Immunohistochemical localization of glucocorticoid receptors in the human cochlea.

In the present study we investigated the localization of glucocorticoid receptors (GCR) in the human inner ear using immunohistochemistry. Celloidin-embedded cochlear sections of patients with normal hearing (n = 5), patients diagnosed with MD (n = 5), and noise induced hearing loss (n = 5) were immunostained using GCR rabbit affinity-purified polyclonal antibodies and secondary fluorescent or HRP labeled antibodies. Digital fluorescent images were acquired using a light sheet laser confocal microscope. In celloidin-embedded sections GCR-IF was present in the cell nuclei of hair cells and supporting cells of the organ of Corti. GCR-IF was detected in cell nuclei of the Reisner's membrane. GCR-IF was seen in cell nuclei of the stria vascularis and the spiral ligament. GCR-IF was found in the spiral ganglia cell nuclei, however, spiral ganglia neurons showed no GCR-IF. Although GCRs were found in most cell nuclei of the cochlea, the intensity of IF was differential among the different cell types being more intense in supporting cells than in sensory hair cells. The differential expression of GCR receptors found in the human cochlea may help to understand the site of action of glucocorticoids in different ear diseases.

13 CO2 labelling as a tool for elucidating the mechanism of cuticle development: a case of Clusia rosea.

The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. Epicuticular differed in composition from IW. The newly synthetised wax was deposited in IW first and later in EW. Both young and mature leaves synthetised IW and EW. The faster dynamics in young leaves were due to lower wax coverage, not a faster synthesis rate. Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of C. rosea. We discuss the ecophysiological significance of the new insights.

Lactose repressor hinge domain independently binds DNA.

The short 8-10 amino acid "hinge" sequence in lactose repressor (LacI), present in other LacI/GalR family members, links DNA and inducer-binding domains. Structural studies of full-length or truncated LacI-operator DNA complexes demonstrate insertion of the dimeric helical "hinge" structure at the center of the operator sequence. This association bends the DNA ∼40° and aligns flanking semi-symmetric DNA sites for optimal contact by the N-terminal helix-turn-helix (HtH) sequences within each dimer. In contrast, the hinge region remains unfolded when bound to nonspecific DNA sequences. To determine ability of the hinge helix alone to mediate DNA binding, we examined (i) binding of LacI variants with deletion of residues 1-50 to remove the HtH DNA binding domain or residues 1-58 to remove both HtH and hinge domains and (ii) binding of a synthetic peptide corresponding to the hinge sequence with a Val52Cys substitution that allows reversible dimer formation via a disulfide linkage. Binding affinity for DNA is orders of magnitude lower in the absence of the helix-turn-helix domain with its highly positive charge. LacI missing residues 1-50 binds to DNA with ∼4-fold greater affinity for operator than for nonspecific sequences with minimal impact of inducer presence; in contrast, LacI missing residues 1-58 exhibits no detectable affinity for DNA. In oxidized form, the dimeric hinge peptide alone binds to O1 and nonspecific DNA with similarly small difference in affinity; reduction to monomer diminished binding to both O1 and nonspecific targets. These results comport with recent reports regarding LacI hinge interaction with DNA sequences.

Electrochemical Assay for a Total Cellulase Activity with Improved Sensitivity.

Electrochemical methods allow fast and inexpensive analysis of enzymatic activities. Here, we report a simple and yet efficient electrochemical assay for the total activity of cellulase, a hydrolytic enzyme widely used in food and textiles industries, and for production of bioethanol. The assay exploits the difference in electrochemical signals from a soluble redox indicator, ferricyanide, on nitrocellulose films treated by cellulases. Ferricyanide electrochemistry is totally inhibited on graphite electrodes modified with an insulating nitrocellulose film and is evoked after the cellulase treatment. Ferricyanide voltammetric responses correlate with the increased permeability of the films and electrochemically active surface area of electrodes becoming accessible to the ferricyanide reaction after nitrocellulose digestion by cellulase. Trichoderma and Aspergillus niger cellulases activities were determined in a 5 min assay with a sensitivity of 10-8 U per assay, being 103-104-fold more sensitive than the standard commercially available optical assays. That makes the developed electrochemical approach the most prospective cost-effective alternative both for research and automated industrial applications.

Bebé colodión autorresolutivo: nueva mutación en el gen ALOX12B / Self-healing Collodion Baby: A New Mutation in the ALOX12BGene

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Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

Deposition of bacteriorhodopsin protein in a purple membrane form on nitrocellulose membranes for enhanced photoelectric response.

Bacteriorhodopsin protein (bR)-based systems are one of the simplest known biological energy converters. The robust chemical, thermal and electrochemical properties of bR have made it an attractive material for photoelectric devices. This study demonstrates the photoelectric response of a dry bR layer deposited on a nitrocellulose membrane with indium tin oxide (ITO) electrodes. Light-induced electrical current as well as potential and impedance changes of dried bR film were recorded as the function of illumination. We have also tested bR in solution and found that the electrical properties are strongly dependent on light intensity changing locally proton concentration and thus pH of the solution. Experimental data support the assumption that bR protein on a positively charged nitrocellulose membrane (PNM) can be used as highly sensitive photo- and pH detector. Here the bR layer facilitates proton translocation and acts as an ultrafast optoelectric signal transducer. It is therefore useful in applications related to bioelectronics, biosensors, bio-optics devices and current carrying junction devices.

Degradation of nitrocellulose-based paint by Desulfovibrio desulfuricans ATCC 13541.

Nitrocellulose is one of the most commonly used compounds in ammunition and paint industries and its recalcitrance to degradation has a negative impact on human health and the environment. In this study the capability of Desulfovibrio desulfuricans ATCC 13541 to degrade nitrocellulose as binder in paint was assayed for the first time. Nitrocellulose-based paint degradation was followed by monitoring the variation in nitrate, nitrite and ammonium content in the culture medium using Ultraviolet-Visible spectroscopy. At the same time cell counts and ATP assay were performed to estimate bacterial density and activity in all samples. Infrared spectroscopy and colorimetric measurements of paint samples were performed to assess chemical and colour changes due to the microbial action. Microscope observations of nitrocellulose-based paint samples demonstrated the capability of the bacterium to adhere to the paint surface and change the paint adhesive characteristics. Finally, preliminary studies of nitrocellulose degradation pathway were conducted by assaying nitrate- and nitrite reductases activity in D. desulfuricans grown in presence or in absence of paint. We found that D. desulfuricans ATCC 13541 is able to transform nitrocellulose as paint binder and we hypothesised ammonification as degradation pathway. The results suggest that D. desulfuricans ATCC 13541 is a good candidate as a nitrocellulose-degrading bacterium.

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease.

BACKGROUND: In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. RESULTS: In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10(-5) dilution of Asia1/JSL/05 (1 × 10(7.2)TCID(50)/50 µL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. CONCLUSION: We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields.

Development of a colloidal gold-based immunochromatographic test strip for screening of microalbuminuria.

A rapid immunochromatography (ICG) assay based on antibody colloidal gold nanoparticles specific to human serum albumin (HSA) was developed, and its applications for primary screening of HSA in the urine were evaluated. A monoclonal antibody (MAb) specific to HSA was produced from cloned hybridoma cells (EMRC1) and used to develop an ICG strip. The nanocolloidal gold, with an average particle diameter of 20 nm, was synthesized and labeled MAb as the detection reagent. An antibody colloidal gold probe was applied on the conjugate pad, and HSA antigen was immobilized to a nitrocellulose membrane as the capture reagent to prepare the ICG strip test. This test required only 10 min to accomplish a semiquantitative detection of albumin. The sensitivity to urinary albumin was found to be approximately 20 µg/mL, and the analytical range was 20-25 µg/mL. The reliability of the testing procedures was examined by carrying out the ICG strip test with 40 urine samples and comparing the results of these tests with those obtained via immunoturbidimetry. The ICG strip was adequately sensitive and accurate for a rapid screening of HSA in the urine.

Discrimination of Alicyclobacillus strains using nitrocellulose membrane filter and attenuated total reflectance fourier transform infrared spectroscopy.

UNLABELLED: Alicyclobacillus spp. are thermoacidophilic, spore-forming bacteria, some of which cause spoilage in pasteurized and heat-treated apple juice products through the production of guaiacol. It would be helpful if a rapid method to detect and discriminate Alicyclobacillus strains was available. A simple and rapid sample preparation method using nitrocellulose membrane filter (NMF) and a single reflection horizontal attenuated total reflection (HATR) accessory with Fourier transform infrared (FT-IR) was developed here. Fourier transform infrared (FT-IR) spectroscopy was used and tested on 8 Alicyclobacillus strains (KF, WAC, NWN-13501, NWN-12697, NWN-12654, NWN-10682, 1016, 1101). A linear discriminant analysis (LDA) was established to discriminate Alicyclobacillus strains. The sample preparation method could successfully separated strains into different groups by principal component analysis (PCA). High identification accuracy (95%) was achieved with the LDA model. PRACTICAL APPLICATION: The method developed in the paper can be used to discriminate different Alicyclobacillus strains from each other making it possible to easily determine whether the strain of Alicyclobacillus present is associated with juice spoilage.

Development of a membrane-bound random DNA sequence combinatorial array recognition surface (CARS).

A partially overlapping population of random sequence 60mer DNA molecules consisting of many concatamers of varied lengths was spatially separated in one and two dimensions by electrophoresis in polyacrylamide and transferred to nitrocellulose membranes. The spatially separated library serves as a potential sensor interface on which many different molecular recognition events or target analyte-binding patterns may emerge, thereby theoretically representing a "universal sensor" surface. The separated DNA library has been referred to as a DNA combinatorial array recognition surface or "CARS." After UV baking and various fluorescence staining or fluorescent probe interactions, the one-dimensional (1-D) and 2-D membrane-bound CARS were digitally photographed and subjected to image analysis with National Institutes of Health Image-Java software. Image analysis demonstrated relatively consistent and more similar spatial fluorescence patterns within CARS analyte treatment groups but noteworthy pattern differences before and after analyte addition and between different analyte treatments. Taken together, these data suggest a potential role for CARS as a novel, inexpensive, self-assembling universal molecular recognition surface that could be coupled to sophisticated Bayesian or other pattern recognition algorithms to classify analytes or make specific identifications, much like the senses of smell or taste.

Nitromatrix provides improved LC-MALDI signals and more protein identifications.

The beneficial use of NC in MALDI-MS has previously been reported to provide better S/N and reproducibility as well as less alkali metal adducts. We have therefore investigated if additional beneficial properties of NC also existed for commonly employed proteomics-based LC-MALDI procedures. Specifically we studied the effects of NC as a matrix cofactor for prestructured sample supports (AnchorChip plates), and compared the performance with several alternative sample preparation methods recently reported in the literature. The work reported here describes a new method of mixing the NC-matrix solution with the LC-eluent prior to sample deposition and shows that a mixture of CHCA and NC in a complex solvent offers superior analytical results in several ways: most striking is the higher signal intensity, and that the signals last much longer, due to the robustness of the matrix formulation. We have tested the use of the nitromatrix on a single LC-MALDI preparation and found that at least ten reiterative analyses could be performed, resulting in total analysis times of more than 75 h (approximately 15 million laser shots). Consequently more than twice as many proteins could be identified than from a single analysis. This combination of longer, and stronger, MALDI signals provided an increase in the number of peptides, greater sequence coverage in MS/MS experiments and ultimately more confident peptide assignments.

Self-healing collodion membrane and mild nonbullous congenital ichthyosiform erythroderma due to 2 novel mutations in the ALOX12B gene.

BACKGROUND: Collodion phenotype is a term applied to the condition affecting a newborn involving a parchmentlike membrane covering the whole body surface (collodion membrane). This presentation is common to several different forms of autosomal recessive congenital ichthyoses, including nonbullous congenital ichthyosiform erythroderma (NCIE), lamellar ichthyosis (LI), and harlequin ichthyosis (HI). Recent years have seen considerable advances in our understanding of the molecular basis of autosomal recessive forms of congenital ichthyosis. Several genetic loci have been identified for LI and NCIE. OBSERVATIONS: We describe the clinical and molecular features of 2 cases of self-healing newborns of collodion phenotype developing mild NCIE. A dramatic improvement of the skin was observed in the first few weeks after birth in both cases. The molecular analysis of the ALOX12B gene demonstrated that both patients were compound heterozygous for previously unreported mutations. CONCLUSIONS: Both patients were compound heterozygous for novel ALOX12B mutations, underscoring the concept that mutations in at least 2 different genes, ALOX12B and TGM1, may result in this unusual clinical phenotype.

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

Fluorescein transport properties across artificial lipid membranes, Caco-2 cell monolayers and rat jejunum.

Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M-S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M-S P(app) values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and alpha-CHC (alpha-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium. One is very likely an MCT (monocarboxylic acid cotransporter) isoform, inhibited by specific MCT inhibitor alpha-CHC, while the involvement of the second one with overlapping substrate/inhibitor specificities (most probably a member of the organic anion-transporting polypeptide family, inhibited at least partially by DIDS) could not be excluded.

Degradation of nitrocellulose by fungi.

Three lignocellulolytic fungi, Trametes versicolor, Pleurotus ostreatus, and Coprinus cinereus, and two cellulolytic fungi Trichoderma reesei and Chaetomium elatum were tested for their ability to degrade nitrocellulose. They were provided with different carbon and nitrogen sources in liquid cultures. Nitrocellulose (N content above 12%) was added as nitrogen source (in solution in acetone) alongside amino acids or as sole N source. Either starch or carboxy-methyl cellulose were provided as carbon sources. After 28 days of growth the highest decrease of nitrocellulose was observed with Chaetomium elatum when up to 43% was degraded in a medium containing nitrocellulose as the only nitrogen source. Coprinus cinereus caused a 37% decrease of nitrocellulose when provided with amino acids and starch as co-substrate. In cultures of Trametes versicolor, Pleurotus ostreatus and Trichoderma reesei, only 10%-22% decrease of nitrocellulose was measured in all media. In the presence of nitrocellulose with N content below 12% supplied as 3 mm pellets as the only carbon source, or with nitrocellulose with carboxy-methyl cellulose, the release of nitrite and nitrate from liquid cultures of Chaetomium elatum was measured. Between 6 and 9 days of growth in these media, an increase in both nitrite and nitrate was observed with a loss in weight of nitrocellulose up to 6% achieved after 34 days. The physical nature of the NC pellets may have reduced the rate of degradation in comparison with supplying NC in solution in the cultures.

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein.

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.

Competition on nitrocellulose-immobilized antibody arrays: from bacterial protein binding assay to protein profiling in breast cancer cells.

Large scale comparative evaluation of protein expression requires miniaturized techniques to provide sensitive and accurate measurements of the abundance of molecules present as individual and/or assembled protein complexes in cells. The principle of competition between target molecules for binding to arrayed antibodies has recently been proposed to assess differential expression of numerous proteins with one-color or two-color fluorescence detection methods. To establish the limiting factors and to validate the use of alternative detection for protein profiling, we performed competitive binding assays under different conditions. A model experimental protocol was developed whereby the competitive displacement of multi-subunit bacterial RNA polymerase and/or its subunits was evaluated through binding to subunit-specific immobilized monoclonal antibodies. We show that the difference in physico-chemical properties of unlabeled and labeled molecules significantly affects the performance of one-color detection, whereas epitope inaccessibility in the protein complex can prohibit the assessment of competition by both detection methods. Our data also demonstrate that antibody cross-reactivity, target protein truncation and abundance, as well as the cellular compartment of origin are major factors that affect protein profiling on antibody arrays. The experimental conditions established for prokaryotic proteins were adopted to compare protein profiles in the breast tumor-derived cell lines MDA MB-231 and SKBR3. Competitive displacement was detected and confirmed for a number of proteins using both detection methods; however, we show that overall the two-color method is better suited for accurate expression profile evaluation of a large, complex set of proteins. Antibody array data confirm the functional linkage between the ErbB2 receptor and AP-2 transcription factors in these cell lines and highlight unexpected differences in G1 cyclin expression.