Coating and selective deposition of nanofilm on silicone rubber for cell adhesion and growth

A recently developed method for surface modification, layer-by-layer (LbL) assembly, has been applied to silicone, and its ability to encourage endothelial cell growth and control cell growth patterns has been examined. The surfaces studied consisted of a precursor, with alternating cationic polyethyleneimine (PEI) and anionic sodium polystyrene sulfonate (PSS) layers followed by alternating gelatin and poly-d-lysine (PDL) layers. Film growth increased linearly with the number of layers. Each PSS/PEI bilayer was 3 nm thick, and each gelatin/PDL bilayer was 5 nm thick. All layers were more hydrophilic than the unmodified silicone rubber surface, as determined from contact angle measurements. The contact angle was primarily dictated by the outermost layer. Of the coatings studied, gelatin was the most hydrophilic. A film of (PSS/PEI)₄/(gelatin/PDL)₄/ gelatin was highly favorable for cell adhesion and growth, in contrast to films of (PSS/PEI)₈ or (PSS/PEI)₈/PSS. Cell growth patterns were successfully controlled by selective deposition of microspheres on silicone rubber, using microcontact printing with a silicone stamp. Cell adhesion was confined to the region of microsphere deposition. These results demonstrate that the LbL self-assembly technique provides a general approach to coat and selectively deposit films with nanometer thickness on silicone rubber. Furthermore, they show that this method is a viable technique for controlling cellular adhesion and growth.     

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K⁺ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K⁺ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.     

Sequential actions of glucose oxidase and peroxidase in molecular films assembled by layer-by-layer alternate adsorption

Molecular films of protein/polyion layers were assembled by means of alternate adsorption through electrostatic interaction. Glucose oxidase (GOD) and peroxidase (POD) were assembled in combination with sodium poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI), respectively. Enzyme activities of those films on specific substrates (glucose and H(2)O(2)) were examined by coloring reaction of dye DA67. A multienzyme film containing GOD layer and POD layer was prepared by alternate adsorption of POD/PSS followed by PEI/GOD. Sequential redox reaction of glucose/H(2)O(2)/DA67 was demonstrated successfully with this supramolecular system. (c) 1996 John Wiley & Sons, Inc.     

The decline in antibodies to hepatitis C virus during antiviral therapy

The goal of the study was to analyze B-cell response to hepatitis C virus during antiviral therapy among responders and non-responders. The content of antibodies to individual structural and non-structural HCV proteins was investigated during two years in three groups of patients: initial responders, non-responders and a reference group (without therapy). Treated patients in all groups exhibited the decrease in antibodies to analyzed HCV proteins, but with different patterns. The first statistically significant differences in the decline of the virus-specific antibodies between initial responders and non-responders were observed within the first three months after the beginning of therapy. Some treated patients demonstrated the decrease in antibody levels to HCV proteins after the end of therapy.     

Differences in Receptor Specificity between the Influenza Viruses of Duck,Chicken, and Human

The affinity of the duck, chicken, and human influenza viruses to the host cell sialosides was determined, and considerable distinctions between duck and chicken viruses were found. Duck viruses bind to a wide range of sialosides, including the short-stem gangliosides. Most of the chicken viruses, like human ones, lose the ability to bind these gangliosides, which strictly correlates with the appearance of carbohydrate at position 158–160. The affinity of the chicken viruses to sialoglycoconjugates of chicken intestine as well as chicken, monkey, and human respiratory epithelial cells exceeds that of the duck viruses. The human influenza viruses have high affinity to the same cells but do not bind at all to the duck epithelial cell. This testifies to the absence of 6"-sialylgalactose residues from the duck cells, in contrast to chicken and monkey cells. The alteration of the receptor specificity of chicken viruses in comparison with duck ones results in the similarity of the patterns of accessible cells for chicken and human influenza viruses. This may be the cause of the appearance of the line of H9N2 viruses from Hong Kong live bird markets with receptor specificity similar to that of H3N2 human viruses, and of the ability of H5N1 and H9N2 chicken influenza viruses to infect humans.     

New nanocomposite films for biosensors: layer-by-layer adsorbed films of polyelectrolytes, proteins or DNA

This report describes the construction of ultrathin multicomponent films with an internal structure on the nanometre scale. In the simplest case, the films are built-up by the subsequent adsorption of polyanions and polycations. They can be fabricated on inorganic substrates such as glass, quartz or silicon wafers, or on various organic materials. The polymeric interlayers can incorporate materials with desired electrical or optical properties. The average thickness of the layers can be fine-tuned with Angstrom precision by the addition of suitable salts. They are temperature stable up to at least 200°C and can be laterally structured using conventional photolithographic techniques. The films provide for a well-defined substrate-independent interface for the immobilization of biological macromolecules, such as proteins or DNA, in their active state. The immobilization of streptavidin enables the controlled attachment of any biotinylated molecule with no resulting loss in its biological activity. Area-selective immobilization provides the possibility of built-in quality control for the fabrication of biosensors with separated reference and sample areas on the same substrate.     

Microcapsule modification with peroxidase-catalyzed phenol polymerization

A biocatalytic polymer synthesis on a surface of polyelectrolyte microcapsules was studied. Horseradish peroxidase assembled in nanoorganized capsule walls by alternate adsorption with linear polyions retains its activity in reactions of enzyme-catalyzed polymerization of 4-oxyphenols. It allowed controllable synthesis of a phenolic polymer layer on microcapsule walls using an outermost surface peroxidase layer as a template. By varying the phenol type, buffer pH, and reaction component concentrations, the phenolic polymer coating of the capsules with a thickness in the range 20-50 nm was formed. The polymeric products are fluorescent, which provided a good opportunity for confocal image analysis of the capsule wall structure and the attached layer. The influence of a phenolic polymer layer on the permeability of the capsule walls was investigated.