; Cho, Sung-Woo; Lehenkari, Petri P; Skrifvars, Mikael; Tuukkanen, Juha

2014-09-01

The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications. Copyright © 2014 Elsevier B.V. All rights reserved.

MUC1, the renaissance molecule.

PubMed

Gendler, S J

2001-07-01

MUC1 is a large, heavily glycosylated mucin expressed on the apical surfaces of most simple, secretory epithelia including the mammary gland, gastrointestinal, respiratory, urinary and reproductive tracts. Although MUC1 was thought to be an epithelial-specific protein, it is now known to be expressed on a variety of hematopoietic cells as well. Mucins function in protection and lubrication of epithelial surfaces. Transmembrane mucins, which contain cytoplasmic tail domains, appear to have additional functions through their abilities to interact with many proteins involved in signal transduction and cell adhesion. The goal of this review is to highlight recent discoveries that suggest that MUC1 may be a multifunctional protein, located on the surfaces of cells as a sensor of the environment, poised to signal to the interior when things go awry.

Salicylate effects on proton gradient dissipation by isolated gastric mucosal surface cells.

PubMed

Olender, E J; Woods, D; Kozol, R; Fromm, D

1986-11-01

The effects of salicylate were examined on Na+/H+ exchange by isolated gastric mucosal surface cells loaded with H+ and resuspended in a buffered medium. Choline salicylate (pH 7.4) increases the dissipation of an intracellular proton gradient which was measured using acridine orange. The exchange of extracellular Na+ with intracellular H+ by surface cells not only remains intact but also is enhanced upon exposure to salicylate. This was confirmed by cellular uptake of 22Na and titration of cellular H+ efflux. Salicylate increases Na+/H+ exchange via a pathway predominantly sensitive to amiloride. However, the data also suggest that salicylate dissipates an intracellular proton gradient by an additional mechanism. The latter is independent of extracellular Na+ and not due to a generalized increase in cellular permeability.

Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

PubMed Central

Maria, Zahra; Campolo, Allison R.; Lacombe, Veronique A.

2015-01-01

Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes. PMID:26720696

Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria.

PubMed

Maria, Zahra; Campolo, Allison R; Lacombe, Veronique A

2015-01-01

Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes.

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

Controlled positioning of analytes and cells on a plasmonic platform for glycan sensing using surface enhanced Raman spectroscopy.

PubMed

Tabatabaei, Mohammadali; Wallace, Gregory Q; Caetano, Fabiana A; Gillies, Elizabeth R; Ferguson, Stephen S G; Lagugné-Labarthet, François

2016-01-01

The rise of molecular plasmonics and its application to ultrasensitive spectroscopic measurements has been enabled by the rational design and fabrication of a variety of metallic nanostructures. Advanced nano and microfabrication methods are key to the development of such structures, allowing one to tailor optical fields at the sub-wavelength scale, thereby optimizing excitation conditions for ultrasensitive detection. In this work, the control of both analyte and cell positioning on a plasmonic platform is enabled using nanofabrication methods involving patterning of fluorocarbon (FC) polymer (C 4 F 8 ) thin films on a plasmonic platform fabricated by nanosphere lithography (NSL). This provides the possibility to probe biomolecules of interest in the vicinity of cells using plasmon-mediated surface enhanced spectroscopies. In this context, we demonstrate the surface enhanced biosensing of glycan expression in different cell lines by surface enhanced Raman spectroscopy (SERS) on these plasmonic platforms functionalized with 4-mercaptophenylboronic acid (4-MPBA) as the Raman reporter. These cell lines include human embryonic kidney (HEK 293), C2C12 mouse myoblasts, and HeLa (Henrietta Lacks) cervical cancer cells. A distinct glycan expression is observed for cancer cells compared to other cell lines by confocal SERS mapping. This suggests the potential application of these versatile SERS platforms for differentiating cancerous from non-cancerous cells.

Release of Membrane-Bound Vesicles and Inhibition of Tumor Cell Adhesion by the Peptide Neopetrosiamide A

PubMed Central

Austin, Pamela; Heller, Markus; Williams, David E.; McIntosh, Lawrence P.; Vogl, A. Wayne; Foster, Leonard J.; Andersen, Raymond J.; Roberge, Michel; Roskelley, Calvin D.

2010-01-01

Background Neopetrosiamide A (NeoA) is a 28-amino acid tricyclic peptide originally isolated from a marine sponge as a tumor cell invasion inhibitor whose mechanism of action is unknown. Methodology/Principal Findings We show that NeoA reversibly inhibits tumor cell adhesion, disassembles focal adhesions in pre-attached cells, and decreases the level of β1 integrin subunits on the cell surface. NeoA also induces the formation of dynamic, membrane-bound protrusions on the surface of treated cells and the release of membrane-bound vesicles into the culture medium. Proteomic analysis indicates that the vesicles contain EGF and transferrin receptors as well as a number of proteins involved in adhesion and migration including: β1 integrin and numerous α integrin subunits; actin and actin-binding proteins such as cofilin, moesin and myosin 1C; and membrane modulating eps15 homology domain (EHD) proteins. Surface labeling, trafficking inhibition, and real-time imaging experiments all suggest that β1 integrin-containing vesicles are released directly from NeoA-induced cell surface protrusions rather than from vesicles generated intracellularly. The biological activity of NeoA is dependent on its disulfide bond pattern and NMR spectroscopy indicates that the peptide is globular with a continuous ridge of hydrophobic groups flanked by charged amino acid residues that could facilitate a simultaneous interaction with lipids and proteins in the membrane. Conclusions/Significance NeoA is an anti-adhesive peptide that decreases cell surface integrin levels through a novel, yet to be elucidated, mechanism that involves the release of adhesion molecule-containing vesicles from the cell surface. PMID:20520768

Urokinase Receptor Counteracts Vascular Smooth Muscle Cell Functional Changes Induced by Surface Topography

PubMed Central

Kiyan, Yulia; Kurselis, Kestutis; Kiyan, Roman; Haller, Hermann; Chichkov, Boris N.; Dumler, Inna

2013-01-01

Current treatments for human coronary artery disease necessitate the development of the next generations of vascular bioimplants. Recent reports provide evidence that controlling cell orientation and morphology through topographical patterning might be beneficial for bioimplants and tissue engineering scaffolds. However, a concise understanding of cellular events underlying cell-biomaterial interaction remains missing. In this study, applying methods of laser material processing, we aimed to obtain useful markers to guide in the choice of better vascular biomaterials. Our data show that topographically treated human primary vascular smooth muscle cells (VSMC) have a distinct differentiation profile. In particular, cultivation of VSMC on the microgrooved biocompatible polymer E-shell induces VSMC modulation from synthetic to contractile phenotype and directs formation and maintaining of cell-cell communication and adhesion structures. We show that the urokinase receptor (uPAR) interferes with VSMC behavior on microstructured surfaces and serves as a critical regulator of VSMC functional fate. Our findings suggest that microtopography of the E-shell polymer could be important in determining VSMC phenotype and cytoskeleton organization. They further suggest uPAR as a useful target in the development of predictive models for clinical VSMC phenotyping on functional advanced biomaterials. PMID:23843899

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains

PubMed Central

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS. PMID:27924253

Lactobacillus acidophilus CP23 with weak immunomodulatory activity lacks anchoring structure for surface layer protein.

PubMed

Yanagihara, Sae; Kato, Shinji; Ashida, Nobuhisa; Yamamoto, Naoyuki

2015-05-01

To determine the reason for the low levels of Surface layer protein A (SlpA) on CP23 cells, which might play a crucial role in the immunomodulatory effect of Lactobacillus acidophilus, the DNA sequence of the slpA gene of CP23 and L-92 strains, including the upstream region, were analyzed. Unexpectedly, there was no significant difference in the predicted amino acid sequence of the C-terminus needed for cell anchoring, and only an additional Ala-Val-Ala sequence inserted in the N-terminal region of the mature CP23 protein. Therefore, anchoring of SlpA on the cell wall of CP23 and L-92 was evaluated by a reconstitution assay, which showed that SlpA released by LiCl treatment from both CP23 and L-92 was successfully anchored on LiCl-treated L-92 cells, but not on LiCl-treated CP23 cells. Moreover, quantitative analysis of SlpA protein in the culture medium of CP23 and L-92 by ELISA revealed higher levels of SlpA secretion in CP23 cells than in L-92 cells. Collectively, these results suggest that the lower levels of SlpA on the surface of CP23 cells might be caused by less cell wall capacity for SlpA anchoring, leading to an accumulation of SlpA in the culture medium of CP23 cells. The present study supports the importance of cell surface structure of L. acidophilus L-92 for SlpA anchoring on the cell surface needed for immunomodulatory effect. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Enhancement of endothelialisation of coronary stents by laser surface engineering.

PubMed

Li, Lin; Mirhosseini, Nazanin; Michael, Alun; Liu, Zhu; Wang, Tao

2013-11-01

Coronary stents have been widely used in the treatment of coronary heart disease. However, complications have hampered the long-term success of the device. Bare-metal stents (BMS) have a high rate of restenosis and poor endothelialisation. The drug-eluting stents (DES), although dramatically reduce restenosis, significantly prevent endothelialisation leading to late thrombosis and behave the same way as BMS after drug releasing. Rapid adhesion and growth of endothelial cells on the stent surface is a key process for early vascular healing after coronary stenting which contributes to the reduction of major complications. Surface properties manipulate cell growth and directly determine the success and life-span of the implants. However, the ideal surface properties of coronary stents are not yet fully understood. The objective of this research is to understand how surface micro/nano textures and associated material chemistry changes generated by a laser beam affect the behavior of endothelial cells on bare metal 316L stents. A high power laser beam was applied to modifying the surface properties of 316L coronary stent material and the commercial coronary stents, followed by examination of the adhesion and proliferation of human coronary endothelial cells that were growing on the surfaces. Surface properties were examined by scanning electron microscopy, contact angle measurement, and X-ray photoelectron spectroscopy. A novel surface with combined micro/nano features was created on stent material 316L and coronary stent with a specific surface chemistry. This surface gives rise to a threefold increase in the adhesion and eightfold increase in the proliferation of endothelial cells. Interestingly, such effects were only observed when the surface texture was produced in the nitrogen atmosphere suggesting the importance of the surface chemistry, including the dramatic increase of chromium nitride, for the interaction of endothelial cells with the material surface. This novel surface is also super-hydrophilic with close to zero water/cell culture fluid contact angles and low cytotoxicity. A novel surface created by laser surface-engineering with a combination of defined surface texture and surface chemistry was found beneficial for the improvement of coronary stent endothelialisation. The technology presented here could work with both DES and BMS with added benefit for the improvement of the biocompatibility of current coronary stents. © 2013 Wiley Periodicals, Inc.

A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: to enhance the efficiency of recombinant DNA utilization.

PubMed

Mozley, Olivia L; Thompson, Ben C; Fernandez-Martell, Alejandro; James, David C

2014-01-01

In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60-70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG-containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl-PEI, was chemically synthesized. Using Design of Experiments-Response Surface Modeling to optimize the transfection process, the function of propyl-PEI was compared to that of unmodified PEI in both parental CHO-S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex cytotoxicity. The combination of propyl-PEI and Clone 4 doubled the efficiency of recombinant DNA utilization and reporter protein production. These data show that for maximal efficacy, strategies to increase polyplex internalization into cells must be used in concert with strategies to offset the inherent cytotoxicity of this process. © 2014 American Institute of Chemical Engineers.

Occludin confers adhesiveness when expressed in fibroblasts.

PubMed

Van Itallie, C M; Anderson, J M

1997-05-01

Occludin is an integral membrane protein specifically associated with tight junctions. Previous studies suggest it is likely to function in forming the intercellular seal. In the present study, we expressed occludin under an inducible promotor in occludin-null fibroblasts to determine whether this protein confers intercellular adhesion. When human occludin is stably expressed in NRK and Rat-1 fibroblasts, which lack endogenous occludin and tight junctions but do have well developed ZO-1-containing adherens-like junctions, occludin colocalizes with ZO-1 to points of cell-cell contact. In contrast, L-cell fibroblasts which lack cadherin-based adherens junctions, target neither ZO-1 nor occludin to sites of cell contact. Occludin-induced adhesion was next quantified using a suspended cell assay. In NRK and Rat-1 cells, occludin expression induces adhesion in the absence of calcium, thus independent of cadherin-cadherin contacts. In contrast, L-cells are nonadhesive in this assay and show no increase in adhesion after induction of occludin expression. Binding of an antibody to the first of the putative extracellular loops of occludin confirmed that this sequence was exposed on the cell surface, and synthetic peptides containing the amino acid sequence of this loop inhibit adhesion induced by occludin expression. These results suggest that the extracellular surface of occludin is directly involved in cell-cell adhesion and the ability to confer adhesiveness correlates with the ability to colocalize with its cytoplasmic binding protein, ZO-1.

Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling

PubMed Central

Diering, Graham H.; Numata, Yuka; Fan, Steven; Church, John; Numata, Masayuki

2013-01-01

To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na+/H+ exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase–Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation. PMID:24006492

Understanding and Portraying the Global Atmospheric Circulation.

ERIC Educational Resources Information Center

Harrington, John, Jr.; Oliver, John E.

2000-01-01

Examines teaching models of atmospheric circulation and resultant surface pressure patterns, focusing on the three-cell model and the meaning of meridional circulation as related to middle and high latitudes. Addresses the failure of the three-cell model to explain seasonal variations in atmospheric circulation. Suggests alternative models. (CMK)

In Vitro Biocompatibility of Si Alloyed Multi-Principal Element Carbide Coatings

PubMed Central

Vladescu, Alina; Titorencu, Irina; Dekhtyar, Yuri; Jinga, Victor; Pruna, Vasile; Balaceanu, Mihai; Dinu, Mihaela; Pana, Iulian; Vendina, Viktorija

2016-01-01

In the current study, we have examined the possibility to improve the biocompatibility of the (TiZrNbTaHf)C through replacement of either Ti or Ta by Si. The coatings were deposited on Si and 316L stainless steel substrates by magnetron sputtering in an Ar+CH4 mixed atmosphere and were examined for elemental composition, chemical bonds, surface topography, surface electrical charge and biocompatible characteristics. The net surface charge was evaluated at nano and macroscopic scale by measuring the electrical potential and work function, respectively. The biocompatible tests comprised determination of cell viability and cell attachment to the coated surface. The deposited coatings had C/(metal+Si) ratios close to unity, while a mixture of metallic carbide, free-carbon and oxidized species formed on the film surface. The coatings’ surfaces were smooth and no influence of surface roughness on electrical charge or biocompatibility was found. The biocompatible characteristics correlated well with the electrical potential/work function, suggesting a significant role of surface charge in improving biocompatibility, particularly cell attachment to coating's surface. Replacement of either Ti or Ta by Si in the (TiZrNbTaHf)C coating led to an enhanced surface electrical charge, as well as to superior biocompatible properties, with best results for the (TiZrNbSiHf)C coating. PMID:27571361

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

PubMed Central

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Microcinematographic analysis of tethered Leptospira illini.

PubMed Central

Charon, N W; Daughtry, G R; McCuskey, R S; Franz, G N

1984-01-01

A model of Leptospira motility was recently proposed. One element of the model states that in translating cells the anterior spiral-shaped end gyrates counterclockwise and the posterior hook-shaped end gyrates clockwise. We tested these predictions by analyzing cells tethered to a glass surface. Leptospira illini was incubated with antibody-coated latex beads (Ab-beads). These beads adhered to the cells, and subsequently some cells became attached to either the slide or the cover glass via the Ab-beads. As previously reported, these cells rapidly moved back and forth across the surface of the beads. In addition, a general trend was observed: cells tethered to the cover glass rotated clockwise around the Ab-bead; cells tethered to the slide rotated counterclockwise around the Ab-bead. A computer-aided microcinematographic analysis of tethered cells indicated that the direction of rotation of cells around the Ab-bead was a function of both the surface of attachment and the shape of the cell ends. The results can best be explained by assuming that the gyrating ends interact with the glass surface to cause rotation around the Ab-beads. The analysis obtained indicates that the hook- and spiral-shaped ends rotate in the directions predicted by the model. In addition, the tethered cell assay permitted detection of rapid, coordinated reversals of the cell ends, e.g., cells rapidly switched from a hook-spiral configuration to a spiral-hook configuration. These results suggest the existance of a mechanism which coordinates the shape of the cell ends of L. illini. Images PMID:6501226

Deoxyspergualin preferentially inhibits the growth and maturation of anti-CD40-activated surface IgD+ B lymphocytes.

PubMed

Morikawa, K; Nemoto, K; Miyawaki, T; Morikawa, S

1998-06-01

Deoxyspergualin (DSG), an analogue of spermidin, is a potent immunosuppressive drug with an action quite distinct from that of cyclosporin, rapamycin, or FK506. In this study we investigated the effect of DSG and methyldeoxyspergualin (MeDSG) on the proliferation and differentiation of human B cells stimulated with anti-CD40 MoAb. Highly purified B cells obtained from tonsillar samples were used as target cells. Both agents inhibited the proliferative response of anti-CD40-stimulated B cells in the absence and presence of IL-4, IL-2 or IL-10 in a dose-dependent manner. This inhibitory effect differed markedly among cell populations based on surface IgD expression: strong inhibition of sIgD+ B cells but little inhibition of sIgD- B cells. The drugs also suppressed the production of IgG, IgM and IgA by unfractionated B cells, which suggests that DSG acts against post-switch (sIgD-) B cells. Although the drugs suppressed immunoglobulin synthesis by both sIgD+ and sIgD- B cells, the effect was more marked in the sIgD+ B cells. Analysis of the subclass of IgG secreted by sIgD+ B cells revealed a decline in IgG1 and IgG3 in the presence of DSG. These results suggest that DSG preferentially inhibits the growth and maturation of sIgD+ naive B cells.

The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins

PubMed Central

Wang, Ran; Jiang, Lun; Zhang, Ming; Zhao, Liang; Hao, Yanling; Guo, Huiyuan; Sang, Yue; Zhang, Hao; Ren, Fazheng

2017-01-01

Lactobacillus salivarius REN, a novel probiotic isolated from Chinese centenarians, can adhere to intestinal epithelial cells and subsequently colonize the host. We show here that the surface-layer protein choline-binding protein A (CbpA) of L. salivarius REN was involved in adherence to the human colorectal adenocarcinoma cell line HT-29. Adhesion of a cbpA deletion mutant was significantly reduced compared with that of wild-type, suggesting that CbpA acts as an adhesin that mediates the interaction between the bacterium and its host. To identify the molecular mechanism of adhesion, we determined the crystal structure of a truncated form of CbpA that is likely involved in binding to its cell-surface receptor. The crystal structure identified CbpA as a peptidase of the M23 family whose members harbor a zinc-dependent catalytic site. Therefore, we propose that CbpA acts as a multifunctional surface protein that cleaves the host extracellular matrix and participates in adherence. Moreover, we identified enolase as the CbpA receptor on the surface of HT-29 cells. The present study reveals a new class of surface-layer proteins as well as the molecular mechanism that may contribute to the ability of L. salivarius REN to colonize the human gut. PMID:28281568

The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins.

PubMed

Wang, Ran; Jiang, Lun; Zhang, Ming; Zhao, Liang; Hao, Yanling; Guo, Huiyuan; Sang, Yue; Zhang, Hao; Ren, Fazheng

2017-03-10

Lactobacillus salivarius REN, a novel probiotic isolated from Chinese centenarians, can adhere to intestinal epithelial cells and subsequently colonize the host. We show here that the surface-layer protein choline-binding protein A (CbpA) of L. salivarius REN was involved in adherence to the human colorectal adenocarcinoma cell line HT-29. Adhesion of a cbpA deletion mutant was significantly reduced compared with that of wild-type, suggesting that CbpA acts as an adhesin that mediates the interaction between the bacterium and its host. To identify the molecular mechanism of adhesion, we determined the crystal structure of a truncated form of CbpA that is likely involved in binding to its cell-surface receptor. The crystal structure identified CbpA as a peptidase of the M23 family whose members harbor a zinc-dependent catalytic site. Therefore, we propose that CbpA acts as a multifunctional surface protein that cleaves the host extracellular matrix and participates in adherence. Moreover, we identified enolase as the CbpA receptor on the surface of HT-29 cells. The present study reveals a new class of surface-layer proteins as well as the molecular mechanism that may contribute to the ability of L. salivarius REN to colonize the human gut.

TMEM2: A missing link in hyaluronan catabolism identified?

PubMed

Yamaguchi, Yu; Yamamoto, Hayato; Tobisawa, Yuki; Irie, Fumitoshi

2018-03-27

Hyaluronan (HA) is a glycosaminoglycan (GAG) composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is an extremely long, unbranched polymer, which often exceeds 10 6  Da and sometimes reaches 10 7  Da. A feature that epitomizes HA is its rapid turnover; one-third of the total body HA is turned over daily. The current model of HA catabolism postulates that high-molecular weight HA in the extracellular space is first cleaved into smaller fragments by a hyaluronidase(s) that resides at the cell surface, followed by internalization of fragments and their degradation into monosaccharides in lysosomes. Over the last decade, considerable research has shown that the HYAL family of hyaluronidases plays significant roles in HA catabolism. Nonetheless, the identity of a hyaluronidase responsible for the initial step of HA cleavage on the cell surface remains elusive, as biochemical and enzymological properties of HYAL proteins are not entirely consistent with those expected of cell surface hyaluronidases. Recent identification of transmembrane 2 (TMEM2) as a cell surface protein that possesses potent hyaluronidase activity suggests that it may be the "missing" cell surface hyaluronidase, and that novel models of HA catabolism should include this protein. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Cell surface marker profiling of human tracheal basal cells reveals distinct subpopulations, identifies MST1/MSP as a mitogenic signal, and identifies new biomarkers for lung squamous cell carcinomas.

PubMed

Van de Laar, Emily; Clifford, Monica; Hasenoeder, Stefan; Kim, Bo Ram; Wang, Dennis; Lee, Sharon; Paterson, Josh; Vu, Nancy M; Waddell, Thomas K; Keshavjee, Shaf; Tsao, Ming-Sound; Ailles, Laurie; Moghal, Nadeem

2014-12-31

The large airways of the lungs (trachea and bronchi) are lined with a pseudostratified mucociliary epithelium, which is maintained by stem cells/progenitors within the basal cell compartment. Alterations in basal cell behavior can contribute to large airway diseases including squamous cell carcinomas (SQCCs). Basal cells have traditionally been thought of as a uniform population defined by basolateral position, cuboidal cell shape, and expression of pan-basal cell lineage markers like KRT5 and TP63. While some evidence suggests that basal cells are not all functionally equivalent, few heterogeneously expressed markers have been identified to purify and study subpopulations. In addition, few signaling pathways have been identified that regulate their cell behavior. The goals of this work were to investigate tracheal basal cell diversity and to identify new signaling pathways that regulate basal cell behavior. We used flow cytometry (FACS) to profile cell surface marker expression at a single cell level in primary human tracheal basal cell cultures that maintain stem cell/progenitor activity. FACS results were validated with tissue staining, in silico comparisons with normal basal cell and lung cancer datasets, and an in vitro proliferation assay. We identified 105 surface markers, with 47 markers identifying potential subpopulations. These subpopulations generally fell into more (~ > 13%) or less abundant (~ < 6%) groups. Microarray gene expression profiling supported the heterogeneous expression of these markers in the total population, and immunostaining of large airway tissue suggested that some of these markers are relevant in vivo. 24 markers were enriched in lung SQCCs relative to adenocarcinomas, with four markers having prognostic significance in SQCCs. We also identified 33 signaling receptors, including the MST1R/RON growth factor receptor, whose ligand MST1/MSP was mitogenic for basal cells. This work provides the largest description to date of molecular diversity among human large airway basal cells. Furthermore, these markers can be used to further study basal cell function in repair and disease, and may aid in the classification and study of SQCCs.

Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.

PubMed

Villanueva-Cabello, Tania M; Mollicone, Rosella; Cruz-Muñoz, Mario E; López-Guerrero, Delia V; Martínez-Duncker, Iván

2015-12-01

CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigen-presenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaα2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction

PubMed Central

Mostofi, Sepideh; Bonyadi Rad, Ehsan; Wiltsche, Helmar; Fasching, Ulrike; Szakacs, Gabor; Ramskogler, Claudia; Srinivasaiah, Sriveena; Ueçal, Muammer; Willumeit, Regine; Weinberg, Annelie-Martina; Schaefer, Ute

2016-01-01

This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd) were analysed by scanning electron microscopy (SEM) and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for in vivo orthopedic applications. PMID:27459513

Pyroelectricity as a possible mechanism for cell membrane permeabilization.

PubMed

García-Sánchez, Tomás; Muscat, Adeline; Leray, Isabelle; Mir, Lluis M

2018-02-01

The effects of pyroelectricity on cell membrane permeability had never been explored. Pyroelectricity consists in the generation of an electric field in the surface of some materials when a change in temperature is produced. In the present study, tourmaline microparticles, which are known to display pyroelectrical properties, were subjected to different changes in temperature upon exposure to cells in order to induce an electric field at their surface. Then, the changes in the permeability of the cell membrane to a cytotoxic agent (bleomycin) were assessed by a cloning efficacy test. An increase in the permeability of the cell membrane was only detected when tourmaline was subjected to a change in temperature. This suggests that the apparition of an induced pyroelectrical electric field on the material could actually be involved in the observed enhancement of the cell membrane permeability as a result of cell electropermeabilization. Copyright © 2017 Elsevier B.V. All rights reserved.

Basic science for the clinician 53: mast cells.

PubMed

Sigal, Leonard H

2011-10-01

Mast cells stand at the interface between the innate immune system and the acquired (adaptive) immune response, serving as sentinels detecting invaders and directing a concerted and coordinated response. Mast cells reside immediately under body surfaces and within lymph nodes, near blood vessels and nerves, perfectly situated to for early detection and defense. They secrete a wide array of prostanoids, cytokines, chemokines, and other proteins mediators and modifiers of a variety of immune and inflammatory functions and bear surface markers suggesting broad functions, including as antigen-presenting cells. Although usually not given their due in medical school lectures, there is great likelihood that mast cells will be implicated in the pathogenesis of rheumatoid arthritis, scleroderma, multiple sclerosis, and perhaps cancer. Thus, better insights into mast cell functions and mast cell-derived effector molecules should command our attention as we move forward in better understanding disease immunopathogenesis and directed intelligent therapeutics development.

Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface.

PubMed

Tan, Shumin; Noto, Jennifer M; Romero-Gallo, Judith; Peek, Richard M; Amieva, Manuel R

2011-05-01

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche.

Helicobacter pylori Perturbs Iron Trafficking in the Epithelium to Grow on the Cell Surface

PubMed Central

Tan, Shumin; Noto, Jennifer M.; Romero-Gallo, Judith; Peek, Richard M.; Amieva, Manuel R.

2011-01-01

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche. PMID:21589900

Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization

PubMed Central

Boyd, Chelsea D.; Smith, T. Jarrod; El-Kirat-Chatel, Sofiane; Newell, Peter D.; Dufrêne, Yves F.

2014-01-01

The localization of the LapA protein to the cell surface is a key step required by Pseudomonas fluorescens Pf0-1 to irreversibly attach to a surface and form a biofilm. LapA is a member of a diverse family of predicted bacterial adhesins, and although lacking a high degree of sequence similarity, family members do share common predicted domains. Here, using mutational analysis, we determine the significance of each domain feature of LapA in relation to its export and localization to the cell surface and function in biofilm formation. Our previous work showed that the N terminus of LapA is required for cleavage by the periplasmic cysteine protease LapG and release of the adhesin from the cell surface under conditions unfavorable for biofilm formation. We define an additional critical region of the N terminus of LapA required for LapG proteolysis. Furthermore, our results suggest that the domains within the C terminus of LapA are not absolutely required for biofilm formation, export, or localization to the cell surface, with the exception of the type I secretion signal, which is required for LapA export and cell surface localization. In contrast, deletion of the central repetitive region of LapA, consisting of 37 repeats of 100 amino acids, results in an inability to form a biofilm. We also used single-molecule atomic force microscopy to further characterize the role of these domains in biofilm formation on hydrophobic and hydrophilic surfaces. These studies represent the first detailed analysis of the domains of the LapA family of biofilm adhesin proteins. PMID:24837291

Hydrogenated amorphous silicon coatings may modulate gingival cell response

NASA Astrophysics Data System (ADS)

Mussano, F.; Genova, T.; Laurenti, M.; Munaron, L.; Pirri, C. F.; Rivolo, P.; Carossa, S.; Mandracci, P.

2018-04-01

Silicon-based materials present a high potential for dental implant applications, since silicon has been proven necessary for the correct bone formation in animals and humans. Notably, the addition of silicon is effective to enhance the bioactivity of hydroxyapatite and other biomaterials. The present work aims to expand the knowledge of the role exerted by hydrogen in the biological interaction of silicon-based materials, comparing two hydrogenated amorphous silicon coatings, with different hydrogen content, as means to enhance soft tissue cell adhesion. To accomplish this task, the films were produced by plasma enhanced chemical vapor deposition (PECVD) on titanium substrates and their surface composition and hydrogen content were analyzed by means of X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectrophotometry (FTIR) respectively. The surface energy and roughness were measured through optical contact angle analysis (OCA) and high-resolution mechanical profilometry respectively. Coated surfaces showed a slightly lower roughness, compared to bare titanium samples, regardless of the hydrogen content. The early cell responses of human keratinocytes and fibroblasts were tested on the above mentioned surface modifications, in terms of cell adhesion, viability and morphometrical assessment. Films with lower hydrogen content were endowed with a surface energy comparable to the titanium surfaces. Films with higher hydrogen incorporation displayed a lower surface oxidation and a considerably lower surface energy, compared to the less hydrogenated samples. As regards mean cell area and focal adhesion density, both a-Si coatings influenced fibroblasts, but had no significant effects on keratinocytes. On the contrary, hydrogen-rich films increased manifolds the adhesion and viability of keratinocytes, but not of fibroblasts, suggesting a selective biological effect on these cells.

The Role of Nanoparticle Surface Functionality in the Disruption of Model Cell Membranes

PubMed Central

Moghadam, Babak Y.; Hou, Wen-Che; Corredor, Charlie; Westerhoff, Paul; Posner, Jonathan D.

2012-01-01

Lipid bilayers are biomembranes common to cellular life and constitute a continuous barrier between cells and their environment. Understanding the interaction of engineered nanomaterials (ENMs) with lipid bilayers is an important step toward predicting subsequent biological effects. In this study, we assess the effect of varying the surface functionality and concentration of 10 nm-diameter gold (Au) and titanium dioxide (TiO2) ENMs on the disruption of negatively charged lipid bilayer vesicles (liposomes) using a dye leakage assay. Our findings show that Au ENMs having both positive and negative surface charge induce leakage that reaches a steady state after several hours. Positively charged particles with identical surface functionality and different core composition show similar leakage effects and result in faster and greater leakage than negatively charged particles, which suggests that surface functionality, not particle core composition, is a critical factor in determining the interaction between ENMs and lipid bilayers. The results suggest that particles permanently adsorb to bilayers and that only one positively charged particle is required to disrupt a liposome and trigger leakage of its entire contents in contrast to mellitin molecules, the most widely studied membrane lytic peptide, which requires hundred of molecules to generate leakage. PMID:22921268

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

PubMed Central

Hubert, Rene S.; Vivanco, Igor; Chen, Emily; Rastegar, Shiva; Leong, Kahan; Mitchell, Steve C.; Madraswala, Rashida; Zhou, Yanhong; Kuo, James; Raitano, Arthur B.; Jakobovits, Aya; Saffran, Douglas C.; Afar, Daniel E. H.

1999-01-01

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging. PMID:10588738

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.

Crucial role of interleukin-4 in the survival of colon cancer stem cells.

PubMed

Francipane, Maria Giovanna; Alea, Mileidys Perez; Lombardo, Ylenia; Todaro, Matilde; Medema, J P; Stassi, Giorgio

2008-06-01

Colon tumors may be maintained by a rare fraction of cancer stem-like cells (CSC) that express the cell surface marker CD133. Self-renewing CSCs exhibit relatively greater resistance to clinical cytotoxic therapies and recent work suggests that this resistance may be mediated in part by an autocrine response to the immune cytokine interleukin 4 (IL-4). Blocking IL-4 signaling can sensitize CSCs to apoptotic stimuli and increase the in vivo efficacy of cytotoxic therapy. These findings suggest that inhibitors of IL-4 signaling may offer a new therapeutic tool in colon carcinoma.

High-efficiency indium tin oxide/indium phosphide solar cells

NASA Technical Reports Server (NTRS)

Li, X.; Wanlass, M. W.; Gessert, T. A.; Emery, K. A.; Coutts, T. J.

1989-01-01

Improvements in the performance of indium tin oxide (ITO)/indium phosphide solar cells have been realized by the dc magnetron sputter deposition of n-ITO onto an epitaxial p/p(+) structure grown on commercial p(+) bulk substrates. The highest efficiency cells were achieved when the surface of the epilayer was exposed to an Ar/H2 plasma before depositing the bulk of the ITO in a more typical Ar/O2 plasma. With H2 processing, global efficiencies of 18.9 percent were achieved. It is suggested that the excellent performance of these solar cells results from the optimization of the doping, thickness, transport, and surface properties of the p-type base, as well as from better control over the ITO deposition procedure.

Recombination phenomena in high efficiency silicon solar cells

NASA Technical Reports Server (NTRS)

Sah, C. T.

1985-01-01

The dominant recombination phenomena which limit the highest efficiency attainable in silicon solar cells under terrestrial sunlight are reviewed. The ultimate achievable efficiency is limited by the two intrinsic recombination mechanisms, the interband Auger recombination and interband Radiative recombination, both of which occur in the entire cell body but principally in the base layer. It is suggested that an optimum (26%) cell design is one with lowly doped 50 to 100 micron thick base, a perfect BSF, and zero extrinsic recombination such as the thermal mechanism at recombination centers the Shockley-Read-Hall process (SRH) in the bulk, on the surface and at the interfaces. The importance of recombination at the interfaces of a high-efficiency cell is demonstrated by the ohmic contact on the back surface whose interface recombination velocity is infinite. The importance of surface and interface recombination is demonstrated by representing the auger and radiative recombination losses by effective recombination velocities. It is demonstrated that the three highest efficiency cells may all be limited by the SRH recombination losses at recombination centers in the base layer.

Fluorescent Nanocrystals Reveal Regulated Portals of Entry into and Between the Cells of Hydra

PubMed Central

Tortiglione, Claudia; Quarta, Alessandra; Malvindi, Maria Ada; Tino, Angela; Pellegrino, Teresa

2009-01-01

Initially viewed as innovative carriers for biomedical applications, with unique photophysical properties and great versatility to be decorated at their surface with suitable molecules, nanoparticles can also play active roles in mediating biological effects, suggesting the need to deeply investigate the mechanisms underlying cell-nanoparticle interaction and to identify the molecular players. Here we show that the cell uptake of fluorescent CdSe/CdS quantum rods (QRs) by Hydra vulgaris, a simple model organism at the base of metazoan evolution, can be tuned by modifying nanoparticle surface charge. At acidic pH, amino-PEG coated QRs, showing positive surface charge, are actively internalized by tentacle and body ectodermal cells, while negatively charged nanoparticles are not uptaken. In order to identify the molecular factors underlying QR uptake at acidic pH, we provide functional evidence of annexins involvement and explain the QR uptake as the combined result of QR positive charge and annexin membrane insertion. Moreover, tracking QR labelled cells during development and regeneration allowed us to uncover novel intercellular trafficking and cell dynamics underlying the remarkable plasticity of this ancient organism. PMID:19888325

Chemical characterization of solid polymer electrolyte membrane surfaces in LiFePO4 half-cells

NASA Astrophysics Data System (ADS)

Kyu, Thein; He, Ruixuan; Peng, Fang; Dunn, William E.; Kyu's Group Team, Dr.

High temperature (60 °C) capacity retention of succinonitrile plasticized solid polymer electrolyte membrane (PEM) in a LiFePO4 half-cell was investigated with or without lithium bis(oxalato)borate (LiBOB) modification. Various symmetric cells and half-cells were studied under different thermal and electrochemical conditions. At room temperature cycling, the unmodified PEM in the half-cell appeared stable up to 50 cycles tested. Upon cycling at 60 °C, the capacity decays rapidly and concurrently the cell resistance increased. The chemical compositions of the solid PEM surfaces on both cathode and anode sides were analyzed. New IR bands (including those belonged to amide) were discerned on the unmodified PEM surface of the Li electrode side at 60 °C suggestive of side reaction, but no new bands develop during room temperature cycling. To our astonishment, the side reaction was effectively suppressed upon LiBOB addition (0.4 wt%) into the PEM, contributing to increased high temperature capacity retention at 60°C. Plausible mechanisms of capacity fading and improved cycling performance due to LiBOB modification are discussed.

CD70 is downregulated by interaction with CD27

PubMed Central

Kuka, Mirela; Munitic, Ivana; Torchia, Maria Letizia Giardino; Ashwell, Jonathan D.

2013-01-01

Engagement of the receptor CD27 by CD70 affects the magnitude and quality of T cell responses in a variety of infection models, and exaggerated signaling via this pathway results in enhanced immune responses and autoimmunity. One means by which signaling is regulated is tight control of cell surface CD70, which is expressed on dendritic, T, and B cells only upon activation. Here we show that there is a second level of regulation. First, although undetectable on the cell surface by flow cytometry, immature dendritic cells (DC) have a small pool of CD70 that continuously recycles from the plasma membrane. In addition, surface levels of CD70 on DC and T cells were higher in mice deficient in CD27, or on DC for which the interaction between CD70 and CD27 was precluded by blocking antibodies. Binding of CD70 by its receptor resulted in downregulation of CD70 transcription and protein levels, suggesting that CD70-mediated “reverse signals” regulate its own levels. Therefore, the ability of CD70 to trigger costimulation is self-regulated when it binds its complementary receptor. PMID:23913967

Differential effect of rebamipide on transmembrane mucin biosynthesis in stratified ocular surface epithelial cells.

PubMed

Uchino, Yuichi; Woodward, Ashley M; Argüeso, Pablo

2016-12-01

Mucins are a group of highly glycosylated glycoproteins responsible for the protection of wet-surfaced epithelia. Recent data indicate that transmembrane mucins differ in their contribution to the protective function of the ocular surface, with MUC16 being the most effective barrier on the apical surface glycocalyx. Here, we investigated the role of the mucoprotective drug rebamipide in the regulation of transmembrane mucin biosynthesis using stratified cultures of human corneal and conjunctival epithelial cells. We find that the addition of rebamipide to corneal, but not conjunctival, epithelial cells increased MUC16 protein biosynthesis. Rebamipide did not affect the levels of MUC1, 4 and 20 compared to control. In these experiments, rebamipide had no effect on the expression levels of Notch intracellular domains, suggesting that the rebamipide-induced increase in MUC16 biosynthesis in differentiated corneal cultures is not regulated by Notch signaling. Overall these findings indicate that rebamipide induces the differential upregulation of MUC16 in stratified cultures of human corneal epithelial cells, which may have implications to the proper restoration of barrier function in ocular surface disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Potential mechanisms for the effects of tea extracts on the attachment, biofilm formation and cell size of Streptococcus mutans.

PubMed

Wang, Yi; Lee, Sui M; Dykes, Gary A

2013-01-01

Tea can inhibit the attachment of Streptococcus mutans to surfaces and subsequent biofilm formation. Five commercial tea extracts were screened for their ability to inhibit attachment and biofilm formation by two strains of S. mutans on glass and hydroxyapatite surfaces. The mechanisms of these effects were investigated using scanning electron microscopy (SEM) and phytochemical screening. The results indicated that extracts of oolong tea most effectively inhibited attachment and extracts of pu-erh tea most effectively inhibited biofilm formation. SEM images showed that the S. mutans cells treated with extracts of oolong tea, or grown in medium containing extracts of pu-erh tea, were coated with tea components and were larger with more rounded shapes. The coatings on the cells consisted of flavonoids, tannins and indolic compounds. The ratio of tannins to simple phenolics in each of the coating samples was ∼3:1. This study suggests potential mechanisms by which tea components may inhibit the attachment and subsequent biofilm formation of S. mutans on tooth surfaces, such as modification of cell surface properties and blocking of the activity of proteins and the structures used by the bacteria to interact with surfaces.

Improving the degradation behavior and in vitro biological property of nano-hydroxyapatite surface- grafted with the assist of citric acid.

PubMed

Jiang, Liuyun; Jiang, Lixin; Xiong, Chengdong; Su, Shengpei

2016-10-01

To obtain ideal nano-hydroxyapatite(n-HA) filler for poly(lactide-co-glycolide) (PLGA), a new surface-grafting with the assist of citric acid for nano-hydroxyapatite (n-HA) was designed, and the effect of n-HA surface-grafted with or without citric acid on in vitro degradation behavior and cells viability was studied by the experiments of soaking in simulated body fluid (SBF) and incubating with human osteoblast-like cells (MG-63). The change of pH value, tensile strength reduction, the surface deposits, cells attachment and proliferation of samples during the soaking and incubation were investigated by means of pH meter, electromechanical universal tester, scanning electron microscope (SEM) coupled with energy-dispersive spectro-scopy (EDS), fluorescence microscope and MTT method. The results showed that the introduction of citric acid not only delayed the strength reduction during the degradation by inhibiting the detachment of n-HA from PLGA, but also endowed it better cell attachment and proliferation, suggesting that the n-HA surface-grafted with the assist of citric acid was an important bioactive ceramic fillers for PLGA used as bone materials. Copyright © 2016 Elsevier B.V. All rights reserved.

Glycocalyx Engineering Reveals a Siglec-Based Mechanism for NK Cell Immunoevasion

PubMed Central

Hudak, Jason E.; Canham, Stephen M.; Bertozzi, Carolyn R.

2013-01-01

The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human NK cell activation through the recruitment of Siglec-7. Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy. PMID:24292068

["Light" epithelial cells of swine and bovine oviducts].

PubMed

Suuroia, T; Aunapuu, M; Arend, A; Sépp, E

2002-01-01

The ultrastructure of oviduct epithelium of clinically healthy cows and 15 sows was investigated using scanning and transmission electron microscopy. In all parts of the oviduct, ciliated and non-ciliated epithelial cells are present, but their number varies in both the investigated animals in different regions of the oviduct, depending on the phase of the estrous cycle. In addition to ciliated cells with numerous cilia on their luminal surface, so-called pale ciliary cells were found in all parts of the oviduct of cows and sows. The cytoplasm of these cells is electron-lucent, their luminal surface carries few cilia and short microvilli. The apical cytoplasm contains species specific secretory granules, which means that these cells have features characteristic of both secretory and ciliated cells. It is suggested that the pale ciliated and non-ciliated secretory cells are functional stages of the same tubar epithelium cell, and that the transformation between these two cell types is regulated by functional requirements of the organ in different phases of the estrous cycle.

T- and B-cell subpopulations in infectious mononucleosis

PubMed Central

Papamichail, M.; Sheldon, P. J.; Holborow, E. J.

1974-01-01

Mononuclear cells separated from the blood in fourteen cases of infectious mononucleosis at various intervals from the onset were tested for the presence of surface immunoglobulin and for ability to form spontaneous rosettes with washed sheep red blood cells. The mononucleosis during the acute phase of the illness consisted largely of a T lymphocytosis. The absolute count of T lymphocytes returned to the normal range approximately 2 months after the onset of the illness. B cells (bearing surface immunoglobulin) were only slightly increased in the acute phase. In four cases appreciable numbers of fluorescent rosetting cells were also present, and investigation suggested that these were T cells coated with anti-T-cell autoantibody. During the first 2 weeks of the illness responsiveness to phytohaemagglutinin was severely depressed, but thereafter returned towards normal. It is thought likely that in infectious mononucleosis the vast majority of atypical mononuclear cells are T cells proliferating in response to E-B virus-infected B cells, and cytotoxic towards them. ImagesFig. 3 PMID:4549622

Preferential invasion of mitotic cells by Salmonella reveals that cell surface cholesterol is maximal during metaphase.

PubMed

Santos, António J M; Meinecke, Michael; Fessler, Michael B; Holden, David W; Boucrot, Emmanuel

2013-07-15

Cell surface-exposed cholesterol is crucial for cell attachment and invasion of many viruses and bacteria, including the bacterium Salmonella, which causes typhoid fever and gastroenteritis. Using flow cytometry and 3D confocal fluorescence microscopy, we found that mitotic cells, although representing only 1-4% of an exponentially growing population, were much more efficiently targeted for invasion by Salmonella. This targeting was not dependent on the spherical shape of mitotic cells, but was instead SipB and cholesterol dependent. Thus, we measured the levels of plasma membrane and cell surface cholesterol throughout the cell cycle using, respectively, brief staining with filipin and a fluorescent ester of polyethylene glycol-cholesterol that cannot flip through the plasma membrane, and found that both were maximal during mitosis. This increase was due not only to the rise in global cell cholesterol levels along the cell cycle but also to a transient loss in cholesterol asymmetry at the plasma membrane during mitosis. We measured that cholesterol, but not phosphatidylserine, changed from a ∼2080 outerinner leaflet repartition during interphase to ∼5050 during metaphase, suggesting this was specific to cholesterol and not due to a broad change of lipid asymmetry during metaphase. This explains the increase in outer surface levels that make dividing cells more susceptible to Salmonella invasion and perhaps to other viruses and bacteria entering cells in a cholesterol-dependent manner. The change in cholesterol partitioning also favoured the recruitment of activated ERM (Ezrin, Radixin, Moesin) proteins at the plasma membrane and thus supported mitotic cell rounding.

Notch Signaling Is Involved in Neurogenic Commitment of Human Periodontal Ligament-Derived Mesenchymal Stem Cells

PubMed Central

Osathanon, Thanaphum; Manokawinchoke, Jeeranan; Nowwarote, Nunthawan; Aguilar, Panuroot; Palaga, Tanapat

2013-01-01

Notch signaling plays critical roles in stem cells by regulating cell fate determination and differentiation. The aim of this study was to evaluate the participation of Notch signaling in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells (hPDLSCs) and to examine the ability to control differentiation of these cells using modified surfaces containing affinity immobilized Notch ligands. Neurogenic induction of hPDLSCs was performed via neurosphere formation. Cells were aggregated and form spheres as early 1 day in culture. In addition, the induced cells exhibited increased mRNA and protein expression of neuronal markers that is, β3-tubulin and neurofilament. During neuronal differentiation, a significant increase of Hes1 and Hey1 mRNA expression was noted. Using pharmacological inhibition (γ-secretase inhibitor) or genetic manipulation (overexpression of dominant negative mastermind-like transcription co-activators), neurosphere formation was attenuated and a marked decrease in neurogenic mRNA expression was observed. To confirm the role of Notch signaling in neuronal differentiation of hPDLSCs, the Notch ligand, Jagged-1, is bound to the surface using an affinity immobilization technique. The hPDLSC cultured on a Jagged-1-modified surface had increased expression of Notch signaling target genes, Hes-1 and Hey-1, confirming the activity and potency of surface-bound Jagged-1. Further, hPDLSC on surface-bound Jagged-1 under serum-free conditions showed multiple long and thin neurite-like extensions, and an increase in the expression of neurogenic mRNA markers was observed. Pretreatment of the cells with γ-secretase inhibitor, DAPT, before seeding on the Jagged-1-modified surface blocked development of the neurite-like morphology. Together, the results in this study suggest the involvement of Notch signaling in neurogenic commitment of hPDLSCs. PMID:23379739

Comparative evaluation of air cell and eggshell temperature measurement methodologies used in broiler hatching eggs during late incubation.

PubMed

Peebles, E D; Zhai, W; Gerard, P D

2012-07-01

The current study was conducted to compare and contrast the uses of 2 devices (temperature transponder or infrared thermometer) and their locations (inner air cell membrane or outer eggshell surface) in Ross × Ross 708 broiler hatching eggs. The air cells of 14 embryonated and 10 nonembryonated eggs were implanted with temperature transponders on d 13.5 of incubation. Likewise, for these same eggs, eggshell surface temperature was detected with the use of transponders and an infrared thermometer. Temperatures were recorded every 12 h between 14.5 and 18 d of incubation, and graphs and corresponding regression values were used to track the temperatures over these time periods. The temperature readings using all methods in embryonated and nonembryonated eggs were positively correlated. In nonembryonated eggs, temperatures in the air cell and on the eggshell surface using transponders were higher than those on the eggshell surface using an infrared thermometer. Mean air cell temperature readings of embryonated eggs using transponders were higher than those of the eggshell, as determined with the use of transponders or an infrared thermometer. Furthermore, the differences in air cell temperature using transponders and eggshell temperature using an infrared thermometer in embryonated eggs increased with embryonic age. These readings confirmed increased embryo heat production during the incubational period examined. It was further concluded that when compared with actual embryo body temperatures determined in previous studies, the use of transponders in the air cells of broiler hatching eggs detected a higher and closer temperature than eggshell surface temperature. It is suggested that the air cell transponders in embryonated eggs circumvented the confounding effects of the thermal barrier properties of the eggshell and the flow of air across its surface.

ERAP1 reduces accumulation of aberrant and disulfide-linked forms of HLA-B27 on the cell surface.

PubMed

Tran, Tri M; Hong, Sohee; Edwan, Jehad H; Colbert, Robert A

2016-06-01

Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) variants contribute to the risk of ankylosing spondylitis in HLA-B27 positive individuals, implying a disease-related interaction between these gene products. The aim of this study was to determine whether reduced ERAP1 expression would alter the cell surface expression of HLA-B27 and the formation of aberrant disulfide-linked forms that have been implicated in the pathogenesis of spondyloarthritis. ERAP1 expression was knocked down in monocytic U937 cells expressing HLA-B27 and endogenous HLA class I. The effect of ERAP1 knockdown on the accumulation HLA-B alleles (B18, B51, and B27) was assessed using immunoprecipitation, isoelectric focusing, and immunoblotting, as well as flow cytometry with antibodies specific for different forms of HLA-B27. Cell surface expression of aberrant disulfide-linked HLA-B27 dimers was assessed by immunoprecipitation and electrophoresis on non-reducing polyacrylamide gels. ERAP1 knockdown increased the accumulation of HLA-B27 on the cell surface including disulfide-linked dimers, but had no effect on levels of HLA-B18 or -B51. Antibodies with unique specificity for HLA-B27 confirmed increased cell surface expression of complexes shown previously to contain long peptides. IFN-γ treatment resulted in striking increases in the expression of disulfide-linked HLA-B27 heavy chains, even in cells with normal ERAP1 expression. Our results suggest that normal levels of ERAP1 reduce the accumulation of aberrant and disulfide-linked forms of HLA-B27 in monocytes, and thus help to maintain the integrity of cell surface HLA-B27 complexes. Published by Elsevier Ltd.

ERAP1 Reduces Accumulation of Aberrant and Disulfide-Linked Forms of HLA-B27 on the Cell Surface

PubMed Central

Tran, Tri; Hong, Sohee; Edwan, Jehad; Colbert, Robert A.

2016-01-01

Objective Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) variants contribute to the risk of ankylosing spondylitis in HLA-B27 positive individuals, implying a disease-related interaction between these gene products. The aim of this study was to determine whether reduced ERAP1 expression would alter the cell surface expression of HLA-B27 and the formation of aberrant disulfide-linked forms that have been implicated in the pathogenesis of spondyloarthritis. Methods ERAP1 expression was knocked down in monocytic U937 cells expressing HLA-B27 and endogenous HLA class I. The effect of ERAP1 knockdown on the accumulation HLA-B alleles (B18, B51, and B27) was assessed using immunoprecipitation, isoelectric focusing, and immunoblotting, as well as flow cytometry with antibodies specific for different forms of HLA-B27. Cell surface expression of aberrant disulfide-linked HLA-B27 dimers was assessed by immunoprecipitation and electrophoresis on non-reducing polyacrylamide gels. Results ERAP1 knockdown increased the accumulation of HLA-B27 on the cell surface including disulfide-linked dimers, but had no effect on levels of HLA-B18 or -B51. Antibodies with unique specificity for HLA-B27 confirmed increased cell surface expression of complexes shown previously to contain long peptides. IFN-γ treatment resulted in striking increases in the expression of disulfide-linked HLA-B27 heavy chains, even in cells with normal ERAP1 expression. Conclusions Our results suggest that normal levels of ERAP1 reduce the accumulation of aberrant and disulfide-linked forms of HLA-B27 in monocytes, and thus help to maintain the integrity of cell surface HLA-B27 complexes. PMID:27107845

Gracilaria lemaneiformis polysaccharide as integrin-targeting surface decorator of selenium nanoparticles to achieve enhanced anticancer efficacy.

PubMed

Jiang, Wenting; Fu, Yuanting; Yang, Fang; Yang, Yufeng; Liu, Ting; Zheng, Wenjie; Zeng, Lilan; Chen, Tianfeng

2014-08-27

The poor permeability of glioma parenchyma represents a major limit for antiglioblastoma drug delivery. Gracilaria lemaneiformis polysaccharide (GLP), which has a high binding affinity to αvβ3 integrin overexpressed in glioma cells, was employed in the present study to functionalize selenium nanoparticles (SeNPs) to achieve antiglioblastoma efficacy. GLP-SeNPs showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. In U87 glioma cell membrane, which has a high integrin expression level, GLP-SeNPs exhibited significantly higher cellular uptake than unmodified SeNPs. As expected, U87 cells exhibited a greater uptake of GLP-SeNPs than C6 cells with low integrin expression level. Furthermore, the internalization of GLP-SeNPs was inhibited by cyclo-(Arg-Gly-Asp-Phe-Lys) peptides, suggesting that cellular uptake into U87 cells and C6 cells occurred via αvβ3 integrin-mediated endocytosis. For U87 cells, the cytotoxicity of SeNPs decorated by GLP was enhanced significantly because of the induction of various apoptosis signaling pathways. Internalized GLP-SeNPs triggered intracellular reactive oxygen species downregulation. Therefore, p53, MAPKs, and AKT pathways were activated to advance cell apoptosis. These findings suggest that surface decoration of nanomaterials with GLP could be an efficient strategy for design and preparation of glioblastoma targeting nanodrugs.

Differential Utilization and Localization of ErbB Receptor Tyrosine Kinases in Skin Compared to Normal and Malignant Keratinocytes1

PubMed Central

Stoll, Stefan W; Kansra, Sanjay; Peshick, Scott; Fry, David W; Leopold, Wilbur R; Wiesen, Jane F; Sibilia, Maria; Zhang, Tong; Werb, Zena; Derynck, Rik; Wagner, Erwin F; Elder, James T

2001-01-01

Abstract Induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1-dependent growth/survival signals, while evading ErbB2-dependent differentiation signals. PMID:11571634

Control of bacterial adhesion and growth on honeycomb-like patterned surfaces.

PubMed

Yang, Meng; Ding, Yonghui; Ge, Xiang; Leng, Yang

2015-11-01

It is a great challenge to construct a persistent bacteria-resistant surface even though it has been demonstrated that several surface features might be used to control bacterial behavior, including surface topography. In this study, we develop micro-scale honeycomb-like patterns of different sizes (0.5-10 μm) as well as a flat area as the control on a single platform to evaluate the bacterial adhesion and growth. Bacteria strains, Escherichia coli and Staphylococcus aureus with two distinct shapes (rod and sphere) are cultured on the platforms, with the patterned surface-up and surface-down in the culture medium. The results demonstrate that the 1 μm patterns remarkably reduce bacterial adhesion and growth while suppressing bacterial colonization when compared to the flat surface. The selective adhesion of the bacterial cells on the patterns reveals that the bacterial adhesion is cooperatively mediated by maximizing the cell-substrate contact area and minimizing the cell deformation, from a thermodynamic point of view. Moreover, study of bacterial behaviors on the surface-up vs. surface-down samples shows that gravity does not apparently affect the spatial distribution of the adherent cells although it indeed facilitates bacterial adhesion. Furthermore, the experimental results suggest that two major factors, i.e. the availability of energetically favorable adhesion sites and the physical confinements, contribute to the anti-bacterial nature of the honeycomb-like patterns. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of Receptor Activity Modifying Protein 1 in Function of the Calcium Sensing Receptor in the Human TT Thyroid Carcinoma Cell Line

PubMed Central

Desai, Aditya J.; Roberts, David J.

2014-01-01

The Calcium Sensing Receptor (CaSR) plays a role in calcium homeostasis by sensing minute changes in serum Ca2+ and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively) and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has occurred. PMID:24454825

Role of receptor activity modifying protein 1 in function of the calcium sensing receptor in the human TT thyroid carcinoma cell line.

PubMed

Desai, Aditya J; Roberts, David J; Richards, Gareth O; Skerry, Timothy M

2014-01-01

The Calcium Sensing Receptor (CaSR) plays a role in calcium homeostasis by sensing minute changes in serum Ca(2+) and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively) and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has occurred.

Did Mineral Surface Chemistry and Toxicity Contribute to Evolution of Microbial Extracellular Polymeric Substances?

PubMed Central

Campbell, Jay M.; Zhang, Nianli; Hickey, William J.

2012-01-01

Abstract Modern ecological niches are teeming with an astonishing diversity of microbial life in biofilms closely associated with mineral surfaces, which highlights the remarkable success of microorganisms in conquering the challenges and capitalizing on the benefits presented by the mineral–water interface. Biofilm formation capability likely evolved on early Earth because biofilms provide crucial cell survival functions. The potential toxicity of mineral surfaces toward cells and the complexities of the mineral–water–cell interface in determining the toxicity mechanisms, however, have not been fully appreciated. Here, we report a previously unrecognized role for extracellular polymeric substances (EPS), which form biofilms in shielding cells against the toxicity of mineral surfaces. Using colony plating and LIVE/DEAD staining methods in oxide suspensions versus oxide-free controls, we found greater viability of wild-type, EPS-producing strains of Pseudomonas aeruginosa PAO1 compared to their isogenic knockout mutant with defective biofilm-producing capacity. Oxide toxicity was specific to its surface charge and particle size. High resolution transmission electron microscopy (HRTEM) images and assays for highly reactive oxygen species (hROS) on mineral surfaces suggested that EPS shield via both physical and chemical mechanisms. Intriguingly, qualitative as well as quantitative measures of EPS production showed that toxic minerals induced EPS production in bacteria. By determining the specific toxicity mechanisms, we provide insight into the potential impact of mineral surfaces in promoting increased complexity of cell surfaces, including EPS and biofilm formation, on early Earth. Key Words: Mineral toxicity—Bacteria—EPS evolution—Biofilms—Cytotoxicity—Silica—Anatase—Alumina. Astrobiology 12, 785–798. PMID:22934560

Glut-1 translocation in FRTL-5 thyroid cells: role of phosphatidylinositol 3-kinase and N-glycosylation.

PubMed

Samih, N; Hovsepian, S; Aouani, A; Lombardo, D; Fayet, G

2000-11-01

It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached the cell surface. Likewise, a decrease in glucose consumption (-50%) after treatment of cells with tunicamycin was accompanied by a decrease (-70% vs. control) in the membrane expression of a 50-kDa form of Glut-1 and an increase in its unglycosylated 41-kDa form. These results suggest that carbohydrate moiety is essential for the biological activity of glucose transport but is not required for the translocation of Glut-1 from the intracellular membrane pool to the plasma membrane.

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.

Matrigel immobilization on the shish-kebab structured poly(ε-caprolactone) nanofibers for skin tissue engineering

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

Jing, Xin, E-mail: jingxinscut@gmail.com; Mi, Hao-Yang; Wisconsin Institutes for Discovery, University of Wisconsin-Madison, 53715

Surface properties of tissue engineering scaffolds such as topography, hydrophilicity, and functional groups play a vital role in cell adhesion, migration, proliferation, and apoptosis. First, poly(ε-caprolactone) (PCL) shish-kebab scaffolds (PCL-SK), which feature a three-dimensional structure comprised of electrospun PCL nanofibers covered by periodic, self-induced PCL crystal lamellae on the surface, was created to mimic the nanotopography of native collagen fibrils in the extracellular matrix (ECM). Second, matrigel was covalently immobilized on the surface of alkaline hydrolyzed PCL-SK scaffolds to enhance their hydrophilicity. This combined approach not only mimics the nanotopography of native collagen fibrils, but also simulates the surface featuresmore » of collagen fibrils for cell growth. To investigate the viability of such scaffolds, HEF1 fibroblast cell assays were conducted and the results revealed that the nanotopography of the PCL-SK scaffolds facilitated cell adhesion and proliferation. The matrigel functionalization on PCL-SK scaffolds further enhanced cellular response, which suggested elevated biocompatibility and greater potential for skin tissue engineering applications.« less

Regenerative Fuel Cell Power Systems for Lunar and Martian Surface Exploration

NASA Technical Reports Server (NTRS)

Guzik, Monica C.; Jakupca, Ian J.; Gilligan, Ryan P.; Bennett, William R.; Smith, Phillip J.; Fincannon, James

2017-01-01

This paper presents the preliminary results of a recent National Aeronautics and Space Administration (NASA) study funded under the Advanced Exploration Systems (AES) Modular Power Systems (AMPS) project. This study evaluated multiple surface locations on both the Moon and Mars, with the goal of establishing a common approach towards technology development and system design for surface power systems that use Regenerative Fuel Cell (RFC) energy storage methods. One RFC design may not be applicable to all surface locations; however, AMPS seeks to find a unified architecture, or series of architectures, that leverages a single development approach to answer the technology need for RFC systems. Early system trades were performed to select the most effective fuel cell and electrolyzer architectures based on current state-of-the-art technology, whereas later trades will establish a detailed system design to enable a near-term ground (non-flight) demonstration. This paper focuses on the initial trade studies, presents the selected fuel cell and electrolyzer architectures for follow-on system design studies, and suggests areas for further technology investment.

Trans-infection but not infection from within endosomal compartments after cell-to-cell HIV-1 transfer to CD4+ T cells.

PubMed

Permanyer, Marc; Ballana, Ester; Badia, Roger; Pauls, Eduardo; Clotet, Bonaventura; Esté, José A

2012-09-14

Cellular contacts between HIV-1-infected donor cells and uninfected primary CD4(+) T lymphocytes lead to virus transfer into endosomes. Recent evidence suggests that HIV particles may fuse with endosomal membranes to initiate a productive infection. To explore the role of endocytosis in the entry and replication of HIV, we evaluated the infectivity of transferred HIV particles in a cell-to-cell culture model of virus transmission. Endocytosed virus led to productive infection of cells, except when cells were cultured in the presence of the anti-gp120 mAb IgGb12, an agent that blocks virus attachment to CD4, suggesting that endocytosed virus was recycled to the outer cell surface. Confocal microscopy confirmed the colocalization of internalized virus antigen and the endosomal marker dynamin. Additionally, virus transfer, fusion, or productive infection was not blocked by dynasore, dynamin-dependent endosome-scission inhibitor, at subtoxic concentrations, suggesting that the early capture of virus into intracellular compartments did not depend on endosomal maturation. Our results suggest that endocytosis is not a mechanism of infection of primary CD4 T cells, but may serve as a reservoir capable of inducing trans-infection of cells after the release of HIV particles to the extracellular environment.

Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications

PubMed Central

Stevenson, G.; Rehman, S.; Draper, E.; Hernández‐Nava, E.; Hunt, J.

2016-01-01

ABSTRACT In this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast‐like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in‐growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre‐clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586–1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:26702609

Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

PubMed

Seiler, C; Nicolson, T

1999-11-15

Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. Copyright 1999 John Wiley & Sons, Inc.

Cross-reactivity between the immunodominant determinant of the antigen I component of Streptococcus sobrinus SpaA protein and surface antigens from other members of the Streptococcus mutans group.

PubMed

Goldschmidt, R M; Curtiss, R

1990-07-01

Most members of the Streptococcus mutans group of microorganisms specify a major cell surface-associated protein, SpaA, that is defined by its antigenic properties. The region of the spaA gene from Streptococcus sobrinus 6715 encoding the immunodominant determinant of the major antigenic component (antigen I) of the SpaA protein has recently been characterized. This study examined whether recognition of the immunodominant determinant is independent of the immunized animal host and whether antibodies elicited by the immunodominant determinant cross-react with cell surface proteins from S. mutans of various serotypes. Mouse and rabbit antisera to the undenatured SpaA protein reacted similarly both with the immunodominant determinant and with other antigenic structures of the protein in Western immunoblots with SpaA polypeptides that were specified by spaA gene fragments expressed in recombinant Escherichia coli. This suggests that the antibody responses of inbred and outbred animals were similar. Furthermore, antibodies raised against both the S. sobrinus SpaA immunodominant determinant expressed by recombinant E. coli and the purified protein from S. sobrinus displayed similar strain specificities and protein band profiles towards cells surface proteins from S. mutans of various serotypes in immunodot and Western blot analyses, respectively. This suggests that for S. sobrinus serotype g, the immune response against the SpaA protein is governed by the immunodominant determinant of antigen I. In addition, it indicates that the SpaA protein domain containing the immunodominant determinant overlaps the domain conferring cross-reactivity to cell surface proteins of S. mutans of various serotypes.

Microwave exposure as a fast and cost-effective alternative of oxygen plasma treatment of indium-tin oxide electrode for application in organic solar cells

NASA Astrophysics Data System (ADS)

Soultati, Anastasia; Kostis, Ioannis; Papadimitropoulos, Giorgos; Zeniou, Angelos; Gogolides, Evangelos; Alexandropoulos, Dimitris; Vainos, Nikos; Davazoglou, Dimitris; Speliotis, Thanassis; Stathopoulos, Nikolaos A.; Argitis, Panagiotis; Vasilopoulou, Maria

2017-12-01

Pre-treatment methods are commonly employed to clean as well as to modify electrode surfaces. Many previous reports suggest that modifying the surface properties of indium tin oxide (ITO) by oxygen plasma treatment is a crucial step for the fabrication of high performance organic solar cells. In this work, we propose a fast and cost-effective microwave exposure step for the modification of the surface properties of ITO anode electrodes used in organic solar cells. It is demonstrated that a short microwave exposure improves the hydrophilicity and reduces the roughness of the ITO surface, as revealed by contact angle and atomic force microscopy (AFM) measurements, respectively, leading to a better quality of the PEDOT:PSS film coated on top of it. Similar results were obtained with the commonly used oxygen plasma treatment of ITO suggesting that microwave exposure is an effective process for modifying the surface properties of ITO with the benefits of low-cost, easy and fast processing. In addition, the influence of the microwave exposure of ITO anode electrode on the performance of an organic solar cell based on the poly(3-hexylthiophene):[6,6]-phenyl C70 butyric acid methyl ester (P3HT:PC70BM) blend is investigated. The 71% efficiency enhancement obtained in the microwave annealed-ITO based device as compared to the device with the as-received ITO was mainly attributed to the improvement in the short circuit current (J sc) and decreased leakage current caused by the reduced series and the increased shunt resistances and also by the higher charge generation efficiency, and the reduced recombination losses.

Correlation of lung surface area to apoptosis and proliferation in human emphysema.

PubMed

Imai, K; Mercer, B A; Schulman, L L; Sonett, J R; D'Armiento, J M

2005-02-01

Pulmonary emphysema is associated with alterations in matrix proteins and protease activity. These alterations may be linked to programmed cell death by apoptosis, potentially influencing lung architecture and lung function. To evaluate apoptosis in emphysema, lung tissue was analysed from 10 emphysema patients and six individuals without emphysema (normal). Morphological analysis revealed alveolar cells in emphysematous lungs with convoluted nuclei characteristic of apoptosis. DNA fragmentation was detected using terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) and gel electrophoresis. TUNEL revealed higher apoptosis in emphysematous than normal lungs. Markers of apoptosis, including active caspase-3, proteolytic fragment of poly (ADP-ribose) polymerase, Bax and Bad, were detected in emphysematous lungs. Linear regression showed that apoptosis was inversely correlated with surface area. Emphysematous lungs demonstrated lower surface areas and increased cell proliferation. There was no correlation between apoptosis and proliferation, suggesting that, although both events increase during emphysema, they are not in equilibrium, potentially contributing to reduced lung surface area. In summary, cell-based mechanisms associated with emphysematous parenchymal damage include increased apoptosis and cell proliferation. Apoptosis correlated with airspace enlargement, supporting epidemiological evidence of the progressive nature of emphysema. These data extend the understanding of cell dynamics and structural changes within the lung during emphysema pathogenesis.

Cell density related H2 consumption in relation to anoxic Fe(0) corrosion and precipitation of corrosion products by Shewanella oneidensis MR-1.

PubMed

De Windt, Wim; Boon, Nico; Siciliano, Steven D; Verstraete, Willy

2003-11-01

In the absence of oxygen, a protective H2 film is formed around an Fe(0) surface, inhibiting the electron flow from this surface. Our study of anoxic corrosion of Fe(0) beads revealed that, in the presence of Shewanella oneidensis MR-1, H2 removal and precipitation of Fe mineral particles on the cell surface are determining processes for corrosion. These two biologically mediated processes were governed by cell density. H2 removal by Shewanella oneidensis was detected at cell concentrations of 1.0 x 10(6) live cells ml-1 and higher and H2 was electron donor for denitrification of NO3-. The removal of the protective H2 layer from Fe(0) beads by Shewanella oneidensis, resulted in an increase of Fe release out of the Fe(0) beads from 153 +/- 25 mg l(-1) to 196 +/- 7 mg l-1 after 20 h. When the cell concentration exceeded 1.0 x 10(8) live cells ml-1, precipitation of iron minerals on the cell surface was characteristic for the greatest percentage of MR-1 cells, whereas micrometre-scale iron precipitates not associated with culturable cell biomass significantly decreased in number. Addition of supernatant of a corrosion assay with high cell concentration induced metabolic activity in a corrosion assay with low cell concentration, resulting in increased H2 consumption and Fe release from Fe(0) beads. Homoserine lactone-like molecules were detected in the supernatant by a bio-assay, suggesting the involvement of a quorum-sensing regulatory mechanism.

Herpes B Virus, Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts

PubMed Central

Vasireddi, Mugdha

2012-01-01

B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8+ T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions. PMID:22973043

Assessing the in vitro toxicity of the lunar dust environment using respiratory cells exposed to Al(2)O(3) or SiO(2) fine dust particles.

PubMed

Jordan, Jacqueline A; Verhoff, Ashley M; Morgan, Julie E; Fischer, David G

2009-12-01

Prior chemical and physical analysis of lunar soil suggests a composition of dust particles that may contribute to the development of acute and chronic respiratory disorders. In this study, fine Al(2)O(3) (0.7 μm) and fine SiO(2) (mean 1.6 μm) were used to assess the cellular uptake and cellular toxicity of lunar dust particle analogs. Respiratory cells, murine alveolar macrophages (RAW 264.7) and human type II epithelial (A549), were cultured as the in vitro model system. The phagocytic activity of both cell types using ultrafine (0.1 μm) and fine (0.5 μm) fluorescent polystyrene beads was determined. Following a 6-h exposure, RAW 264.7 cells had extended pseudopods with beads localized in the cytoplasmic region of cells. After 24 h, the macrophage cells were rounded and clumped and lacked pseudopods, which suggest impairment of phagocytosis. A549 cells did not contain beads, and after 24 h, the majority of the beads appeared to primarily coat the surface of the cells. Next, we investigated the cellular response to fine SiO(2) and Al(2)O(3) (up to 5 mg/ml). RAW 264.7 cells exposed to 1.0 mg/ml of fine SiO(2) for 6 h demonstrated pseudopods, cellular damage, apoptosis, and necrosis. A549 cells showed slight toxicity when exposed to fine SiO(2) for the same time and dose. A549 cells had particles clustered on the surface of the cells. Only a higher dose (5.0 mg/ml) of fine SiO(2) resulted in a significant cytotoxicity to A549 cells. Most importantly, both cell types showed minimal cytotoxicity following exposure to fine Al(2)O(3). Overall, this study suggests differential cellular toxicity associated with exposure to fine mineral dust particles.

Aspergillus terreus accessory conidia are unique in surface architecture, cell wall composition and germination kinetics.

PubMed

Deak, Eszter; Wilson, Selwyn D; White, Elizabeth; Carr, Janice H; Balajee, S Arunmozhi

2009-10-30

Infection with Aspergillus terreus is more likely to result in invasive, disseminated disease when compared to other Aspergillus species; importantly this species appears to be less susceptible to the antifungal drug amphotericin B. Unique to this species is the ability to produce specialized structures denoted as accessory conidia (AC) directly on hyphae both in vitro and in vivo. With the hypothesis that production of AC by A. terreus may enhance virulence of this organism, we analyzed the phenotype, structure and metabolic potential of these conidia. Comparison of A. terreus phialidic conidia (conidia that arise from conidiophores, PC) and AC architecture by electron microscopy revealed distinct morphological differences between the two conidial forms; AC have a smoother, thicker outer cell surface with no apparent pigment-like layer. Further, AC germinated rapidly, had enhanced adherence to microspheres, and were metabolically more active compared to PC. Additionally, AC contained less cell membrane ergosterol, which correlated with decreased susceptibility to AMB as determined using a flow cytometry based analysis. Furthermore, AC exhibited surface patches of beta1-3 glucan, suggestive of attachment scarring. Collectively, the findings of this study suggest a possible role for AC in A. terreus pathogenesis.

The contribution of B-cell proliferation to spleen enlargement in Babesia microti-infected mice.

PubMed Central

Inchley, C J

1987-01-01

Flow cytofluorimetric analysis showed that B-cell proliferation makes a major contribution to the enlargement and increased cellularity of the spleen, which are characteristic of Babesia microti infections in mice. Expansion of the B-cell population was accompanied by modulation of the cell surface, which affected most B lymphocytes, and which was detected as a reduction in the density of surface immunoglobulin. This effect was noted as early as Day 7, shortly after the appearance of parasites in the circulation and the onset of gross spleen changes. In contrast to the results for B cells, the frequency of splenic T cells declined, and when the data were transformed into absolute numbers it became clear that only limited T-cell proliferation had occurred. There was no evidence to suggest that the balance of T-cell subsets was shifted in favour of suppressor T cells. The relationships of these results to reports of immunosuppression by this parasite are discussed. Images Figure 2 Figure 5 PMID:3493207

A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?

PubMed Central

Thygesen, Uffe H.; Riemann, Lasse; Stedmon, Colin A.

2015-01-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. PMID:26253668

In vitro investigation of anodization and CaP deposited titanium surface using MG63 osteoblast-like cells

NASA Astrophysics Data System (ADS)

Lee, J. M.; Lee, J. I.; Lim, Y. J.

2010-03-01

The aim of the present study was to investigate surface characteristics in four different titanium surfaces (AN: anodized at 270 V; AN-CaP: anodic oxidation and CaP deposited; SLA: sandblasted and acid etched; MA: machined) and to evaluate biological behaviors such as cell adhesion, cell proliferation, cytoskeletal organization, and osteogenic protein expression of MG63 osteoblast-like cells at the early stage. Surface analysis was performed using scanning electron microscopy, thin-film X-ray diffractometry, and a confocal laser scanning microscope. In order to evaluate cellular responses, MG63 osteoblast-like cells were used. The cell viability was evaluated by MTT assay. Immunofluorescent analyses of actin, type I collagen, osteonectin and osteocalcin were performed. The anodized and CaP deposited specimen showed homogeneously distributed CaP particles around micropores and exhibited anatase type oxides, titanium, and HA crystalline structures. This experiment suggests that CaP particles on the anodic oxidation surface affect cellular attachment and spreading. When designing an in vitro biological study for CaP coated titanium, it must be taken into account that preincubation in medium prior to cell seeding and the cell culture medium may affect the CaP coatings. All these observations illustrate the importance of the experimental conditions and the physicochemical parameters of the CaP coating. It is considered that further evaluations such as long-term in vitro cellular assays and in vivo experiments should be necessary to figure out the effect of CaP deposition to biological responses.

Comparative study on morphologic changes and cell attachment of periodontitis-affected root surfaces following conditioning with CO2 and Er:YAG laser irradiations.

PubMed

Belal, Mahmoud Helmy; Watanabe, Hisashi

2014-10-01

Clinical application of lasers in periodontal therapy has continued to expand in last decades; however there are still some controversies. The present study aimed to compare the conditioning effects of the carbon dioxide (CO2) or erbium-doped: yttrium, aluminum and garnet (Er:YAG) laser on periodontally diseased root surfaces following scaling and root planing (SRP) in terms of the alteration of morphologies as well as the attachment of periodontal ligament cells. Forty-five periodontally affected root specimens were prepared and randomly assigned into three groups: I control (untreated diseased), II. SRP+CO2 laser (pulsed, noncontact mode), and III. SRP+Er:YAG laser (slight contact mode). After treatment, five specimens in each group were used for surface topographic examination. The remaining 10 specimens in each group were incubated with human periodontal ligament cell suspension. All the specimens were finally evaluated by scanning electron microscopy. The control specimens showed the lowest number of cultured cells, mostly in oval shape, with no tightly attached cells. The CO2 lased specimens showed a significant increase in the number of attached cells compared with controls, but demonstrated some major thermal alterations on the surfaces. The Er:YAG lased specimens showed the significantly highest number of attached cells, mostly in flat form, and did not show distinct thermal damage. The present study suggests that compared with the CO2 laser, the Er:YAG laser may constitute a more useful conditioning tool for enhancing periodontal cell attachment to periodontally diseased root surfaces, with fewer undesirable thermal side effects.

Protein C Inhibitor (PCI) Binds to Phosphatidylserine Exposing Cells with Implications in the Phagocytosis of Apoptotic Cells and Activated Platelets

PubMed Central

Rieger, Daniela; Assinger, Alice; Einfinger, Katrin; Sokolikova, Barbora; Geiger, Margarethe

2014-01-01

Protein C Inhibitor (PCI) is a secreted serine protease inhibitor, belonging to the family of serpins. In addition to activated protein C PCI inactivates several other proteases of the coagulation and fibrinolytic systems, suggesting a regulatory role in hemostasis. Glycosaminoglycans and certain negatively charged phospholipids, like phosphatidylserine, bind to PCI and modulate its activity. Phosphatidylerine (PS) is exposed on the surface of apoptotic cells and known as a phagocytosis marker. We hypothesized that PCI might bind to PS exposed on apoptotic cells and thereby influence their removal by phagocytosis. Using Jurkat T-lymphocytes and U937 myeloid cells, we show here that PCI binds to apoptotic cells to a similar extent at the same sites as Annexin V, but in a different manner as compared to live cells (defined spots on ∼10–30% of cells). PCI dose dependently decreased phagocytosis of apoptotic Jurkat cells by U937 macrophages. Moreover, the phagocytosis of PS exposing, activated platelets by human blood derived monocytes declined in the presence of PCI. In U937 cells the expression of PCI as well as the surface binding of PCI increased with time of phorbol ester treatment/macrophage differentiation. The results of this study suggest a role of PCI not only for the function and/or maturation of macrophages, but also as a negative regulator of apoptotic cell and activated platelets removal. PMID:25000564

Cellulose/soy protein isolate composite membranes: evaluations of in vitro cytocompatibility with Schwann cells and in vivo toxicity to animals.

PubMed

Luo, Lihua; Gong, Wenrong; Zhou, Yi; Yang, Lin; Li, Daokun; Huselstein, Celine; Wang, Xiong; He, Xiaohua; Li, Yinping; Chen, Yun

2015-01-01

To evaluate the in vitro cytocompatibility of cellulose/soy protein isolate composite membranes (CSM) with Schwann cells and in vivo toxicity to animals. A series of cellulose/soy protein isolate composite membranes (CSM) were prepared by blending, solution casting and coagulation process. The cytocompatibility of the CSM to Schwann cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by direct cells culture of Schwann cells on the surfaces of the CSM, respectively. The in vivo toxicity of the CSM to animals were also evaluated by acute toxicity testing, skin sensitization testing, pyrogen testing and intracutaneous stimulation testing, respectively, according to the ISO 10993 standard. The MTT assay showed that the cell viability of Schwann cells cultured in extracts from the CSM was higher than that from the neat cellulose membrane without containing SPI component. The direct cells culture indicated that the Schwann cells could attach and grow well on the surface of the CSM and the incorporation of SPI into cellulose contributed to improvement of cell adhesion and proliferation. The evaluations of in vivo biological safety suggested that the CSM showed no acute toxicity, no skin sensitization and no intracutaneous stimulation to the experimental animals. The CSM had in vitro cytocompatibility with Schwann cells and biological safety to animals, suggesting potential for the applications as nerve conduit for the repair of nerve defect.

The nature of inherent bactericidal activity: insights from the nanotopology of three species of dragonfly

NASA Astrophysics Data System (ADS)

Mainwaring, David E.; Nguyen, Song Ha; Webb, Hayden; Jakubov, Timur; Tobin, Mark; Lamb, Robert N.; Wu, Alex H.-F.; Marchant, Richard; Crawford, Russell J.; Ivanova, Elena P.

2016-03-01

While insect wings are widely recognised as multi-functional, recent work showed that this extends to extensive bactericidal activity brought about by cell deformation and lysis on the wing nanotopology. We now quantitatively show that subtle changes to this topography result in substantial changes in bactericidal activity that are able to span an order of magnitude. Notably, the chemical composition of the lipid nanopillars was seen by XPS and synchrotron FTIR microspectroscopy to be similar across these activity differences. Modelling the interaction between bacterial cells and the wing surface lipids of 3 species of dragonflies, that inhabit similar environments, but with distinctly different behavioural repertoires, provided the relationship between surface structure and antibacterial functionality. In doing so, these principal behavioural patterns correlated with the demands for antimicrobial efficiency dictated by differences in their foraging strategies. This work now reveals a new feature in the design elegance of natural multi-functional surfaces as well providing insights into the bactericidal mechanism underlying inherently antimicrobial materials, while suggesting that nanotopology is related to the evolutionary development of a species through the demands of its behavioural repertoire. The underlying relationship between the processes of wetting, adhesion and capillarity of the lipid nanopillars and bactericidal efficiency suggests new prospects for purely mechano-responsive antibacterial surfaces.While insect wings are widely recognised as multi-functional, recent work showed that this extends to extensive bactericidal activity brought about by cell deformation and lysis on the wing nanotopology. We now quantitatively show that subtle changes to this topography result in substantial changes in bactericidal activity that are able to span an order of magnitude. Notably, the chemical composition of the lipid nanopillars was seen by XPS and synchrotron FTIR microspectroscopy to be similar across these activity differences. Modelling the interaction between bacterial cells and the wing surface lipids of 3 species of dragonflies, that inhabit similar environments, but with distinctly different behavioural repertoires, provided the relationship between surface structure and antibacterial functionality. In doing so, these principal behavioural patterns correlated with the demands for antimicrobial efficiency dictated by differences in their foraging strategies. This work now reveals a new feature in the design elegance of natural multi-functional surfaces as well providing insights into the bactericidal mechanism underlying inherently antimicrobial materials, while suggesting that nanotopology is related to the evolutionary development of a species through the demands of its behavioural repertoire. The underlying relationship between the processes of wetting, adhesion and capillarity of the lipid nanopillars and bactericidal efficiency suggests new prospects for purely mechano-responsive antibacterial surfaces. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08542j

INVOLVEMENT OF TOLL-LIKE RECEPTOR 4 AND MAPK PATHWAYS IN LPS-INDUCED CD40 EXPRESSION IN MONOCYTIC CELLS

EPA Science Inventory

CD40 is a co-stimulatory surface molecule actively expressed on mature dendritic cells (DC). Recent studies suggest that endotoxin (LPS) inhalation induces DC maturation in the airways of healthy volunteers. To characterize the effect of LPS on CD40 expression and underlying mech...

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.

Design principles for high efficiency small-grain polysilicon solar cells, with supporting experimental studies

NASA Technical Reports Server (NTRS)

Lindholm, F. A.; Neugroschel, A.; Sah, C. T.

1982-01-01

Design principles suggested here aim toward high conversion efficiency (greater than 15 percent) in polysilicon cells. The principles seek to decrease the liabilities of both intragranular and grain-boundary-surface defects. The advantages of a phosphorus atom concentration gradient in a thin (less than 50 microns) base of a p(+)/n(x)/n(+) drift-field solar cell, which produces favorable gradients in chemical potential, minority-carrier mobility and diffusivity, and recombination lifetime (via phosphorus gettering) are suggested. The degrading effects of grain boundaries are reduced by these three gradients and by substituting atoms (P, H, F or Li) for vacancies on the grain-boundary surface. From recent experiments comes support for the benefits of P diffusion down grain boundaries and, for quasi-grain-boundary-free and related structures. New analytic solutions for the n(x)-base include the effect of a power-law dependence between P concentration and lifetime. These provide an upper-bound estimate on the open circuit voltage. Finite-difference numerical solutions of the six Shockley equations furnish complete information about all solar-cell parameters and add insight concerning design.

Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding.

PubMed

Jolly, Amber L; Agarwal, Paresh; Metruccio, Matteo M E; Spiciarich, David R; Evans, David J; Bertozzi, Carolyn R; Fleiszig, Suzanne M J

2017-06-01

Cell surface glycosylation is thought to be involved in barrier function against microbes at mucosal surfaces. Previously we showed that the epithelium of healthy mouse corneas becomes vulnerable to Pseudomonas aeruginosa adhesion if it lacks the innate defense protein MyD88 (myeloid differentiation primary response gene 88), or after superficial injury by blotting with tissue paper. Here we explored their effect on corneal surface glycosylation using a metabolic label, tetra-acetylated N -azidoacetylgalactosamine (Ac 4 GalNAz). Ac 4 GalNAz treatment labeled the surface of healthy mouse corneas, leaving most cells viable, and bacteria preferentially associated with GalNAz-labeled regions. Surprisingly, corneas from MyD88 -/- mice displayed similar GalNAz labeling to wild-type corneas, but labeling was reduced and patchy on IL-1 receptor (IL-1R)-knockout mouse corneas ( P < 0.05, ANOVA). Tissue paper blotting removed GalNAz-labeled surface cells, causing DAPI labeling (permeabilization) of underlying cells. MS of material collected on the tissue paper blots revealed 67 GalNAz-labeled proteins, including intracellular proteins. These data show that the normal distribution of surface glycosylation requires IL-1R, but not MyD88, and is not sufficient to prevent bacterial binding. They also suggest increased P. aeruginosa adhesion to MyD88 -/- and blotted corneas is not due to reduction in total surface glycosylation, and for tissue paper blotting is likely due to cell permeabilization.-Jolly, A. L., Agarwal, P., Metruccio, M. M. E., Spiciarich, D. R., Evans, D. J., Bertozzi, C. R., Fleiszig, S. M. J. Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding. © FASEB.

Transport of surface engineered polyamidoamine (PAMAM) dendrimers across IPEC-J2 cell monolayers.

PubMed

Pisal, Dipak S; Yellepeddi, Venkata K; Kumar, Ajay; Palakurthi, Srinath

2008-11-01

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAM(G4) dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P(app)) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.

Epithelial Microvilli Establish an Electrostatic Barrier to Microbial Adhesion

PubMed Central

Bennett, Kaila M.; Walker, Sharon L.

2014-01-01

Microvilli are membrane extensions on the apical surface of polarized epithelia, such as intestinal enterocytes and tubule and duct epithelia. One notable exception in mucosal epithelia is M cells, which are specialized for capturing luminal microbial particles; M cells display a unique apical membrane lacking microvilli. Based on studies of M cell uptake under different ionic conditions, we hypothesized that microvilli may augment the mucosal barrier by providing an increased surface charge density from the increased membrane surface and associated glycoproteins. Thus, electrostatic charges may repel microbes from epithelial cells bearing microvilli, while M cells are more susceptible to microbial adhesion. To test the role of microvilli in bacterial adhesion and uptake, we developed polarized intestinal epithelial cells with reduced microvilli (“microvillus-minus,” or MVM) but retaining normal tight junctions. When tested for interactions with microbial particles in suspension, MVM cells showed greatly enhanced adhesion and uptake of particles compared to microvillus-positive cells. This preference showed a linear relationship to bacterial surface charge, suggesting that microvilli resist binding of microbes by using electrostatic repulsion. Moreover, this predicts that pathogen modification of electrostatic forces may contribute directly to virulence. Accordingly, the effacement effector protein Tir from enterohemorrhagic Escherichia coli O157:H7 expressed in epithelial cells induced a loss of microvilli with consequent enhanced microbial binding. These results provide a new context for microvillus function in the host-pathogen relationship, based on electrostatic interactions. PMID:24778113

Calcium carbonate mineralization mediated by in vitro cultured mantle cells from Pinctada fucata.

PubMed

Kong, Wei; Li, Shiguo; Xiang, Liang; Xie, Liping; Zhang, Rongqing

2015-08-07

Formation of the molluscan shell is believed to be an extracellular event mediated by matrix proteins. We report calcium carbonate mineralization mediated by Pinctada fucata mantle cells. Crystals only appeared when mantle cells were present in the crystallization solution. These crystals were piled up in highly ordered units and showed the typical characteristics of biomineralization products. A thin organic framework was observed after dissolving the crystals in EDTA. Some crystals had etched surfaces with a much smoother appearance than other parts. Mantle cells were observed to be attached to some of these smooth surfaces. These results suggest that mantle cells may be directly involved in the nucleation and remodeling process of calcium carbonate mineralization. Our result demonstrate the practicability of studying the mantle cell mechanism of biomineralization and contribute to the overall understanding of the shell formation process. Copyright © 2015 Elsevier Inc. All rights reserved.

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

Targeting prostate cancer cells with hybrid elastin-like polypeptide/liposome nanoparticles

PubMed Central

Zhang, Wei; Song, Yunmei; Eldi, Preethi; Guo, Xiuli; Hayball, John D; Garg, Sanjay; Albrecht, Hugo

2018-01-01

Prostate cancer cells frequently overexpress the gastrin-releasing peptide receptor, and various strategies have been applied in preclinical settings to target this receptor for the specific delivery of anticancer compounds. Recently, elastin-like polypeptide (ELP)-based self-assembling micelles with tethered GRP on the surface have been suggested to actively target prostate cancer cells. Poorly soluble chemotherapeutics such as docetaxel (DTX) can be loaded into the hydrophobic cores of ELP micelles, but only limited drug retention times have been achieved. Herein, we report the generation of hybrid ELP/liposome nanoparticles which self-assembled rapidly in response to temperature change, encapsulated DTX at high concentrations with slow release, displayed the GRP ligand on the surface, and specifically bound to GRP receptor expressing PC-3 cells as demonstrated by flow cytometry. This novel type of drug nanocarrier was successfully used to reduce cell viability of prostate cancer cells in vitro through the specific delivery of DTX. PMID:29391790

Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

PubMed Central

Scurti, Gina; Thyagarajan, Krishnamurthy; Kaur, Navtej; Husain, Shahid; Fang, Quan; Naga, Osama S.; Simms, Patricia; Beeson, Gyda; Voelkel-Johnson, Christina; Garrett-Mayer, Elizabeth; Beeson, Craig C.; Nishimura, Michael I.; Mehrotra, Shikhar

2014-01-01

Ex vivo-expanded CD8+ T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced anti-tumor efficacy, relative to CD8+ T cells with a central memory-like phenotype (TCM cells). Further, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM possess relatively greater anti-oxidative capacity than TEM cells. Here we report that TCM cells exhibit a relative increase compared to TEM cells in expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity and glycolytic enzyme levels. Notably, TCR-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, up-regulated thiol levels and antioxidant genes. A comparison of anti-tumor CD8+ T cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth, and that profiling this biomarker may have benefits to T cell adoptive immunotherapy protocols. PMID:25164014

Roles of reaction kinetics of CO 2 on a PrBaCo 2O 5.5+δ surfaces

DOE PAGES

Xu, Xing; Mace, Brennan; Enriquez, Erik; ...

2017-08-18

A symmetric PBCO/YSZ/PBCO cell (where PBCO refers to PrBaCo 2O 5.5+δ, and YSZ to yttria stabilized zirconia) was designed and fabricated for the investigation of the catalytic nature and reaction kinetics of CO 2 on PBCO surfaces. The electrochemical impedance spectroscopy was employed to probe the CO 2 reaction behavior on the PBCO electrode. The symmetric equivalent circuit cell characterization reveals that the surface resistance of CO 2 on PBCO is only 30.2 Ω cm 2 and the maximum surface exchange coefficient k chem is 2.0 × 10 -4 cm s -1 at 1147 K, suggesting that PBCO can bemore » an excellent candidate for CO 2 reduction.« less

The effect of surface tension on steadily translating bubbles in an unbounded Hele-Shaw cell

PubMed Central

2017-01-01

New numerical solutions to the so-called selection problem for one and two steadily translating bubbles in an unbounded Hele-Shaw cell are presented. Our approach relies on conformal mapping which, for the two-bubble problem, involves the Schottky-Klein prime function associated with an annulus. We show that a countably infinite number of solutions exist for each fixed value of dimensionless surface tension, with the bubble shapes becoming more exotic as the solution branch number increases. Our numerical results suggest that a single solution is selected in the limit that surface tension vanishes, with the scaling between the bubble velocity and surface tension being different to the well-studied problems for a bubble or a finger propagating in a channel geometry. PMID:28588410

Evidence for Direct Electron Transfer by a Gram-Positive Bacterium Isolated from a Microbial Fuel Cell▿†

PubMed Central

Wrighton, K. C.; Thrash, J. C.; Melnyk, R. A.; Bigi, J. P.; Byrne-Bailey, K. G.; Remis, J. P.; Schichnes, D.; Auer, M.; Chang, C. J.; Coates, J. D.

2011-01-01

Despite their importance in iron redox cycles and bioenergy production, the underlying physiological, genetic, and biochemical mechanisms of extracellular electron transfer by Gram-positive bacteria remain insufficiently understood. In this work, we investigated respiration by Thermincola potens strain JR, a Gram-positive isolate obtained from the anode surface of a microbial fuel cell, using insoluble electron acceptors. We found no evidence that soluble redox-active components were secreted into the surrounding medium on the basis of physiological experiments and cyclic voltammetry measurements. Confocal microscopy revealed highly stratified biofilms in which cells contacting the electrode surface were disproportionately viable relative to the rest of the biofilm. Furthermore, there was no correlation between biofilm thickness and power production, suggesting that cells in contact with the electrode were primarily responsible for current generation. These data, along with cryo-electron microscopy experiments, support contact-dependent electron transfer by T. potens strain JR from the cell membrane across the 37-nm cell envelope to the cell surface. Furthermore, we present physiological and genomic evidence that c-type cytochromes play a role in charge transfer across the Gram-positive bacterial cell envelope during metal reduction. PMID:21908627

Effect of biocompatible polymers on the structural integrity of lipid bilayers under external stimuli

NASA Astrophysics Data System (ADS)

Wang, Jia-Yu; Kausik, Ravinath; Chen, Chi-Yuan; Han, Song-I.; Marks, Jeremy; Lee, Ka Yee

2010-03-01

Cell membrane dysfunction due to loss of structural integrity is the pathology of tissue death in trauma and common diseases. It is now established that certain biocompatible polymers, such as Poloxamer 188, Poloxamine 1107 and polyethylene glycol (PEG), are effective in sealing of injured cell membranes, and able to prevent acute necrosis. Despite these broad applications of these polymers for human health, the fundamental mechanisms by which these polymers interact with cell membranes are still under debate. Here, the effects of a group of biocompatible polymers on phospholipid membrane integrity under osmotic and oxidative stress were explored using giant unilamellar vesicles as model cell membranes. Our results suggest that the adsorption of the polymers on the membrane surface is responsible for the cell membrane resealing process due to its capability of slowing down the surface hydration dynamics.

Identification and quantification of the rat hepatocyte asialoglycoprotein receptor.

PubMed Central

Schwartz, A L; Marshak-Rothstein, A; Rup, D; Lodish, H F

1981-01-01

The asialoglycoprotein receptor from rat liver was purified by solubilization and affinity chromatography on asialoorosomucoid-Sepharose. The preparation yielded four distinct polypeptides of Mr 40,000-120,000. We prepared a monoclonal antibody that both immunoprecipitates solubilized receptor activity and blocks the binding of galactose-terminal glycoproteins to immobilized receptor. The monoclonal antibody and a rabbit antireceptor antiserum immunoprecipitated all four polypeptide species. Peptide analysis by two-dimensional chromatography of the individual 125I-labeled species showed nearly identical patterns, which also suggested that the four polypeptides have a similar primary structure. To identify and quantitate the asialoglycoprotein receptor on the hepatocyte cell surface, intact cells were iodinated with lactoperoxidase, and the solubilized membranes were treated with antireceptor antibody. The Mr 55,000 and Mr 65,000 species were the major species found. Our results suggest that the Mr of the surface receptor is at least 55,000 and that it comprises between 1-2% of the iodinated hepatocyte surface protein. Images PMID:6267585

Retention of CXCR4 in the endoplasmic reticulum blocks dissemination of a T cell hybridoma

PubMed Central

Zeelenberg, Ingrid S.; Stalle, Lisette Ruuls-Van; Roos, Ed

2001-01-01

The dissemination of T cell hybridomas to multiple nonhematopoietic tissues is blocked by pertussis toxin, suggesting the involvement of a chemokine. To study whether this chemokine is SDF-1, we employed a strategy proposed previously for gene therapy of AIDS, whereby the SDF-1 receptor CXCR4 (also a coreceptor for HIV) is retained in the endoplasmic reticulum (ER) and fails to reach the cell surface. We transfected SDF-1, carrying an ER retention sequence, into a T cell hybridoma. This altered chemokine is retained in the ER, where it binds CXCR4 and prevents the latter protein from reaching the surface. These cells failed to migrate toward SDF-1 or to invade fibroblast monolayers, although they could still migrate toward thymus and activation-regulated chemokine (TARC) and invade TARC-treated monolayers. Furthermore, the ability of the transfected cells to disseminate to multiple organs upon intravenous injection into mice was abolished. This dissemination reflects the in vivo migration patterns of activated and memory T cells into nonhematopoietic tissues, which is thus likely to depend on CXCR4. Attempts to block CXCR4 function as a therapy for AIDS may affect this migration with consequences for T cell function. Our results also suggest a decisive role for CXCR4 in the dissemination of hematopoietic malignancies expressing this receptor. PMID:11457880

Retention of CXCR4 in the endoplasmic reticulum blocks dissemination of a T cell hybridoma.

PubMed

Zeelenberg, I S; Ruuls-Van Stalle, L; Roos, E

2001-07-01

The dissemination of T cell hybridomas to multiple nonhematopoietic tissues is blocked by pertussis toxin, suggesting the involvement of a chemokine. To study whether this chemokine is SDF-1, we employed a strategy proposed previously for gene therapy of AIDS, whereby the SDF-1 receptor CXCR4 (also a coreceptor for HIV) is retained in the endoplasmic reticulum (ER) and fails to reach the cell surface. We transfected SDF-1, carrying an ER retention sequence, into a T cell hybridoma. This altered chemokine is retained in the ER, where it binds CXCR4 and prevents the latter protein from reaching the surface. These cells failed to migrate toward SDF-1 or to invade fibroblast monolayers, although they could still migrate toward thymus and activation-regulated chemokine (TARC) and invade TARC-treated monolayers. Furthermore, the ability of the transfected cells to disseminate to multiple organs upon intravenous injection into mice was abolished. This dissemination reflects the in vivo migration patterns of activated and memory T cells into nonhematopoietic tissues, which is thus likely to depend on CXCR4. Attempts to block CXCR4 function as a therapy for AIDS may affect this migration with consequences for T cell function. Our results also suggest a decisive role for CXCR4 in the dissemination of hematopoietic malignancies expressing this receptor.

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

Indentation of Graphene-Covered Atomic Force Microscopy Probe Across a Lipid Bilayer Membrane: Effect of Tip Shape, Size, and Surface Hydrophobicity.

PubMed

Lv, Kang; Li, Yinfeng

2018-06-21

Understanding the interaction of graphene with cell membranes is crucial to the development of graphene-based biological applications and the management of graphene safety issues. To help reveal the key factors controlling the interaction between graphene and cell membranes, here we adopt the dissipative particle dynamics method to analyze the evolution of interaction force and free energy as the graphene-covered atomic force microscopy (AFM) probe indents across a lipid bilayer. The simulation results show that the graphene-covered AFM probe can cause severe deformation of the cell membrane which drives the lipid molecule to adsorb and diffuse at the surface of graphene. The breakthrough force and free energy are calculated to study the effects of the tip shape, size, and surface hydrophobicity on the piercing behaviors of graphene-covered AFM. In addition, the deformation of cell membrane can decrease the dependency of the breakthrough force on the tip shape. The analysis of surface functionalization suggests that the horizontal patterns on graphene can change the preferred orientation in the penetration process, but the vertical patterns on graphene may disrupt the cell membrane. What's more, the bending stiffness of graphene has little influence on the penetration process as graphene pierces into the cell membrane. These results provide useful guidelines for the molecular design of graphene materials with controllable cell penetrability.

XC-cell fusion induced by murine plasmocytoma cells. II. Cytological and ultrastructural study.

PubMed

Lemay, P; Torpier, G; Lefebvre, J C; Samaille, J

1975-06-10

The MF2 strain, a mouse myeloma derived cell line, was found to induce the mixed culture cytopathogenicity test when cocultured with XC cells. Only one MF2 cell was present per syncytium, as shown by autoradiography. Pretreatment of cells with inhibitors of DNA, RNA or protein synthesis suggested that a normal RNA synthesis was required to obtain optimal polykaryon growth. Immunoelectron microscopy using a syngenic mouse MF2 cell antiserum and peroxydase labeling revealed a complete mixing and redistribution of the respective plasma membrane sites of MF2 and XC cells on polykaryon surface.

Target-specific cellular uptake of PLGA nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate.

PubMed

Kim, Sun Hwa; Jeong, Ji Hoon; Chun, Ki Woo; Park, Tae Gwan

2005-09-13

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with anionic surface charge were surface coated with cationic di-block copolymer, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL) conjugate, for enhancing their site-specific intracellular delivery against folate receptor overexpressing cancer cells. The PLGA nanoparticles coated with the conjugate were characterized in terms of size, surface charge, and change in surface composition by XPS. By employing the flow cytometry method and confocal image analysis, the extent of cellular uptake was comparatively evaluated under various conditions. PLL-PEG-FOL coated PLGA nanoparticles demonstrated far greater extent of cellular uptake to KB cells, suggesting that they were mainly taken up by folate receptor-mediated endocytosis. The enhanced cellular uptake was also observed even in the presence of serum proteins, possibly due to the densely seeded PEG chains. The PLL-PEG-FOL coated PLGA nanoparticles could be potentially applied for cancer cell targeted delivery of various therapeutic agents.

The effect of dry shear aligning of nanotube thin films on the photovoltaic performance of carbon nanotube-silicon solar cells.

PubMed

Stolz, Benedikt W; Tune, Daniel D; Flavel, Benjamin S

2016-01-01

Recent results in the field of carbon nanotube-silicon solar cells have suggested that the best performance is obtained when the nanotube film provides good coverage of the silicon surface and when the nanotubes in the film are aligned parallel to the surface. The recently developed process of dry shear aligning - in which shear force is applied to the surface of carbon nanotube thin films in the dry state, has been shown to yield nanotube films that are very flat and in which the surface nanotubes are very well aligned in the direction of shear. It is thus reasonable to expect that nanotube films subjected to dry shear aligning should outperform otherwise identical films formed by other processes. In this work, the fabrication and characterisation of carbon nanotube-silicon solar cells using such films is reported, and the photovoltaic performance of devices produced with and without dry shear aligning is compared.

The effect of dry shear aligning of nanotube thin films on the photovoltaic performance of carbon nanotube–silicon solar cells

PubMed Central

Stolz, Benedikt W; Tune, Daniel D

2016-01-01

Summary Recent results in the field of carbon nanotube–silicon solar cells have suggested that the best performance is obtained when the nanotube film provides good coverage of the silicon surface and when the nanotubes in the film are aligned parallel to the surface. The recently developed process of dry shear aligning – in which shear force is applied to the surface of carbon nanotube thin films in the dry state, has been shown to yield nanotube films that are very flat and in which the surface nanotubes are very well aligned in the direction of shear. It is thus reasonable to expect that nanotube films subjected to dry shear aligning should outperform otherwise identical films formed by other processes. In this work, the fabrication and characterisation of carbon nanotube–silicon solar cells using such films is reported, and the photovoltaic performance of devices produced with and without dry shear aligning is compared. PMID:27826524

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

Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle.

PubMed

Muñoz, P; Rosemblatt, M; Testar, X; Palacín, M; Zorzano, A

1995-04-01

1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented.

Increased Tolerance to Heavy Metals Exhibited by Swarming Bacteria

NASA Astrophysics Data System (ADS)

Anyan, M.; Shrout, J. D.

2014-12-01

Pseudomonas aeruginosa is a ubiquitous, Gram-negative bacterium that utilizes several different modes of motility to colonize surfaces, including swarming, which is the coordinated movement of cells over surfaces in groups. Swarming facilitates surface colonization and biofilm development for P. aeruginosa, and it is known that swarming behavior is influenced by changes in nutrient composition and surface moisture. To understand the fate and cycling of heavy metals in the environment, it is important to understand the interaction and toxicity of these metals upon bacteria. While previous studies have shown surface-attached bacterial biofilms to be highly resistant to heavy metal toxicity, little is known about the influence of heavy metals upon surface motile bacteria and developing biofilms. Using a combination of laboratory assays we examined differences in bacterial behavior in response to two metals, Cd and Ni. We find that surface swarming bacteria are able to grow on 4x and 2.5x more Cd and Ni, respectively, than planktonic cells (i.e., test tube cultures). P. aeruginosa was able to swarm in the presence ≤0.051mM Ni and ≤0.045mM Cd. To investigate the bioavailability of metals to bacteria growing under our examined conditions, we separated cell and supernatant fractions of P. aeruginosa cultures, and used ICP-MS techniques to measure Cd and Ni sorption. A greater percentage of Cd than Ni was sorbed by both cells and supernatant (which contains rhamnolipid, a surfactant known to sorb some metals and improve swarming). While we show that cell products such as rhamnolipid bind heavy metals (as expected) and should limit metal bioavailability, our results suggest at least one additional mechanism (as yet undetermined) that promotes cell survival during swarming in the presence of these heavy metals.

Ultrasonic and electromagnetic enhancement of a culture of human SAOS-2 osteoblasts seeded onto a titanium plasma-spray surface.

PubMed

Fassina, Lorenzo; Saino, Enrica; Sbarra, Maria Sonia; Visai, Livia; Cusella De Angelis, Maria Gabriella; Mazzini, Giuliano; Benazzo, Francesco; Magenes, Giovanni

2009-06-01

Several studies suggest that the surface coating of titanium could play an important role in bone tissue engineering. In the present study, we have followed a particular biomimetic strategy where ultrasonically or electromagnetically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix on a titanium plasma-spray surface. In comparison with control conditions, the ultrasonic stimulation (average power, 149 mW; frequency, 1.5 MHz) and the electromagnetic stimulation (magnetic field intensity, 2 mT; frequency, 75 Hz) caused higher cell proliferation, and increased surface coating with decorin, osteocalcin, osteopontin, and type I collagen together with higher incorporation of calcium and phosphorus inside the extracellular matrix. The immunofluorescence related to the preceding bone matrix proteins showed their colocalization in the cell-rich areas. The use of the two physical stimulations aimed at obtaining the coating of the rough titanium plasma-spray surface in terms of cell colonization and deposition of extracellular matrix. The superficially cultured biomaterial could be theoretically used, in clinical applications, as an implant for bone repair.

Enhanced charge carrier transport properties in colloidal quantum dot solar cells via organic and inorganic hybrid surface passivation† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ta06835a Click here for additional data file.

PubMed Central

Hong, John; Hou, Bo; Lim, Jongchul; Pak, Sangyeon; Kim, Byung-Sung; Cho, Yuljae; Lee, Juwon; Lee, Young-Woo; Giraud, Paul; Lee, Sanghyo; Park, Jong Bae; Morris, Stephen M.; Snaith, Henry J.; Kim, Jong Min

2016-01-01

Colloidal quantum dots (CQDs) are extremely promising as photovoltaic materials. In particular, the tunability of their electronic band gap and cost effective synthetic procedures allow for the versatile fabrication of solar energy harvesting cells, resulting in optimal device performance. However, one of the main challenges in developing high performance quantum dot solar cells (QDSCs) is the improvement of the photo-generated charge transport and collection, which is mainly hindered by imperfect surface functionalization, such as the presence of surface electronic trap sites and the initial bulky surface ligands. Therefore, for these reasons, finding effective methods to efficiently decorate the surface of the as-prepared CQDs with new short molecular length chemical structures so as to enhance the performance of QDSCs is highly desirable. Here, we suggest employing hybrid halide ions along with the shortest heterocyclic molecule as a robust passivation structure to eliminate surface trap sites while decreasing the charge trapping dynamics and increasing the charge extraction efficiency in CQD active layers. This hybrid ligand treatment shows a better coordination with Pb atoms within the crystal, resulting in low trap sites and a near perfect removal of the pristine initial bulky ligands, thereby achieving better conductivity and film structure. Compared to halide ion-only treated cells, solar cells fabricated through this hybrid passivation method show an increase in the power conversion efficiency from 5.3% for the halide ion-treated cells to 6.8% for the hybrid-treated solar cells. PMID:29308200

Cell-Surface Expression of Neuron-Glial Antigen 2 (NG2) and Melanoma Cell Adhesion Molecule (CD146) in Heterogeneous Cultures of Marrow-Derived Mesenchymal Stem Cells

PubMed Central

Russell, Katie C.; Tucker, H. Alan; Bunnell, Bruce A.; Andreeff, Michael; Schober, Wendy; Gaynor, Andrew S.; Strickler, Karen L.; Lin, Shuwen; Lacey, Michelle R.

2013-01-01

Cellular heterogeneity of mesenchymal stem cells (MSCs) impedes their use in regenerative medicine. The objective of this research is to identify potential biomarkers for the enrichment of progenitors from heterogeneous MSC cultures. To this end, the present study examines variation in expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) on the surface of MSCs derived from human bone marrow in response to culture conditions and among cell populations. Multipotent cells isolated from heterogeneous MSC cultures exhibit a greater than three-fold increase in surface expression for NG2 and greater than two-fold increase for CD146 as compared with parental and lineage-committed MSCs. For both antigens, surface expression is downregulated by greater than or equal to six-fold when MSCs become confluent. During serial passage, maximum surface expression of NG2 and CD146 is associated with minimum doubling time. Upregulation of NG2 and CD146 during loss of adipogenic potential at early passage suggests some limits to their utility as potency markers. A potential relationship between proliferation and antigen expression was explored by sorting heterogeneous MSCs into rapidly and slowly dividing groups. Fluorescence-activated cell sorting revealed that rapidly dividing MSCs display lower scatter and 50% higher NG2 surface expression than slowly dividing cells, but CD146 expression is comparable in both groups. Heterogeneous MSCs were sorted based on scatter properties and surface expression of NG2 and CD146 into high (HI) and low (LO) groups. ScLONG2HI and ScLONG2HICD146HI MSCs have the highest proliferative potential of the sorted groups, with colony-forming efficiencies that are 1.5–2.2 times the value for the parental controls. The ScLO gate enriches for rapidly dividing cells. Addition of the NG2HI gate increases cell survival to 1.5 times the parental control. Further addition of the CD146HI gate does not significantly improve cell division or survival. The combination of low scatter and high NG2 surface expression is a promising selection criterion to enrich a proliferative phenotype from heterogeneous MSCs during ex vivo expansion, with potentially numerous applications. PMID:23611563

Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

PubMed

Hönig, Ellena; Ringer, Karina; Dewes, Jenny; von Mach, Tobias; Kamm, Natalia; Kreitzer, Geri; Jacob, Ralf

2018-05-10

Epithelial cells require a precise intracellular transport and sorting machinery in order to establish and maintain their polarized architecture. This machinery includes beta-galactoside binding galectins for glycoprotein targeting to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analyzed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, galectin-3 knockout results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells. © 2018. Published by The Company of Biologists Ltd.

Different processing of LH/hCG receptors in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1)

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

Kellokumpu, S.

1987-02-01

The metabolic fate of LH/hCG receptors after exposure to human chorionic gonadotropin (hCG) was examined in cultured rat luteal cells and murine Leydig tumor cells (MLTC-1). Kinetic studies performed after pulse-labelling of the cells with (/sup 125/I)hCG indicated that the bound hormone was lost much more rapidly from the tumor cells than from the luteal cells. The tumor cells were also found to internalize and degrade the hormone more effectively than the luteal cells. Chemical cross-linking and analyses by SDS-PAGE of this material revealed that both cell types also released, in addition to intact hCG, two previously characterized receptor fragment-(/supmore » 125/I)hCG complexes (M/sub r/ 96,000 and 74,000) into the medium, although their amount was negligible in MLTC-1 cells. Possibly due to rapid discharge of the ligand from its receptor, no similar complexes could be detected inside the MLTC-1 cells, suggesting that they were released directly from the cell surface. However, the M/sub r/ 74,000 complex was observed inside MLTC-1 cells if chloroquine, a lysosomotropic agent, was present during the incubations. This suggests that the internalized receptor also becomes degraded, at least when complexed to hCG. The results thus provide evidence that there exist two different mechanisms for proteolytic processing of LH/hCG receptors in these target cells. In tumor cells, the degradation seems to occur almost exclusively intracellularly, whereas in luteal cells a substantial portion of the receptors is also degraded at the cell surface.« less

Evidence for Ig Light Chain Isotype Exclusion in Shark B Lymphocytes Suggests Ordered Mechanisms.

PubMed

Iacoangeli, Anna; Lui, Anita; Haines, Ashley; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

2017-09-01

Unlike most vertebrates, the shark IgL gene organization precludes secondary rearrangements that delete self-reactive VJ rearranged genes. Nurse sharks express four L chain isotypes, κ, λ, σ, and σ-2, encoded by 35 functional minigenes or clusters. The sequence of gene activation/expression and receptor editing of these isotypes have not been studied. We therefore investigated the extent of isotypic exclusion in separated B cell subpopulations. Surface Ig (sIg)κ-expressing cells, isolated with mAb LK14 that recognizes Cκ, carry predominantly nonproductive rearrangements of other L chain isotypes. Conversely, after depletion with LK14, sIgM + cells contained largely nonproductive κ and enrichment for in-frame VJ of the others. Because some isotypic inclusion was observed at the mRNA level, expression in the BCR was examined. Functional λ mRNA was obtained, as expected, from the LK14-depleted population, but was also in sIgκ + splenocytes. Whereas λ somatic mutants from the depleted sample displayed evidence of positive selection, the λ genes in sIgκ + cells accumulated bystander mutations indicating a failure to express their products at the cell surface in association with the BCR H chain. In conclusion, a shark B cell expresses one L chain isotype at the surface and other isotypes as nonproductive VJ, sterile transcripts, or in-frame VJ whose products may not associate with the H chain. Based on the mRNA content found in the B cell subpopulations, an order of L chain gene activation is suggested as: σ-2 followed by κ, then σ and λ. Copyright © 2017 by The American Association of Immunologists, Inc.

Prion propagation in cells expressing PrP glycosylation mutants.

PubMed

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction

PubMed Central

Schröder, Andreas

2018-01-01

The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus. PMID:29630597

A novel 2,6-diisopropylphenyl-docosahexaenoamide conjugate induces apoptosis in T cell acute lymphoblastic leukemia cell lines

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

Altenburg, Jeffrey D.; Harvey, Kevin A.; McCray, Sharon

2011-07-29

Highlights: {yields} 2,6-Diisopropylphenyl-docosahexaenoamide conjugates (DIP-DHA) inhibits the proliferation of T-cell leukemic cell lines. {yields} DIP-DHA resulted in increased activation of caspase-3, and caspase-7. {yields} DIP-DHA significantly downregulated CXCR4 surface expression. -- Abstract: We have previously characterized the effects of 2,6-diisopropylphenyl-docosahexaenoamide (DIP-DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP-DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP-DHA resulted in significantly greater inhibition of proliferation and induction of apoptosis than thatmore » of parent compounds, 2,6-diisopropylphenol (DIP) or docosahexaenoate (DHA). Treatment of the cells with DIP-DHA resulted in increased activation of caspase-3, and caspase-7. Furthermore, induction of apoptosis in both cell lines was reversed in the presence of a caspase family inhibitor. Treatment with DIP-DHA reduced mitochondrial membrane potential. These observations suggest that the effects are driven by intrinsic apoptotic pathways. DIP-DHA treatment also downregulated surface CXCR4 expression, an important chemokine receptor involved in cancer metastasis that is highly expressed in both CEM and Jurkat cells. In conclusion, our data suggest that the DIP-DHA conjugate exhibits significantly more potent effects on CEM and Jurkat cells than that of DIP or DHA alone. These conjugates have potential use for treatment of patients with T cell acute lymphoblastic leukemia.« less

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

Characterization and cell behavior of titanium surfaces with PLL/DNA modification via a layer-by-layer technique.

PubMed

Gao, Wenli; Feng, Bo; Lu, Xiong; Wang, Jianxin; Qu, Shuxin; Weng, Jie

2012-08-01

This study describes the fabrication of two types of multilayered films onto titanium by layer-by-layer (LBL) self-assembly, using poly-L-lysine (PLL) as the cationic polyelectrolyte and deoxyribonucleic acid (DNA) as the anionic polyelectrolyte. The assembling process of each component was studied using atomic force microscopy (AFM) and quartz crystal balance (QCM). Zeta potential of the LBL-coated microparticles was measured by dynamic light scattering. Titanium substrates with or without multilayered films were used in osteoblast cell culture experiments to study cell proliferation, viability, differentiation, and morphology. Results of AFM and QCM indicated the progressive build-up of the multilayered coatings. The surface morphology of three types of multilayered films showed elevations in the nanoscale range. The data of zeta potential showed that the surface terminated with PLL displayed positive charge while the surface terminated with DNA displayed negative charge. The proliferation of osteoblasts on modified titanium films was found to be greater than that on control (p < 0.05) after 3 and 7 days culture, respectively. Alamar blue measurement showed that the PLL/DNA-modified films have higher cell viability (p < 0.05) than the control. Still, the alkaline phosphatase activity assay revealed a better differentiated phenotype on three types of multilayered surfaces compared to noncoated controls. Collectively our results suggest that PLL/DNA were successfully used to surface engineer titanium via LBL technique, and enhanced its cell biocompatibility. Copyright © 2012 Wiley Periodicals, Inc.

An MHC class I immune evasion gene of Marek׳s disease virus.

PubMed

Hearn, Cari; Preeyanon, Likit; Hunt, Henry D; York, Ian A

2015-01-15

Marek׳s disease virus (MDV) is a widespread α-herpesvirus of chickens that causes T cell tumors. Acute, but not latent, MDV infection has previously been shown to lead to downregulation of cell-surface MHC class I (Virology 282:198-205 (2001)), but the gene(s) involved have not been identified. Here we demonstrate that an MDV gene, MDV012, is capable of reducing surface expression of MHC class I on chicken cells. Co-expression of an MHC class I-binding peptide targeted to the endoplasmic reticulum (bypassing the requirement for the TAP peptide transporter) partially rescued MHC class I expression in the presence of MDV012, suggesting that MDV012 is a TAP-blocking MHC class I immune evasion protein. This is the first unique non-mammalian MHC class I immune evasion gene identified, and suggests that α-herpesviruses have conserved this function for at least 100 million years. Copyright © 2014 Elsevier Inc. All rights reserved.

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

PubMed Central

Buchanan, Susan K; Lukacik, Petra; Grizot, Sylvestre; Ghirlando, Rodolfo; Ali, Maruf M U; Barnard, Travis J; Jakes, Karen S; Kienker, Paul K; Esser, Lothar

2007-01-01

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 Å above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane. PMID:17464289

Mechanism for the antibacterial action of epigallocatechin gallate (EGCg) on Bacillus subtilis.

PubMed

Nakayama, Motokazu; Shimatani, Kanami; Ozawa, Tadahiro; Shigemune, Naofumi; Tomiyama, Daisuke; Yui, Koji; Katsuki, Mao; Ikeda, Keisuke; Nonaka, Ai; Miyamoto, Takahisa

2015-01-01

Catechins are a class of polyphenols and have high anti-bacterial activity against various microorganisms. Here, we report the mechanism for antibacterial activity of epigallocatechin gallate (EGCg) against Gram-positive bacteria Bacillus subtilis, which is highly sensitive to EGCg. Transmission electron microscope analysis revealed that deposits containing EGCg were found throughout the cell envelope from the outermost surface to the outer surface of cytoplasmic membrane. Aggregating forms of proteins and EGCg were identified as spots that disappeared or showed markedly decreased intensity after the treatment with EGCg compared to the control by two-dimensional electrophoresis. Among the identified proteins included 4 cell surface proteins, such as oligopeptide ABC transporter binding lipoprotein, glucose phosphotransferase system transporter protein, phosphate ABC transporter substrate-binding protein, and penicillin-binding protein 5. Observations of glucose uptake of cells and cell shape B. subtilis after the treatment with EGCg suggested that EGCg inhibits the major functions of these proteins, leading to growth inhibition of B. subtilis.

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.

Characteristics of Sputtered Cr Thin Films and Application as a Working Electrode in Transparent Conductive Oxide-Less Dye-Sensitized Solar Cells.

PubMed

Park, Yong Seob; Kang, Ki-Noh; Kim, Young-Baek; Hwang, Sung Hwan; Lee, Jaehyeong

2018-09-01

Cr metal electrode was suggested as the working electrode material to fabricate DSSCs without the TCO, and thin films were fabricated by an unbalanced magnetron sputtering system. The surface morphologies show uniform and smooth surfaces regardless of various film thicknesses, and the small crystallites of various sizes were showed with the vertical direction on the surface of Cr thin films with the increase of film thickness. And also, the root mean square (RMS) surface roughness value of Cr thin films increased, and the sheet resistance is decreased with the increase of film thickness. The maximum cell efficiency of the TCO-less DSSC was observed when a Cr working electrode with a thickness of 80 nm was applied to the TCO-less DSSC. Consequently, these results are related to the result of the optimization of conduction characteristics, transmission properties and surface properties of Cr thin films.

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

Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

NASA Astrophysics Data System (ADS)

Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

2016-11-01

Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

Immunohistochemical study of dental pulp applied with 4-META/MMA-TBB adhesive resin after pulpotomy.

PubMed

Nakamura, M; Inoue, T; Shimono, M

2000-08-01

The purpose of this study was to investigate nerve regeneration and proliferative activity in amputated pulp tissue after the application of 4-META/MMA-TBB adhesive resin (4-META resin). Calcium hydroxide was used as a control material. At 3 days, fibroblast-like cells were positive for proliferating cell nuclear antigen (PCNA) in both 4-META resin- and calcium hydroxide-treated groups and were located mainly within 0.5 mm from the cut surface. Only a few fragmented neurofilament protein (NFP)-positive nerve fibers were observed in this area. At 7 and 14 days, the number of PCNA-positive cells had gradually decreased and regenerated NFP-positive nerve fibers were observed close to the cut surface of the pulp in both groups. At 21 days in the experimental group, several PCNA-positive cells were still found in the area 0.5 mm from the cut surface, and NFP-positive nerve fibers were detected about 0.15-;0.2 mm from the cut surface. In contrast, a dentin bridge was produced under the necrotic layer at 21 days in the control group. PCNA-positive cells were not found underneath the dentin bridge, but NFP-positive nerve fibers had regenerated close to it. These results suggest that although cell differentiation and nerve regeneration are delayed, wound healing occurred even after the application of 4-META resin to exposed pulp surface the same as calcium hydroxide application. Copyright 2000 John Wiley & Sons, Inc.

In situ proteolysis of the Vibrio cholerae matrix protein RbmA promotes biofilm recruitment.

PubMed

Smith, Daniel R; Maestre-Reyna, Manuel; Lee, Gloria; Gerard, Harry; Wang, Andrew H-J; Watnick, Paula I

2015-08-18

The estuarine gram-negative rod and human diarrheal pathogen Vibrio cholerae synthesizes a VPS exopolysaccharide-dependent biofilm matrix that allows it to form a 3D structure on surfaces. Proteins associated with the matrix include, RbmA, RbmC, and Bap1. RbmA, a protein whose crystallographic structure suggests two binding surfaces, associates with cells by means of a VPS-dependent mechanism and promotes biofilm cohesiveness and recruitment of cells to the biofilm. Here, we show that RbmA undergoes limited proteolysis within the biofilm. This proteolysis, which is carried out by the hemagglutinin/protease and accessory proteases, yields the 22-kDa C-terminal polypeptide RbmA*. RbmA* remains biofilm-associated. Unlike full-length RbmA, the association of RbmA* with cells is no longer VPS-dependent, likely due to an electropositive surface revealed by proteolysis. We provide evidence that this proteolysis event plays a role in recruitment of VPS(-) cells to the biofilm surface. Based on our findings, we propose that association of RbmA with the matrix reinforces the biofilm structure and leads to limited proteolysis of RbmA to RbmA*. RbmA*, in turn, promotes recruitment of cells that have not yet initiated VPS synthesis to the biofilm surface. The assignment of two functions to RbmA, separated by a proteolytic event that depends on matrix association, dictates an iterative cycle in which reinforcement of recently added biofilm layers precedes the recruitment of new VPS(-) cells to the biofilm.

CD24 cell surface expression in Mvt1 mammary cancer cells serves as a biomarker for sensitivity to anti-IGF1R therapy.

PubMed

Rostoker, Ran; Ben-Shmuel, Sarit; Rashed, Rola; Shen Orr, Zila; LeRoith, Derek

2016-05-14

The pro-tumorigenic effects of the insulin-like growth factor receptor (IGF1R) are well described. IGF1R promotes cancer cell survival and proliferation and prevents apoptosis, and, additionally it was shown that IGF1R levels are significantly elevated in most common human malignancies including breast cancer. However, results from phase 3 clinical trials in unselected patients demonstrated lack of efficacy for anti-IGF1R therapy. These findings suggest that predictive biomarkers are greatly warranted in order to identify patients that will benefit from anti-IGF1R therapeutic strategies. Using the delivery of shRNA vectors into the Mvt1 cell line, we tested the role of the IGF1R in the development of mammary tumors. Based on CD24 cell surface expression, control and IGF1R-knockdown (IGF1R-KD) cells were FACS sorted into CD24(-) and CD24(+) subsets and further characterized in vitro. The tumorigenic capacity of each was determined following orthotopic inoculation into the mammary fat pad of female mice. Tumor cells were FACS characterized upon sacrifice to determine IGF1R effect on the plasticity of this cell's phenotype. Metastatic capacity of the cells was assessed using the tail vein assay. In this study we demonstrate that downregulation of the IGF1R specifically in cancer cells expressing CD24 on the cell surface membrane affect both their morphology (from mesenchymal-like into epithelial-like morphology) and phenotype in vitro. Moreover, we demonstrate that IGF1R-KD abolished both CD24(+) cells capacity to form mammary tumors and lung metastatic lesions. We found in both cells and tumors a marked upregulation in CTFG and a significant reduction of SLP1 expression in the CD24(+)/IGF1R-KD; tumor-suppressor and tumor-promoting genes respectively. Moreover, we demonstrate here that the IGF1R is essential for the maintenance of stem/progenitor-like cancer cells and we further demonstrate that IGF1R-KD induces in vivo differentiation of the CD24(+) cells toward the CD24(-) phenotype. This further supports the antitumorigenic effects of IGF1R-KD, as we recently published that these differentiated cells demonstrate significantly lower tumorigenic capacity compared with their CD24(+) counterparts. Taken together these findings suggest that CD24 cell surface expression may serve as a valuable biomarker in order to identify mammary tumors that will positively respond to targeted IGF1R therapies.

Surface complexation modeling of proton and Cd adsorption onto an algal cell wall.

PubMed

Kaulbach, Emily S; Szymanowski, Jennifer E S; Fein, Jeremy B

2005-06-01

This study quantifies Cd adsorption onto the cell wall of the algal species Pseudokirchneriella subcapitata by applying a surface complexation approach to model the observed adsorption behavior. We use potentiometric titrations to determine deprotonation constants and site concentrations for the functional groups on the algal cell wall. Adsorption and desorption kinetics experiments illustrate that adsorption of Cd onto the cell wall is rapid and reversible, except under low pH conditions. Adsorption experiments conducted as a function of pH and total Cd concentration yield the stoichiometry and site-specific stability constants for the important Cd-algal surface complexes. We model the acid/base properties of the algal cell wall by invoking four discrete surface functional group types, with pKa values of 3.9 +/- 0.3, 5.4 +/- 0.1, 7.6 +/- 0.3, and 9.6 +/- 0.4. The results of the Cd adsorption experiments indicate that the first, third, and fourth sites contribute to Cd adsorption under the experimental conditions, with calculated log stability constant values of 4.1 +/- 0.5, 5.4 +/- 0.5, and 6.1 +/- 0.4, respectively. Our results suggest that the stabilities of the Cd-surface complexes are high enough for algal adsorption to affect the fate and transport of Cd under some conditions and that on a per gram basis, algae and bacteria exhibit broadly similar extents of Cd adsorption.

The influence of size and charge of chitosan/polyglutamic acid hollow spheres on cellular internalization, viability and blood compatibility.

PubMed

Dash, Biraja C; Réthoré, Gildas; Monaghan, Michael; Fitzgerald, Kathleen; Gallagher, William; Pandit, Abhay

2010-11-01

Polymeric hollow spheres can be tailored as efficient carriers of various therapeutic molecules due to their tunable properties. However, the entry of these synthetic vehicles into cells, their cell viability and blood compatibility depend on their physical and chemical properties e.g. size, surface charge. Herein, we report the effect of size and surface charge on cell viability and cellular internalization behaviour and their effect on various blood components using chitosan/polyglutamic acid hollow spheres as a model system. Negatively charged chitosan/polyglutamic acid hollow spheres of various sizes 100, 300, 500 and 1000 nm were fabricated using a template based method and covalently surface modified using linear polyethylene glycol and methoxyethanol amine to create a gradient of surface charge from negative to neutrally charged spheres respectively. The results here suggest that both size and surface charge have a significant influence on the sphere's behaviour, most prominently on haemolysis, platelet activation, plasma recalcification time, cell viability and internalization over time. Additionally, cellular internalization behaviour and viability was found to vary with different cell types. These results are in agreement with those of inorganic spheres and liposomes, and can serve as guidelines for tailoring polymeric solid spheres for specific desired applications in biological and pharmaceutical fields, including the design of nanometer to submicron-sized delivery vehicles. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

The origin of high electrolyte-electrode interfacial resistances in lithium cells containing garnet type solid electrolytes.

PubMed

Cheng, Lei; Crumlin, Ethan J; Chen, Wei; Qiao, Ruimin; Hou, Huaming; Franz Lux, Simon; Zorba, Vassilia; Russo, Richard; Kostecki, Robert; Liu, Zhi; Persson, Kristin; Yang, Wanli; Cabana, Jordi; Richardson, Thomas; Chen, Guoying; Doeff, Marca

2014-09-14

Dense LLZO (Al-substituted Li7La3Zr2O12) pellets were processed in controlled atmospheres to investigate the relationships between the surface chemistry and interfacial behavior in lithium cells. Laser induced breakdown spectroscopy (LIBS), scanning electron microscopy (SEM), X-ray diffraction (XRD), Raman spectroscopy, synchrotron X-ray photoelectron spectroscopy (XPS) and soft X-ray absorption spectroscopy (XAS) studies revealed that Li2CO3 was formed on the surface when LLZO pellets were exposed to air. The distribution and thickness of the Li2CO3 layer were estimated by a combination of bulk and surface sensitive techniques with various probing depths. First-principles thermodynamic calculations confirmed that LLZO has an energetic preference to form Li2CO3 in air. Exposure to air and the subsequent formation of Li2CO3 at the LLZO surface is the source of the high interfacial impedances observed in cells with lithium electrodes. Surface polishing can effectively remove Li2CO3 and dramatically improve the interfacial properties. Polished samples in lithium cells had an area specific resistance (ASR) of only 109 Ω cm(2) for the LLZO/Li interface, the lowest reported value for Al-substituted LLZO. Galvanostatic cycling results obtained from lithium symmetrical cells also suggest that the quality of the LLZO/lithium interface has a significant impact on the device lifetime.

Oligonucleotide and Parylene Surface Coating of Polystyrene and ePTFE for Improved Endothelial Cell Attachment and Hemocompatibility

PubMed Central

Schleicher, Martina; Hansmann, Jan; Elkin, Bentsian; Kluger, Petra J.; Liebscher, Simone; Huber, Agnes J. T.; Fritze, Olaf; Schille, Christine; Müller, Michaela; Schenke-Layland, Katja; Seifert, Martina; Walles, Heike; Wendel, Hans-Peter; Stock, Ulrich A.

2012-01-01

In vivo self-endothelialization by endothelial cell adhesion on cardiovascular implants is highly desirable. DNA-oligonucleotides are an intriguing coating material with nonimmunogenic characteristics and the feasibility of easy and rapid chemical fabrication. The objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on vascular implant surfaces. DNA-oligonucleotides immobilized by adsorption on parylene (poly(monoaminomethyl-para-xylene)) coated polystyrene and ePTFE were resistant to high shear stress (9.5 N/m2) and human blood serum for up to 96 h. Adhesion of murine endothelial progenitor cells, HUVECs and endothelial cells from human adult saphenous veins as well as viability over a period of 14 days of HUVECs on oligonucleotide coated samples under dynamic culture conditions was significantly enhanced (P < 0.05). Oligonucleotide-coated surfaces revealed low thrombogenicity and excellent hemocompatibility after incubation with human blood. These properties suggest the suitability of immobilization of DNA-oligonucleotides for biofunctionalization of blood vessel substitutes for improved in vivo endothelialization. PMID:22481939

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

The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

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

Fujiwara, Daisuke; Kato, Kazunori, E-mail: kzkatou@juntendo.ac.jp; Department of Atopy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421

2013-05-17

Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present inmore » esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression was enhanced, suggesting that hypoxia had been induced. Comparison of cancer drug resistance using cisplatin and doxorubicin in 3-D-cultured esophageal cancer cells showed that cancer drug resistance had increased. These results indicate that 3-D culture of esophageal squamous cell carcinoma lines is a useful method for inducing cancer stem cells.« less

Release of Membrane-associated Mucins from Ocular Surface Epithelia

PubMed Central

Blalock, Timothy D.; Spurr-Michaud, Sandra J.; Tisdale, Ann S.; Gipson, Ilene K.

2008-01-01

Purpose Three membrane-associated mucins (MAMs)—MUC1, MUC4 and MUC16—are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. Methods An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to MUC16’s cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in release/shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor (TNF), TNF-α-converting enzyme, and matrix metalloproteinases-7 and –9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. Results MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced release of MUC16 but not MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16—78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. Conclusions Results suggest that extracellular domains of MUC1, 4, and 16 can be released from the ocular surface by agents present in tears. Neutrophil elastase and TNF present in higher amounts in dry eye patients’ tears may cause MAM release—allowing rose bengal staining. PMID:18436821

Conditionally immortal ovarian cell lines for investigating the influence of ovarian stroma on the estrogen sensitivity and tumorigenicity of ovarian surface epithelial cells.

PubMed

Jiang, Feng; Saunders, Beatriz O; Haller, Edward; Livingston, Sandra; Nicosia, Santo V; Bai, Wenlong

2003-01-01

The tendency of the ovarian surface epithelium (OSE) to undergo metaplastic and morphogenetic changes during the life cycle, at variance with the adjacent peritoneal mesothelial cells, suggests that its biology may be regulated by underlying ovarian stromal cues. However, little is known about the role that the ovarian stroma plays in the pathobiology of the OSE, largely because of the lack of a suitable in vitro model. Here, we describe the establishment and characterization of conditionally immortalized ovarian stromal and surface epithelial cell lines from H-2K(b)-tsA58 transgenic mice that carry the thermolabile mutant of SV-40 large T antigen under the control of an interferon-gamma (IFN-gamma)-inducible promoter. These cells express functional T antigens, grow continuously under permissive conditions at 33 degrees C in the presence of IFN-gamma, and stop dividing when the activity and expression of the tumor antigen is suppressed by restrictive conditions without IFN-gamma at 39 degrees C. Morphological, immunohistochemical, and ultrastructural analyses show that conditionally immortal OSE cells form cobblestone-like monolayers, express cytokeratin and vimentin, contain several microvilli, and develop tight junctions, whereas stromal cells are spindle-like, express vimentin but not cytokeratin, and contain rare microvilli, thus exhibiting epithelial and stromal phenotypes, respectively. At variance with the reported behavior of rat epithelial cells, conditionally immortal mouse epithelial cells are not spontaneously transformed after continuous culture in vitro. More importantly, conditioned media from stromal cells cultured under permissive conditions increase the specific activity of the endogenous estrogen receptor in BG-1 human ovarian epithelial cancer cells and promote these cells' anchorage-independent growth, suggesting the paracrine influence of a stromal factor. In addition, stromal cells cultured under restrictive conditions retain this growth-stimulatory activity, which, therefore, appears to be independent of T antigen expression. These established cell lines should provide a useful in vitro model system for studying the role of cellular interactions in OSE cell growth and tumorigenesis.

IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

PubMed

Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

2017-10-10

Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP + mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP + cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP + mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways.

PubMed

Bezerra, Fábio; Ferreira, Marcel R; Fontes, Giselle N; da Costa Fernandes, Célio Jr; Andia, Denise C; Cruz, Nilson C; da Silva, Rodrigo A; Zambuzzi, Willian F

2017-08-01

Although, intracellular signaling pathways are proposed to predict the quality of cell-surface relationship, this study addressed pre-osteoblast behavior in response to nano hydroxyapatite (HA)-blasted titanium (Ti) surface by exploring critical intracellular pathways and pre-osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy (AFM) and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid-etching (DAE), and nano hydroxyapatite-blasted (nHA). The results revealed critical differences in surface topography, impacting the water contact angle and later the osteoblast performance. In order to evaluate the effect of those topographical characteristics on biological responses, we have seeded pre-osteoblast cells on the Ti discs for up to 4 h and subjected the cultures to biological analysis. First, we have observed pre-osteoblasts morphological changes resulting from the interaction with the Ti texturized surfaces whereas the cells cultured on nHA presented a more advanced spreading process when compared with the cells cultured on the other surfaces. These results argued us for analyzing the molecular machinery and thus, we have shown that nHA promoted a lower Bax/Bcl2 ratio, suggesting an interesting anti-apoptotic effect, maybe explained by the fact that HA is a natural element present in bone composition. Thereafter, we investigated the potential effect of those surfaces on promoting pre-osteoblast adhesion and survival signaling by performing crystal violet and immunoblotting approaches, respectively. Our results showed that nHA promoted a higher pre-osteoblast adhesion supported by up-modulating FAK and Src activations, both signaling transducers involved during eukaryotic cell adhesion. Also, we have shown Ras-Erk stimulation by the all evaluated surfaces. Finally, we showed that all Ti-texturing surfaces were able to promote osteoblast differentiation up to 10 days, when alkaline phosphatase (ALP) activity and osteogenic transcription factors were up-modulated. Altogether, our results showed for the first time that nano hydroxyapatite-blasted titanium surface promotes crucial intracellular signaling network responsible for cell adapting on the Ti-surface.Biotechnol. Bioeng. 2017;114: 1888-1898. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Protein profile of basal prostate epithelial progenitor cells--stage-specific embryonal antigen 4 expressing cells have enhanced regenerative potential in vivo.

PubMed

Höfner, Thomas; Klein, Corinna; Eisen, Christian; Rigo-Watermeier, Teresa; Haferkamp, Axel; Sprick, Martin R

2016-04-01

The long-term propagation of basal prostate progenitor cells ex vivo has been very difficult in the past. The development of novel methods to expand prostate progenitor cells in vitro allows determining their cell surface phenotype in greater detail. Mouse (Lin(-)Sca-1(+) CD49f(+) Trop2(high)-phenotype) and human (Lin(-) CD49f(+) TROP2(high)) basal prostate progenitor cells were expanded in vitro. Human and mouse cells were screened using 242 anti-human or 176 antimouse monoclonal antibodies recognizing the cell surface protein profile. Quantitative expression was evaluated at the single-cell level using flow cytometry. Differentially expressed cell surface proteins were evaluated in conjunction with the known CD49f(+)/TROP2(high) phenotype of basal prostate progenitor cells and characterized by in vivo sandwich-transplantation experiments using nude mice. The phenotype of basal prostate progenitor cells was determined as CD9(+)/CD24(+)/CD29(+)/CD44(+)/CD47(+)/CD49f(+)/CD104(+)/CD147(+)/CD326(+)/Trop2(high) of mouse as well as human origin. Our analysis revealed several proteins, such as CD13, Syndecan-1 and stage-specific embryonal antigens (SSEAs), as being differentially expressed on murine and human CD49f(+) TROP2(+) basal prostate progenitor cells. Transplantation experiments suggest that CD49f(+) TROP2(high) SSEA-4(high) human prostate basal progenitor cells to be more potent to regenerate prostate tubules in vivo as compared with CD49f(+) TROP2(high) or CD49f(+) TROP2(high) SSEA-4(low) cells. Determination of the cell surface protein profile of functionally defined murine and human basal prostate progenitor cells reveals differentially expressed proteins that may change the potency and regenerative function of epithelial progenitor cells within the prostate. SSEA-4 is a candidate cell surface marker that putatively enables a more accurate identification of the basal PESC lineage. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Lytic agents, cell permeability, and monolayer penetrability.

PubMed

Salton, M R

1968-07-01

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20-30 % lipid and 50-75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2-3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.

The role of basal cells in adhesion of columnar epithelium to airway basement membrane.

PubMed

Evans, M J; Plopper, C G

1988-08-01

In this report, we present a new concept of the role of the basal cell in airway epithelium. Previously, the basal cell was thought to be the progenitor cell for the columnar epithelium. However, several studies have shown that this concept may not be correct. The morphologic aspects of the basal cell suggest that it could play a role in adhesion of the columnar epithelium to the basement membrane. Basal cells form attachments with columnar cells (desmosomes) and with the basement membrane (hemidesmosomes). Columnar cells do not form hemidesmosome attachments with the basement membrane. Basal cells could strengthen the adhesion of columnar cells to the basement membrane by forming hemidesmosome attachments to the basement membrane and desmosome attachments with adjacent columnar cells. Incidental evidence from 2 existing publications concerning airway microanatomy support this concept. As columnar cells grow taller, the proportion of the cell surface in contact with the basement membrane becomes progressively smaller, and thus the cell surface area related to adhesion also becomes smaller. It was found that the number of basal cells per millimeter of basement membrane was closely related to the height of the columnar cell epithelium (r = 0.98), but not to the number of columnar cells (r = 0.42). The consistency of the relationship between increased columnar cell height (and thus decreased surface area for adhesion) and the number of basal cells present (r = 0.98) supports the concept that the basal cell plays a role in adhesion of columnar cells to the basement membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications.

PubMed

Stevenson, G; Rehman, S; Draper, E; Hernández-Nava, E; Hunt, J; Haycock, J W

2016-07-01

In this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast-like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in-growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre-clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586-1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

In Vitro Evidence of the Presence of Mesenchymal Stromal Cells in Cervical Cancer and Their Role in Protecting Cancer Cells from Cytotoxic T Cell Activity

PubMed Central

Montesinos, Juan J.; Mora-García, María de L.; Mayani, Héctor; Flores-Figueroa, Eugenia; García-Rocha, Rosario; Fajardo-Orduña, Guadalupe R.; Castro-Manrreza, Marta E.; Weiss-Steider, Benny

2013-01-01

Mesenchymal stromal cells (MSCs) have been isolated from different tumors and it has been suggested that they support tumor growth through immunosuppression processes that favor tumor cell evasion from the immune system. To date, however, the presence of MSCs in cervical cancer (CeCa) and their possible role in tumor growth remains unknown. Herein we report on the presence of MSCs in cervical tissue, both in normal conditions (NCx-MSCs) and in CeCa (CeCa-MSCs), and described several biological properties of such cells. Our study showed similar patterns of cell surface antigen expression, but distinct differentiation potentials, when we compared both cervical MSC populations to MSCs from normal bone marrow (BM-MSCs, the gold standard). Interestingly, CeCa-MSCs were negative for the presence of human papiloma virus, indicating that these cells are not infected by such a viral agent. Also, interestingly, and in contrast to NCx-MSCs, CeCa-MSCs induced significant downregulation of surface HLA class I molecules (HLA-A*0201) on CaSki cells and other CeCa cell lines. We further observed that CeCa-MSCs inhibited antigen-specific T cell recognition of CaSki cells by cytotoxic T lymphocytes (CTLs). HLA class I downregulation on CeCa cells correlated with the production of IL-10 in cell cocultures. Importantly, this cytokine strongly suppressed recognition of CeCa cells by CTLs. In summary, this study demonstrates the presence of MSCs in CeCa and suggests that tumor-derived MSCs may provide immune protection to tumor cells by inducing downregulation of HLA class I molecules. This mechanism may have important implications in tumor growth. PMID:23656504

Overexpression of sialomucin complex, a rat homologue of MUC4, inhibits tumor killing by lymphokine-activated killer cells.

PubMed

Komatsu, M; Yee, L; Carraway, K L

1999-05-01

Sialomucin complex (SMC) is a large heterodimeric glycoprotein complex composed of a mucin subunit ascites sialoglycoprotein-1 and a transmembrane subunit ascites sialoglycoprotein-2. It is a rat homologue of human mucin gene MUC4 and is abundantly expressed on the cell surface of highly metastatic ascites 13762 rat mammary adenocarcinoma cells. Because of their extended and rigid structures, mucin-type glycoproteins are suggested to have suppressing effects on cell-cell and cell-matrix interactions. During the metastatic process, these effects presumably cause tumor cell detachment from the primary tumor mass and facilitate escape of the tumor cells from immunosurveillance. Analyses of human breast cancer cells in solid tumors and tumor effusions showed that the more aggressive cells in effusions are stained with polyclonal antibodies against SMC more frequently than cells in solid tumors, suggesting a role for MUC4/SMC in tumor progression and metastasis. Previously, we generated recombinant cDNAs for SMC that vary in the number of mucin repeats to study the putative functions of SMC in tumor metastasis. These cDNAs were transfected into human cancer cell lines and tested for the effect of the expression of this gene. Here, using a tetracycline-responsive inducible expression system, we demonstrate that overexpression of SMC masks the surface antigens on target tumor cells and effectively suppresses tumor cell killing by cytotoxic lymphocytes. This effect results from the ability of SMC to block killer cell binding to the tumor cells and is dependent on both overexpression of the mucin and the number of mucin repeats in the expressed SMC. These results provide an explanation for the proposed role of SMC/MUC4 in tumor progression.

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

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

Effect of inversion layer at iron pyrite surface on photovoltaic device

NASA Astrophysics Data System (ADS)

Uchiyama, Shunsuke; Ishikawa, Yasuaki; Uraoka, Yukiharu

2018-03-01

Iron pyrite has great potential as a thin-film solar cell material because it has high optical absorption, low cost, and is earth-abundant. However, previously reported iron pyrite solar cells showed poor photovoltaic characteristics. Here, we have numerically simulated its photovoltaic characteristics and band structures by utilizing a two-dimensional (2D) device simulator, ATLAS, to evaluate the effects of an inversion layer at the surface and a high density of deep donor defect states in the bulk. We found that previous device structures did not consider the inversion layer at the surface region of iron pyrite, which made it difficult to obtain the conversion efficiency. Therefore, we remodeled the device structure and suggested that removing the inversion layer and reducing the density of deep donor defect states would lead to a high conversion efficiency of iron pyrite solar cells.

A slow “catch and release” process prolongs immune attack on cancer cells | Center for Cancer Research

Cancer.gov

Center for Cancer Research investigators have discovered that some cancer cells catch immune signaling molecules called cytokines on their surfaces then slowly release the molecules. The results suggest that the immune system may exploit this weak spot to mount a prolonged attack on the tumor. Read more...

A novel phenoxazine derivative suppresses surface IgM expression in DT40 B cell line

PubMed Central

Gao, Sanyang; Takano, Tomoko; Sada, Kiyonao; He, Jinsong; Noda, Chiseko; Hori-Tamura, Naoko; Tomoda, Akio; Yamamura, Hirohei

2002-01-01

2-amino-4, 4α-dihydro-4α, 7-dimethyl-3H-phenoxazine-3-one (Phx) has been demonstrated to be an actinomycin D-like phenoxazine, and to display anti-tumour activity. In this study, we report on the effect of Phx on B cell antigen receptor (BCR) and receptor-mediated signalling in DT40 B cells. Treatment of B cells with Phx for 12 h inhibited BCR-stimulated tyrosine phosphorylation of cellular proteins. B cells exposed to Phx exhibited down-regulation of surface IgM which is part of BCR. In contracts with actinomycin D, Phx rapidly reduced the expression of IgM without decreasing the expression of other signalling molecules. Analysis with confocal microscopy demonstrated that Phx treatment reduced IgM expression both at the cell surface and inside the cell. Treatment of B cells with Phx resulted in the reduction of IgM secretion. Since MG-132, a proteasomal inhibitor, restored IgM contents to the control levels, Phx has the specific effect of accelerating IgM degradation. These results suggest that Phx down-regulates the expression of IgM and inhibits BCR-mediated signalling and IgM secretion. Phx may be useful as an immunosuppressive agent for therapeutic purposes. PMID:12411404

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex.

PubMed

Wise, Jillian F; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K; Tao, Rong-Hua; Sabichi, Anita L; Ao, Xue; Maeng, Hoyoung; Samaniego, Felipe

2013-06-06

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex

PubMed Central

Wise, Jillian F.; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K.; Tao, Rong-Hua; Sabichi, Anita L.; Ao, Xue; Maeng, Hoyoung

2013-01-01

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL–Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target. PMID:23599269

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

[Effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterial on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells].

PubMed

Li, Jingfeng; Zheng, Qixin; Guo, Xiaodong; Chen, Liaobin

2014-10-01

In the present research, the effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterials on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells were investigated. The experiments were divided into three groups due to biomaterials used: Group A (composite materials of sintered bone modified with surface mineralization and P24, a peptide of bone morphogenetic protein-2); Group B (sintered bone modified with surface mineralization) and Group C (sintered bone only). The three groups were observed by scanning electron microscopy (SEM) before the experiments, respectively. Then MC3T3-E1 cells were cultured on the surfaces of the three kinds of material, respectively. The cell adhesion rate was assessed by precipitation method. The proliferative ability of MC3T3-E1 cells were measured with MTT assay. And the ALP staining and measurement of alkaline phosphatase (ALP) activity were performed to assess the differentiation of cells into osteoblasts. The SEM results showed that the materials in the three groups retained the natural pore structure and the pore sizes were in the range between 200-850 μm. The adhesive ratio measurements and MTT assay suggested that adhesion and proliferation of MC3T3-E1 cells in Group A were much higher than those in Group B and Group C (P < 0.05). The ALP staining and ALP activity of MC3T3-E1 cells in Group A were significantly higher than those in Group B and Group C (P < 0.05). The sintered bone modified with surface mineralization/P24 composite material was confirmed to improve the adhesion rate and proliferation and osteodifferentiation of MC3T3-E1 cells, and maintained their morphology.

CCR2-64I polymorphism is not associated with altered CCR5 expression or coreceptor function.

PubMed

Mariani, R; Wong, S; Mulder, L C; Wilkinson, D A; Reinhart, A L; LaRosa, G; Nibbs, R; O'Brien, T R; Michael, N L; Connor, R I; Macdonald, M; Busch, M; Koup, R A; Landau, N R

1999-03-01

A polymorphism in the gene encoding CCR2 is associated with a delay in progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. The polymorphism, CCR2-64I, changes valine 64 of CCR2 to isoleucine. However, it is not clear whether the effect on AIDS progression results from the amino acid change or whether the polymorphism marks a genetically linked, yet unidentified mutation that mediates the effect. Because the gene encoding CCR5, the major coreceptor for HIV type 1 primary isolates, lies 15 kb 3' to CCR2, linked mutations in the CCR5 promoter or other regulatory sequences could explain the association of CCR2-64I with slowed AIDS pathogenesis. Here, we show that CCR2-64I is efficiently expressed on the cell surface but does not have dominant negative activity on CCR5 coreceptor function. A panel of peripheral blood mononuclear cells (PBMC) from uninfected donors representing the various CCR5/CCR2 genotypes was assembled. Activated primary CD4(+) T cells of CCR2 64I/64I donors expressed cell surface CCR5 at levels comparable to those of CCR2 +/+ donors. A slight reduction in CCR5 expression was noted, although this was not statistically significant. CCR5 and CCR2 mRNA levels were nearly identical for each of the donor PBMC, regardless of genotype. Cell surface CCR5 and CCR2 levels were more variable than mRNA transcript levels, suggesting that an alternative mechanism may influence CCR5 cell surface levels. CCR2-64I is linked to the CCR5 promoter polymorphisms 208G, 303A, 627C, and 676A; however, in transfected promoter reporter constructs, these did not affect transcriptional activity. Taken together, these findings suggest that CCR2-64I does not act by influencing CCR5 transcription or mRNA levels.

Hybrid Antibody-Induced Topographical Redistribution of Surface Immunoglobulins, Alloantigens, and Concanavalin A Receptors on Mouse Lymphoid Cells

PubMed Central

Stackpole, Christopher W.; De Milio, Lawrence T.; Hämmerling, Ulrich; Jacobson, Janet B.; Lardis, Michael P.

1974-01-01

Redistribution of surface immunoglobulins, H-2b, Thy-1.2, and TL.1,2,3 alloantigens, and concanavalin A receptors on mouse lymphoid cells induced by hybrid rabbit F(ab′)2 antibody (anti-mouse immunoglobulin/anti-visual marker or anti-concanavalin A/anti-visual marker) was studied by immunofluorescence. When used directly to label surface immunoglobulin, and indirectly to label alloantigens and concanavalin A receptors, hybrid antibodies induced similar displacement of all surface components from a uniform distribution into “patches” and “caps” at 37°. One hybrid antibody preparation, antimouse immunoglobulin/anti-ferritin, contained negligible amounts of bivalent anti-mouse immunoglobulin antibody, and was therefore “monovalent” for the antimouse immunoglobulin specificity. This observation suggests that factors other than multivalent crosslinking are responsible for hybrid antibody-induced redistribution of cell-surface components. Cap formation induced by hybrid antibody was enhanced markedly by attachment of the visual marker, either ferritin or southern bean mosaic virus, at 37°. At -5°, hybrid antibody does not displace uniformly distributed H-2b alloantigen-alloantibody complexes, but patches of label develop when ferritin attaches to the hybrid antibody. These results explain the patchy distribution of cell-surface components, which is a temperature-independent characteristic of labeling with hybrid antibodies and visual markers for electron microscopy. Images PMID:4595577

Significance of a Posttranslational Modification of the PilA Protein of Geobacter sulfurreducens for Surface Attachment, Biofilm Formation, and Growth on Insoluble Extracellular Electron Acceptors.

PubMed

Richter, Lubna V; Franks, Ashley E; Weis, Robert M; Sandler, Steven J

2017-04-15

Geobacter sulfurreducens , an anaerobic metal-reducing bacterium, possesses type IV pili. These pili are intrinsic structural elements in biofilm formation and, together with a number of c -type cytochromes, are thought to serve as conductive nanowires enabling long-range electron transfer (ET) to metal oxides and graphite anodes. Here, we report that a posttranslational modification of a nonconserved amino acid residue within the PilA protein, the structural subunit of the type IV pili, is crucial for growth on insoluble extracellular electron acceptors. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the secreted PilA protein revealed a posttranslational modification of tyrosine-32 with a moiety of a mass consistent with a glycerophosphate group. Mutating this tyrosine into a phenylalanine inhibited cell growth with Fe(III) oxides as the sole electron acceptor. In addition, this amino acid substitution severely diminished biofilm formation on graphite surfaces and impaired current output in microbial fuel cells. These results demonstrate that the capability to attach to insoluble electron acceptors plays a crucial role for the cells' ability to utilize them. The work suggests that glycerophosphate modification of Y32 is a key factor contributing to the surface charge of type IV pili, influencing the adhesion of Geobacter to specific surfaces. IMPORTANCE Type IV pili are bacterial appendages that function in cell adhesion, virulence, twitching motility, and long-range electron transfer (ET) from bacterial cells to insoluble extracellular electron acceptors. The mechanism and role of type IV pili for ET in Geobacter sulfurreducens is still a subject of research. In this study, we identified a posttranslational modification of the major G. sulfurreducens type IV pilin, suggested to be a glycerophosphate moiety. We show that a mutant in which the glycerophosphate-modified tyrosine-32 is replaced with a phenylalanine has reduced abilities for ET and biofilm formation compared with those of the wild type. The results show the importance of the glycerophosphate-modified tyrosine for surface attachment and electron transfer in electrode- or Fe(III)-respiring G. sulfurreducens cells. Copyright © 2017 American Society for Microbiology.

Significance of a Posttranslational Modification of the PilA Protein of Geobacter sulfurreducens for Surface Attachment, Biofilm Formation, and Growth on Insoluble Extracellular Electron Acceptors

PubMed Central

Franks, Ashley E.; Weis, Robert M.; Sandler, Steven J.

2017-01-01

ABSTRACT Geobacter sulfurreducens, an anaerobic metal-reducing bacterium, possesses type IV pili. These pili are intrinsic structural elements in biofilm formation and, together with a number of c-type cytochromes, are thought to serve as conductive nanowires enabling long-range electron transfer (ET) to metal oxides and graphite anodes. Here, we report that a posttranslational modification of a nonconserved amino acid residue within the PilA protein, the structural subunit of the type IV pili, is crucial for growth on insoluble extracellular electron acceptors. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the secreted PilA protein revealed a posttranslational modification of tyrosine-32 with a moiety of a mass consistent with a glycerophosphate group. Mutating this tyrosine into a phenylalanine inhibited cell growth with Fe(III) oxides as the sole electron acceptor. In addition, this amino acid substitution severely diminished biofilm formation on graphite surfaces and impaired current output in microbial fuel cells. These results demonstrate that the capability to attach to insoluble electron acceptors plays a crucial role for the cells' ability to utilize them. The work suggests that glycerophosphate modification of Y32 is a key factor contributing to the surface charge of type IV pili, influencing the adhesion of Geobacter to specific surfaces. IMPORTANCE Type IV pili are bacterial appendages that function in cell adhesion, virulence, twitching motility, and long-range electron transfer (ET) from bacterial cells to insoluble extracellular electron acceptors. The mechanism and role of type IV pili for ET in Geobacter sulfurreducens is still a subject of research. In this study, we identified a posttranslational modification of the major G. sulfurreducens type IV pilin, suggested to be a glycerophosphate moiety. We show that a mutant in which the glycerophosphate-modified tyrosine-32 is replaced with a phenylalanine has reduced abilities for ET and biofilm formation compared with those of the wild type. The results show the importance of the glycerophosphate-modified tyrosine for surface attachment and electron transfer in electrode- or Fe(III)-respiring G. sulfurreducens cells. PMID:28138101

Mechanical algal disruption for efficient biodiesel extraction

NASA Astrophysics Data System (ADS)

Krehbiel, Joel David

Biodiesel from algae provides several benefits over current biodiesel feedstocks, but the energy requirements of processing algae into a useable fuel are currently so high as to be prohibitive. One route to improving this is via disruption of the cells prior to lipid extraction, which can significantly increase energy recovery. Unfortunately, several obvious disruption techniques require more energy than can be gained. This dissertation examines the use of microbubbles to improve mechanical disruption of algal cells using experimental, theoretical, and computational methods. New laboratory experiments show that effective ultrasonic disruption of algae is achieved by adding microbubbles to an algal solution. The configuration studied flows the solution through a tube and insonifies a small section with a high-pressure ultrasound wave. Previous biomedical research has shown effective cell membrane damage on animal cells with similar methods, but the present research is the first to extend such study to algal cells. Results indicate that disruption increases with peak negative pressure between 1.90 and 3.07 MPa and with microbubble concentration up to 12.5 x 107 bubbles/ml. Energy estimates of this process suggest that it requires only one-fourth the currently most-efficient laboratory-scale disruption process. Estimates of the radius near each bubble that causes disruption (i.e. the disruption radius) suggest that it increases with peak negative pressure and is near 9--20 microm for all cases tested. It is anticipated that these procedures can be designed for better efficiency and efficacy, which will be facilitated by identifying the root mechanisms of the bubble-induced disruption. We therefore examine whether bubble expansion alone creates sufficient cell deformation for cell rupture. The spherically-symmetric Marmottant model for bubble dynamics allows estimation of the flow regime under experimental conditions. Bubble expansion is modeled as a point source of mass at the bubble center, and if the bubble-to-cell spacing is much larger than the cell radius, the flow around the cell is approximately extensional in the cell's frame of reference. It is known that the present algae are quasi-spherical with cytoplasmic viscosity approximately 100 times that of water, so the cell is approximated as a viscous sphere. Thus, conditions that cause cell disruption from an expanding microbubble are modeled as either steady inviscid extensional flow or steady point source flow over a viscous sphere. In the inviscid extensional flow model, the flow inside the sphere is dominated by viscous forces so the Stokes equation is solved with matched stresses at the sphere surface from the exterior inviscid extensional flow. The short-time deformation of the sphere surface suggests that inviscid extensional flow is insufficient to disrupt cells. This indicates that asymmetry of the flow over the sphere may be required to provide sufficient surface areal strain to rupture the cell. In a more detailed model, the bubble expansion is modeled as an expansion near a viscous sphere using finite element software. For conditions similar to those seen in the experiment, deformation shows similar scaling to disruption. The deformation in this model is significantly higher than predicted from the inviscid extensional flow model due to the effect of asymmetric flow on the cell membrane. Estimates suggest 21% average areal strain on the algal membrane is required to disrupt algal cells, and this result agrees well with areal strains typically required to disrupt cell membranes although the actual value would be lessened by the effect of an elastic membrane, which is neglected in the present model. The local areal strain on the sphere surface is a maximum closest to the point source, and there is compressive strain near theta = +/-pi/4 with theta the angle from the line between the cell center and the point source. The maximum local areal strain shows less sensitivity to the viscosity of the interior fluid than the average areal strain. Overall, the dissertation lays the groundwork for more efficient algal disruption through the judicious use of microbubbles. Separation of bubble generation and bubble growth provides the ability to improve the efficiency of each process and localize energy. Results suggest that effective disruption can occur by pulsing high-pressure ultrasound waves to a solution of cells co-suspended with microbubbles. The models are thought to represent basic phenomenological mechanisms of disruption that could be exploited to improve the overall energy efficiency of schemes. Analysis suggests that extensional flow alone cannot be the cause of cell disruption near an expanding microbubble. Additionally, this work provides an estimate of the areal strain required disrupt an algal cell membrane. This research suggests a couple routes toward reducing the energy required for production of algal biodiesel.

Ciliary metachronal wave propagation on the compliant surface of Paramecium cells.

PubMed

Narematsu, Naoki; Quek, Raymond; Chiam, Keng-Hwee; Iwadate, Yoshiaki

2015-12-01

Ciliary movements in protozoa exhibit metachronal wave-like coordination, in which a constant phase difference is maintained between adjacent cilia. It is at present generally thought that metachronal waves require hydrodynamic coupling between adjacent cilia and the extracellular fluid. To test this hypothesis, we aspirated a Paramecium cell using a micropipette which completely sealed the surface of the cell such that no fluid could pass through the micropipette. Thus, the anterior and the posterior regions of the cell were hydrodynamically decoupled. Nevertheless, we still observed that metachronal waves continued to propagate from the anterior to the posterior ends of the cell, suggesting that in addition to hydrodynamic coupling, there are other mechanisms that can also transmit the metachronal waves. Such transmission was also observed in computational modeling where the fluid was fully decoupled between two partitions of a beating ciliary array. We also imposed cyclic stretching on the surface of live Paramecium cells and found that metachronal waves persisted in the presence of cyclic stretching. This demonstrated that, in addition to hydrodynamic coupling, a compliant substrate can also play a critical role in mediating the propagation of metachronal waves. © 2015 Wiley Periodicals, Inc.

Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3*

PubMed Central

Roy, Adhiraj; Kim, Jeong-Ho

2014-01-01

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations. PMID:24451370

Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye.

PubMed

Argüeso, Pablo; Tisdale, Ann; Spurr-Michaud, Sandra; Sumiyoshi, Mika; Gipson, Ilene K

2006-01-01

Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.

Mucin Characteristics of Human Corneal-Limbal Epithelial Cells that Exclude the Rose Bengal Anionic Dye

PubMed Central

Argüeso, Pablo; Tisdale, Ann; Spurr-Michaud, Sandra; Sumiyoshi, Mika; Gipson, Ilene K.

2005-01-01

Purpose Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. Methods HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. Results The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. Conclusions These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia. PMID:16384952

Surface apposition and multiple cell contacts promote myoblast fusion in Drosophila flight muscles

PubMed Central

Dhanyasi, Nagaraju; Segal, Dagan; Shimoni, Eyal; Shinder, Vera

2015-01-01

Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell–cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast–myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell–cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process. PMID:26459604

Biophysical regulation of epigenetic state and cell reprogramming

NASA Astrophysics Data System (ADS)

Downing, Timothy L.; Soto, Jennifer; Morez, Constant; Houssin, Timothee; Fritz, Ashley; Yuan, Falei; Chu, Julia; Patel, Shyam; Schaffer, David V.; Li, Song

2013-12-01

Biochemical factors can help reprogram somatic cells into pluripotent stem cells, yet the role of biophysical factors during reprogramming is unknown. Here, we show that biophysical cues, in the form of parallel microgrooves on the surface of cell-adhesive substrates, can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells’ epigenetic state. Specifically, decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves, suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications.

Penicillium purpurogenum cultures under ethanol-induced stress and its correlation with fungal adhesion and biodegrading ability.

PubMed

Gomaa, Ola M; Husseiny, Sherif M; Abd El Kareem, Hussein; Talaat, Riham

2016-10-01

Fungi are known to be affected by external environmental stimuli, resulting in different stress response effects, which in turn could be used to enhance its biodegrading ability. In a previous study, ethanol was used to manipulate cell-cell and cell-surface interaction to prevent cell loss and maximize the usage of Penicillium purpurogenum cells in the media, a correlation was drawn between ethanol oxidative stress, surface-bound proteins and fungal adhesion. The present study focuses on a more detailed study of the effect of ethanol on the same fungus. The results show that the presence of Yap1p gene and the detection of an oxidized form of glutathione (GSSG) suggest that a stress response might be involved in the adhesion process. The process of adhesion could be described as a signaling process and it is affected by the germ tube formation as an initial step in adhesion. Protein profile showed polymorphism in surface-bound proteins for cultures amended with ethanol when compared to control cultures. Ethanol also affected the DNA polymorphic profile of DNA, rendering the fungus genetically variable. P. purpurogenum produced phenol oxidase enzyme and could be used to degrade total phenols in olive mill waste water without the formation of biofilm on the surface of the containers.

Ulex europaeus 1 lectin targets microspheres to mouse Peyer's patch M-cells in vivo.

PubMed

Foster, N; Clark, M A; Jepson, M A; Hirst, B H

1998-03-01

The interaction of latex microspheres with mouse Peyer's patch membranous M-cells was studied in a mouse gut loop model after the microspheres were coated with a variety of agents. Carboxylated microspheres (diameter 0.5 micron) were covalently coated with lectins Ulex europaeus 1, Concanavalin A, Euonymus europaeus and Bandeiraea simplicifolia 1 isolectin-B4, human immunoglobulin A or bovine serum albumin. Of the treatments examined, only Ulex europaeus (UEA1) resulted in significant selective binding of microspheres to M-cells. UEA1-coated microspheres bound to M-cells at a level 100-fold greater than BSA-coated microspheres, but binding to enterocytes was unaffected. Incubation of UEA1-coated microspheres with alpha-L-fucose reduced M-cell binding to a level comparable with BSA-coated microspheres. This indicated that targeting by UEA1 was via a carbohydrate receptor on the M-cell surface. Adherence of UEA1-coated microspheres to M-cells occurred within 10 min of inoculation into mouse gut loops and UEA1-coated microspheres were transported to 10 microns below the apical surface of M-cells within 60 min of inoculation. UEA1-coated microspheres also targeted mouse Peyer's patch M-cells after intragastric administration. These results demonstrated that altering the surface chemistry of carboxylated polystyrene microspheres increased M-cell targeting, suggesting a strategy to enhance delivery of vaccine antigens to the mucosal immune system.

Basal Immunoglobulin Signaling Actively Maintains Developmental Stage in Immature B Cells

PubMed Central

Tze, Lina E; Schram, Brian R; Lam, Kong-Peng; Hogquist, Kristin A; Hippen, Keli L; Liu, Jiabin; Shinton, Susan A; Otipoby, Kevin L; Rodine, Peter R; Vegoe, Amanda L; Kraus, Manfred; Hardy, Richard R; Schlissel, Mark S; Rajewsky, Klaus

2005-01-01

In developing B lymphocytes, a successful V(D)J heavy chain (HC) immunoglobulin (Ig) rearrangement establishes HC allelic exclusion and signals pro-B cells to advance in development to the pre-B stage. A subsequent functional light chain (LC) rearrangement then results in the surface expression of IgM at the immature B cell stage. Here we show that interruption of basal IgM signaling in immature B cells, either by the inducible deletion of surface Ig via Cre-mediated excision or by incubating cells with the tyrosine kinase inhibitor herbimycin A or the phosphatidylinositol 3-kinase inhibitor wortmannin, led to a striking “back-differentiation” of cells to an earlier stage in B cell development, characterized by the expression of pro-B cell genes. Cells undergoing this reversal in development also showed evidence of new LC gene rearrangements, suggesting an important role for basal Ig signaling in the maintenance of LC allelic exclusion. These studies identify a previously unappreciated level of plasticity in the B cell developmental program, and have important implications for our understanding of central tolerance mechanisms. PMID:15752064

Extensions and applications of a second-order landsurface parameterization

NASA Technical Reports Server (NTRS)

Andreou, S. A.; Eagleson, P. S.

1983-01-01

Extensions and applications of a second order land surface parameterization, proposed by Andreou and Eagleson are developed. Procedures for evaluating the near surface storage depth used in one cell land surface parameterizations are suggested and tested by using the model. Sensitivity analysis to the key soil parameters is performed. A case study involving comparison with an "exact" numerical model and another simplified parameterization, under very dry climatic conditions and for two different soil types, is also incorporated.

Cytogenetic and molecular genetic characterization of immortalized human ovarian surface epithelial cell lines: consistent loss of chromosome 13 and amplification of chromosome 20.

PubMed

Jin, Yuesheng; Zhang, Hao; Tsao, Sai Wah; Jin, Charlotte; Lv, Mei; Strömbeck, Bodil; Wiegant, Joop; Wan, Thomas Shek Kong; Yuen, Po Wing; Kwong, Yok-Lam

2004-01-01

This study aimed at identifying the genetic events involved in immortalization of ovarian epithelial cells, which might be important steps in ovarian carcinogenesis. The genetic profiles of five human ovarian surface epithelial (HOSE) cell lines immortalized by retroviral transfection of the human papillomavirus (HPV) E6/E7 genes were thoroughly characterized by chromosome banding and fluorescence in situ hybridization (FISH), at various passages pre- and post-crisis. In pre-crisis, most cells had simple, non-clonal karyotypic changes. Telomere association was the commonest aberration, suggesting that tolermase dysfunction might be an important genetic event leading to cellular crisis. After immortalization post-crisis, however, the karyotypic patterns were non-random. Loss of genetic materials was a characteristic feature. The commonest numerical aberrations were -13, -14, -16, -17, -18, and +5. Among them, loss of chromosome 13 was common change observed in all lines. The only recurrent structural aberration was homogeneously staining regions (hsr) observed in three lines. FISH and combined binary ratio labeling (COBRA)-FISH showed in two cases that the hsrs were derived from chromosome 20. Clonal evolution was observed in four of the lines. In one line, hsr was the only change shared by all subclones, suggesting that it might be a primary event in cell immortalization. The results of the present study suggested that loss of chromosome 13 and the amplification of chromosome 20 might be early genetic events involved in ovarian cell immortalization, and might be useful targets for the study of genomic aberrations in ovarian carcinogenesis.

Resistant tissues of modern marchantioid liverworts resemble enigmatic Early Paleozoic microfossils

PubMed Central

Graham, Linda E.; Wilcox, Lee W.; Cook, Martha E.; Gensel, Patricia G.

2004-01-01

Absence of a substantial pretracheophyte fossil record for bryophytes (otherwise predicted by molecular systematics) poses a major problem in our understanding of earliest land-plant structure. In contrast, there exist enigmatic Cambrian–Devonian microfossils (aggregations of tubes or sheets of cells or possibly a combination of both) controversially interpreted as an extinct group of early land plants known as nematophytes. We used an innovative approach to explore these issues: comparison of tube and cell-sheet microfossils with experimentally degraded modern liverworts as analogues of ancient early land plants. Lower epidermal surface tissues, including rhizoids, of Marchantia polymorpha and Conocephalum conicum were resistant to breakdown after rotting for extended periods or high-temperature acid treatment (acetolysis), suggesting fossilization potential. Cell-sheet and rhizoid remains occurred separately or together depending on the degree of body degradation. Rhizoid break-off at the lower epidermal surface left rimmed pores at the centers of cell rosettes; these were similar in structure, diameter, and distribution to pores characterizing nematophyte cell-sheet microfossils known as Cosmochlaina. The range of Marchantia rhizoid diameters overlapped that of Cosmochlaina pores. Approximately 14% of dry biomass of Marchantia vegetative thalli and 40% of gametangiophores was resistant to acetolysis. Pre- and posttreatment cell-wall autofluorescence suggested the presence of phenolic compounds that likely protect lower epidermal tissues from soil microbe attack and provide dimensional stability to gametangiophores. Our results suggest that at least some microfossils identified as nematophytes may be the remains of early marchantioid liverworts similar in some ways to modern Marchantia and Conocephalum. PMID:15263095

Functional Anchoring Lipids for Drug Delivery Carrier Fabrication and Cell Surface Re-Engineering Applications

NASA Astrophysics Data System (ADS)

Vabbilisetty, Pratima

For decades, lipid vesicular bodies such as liposomes have been widely used and explored as biomimetic models of cell membranes and as drug/gene delivery carrier systems. Similarly, micellar iron oxide nanoparticles have also been investigated as potential MRI agents as well as drug delivery carrier systems. Cell surface carbohydrate-protein interactions allow them to serve as markers for recognition of many molecular and cellular activities thereby, are exploited as attractive molecules for surface modification of nanocarrier systems with purpose for tissues specific targeting and biocompatibility. In addition, the cell lipid membrane serves as an important platform for occurrence of many biological processes that are governed and guided by cell surface receptors. Introduction of chemoselective functional groups, via bio-orthogonal conjugation strategies, at the cell surface facilitates many cellular modifications and paves path for novel and potential biomedical applications. Anchoring lipids are needed for liposome surface functionalization with ligands of interest and play important roles in ligand grafting density, liposomes stability and biological activity. On the other hand, anchoring lipids are also needed for cell surface re-engineering by lipid fusion approach and have high impact for ligand insertion efficiency and biological activity. Overall, in this dissertation study, functional anchoring lipids for glyco-functionalized carrier systems and for efficient cell surface re-engineering applications were systematically investigated, respectively. Firstly, investigation of the synthesis of glyco-functionalized liposome systems based on phosphatidylethonalamine (PE) and cholesterol (Chol) anchoring lipids, prepared by post chemically selective functionalization via Staudinger ligation were carried out. The effect of anchor lipids on the stability, encapsulation and releasing capacity of the glycosylated liposomes were investigated by dynamic light scattering (DLS) technique and by entrapping 5, 6-carboxyfluorescein (CF) dye and monitoring the fluorescence leakage, respectively. Overall, the Chol-anchored liposomes showed faster releasing rate than DSPE-anchored liposomes. This could be due to the increase in rigidity of the lipid membrane upon inclusion of Chol, thereby, leading to fast leakage of liposomes. Second, the potential effects of phospholipid (PE) and cholesterol (Chol)-based anchor lipids on cell surface re-engineering via copper free click chemistry were assessed with RAW 264.7 cells as model. The confocal microscopy and flow cytometry results indicated the successful incorporation of biotinylated Chol-based anchor lipids after specific streptavidin-FITC binding onto the cell surface. Higher fluorescence intensities from the cell membrane were observed for Chol-based anchor lipids when compared to DSPE as anchoring lipid. Furthermore, cytotoxicity of the synthesized biotinylated anchor lipids on the RAW 264.7 cells was assessed by MTT assay. The MTT assay results further confirmed that cell surface re-engineering via lipid anchoring approach strategy has very little or negligible amount of cytotoxicity on the cell viability. Thus, this study suggests the possible use of these lipids for potential cell surface re-engineering applications. In addition, synthesis of lipid coated iron oxide nanoparticles via dual solvent exchange approach and their glyco-functionalization via Staudinger ligation were investigated and characterized by FT-IR and TEM techniques. The stability of iron oxide nanoparticles with varying compositions of lipid anchors was evaluated by dynamic light scattering technique.

On the Ni-Ion release rate from surfaces of binary NiTi shape memory alloys

NASA Astrophysics Data System (ADS)

Ševčíková, Jana; Bártková, Denisa; Goldbergová, Monika; Kuběnová, Monika; Čermák, Jiří; Frenzel, Jan; Weiser, Adam; Dlouhý, Antonín

2018-01-01

The study is focused on Ni-ion release rates from NiTi surfaces exposed in the cell culture media and human vascular endothelial cell (HUVEC) culture environments. The NiTi surface layers situated in the depth of 70 μm below a NiTi oxide scale are affected by interactions between the NiTi alloys and the bio-environments. The finding was proved with use of inductively coupled plasma mass spectrometry and electron microscopy experiments. As the exclusive factor controlling the Ni-ion release rates was not only thicknesses of the oxide scale, but also the passivation depth, which was two-fold larger. Our experimental data strongly suggested that some other factors, in addition to the Ni concentration in the oxide scale, admittedly hydrogen soaking deep below the oxide scale, must be taken into account in order to rationalize the concentrations of Ni-ions released into the bio-environments. The suggested role of hydrogen as the surface passivation agent is also in line with the fact that the Ni-ion release rates considerably decrease in NiTi samples that were annealed in controlled hydrogen atmospheres prior to bio-environmental exposures.

Expansion of divergent SEA domains in cell surface proteins and nucleoporin 54.

PubMed

Pei, Jimin; Grishin, Nick V

2017-03-01

SEA (sea urchin sperm protein, enterokinase, agrin) domains, many of which possess autoproteolysis activity, have been found in a number of cell surface and secreted proteins. Despite high sequence divergence, SEA domains were also proposed to be present in dystroglycan based on a conserved autoproteolysis motif and receptor-type protein phosphatase IA-2 based on structural similarity. The presence of a SEA domain adjacent to the transmembrane segment appears to be a recurring theme in quite a number of type I transmembrane proteins on the cell surface, such as MUC1, dystroglycan, IA-2, and Notch receptors. By comparative sequence and structural analyses, we identified dystroglycan-like proteins with SEA domains in Capsaspora owczarzaki of the Filasterea group, one of the closest single-cell relatives of metazoans. We also detected novel and divergent SEA domains in a variety of cell surface proteins such as EpCAM, α/ε-sarcoglycan, PTPRR, collectrin/Tmem27, amnionless, CD34, KIAA0319, fibrocystin-like protein, and a number of cadherins. While these proteins are mostly from metazoans or their single cell relatives such as choanoflagellates and Filasterea, fibrocystin-like proteins with SEA domains were found in several other eukaryotic lineages including green algae, Alveolata, Euglenozoa, and Haptophyta, suggesting an ancient evolutionary origin. In addition, the intracellular protein Nucleoporin 54 (Nup54) acquired a divergent SEA domain in choanoflagellates and metazoans. © 2016 The Protein Society.

Nanotubular topography enhances the bioactivity of titanium implants.

PubMed

Huang, Jingyan; Zhang, Xinchun; Yan, Wangxiang; Chen, Zhipei; Shuai, Xintao; Wang, Anxun; Wang, Yan

2017-08-01

Surface modification on titanium implants plays an important role in promoting mesenchymal stem cell (MSC) response to enhance osseointegration persistently. In this study, nano-scale TiO 2 nanotube topography (TNT), micro-scale sand blasted-acid etched topography (SLA), and hybrid sand blasted-acid etched/nanotube topography (SLA/TNT) were fabricated on the surfaces of titanium implants. Although the initial cell adherence at 60 min among TNT, SLA and TNT/SLA was not different, SLA and SLA/TNT presented to be rougher and suppressed the proliferation of MSC. TNT showed hydrophilic surface and balanced promotion of cellular functions. After being implanted in rabbit femur models, TNT displayed the best osteogenesis inducing ability as well as strong bonding strength to the substrate. These results indicate that nano-scale TNT provides favorable surface topography for improving the clinical performance of endosseous implants compared with micro and hybrid micro/nano surfaces, suggesting a promising and reliable surface modification strategy of titanium implants for clinical application. Copyright © 2017 Elsevier Inc. All rights reserved.

Human Leukocyte Antigen-Presented Macrophage Migration Inhibitory Factor is a Surface Biomarker and Potential Therapeutic Target for Ovarian Cancer

PubMed Central

Patterson, Andrea M; Kaabinejadian, Saghar; McMurtrey, Curtis P; Bardet, Wilfried; Jackson, Ken W; Zuna, Rosemary E; Husain, Sanam; Adams, Gregory P; MacDonald, Glen; Dillon, Rachelle L.; Ames, Harold; Buchli, Rico; Hawkins, Oriana E; Weidanz, Jon A; Hildebrand, William H

2015-01-01

T cells recognize cancer cells via human leukocyte antigen (HLA)/peptide complexes and, when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2-presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n=27) and normal fallopian tube (n=24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared to normal fallopian tube epithelium (p<0.0001), with minimal staining of normal stroma and blood vessels (p<0.0001 and p<0.001 compared to tumor cells) suggesting a therapeutic window. We then demonstrated the anti-cancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2-presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy. PMID:26719579

Non-enzymatic palladium recovery on microbial and synthetic surfaces.

PubMed

Rotaru, Amelia-Elena; Jiang, Wei; Finster, Kai; Skrydstrup, Troels; Meyer, Rikke Louise

2012-08-01

The use of microorganisms as support for reduction of dissolved Pd(II) to immobilized Pd(0) nanoparticles is an environmentally friendly approach for Pd recovery from waste. To better understand and engineer Pd(0) nanoparticle synthesis, one has to consider the mechanisms by which Pd(II) is reduced on microbial surfaces. Escherichia coli, Shewanella oneidensis, and Pseudomonas putida were used as model organisms in order to elucidate the role of microbial cells in Pd(II) reduction under acidic conditions. Pd(II) was reduced by formate under acidic conditions, and the process occurred substantially faster in the presence of cells as compared to cell-free controls. We found no difference between native (untreated) and autoclaved cells, and could demonstrate that even a non-enzymatic protein (bovine serum albumin) stimulated Pd(II) reduction as efficiently as bacterial cells. Amine groups readily interact with Pd(II), and to specifically test their role in surface-assisted Pd(II) reduction by formate, we replaced bacterial cells with polystyrene microparticles functionalized with amine or carboxyl groups. Amine-functionalized microparticles had the same effect on Pd(II) reduction as bacterial cells, and the effect could be hampered if the amine groups were blocked by acetylation. The interaction with amine groups was confirmed by infrared spectroscopy on whole cells and amine-functionalized microparticles. In conclusion, bio-supported Pd(II) reduction on microbial surfaces is possibly mediated by a non-enzymatic mechanism. We therefore suggest the use of amine-rich biomaterials rather than intact cells for Pd bio-recovery from waste. Copyright © 2012 Wiley Periodicals, Inc.

Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system.

PubMed

Das, Krishna; Eisel, David; Lenkl, Clarissa; Goyal, Ashish; Diederichs, Sven; Dickes, Elke; Osen, Wolfram; Eichmüller, Stefan B

2017-01-01

In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor.

Hemocompatibility and biocompatibility of antibacterial biomimetic hybrid films

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

Coll Ferrer, M. Carme; Department of Materials Science and Engineering, University of Pennsylvania, Philadelphia, PA 19104; Eckmann, Uriel N.

In previous work, we developed novel antibacterial hybrid coatings based on dextran containing dispersed Ag NPs (∼ 5 nm, DEX-Ag) aimed to offer dual protection against two of the most common complications associated with implant surgery, infections and rejection of the implant. However, their blood-material interactions are unknown. In this study, we assess the hemocompatibility and biocompatibility of DEX-Ag using fresh blood and two cell lines of the immune system, monocytes (THP-1 cells) and macrophages (PMA-stimulated THP-1 cells). Glass, polyurethane (PU) and bare dextran (DEX) were used as reference surfaces. PU, DEX and DEX-Ag exhibited non-hemolytic properties. Relative to glassmore » (100%), platelet attachment on PU, DEX and DEX-Ag was 15%, 10% and 34%, respectively. Further, we assessed cell morphology and viability, pro-inflammatory cytokines expression (TNF-α and IL-1β), pro-inflammatory eicosanoid expression (Prostaglandin E{sub 2}, PGE{sub 2}) and release of reactive oxygen species (ROS, superoxide and H{sub 2}O{sub 2}) following incubation of the cells with the surfaces. The morphology and cell viability of THP-1 cells were not affected by DEX-Ag whereas DEX-Ag minimized spreading of PMA-stimulated THP-1 cells and caused a reduction in cell viability (16% relative to other surfaces). Although DEX-Ag slightly enhanced release of ROS, the expression of pro-inflammatory cytokines remained minimal with similar levels of PGE{sub 2}, as compared to the other surfaces studied. These results highlight low toxicity of DEX-Ag and hold promise for future applications in vivo. - Highlights: • We examined specific blood-contact reactions of dextran doped with Ag NPs coatings. • Biocompatibility was assessed with THP-1 cells and PMA-stimulated THP-1 cells. • Glass, polyurethane and dextran were used as reference surfaces. • Hybrid coatings exhibited non-hemolytic properties. • Low toxicity, inflammatory response and ROS suggest potential for in vivo use.« less

SNAP23-Dependent Surface Translocation of Leukotriene B4 (LTB4) Receptor 1 Is Essential for NOX2-Mediated Exocytotic Degranulation in Human Mast Cells Induced by Trichomonas vaginalis-Secreted LTB4

PubMed Central

Lee, Young Ah; Kim, Kyeong Ah; El-Benna, Jamel

2016-01-01

ABSTRACT Trichomonas vaginalis is a sexually transmitted parasite that causes vaginitis in women and itself secretes lipid mediator leukotriene B4 (LTB4). Mast cells are important effector cells of tissue inflammation during infection with parasites. Membrane-bridging SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes are critical for fusion during exocytosis. Although T. vaginalis-derived secretory products (TvSP) have been shown to induce exocytosis in mast cells, information regarding the signaling mechanisms between mast cell activation and TvSP is limited. In this study, we found that SNAP23-dependent surface trafficking of LTB4 receptor 1 (BLT1) is required for nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-mediated exocytotic degranulation of mast cells induced by TvSP. First, stimulation with TvSP induced exocytotic degranulation and reactive oxygen species (ROS) generation in HMC-1 cells. Next, TvSP-induced ROS generation and exocytosis were strongly inhibited by transfection of BLT1 small interfering RNA (siRNA). TvSP induced trafficking of BLT1 from the cytosol to the plasma membrane. We also found that knockdown of SNAP23 abrogated TvSP-induced ROS generation, exocytosis, and surface trafficking of BLT1 in HMC-1 cells. By coimmunoprecipitation, there was a physical interaction between BLT1 and SNAP23 in TvSP-stimulated HMC-1 cells. Taken together, our results suggest that SNAP23-dependent surface trafficking of BLT1 is essential for exocytosis in human mast cells induced by T. vaginalis-secreted LTB4. Our data collectively demonstrate a novel regulatory mechanism for SNAP23-dependent mast cell activation of T. vaginalis-secreted LTB4 involving surface trafficking of BLT1. These results can help to explain how the cross talk mechanism between parasite and host can govern deliberately tissue inflammatory responses. PMID:27795355

SNAP23-Dependent Surface Translocation of Leukotriene B4 (LTB4) Receptor 1 Is Essential for NOX2-Mediated Exocytotic Degranulation in Human Mast Cells Induced by Trichomonas vaginalis-Secreted LTB4.

PubMed

Min, Arim; Lee, Young Ah; Kim, Kyeong Ah; El-Benna, Jamel; Shin, Myeong Heon

2017-01-01

Trichomonas vaginalis is a sexually transmitted parasite that causes vaginitis in women and itself secretes lipid mediator leukotriene B 4 (LTB 4 ). Mast cells are important effector cells of tissue inflammation during infection with parasites. Membrane-bridging SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes are critical for fusion during exocytosis. Although T. vaginalis-derived secretory products (TvSP) have been shown to induce exocytosis in mast cells, information regarding the signaling mechanisms between mast cell activation and TvSP is limited. In this study, we found that SNAP23-dependent surface trafficking of LTB 4 receptor 1 (BLT1) is required for nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-mediated exocytotic degranulation of mast cells induced by TvSP. First, stimulation with TvSP induced exocytotic degranulation and reactive oxygen species (ROS) generation in HMC-1 cells. Next, TvSP-induced ROS generation and exocytosis were strongly inhibited by transfection of BLT1 small interfering RNA (siRNA). TvSP induced trafficking of BLT1 from the cytosol to the plasma membrane. We also found that knockdown of SNAP23 abrogated TvSP-induced ROS generation, exocytosis, and surface trafficking of BLT1 in HMC-1 cells. By coimmunoprecipitation, there was a physical interaction between BLT1 and SNAP23 in TvSP-stimulated HMC-1 cells. Taken together, our results suggest that SNAP23-dependent surface trafficking of BLT1 is essential for exocytosis in human mast cells induced by T. vaginalis-secreted LTB 4 Our data collectively demonstrate a novel regulatory mechanism for SNAP23-dependent mast cell activation of T. vaginalis-secreted LTB 4 involving surface trafficking of BLT1. These results can help to explain how the cross talk mechanism between parasite and host can govern deliberately tissue inflammatory responses. Copyright © 2016 American Society for Microbiology.

Bone regeneration performance of surface-treated porous titanium.

PubMed

Amin Yavari, Saber; van der Stok, Johan; Chai, Yoke Chin; Wauthle, Ruben; Tahmasebi Birgani, Zeinab; Habibovic, Pamela; Mulier, Michiel; Schrooten, Jan; Weinans, Harrie; Zadpoor, Amir Abbas

2014-08-01

The large surface area of highly porous titanium structures produced by additive manufacturing can be modified using biofunctionalizing surface treatments to improve the bone regeneration performance of these otherwise bioinert biomaterials. In this longitudinal study, we applied and compared three types of biofunctionalizing surface treatments, namely acid-alkali (AcAl), alkali-acid-heat treatment (AlAcH), and anodizing-heat treatment (AnH). The effects of treatments on apatite forming ability, cell attachment, cell proliferation, osteogenic gene expression, bone regeneration, biomechanical stability, and bone-biomaterial contact were evaluated using apatite forming ability test, cell culture assays, and animal experiments. It was found that AcAl and AnH work through completely different routes. While AcAl improved the apatite forming ability of as-manufactured (AsM) specimens, it did not have any positive effect on cell attachment, cell proliferation, and osteogenic gene expression. In contrast, AnH did not improve the apatite forming ability of AsM specimens but showed significantly better cell attachment, cell proliferation, and expression of osteogenic markers. The performance of AlAcH in terms of apatite forming ability and cell response was in between both extremes of AnH and AsM. AcAl resulted in significantly larger volumes of newly formed bone within the pores of the scaffold as compared to AnH. Interestingly, larger volumes of regenerated bone did not translate into improved biomechanical stability as AnH exhibited significantly better biomechanical stability as compared to AcAl suggesting that the beneficial effects of cell-nanotopography modulations somehow surpassed the benefits of improved apatite forming ability. In conclusion, the applied surface treatments have considerable effects on apatite forming ability, cell attachment, cell proliferation, and bone ingrowth of the studied biomaterials. The relationship between these properties and the bone-implant biomechanics is, however, not trivial. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

Pectin Metabolism and Assembly in the Cell Wall of the Charophyte Green Alga Penium margaritaceum1[W][OPEN

PubMed Central

Domozych, David S.; Sørensen, Iben; Popper, Zoë A.; Ochs, Julie; Andreas, Amanda; Fangel, Jonatan U.; Pielach, Anna; Sacks, Carly; Brechka, Hannah; Ruisi-Besares, Pia; Willats, William G.T.; Rose, Jocelyn K.C.

2014-01-01

The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants. PMID:24652345

Activation of human B cells by phosphorothioate oligodeoxynucleotides.

PubMed Central

Liang, H; Nishioka, Y; Reich, C F; Pisetsky, D S; Lipsky, P E

1996-01-01

To investigate the potential of DNA to elicit immune responses in man, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell-depleted human peripheral blood B cells. Among 47 ODNs of various sequences tested, 12 phosphorothioate oligodeoxynucleotides (sODNs) induced marked B cell proliferation and Ig production. IL-2 augmented both proliferation and production of IgM, IgG, and IgA, as well as IgM anti-DNA antibodies, but was not necessary for B cell stimulation. Similarly, T cells enhanced stimulation, but were not necessary for B cell activation. After stimulation with the active sODNs, more than 95% of B cells expressed CD25 and CD86. In addition, B cells stimulated with sODNs expressed all six of the major immunoglobulin VH gene families. These results indicate that the human B cell response to sODN is polyclonal. Active sODN coupled to Sepharose beads stimulated B cells as effectively as the free sODN, suggesting that stimulation resulted from engagement of surface receptors. These data indicate that sODNs can directly induce polyclonal activation of human B cells in a T cell-independent manner by engaging as yet unknown B cell surface receptors. PMID:8787674

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

A functional glycoprotein competitive recognition and signal amplification strategy for carbohydrate-protein interaction profiling and cell surface carbohydrate expression evaluation

NASA Astrophysics Data System (ADS)

Wang, Yangzhong; Chen, Zhuhai; Liu, Yang; Li, Jinghong

2013-07-01

A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes.A simple and sensitive carbohydrate biosensor has been suggested as a potential tool for accurate analysis of cell surface carbohydrate expression as well as carbohydrate-based therapeutics for a variety of diseases and infections. In this work, a sensitive biosensor for carbohydrate-lectin profiling and in situ cell surface carbohydrate expression was designed by taking advantage of a functional glycoprotein of glucose oxidase acting as both a multivalent recognition unit and a signal amplification probe. Combining the gold nanoparticle catalyzed luminol electrogenerated chemiluminescence and nanocarrier for active biomolecules, the number of cell surface carbohydrate groups could be conveniently read out. The apparent dissociation constant between GOx@Au probes and Con A was detected to be 1.64 nM and was approximately 5 orders of magnitude smaller than that of mannose and Con A, which would arise from the multivalent effect between the probe and Con A. Both glycoproteins and gold nanoparticles contribute to the high affinity between carbohydrates and lectin. The as-proposed biosensor exhibits excellent analytical performance towards the cytosensing of K562 cells with a detection limit of 18 cells, and the mannose moieties on a single K562 cell were determined to be 1.8 × 1010. The biosensor can also act as a useful tool for antibacterial drug screening and mechanism investigation. This strategy integrates the excellent biocompatibility and multivalent recognition of glycoproteins as well as the significant enzymatic catalysis and gold nanoparticle signal amplification, and avoids the cell pretreatment and labelling process. This would contribute to the glycomic analysis and the understanding of complex native glycan-related biological processes. Electronic supplementary information (ESI) available: Experimental details; characterization of probes; the influence of electrolyte pH; probe concentration and glucose concentration on the electrode ECL effect. See DOI: 10.1039/c3nr01598j

A model of extracellular enzymes in free-living microbes: which strategy pays off?

PubMed

Traving, Sachia J; Thygesen, Uffe H; Riemann, Lasse; Stedmon, Colin A

2015-11-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

CD24-Positive Cells from Normal Adult Mouse Liver Are Hepatocyte Progenitor Cells

PubMed Central

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M.; Rao, Pulivarthi H.

2011-01-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45−, Ter119−) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes. PMID:21361791

CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells.

PubMed

Qiu, Qiong; Hernandez, Julio Cesar; Dean, Adam M; Rao, Pulivarthi H; Darlington, Gretchen J

2011-12-01

The identification of specific cell surface markers that can be used to isolate liver progenitor cells will greatly facilitate experimentation to determine the role of these cells in liver regeneration and their potential for therapeutic transplantation. Previously, the cell surface marker, CD24, was observed to be expressed on undifferentiated bipotential mouse embryonic liver stem cells and 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced oval cells. Here, we describe the isolation and characterization of a rare, primary, nonhematopoietic, CD24+ progenitor cell population from normal, untreated mouse liver. By immunohistochemistry, CD24-expressing cells in normal adult mouse liver were colocalized with CK19-positive cholangiocytes. This nonhematopoietic (CD45-, Ter119-) CD24+ cell population isolated by flow cytometry represented 0.04% of liver cells and expressed several markers of liver progenitor/oval cells. The immunophenotype of nonhematopoietic CD24+ cells was CD133, Dlk, and Sca-1 high, but c-Kit, Thy-1, and CD34 low. The CD24+ cells had increased expression of CK19, epithelial cell adhesion molecule, Sox 9, and FN14 compared with the unsorted cells. Upon transplantation of nonhematopoietic CD24+ cells under the sub-capsule of the livers of Fah knockout mice, cells differentiated into mature functional hepatocytes. Analysis of X and Y chromosome complements were used to determine whether or not fusion of the engrafted cells with the recipient hepatocytes occurred. No cells were found that contained XXXY or any other combination of donor and host sex chromosomes as would be expected if cell fusion had occurred. These results suggested that CD24 can be used as a cell surface marker for isolation of hepatocyte progenitor cells from normal adult liver that are able to differentiate into hepatocytes.