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Flow cytometric analysis of variations in the DNA content of superficial and deep layers in colorectal cancer.

We investigated variations in the DNA content of multiple samples from both superficial and deep layers of tumors from 81 colorectal cancer cases. The findings were only diploid in 21 cases (25.9%) and only aneuploid in 49 cases (60.5%). On the whole, the DNA ploidy agreed in 70 cases (86.4%) between both layers. In the 49 cases with only aneuploid tumors in both layers, the DNA index (mean +/- SD) was 1.58 +/- 0.25 in the superficial layer and 1.59 +/- 0.25 in the deep layer, with the values being almost identical. In contrast, a variation in the DNA ploidy of 11 cases (13.6%) was observed. These findings show that DNA ploidy remains stable during tumor progression from the superficial into the deep layers in most cases of colorectal cancer. However, the existence of heterogeneity in the DNA ploidy also shows that several specimens should be collected from both layers before making a determination of DNA ploidy.

Adult↗

Controlled delivery achieved with bi-layer matrix devices produced by co-injection moulding.

The aim of this study was to design new soy protein-based bi-layered co-injection moulded matrix systems aimed to achieve controlled drug delivery. The devices consisted of a drug-free outer layer (skin) and a drug-containing core. The systems overcame the inherent disadvantage of non-linear release associated with diffusion-controlled single-layer matrix devices by providing additional releasing area with time to compensate for the decreasing release rate. As expected, the bi-layer devices presented a significant decrease in drug release rate when compared with a correspondent single layer matrix system. The skin thickness and the degree of crosslinking of the core appeared to be very important tools to tailor the release patterns. Furthermore, due to the amphoteric nature of the soy protein, the developed devices evidenced a pH-dependent behaviour. The mechanisms of drug release were also elucidated at two different pH values: i) pH 5.0, near the isoelectric point of soy (low matrix solubility); and ii) pH 7.4, physiological pH (high matrix solubility). Consequently, changing the release medium from pH 5.0 to pH 7.4 after two hours, led to an abrupt increase in drug release and the devices presented a typical controlled drug delivery profile: slow release/fast release. These evidences may provide for the development of individual systems with different release onsets that in combination may exhibit drug releases at predetermined times in a pre-programmed way. Another possibility is the production of three-layer devices presenting bimodal release profiles (fast release/slow release/fast release) by similar technologies. Scanning electron micrograph of a developed bi-layer device.

Biocompatible Materials↗

Interaction between a Solute Molecule and a Fluorocarbon Bonded Layer in Reversed-Phase High-Performance Liquid Chromatography.

Glassesand silica-gel packings modified with fluorocarbon and hydrocarbon silylation agents were prepared to investigate the separation mechanism of a fluorocarbon bonded layer in reversed-phase high-performance liquid chromatography (RP-HPLC). On the hydrocarbon layer the contact angles of benzene (Be) and hexafluorobenzene (FB) decreased with increasing carbon chain length of the modifier, and the mobile phase (MP) was independent of the modifier. The fluorocarbon layer gave a larger contact angle for each liquid than the hydrocarbon layer. The retention factor, k, of Be and FB on hydrocarbon packings increased with decreasing contact angle. It was demonstrated that the separation of Be and FB on hydrocarbon packings is caused by solvophobic interaction between solute molecules and the stationary layer. In contrast, k increased with increasing contact angle at the fluorocarbon layer. The retention of Be and FB on fluorocarbon packings may be attributed to the penetration of the solute molecules from the mobile phase into the fluorocarbon stationary layer. Copyright 1999 Academic Press.

Journal Article↗

Influence of soft drinks on the thickness and morphology of in situ acquired pellicle layer on enamel.

Diet-induced demineralization is a key factor in tooth enamel loss, with soft drinks being a significant aetiological agent. In the oral environment, the acquired pellicle layer might modify the demineralization potential of drinks. The objectives of this exploratory in situ study were to determine the influence of soft drinks, volunteers, and exposure times on the thickness and morphology of pellicle layers using an atomic force microscope (AFM). Polished uninterrupted third molars were partly taped and exposed to the oral environment for periods of 2 and 5 days, respectively. Each day 1L of either mineral water, a "toothkind" blackcurrant drink, a "prototype" blackcurrant drink, or orange juice was consumed in this four-way crossover, single-center study. Subsequently the tape was removed and the surfaces imaged with an AFM before and after chemical cleaning. The pellicle layer morphology and thickness were investigated. The maximum pellicle layer thickness was observed after the consumption of the prototype blackcurrant drink, followed by the toothkind blackcurrant drink, orange juice, and water on days 2 and 5, respectively. A three-way ANOVA at the 95% confidence level showed statistically significant differences between the pellicle layer thicknesses, factors being drinks (p=0.0046), exposure time (p=0.0009), and volunteers (p=0.0244). Depending on the volunteer either a uniform or a patchy pellicle layer was observed. This demonstrates that AFM is a useful tool in obtaining simultaneously qualitative and quantitative data of in situ acquired pellicle layers.

Beverages↗

Time profile of eosinophilic neurons in the cortical layers and cortical atrophy.

Eosinophilic neurons (ENs) appear in the post-ischemic cortex; however, whether there are differences in the time profile for different cortical layers and the fate of the cortex with ENs is largely unknown. We examined the time profile of ENs in different cortical layers and evolution of cortical atrophy after transient cerebral ischemia in Mongolian gerbils. Unilateral forebrain ischemia was induced twice by 10-minute unilateral common carotid artery occlusions. Brains at 24 hours, 4 days, and 2, 4, and 16 weeks post-ischemia were prepared for morphometric analysis. Quantitative analysis of ENs in regions of interest in the rostral and caudal cortex showed the highest number of ENs at 4 days post-ischemia in layers 3 and 6. Reduction in ENs after this peak was slower in layer 6 than in layer 3 in both rostral and caudal cortex, and this difference was significant in layer 6 of the caudal cortex. Infarcts with significant atrophy appeared in the rostral cortex. In the caudal cortex, only selective neuronal death with mild but distinct atrophy was observed. We observed a significant difference between cortical layers in the time profile of ENs in the post-ischemic cortex. Selective neuronal death without infarction was sufficient to induce cortical atrophy after transient cerebral ischemia.

Animals↗

The mechanism of action of the two-layer (Euro-Collins' solution/perfluorochemical) cold storage method in canine pancreas preservation.

To clarify the mechanism of action of a two-layer [Euro-Collins' solution (EC)/perfluorochemical (PFC)] cold storage method in the preservation of the pancreas, pancreatic viability and tissue concentrations of adenosine triphosphate (ATP) were examined in the canine model of pancreatic autotransplantation after preservation for 24 and 48 h by simple cold storage in EC (group 1), the two-layer, EC/PFC, method (group 2) and the two-layer, EC+2, 4 dinitrophenol (DNP)/PFC, method (group 3). DNP is an uncoupler of oxidative phosphorylation. Maintenance of normoglycemia for at least 5 days after transplantation was considered a successful preservation. After preservation for 24 h, the functional success rates of groups 1, 2 and 3 were 100% (4/4), 100% (5/5) and 80% (4/5) respectively. One of five dogs in group 3 died of a cause unrelated to the pancreas. ATP tissue concentrations in group 2 were significantly higher than in group 1 (7.47 +/- 0.47 micromol/g dry weight vs 1.41 +/- 0.53 micromol/g dry weight, P < 0.01) and ATP tissue concentrations in group 3 were significantly lower than in group 2 (1.25 +/- 0.37 micromol/g dry weight vs 7.47 +/- 0.47 micromol/g dry weight, P < 0.01). It was apparent that ATP was not an essential factor for successful 24-hour preservation of the canine pancreas in EC because all the pancreatic grafts except one of five grafts in group 3 remained viable after preservation for 24 h, regardless of ATP tissue concentrations. On the other hand, after preservation for 48 h, the functional success rates for groups 1, 2 and 3 were 0% (0/4), 100% (4/4) and 0% (0/3) respectively. ATP tissue concentrations in group 2 were significantly higher than in group 1 (7.91 +/- 1.21 micromol/g dry weight vs 1.21 +/- 0.31 micromol/g dry weight, P < 0.01) and ATP tissue concentrations in group 3 were significantly lower than in group 2 (0.61 +/- 0.07 micromol/g dry weight vs 7.91 +/- 1.21 micromol/g dry weight, P < 0.01). It was clear that preservation of the pancreas for 48 h was unsuccessful by simple cold storage in EC (group 1) and the two-layer method (group 2) made preservation for 48 h possible by increasing ATP tissue concentrations. However, DNP (group 3) inhibited the synthesis of ATP and the effectiveness of the two-layer method for 48-hour preservation of the pancreas. It was clear that maintenance of high ATP tissue concentrations during preservation was essential for the successful preservation of the canine pancreas in EC by the two-layer method for more than 48 h. We concluded that an adequate supply of oxygen to the pancreas during preservation by the two-layer method led to sufficient production of ATP to maintain cellular integrity and permitted the improvement of pancreatic preservation.

Animals↗

Introduction of sulphhydryl groups into the crystalline bacterial cell surface layer protein from Bacillus stearothermophilus PV72 and its application as an immobilization matrix.

The crystalline cell surface layer (S-layer) from Bacillus stearothermophilus PV72 was used as a matrix for reversible immobilization of beta-D-galactosidase via disulphide bonds. In order to obtain an immobilization matrix stable towards acid, alkali and reducing agents such as dithiothreitol (DTT), the S-layer subunits were first cross-linked with glutaraldehyde. This was done in a way whereby 75% of the free amino groups remained unmodified, and then could be completely converted into sulphhydryl groups upon reaction with the monofunctional imidoester iminothiolane. After activation of the sulphhydryl groups with 2,2'-dipyridyldisulphide, 550 micrograms beta-D-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one beta-D-galactosidase molecule [relative molecular mass (M(r)), 116,000] per two S-layer subunits (M(r), 130,000). At least 90% of the sulphhydryl groups from the S-layer protein could be regenerated for further activation by cleaving the disulphide bonds with DTT. In comparative studies beta-D-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.

Cell Wall↗

Studies on the ultrastructure and permeability of the hemotrichorial placenta. I. Intercellular junctions of layer I and tracer administration into the maternal compartment.

The distribution of lanthanum chloride and horseradish peroxidase within the full-term chorioallantoic placenta of the rat was investigated 1 to 20 min after administration of these tracers into the maternal blood circulation. Both tracers rapidly penetrate trophoblastic layer I and diffuse into the interspace between layers I and II. They are localized in extensive infoldings and caveolae of the outer surface of layer II. The syncytial character of layer II is confirmed at this development stage of the placenta. There is no vesicular uptake or penetration beyond layer II until 20 min after tracer administration. Our results indicate that trophoblastic layer II is the main barrier in the chorioallantoic rat placenta preventing the permeation of macromolecules from maternal to fetal compartments. With freeze-fracturing, particulate strutures of variable arrangement and size are found between adjacent cells of layer I. With goniometric analysis small gaps between the appositions of the membrane are observed. These structures are interpreted as tight and/or gap junctions during stages of assembly or disassembly.

Animals↗

Ultrastructural characterization of exocytotic release sites in different layers of the median eminence of the rat.

The release of neuronal secretory products by exocytosis in different layers of the median eminence of the rat was investigated ultrastructurally after perfusion with Ringer solution containing tannic acid. Exocytotic images were observed in all layers studied. Neurohaemal release sites were found in the pars externa of the palisade layer, where they occurred not only against the basal lamina of the pericapillary space, but also opposite, adjacent to neuronal and glial elements. In the lateral portion of the pars externa of the palisade layer most release sites were separated from the pericapillary space or the pial surface by ependymal or glial processes. In the pars interna of the palisade layer, and in the reticular, fibre and subependymal layers, release was observed in different types of axonal processes without morphological synaptic specializations. We suggest that products released in the pars externa of the palisade layer are destined to reach the capillaries of the primary portal plexus. Although the non-vascular release sites may serve a similar hormonal function, they may alternatively represent the morphological correlate of axo-axonal contacts or of paracrine, non-synaptic release sites.

Animals↗

Enveloping layer and periderm of the trout embryo (Salmo trutta fario L.)20U.

The origin of pericarderm and epidermis has been studied in trout embryos from stage 20(2 days after fertilization at 10degrees c) to hatching (stage 410). Between stages 20 and 50, the blastodisc consists of an inner mass of blastomeres covered by a superficial layer of closely packed blastomeres, the peripheral layer. This layer gives rise to both peripheral cells and to cells joining the inner mass of blastomeres. Between stages 50 and 110, junctions differentiate between peripheral cells. This newly formed superficial epithelium the enveloping layer, no longer gives rise to inward migrating cells. From stage 110 on, a basement membrane differentiates beneath a one-cell thick subperipheral layer, which thus becomes the ectodermal basal layer, the prospective epidermal basal layer. From these and ultrastructural observations, it is concluded that 1)the epidermis apparently originates, at least in part from the peripheral cells, between stages 20 and 50; 2) the periderm assumes a protective function over the body of the embryo and also a secretory function over the yolk sac (probably producing the hatching enzymes); 3)the periderm, which is a temporary structure, appears to play the role of an embryonic membrane in teleosts.

Animals↗

A determination of the volumes of the layers of the rat hippocampal region.

The absolute volumes of the hippocampal and subicular cortical layers in the rat were determined. The boundaries of the various layers were defined on series of sections made through the entire hippocampal region of five rats and stained according to the Timm sulfide silver technique. Coordinates representing the boundaries of the layers on selected sections were fed into a mini-computer programmed to calculate the volume of the layers from the areas of the profiles and the distances between the sections. The distribution of the layers indicates that they constitute the same proportion of the volume of the dorsal and ventral divisions of the hippocampal region, with the exception of the structures lying in regio inferior and regio superior. Although the combined regio superior and regio inferior components of the layers of Ammon's horn occupy the same percentage of the volume of the dorsal and ventral hippocampal regions, the regio superior components occupy a larger percentage of Ammon's horn in the dorsal region than they do in the ventral region. The inter-animal variations in the volumes of the various layers indicate that it is possible to describe quantitatively the subdivisions of the hippocampal region with a precision that is compatible with comparative studies.

Animals↗

Afferents to the seizure-sensitive neurons in layer III of the medial entorhinal area: a tracing study in the rat.

Neurons in layer III of the medial entorhinal area (MEA) in the rat are extremely vulnerable to local injections of amino-oxyacetic acid and to experimentally induced limbic seizures. A comparable specific pathology has been noted in surgical specimens from patients with temporal lobe epilepsy. Efforts to understand this preferential neuronal vulnerability led us to study the neural input to this layer in the rat. Iontophoretic injection of the retrograde tracer fast blue, aimed at layer III of the MEA, resulted in retrogradely labeled neurons in the presubiculum in all the injected hemispheres. The nucleus reuniens thalami, the anteromedial thalamic nucleus, the ventral portion of the claustrum (endopiriform nucleus), the dorsomedial parts of the anteroventral thalamic nucleus, and the septum-diagonal band complex were labeled less frequently. In only one experiment, retrogradely labeled neurons were observed in the ventrolateral hypothalamus and in the brainstem nucleus raphe dorsalis. Since projections from claustrum to the entorhinal cortex has not been studied in the rat with modern sensitive anterograde tracing techniques, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were placed into the ventral portion of the claustrum. Anterogradely labeled fibers in the entorhinal area proved not to be confined to the MEA, since a prominent projection distributed to the lateral entorhinal area as well. In both areas, the densest terminal labeling was present in layers IV-VI, whereas layer III appeared to be only sparsely labeled. The present data indicate that of all potential afferents only those from the presubiculum distribute preferentially to layer III of the MEA. This, in turn, suggests a potentially important role of the presubiculum in the seizure-related degeneration of neurons in layer III of the MEA.

Afferent Pathways↗

Influence of an S-layer on surface properties of Bacillus stearothermophilus.

Various aspects of surface properties of the S-layer-carrying Bacillus stearothermophilus PV72 and of an S-layer-deficient mutant (strain PV72/T5) have been tested by adsorption assays on solid surfaces, electrostatic interaction chromatography and hydrophobic interaction chromatography. The adsorption assays have shown that cell adhesion of the S-layer-carrying strain was less influenced by environmental changes than it was with the S-layer-deficient mutant. Electrostatic interaction chromatography indicated that both strains have positively and negatively charged groups exposed on the cell surface but the S-layer-carrying strain reveals more positively charged groups than does the S-layer-deficient mutant. Hydrophobic interaction chromatography showed that both strains have a hydrophilic surface but that the hydrophilic properties are more pronounced with the strain lacking an S-layer.

Bacterial Adhesion↗

On the developing rat placenta. I. Differentiation and junctional alterations of labyrinthine layers II and III.

The differentiation of chorionic layers II and III and alterations of intercellular junctions within the developing placental labyrinth were investigated in rats. Between the 14th and 16th day of gestation, the trabecular structures of the labyrinth alter and increasing numbers of primary, secondary, and tertiary lamellae develop. Trophoblastic layer I remains multicellular. Layer III rearranges and cytoplasmic fusions result in a continuous cytoplasmic barrier intervening between the already syncytial trophoblastic layer II and fetal capillaries. Simultaneously, most of the "undifferentiated" cells within the labyrinth disappear. Thin sectioning anf freeze-fracturing reveal multiple images of intercellular junctions, particularly gap and tight junctions in the cellular and syncytial parts of layers II and III. Continuous and regularly arranged tight junctions occur as well as fragmented structures which are spaced irregularly. Gap junctions are occasionally found between the juxtasinusoidal regions of layers II and III on the 14th day. More often they occur within restricted areas near the center of the trabeculae, integrated within extensive tight junctional strands. Gap junctions increase in later stages of pregnancy and eventually become distributed between the interfaces of layers II and III in the primary lamellae.

Animals↗

On three types of large nerve cells in the granular layer of the human cerebellar cortex.

The large nerve cells of the granular layer of the human cerebellar cortex have been investigated by means of a newly developed Golgi technique that reveals the pigmentation pattern of individual nerve cells through transparent impregnations of their cell bodies and cellular processes. Three types of large nerve cells have been distinguished: Type I cells correspond to the Golgi cells. They have a rounded or polygonal cell body with only a few dendrites that radiate in all directions. The axon ramifies profusely, close to the parent soma. Cells of this type are most frequently encountered among the large cells of the granular layer. Type I cells contain only a few pigment granules. Type II cells have a fusiform or triangular cell body with a few rather extended dendrites that rarely ramify. Cells of this type are either devoid of pigment or contain a few large pigment granules that can be stained intensely by aldehydefuchsin. Type III cells are multipolar neurons with a fair number of dendrites originating from any point of the soma. The dendrites extend into the deep portions of the molecular layer. Here and also within the Purkinje cell layer they repeatedly branch off forming a dense dendritic arborization. Cells of this type are evenly distributed throughout the entire granular layer and throughout the adjacent portions of the Purkinje cell layer and the white matter as well. Type III cells are filled with tightly packed lipofuscin granules. The pattern of pigmentation is characteristic for each of these neuronal types. The three types of large nerve cells within the granular layer of the human cerebellar cortex can therefore be distinguished not only in Golgi impregnations but also in pigment-Nissl preparations.

Adult↗

Formation by the uterus of a peripheral layer of the sheath in microfilariae of Litomosoides carinii and Brugia malayi.

The eggshells of young developmental stages in the uterus are rather thin and homogenous. In the brezel stage of Brugia malayi they are 35 nm thick and 20 nm in Litomosoides carinii. In young developmental stages up to brezel stages the eggshells bind the lectins WGA, DBA and PNA labelled with colloidal gold. This shows that GlcNAc, GalNAc and Gal residues are present at the surface of the sheath. In intrauterine microfilariae of B. malayi the original sheath is reduced to a thickness of 7 nm. It is reinforced by secretions from a specialized area of the epithelium of the uterus which do not appear as a homogeneous layer but look like a string of pearls. This layer may be called the "uterine layer". It has a thickness of 40-80 nm. In the microfilaria of L. carinii, the thickness of the original sheath is reduced to 2-3 nm and the uterine layer has a thickness of 7 nm. The uterine layer does not react with any of the lectins, which shows that the surface lacks N-acetylglucosamine, N-acetylgalactosamine and galactose residues. The uterine layer appears to be an ancestral (plesiomorphic) feature which is present in free-living nematodes and the highly specialized bloodforms of filariae. The uterine layer seems to protect and disguise the original sheath against the immune reactions of the host.

Animals↗

Proton dissociation dynamics in the aqueous layer of multilamellar phospholipid vesicles.

The water layers interspacing between the phospholipid membranes of a multilamellar vesicle are 3-10 water layers across and their width is adjusted by osmotic pressure (Parsegian, V.A., et al., 1986. Methods Enzymol. 127:400-416). In these thin water layers we dissolved pyranine (8 hydroxypyrene 1,3,6 trisulfonate), a compound which, upon photo excitation, ejects it hydroxy proton with time constant of 100 psec. (Gutman, M. 1986. Methods Enzymol. 127:522-538). In the present study we investigated how the width of the aqueous layer, the density of phosphomoieties on the membrane's surface and the activity of water in the layer affect the capacity of protons to diffuse out from the electrostatic cage of the excited anion before it decays to the ground state. Using a combination of steady-state and subnanosecond time-resolved fluorescence measurements we determined the average number of proton excited-anion recombinations before the proton escapes from the Coulomb cage. The probability of recombination in thin water layer is significantly higher than in bulk. The factor contributing most to enhancement of recombination is the diminished water activity of the thin aqueous layer. The time frame for proton escape from an electrostatic trap as big as a membrane-bound protein is 3 orders of magnitude shorter than turnover time of membrane-bound enzymes. Thus the effects of local forces on proton diffusion, at the time scale of physiological processes, is negligible.

Arylsulfonates↗

Water sorption properties of carbonized layers produced by controlled B+ bombardment of thin polyimide and polysulfone films.

Thin polyimide (PI) and polyethersulfone (PES) films are widely used as functional layers for microelectronic sensors. Ion implantation modifies the layer structure and morphology of these polymers and hence results in new mechanical and optical properties. However, ion-modified layers also show a change in sensitivity to moisture uptake under specific conditions. This is important for developing humidity sensors. Therefore, the water sorption ability of such modified polymer layers is studied by spectroscopic ellipsometry under definite relative humidity conditions (1-95%). Swelling data were obtained by fitting procedures based on changes of effective layer thickness and optical constants due to water uptake. Irradiation doses from 0.5 to 5x10(15) B+ cm(-2) at an energy of 180 keV were used for polymer modification. At irradiation doses from 0.5 to 0.7x10(15) B+ cm(-2), the maximum out-of-plane swelling is reached. At higher doses >2x10(15) B+ cm(-2), the swelling decreases and corresponds to values of the pure polymer layers. The wetting properties of the layer surfaces determined by contact angle measurements are important to explain this behavior.

Journal Article↗