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At least 55 records · Page 3Linked to original sources
PubMed2003

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing.

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K(a): approximately 10(15) M(-1)) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.

Animals↗
PubMedDate not supplied

An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology.

The bacterial cell surface layer (S-layer) protein of Bacillus sphaericus CCM 2177 assembles into a square lattice structure and recognizes a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure in the rigid cell wall layer. For generating a nanopatterned sensing layer with high density and well defined distance of the ligand on the outermost surface, an S-layer fusion protein incorporating the sequence of a variable domain of a heavy chain camel antibody directed against prostate-specific antigen (PSA) was constructed, produced, and recrystallized on gold chips precoated with thiolated SCWP. The S-layer protein moiety consisted of the N-terminal part which specifically recognized the SCWP as binding site and the self-assembly domain. The PSA-specific variable domain of the camel heavy chain antibody was selected by several rounds of panning from a phage display library of an immunized dromedary, and was produced by heterologous expression in Escherichia coli. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-PSA-N7. The S-layer fusion protein had retained the ability to self-assemble into the square lattice structure. According to the selected fusion site in the SbpA sequence, the cAb-PSA-N7 moiety remained located on the outer surface of the protein lattice. After recrystallization of the S-layer fusion protein on gold chips precoated with thiolated SCWP, the monomolecular protein lattice was exploited as sensing layer in surface plasmon resonance biochips to detect PSA.

Animals↗
PubMed2002

Molecular characterization of the S-layer gene, sbpA, of Bacillus sphaericus CCM 2177 and production of a functional S-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen.

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.

Bacillus↗
PubMed2005

Fluorescent phospholipid analogs as microscopic probes for detection of the mycolic acid-containing layer in Corynebacterium glutamicum: detecting alterations in the mycolic acid-containing layer following ethambutol treatment.

Corynebacterium glutamicum belongs to the mycolic acid-containing actinomycetes, which also include Mycobacterium, Nocardia, and Rhodococcus. The cells of this group possess a cell wall with a thick outer layer composed primarily of mycolic acid, which functions as a permeability barrier. To investigate the mechanism of mycolic acid-containing layer (mycolate layer) formation, we have developed a fluorescence microscopic technique detecting the mycolate layer in situ. The staining specificity of fluorescence-labeled phospholipid analogs was determined by simultaneous staining with the hydrophobic fluorescent dye Nile Red and peptidoglycan-staining fluorescence-conjugated vancomycin. We found that fluorescence-labeled phospholipid analogs preferentially stain the mycolate layer. Using this technique, we observed the effect of the anti-mycobacterial drug ethambutol on C. glutamicum mycolate-layer formation. Ethambutol interfered specifically with mycolate-layer formation on the division planes and cell poles, while the side-wall mycolate layer was not severely affected. This indicates that mycolate-layer formation occurs mainly on division planes and cell poles in C. glutamicum, where the peptidoglycan layer is actively synthesized.

Anti-Infective Agents↗
PubMed1987

Distribution and size of thalamic neurons projecting to layer I of the auditory cortical fields of the cat compared to those projecting to layer IV.

The distribution of thalamocortical neurons projecting to layer I of the cat auditory cortical fields was examined by the horseradish peroxidase (HRP) method. After HRP injection into layer I of the primary auditory cortex (AI), HRP-labeled neuronal cell bodies were distributed mainly in the medial, dorsal, and ventrolateral divisions of the medial geniculate nucleus (MGN) and suprageniculate nucleus (Sg), and additionally in the lateral and medial divisions of the posterior group of the thalamus (Pol and Pom), lateroposterior thalamic nucleus (Lp), and nucleus of the brachium of the inferior colliculus (BIN). After HRP injection into layer I of the second auditory cortex (AII), labeled neurons were seen mainly in the medial, dorsal, and ventrolateral divisions of the MGN and Sg and additionally in the Pom, Lp, and BIN. After HRP injection into layer I of the anterior auditory field (AAF), labeled neurons were located mainly in the medial and dorsal divisions of the MGN, Sg, Pol, and BIN, and additionally in the ventrolateral divisions of the MGN, Pom, and Lp. After HRP injection into layer I of the dorsal part of the posterior ectosylvian gyrus (Epd), labeled neurons were observed chiefly in the medial and dorsal divisions of the MGN, Sg, and Lp and additionally in the ventrolateral division of the MGN, Pom, and BIN. After HRP injection into layer I of the ventral part of the posterior ectosylvian gyrus (Epv), labeled neurons were distributed chiefly in the medial and dorsal divisions of the MGN and Pol and additionally in the ventrolateral division of the MGN, Sg, and BIN. Thus no labeled neurons were found in the ventral division of the MGN after HRP injection into layer I of all auditory cortical fields examined in the present study. The average soma diameters of neurons that were labeled after HRP injection into layer I were statistically smaller than those of neurons that were labeled after HRP injection into layer IV.

Animals↗
PubMed1985

Demonstration of the granular layer and the fate of the hyaline layer during the development of a sea urchin (Lytechinus variegatus).

Employing electron-microscopic methods that help retain polyanionic materials, we describe the extracellular coverings of a sea urchin (Lytechinus variegatus) throughout ontogeny. The surface of the embryo is covered by a two-layered cuticle (commonly called the hyaline layer), which in turn is covered by a granular layer. The granular layer is retained after addition of alcian blue to the fixative solutions, and has not been previously described for any sea urchin. After hatching, the granular layer disappears, but the hyaline layer continues to cover most of the larval surface until settlement and metamorphosis. A few days before metamorphosis, the hyaline layer lining the vestibular invagination of the competent pluteus larva is replaced by a three-layered cuticle resembling that of the adult sea urchin. The hyaline layer covering the rest of the larva is evidently lost at metamorphosis during the involution of the general epidermis.

Animals↗
PubMedDate not supplied

Overpressured layer chromatographic study of retention behaviour of various benzodiazepine derivatives on layers impregnated with tricaprylmethylammonium chloride.

The retention behaviour of various benzodiazepine derivatives was investigated on silica gel layers impregnated with tricaprylmethylammonium chloride (TCMA). The chromatograms were developed by means of overpressured layer chromatography (OPLC). As for the case of amino- and nitrosalicylic acids, pyrimidine derivatives, barbiturates, penicillins, cephalosporins and tetracyclines, the retention of benzodiazepine derivatives increased with increasing layer TCMA concentration with eluents containing methanol and water, but not TCMA. On increase of the methanol content of the eluent, a retention-decreasing effect was observed. On layers impregnated with TCMA, a linear relationship existed between the RM values of the benzodiazepines and the methanol content of the eluent. A similar relationship held for silica gel layers impregnated with paraffin oil (traditional reversed-phase). There was no correlation between the results obtained on layers treated with TCMA or with paraffin oil. On TCMA-impregnated layers, the retention of compounds having different chemical structures showed no dependence on the pH of the eluent. There were two reasons for this. Firstly, as it was established, above a certain RF value, the pH of the layer in the presence of TCMA was almost identical irrespective of the original pH of the buffer. Secondly, below this RF value, the actual pH of the layer did not have a strong enough effect to cause appreciable differences between the retentions of the dissociated and undissociated species of the analytes. The conditions for optimum separation are given.

Anti-Anxiety Agents↗
PubMedDate not supplied

The influence of input from the lower cortical layers on the orientation tuning of upper layer V1 cells in a primate.

The receptive fields of cells in the primary visual cortex (area 17 or V1) show clear orientation selectivity, unlike those of the lateral geniculate nucleus (LGN) cells which provide their visual input. The intrinsic circuitry of V1 cells is believed to be partly responsible for this selectivity. We investigated the influence of ascending projections from neurons in the lower layers (5 and 6) of V1 on the orientation selectivity of single neurons in the upper layers (2,3, and 4) by reversibly inactivating ("blocking") lower layer neural activity with iontophoretic application of gamma-aminobutyric acid (GABA) while recording from upper layer cells in the prosimian primate, Galago crassicaudatus. During lower layer blocking, the majority (20/28 = 71.4%) of upper layer neurons exhibited a change in the orientation of their preferred stimulus, a reduction in their orientation tuning, and/or an increase in their response amplitude. Twelve (42.9%) neurons exhibited shifts in their preferred orientation averaging 11 (+/- 4) deg. These neurons were located on average, 272 (+/- 120) microns tangential from the vertical axis of the pipette center. Eleven neurons (39.2%) exhibited an average reduced orientation tuning of 52.5%. Their average location was 230 +/- (115) microns away from the vertical axis of the pipette. Five (17.9%) neurons with average location 145 (+/- 75) microns from the vertical axis exhibited both effects. Two (7.1%) neurons that exhibited significant increases in response amplitude to stimulus angles within 10 deg of the peak excitatory stimulus without changes in orientation selectivity or tuning were located less than 100 microns from the vertical axis. The effects on the orientation tuning of cells were restricted in all cases to within +/- 30 deg of the preferred stimulus orientation. This suggests that layer blocking affects cells with preferred stimulus orientations similar to those of the recorded neurons. Only cells located within 500 microns tangential to the vertical axis of the injection site exhibited these effects. These results suggest that cells within layers 5 and 6 provide organized, orientation-tuned inhibition that sharpens the orientation tuning of cells in the upper cortical layers within the same, or closely neighboring, cell columns.

Animals↗
PubMed2006

Layer-by-layer enzyme/polyelectrolyte films as a functional protective barrier in oxidizing media.

The influence of a catalase (Cat) layer located at different depths in the layer-by-layer hemoglobin/polystyrene sulfonate films with an (Hb/PSS)(20)(-)(x)/(Cat/PSS)/(Hb/PSS)(x) (x = 0-20) architecture on kinetics of hemoglobin degradation under treatment with hydrogen peroxide solutions of different concentrations and features of H(2)O(2) decay in surrounding solutions has been studied. While assembled on the top of the multilayers, the catalase layer shows the highest activity in hydrogen peroxide decomposition. Hemoglobin in such films retains its nativity for a longer period of time. The effect of catalase layers is compared with that of protamine, horseradish peroxidase, and inactivated catalase. Positioning an active layer with catalytic properties as an outer layer is the best protection strategy for layer-by-layer assembled films in aggressive media.

Electrolytes↗
PubMed2005

Single-layer versus double-layer closure of facial lacerations: a randomized controlled trial.

BACKGROUND: The objective of this study was to compare the cosmetic outcome of facial lacerations closed with a single or double layer of sutures. METHODS: Patients aged 1 year or older presenting to a university-based emergency department with nongaping (width, < 10 mm), simple, nonbite, facial lacerations were randomized to closure with a single layer of simple interrupted 6-0 polypropylene sutures or a double layer of simple interrupted 6-0 polypropylene plus inverted deep dermal 5-0 polyglactin sutures. At 90 days, the scar width and cosmetic appearance were determined using a validated 100-mm visual analogue scale ranging from 0 (worst) to 100 (best) and a validated wound evaluation score ranging from 0 (worst) to 6 (best). RESULTS: Sixty-five patients were randomized to single-layer (n = 32) or double-layer (n = 33) closure. Mean age (SD) was 18.5 years (20.0), and 14 percent were female. Groups were similar in baseline patient and wound characteristics. Length of single-layer closure was 7 minutes shorter (95 percent CI, 2 to 11 minutes) than double-layer closure. There were no infections or dehiscences in either group. There were no between-group differences in patient (mean difference, 0.5 mm; 95 percent CI, -5.7 to 6.6 mm) or practitioner (mean difference, 1.0 mm; 95 percent CI, -4.8 to 6.7 mm) visual analogue scale scores. All but one patient had an optimal wound evaluation score of 6 (p = not significant). Scar width was similar at 90 days (mean difference, 0.2 mm; 95 percent CI, -0.05 to 0.5). CONCLUSIONS: Single-layer closure of nongaping, minor (< 3 cm) facial lacerations is faster than double-layer closure. Cosmetic outcome and scar width are similar in sutured wounds whether or not deep dermal sutures are used.

Adolescent↗
PubMed2002

Analysis of variance study of the rat cortical layer 4 barrel and layer 5b neurones.

Unique formation of rodent cortical barrels by layer 4 neurones attracts study of the sensory function of cortical input stage neurones (layer 4) compared with that of output stage neurones (layer 5). We have recorded extracellular responses from rat somatosensory cortical neurones to deflections of contralateral vibrissae. Thirty-two layer 4 barrel neurones and 29 layer 5b neurones were studied. Whisker stimulations were ramp-and-hold deflections with one of six different ramp velocities (100-2.5 mm s(-1)) and one of four different plateau amplitudes (2000-200 microm). Twenty-four (6 x 4) different stimulus forms were applied to the tip of a whisker trimmed to 10 mm in a predetermined order in stimulus cycles of 20-50 repetitions. Spike counts for a period of 2560 ms in 10 ms bins were summed to construct a matrix of 24 peristimulus histograms for each neurone. Twenty-four amplitude and 24 velocity values were computed from counts during the plateau and ramp phases, respectively. To determine the amplitude- and velocity dependence of a neurone, an amplitude F value (the ratio of variations among-/within-amplitude of the amplitude value) and a velocity F value (ratio of variations among-/within-velocity of the velocity value) were derived by analysis of variance. The amplitude F value of the layer 4 barrel neurones was greater than that of the layer 5b neurones (P < 0.0001). The velocity F value of the barrel neurones was smaller than that of the layer 5b neurones (P = 0.0226). The results suggests that barrel neurones and layer 5b neurones tend to detect amplitude and velocity components of whisker deflection, respectively.

Analysis of Variance↗
PubMed2000

On ultrasonic guided waves in a thin anisotropic layer lying between two isotropic layers

In this paper, dispersion of guided waves in a three-layered sandwich plate is considered. The focus here is on a configuration consisting of a thin anisotropic layer sandwiched between two identical isotropic layers. This configuration could model, for example, a superconducting tape where the middle layer is a brittle superconductor and the surrounding layers are isotropic and ductile. An approximate dispersion relation correct to O(h) governing the guided waves is obtained by expanding the field inside the thin middle layer in powers of the small thickness h of the layer. Numerical examples are given for two specific systems with superconducting middle layers. Some characteristic features, particularly at low frequencies, are investigated. Comparison between the exact and approximate dispersion relations are made to show that the approximation works well in the frequency interval of interest. The characteristic features may be useful for ultrasonic measurements of the anisotropic elastic constants of the thin layer.

Journal Article↗
PubMed1996

Characterization and replicase activity of double-layered and single-layered rotavirus-like particles expressed from baculovirus recombinants.

Rotavirus has a capsid composed of three concentric protein layers. We coexpressed various combinations of the rotavirus structural proteins of single-layered (core) and double-layered (single-shelled) capsids from baculovirus vectors in insect cells and determined the ability of the various combinations to assemble into viruslike particles (VLPs). VLPs were purified by centrifugation, their structure was examined by negative-stain electron microscopy, their protein content was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and GTP binding assays, and their ability to support synthesis of negative-strand RNAs on positive-sense template RNAs was determined in an in vitro replication system. Coexpression of all possible combinations of VP1, VP2, VP3, and VP6, the proteins of double-layered capsids, resulted in the formation of VP1/2/3/6, VP1/2/6, VP2/3/6, and VP2/6 double-layered VLPs. These VLPs had the structural characteristics of empty rotavirus double-layered particles and contained the indicated protein species. Only VPI/2/3/6 and VP1/2/6 particles supported RNA replication. Coexpression of all possible combinations of VPl, VP2, and VP3, the proteins of single-layered capsids, resulted in the formation of VP1/2/3, VP1/2, VP2/3, and VP2 single-layered VLPs. These VLPs had the structural characteristics of empty single-layered rotavirus particles and contained the indicated protein species. Only VP1/2/3 and VP1/2 VLPs supported RNA replication. We conclude that (i) the assembly of VP1 and VP3 into VLPs requires the presence of VP2, (ii) the role of VP2 in the assembly of VP1 and VP3 and in replicase activity is most likely structural, (iii) VP1 is required and VP3 is not required for replicase activity of VLPs, and (iv) VP1/2 VLPs constitute the minimal replicase particle in the in vitro replication system.

Animals↗
PubMed2002

Expressions for determination of layer absorbances of a weakly absorbing double-layer sample through reflectance at the Brewster angles.

A new technique for determining absorbances of interferenceless (incoherent) layers based on measurements of the ratio of the front and the back reflectivities of a double-layer sample at the Brewster angles is proposed. A double-layer stack must have at least one absorbing layer, and the two layers should be interferenceless and should be thicker than the wavelength of the incident light. We found that under these conditions the ratio of the front and the back reflectivities at the Brewster angle of a sample surface is directly related to layer absorbance. For a layer with a known thickness this means finding the extinction coefficient of the layer material. In comparison with the conventional method for measuring transmittance, the advantage of this approach is that it affords an opportunity to get rid of the influence of surface effects on the measuring volume absorption coefficient. For a thick layer with known thickness, it makes possible the determination of a small bulk absorption on a background with even greater surface effects. We trust that this technique will prove to be powerful for measuring the extinction coefficients of weakly absorbing materials.

Journal Article↗
PubMed2007

Monolithic porous polymer layer for the separation of peptides and proteins using thin-layer chromatography coupled with MALDI-TOF-MS.

Plates for thin-layer chromatography (TLC) with an attached layer of porous polymer monolith have been prepared and used for the separation of small molecules, peptides, and proteins. The 50-200-mum. thin poly(butyl methacrylate-co-ethylene dimethacrylate) layers were prepared in situ using UV-initiated polymerization. Precise control of the reaction conditions enables the preparation of monolithic layers with a well-defined porous structure that determines the chromatographic performance. Compared to conventional TLC and high-performance TLC using precoated layers based on silica, the small layer thickness and absence of any binder is expected to improve both retention characteristics and separation efficiency of the polymer-based monolithic thin-layer chromatographic plates. Spots of the separated compounds were first detected using typical UV imaging. Since the monolithic thin layers can be also prepared directly on the stainless steel MALDI carrier plate, the separation in TLC format can be coupled with MALDI-TOF-MS. Application of a conventional MALDI matrix facilitated desorption and ionization of peptides and proteins for molecular weight determination of the separated compounds.

Chromatography, Thin Layer↗
PubMed2001

Characterization of an S-layer glycoprotein produced in the course of S-layer variation of Bacillus stearothermophilus ATCC 12980 and sequencing and cloning of the sbsD gene encoding the protein moiety.

The cell surface of Bacillus stearothermophilus ATCC 12980 is completely covered by an oblique lattice which consists of the S-layer protein SbsC. On SDS-polyacrylamide gels, the mature S-layer protein migrates as a single band with an apparent molecular mass of 122 kDa. During cultivation of B. stearothermophilus ATCC 12980 at 67 degrees C instead of 55 degrees C, a variant developed that had a secondary cell wall polymer identical to that of the wild-type strain, but it carried an S-layer glycoprotein that could be separated on SDS-polyacrylamide gels into four bands with apparent molecular masses of 92, 118, 150 and 175 kDa. After deglycosylation, only a single protein band with a molecular mass of 92 kDa remained. The complete nucleotide sequence encoding the protein moiety of this S-layer glycoprotein, termed SbsD, was established by PCR and inverse PCR. The sbsD gene of 2,709 bp is predicted to encode a protein of 96.2 kDa with a 30-amino-acid signal peptide. Within the 807 bp encoding the signal peptide and the N-terminal sequence (amino acids 31-269), different nucleotides for sbsD and sbsC were observed in 46 positions, but 70% of these mutations were silent, thus leading to a level of identity of 95% for the N-terminal parts. The level of identity of the remaining parts of SbsD and SbsC was below 10%, indicating that the lysine-, tyrosine- and arginine-rich N-terminal region in combination with a distinct type of secondary cell wall polymer remained conserved upon S-layer variation. The sbsD sequence encoding the mature S-layer protein cloned into the pET28a vector led to stable expression in Escherichia coli HMS174(DE3). This is the first example demonstrating that S-layer variation leads to the synthesis of an S-layer glycoprotein.

Amino Acid Sequence↗