Coated Glass Coverslips Research Articles

Pyelonephritic Escherichia coli Expressing P Fimbriae Decrease Immune Response of the Mouse Kidney

P fimbriae are proteinaceous appendages on the surface of Escherichia coli bacteria that mediate adherence to uroepithelial cells. E. coli that express P fimbriae account for the majority of ascending urinary tract infections in women with normal urinary tracts. The hypothesis that P fimbriae on uropathic E. coli attach to renal epithelia and may regulate the immune response to establish infection was investigated. The polymeric Ig receptor (pIgR), produced by renal epithelia, transports IgA into the urinary space. Kidney pIgR and urine IgA levels were analyzed in a mouse model of ascending pyelonephritis, using E. coli with (P+) and without (P-) P fimbriae, to determine whether P(+) E. coli regulate epithelial pIgR expression and IgA transport into the urine. (P+) E. coli establish infection and persist to a greater amount than P(-) E. coli. P(+)-infected mice downregulate pIgR mRNA and protein levels compared with P(-)-infected or PBS controls at > or =48 h. The decrease in pIgR was associated with decreased urinary IgA levels in the P(+)-infected group at 48 h. pIgR mRNA and protein also decline in P(+) E. coli-infected LPS-hyporesponsive mice. These studies identify a novel virulence mechanism of E. coli that express P fimbriae. It is proposed that P fimbriae decrease pIgR expression in the kidney and consequently decrease IgA transport into the urinary space. This may explain, in part, how E. coli that bear P fimbriae exploit the immune system of human hosts to establish ascending pyelonephritis.

β-Catenin Interacts With the FUS Proto-oncogene Product and Regulates Pre-mRNA Splicing

beta-Catenin is a downstream effector of the Wnt signaling pathway and is believed to exert its oncogenic function by activating T-cell factor (TCF)/lymphoid enhancer factor (LEF) family transcriptional factors. However, it is still uncertain whether the diverse effects of beta-catenin are caused solely by aberrant gene transactivation. In this study, we used a proteomics approach to obtain further insight into the functional properties of nuclear beta-catenin. The protein assembly of a native beta-catenin-containing complex in nuclear extracts from a colorectal cancer cell line, DLD-1, was identified using immunoprecipitation and mass spectrometry. beta-Catenin physically interacted with fusion (FUS)/translocated in liposarcoma (TLS) and various RNA-binding proteins. The expression of FUS/TLS was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. The transient transfection of FUS suppressed beta-catenin-evoked gene transactivation of TCF/LEF, and beta-catenin transfection affected the splicing pattern of the E1A minigene and induced a novel splicing variant of estrogen receptor (ER)-beta exerting a dominant-negative activity. Human cancer expresses a large variety of alternatively spliced messenger RNA (mRNA), but the precise molecular mechanisms responsible for cancer-related alternative splicing are largely unknown. In this study, we demonstrated the interaction of beta-catenin with FUS/TLS and other RNA-binding proteins involved in the regulation of pre-mRNA splicing. Certain mRNA splicing abbreviations seen in human cancers may be induced by the activation of the Wnt signaling pathway.

Indirect activation of adenosine A1 receptors in cultured rat hippocampal neurons by volatile anaesthetics

Volatile anaesthetics depress excitatory signal transmission by potentiating the inhibitory action of GABAA receptors and there is strong evidence that this is related with anaesthesia. Using primary hippocampal cultures we analyzed the possibility that the volatile anaesthetics enflurane and sevoflurane depress excitatory signal transmission by activation of adenosine A1 receptors. Primary rat hippocampal cultures on 4 cm poly-L-lysine coated glass coverslips were loaded with the Ca2+-indicator fluo-3 and incorporated in a gastight, temperature-controlled perfusion chamber. The intracellular Ca2+-concentration was monitored with a confocal laser-scanning microscope (BioRad) using the 488 nm laser line of a krypton-argon laser for excitation and the Lasersharp Acquisition software for analysis. Continuous perfusion in Mg2+-free medium generated spontaneous synchronized calcium oscillations, which were dose dependently depressed by the volatile anaesthetics enflurane and sevoflurane (0.25-1 minimum alveolar concentration). Addition of 100 nmol of 2-chloro-N6-cyclopentyladenosine, a specific adenosine A1 receptor antagonist, partly reversed the anaesthetic-induced inhibition of the oscillation amplitude of the oscillating cells. The effect of the anaesthetics was mimicked by the addition of S-(p-nitrobenzyl)-6-thioguanosine, an adenosine transport inhibitor and by the addition of 5-amino-5-deoxyadenosine, an inhibitor of adenosine kinase. The volatile anaesthetics sevoflurane and enflurane activate adenosine A1 receptors in primary rat hippocampal cultures. This effect is mediated by liberation of adenosine most likely by an interaction of the volatile anaesthetics with adenosine transport or key enzymes in adenosine metabolism.

Identification of a Novel Recognition Sequence for Fibronectin within the NH2-terminal β-Sandwich Domain of Tissue Transglutaminase

Tissue transglutaminase belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. Unlike other transglutaminases, it is involved in cell-matrix interactions and serves as an adhesion co-receptor for fibronectin. Previous work established that the fibronectin-binding motif(s) is located within the NH2-terminal proteolytic fragment of the protein consisting of residues 1-272. Here we identify a novel fibronectin recognition site within this sequence of tissue transglutaminase. Substitution of individual domains of tissue transglutaminase with those from homologous factor XIIIA showed that the major fibronectin-binding site is present within the first beta-sandwich domain of the protein. Experiments with deletion mutants of the first domain revealed that amino acids 81-140 of tissue transglutaminase are involved in fibronectin binding. Using synthetic peptides encompassing this region, we found that the peptide 88WTATVVDQQDCTLSLQLTT106 inhibited the interaction of tissue transglutaminase with fibronectin and decreased transglutaminase-dependent cell adhesion and spreading. In the three-dimensional structure of the first domain, amino acids 88-106 comprise an extended hairpin formed by antiparallel beta strands 5 and 6. Mutations of Asp94 and Asp97 within the beta5/beta6 hairpin to Ala significantly reduced the affinity of tissue transglutaminase for fibronectin, indicating that these residues are critical for fibronectin binding. Identification of the fibronectin-binding site on tissue transglutaminase will help to dissect the role of this protein in cell-matrix interactions.

Characterization of the Protein Dimerization Domain Responsible for Assembly of Functional Selenodeiodinases

Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.

Sizing of single fluorescently stained DNA fragments by scanning microscopy

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-L-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7-14%. The proposed method is straightforward and can be applied to standardized microtiter plates.

Adhesion, cytoskeletal architecture and activation status of primary human macrophages on a diamond-like carbon coated surface

Diamond-like carbon is a promising surface coating for biomedicinal implants like coronary stents or hip joints. Before widespread clinical use of this material, its biocompatibility has to be thoroughly assessed. Cells likely to encounter a diamond-like coated implant in the human body are cells of the monocytic lineage. Their interaction with the diamond-like carbon coated surface will probably critically influence the fate of the implant, as monocytes orchestrate inflammatory reactions and also affect osseointegration of implants. We therefore investigated adhesion, cytoarchitecture and activation status of primary human monocytes and their differentiated derivatives, macrophages, on diamond-like coated glass coverslips using immunofluorescence technique. We show that adhesion of primary monocytes to a diamond-like-coated coverslip is slightly, but not significantly, enhanced in comparison to uncoated coverslips, while the actin and microtubule cytoskeletons of mature macrophages show a normal development. The activation status of macrophages, as judged by polarization of the cell body, was not affected by growth on a diamond-like carbon surface. We conclude that diamond-like carbon shows good indications for biocompatibility to blood monocytes in vitro. It is therefore unlikely that contact with a diamond-like carbon coated surface in the human body will elicit inflammatory signals by these cells.

Cleavage of the Actin-capping Protein α-Adducin at Asp-Asp-Ser-Asp633-Ala by Caspase-3 Is Preceded by Its Phosphorylation on Serine 726 in Cisplatin-induced Apoptosis of Renal Epithelial Cells

Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328-13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of alpha-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human alpha-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp(29)-Glu, Asp-Ile-Val-Asp(208)-Arg, and Asp-Asp-Ser-Asp(633)-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp(633)-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of alpha-adducin and gamma-adducin in the MARCKS domain that preceded alpha-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of alpha-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of alpha-adducin at Asp-Asp-Ser-Asp(633)-Ala.

Molecular barriers to biomaterial thrombosis by modification of surface proteins with polyethylene glycol

For cardiovascular biomaterials, thrombosis, thromboembolism and vascular graft occlusion are believed to be precipitated by the adsorption of proteins containing adhesive ligands for platelets. Polyethylene-glycol-diisocyanate (PEG-diisocyanate, 3400 MW) may potentially react with protein amines to form molecular barriers on adsorbed proteins on biomaterials, thereby masking adhesive ligands and preventing acute surface thrombosis. To test this notion, PE, PTFE, and glass microconduits were pre-adsorbed with fibrinogen and treated with PEG-diisocyanate, non-reactive PEG-dihydroxyl, or remained untreated. Following perfusion of 111In-labeled platelets in whole human blood for 1 min (wall shear rate=312 s -1), PEG-diisocyanate treated surfaces experienced 96% (PE), 97% (PTFE) and 94% (glass) less platelet deposition than untreated surfaces. Similar reductions were seen for PEG-diisocyanate versus PEG-dihydroxyl treatment. Low shear perfusions of plasma for one hour prior to blood contact did not reduce the inhibitory effect of PEG-diisocyanate. Platelet adhesion onto collagen coated glass coverslips and platelet deposition onto preclotted Dacron was also reduced by treatment with PEG-diisocyanate (93 and 91%, respectively). Protein-reactive PEG may thus have utility in forming molecular barriers on surface associated proteins to inhibit acute thrombosis on cardiovascular biomaterials.

Prolonged Bleeding Time With Defective Platelet Filopodia Formation in the Wistar Furth Rat

Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine–coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF rats.

Prolonged Bleeding Time With Defective Platelet Filopodia Formation in the Wistar Furth Rat

AbstractHereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine–coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF rats.

Human chorionic gonadotropin (hCG) increases cytosolic free calcium in adult rat Leydig cells

Regulation of steroidogenesis by luteinizing hormone is mediated by cAMP and calcium. We have investigated changes in cytosolic free calcium ([Ca 2+] i) in Leydig cells by using Fura-2 as the fluorescent calcium indicator. Purified Leydig cells were plated on polylysine coated glass coverslips, cultured for 24 h in DMEM F12 and loaded with Fura-2 at 37°C. [Ca 2+] i measurements were made fluorometrically by placing coverslips into 3 ml cuvettes with PBS + calcium. Addition of hCG increased [Ca 2+] i gradually after a lag of about 2–3 min and plateaued by 5–6 min. The plateau level was sustained for at least 15 min. Absence of external Ca 2+ in the medium or presence of diltiazem or nicardipine or cobalt chloride abolished the rise. Addition of BAY K 8644 or KCl caused a small but significant increase of [Ca 2+] i. 8-Br-cAMP, forskolin or cholera toxin produced a gradual rise in [Ca 2+] i that plateaued after 5–6 min similar to that observed with hCG. The action of hCG was inhibited by protein kinase A inhibitor (20-residues peptide) but not by protein kinase C inhibitor (staurosporine). We conclude that binding of hCG to its receptors would transmit the signal through G proteins to adenylyl cyclase to increase cAMP which would increase Ca 2+ influx into cytosol across plasma membrane Ca 2+ channels. Therefore, it appears that the primary action of hCG is to increase cytosolic cAMP which would then regulate [Ca 2+] i as well as steroidogenesis.

Characterization of the Growth of Cultured Cortical Neurons on Sections of Adult Rat Cortex

Cultured neurons isolated from the embryonic day 18 (E18) rat cortex were plated onto cryostat sections of adult CNS and peripheral nerve. The ability of these sections to support neuronal attachment and neurite extension was compared to that of neurons on the adjacent poly-L-lysine (PLL)-coated glass coverslips. A quantitative analysis of the data demonstrated that sections of cerebral cortex provide a good substrate for neuronal attachment and growth of neurites, similar to that of the PLL-coated glass coverslips. Sections of sciatic nerve demonstrated a decreased ability to support neuronal attachment and the growth of processes as compared to sections of cortex. Sections of optic nerve supported limited neuronal attachment, and the few neurons that were present on top of the sections were devoid of processes. Control experiments showed that the ability of the cortical sections to support the attachment and growth of cultured neurons was due to the molecular composition of the sections and not to influences of the tissue culture environment. The interactions of neurons with the sections of CNS cortex were partially dependent on a member of the beta 1 integrin family of extracellular matrix receptors present on cultured neurons. This in vitro system allowed for the definition of some of the molecular and the cellular interactions that may occur between growing axons and the environment of the adult cortex.

A new medium for in vitro peripheral nervous tissue myelination without the use of antimitotics

This paper describes a new medium which promotes myelin formation in cultures of rat dorsal root ganglia, and controls fibroblast proliferation. The medium, medium ‘M’, contains d-valine rather than l-valine and is added to cultures after seven days in vitro (DIV). The presence of d-amino acid oxidase in Schwann cells and neurons permits these cells to convert d-valine to l-valine, an essential amino-acid. Fibroblasts however lack d-amino acid oxidase but as some l-valine is present in the fetal calf serum component of medium ‘M’ those fibroblasts already present in the cultures by day 7 are maintained. Further fibroblast proliferation is however inhibited. The use of medium ‘M’ does not require the use of specific antimitotic agents making it cheaper and quicker to obtain myelinated cultures than with previous methods. The medium results in widespread myelin formation by 21 DIV. Cultures may be grown on plastic or collagen coated glass coverslips.

GACH-prepared specimens for sem and confocal reflected light microscopy

Biological specimens prepared for SEM are difficult to observe in detail by means of light microscopy. Conventional epi-illuminescence produces relatively poor images with reflected light due to poor contrast and multiple scattering of the light (1). However, we have found that mammalian tissue culture cells prepared for SEM by means of the co-polymerized glutaraldehyde-carbohydrazide (GACH) technique (2), can be alternately observed by means of SEM and reflected light microscopy if the light optical instrument is a confocal tandem scanning microscope (TSM). The images produced with TSM show resolution comparable to that of the SEM at magnifications ≤ 1,000 X. Furthermore, due to the extremely small depth of focus containing high contrast and brightness, optical sections can be produced which bear detail similar to that in conventional TEM observations.Fibroblasts and myotobes, from newborn rat hindlimb muscles were grown in Eagle's minimum essential medium with 10% fetal calf serum on collagen- coated glass cover slips for a period of 7 d (3).

Transmission and scanning electron microscopy of cell monolayers grown on polymethylpentene coverslips.

Plastic coverslips made of polymethylpentene serve as excellent substrates for growth of bovine endothelial cells, and are easily processed for both transmission (TEM) and scanning (SEM) electron microscopy. Portions of the same coverslip (monolayer) are used for both SEM and TEM examination and are fixed, postfixed, and dehydrated as a single entity. The portion of the coverslip for SEM is then excised, critical point dried, and mounted for sputter coating prior to viewing. The remaining piece of coverslip used for TEM is Epon-Araldite embedded, polymerized, separated from the coverslip by liquid nitrogen immersion, and sectioned either "en face" or in cross section for viewing. Coated glass coverslips are not required and organic solvents such as propylene oxide, acetone, and amyl acetate can be used for dehydration and infiltration. Furthermore, specimens do not require re-embedding or blocks to be glued onto blank capsules before sectioning. The number of cells needed to achieve a monolayer is significantly reduced compared to the usual culture flasks, but are abundant enough to assess ultrastructural changes accurately. Support films may be required to prevent folding of the ultrathin section which can obstruct viewing of cells located on the edge of the section.