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PubMed2000

S-layer-coated liposomes as a versatile system for entrapping and binding target molecules.

In the present study, unilamellar liposomes coated with the crystalline bacterial cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were used as matrix for defined binding of functional molecules via the avidin- or streptavidin-biotin bridge. The liposomes were composed of dipalmitoyl phosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4 and they had an average size of 180 nm. For introducing specific functions into the S-layer lattice without affecting substances encapsulated within the liposomes, crosslinking and activation reagents had to be identified which did not penetrate the liposomal membrane. Among different reagents, a hydrophilic dialdehyde generated by periodate cleavage of raffinose and a sulfo-succinimide activated dicarboxylic acid were found to be impermeable for the liposomal membrane. Both reagents completely crosslinked the S-layer lattice without interfering with its regular structure. Biotinylation of S-layer-coated liposomes was achieved by coupling p-diazobenzoyl biocytin which preferably reacts with the phenolic residue of tyrosine or with the imidazole ring of histidine. By applying this method, two biotin residues accessible for subsequent avidin binding were introduced per S-layer subunit. As visualized by labeling with biotinylated ferritin, an ordered monomolecular layer of streptavidin was formed on the surface of the S-layer-coated liposomes. As a second model system, biotinylated anti-human IgG was attached via the streptavidin bridge to the biotinylated S-layer-coated liposomes. The biological activity of the bound anti-human IgG was confirmed by ELISA.

Amines↗
PubMed2000

Physiological characterization of layer III non-pyramidal neurons in piriform (olfactory) cortex of rat.

We performed whole-cell recordings of layer III non-pyramidal neurons in the piriform cortex of Sprague-Dawley rats. For comparison purposes, recordings were made from deep pyramidal cells, which are also present in layer III. These two cell types could be distinguished both anatomically and physiologically. Anatomically, the layer III non-pyramidal neuron displayed smooth beady dendrites, while deep pyramidal cells showed thicker dendrites with spines. The dendrites of the layer III non-pyramidal neuron also tended to be restricted to layer III while deep pyramidal cells had long apical dendrites that spanned layers I and II. Although the resting membrane potentials of both cell types were very similar, significant differences were noted in other physiological measures. Layer III non-pyramidal neurons typically displayed higher input resistances, faster time constants, smaller spike amplitudes, shorter spike widths, and higher spike thresholds. In addition, layer III non-pyramidal neurons were able to spike at much higher rates when stimulated with the same level of threshold normalized current injection. The most dramatic differences in physiology were seen in the pattern of spiking in response to increasing levels of positive constant current pulses. Layer III non-pyramidal neurons showed qualitatively different responses at low and high levels of stimulation. At low levels, spikes occurred with long latency and the firing frequency increased throughout the duration of the current pulse. At high levels, non-pyramidal neurons started spiking with short latency, followed by a decrease in firing frequency, which in turn was followed by an increase in firing frequency. Deep pyramidal neurons differed dramatically from this pattern, displaying a qualitatively similar response at all levels of current injection. This response was characterized by short latency spikes and spike adaptation for the duration of the current pulse.

Action Potentials↗
PubMed1998

The effect of ACPD on the responses to NMDA and AMPA varies with layer in slices of rat visual cortex.

The effect of 1S,3R-aminocyclopentane dicarboxylic acid (ACPD) was measured on cells from various layers in slices of the rat visual cortex using whole-cell recording techniques. The position of the recorded cell was estimated by distance from pia to the layer VI/white matter boundary, and verified in 34/97 cells by staining with biocytin. Potentiation or depression of the responses to NMDA and AMPA by the metabotropic glutamate agonist ACPD was examined by iontophoresis of the drugs close to the cell body. Iontophoresis of ACPD had different effects in different layers. In layer VI, ACPD produced a substantial depolarization, which augmented the responses to NMDA and AMPA. In layer V, ACPD did not produce a significant depolarization, but potentiated the response to NMDA and AMPA. In layer IV, ACPD produced a small hyperpolarization, and depressed the response to NMDA. In layers II and III, the results were small and variable. Most recordings from stained cells were from pyramidal cells. Where recordings from non-pyramidal cells were obtained (3/34), results were the same as from pyramidal cells in the same layer. The same results were obtained when tetrodotoxin was in the bath solution. We conclude that the potentiation or depression of the response to NMDA and AMPA by ACPD varies with layer in rat visual cortex.

Animals↗
PubMed1999

Thalamocortical afferents of Lorente de Nó: medial geniculate axons that project to primary auditory cortex have collateral branches to layer I.

The injection of anterograde tracers into the ventral division of the medial geniculate body (MGV) of both rats and rabbits labels terminal axons in layer I of auditory cortex as well as the more conventional terminal arbors in layers III/IV. Whether these layer I projections represent a separate lemniscal pathway to the molecular layer or arise as collaterals of axons terminating in III/IV has not been addressed. Focal injections of the anterograde tracers biocytin or biotinylated dextran amine were made into the MGV of young rabbits. Serial section reconstruction of single MGV axons in auditory cortex revealed that layer I axons were collaterals of thalamocortical afferents that formed multiple divergent patches within III/IV. MGV collaterals to layer I often coursed tangentially for several millimeters before terminating. In some cases, the layer I collaterals descended to arborize within a thalamocortical patch in layers III/IV. These results suggest considerable radial and tangential divergence in the auditory thalamocortical pathway and argue for an expanded role for layer I in the processing of specific sensory stimuli.

Afferent Pathways↗
PubMed2000

High molecular mass glycans are major structural elements associated with the laminated layer of in vitro cultivated Echinococcus multilocularis metacestodes.

The laminated layer of the larval stage (metacestode) of the cestode parasite Echinococcus multilocularis is composed largely of carbohydrates, which form a tight microfibrillar meshwork around the entire metacestode. Since this laminated layer is the only parasite structure which is in constant contact with host immune and non-immune cells, and appears largely resistant to physiological and immunological reactions of the host, it most likely carries out important functions with regard to host-parasite interactions. In infected hosts, the metacestode is usually concentrically covered by host connective tissue cells and large amounts of collagen, causing a dense scar-like fibrosis, and it is likely that host-derived components are incorporated into the laminated layer at the host-parasite interface. Therefore, in order to obtain information on the molecular composition of this structure, we used parasite larvae which were generated through in vitro cultivation and thus were largely devoid of interfering host components. Lectin fluorescence on section-labelling of metacestodes embedded in LR-White suggested that the laminated layer is largely composed of N-acetyl-beta-D-galactosaminyl, and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1.3-galactose, while N-linked glycopeptides and alpha-D-mannosyl residues and/or glucosyl residues were found mainly within the germinal layer, and within the cellular mass and the surface of developing protoscoleces. Lectin-gold EM confirmed these findings. The laminated layer was isolated from in vitro cultivated metacestodes by urea extraction, and the ultrastructure of the purified laminated layer was assessed comparatively with respect to the laminated layer of intact parasites. The glycan composition was determined using SDS-PAGE and lectin blotting. This work has laid the basis for a more detailed dissection of the molecular composition of the laminated layer.

Animals↗
PubMed2004

Modelling the fibrous tissue layer in cemented hip replacements: experimental and finite element methods.

The long-term fixation of cemented femoral components may be jeopardised by the presence of a fibrous tissue layer at the bone-cement interface. This study used both experimental and finite element (FE) methods to investigate the load transfer characteristics of two types of cemented hip replacements (Lubinus SPII and Müller-Curved) with a fibrous tissue layer. The experimental part investigated six stems of each type, where these were implanted in composite femurs with a specially selected silicone elastomer modelling the soft interfacial layer. Two fibrous tissue conditions were examined: a layer covering the full cement mantle, representing a revision condition; and a layer covering the proximal portion of the cement mantle, representing a non-revised implant with partial debonding and fibrous tissue formation. The FE method was used to model the full fibrous tissue layer condition, for both implants. The layer was modelled as a homogeneous, linearly isotropic material. A cross-comparison was performed of the experimental and FE findings. Agreement between experimental and FE models was verified to be within 15%. Varying the stiffness parameter of the FE soft tissue layer had little influence on the cortical bone strains, though had considerable effect on the cement strains. Stress shielding occurred for both stems under both fibrous tissue conditions, with the greatest reduction around the calcar. However, the cortical bone strains were generally larger than those for the equivalent well-fixed stems. The fibrous tissue layer was not found to increase the general strain pattern of the cement mantle, though localised regions of high stress were detected.

Arthroplasty, Replacement, Hip↗
PubMed2001

Fracture resistance of dentin-composite interfaces using different adhesive resin layers.

OBJECTIVES: The objective of this study was to study the effect of different adhesive layers on the interfacial fracture toughness (K(ICi)) of dentin-resin composite interfaces. METHODS: Miniature short-rod fracture toughness specimens containing a chevron-shaped dentin-bonded interface along their midplane were used for testing. Each interface zone contained a thinned (one coat of unfilled adhesive resin, air-thinned), one-layer (one coat of unfilled adhesive resin, brush-thinned), two-layer (two coats of unfilled adhesive resin, brush-thinned), 10% filled or 45% filled adhesive resin layer. After storage in distilled water at 37 degrees C for 24h, the fracture toughness test specimens were loaded in tension at an extension rate of 0.5mm/min until fracture and the K(ICi) were determined. The results were analysed by ANOVA and Fisher's LSD test (p<0.05). Scanning electron microscopy was used to examine representative fracture surfaces. RESULTS: There were no significant differences in mean K(ICi) among the different unfilled adhesive resin layer groups. SEM examination of these specimens showed that fracture generally occurred between the resin-infiltrated layer and adhesive resin layer during interfacial fracture toughness testing. The mean K(ICi) for the 10% filled groups was not significantly different from the unfilled groups. The 45% filled group, however, demonstrated the highest K(ICi) values, the thickest adhesive resin layer under SEM examination, and a fracture path through the adhesive resin layer. CONCLUSIONS: There were no significant differences in K(ICi) when the unfilled adhesive resin was used despite different application methods. The 45% filled adhesive resin improved the properties of the dentin-composite interface with respect to both interfacial fracture resistance and dentinal seal after fracture.

Acid Etching, Dental↗
PubMed1996

The structure of aquareovirus shows how the different geometries of the two layers of the capsid are reconciled to provide symmetrical interactions and stabilization.

BACKGROUND: Aquareoviruses are important pathogens of aquatic animals and have severe consequences in aquaculture. These viruses belong to the family Reoviridae. A structural feature common to members of the Reoviridae is a multilayered capsid, formed by several concentric icosahedral shells with different protein compositions. How these proteins, which often are present in unequal stoichiometries, interact between icosahedral layers to stabilize the capsid is not well understood. RESULTS: We have determined the three-dimensional structure of aquareovirus to 23 A resolution using electron cryomicroscopy and computer image analysis. The protein capsid is composed of two structurally distinct icosahedral layers: an outer layer approximately 100 A thick, with incomplete T=13 left-handed symmetry, surrounds an inner layer 600 A in diameter that has T=1 symmetry and is perforated by channels near the fivefold axes. There are 120 subunits, arranged in dimers, in the inner layer, each of which interacts with two of the 600 subunits in the outer layer. A separate set of closely interacting proteins forms the fivefold axes of the virus structure, forming continuous density throughout both layers of the capsid. Comparison of full and empty (lacking RNA) virus structures reveals an RNA shell that lies directly beneath the inner layer. CONCLUSIONS: Our aquareovirus structure displays marked similarity to the mammalian reovirus intermediate subviral particles, suggesting a close evolutionary relationship. However, the noticeable distinction is that aquareovirus lacks the hemagglutinin spike observed in reovirus. The T=1 inner layer organization observed in the aquareovirus appears to be common to other members of the Reoviridae. Such organization may be of fundamental significance in the endogenous transcription of the genome in these viruses.

Animals↗
PubMed1997

An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy.

BACKGROUND: Bluetongue virus (BTV), which belongs to the Reoviridae family and orbivirus genus, is a non-enveloped, icosahedral, double-stranded RNA virus. Several protein layers enclose its genome; upon cell entry the outer layer is stripped away leaving a core, the surface of which is composed of VP7. The structure of the trimeric VP7 molecule has previously been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one which is largely alpha-helical and the other, smaller domain, is a beta barrel with jelly-roll topology. The relative orientations of these two domains vary in different crystal forms. The structure of VP7 and the organizations of 780 subunits of this molecule in the core of virus is central to the assembly and function of BTV. RESULTS: A 23 A resolution map of the core, determined using electron cryomicroscopy (cryoEM) data, reveals that the 260 trimers of VP7 are organized on a rather precise T = 13 laevo icosahedral lattice, in accordance with the theory of quasi-equivalence. The VP7 layer occupies a shell that is between 260 A and 345 A from the centre of the core. Below this radius (230-260 A) lies the T = 1 layer of 120 molecules of VP3. By fitting the X-ray structure of an individual VP7 trimer onto the cryoEM BTV core structure, we have generated an atomic model of the VP7 layer of BTV. This demonstrates that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the in vivo conformation of the molecule in the core. CONCLUSIONS: The beta-barrel domains of VP7 are external to the core and interact with protein in the outer layer of the mature virion. The lower, alpha-helical domains of VP7 interact with VP3 molecules which form the inner layer of the BTV core. Adjacent VP7 trimer-trimer interactions in the T = 13 layer are mediated principally through well-defined regions in the broader lower domains, to form a structure that conforms well with that expected from the theory of quasi-equivalence with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth surface upon which the two-dimensional lattice of VP7 trimers is laid down.

Bluetongue virus↗
PubMed2002

High-resolution EEG: on the cortical equivalent dipole layer imaging.

BACKGROUND: Brain electrical activity is a spatio-temporally distributed process. Cortical imaging techniques have been developed to reconstruct cortical activity from the scalp electroencephalographic or magnetoencephalographic measurements. Several cortical imaging approaches, such as the epicortical potentials and a dipole layer accounting for the cortical activity, have been used to represent brain electrical activity. METHODS: A closed cortical dipole layer source model is used to equivalently represent brain electrical activity. The relationship between the primary brain electrical sources and the cortical equivalent dipole layer is derived from the theory of electromagnetics. Computer simulation studies were conducted using a 3-concentric-sphere head model to validate the proposed theory. The cortical equivalent dipole layer imaging approach was tested in both computer simulation and human visual evoked potential (VEP) experiments. RESULTS: The strength of the cortical equivalent dipole layer is shown to be proportional to the electrical potential over the same surface generated by primary electrical sources, had the outer medium been replaced by air. The proposed theory was validated by computer simulation in a discrete system. Simulation and VEP experimental studies suggest the feasibility of applying the cortical equivalent dipole layer imaging approach for brain imaging. CONCLUSIONS: The cortical equivalent dipole layer model can equivalently represent the primary brain electrical sources throughout the entire brain surrounded by the dipole layer. The strength of the cortical equivalent dipole layer due to primary sources can be directly calculated according to the theory developed in the present study.

Brain Mapping↗
PubMedDate not supplied

Layer differences in the effect of monocular vision in light- and dark-reared kittens.

We compared the effect of 2 days of monocular vision on the ocular dominance of cells in the visual cortex of light-reared kittens with the effect in dark-reared kittens at 6, 9, and 14 weeks of age, and analyzed the results by layer. The size of the ocular-dominance shift declined with age in all layers in light-reared animals. There was not a large change in the ocular-dominance shift with age in dark-reared animals in any layer, suggesting that dark rearing largely keeps the cortex in the immature 6-week state until 14 weeks or longer, although there was a slight decrease in layers II, III, and IV, and a slight increase in layers V and VI. At 14 weeks, the difference between light- and dark-reared animals was smallest in layer IV, larger in layers II/III, and largest in layers V/VI, suggesting that dark rearing has a large effect on intracortical synapses and a small effect on geniculocortical synapses. There was a significant ocular-dominance shift in layer IV at 14 weeks of age in both light- animals and dark-reared animals, showing that the critical period for ocular-dominance plasticity is not ended at this age. While the ocular-dominance shift after 26 h of monocular deprivation in 6-week animals was similar in light- and dark-reared animals, after 14 h it was smaller in dark-reared animals, showing that ocular-dominance changes occur more slowly in dark-reared animals at this age, in agreement with Mower (1991). Increases in selectivity for axis of movement after 26 h of monocular vision were seen in dark-reared animals at 6 weeks of age, but not at 9 or 14 weeks of age, showing that the critical period for axial selectivity ends earlier than the critical period for ocular dominance in dark-reared animals, as it does in light-reared animals.

Aging↗
PubMedDate not supplied

Cell types and response timings in the medial interlaminar nucleus and C-layers of the cat lateral geniculate nucleus.

Previous evidence concerning the physiological cell classes in the medial interlaminar nucleus (MIN) has been conflicting. We reexamined the MIN using standard functional tests to distinguish X-, Y- and W-cells. Discharge patterns to flashing spots also were used to identify some cells as lagged or nonlagged, as previously done for the geniculate A-layers. Also, each cell's response timing (latency and absolute phase) was measured from discharges to a spot undergoing sinusoidal luminance modulation. Of 71 MIN cells, 48% were Y, 27% were W, 8% were X, and 17% were unclassifiable. Lagged and nonlagged discharge profiles were observed in each cell group, with 28% of all cells being lagged. Lagged cells displayed a response suppression and long latency to discharge following spot onset, and a slow decay in firing at spot offset that was often preceded by a transient discharge. These profiles were indistinguishable from those of lagged cells in the A-layers. MIN cells also were heterogeneous in response timing, displaying a range of latency and absolute phase values similar to that in the A-layers. We extended these analyses to 27 cells in the geniculate C-layers. In layer C, 35% of cells were Y, 10% were X, 25% were W, and 30% were unclassifiable. About 11% had lagged profiles, and were X-cells or unclassifiable cells. Layers C1 and C2 contained only W-cells and no lagged profiles. The range of timings in the C-layers was somewhat narrower than in the MIN. Overall, these results show that the MIN contains a greater variety of functional cell classes than heretofore appreciated. Further, it appears that mechanisms which create different timing delays in the A-layers also exist in the MIN and layer C. These timings may contribute to direction selectivity in extrastriate cortex.

Animals↗
PubMed2002

Layered paving of vesicular nanoparticles formed with cerasome as a bioinspired organic-inorganic hybrid.

A three-dimensional packed vesicular assembly was successfully prepared by using an organic-inorganic hybrid, the Cerasome. This assembly was achieved by using an alternate layer-by-layer adsorption technique with the anionic and the cationic Cerasome derived from corresponding organoalkoxysilane amphiphiles. Adsorption quantities of each Cerasome layer were evaluated by employing a quartz crystal microbalance. The surface structure of the Cerasome paving on a substrate obtained in this way was observed by atomic force microscopy. The Cerasome particles closely packed like a stone pavement were clearly observed in both layers. In addition, the difference in the particle size for each layer indicates the cationic and anionic Cerasomes undoubtedly formed the layer-by-layer assembly. The layered paving of the vesicular nanoparticles was seen in every layer at least up to ten adsorption steps.

Ceramics↗
PubMed2005

Photoconductive properties of organic-inorganic hybrid films of layered perovskite-type niobate.

A hybrid film of layered niobate and an organic amphiphile was prepared by the Langmuir-Blodgett (LB) method. Trimethylammonium-exchanged perovskite-type niobates ((CH(3))(3)NHSr(2)Nb(3)O(10)) were exfoliative to form an aqueous suspension. A monolayer of octadecylamine was produced on such an aqueous dispersion as a template for a hybrid film. A hybrid film was transferred as a Y-type LB film onto a hydrophilic glass plate or an ITO substrate. The structure of a deposited film was investigated with X-ray diffraction (XRD), Fourier transform infrared (FT-IR), and atomic force microscopy (AFM) measurements, indicating a layer-by-layer structure with a single or double sheet of niobate as an inorganic composite. From the cyclic voltammogram on an ITO electrode modified with the Y-type 10 layered film, the lower edge of the conduction band of a niobate layer was determined to be - 0.6 V (vs Ag/AgCl). ac impedance and dc measurements were carried out on 1, 5, and 10-layered LB films (2 mm (electrode spacing) x 8 mm (width)) with aluminum electrodes. The freshly deposited samples behaved as an insulator under the illumination of 280 nm light (2.04 x 10(16) quanta s(-1)). Photoconductivities appeared, however, when they were preirradiated with a 150 W Xe lamp (ca. 2 x 10(18) quanta s(-1)) for 0.5-8.5 h. The process was denoted as photomodification. From the FT-IR and XRD results, it was deduced that the photomodification of LB films caused the decomposition of organic templates (octadecylammonium) accompanied by the collapse of layer-by-layer structures. dc analyses on the 5- and 10-layered films after photomodification also showed that they behaved as a photosemiconductor under UV light illumination.

Journal Article↗
PubMed2006

Dynamic diffuse double-layer model for the electrochemistry of nanometer-sized electrodes.

A dynamic diffuse double-layer model is developed for describing the electrode/electrolyte interface bearing a redox reaction. It overcomes the dilemma of the traditional voltammetric theories based on the depletion layer and Frumkin's model for double-layer effects in predicating the voltammetric behavior of nanometer-sized electrodes. Starting from the Nernst-Planck equation, a dynamic interfacial concentration distribution is derived, which has a similar form to the Boltzmann distribution equation but contains the influence of current density. Incorporation of the dynamic concentration distribution into the Poisson and Butler-Volmer equations, respectively, produces a dynamic potential distribution equation containing the influence of current and a voltammetric equation containing the double-layer effects. Computation based on these two equations gives both the interfacial structure (potential and concentration profiles) and voltammetric behavior. The results show that the electrochemical interface at electrodes of nanometer scales is more like an electric-double-layer, whereas the interface at electrodes larger than 100 nm can be treated as a concentration depletion layer. The double-layer nature of the electrode/electrolyte interface of nanometer scale causes the voltammetric responses to vary with electrode size, reactant charge, the value of formal redox potential, and the dielectric properties of the compact double-layer. These voltammetric features are novel in comparison to the traditional voltammetric theory based on the transport of redox molecules in the depletion layer.

Journal Article↗
PubMed2004

Effect of layer integrity of spin self-assembled multilayer films on surface wettability.

We investigated the correlation between surface wettability and internal structure of polyelectrolyte (PE)/PE and PE/inorganic multilayer films prepared by the spin self-assembly (SA) method. Spin self-assembled poly(allylamine hydrochloride) (PAH)/poly(sodium 4-styrenesulfonate) (PSS) multilayer films deposited from PE solutions of 10 mM show the distinct oscillation in contact angles with variation of the outermost PE layer, representing the saturated values in contact angles of individual PAH and PSS layers. These contact angles are also well consistent with the angles measured from respective PE layers (i.e., PAH and PSS) of the spin SA (PAH/CdS-COO-) and (CdS-NH3+/PSS) films carrying the flat interface between PE and inorganic CdS nanoparticle layers as confirmed by X-ray reflectivity. Furthermore, based on the contact angle of CdS-NH3+ layer in the ordered (CdS-NH3+/PSS) films, the change in surface wettability of CdS-NH3+ layers of two different spin SA (CdS-NH3+/poly(methacrylic acid) (PMAA)) multilayer films with ordered and disordered internal structure is also investigated. The films with ordered and disordered internal structure were fabricated by the pH adjustment of PMAA. The CdS-NH3+ layer in both CdS-NH3+/PSS and CdS-NH3+/PMAA multilayer films with the ordered internal structure has the contact angle of about 25 +/- 2 degrees irrespective of the PSS or PMAA sublayer. As a result, the same surface wettability of PE or inorganic layers, despite different sublayers, strongly indicates that the spin SA method in optimum condition allows the top surface to be completely covered with a low level ofinterdigitation with a sublayer at each deposition step, and this leads to the conclusion that physical and chemical characteristics of the sublayers have no significant influence on those of the outermost layer.

Journal Article↗
PubMed2004

A streptavidin linker layer that functions after drying.

The ability of streptavidin (SA) to simultaneously bind four biotins is often used in linker layers, where a biotinylated molecule is linked to a biotin-functionalized surface via SA. For biosensor and array applications, it is desirable that the SA linker layer be stable to drying and rehydration. In this study it was observed that a significant decrease in binding capacity of a SA layer occurred when that layer was dried. For this study a SA linker layer was constructed by binding SA to a biotin-containing alkylthiolate monolayer (BAT/OEG) self-assembled onto gold. Its stability after drying was investigated using surface plasmon resonance (SPR). Approximately a quarter of the SA layer was removed from the BAT/OEG surface upon drying and rehydration, suggesting disruption of SA-biotin binding when dry. This resulted in the dried SA layer losing approximately 40% of its biotinylated ferritin (BF) binding capacity. Coating the layer with trehalose before drying was found to inhibit the loss of SA from the BAT/OEG surface. SPR showed that the trehalose-protected SA linker layer retained approximately 91% of its original BF binding capacity after drying and rehydration. Atomic force microscopy, which was used to image individual surface-bound SA and BF molecules, qualitatively confirmed these observations.

Biotin↗
PubMed2004

Genetic organization of chromosomal S-layer glycan biosynthesis loci of Bacillaceae.

S-layer glycoproteins are cell surface glycoconjugates that have been identified in archaea and in bacteria. Usually, S-layer glycoproteins assemble into regular, crystalline arrays covering the entire bacterium. Our research focuses on thermophilic Bacillaceae, which are considered a suitable model system for studying bacterial glycosylation. During the past decade, investigations of S-layer glycoproteins dealt with the elucidation of the highly variable glycan structures by a combination of chemical degradation methods and nuclear magnetic resonance spectroscopy. It was only recently that the molecular characterization of the genes governing the formation of the S-layer glycoprotein glycan chains has been initiated. The S-layer glycosylation (slg) gene clusters of four of the 11 known S-layer glycan structures from members of the Bacillaceae have now been studied. The clusters are approximately 16 to approximately 25 kb in size and transcribed as polycistronic units. They include nucleotide sugar pathway genes that are arranged as operons, sugar transferase genes, glycan processing genes, and transporter genes. So far, the biochemical functions only of the genes required for nucleotide sugar biosynthesis have been demonstrated experimentally. The presence of insertion sequences and the decrease of the G + C content at the slg locus suggest that the investigated organisms have acquired their specific S-layer glycosylation potential by lateral gene transfer. In addition, S-layer protein glycosylation requires the participation of housekeeping genes that map outside the cluster. The gene encoding the respective S-layer target protein is transcribed monocistronically and independently of the slg cluster genes. Its chromosomal location is not necessarily in close vicinity to the slg gene cluster.

Bacillaceae↗