One-Pot Reducing Agent-Free Synthesis of Silver Nanoparticles/Nitrocellulose Composite Surface Coating with Antimicrobial and Antibiofilm Activities.

Nitrocellulose with silver nanoparticle (AgNP/NC) composite was prepared in situ using Ag(CH3CO2) and nitrocellulose without any reducing agent. The composite materials synthesized were spray coated onto glass substrates to obtain thin films. The AgNPs/NC composites were characterized by ultraviolet-visible, Fourier transform infrared, X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The antimicrobial activity of AgNPs/NC composite was investigated by tube method and time-kill kinetic studies against three microbial species, including Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and Candida albicans (ATCC 10231). The antibiofilm activities were qualitatively determined against all three organisms. Prepared AgNPs/NC films exhibited good antimicrobial activity and significant inhibition of biofilm development against all three microbial species. The effective dispersion of AgNPs/NC in biofilm was responsible for the significant antibiofilm activity of the prepared material. The reported AgNPs/NC composite can be used as coating additive in bacteriocidal paint which can be applied onto surfaces such as in healthcare environments.

Improving the Patency of Jugular Vein Catheters in Sprague-Dawley Rats by Using an Antiseptic Nitrocellulose Coating.

Preclinical studies in animals often require frequent blood sampling over prolonged periods. A preferred method in rats is the implantation of a polyurethane catheter into the jugular vein, with heparinized glycerol as a lock solution. However, analysis of various biologic compounds (for example, microRNA) precludes the use of heparin. We used sodium citrate as an alternative to heparin but observed more frequent loss of catheter patency. We hypothesized that this effect was due to evaporation of lock solution at the exteriorized portion of the catheter, subsequent blood infiltration into the catheter, and ultimately clot formation within the catheter. We therefore tested evaporation and its variables in vitro by using 5 common catheter materials. We used the migration of dye into vertically anchored catheters as a measure of lock displacement due to evaporation. Exposure to dry room-temperature air was sufficient to cause dye migration against gravity, whereas a humid environment and adding glycerol to the lock solution mitigated this effect, thus confirming loss of the lock solution from the catheter by evaporation. We tested 4 catheter treatments for the ability to reduce lock evaporation. Results were validated in vivo by using male Sprague-Dawley rats (n = 12) implanted with polyurethane jugular vein catheters and randomized to receive a nitrocellulose-based coating on the exteriorized portion of the catheter. Coating the catheters significantly improved patency, as indicated by a Kaplan-Meier log-rank hazard ratio greater than 5 in untreated catheters. We here demonstrate that a simple nitrocellulose coating reduces evaporation from and thus prolongs the patency of polyurethane catheters in rats.

Nano-porous nitrocellulose liquid bandage modulates cell and cytokine response and accelerates cutaneous wound healing in a mouse model.

Nitrocellulose liquid bandage (L-Bandage) is extensively used in hard-to-cover cuts and wounds management, owing to its flexibility, softness, transparency, and conformability. However, evidence supporting their mechanisms of action as wound dressing is scanty. This study introduces a novel nano-porous L-Bandage, and provides results from a mouse full-thickness wound model investigating its mechanism of action on wound healing. Different characteristics, such as porosity, mechanical properties and water vapor transmission rate (WVTR) were determined. The L-Bandage formed film had a porous network structure with mean diameter of 18 nm that could effectively prevent the bacterial invasion, and favorable properties of tensile strength, elongation, and WVTR. The L-Bandage treated wound exhibited accelerated healing, with reduced inflammations, enhanced wound re-epithelialization, contraction, granulation tissue formation, and rapid angiogenesis. Our data suggested that L-Bandage could serve as a promising wound dressing, because of its desirable properties for wound healing.

Techniques of celloidin removal from temporal bone sections.

OBJECTIVES: We sought to determine whether the technique of celloidin removal influences the results of immunostaining in celloidin-embedded cochleae. METHODS: We compared four protocols of celloidin removal, including those using clove oil, acetone, ether-alcohol, and methanol saturated with sodium hydroxide. By optimally fixing our tissue (perfused mice), and keeping constant the fixative type (formalin plus acetic acid), fixation time (25 hours), and decalcification time (ethylenediaminetetraacetic acid for 7 days), we determined whether the technique of celloidin removal influenced the immunostaining results. Six antibodies were used with each removal method: prostaglandin D synthase, sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase), aquaporin 1, connective tissue growth factor, tubulin, and 200 kd neurofilament. RESULTS: Clove oil, acetone, and ether-alcohol resulted in incomplete removal of the celloidin, thereby negatively affecting the results of immunostaining. The methanol-sodium hydroxide method was effective in completely removing the celloidin; it produced the cleanest and most reproducible immunostaining for all six antibodies. CONCLUSIONS: Freshly prepared methanol saturated with sodium hydroxide and diluted 1:2 with methanol was the best solvent for removing celloidin from mouse temporal bone sections, resulting in consistent and reproducible immunostaining with the six antibodies tested.

Substitution of certain amino acids in a short peptide causes a significant difference in their immunoreactivities with antibodies against different epitopes: evidence for possible folding of the peptide on a nitrocellulose or PVDF membrane.

Substitution of amino acids in a peptide caused remarkable differences in its immunoreactivities with antibodies against 3 epitopes in the immobilized peptide. The observed differences in immunoreactivities among the peptides were not due to the differences in efficiencies of their transfer onto nitrocellulose or PVDF membranes. Rather, possible folding of the peptide on the membrane was considered to be the reason for their distinct immunoreactivities with the antibodies.

TGF-beta1 induction of the adenine nucleotide translocator 1 in astrocytes occurs through Smads and Sp1 transcription factors.

BACKGROUND: The adenine nucleotide translocator 1 (Ant1) is an inner mitochondrial membrane protein involved with energy mobilization during oxidative phosphorylation. We recently showed that rodent Ant1 is upregulated by transforming growth factor-beta (TGF-beta) in reactive astrocytes following CNS injury. In the present study, we describe the molecular mechanisms by which TGF-beta1 regulates Ant1 gene expression in cultured primary rodent astrocytes. RESULTS: Transcription reporter analysis verified that TGF-beta1 regulates transcription of the mouse Ant1 gene, but not the gene encoding the closely related Ant2 isoform. A 69 basepair TGF-beta1 responsive element of the Ant1 promoter was also identified. Electrophoretic mobility shift assays demonstrated that astrocyte nuclear proteins bind to this response element and TGF-beta1 treatment recruits additional nuclear protein binding to this element. Antibody supershift and promoter deletion analyses demonstrated that Sp1 consensus binding sites in the RE are important for TGF-beta1 regulation of Ant1 in astrocytes. Additionally, we demonstrate that Smad 2, 3 and 4 transcription factors are expressed in injured cerebral cortex and in primary astrocyte cultures. TGF-beta1 activated Smad transcription factors also contribute to Ant1 regulation since transcription reporter assays in the presence of dominant negative (DN)-Smads 3 and 4 significantly reduced induction of Ant1 by TGF-beta1. CONCLUSION: The specific regulation of Ant1 by TGF-beta1 in astrocytes involves a cooperative interaction of both Smad and Sp1 binding elements located immediately upstream of the transcriptional start site. The first report of expression of Smads 2, 3 and 4 in astrocytes provided here is consistent with a regulation of Ant1 gene expression by these transcription factors in reactive astrocytes. Given the similarity in TGF-beta1 regulation of Ant1 with other genes that are thought to promote neuronal survival, this interaction may represent a general mechanism that underlies the neuroprotective effects of TGF-beta1.

Nova regulates GABA(A) receptor gamma2 alternative splicing via a distal downstream UCAU-rich intronic splicing enhancer.

Nova is a neuron-specific RNA binding protein targeted in patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia, which is characterized by failure of inhibition of brainstem and spinal motor systems. Here, we have biochemically confirmed the observation that splicing regulation of the inhibitory GABA(A) receptor gamma2 (GABA(A)Rgamma2) subunit pre-mRNA exon E9 is disrupted in mice lacking Nova-1. To elucidate the mechanism by which Nova-1 regulates GABA(A)Rgamma2 alternative splicing, we systematically screened minigenes derived from the GABA(A)Rgamma2 and human beta-globin genes for their ability to support Nova-dependent splicing in transient transfection assays. These studies demonstrate that Nova-1 acts directly on GABA(A)Rgamma2 pre-mRNA to regulate E9 splicing and identify an intronic region that is necessary and sufficient for Nova-dependent enhancement of exon inclusion, which we term the NISE (Nova-dependent intronic splicing enhancer) element. The NISE element (located 80 nucleotides upstream of the splice acceptor site of the downstream exon E10) is composed of repeats of the sequence YCAY, consistent with previous studies of the mechanism by which Nova binds RNA. Mutation of these repeats abolishes binding of Nova-1 to the RNA in vitro and Nova-dependent splicing regulation in vivo. These data provide a molecular basis for understanding Nova regulation of GABA(A)Rgamma2 alternative splicing and suggest that general dysregulation of Nova's splicing enhancer function may underlie the neurologic defects seen in Nova's absence.

RNA-structural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon.

Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction. Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site. Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions. Mutational analysis of the mRNA binding site is compatible with the structural model. In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how protein L4 may provoke transcription termination.

Nitrocellulose particles adsorbed to immunoglobulins are a new and effective approach to induce cell activation dependent on receptor aggregation.

Nitrocellulose (NC) has proved to be a versatile tool for the isolation and characterization of various biomolecules. In this report we extend its scope by using antibody-coated NC particles to cross-link molecules on the surface of living cells. Ligation of receptors in Jurkat cells with NC-bound specific antibodies induced protein tyrosine phosphorylation patterns of cellular proteins comparable to conventional antibody cross-linking. In addition, the present study shows that application of NC particles coated with human IgA significantly activated monocytic cells via the Fc alpha receptor (Fc alphaR), whereas cross-linking of receptor-ligand complexes with isotype-specific antibody was less efficient. Subsequent immunoprecipitation and immunoblot analysis of aggregated Fc receptors (FcRs) complexed to Ig-adsorbed particles permits fast identification of molecules involved in the transmission of signals. Therefore, ligand-coated NC particles can be used to examine receptor-mediated cell activation events dependent upon extensive receptor aggregation.

Molecular analysis of animal and plant cells is facilitated by their attachment to collodion (cellulose nitrate) films.

A procedure for the efficient transfer of cell monolayers, cultured on glass coverslips, to microscopy slides has been developed. This technique involves the coating of the upper surface of an ethanol-fixed cultured cell layer with a film of collodion dissolved in n-amyl acetate. The dry collodion-coated cell layer can then be detached by rehydrating it for 1 h under water or phosphate-buffered saline and then carefully peeling it away from the coverslip using a pair of tweezers. Once a cell layer has been so mounted, it can be subjected to rough treatment such as proteolytic degradation (which greatly improves the signal-to-noise ratio in procedures like in situ hybridization [ISH] or primed in situ synthesis [PRINS]) without running the risk of cell detachment because of the partial or total degradation of the extracellular matrix. As an example of its application, we show a PRINS of telomeres from mouse fibroblasts. The high mechanical strength of collodion ensures that the structural integrity and morphology of the cell layer is maintained under experimental conditions where the collodion itself is insoluble. In addition to its use on cell layers, collodion can be used for the production of support films for (i) attaching suspension cell cultures, (ii) immobilizing cells normally cultured in suspension (such as tobacco BY2 cells or germinating tobacco pollen grains) or (iii) planting cryostat sections to microscopy slides. The value of this technique lies in its ease of use and the large number of different applications, in both the plant and animal fields of research, to which it may be applied.

Growth of injured rabbit optic axons within their degenerating optic nerve.

Spontaneous growth of axons after injury is extremely limited in the mammalian central nervous system (CNS). It is now clear, however, that injured CNS axons can be induced to elongate when provided with a suitable environment. Thus injured CNS axons can elongate, but they do not do so unless their environment is altered. We now show apparent regenerative growth of injured optic axons. This growth is achieved in the adult rabbit optic nerve by the use of a combined treatment consisting of: (1) supplying soluble substances originating from growing axons to be injured rabbit optic nerves (Schwartz et al., Science, 228:600-603, 1985), and (2) application of low energy He-Ne laser irradiation, which appears to delay degenerative changes in the injured axons (Schwartz et al., Lasers Surg. Med., 7:51-55, 1985; Assia et al., Brain Res., 476:205-212, 1988). Two to 8 weeks after this treatment, unmyelinated and thinly myelinated axons are found at the lesion site and distal to it. Morphological and immunocytochemical evidence indicate that these thinly myelinated and unmyelinated axons are growing in close association with glial cells. Only these axons are identified as being growing axons. These newly growing axons transverse the site of injury and extend into the distal stump of the nerve, which contains degenerating axons. Axons of this type could be detected distal to the lesion only in nerves subjected to the combined treatment. No unmyelinated or thinly myelinated axons in association with glial cells were seen at 6 or 8 weeks postoperatively in nerves that were not treated, or in nerves in which the two stumps were completely disconnected. Two millimeters distal to the site of injury, the growing axons are confined to a compartment comprising 5%-30% of the cross section of the nerve. A temporal analysis indicates that axons have grown as far as 6 mm distal to the site of injury, by 8 weeks postoperatively. Anterograde labeling with horseradish peroxidase, injected intraocularly, indicates that some of these newly growing axons arise from retinal ganglion cells.

[Physicochemical mechanisms of the action of gramicidin S on a model membrane]. / Izuchenie fiziko-khimicheskikh mekhanizmov deistviia gramitsidina S na model'nom membrane.

Influence of gramicidin S on electric parameters of nitrocellulose ultrafilter as a biomembrane model was studied, the ultrafilters being impregnated with fatty acids or their ethers. It was shown that addition of the antibiotic to the solution over one side of the model membrane resulted in generation of electric potential. With increasing of the drug concentration by one order there was observed more than a 10-fold drop in the membrane resistance while the electric capacitance actually remained unchanged. It was suggested that gramicidin S was localized in thin water layers covering the surface of the ultrafilter pores and separating the polymer matrix and impregnating liquid filling the pores. Such incorporation led to changes in the state of water and water channel surfaces which defined the increase in the model membrane electric conductivity.

Wet cleaving of cells: a method to introduce macromolecules into the cytoplasm. Application for immunolocalization of cytosol-exposed antigens.

A new method, called "wet cleaving," has been introduced to allow direct exposure of cytoplasm to externally supplied macromolecules by mechanical rupture of the plasma membrane. Monolayers of adherent cells or poly-L-lysine-attached suspension cells are overlayed with nitrocellulose sheets. By subsequent removal of the sheets, cells are cleaved, thereby exposing the cytoplasm. The method allows bulk quantities of cells to be cleaved in an efficient manner. Cleavage, although imposing some mechanical stress on the cells, leaves most if not all organelles morphologically intact, as shown by electron microscopy. Mechanically ruptured cells are well suited for use in immunocytochemical studies, as is demonstrated with the immunofluorescence localization of vinculin in chicken embryo fibroblasts.

Assay and characterization of carbohydrate binding by the lectin discoidin I immobilized on nitrocellulose.

Discoidin I is the most abundant galactose binding lectin produced by the cellular slime mold Dictyostelium discoideum and has been implicated in cell-substratum adhesion. We have developed an assay of carbohydrate binding activity utilizing binding of 125I-asialofetuin to discoidin I, or to other lectins, immobilized on nitrocellulose. Among the proteins examined, only lectins exhibited the ability to bind asialofetuin. Specificity of asialofetuin binding was demonstrated by competition with monosaccharides, which inhibited binding consistent with the known sugar specificity of the lectins examined. Experiments with fetuin and derivatives differing in their oligosaccharide structure indicated a requirement for terminal galactosyl residues for probe binding to discoidin I. We have used this assay to characterize the carbohydrate binding behavior of discoidin I. The extent of asialofetuin binding to discoidin I was dependent on the concentrations of both lectin and ligand. Interpretation of equilibrium binding data suggested that, under saturating conditions, 1 mol of oligosaccharide was bound per mole discoidin I monomer. Furthermore, discoidin I in solution and discoidin I on nitrocellulose were equally effective at competing for soluble asialofetuin, suggesting that immobilization had no effect on the carbohydrate binding behavior of discoidin I. Binding was strongly inhibited by ethylenediaminetetraacetic acid; both Ca2+ and Mn2+ could overcome that inhibition, but Mg2+ could not. Preincubation of discoidin I at 60 degrees C stimulated asialofetuin binding 2-fold by increasing the affinity, while preincubation at higher temperatures resulted in a complete loss of activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of human factor VIII by avidin-biotin dot immunobinding ELISAs.

Hemophilia A is a congenital bleeding disorder which is characterized by a functional deficiency of the coagulation protein factor VIII. We have developed sensitive enzyme-linked immunosorbent assays (ELISAs) for measuring the antigenic reactivity of factor VIII. The assays utilize dot immunobinding techniques, commercial monoclonal antibodies, and a detection system enhanced by the interaction of avidin and biotin. The dot immunobinding ELISAs were optimized for measuring factor VIII in normal and hemophilic plasma, and in partially and highly purified preparations of factor VIII. Linear standard curves were established for all samples, defining the range for accurate measurement. Factor VIII was detected at concentrations as low as 0.0005 U/ml, which represents 0.1 pg of protein.

Nitrocellulose and polyvinyl coatings prevent sperm adhesion to glass without affecting the motility of intact and demembranated human spermatozoa.

The effectiveness of several glass coating agents in preventing sperm adherence to glass surfaces for both intact and demembranated human spermatozoa was investigated. These agents included bovine serum albumin (BSA), Sigmacote, poly glu-lys, Collodion and Formvar. The presence of at least 5% of seminal plasma in sperm suspensions prevented sperm adhesion to glass surfaces. On the other hand, 56% of washed spermatozoa resuspended in an isoosmotic buffer containing 1 mg/ml of BSA attached to glass. Collodion, Formvar and Sigmacote reduced sperm attachment to glass to 2, 5 and 7%, respectively. BSA was partially effective, with 20% sperm adherence to glass, and poly glu-lys was totally ineffective. Whereas Sigmacote and BSA coatings lacked transparency, Collodion always achieved the best light transmission. Demembranated reactivated spermatozoa were all attached to glass within 90 seconds of contact. This adhesion was prevented by Collodion and Formvar. Other agents were less effective or interfered with motility. In contrast to intact spermatozoa, demembranated spermatozoa have a very low progressiveness ratio (vector speed/track speed), a wide beating amplitude and because of their whiplash flagellar movement, their motility resembles that of hamster capacitated spermatozoa.

Nitrocellulose-based RAST to detect IgE antibodies in workers hypersensitive to diphenylmethane-4,4'-diisocyanate.

RAST was performed using both nitrocellulose (NC) and paper discs to compare these solid supports for ability to detect IgE antibodies in sera from two patients with hypersensitivity to the industrial chemical, diphenylmethane-4,4'-diisocyanate (MDI). NC disc had distinct advantages in ease of coupling the hapten-conjugate antigen to discs, and in use of lesser amounts of antigen. With regard to sensitivity, NC discs were better able to distinguish the positive sera from 34 control sera including those with elevated levels of total IgE. In both RAST systems, inhibition studies indicated high specificity of antibodies for MDI, with no reactivity detected toward the serum albumin portion of the MDI-HSA conjugate antigen. However, only in the NC RAST did antibodies from both patients react with p-tolyl isocyanate inhibitor. Based on the above results, NC discs displayed several advantages in RAST and are recommended for routine serological assay of isocyanate-specific antibodies.

Migratory capacity of large granular lymphocytes from T-gamma-lymphoproliferative disorders.

We have investigated 10 patients with T gamma-lymphoproliferative disorders (T gamma LPD) for cell migratory capacity. Large granular lymphocytes (LGLs) expanding in these subjects expressed natural killer cell markers (HNKI, B73.1, AB8.28, N901, OKM1) to a variable extent. The patients' LGLs mediated antibody-dependent cellular cytotoxicity, while appreciable natural killer activity was only measurable in 3 patients. The migratory capacity of T gamma LPD was examined by using nitrocellulose filters. The patients' LGLs migrated into filters and showed responsiveness (in 9 of 10 patients) to activated serum, used as chemoattractant, as normal LGLs do. No clearcut correlation emerged between in vitro migratory capacity and disease aggressiveness or involvement of abdominal organs. These results confirm in T gamma LPD the migratory potential of LGLs and suggest the possibility that acquisition of enhanced locomotor capacity is not a crucial determinant of disease aggressiveness in T gamma LPD.

[A new embedding medium for making histological preparations]. / Novaia zalivochnaia sreda dlia prigotovleniia gistologicheskikh preparatov.

A new embedding and infiltrating medium, based on polyethylene glycol and celloidine, is suggested in addition to a new technique for treatment of fixed biological material for morphological and histochemical studies. The technique enables us to make sections 3-10 mkm thick with a well preserved distribution of substances in cellular and tissue structures.