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PubMed1984

Cyclic nucleotide distribution in identified layers of suprafused rabbit retinas.

To assess the effects of pharmacologic agents on metabolite levels in identified retinal layers we have devised an apparatus that allows suprafusion of everted eye cups (vitreal surface exposed) or isolated retinas (photoreceptor surface exposed), recording of electroretinograms (ERG), and rapid freezing in a configuration which permits obtaining frozen tangential sections of the retina. Samples from identified layers of the freeze-dried sections can be subsequently weighed and processed for biochemistry by Lowry methods (Lowry and Passonneau , 1972), or cyclic nucleotides can be extracted from weighed samples and assayed. To test the usefulness of the apparatus, we have compared levels of guanosine 3',5'-monophosphate (cGMP) and adenosine 3',5'-monophosphate (cAMP), measured in defined layers of suprafused rabbit retinas, to concentrations reported for retinal layers from eyes removed from light- and dark-adapted rabbits and quickly frozen. For most layers the concentrations of cyclic nucleotides were similar in both the suprafused and whole eye preparations. In experimental applications we suprafused dark-adapted eye cups and retinas with a physiological saline whose free calcium level was sharply reduced by chelation, and observed that cGMP levels were elevated only in those retinal layers containing photoreceptor portions. Ethyleneglycol bis-(beta-amino-ethyl ether)N,N'-tetraacetic acid (EGTA) (3 mM) yielded a threefold increase in cGMP in 10 min when applied to the photoreceptor surface of isolated retinas. However, when the vitreal surfaces of everted eye cups were suprafused with calcium-chelated medium for 10 min, a threefold increase in cGMP required 20 mM EGTA, 3 mM EGTA being ineffective. In another study, vitreal surfaces of eye cups or photoreceptor surfaces of retinas were suprafused with physiological saline containing 3 mM 3-isobutyl-1-methylxanthine (IBMX) in order to alter cAMP retinal levels. In both cases, eightfold increases in cAMP were measured in the inner nuclear and inner plexiform layers, while three- to fourfold increases occurred in the outer nuclear and outer plexiform layers. This agent also caused a large increase in cAMP in the pigment epithelium.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Adenine nucleotide and P-creatine levels in layers of frog retina as a function of dark and light adaptation.

Eight layers of frog retina were analyzed for ATP, P-creatine, ATP + ADP, and AMP under conditions of dark, 2 sec, 2 min, and in the case of AMP, 2 hr of light adaptation. Samples of each layer, usually ranging between 5 and 50 ng, were dissected from lyophilized frozen sections. After brief light exposure, ATP dropped while ADP rose sharply in the pigment epithelium, outer segments, and inner segments; ADP was too low to be measured accurately in the inner retina. The profile of ATP, P-creatine, and ATP + ADP concentrations showed peaks in the inner nuclear and ganglion layers. AMP, by contrast, was highest in the two plexiform layers. Levels in the inner retina dropped after only 2 sec of light but rose after 2 hr to levels that were higher than dark values in all retinal layers. AMP was often characterized by a non-uniform distribution: adjacent areas of a layer agreed very closely in value to each other but could vary several-fold from a different section of the same frog or from another frog exposed to the same conditions. This distribution produced clusters of values, particularly prominent in dark-adapted animals, something not observed with the other metabolites measured. The peaks of AMP in the plexiform layers suggest that AMP may be a by-product of dopamine-stimulated adenylate cyclase which also has peaks in these same layers.

Adenine Nucleotides↗
PubMed1995

Effect of adhesive layer thickness on the bond strength of a zinc polycarboxylate dental cement.

This study reports the effect of varying the thickness of a commercial zinc polycarboxylate dental cement on its adhesion in a metal-to-metal single lap joint. The thickness was controlled externally in a specially designed apparatus, rather than by the inclusion of isodimensional glass spheres, as has been done previously. The results show that both the tensile shear bond strength and the mode of failure of the cement are influenced by the thickness of the cement layer in the bonded joints. Hence, these results confirm previous findings that there is an optimum thickness for the zinc polycarboxylate layer. They also show that failure is more likely to be cohesive with thicker layers. The optimum thickness of the adhesive layer was 205 microns for the particular cement studied, PolyF Plus (De Trey, Dentsply, Weybridge, Surrey, UK), for which the average shear bond strength was found to be 4.03 MPa. Using stress analysis, the maximum shear and tensile stresses, tau 12(max) and sigma 11(max), have also been obtained. These analyses showed that joints failed at the interface between the cement layers and the lap joint at adhesive layer thicknesses of below 100 microns. This resulted from the high stress concentrations in bonded joints with thin adhesive layers. On the other hand, thicker joints failed cohesively within the adhesive layer because of greater contributions from tensile stresses in these joints.

Adhesiveness↗
PubMed1995

Cytokine expression in adherent layers from patients with myelodysplastic syndrome and acute myelogenous leukemia.

We have previously shown that long-term cultures of adherent layers derived from patients with chronic myelogenous leukemia in blast crisis express high levels of interleukin (IL)-1 beta and that this cytokine may participate in disease progression. In this study, we analyzed cytokine expression in bone marrow adherent layers derived from patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). IL-6 messenger RNA (mRNA) was expressed in adherent layers established from four of nine MDS patients, and from 10 of 17 AML patients (including all four individuals in whom AML had evolved from an antecedent MDS state). Similarly, IL-1 beta mRNA was expressed in adherent layers derived from two of nine MDS patients and from three of 17 AML patients. Cultures from two of 10 AML patients who expressed IL-6 also expressed granulocyte (G) colony-stimulating factor (CSF) mRNA. In contrast, IL-1 beta, IL-6, and G-CSF mRNA were not discernible in adherent layers from any of 14 normal volunteers. Transforming growth factor-beta 1, macrophage (M) CSF, IL-7, and leukemia inhibitory factor mRNA as well as IL-6 protein were constitutively expressed in adherent layers derived from both MDS patients, AML patients, and normal bone marrows, whereas IL-1 alpha, tumor necrosis factor-alpha, and GM-CSF were not expressed in either the normal-, MDS- or AML-derived adherent layers. These results indicate that cultured stroma from a subset of MDS and AML patients produce IL-1 beta and/or IL-6. Although, exposure of adherent layers to exogenous IL-1 beta was able to induce IL-6 expression, in 9 of the 14 samples constitutively expressing cytokines, IL-6 transcript levels were elevated without a concomitant increase in IL-1 beta, suggesting that IL-6 transcription was independently dysregulated.

Adolescent↗
PubMed1989

Loss of HCH from surface soil layers under subtropical conditions.

The loss of HCH (hexachlorocyclohexane) at an application rate of 25 kg ha(-1) was studied under field conditions from two surface soil layers, each of 7.5 cm, at two sites in Delhi. The soil at both sites was sandy loam type, with a pH of 8.2, and 0.8 to 1.0% organic matter content. At site 1, which was kept fallow and not watered, the upper 7.5-cm layer of soil initially lost HCH more rapidly than the lower layer. The half-life of the HCH in the upper and lower 7.5-cm layers was 21 and 41 days, respectively, and it was 26 days for the total HCH in the combined 15-cm soil layer. At site 2, which contained ornamental plants and was watered regularly, there was not much difference in the loss of HCH between the upper and lower layers. The half-life of HCH was 17 and 25 days for the upper and lower 7.5-cm layers, respectively, and it was 20 days for the total 15-cm soil layer, at this site. The loss was greatest initially at the sites, and was faster in wet soil than in dry soil.

Journal Article↗
PubMed1990

Kainic acid lesioning alters development of the outer plexiform layer in neonatal rabbit retina.

The first synaptic relay in the primary visual pathway occurs between terminals of photoreceptors and second-order neurons within the outer plexiform layer of the retina. During development, one of these types of second-order neurons, the type A horizontal cell, differentiates and assumes mature characteristics several days before any other cells ramifying in that synaptic layer. In neonates, horizontal cells appear to be GABAergic during the first 5 days of postnatal life and in addition they also are responsive to kainic acid. We have previously suggested that they may play a pioneering role in the postnatal development of the outer plexiform layer, perhaps providing structural guidance or trophic substances such as GABA, for synaptic development. To test this hypothesis, we first demonstrated that a single intraocular injection of kainic acid within 24 hr of birth results in a permanent and selective loss of type A horizontal cells in the outer retina. Retinas from animals maintained for 5 days postinjection were harvested for analysis of postnatal development of the outer plexiform layer in the absence of horizontal cells. One of the major findings was that kainic acid treatment caused a reversal of the normal complement of photoreceptor cell types, resulting in an abnormally high rod/cone ratio. The distribution of cell processes within the outer plexiform layer was also altered and normal synaptic connections were not made. In spite of these changes in the constituents of the outer plexiform layer, the normal position of the synaptic layer was not affected by the loss of horizontal cells. These results rule out the possibility that horizontal cells provide a structural barrier which is an absolute requirement for establishing the location of the outer plexiform layer. Rather, these cells may be more involved in cell differentiation and synaptogenesis.

Animals↗
PubMed2006

The blood vessel system in the periodontal ligament of the equine cheek teeth--part I: The spatial arrangement in layers.

Corrosion casts of blood vessels in the periodontium of cheek teeth from eight horses were observed three-dimensionally with a dissection microscope. Selected specimens were examined in a scanning electron microscope. Periodontal blood vessels communicated with those from the gingiva, the alveolar bone, and the apical region. In the upper jaw, there were anastomoses with the blood vessels of the mucosa of the maxillary sinus. The periodontal vascular system was organized in two or three layers. The peripheral layer was mainly composed of large venules, the inner one consisted of capillaries. In the intermediate layer, blood vessels were post-capillary venules. This layer was developed only in horses under 10 years of age. In all layers the vascular orientation was mainly occluso-apical, this was defined as the standard pattern. There were many variations displayed in different courses of certain blood vessels. The vascular organization is discussed with regard to the specialized functions of the periodontal ligament (PDL). The wide vessels of the outer layer are thought to play a mechanical role as part of a shock absorbing system. The capillaries of the inner layer meet nutritional requirements. The disappearance of the intermediate layer in horses older than 10 years is taken as an adaptation to the remodelling of the PDL. Modifications in the standard pattern of vascular arrangements are also interpreted as adaptations to life-long changes in the periodontal space. Anastomoses between the periodontal vasculature and the blood vessels of the maxillary sinus indicate that periodontal disease may be transferred into the sinus.

Animals↗
PubMed2005

Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique.

Aiming to develop a scaffold architecture mimicking morphological and mechanically that of a blood vessel, a sequential multi-layering electrospinning (ME) was performed on a rotating mandrel-type collector. A bi-layered tubular scaffold composed of a stiff and oriented PLA outside fibrous layer and a pliable and randomly oriented PCL fibrous inner layer (PLA/PCL) was fabricated. Control over the level of fibre orientation of the different layers was achieved through the rotation speed of the collector. The structural and mechanical properties of the scaffolds were examined using scanning electron microscopy (SEM) and tensile testing. To assess their capability to support cell attachment, proliferation and migration, 3T3 mouse fibroblasts and later human venous myofibroblasts (HVS) were cultured, expanded and seeded on the scaffolds. In both cases, the cell-polymer constructs were cultured under static conditions for up to 4 weeks. Environmental-scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), histological examination and biochemical assays for cell proliferation (DNA) and extracellular matrix production (collagen and glycosaminoglycans) were performed. The findings suggest the feasibility of ME to design scaffolds with a hierarchical organization through a layer-by-layer process and control over fibre orientation. The resulting scaffolds achieved the desirable levels of pliability (elastic up to 10% strain) and proved to be capable to promote cell growth and proliferation. The electrospun PLA/PCL bi-layered tube presents appropriate characteristics to be considered a candidate scaffold for blood vessel tissue engineering.

3T3 Cells↗
PubMed2004

PAMPA--a drug absorption in vitro model 11. Matching the in vivo unstirred water layer thickness by individual-well stirring in microtitre plates.

Many plate-based in vitro assays of membrane permeability (e.g., Caco-2, MDCK, PAMPA) of sparingly soluble candidate molecules report permeability of water, and not of the intended membrane barrier. This is so because the unstirred water layer on both sides of the membrane barrier is rate limiting for these highly permeable molecules. The thickness of this water layer can be 1500-4000 microm in unstirred assays. Under in vivo conditions, however, the unstirred water layer is believed to be 30-100 microm thick. Lightly stirred in vitro assays, using plate shakers, cannot lower the thickness of the water layer to match that found in vivo. In this study, 55 lipophilic drugs were employed to characterize the effect of stirring in parallel artificial membrane permeability assay (PAMPA). Highly efficient individual-well magnetic stirring at speeds greater than 110 rpm has been demonstrated to lower the unstirred water layer thickness to the in vivo range. Stirring at 622 rpm has lowered the layer thickness to 13 microm in some cases, which had not been previously achieved for plate-based permeability assays. With diminished water layer contribution at 622 rpm, for example, the effective permeability of progesterone is 2754 x 10(-6) cm/s. The new stirring apparatus used in this study is not only suitable for PAMPA, but can also be used in Caco-2 assays. Because of the diminished resistance of the thinner water layer, the stirred PAMPA permeation time has decreased from the usual 15 h to about 15 min for lipophilic compounds.

Absorption↗
PubMed2007

Immediate hemodynamic effect of the additional use of the SCD EXPRESS Compression System in patients with venous ulcers treated with the four-layer compression bandaging system.

OBJECTIVES: To test the hypothesis that the SCD EXPRESS intermittent pneumatic compression applied in combination with a four-layer bandage in patients with venous ulcers increases popliteal vein volume flow and velocity. DESIGN: Twenty limbs of 18 patients with venous leg ulcers were studied, median age 76 years. The Total Volume Flow (TVF) and the Peak Systolic Velocity (PSV) were recorded in the popliteal vein using duplex ultrasonography. Measurements were made (i) without bandage, (ii) with four layer bandage and (iii) following the application of the SCD Compression System on top of a four-layer bandage for at least 15 minutes. RESULTS: The median VCSS was 17 (range, 12-22) while the median VSDS for reflux was 4.5 (range, 1-7.5). The median TVF was 71 mL/min (inter-quartile range 57-101) without bandage, 112 (IQR 89-148) with four-layer bandage and 291 (IQR 241-392) with the addition of the SCD System (P<.001, Wilcoxon signed ranks test). The median PSV was 8.4 cm/sec (IQR 6.8-14) without bandage, 13 (9.0-19) with four-layer bandage and 27 (21-31) with the addition of the SCD System (P<.001, Wilcoxon signed ranks test). Both TVF and PSV increased slightly with the addition of the four-layer bandage. However, with the addition of the SCD System these parameters increased three fold. CONCLUSIONS: The SCD EXPRESS Compression System accelerates venous flow in the legs of patients with venous ulcers already treated with a four-layer bandage. The combination of four-layer compression with the SCD System on healing venous ulcers needs to be tested by a clinical effectiveness study.

Aged↗
PubMed2005

Lactobacillus surface layers and their applications.

Surface (S-) layers are crystalline arrays of proteinaceous subunits present as the outermost component of cell wall in several species of the genus Lactobacillus, as well as in many other bacteria and Archaea. Despite the high similarity of the amino acid composition of all known S-layer proteins, the overall sequence similarity is, however, surprisingly small even between the Lactobacillus S-layer proteins. In addition, the typical characteristics of Lactobacillus S-layer proteins, distinguishing them from other S-layer proteins, are small size and high-predicted pI value. Several lactobacilli possess multiple S-layer protein genes, which can be differentially or simultaneously expressed. To date, the characterized functions of Lactobacillus S-layers are involved in mediating adhesion to different host tissues. A few applications for the S-layer proteins of lactobacilli already exist, including their use as antigen delivery vehicles.

Bacterial Outer Membrane Proteins↗
PubMed2004

Characteristics of erythritol and formulation of a novel coating with erythritol termed thin-layer sugarless coating.

The purpose of this study was to clarify the characteristics of erythritol and to develop the optimum basic formulation of a novel coating with erythritol termed thin-layer sugarless coating. Characteristics of erythritol were investigated compared with maltitol, mannitol, sorbitol, xylitol, and sucrose. Furthermore, the optimum basic formulation of thin-layer sugarless coating with erythritol was determined by coating glass beads. We selected a continuous spray mist method for thin-layer sugarless coating due to the formation of a thin sugarless coating layer by a simple method. We demonstrated that erythritol is a suitable coating material for thin-layer sugarless coating compared with maltitol, mannitol, sorbitol, xylitol, and sucrose because of its high water solubility, low hygroscopicity, instant crystallization, and low tackiness. We also demonstrated that thin-layer sugarless coating with erythritol can reduce coating time compared with the coating with maltitol or sucrose due to its characteristics. We developed the optimum basic formulation of thin-layer sugarless coating consists of erythritol, powdered acacia, and talc. We confirmed that a smooth coating layer and high coating efficiency were achieved using the formulation.

Drug Stability↗
PubMed2005

Interfacial characteristics of heterogeneous layers of linear polyvinylamine and branched polyethyleneimine or polyethyleneimine grafted with C12-C22 alkyl chains.

We investigated the characteristics of heterogeneous layers composed of linear hydrolyzed polyvinylamine and branched polyethyleneimine adsorbed at silica/water interfaces. The studies also included heterogeneous layers where branched polyethyleneimine was replaced by polyethyleneimine modified by grafting with C12-C22 alkyl chains. Surface area exclusion chromatography was used to determine the interfacial relaxation and surface affinity of the polymer molecules within homogeneous layers. The relaxation of bare and grafted polyethyleneimine was found to be small and of equal extent but to develop at different rates. Comparatively, the relaxation of hydrolyzed polyvinylamine was faster and of greater extent. Within heterogeneous layers composed of polyvinylamine and bare or grafted polyethyleneimine, the relaxation of the different molecules was strongly increased as compared to that prevailing in homogeneous layers. The chromatographic method was then used to determine the mode of layer establishment. The polymer coating profiles on successive glass fiber filters were found to depend on the sequence of injection of the two polymers, due to the interfacial stability or instability of the initially established layer. It was shown that a previously established extremely thin layer of bare or grafted polyethyleneimine molecules strongly modified the adsorption profile of subsequently adsorbed polyvinylamine molecules.

Journal Article↗
PubMed2006

Characterization and removal of DNAPL from sand and clay layered media.

Important characterization and remediation techniques (e.g. partitioning tracer tests (PTT), cosolvent and surfactant flushing) have been developed over the years to deal with dense nonaqueous phase liquid (DNAPL) sources in the saturated zone. Unfortunately, subsurface media layering and heterogeneity pose a major challenge to the efficiency of these and other techniques that rely on flushing fluids through porous media. Using laboratory column experiments with both single media and layered-media columns and computer modeling of tracer breakthrough results, we examined the difficulty that layering poses to subsurface remediation and characterization techniques. Quantifying tetrachloroethylene (PCE) saturation in layered media was determined using PTT and effluent mass determinations. Conservative tracer breakthrough curves were used to determine permeability and the flow through layers before and after PCE contamination. The removal efficiency of alcohol flushing in the layered systems was also determined. Results showed that even a relatively simple layering with less than an order magnitude difference in permeability leads to difficulty in characterization and remediation using flushing technologies. These results suggest that much greater volumes of flushing solutions will be needed in heterogeneous environments to ensure adequate flushing of lower permeable lenses and layers.

Alcohols↗
PubMed2005

The relative contributions of different skin layers to the mechanical behavior of human skin in vivo using suction experiments.

Although the mechanical behavior of the top layer of the skin, the epidermis, is an important consideration in several clinical and cosmetic applications, there are few reported studies on this layer. The in vivo mechanical behavior of the upper skin layer (here defined as epidermis and papillar dermis) was characterized using a combined experimental and modeling approach. The work was based on the hypothesis that experiments with different length scales represent the mechanical behavior of different skin layers. Suction measurements with aperture diameters of 1, 2 and 6 mm were combined with ultrasound and optical coherence tomography to study the deformation of the skin layers. The experiments were simulated for small displacements with a two-layered finite element model representing the upper layer and the reticular dermis. An identification method compared the experimental and numerical results to identify the material parameters of the model. For one subject the whole parameter estimation procedure was completed, leading to a stiffness of C(10,ul) = 0.11 kPa for the top-layer and C(10,rd) = 0.16 MPa for the reticular dermis. This unexpected, extreme stiffness ratio of the material parameters let to convergence problems of the finite element software for most of the individuals.

Adult↗
PubMed2006

Alterations of M(2)-muscarinic receptor protein and mRNA expression in the urothelium and muscle layer of the streptozotocin-induced diabetic rat urinary bladder.

Diabetes associated alterations of M2-muscarinic receptors (M2-mAChR) in the urothelium and muscle layer of the urinary bladder were studied using streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats, 2 weeks after induction. The bladder was divided into urothelium and muscle layer by microdissection. Tissue M2-mAChR protein levels were measured by Western blotting. Expression of the mRNA that encoded M2-mAChR was estimated using the method of reverse transcription combined with polymerase chain reaction (RT-PCR). M2-mAChR protein and mRNA expressions were found in both the urothelium and muscle layer of the rat urinary bladder. In control rats, the M2-mAChR protein expression ratio in the urothelium and muscle layer was 1:1.66; that for mRNA was 1:0.97. Two weeks after induction of diabetes, the M2-mAChR mRNA expression in the urothelium and muscle layer were significantly increased by 44.4% (P<0.01, n=8) and 28.6% (P<0.01, n=8), respectively. Correspondently, the bladder M2-mAChR protein levels were significantly increased by 33.3% (P<0.001, n=8) in the urothelium and 25.3% (P<0.01, n=8) in the muscle layer of the diabetic rats. In conclusion, M2-mAChR mRNA and protein are expressed in both the urothelium and muscle layer of the rat urinary bladder. STZ-induced diabetes increases mRNA and protein expression of the M2-mAChR in the urothelium as well as the muscle layer.

Animals↗
PubMed2006

Regulation of layer-specific targeting by reciprocal expression of a cell adhesion molecule, capricious.

Layer-specific innervation is a major form of synaptic targeting in the central nervous system. In the Drosophila visual system, photoreceptors R7 and R8 connect to targets in distinct layers of the medulla, a ganglion of the optic lobe. We show here that Capricious (CAPS), a transmembrane protein with leucine-rich repeats (LRRs), is a layer-specific cell adhesion molecule that regulates photoreceptor targeting in the medulla. During the period of photoreceptor targeting, caps is specifically expressed in R8 and its target layer but not in R7 or its recipient layer. caps loss-of-function mutations cause local targeting errors by R8 axons, including layer change. Conversely, ectopic expression of caps in R7 redirects R7 axons to terminate in the CAPS-positive R8 recipient layer. CAPS promotes homophilic cell adhesion in transfected S2 cells. These results suggest that CAPS regulates layer-specific targeting by mediating specific axon-target interaction.

Animals↗
PubMed2005

Er81 is expressed in a subpopulation of layer 5 neurons in rodent and primate neocortices.

Laminar organization is a fundamental cytoarchitecture in mammalian CNS and a striking feature of the neocortex. ER81, a transcription factor, has recently been utilized as a marker of cells in the layer 5 of the neocortex. We further pursued the distribution of ER81 to investigate the identity of the ER81-expressing cells in the brain. Er81 transcript was expressed in a subset of pyramidal cells that were scattered throughout the entire width of layer 5. In the rat cortex, Er81 transcripts were first detected in the ventricular zone at E15, remained expressed in putative prospective layer 5 neurons during infant and juvenile stages. The ER81-expressing subpopulation in adult layer 5 neurons did not segregate with the phenotypes of the projection targets. By retrograde labeling combined with immunohistochemistry or reverse transcription-polymerase chain reaction analysis, we found ER81 expression in nearly all of the layer 5 neurons projecting to the spinal cord or to the superior colliculus, while in only one-third of the layer 5 neurons projecting to the contralateral cortex. Er81 was also detected in layer 5 neurons in a P2 Japanese macaque monkey but not in adult monkey cortices. These findings suggest that a neuron class defined by a molecular criterion does not necessarily segregate with that defined by an anatomical criterion, that ER81 is involved in cell differentiation of a subset of layer 5 projection neurons and that this mechanism is conserved among rodents and primates.

Aging↗