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Charting and unzipping the surface layer of Corynebacterium glutamicum with the atomic force microscope.

Bacterial surface layers (S-layers) are extracellular protein networks that act as molecular sieves and protect a large variety of archaea and bacteria from hostile environments. Atomic force microscopy (AFM) was used to asses the S-layer of Coryne-bacterium glutamicum formed of PS2 proteins that assemble into hexameric complexes within a hexagonal lattice. Native and trypsin-treated S-layers were studied. Using the AFM stylus as a nanodissector, native arrays that adsorbed to mica as double layers were separated. All surfaces of native and protease-digested S-layers were imaged at better than 1 nm lateral resolution. Difference maps of the topographies of native and proteolysed samples revealed the location of the cleaved C-terminal fragment and the sidedness of the S-layer. Because the corrugation depths determined from images of both sides span the total thickness of the S-layer, a three-dimensional reconstruction of the S-layer could be calculated. Lattice defects visualized at 1 nm resolution revealed the molecular boundaries of PS2 proteins. The combination of AFM imaging and single molecule force spectroscopy allowed the mechanical properties of the Corynebacterium glutamicum S-layer to be examined. The results provide a basis for understanding the amazing stability of this protective bacterial surface coat.

Bacterial Outer Membrane Proteins↗

Hippocampal layers on high resolution magnetic resonance images: real or imaginary?

To evaluate whether the lines occasionally detected on clinical magnetic resonance (MR) images are genuine hippocampal layers, a formalin fixed hippocampal specimen was scanned using T2 weighted sequences at 7 Tesla (voxel dimensions 0.064 x 0.064 x 1 mm) and at 1.5 Tesla (voxel dimensions: 0.156 x 0.156 x 1 mm) and compared with the results of histological examination. In addition, a healthy volunteer was scanned with a T2 weighted sequence at 1.5 Tesla (voxel dimensions: 0.469 x 0.469 x 2 mm). On 7 Tesla images hippocampal layers and the granule cell layer of the dentate were visible. On 1.5 Tesla images of the specimen, the hippocampal layers were again identified, but the granule cell layer of the dentate was not detectable. On 1.5 Tesla images of the hippocampus in vivo, 3 layers could be distinguished in the hippocampus on some slices. These mainly represented the alveus, pyramidal cell layer and stratum radiatum. A dark line consisting of a few pixels possibly represented the dentate gyrus. Our results show that the lines occasionally detected on clinical MR images are likely to be real hippocampal layers. However, the resolution currently used in clinical imaging (typically 0.469 x 0.469 x 2 mm or lower) is not sufficient for the detection of all hippocampal layers. For the reliable detection of all hippocampal layers on MR images an increase by a factor of approximately 20 would be necessary.

Adult↗

Layer-specific programs of development in neocortical projection neurons.

How are long-range axonal projections from the cerebral cortex orchestrated during development? By using both passively and actively transported axonal tracers in fetal and postnatal ferrets, we have analyzed the development of projections from the cortex to a number of thalamic nuclei. We report that the projections of a cortical area to its corresponding thalamic nuclei follow highly cell-specific programs of development. Axons from cells in the deepest layers of the cerebral cortex (layer 6 and superficial subplate neurons) appear to grow very slowly and be delayed for several weeks in the cerebral white matter, reaching the thalamus over a protracted period. Neurons of layer 5, on the other hand, develop their projections much faster; despite being born after the neurons of deeper layers, layer 5 neurons are the first to extend their axons out of the cortical hemisphere and innervate the thalamus. Layer 5 projections are massive in the first postnatal weeks but may become partly eliminated later in development, being overtaken in number by layer 6 cells that constitute the major corticothalamic projection by adulthood. Layer 5 projections are area-specific from the outset and arise as collateral branches of axons directed to the brainstem and spinal cord. Our findings show that the early development of corticofugal connections is determined not by the sequence of cortical neurogenesis but by developmental programs specific for each type of projection neuron. In addition, they demonstrate that in most thalamic nuclei, layer 5 neurons (and not subplate or layer 6 neurons) establish the first descending projections from the cerebral cortex.

Animals↗

Use of multiple layers of Gafchromic film to increase sensitivity.

This note investigates the increase of the sensitivity of radiochromic film by layering the film together. Investigations with up to five layers of radiochromic film placed together as a single dosimeter have allowed accurate two-dimensional dosimetry at low radiotherapy doses. The sensitivity of the response of the layered film increases with the number of layers, with the five-layer dosimeter increasing the change in optical density by 4.3 +/- 0.2 times that for a single layer film at 670 nm readout wavelength. Measurements in the penumbral region of a 6 MV x-ray beam show that the spatial resolution is slightly reduced with the layered film. The distance across the region from 20 to 80% of the maximum dose at the beam edge (20/80% penumbra) measured at 1.5 cm depth was measured as 3.1 +/- 0.3 and 3.5 +/- 0.3 mm for one and five layers, respectively. Thus no major reduction in spatial resolution was found by layering the film to five layers but the sensitivity was increased by 430% at 670 nm readout wavelength.

Particle Accelerators↗

An iterative method for calculating gamma-ray build-up factors in multi-layer shields.

Point kernel codes that simulate gamma-ray transport often use build-up factors to take scattered photons into account. This study introduces a new method, for computing multi-layer shield build-up factors. This method, based on an empirical formula for calculating double-layer shield build-up factors, is iterative. For an N-layer shield, each iteration of the method treats the first and the second layer of the shield. It replaces these layers by a single equivalent layer composed of an appropriate material and, hence, it turns the N-layer shield into an (N - 1)-layer shield. In order to determine the equivalent layer of an appropriate material, a neural network approach is developed: some neural networks trained on a large set of various configurations provide the equivalent material for any double-layer configuration. The method is implemented into MERCURE-6.3 straight-line attenuation code and is validated by comparison between MERCURE-6.3 results and reference data for one-dimensional geometries. Reference data obtained from transport calculations performed using the Sn transport code TWODANT. The comparisons prove the accuracy and sturdiness of the method.

Algorithms↗

Steroid concentrations in isolated theca and granulosa layers of preovulatory follicles during the ovulatory cycle of the domestic hen.

The purpose of the present investigation was to measure the concentrations of progesterone (P4), testosterone (T) and estradiol-17 beta (E2) in isolated theca and granulosa layers of the five preovulatory follicles of the domestic hen. The largest follicle (F1), the second largest (F2), third largest (F3), fourth largest (F4) and fifth largest (F5) follicles were removed at 24, 18, 12, 6 and 2 h before the expected ovulation. Theca and granulosa layers were isolated and P4, T, E2 and protein concentrations determined. Protein concentrations of the granulosa and theca layers increased 5- and 15-fold, respectively, during the five ovulatory cycles prior to ovulation. As the follicle approached ovulation, there was a linear decrease in E2 concentration of the theca layer with the most significant decrease (P less than 0.001) occurring between 24 and 18 h of the ovulatory cycle. In the F3 and F4 theca layers, there was a significant increase (P less than 0.005) in E2 at 6 h of the ovulatory cycle. Fluctuations in T concentrations in theca and granulosa layers were similar. There was a significant increase (P less than 0.05) in T in both layers of the F2, F3 and F4 follicles at 6 h followed by a decrease (P less than 0.005) in the theca layers at 2 h of the ovulatory cycle. The P4 concentration of the granulosa layer increased gradually during follicular maturation, with the greatest increase occurring in the F2 and F1 granulosa layers between 18 and 12 h of the ovulatory cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

ISBst12, a novel type of insertion-sequence element causing loss of S-layer-gene expression in Bacillus stearothermophilus ATCC 12980.

The cell surface of the surface layer (S-layer)-carrying strain of Bacillus stearothermophilus ATCC 12980 is completely covered with an oblique lattice composed of the S-layer protein SbsC. In the S-layer-deficient strain, theS-layer gene sbsC was still present but was interrupted by a novel type of insertion sequence (IS) element designated ISBst12. The insertion site was found to be located within the coding region of the sbsC gene, 199 bp downstream from the translation start of SbsC. ISBst12 is 1612 bp long, bounded by 16 bp imperfect inverted repeats and flanked by a directly repeated 8 bp target sequence. ISBst12 contains an ORF of 1446 bp and is predicted to encode a putative transposase of 482 aa with a calculated theoretical molecular mass of 55562 Da and an isoelectric point of 9.13. The putative transposase does not exhibit a typical DDE motif but displays aHis-Arg-Tyr triad characteristic of the active site of integrases from the bacteriophage lambda Int family. Furthermore, two overlapping leucine-zipper motifs were identified at the N-terminal part of the putative transposase. As revealed by Southern blotting, ISBst12 was present in multiple copies in the S-layer-deficient strain as well as in the S-layer-carrying strain. Northern blotting indicated that S-layer gene expression is already inhibited at the transcriptional level, since no sbsC-specific transcript could be identified in the S-layer-deficient strain. By using PCR, ISBst12 was also detected in B. stearothermophilus PV72/p6, in its oxygen-induced strain variant PV72/p2 and in the S-layer-deficient strain PV72/T5.

Amino Acid Sequence↗

Multi-layered PZT/polymer composites to increase signal-to-noise ratio and resolution for medical ultrasound transducers part II: thick film technology.

Increasing transducer bandwidth and signal-to-noise ratio (SNR) is fundamental to improving the quality of medical ultrasound images. In previous work, we have proposed the use of multi-layer 1-3 PZT/polymer composites to increase both, but have encountered significant fabrication challenges [19]. These difficulties include making the bond thickness between the layers small relative to the ultrasound wavelength and aligning the posts of the composite to increase coupling coefficient. Thus, we have developed a multi-layer composite hybrid array that will not require post alignment. Starting from a 2-MHz, three-layer PZT-5H, thick film transducer designed for 1.5-D arrays, cuts are made only through the top layer and back-filled with epoxy, forming a composite layer on top of two ceramic layers. Finite element (PZFlex) simulations show that for a 2-MHz phased-array element with a single matching layer, the three-layer hybrid structure increases the pulse echo SNR by 11 dB versus a single layer PZT element and improves -6 dB pulse echo fractional bandwidth by a factor of 1.4. Composite hybrid arrays fabricated in our laboratory showed an improvement in SNR of 6 to 11 dB over a PZT control and an increase in -6 dB bandwidth by a factor of 1.1. Images from a phased-array scanner confirmed these improvements.

Ceramics↗

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

ATP-Binding Cassette Transporters↗

Oxygen-inhibited layer in adhesion dentistry.

PURPOSE: Characteristics of the oxygen-inhibited layer, including bond strength, photoinitiator decomposition, and post-curing degree of conversion, were investigated. MATERIALS AND METHODS: To investigate shear bond strength, BisCover (Bisco, Inc., Schaumburg, IL, USA) and D/E Resin (Bisco, Inc.) were placed on disks of Renew composite (Bisco, Inc.) and cured both with and without an oxygen-inhibited layer. Light-Bond composite (Reliance Orthodontic, Itasca, IL, USA) was placed in a gelatin capsule and light cured over the cured resin. After soaking in water for 2 hours at 37 degrees C, specimens were sheared to failure using a universal testing machine (Model 4466, Instron Inc., Canton, MA, USA). To investigate microtensile bond strength, composite substrates prepared using Renew A2 composite were light cured either in air or under nitrogen. Light-Core (Bisco, Inc.) was placed on each substrate and light cured. The resulting specimens were sectioned into composite beams and stressed to failure using a microtensile tester (built by Bisco, Inc.). To determine camphorquinone (CQ) decomposition, an experimental CQ resin was placed between two glass plates and irradiated for different time intervals. The absorption spectrum was obtained using a Cary 50 Bio UV-Visible Spectrometer (Varian, Mulgrave, Australia). To explore the degree of conversion, polyester film strips (Mylar, DuPont, Wilmington, DE, USA) coated with the CQ resin were pre-cured in air for different time periods, and then post-cured at low intensity for 5 minutes under nitrogen. A Spectrum 1000FTIR Spectrometer (Perkin Elmer, Norwalk, CT, USA) was used to measure the degree of conversion. RESULTS: Bond strength tests resulted in no significant difference between samples with or without an oxygen-inhibited layer. The oxygen-inhibited layer contained reduced amounts of photoinitiator. The degree of conversion of post-cured oxygen-inhibited layers was lower than that for the control. CONCLUSION: An oxygen-inhibited layer is not necessary for bonding with composite resin. CLINICAL SIGNIFICANCE: It was believed that an oxygen-inhibited layer was required for bonding of composite resin. Studies show that composites bond even in the absence of an oxygen-inhibited layer, including recent products that cure without an oxygen-inhibited layer. With many simplified adhesives, especially self-etching, all-in-one adhesives, the oxygen-inhibited layer is acidic. This acidity interferes with self-curing reactions, making these adhesives incompatible with self-cured composites.

Acid Etching, Dental↗

The distribution of on- and off-centre X- and Y-like cells in the A layers of the cat's lateral geniculate nucleus.

Responses to light were recorded extracellularly from single cells in the A laminae of the dorsal lateral geniculate nucleus (d.l.g.n.) in the cat. Micro-electrodes were passed through the nucleus parallel to the laminar borders allowing the depths of the cells to be measured accurately and the depth distributions of the different cell types to be determined. 1200 cells were recorded in thirty cats along ninety-nine tracks through layers A and A1. The cells were identified physiologically as on- or off-centre cells (hereafter called on or off cells) and most (n = 960) were classified as X- or Y-like cells (hereafter called X or Y cells). Both on and off cells were found throughout layers A and A1 but their proportions changed in a graded fashion with depth. On cells were the predominant type at the tops of the layers, the on and off cells were in balance in the centres, and off cells were the predominant type at the bottoms. The steepest gradient and the maximum differences in proportions occurred in layer A while in A1 the same pattern appeared to be present but was weak and not statistically significant in the present sample. X and Y cells were also distributed differently through the depths of the layers but in patterns that differed from those of the on and off cells. The Y cells were concentrated at the borders and there was a complementary increase of X cells toward the centres of the layers. Again, the pattern was strong in A but weak and not statistically significant in A1. The distribution of X-on cells peaked near the top of layer A while the distribution of X-off cells peaked near the bottom. This was not so in A1 where the distributions of X-on and X-off cells were nearly uniform and similar to one another except for a slight predominance (56%) of X-on over X-off cells that was not related to depth. The Y-on and Y-off cells showed similar patterns of distribution in both layers A and A1 with Y-on cells predominant at the tops of the layers and Y-off cells predominant at the bottoms. The differences in the distributions of the four cell types (X-on, X-off, Y-on, Y-off) through the depths of the layers result in unique combinations of cells at different depths. It is suggested in the Discussion that the changing balance between the cells may have functional consequences.

Action Potentials↗

Three-dimensional structure of the regular tetragonal surface layer of Azotobacter vinelandii.

Fragments of the Azotobacter vinelandii tetragonal surface (S) layer, free of outer membrane material, were obtained by treating whole cells with 100 microM EDTA. The three-dimensional structure of the S layer was reconstructed from tilted-view electron micrographs of the S-layer fragments, after computer-assisted image processing by correlation averaging. At a resolution of 1.7 nm, the S layer exhibited funnel-shaped subunits situated at one fourfold-symmetry axis and interconnected at the other fourfold-symmetry axis to form prominent cruciform linking structures. These data, in conjunction with a relief reconstruction of the surface of freeze-etched whole cells, indicated that the apex of the funnel-shaped subunit was associated with the outer membrane, while the funnel "opening" faced the environment; the cruciform linking structures were formed at the outermost surface of the S layer. Electron microscopy and image enhancement were used to compare the structure of the outer membrane-associated S layer with that of fragments of the S layer dislodged from the outer membrane. This analysis revealed an increase in the lattice constant of the S layer from 12.5 to 13.6 nm and an alteration in the position of the cruciform linking structures in the z direction. These conformational changes resulted in a reduction in the thickness of the S layer (minimum estimate, 5 nm) and an apparent increase in the size of the gaps between the subunits. In terms of the porosity of the S layer, this gave the appearance of a transition from a closed to a more open structure.

Azotobacter↗

Permeability and charge-dependent adsorption properties of the S-layer lattice from Bacillus coagulans E38-66.

We investigated the permeability properties of the oblique S-layer lattice from Bacillus coagulans E38-66 after depositing cell wall fragments on a microfiltration membrane, cross-linking the S-layer protein with glutaraldehyde, and degrading the peptidoglycan with lysozyme. Comparative permeability studies on such multilayered S-layer membranes and suspended S-layer vesicles from thermophilic members of the family Bacillaceae with use of the space technique (M. Sára and U. B. Sleytr, J. Bacteriol. 169:4092-4098, 1987) revealed identical molecular exclusion limits (M. Sára and U. B. Sleytr, J. Membr. Sci. 33:27-49, 1987). Examination of the S-layer lattice from B. coagulans E38-66 with the S-layer membrane technique revealed unhindered passage for molecules up to the size of myoglobin (M(r) 17,000). The molecular dimensions of this protein (2.8 by 3.2 by 4.5 nm) correspond approximately to the size of the ovoid-shaped pore previously shown by high-resolution electron microscopy of negatively stained S-layer self-assembly products (D. Pum, M. Sára, and U. B. Sleytr, J. Bacteriol. 171:5296-5303, 1989). Chemical modification of the S-layer protein and comparative labeling, adsorption, and permeability studies clearly demonstrated that (i) in the native state, free amino and carboxyl groups are present on the outer S-layer face and in the interior of the pores and (ii) electrostatic interactions between these groups prevent unspecific adsorption of the S-layer in vivo.

Adsorption↗

Isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters.

Several methods for isolation of the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A were evaluated. Treatment of cells with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 2 was the most effective means of selectively removing RsaA from cells, and after neutralization, the protein was capable of reassembling into a paracrystalline structure. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid treatment could also be used to extract RsaA and yielded protein capable of reassembly. The success of the methods was likely related to disruption of calcium-mediated bonding; calcium was required for recrystallization, while magnesium and strontium ions were ineffective. Antibody was raised against purified RsaA and, along with the S-layer extraction techniques, was used to evaluate 42 strains of caulobacters isolated from a variety of aquatic and wastewater treatment locations. A single characteristic protein could be isolated from the 35 strains that produced an S layer; with one exception, no proteins were extracted from strains that had no S layer. The presumed S-layer proteins ranged in size from 100 to 193 kDa. All of these proteins specifically reacted with anti-RsaA serum by Western immunoblot analysis. In strain CB15A, a specific S-layer-associated oligosaccharide has been proposed to be involved in a calcium-mediated attachment of the S layer to the cell surface. This molecule was detected by Western immunoblotting with a specific antiserum and on polyacrylamide gels stained for polysaccharides. A comparable band was found in all S-layer-producing strains and for most, S-layer-associated oligosaccharide-specific antibody reacted with them in Western analysis. Overall, in freshwater caulobacters at least portions of their S-layer structures appear to be strongly conserved entities, as well as the means of attachment to the cell surface.

Bacterial Proteins↗

Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.

Campylobacter fetus strains possess regular paracrystalline surface layers (S-layers) composed of high-molecular-weight proteins and can change the size and crystalline structure of the predominant protein expressed. Polyclonal antisera demonstrate antigenic cross-reactivity among these proteins but suggest differences in epitopes. Monoclonal antibodies to the 97-kDa S-layer protein of Campylobacter fetus subsp. fetus strain 82-40LP showed three different reactivities. Monoclonal antibody 1D1 recognized 97-kDa S-layer proteins from all C. fetus strains studied; reactivity of monoclonal antibody 6E4 was similar except for epitopes in S-layer proteins from reptile strains and strains with type B lipopolysaccharide. Monoclonal antibody 2E11 only recognized epitopes on S-layer proteins from strains with type A lipopolysaccharide regardless of size. In vitro shift from a 97-kDa S-layer protein to a 127-kDa S-layer protein resulted in different reactivity, indicating that size change was accompanied by antigenic variation. To examine in vivo variation, heifers were genetically challenged with Campylobacter fetus subsp. venerealis strains and the S-layer proteins from sequential isolates were characterized. Analysis with monoclonal antibodies showed that antigenic reactivities of the S-layer proteins were varied, indicating that these proteins represent a system for antigenic variation.

Animals↗

Host cell invasion and intracellular residence by Aeromonas salmonicida: role of the S-layer.

Virulent strains of the fish pathogen Aeromonas salmonicida, which have surface S-layers (S+), efficiently adhere to, enter, and survive within macrophages. Here we report that S+ bacteria were 10- to 20-fold more adherent to non-phagocytic fish cell lines than S-layer-negative (S-) mutants. When reconstituted with exogenous S-layers, these S- mutants regained adherence. As well, latex beads coated with purified S-layers were more adherent to fish cell lines than uncoated beads, or beads coated with disorganized S-layers, suggesting that purified S-layers were sufficient to mediate high levels of adherence, and that this process relied on S-layer structure. Gentamicin protection assays and electron microscopy indicated that both S+ and S- A. salmonicida invaded non-phagocytic fish cells. In addition, these fish cells were unable to internalize S-layer-coated beads, clearly suggesting that the S-layer is not an invasion factor. Lipopolysaccharide (which is partially exposed in S+ bacteria) appeared to mediate invasion. Surprisingly, A. salmonicida did not show net growth inside fish cells cultured in the presence of gentamicin, as determined by viable bacterial cell counts. On the contrary, bacterial viability sharply decreased after cell infection. We thus concluded that the S-layer is an adhesin that promotes but does not mediate invasion of non-phagocytic fish cell lines. These cell lines should prove useful in studies aimed at characterizing the invasion mechanisms of A. salmonicida, but of limited value in studying the intracellular residence and replication of this invasive bacterium in vitro.

Aeromonas↗

Comparison of S-layer secretion genes in freshwater caulobacters.

Our freshwater caulobacter collection contains about 40 strains that are morphologically similar to Caulobacter crescentus. All elaborate a crystalline protein surface (S) layer made up of protein monomers 100-193 kDa in size. We conducted a comparative study of S-layer secretion in 6 strains representing 3 size groups of S-layer proteins: small (100-108 kDa), medium (122-151 kDa), and large (181-193 kDa). All contained genes predicted to encode ATP-binding cassette transporters and membrane fusion proteins highly similar to those of C. crescentus, indicating that the S-layer proteins were all secreted by a type I system. The S-layer proteins' C-termini showed unexpectedly low sequence similarity but contained conserved residues and predicted secondary structure features typical of type I secretion signals. Cross-expression studies showed that the 6 strains recognized secretion signals from C. crescentus and Pseudomonas aeruginosa and similarly that C. crescentus was able to secrete the S-layer protein C-terminus of 1 strain examined. Inactivation of the ATP-binding cassette transporter abolished S-layer protein secretion, indicating that the type I transporter is necessary for S-layer protein secretion. Finally, while all of the S-layer proteins of this subset of strains were secreted by type I mechanisms, there were significant differences in genome positions of the transporter genes that correlated with S-layer protein size.

ATP-Binding Cassette Transporters↗

Electrical and mechanical interactions between the muscle layers of canine proximal colon.

Electrical and mechanical interactions between the two smooth muscle layers of canine colon have been studied using a dual sucrose gap apparatus. Muscle samples were dissected into an L-shape, with one leg cut in the circular direction and the other cut in the longitudinal direction. Longitudinal muscle was removed from the circular leg and circular muscle was removed from the longitudinal leg. The bend of the L contained both layers. The activity of the two layers was studied simultaneously under basal conditions, after stimulation by neostigmine and carbachol, and in the presence of tetrodotoxin. Interactions were more common after stimulation and were marked by modification of one layer's mechanical and electrical activity during increased activity in the other layer. Two patterns were commonly observed. First, during a burst of membrane potential oscillations and spike potentials in the longitudinal layer, slow waves in the circular layer developed spike potentials and some slow waves were also prolonged. Second, during a slow-wave cycle in the circular layer, the amplitude of membrane potential oscillations in the longitudinal layer was increased with an associated increase in the incidence of spike potentials. These interactions were associated with contractions of increased strength, which were similar in both layers. All interactions continued after nerve-conduction blockade by tetrodotoxin.

Animals↗