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PubMed1990

The stellate layer and rhythmic contractions of the Oryzias latipes embryo.

The objective of this study was to determine which cells in the medaka (Oryzias latipes) embryo participate in the rhythmic contraction waves that propagate slowly across the yolk sac throughout most of embryonic development. To facilitate observation of the cells, we inhibited the contractions temporarily by incubating the embryos with o-nitrobenzylacetate, n-heptanol, or n-octanol. After we washed out the inhibitor, isolated cells in a subepithelial layer (similar to the stellate layer in Fundulus heteroclitus) began to pulse. Stellate cells are much smaller than cells in the surface epithelium (enveloping layer) and are present throughout the developmental period during which the contractions occur, stage 14 to stage 26. We conclude that the active force for the rhythmic contraction waves is provided by cells in the stellate layer and that cells in the enveloping layer are passively deformed by the contraction of cells in the closely apposed stellate layer.

Animals↗
PubMed2001

Mineralized dermal layer of the Brazilian tree-frog Corythomantis greeningi.

Some species of anuran amphibians possess a calcified dermal layer (the Eberth-Kastschenko layer) located between the "stratum spongiosum" and the "stratum compactum." This layer consists of calcium phosphate deposits, proteoglycans, and glycosaminoglycans. Although regarded as a protective layer against desiccation, a calcium reservoir, or possibly a remnant of a dermal skeleton present in anuran ancestors, very little is known about its origin, structure, and function. Thus, we studied the structure and composition of the mineralized dermal layer of Corythomantis greeningi, a peculiar hylid from the Brazilian semiarid region (caatinga), using conventional and cryosubstitution methods combined with transmission, scanning, and analytical electron microscopy. Results show that the dermal layer consists of dense, closely juxtaposed, globular structures. Although the electron opacity of the globules was variable, depending on the type of preparation, crystal-like inclusions were present in all of them, as confirmed by dark field microscopy. Electron probe X-ray microanalysis showed calcium, phosphorus, and oxygen, and electron diffraction revealed a crystalline structure comparable to that of a hydroxyapatite.

Animals↗
PubMedDate not supplied

Optical biosensor investigation of interactions of biomembrane and water-soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers.

A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer. However, electrostatic forces were also but not always involved in the incorporation process. They promoted d-Fp incorporation but had no effect on d-b5 incorporation. In low ionic strength buffer, no incorporation of these two proteins into the DSPE lipid layer was observable. Incorporation of d-b5 into the DLPE layer was abruptly increased at temperatures exceeding phospholipid phase transition point. Incorporation of d-2B4 was dependent on its aggregation state and decreased with increasing protein aggregability. Water-soluble proteins either would not interact with the phospholipid layer (adrenodoxin) or would bind to the layer at the cost of only electrostatic (albumin) or both electrostatic and hydrophobic (P450cam) interactions.

Biosensing Techniques↗
PubMed2001

Compression forces and amount of outer coating layer affecting the time-controlled disintegration of the compression-coated tablets prepared by direct compression with micronized ethylcellulose.

The influence of compression force to inner core tablet or to outer coating layer of the compression-coated tablet on the function of time-controlled disintegration was investigated. The inner core tablet was directly compacted by sodium diclofenac (model drug) and ethylcellulose (EC) with 4.6-microm particle size was used as an outer coating layer. The immersion time of the compression-coated tablet previously soaked in pH 1.2 solution to simulate the residence time of the tablet in the GI tract affecting the dissolution behavior of the compression-coated tablet was also investigated. The effect of the amount of the outer coating layer used on the drug release was examined. The results indicate that sodium diclofenac released from these compression-coated tablets exhibited a longer lag of a period about 16.3 h in both distilled water and pH 6.8 buffer solution, followed by a different drug release behavior. The lag time was independent of the pH of dissolution medium, and the immersion time in pH 1.2 solution. After that lag time, the outer shell of the compression-coated tablets broke into two halves to make a rapid drug release. However, the drug release behavior of the soaked tablet in pH 6.8 buffer solution was dependent on the immersion time. The compression force < 200 kg/cm(2) to the inner core tablet influenced the release behavior of drug less, but > 200 kg/cm(2) might delay the lag time. The lag time of the compression-coated tablets was linearly correlated with the compression force to the outer coating layer (r = 0.9896). We also found that the more the amount of outer coating layer added, the longer the lag time obtained. The study demonstrates that the time-controlled disintegration of the compression-coated tablet was effectively controlled by the compression force applied and the amount of outer coating layer added.

Cellulose↗
PubMed2006

Microstructural imaging of early gel layer formation in HPMC matrices.

A real-time confocal fluorescence imaging method has been developed which allows the critical early stages of gel layer formation in hydroxypropylmethylcellulose (HPMC) matrices to be examined. Congo Red, a fluorophore whose fluorescence is selectively intensified when bound to beta-D-glucopyranosyl sequences, has allowed mapping of hydrated polymer regions within the emerging gel layer, and revealed for the first time, the microstructural sequence of polymer hydration during development of the early gel layer. Liquid penetration and swelling can be examined in unprecedented detail. The earliest images revealed an initial phase of liquid ingress into the tablet pore network, followed by the progressive formation of a coherent gel layer by outward columnar swelling and coalescence of hydrated HPMC particles. Salts can markedly affect HPMC matrix behaviour. Gel layer growth in 0.1-0.5 M NaCl was progressively suppressed until at 0.75 M, particles clearly failed to coalesce into a gel layer, although with considerable polymer swelling. The failure to form a limiting diffusion barrier resulted in enhanced liquid penetration of the core, and the swelling of particles that did not coalesce culminated in surface disintegration. This provides direct evidence of physical mechanisms that contribute to salts accelerating drug release from HPMC matrices.

Congo Red↗
PubMed1990

Sustained release of insulin by double-layered implant using poly(D,L-lactic acid).

This report describes the advantage of double-layered implants using low molecular weight poly(DL-lactic acid) in the sustained release of insulin. The double-layered implant consisted of a polymer matrix containing insulin and a polylactic acid layer which was coated partially on one of the surfaces of the insulin:polymer matrix. The double-layered implants were compared with single-matrix implants from the standpoint of the in vitro dissolution test and in vivo performance. In vitro release rates were controlled by changing the amount of poly(DL-lactic acid) used in the polymer layer. In an in vivo test using diabetic animals, the double-layered implants provided a sustained release of insulin for 19 d, as judged by the changes in blood glucose levels and serum insulin levels after the subcutaneous implantation.

Animals↗
PubMed2003

Gold-coated fused-silica sheathless electrospray emitters based on vapor-deposited titanium adhesion layers.

Gold-coated fused-silica electrospray (ES) emitters based on vapor-deposited adhesion layers of titanium have been manufactured to investigate the possibilities of producing durable ES emitters applicable in chip-based analytical devices. The stabilities of the emitters were studied by both electrospray and electrochemical experiments and a marked increase in the emitter lifetime, compared to that for Cr/Au coated emitters, was found for the Ti/Au emitters in the ES durability tests. This indicates that Ti (rather than Cr) adhesion layers should be used in association with large-scale fabrication of ES emitters by vapor-deposition techniques. The lifetime of about 500-700 hours also allowed the Ti/Au-coated emitter to be used as an integrated part of a capillary liquid chromatography column coupled to a mass spectrometer in a series of LC/MS experiments. The Ti/Au coating was further studied by electrochemical techniques and scanning electron microscopy in conjunction with X-ray spectroscopy. It is shown that the eventual failure of the Ti/Au emitters in ES experiments was due to an almost complete detachment of the gold layer. Experimental evidence suggests that the detachment of the gold coating was due to a reduced adhesion to the titanium layer during oxidation in positive electrospray. Most likely, this was caused by the formation of an oxide layer on the titanium film. It is thus shown that unlimited emitter stabilities are not automatically obtained even if the metallic adhesion layer is stabilized by an oxide formation under positive electrospray conditions.

Journal Article↗
PubMed1997

Motion of a Colloidal Particle Coated with a Layer of Adsorbed Polymers in a Spherical Cavity

An analytical study is presented for the quasisteady translation and steady rotation of a spherical particle covered by a layer of adsorbed polymers located at the center of a spherical cavity that may also have an adsorbed polymer layer on its inside wall. The Reynolds number is assumed to be small, and the surface polymer layers are assumed to be thin with respect to the particle radius and the spacing between solid surfaces. To solve the Stokes flow equations within and outside the polymer layers a method of matched asymptotic expansions in small parameters lambda1 and lambda2 is used, where lambda1 and lambda2 are the ratios of the polymer-layer length scale to the radius of curvature at the particle surface and at the cavity wall, respectively. The results for the hydrodynamic force and torque exerted on the particle are expressed as an effective hydrodynamic thickness (L) of the adsorbed polymer layer surrounding the particle, which are accurate to O(lambda21). The O(lambda1) term for L normalized by its value in the absence of the cavity is found to be independent of the polymer segment distribution, the hydrodynamic interactions among the segments, and the volume fraction of the segments. The O(lambda21) term for L, however, is a sensitive function of the polymer segment distribution and the volume fraction of the segments. In general, the boundary effects on the motion of a polymer-coated particle can be quite significant in appropriate situations.

Journal Article↗
PubMed1998

Effect of Interparticle Electrostatic Double Layer Interactions on Permeate Flux Decline in Crossflow Membrane Filtration of Colloidal Suspensions: An Experimental Investigation.

A systematic study on the effect of electrostatic double layer interaction on permeate flux decline and deposit cake formation in crossflow membrane filtration of colloidal suspensions is reported. Three monodisperse silica suspensions with diameters of 47, 110, and 310 nm were used as model colloids, and a tubular zirconia membrane with an average pore diameter of 20 nm was used as a model membrane. The magnitude and range of the electrostatic double layer interactions were controlled via changes in solution ionic strength and pH. The coupling between colloidal interactions and hydrodynamic forces was investigated by changing the transmembrane pressure and particle size. The results indicate that the rate of flux decline is strongly dependent on solution ionic strength and, to a much lesser degree, on solution pH (for the investigated pH range 6.1-10.0). Variations in flux decline rate with solution ionic strength are especially significant as the particle size decreases. Particle cake thickness, permeability, and porosity generally increased with a decrease in solution ionic strength for a given particle size. For given physical and chemical conditions, the cake layer porosity increased with decreasing particle size, while cake permeability decreased with decreasing particle size. These trends are consistent with the increased importance of double layer repulsive forces in controlling the cake layer structure as the solution ionic strength and particle size decrease. Pressure relaxation experiments indicated that the particle cake layer is reversible, implying no irreversible deposition (attachment) of silica colloids onto the zirconia membrane surface. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Molecular Dynamic Simulations of Single, Interacting, and Sheared Double Layers.

This article presents the results of molecular dynamic simulations of double layers developing on a flat mineral surface in aqueous sodium chloride solutions. The first part considered the configuration of the double layer in comparison to the classical point of view. This part investigates the influences of changed system properties on the double layer. For this purpose systems of different temperature, electrolyte concentration, and surface charge were simulated. The interaction of two overlapping double layers was investigated in comparison to that of single double layers. Nonequilibrium investigations using electrokinetic and rheological measurements provide findings regarding the microscopic behavior of a double layer under shear. Copyright 1998 Academic Press.

Journal Article↗
PubMed2001

Counterion Triple Layer in Solid/Solution Interface: Stirring and Temperature Effects on pH Measurements.

This paper reports on the pH glass electrode potential based on the double-capacitor theory and the discovery of a mobile counterion triple layer next to the double layer to explain the electrode potential changes caused by stirring and temperature increase. This mobile triple layer behaves like a rubberband, which may be loosened by stirring (or agitation) and temperature increase and tightened after stirring stops or temperature lowers. Stirring and temperature exert different effects on the potential of the pH and SCE electrodes via their different mechanisms. In contrast to the past, results reported here were obtained from experiments designed with the glass electrode and SCE in separate beakers connected with a conducting wire, so that one electrode could be stirred while keeping the other unstirred. There is a weak charge attraction between the double and triple layers, not a strong covalent bonding for complexation, so that the latter can be removed by stirring or temperature increase. In light of present results and the new concept, the Boltzmann equation has been modified to include the net charge density and the triple-layer potential. The counterion triple layer concept will have a tremendous impact on our understanding of the interface structure and properties, and stimulate further studies and applications of ion-selective electrodes. Copyright 2001 Academic Press.

Journal Article↗
PubMed2002

Impurity distribution in a solid-liquid interface during static layer crystallization.

The impurity distribution inside crystalline layers in static layer crystallizations was explored theoretically and experimentally for a binary eutectic mixture of caprolactam and water. The impurity distribution in the layer is modeled by a mass balance equation coupled with an energy balance equation. The mass balance equation is established with a combination of the growth of the layer and the diffusional movement of impurity. The mathematical model was solved by a numerical method. The predictions are discussed with respect to the interfacial distribution coefficient, concentration, and temperature at the solid-liquid interface, crystalline layer thickness, and concentration of the layer. The impurity distribution obtained by the model was appropriately verified with the results obtained by the experiments.

Journal Article↗
PubMed2001

Formation of the single-layer beta-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain.

Borrelia outer surface protein A (OspA) contains a unique single-layer beta-sheet that connects N and C-terminal globular domains. This single-layer beta-sheet segment (beta-strands 8-10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer beta-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to beta-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta-strand. (1)H-(15)N heteronuclear NOE measurements and amide H-(2)H exchange revealed that the single-layer beta-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta-sheet structure.

Amino Acid Sequence↗
PubMed1995

Afferents to different layers of the dorsolateral isocortex in rats.

Fluorescent somatopetal tracers were used to infiltrate, by diffusion rather than injections, the dorsolateral cortex of one hemisphere in rats. In different animals the tracers penetrated into the cortex to different depths. We found several interesting features of the commissural system: first, there were no areas without commissural neurons. At least a few labelled cell bodies were present in a single-cell layer also in "acallosal" cortical areas. Secondly, there is a considerable variety of laminar distribution patterns of labelled perikarya in different areas. Thirdly, some cortical fields, which cytoarchitecturally appear uniform, can be subdivided according to different distributions of cell bodies with commissural projections. Fourthly, when only supragranular layers were infiltrated, labelled cell bodies were present mainly in the supragranular layers of the contralateral cortex. Infiltration of the first layer alone did not label any neurons in the contralateral cortex but did label neurons in layer VIb ipsilaterally. In the subcortex, the labelled perikarya were found in the structures already known to project directly to the cortex. In rats with the tracer restricted mainly to the supragranular layers, a conspicuously reduced labelling was found in the basal forebrain and the thalamus. In the thalami of those animals, labelled neurons were found only in paralamellar nuclei. The high sensitivity of the tracer used, together with infiltration of the entire dorsolateral cortex, allows us to conclude that probably all sources of innervation of the isocortex in rats have been seen.

Afferent Pathways↗
PubMed1996

Ultrastructural and cytochemical identification of apoptotic cell death accompanying development of the fetal rat olfactory nerve layer.

It has been previously shown that the embryonic olfactory nerve contains, in addition to glial ensheathing cells, a large population of differentiated neurons that migrate from the developing olfactory epithelium, in close association with the olfactory axon fascicles. The purpose of our study was to verify the hypothesis according to which a process of physiological cell death might be involved in the progressive disappearance of these migrating neurons that has been reported during late embryonic stages in several immunocytochemical studies. To do so, we have investigated the development of the olfactory nerve layer in rat embryos by using light and electron microscopy, with special reference to the presence of cell death processes within this structure. We have also applied the histochemical TUNEL method allowing in situ visualization of cells degenerating by apoptosis. In order to determine if neurons were present among dying cells, a procedure of double-labeling was performed by combining the DNA-specific bisbenzimide with two neuronal markers, the protein B-50/GAP-43 and the lectin Ulex europaeus I. Results brought out the precise temporal and spatial patterns of programmed cell death accompanying the morphogenesis of the olfactory nerve layer. A cell death process was observed within the olfactory nerve layer from its onset at embryonic day 13 (E13). While only few pycnotic cells were observed in E13 and E14 embryos, their number increased from E15 to reach a maximum at E16 and then diminished. Few dying cells were also observed along the olfactory axon fascicles when they penetrated the olfactory nerve layer. Degenerating cells appeared strongly TUNEL-labeled and exhibited morphological features of cell death by apoptosis. Double-labeling experiments revealed that some of the apoptotic cells were neurons. These observations indicate that apoptosis may account for the progressive decrease in the number of migrating neurons present within the embryonic olfactory nerve layer. Otherwise, a zone of massive cell death by apoptosis was observed at E14 within the nasal mesenchyme located ventrally and caudally to the olfactory nerve layer. Double-labeling experiments showed that apoptotic cells present within this zone were not neurons. Our findings strongly suggest that apoptotic cell death of migrating neurons may allow the elimination of non-functional cells whereas that of mesenchymal cells may facilitate outgrowth of the newly formed olfactory axon fascicles by pathway formation.

Animals↗
PubMed1987

Functional and structural differences between cultured outer and inner layer cells of bovine adrenal cortex.

Outer and inner layer cells of bovine adrenal cortex were cultured separately to compare cellular structural characteristics and functional differences. Outer layer cells were polygonal in shape with radially distributed lipid droplets in the cytoplasm, and produced mainly aldosterone and cortisol. The aldosterone production increased upon stimulation with angiotensin II or dibutyryl-cAMP. In contrast, inner layer cells were spindle-shaped and had fine diffused lipid droplets. They produced four times as much cortisol as outer layer cells but no aldosterone. Cortisol production increased with ACTH or dibutyryl-cAMP stimulation. When stimulated by ACTH or by dibutyryl-cAMP, both types of adrenocortical cells showed "cellular retraction" whereby the number of cytoplasmic lipid droplets decreased and microvilli on the cellular surface increased. At the same time, the transverse distribution of actin fibers disappeared and the microtubules changed their distribution pattern from circular to radial. Stimulation by angiotensin II, on the other hand, brought no marked structural changes. These results indicate that, in functional terms, the outer layer cells and the inner layer cells in this culture system reflect zona glomerulosa and zona fasciculata-reticularis, respectively.

Adrenal Cortex↗
PubMed1988

Layer-specific innervation of the dopamine-deficient frontal cortex in weaver mutant mice by grafted mesencephalic dopaminergic neurones.

The dopamine innervation of the frontal cortex originates in the A9 and A10 mesencephalic dopamine cell groups. In weaver mutant mice, there is a 77% frontocortical dopamine deficiency associated with losses of dopamine neurones in areas A9 and A10. The dopamine-depleted cortical areas of weaver mutant mice are receptive to reinnervation by afferent fibres originating in dopamine-containing mesencephalic grafts from normal donor embryos. In the anteromedial frontal lobe, reinnervation by tyrosine hydroxylase immunoreactive fibres is largely confined to the basal cortical layers whereas in the anterior cingulate cortex, tyrosine hydroxylase immunoreactive fibres also occupy superficial layers, including the molecular layer. Normally, the dopaminergic innervation of the anteromedial frontal lobe is distributed among the basal cortical layers (IV-VI), and the dopaminergic innervation of the cingulate cortex occupies both basal and superficial cortical layers. The pattern of innervation following transplantation indicates that, in repopulating dopamine-deficient cortical areas of recipient weaver mutants, graft-derived dopamine fibres show a preference for those layers which are normally invested by dopamine afferents.

Animals↗
PubMed1975

On the fine structure of the external glial layer in the isocortex of man.

The surface of the external glial layer of the isocortex in the human temporal lobe is generally slightly undulated, with a few protrusions and indentations. The surface is formed by an uninterrupted basement membrane which is continuous over the surface no matter how tortuous it becomes. The overall thickness of the glial layer is generally 15 to 25 mum, but diminishes to about 5 mum immediately beneath blood vessels. It consists mainly of a variable number of stacked glial cell processes. Two groups of cell bodies are encountered particularly in the middle and lower levels of the glial layer. Most of the cells are specialized fibrous astrocytes. They are characterized by eccentrically placed, rounded nuclei with homogeneously dispersed chromatin, and electron-lucent cytoplasm rich in filaments. Lipofuscin pigment granules occupy large areas of the perikaryon. The astroglial cells give rise to four types of processes: foot-processes, tangential and radial processes, and processes irregular in outline. The foot-processes ascend towards the cortical surface and terminate as flat expansions spreading out immediately beneath the basement membrane. Contiguous terminal expansions are connected by gap junctions. The individual profiles are irregular in form and fit together like in a jig-saw puzzle. The plasmalemma beneath the basement membrane is underlined by a fuzzy material, which is penetrated by glial filaments. In the terminal expansions individual or groups of mitochondria are abundant. The tangential processes are straight and slender and form a lattice within the middle and deep level of the external glial layer. They contain numerous filaments, evenly distributed or fasciculated. The remainder of the lattice is filled up by a considerable number of processes irregular in outline and varying greatly in size. They contain fewer filaments than the tangential processes, coursing in all directions, and glycogen particles. In both types of processes only a few mitochondria are present. These processes are also connected by gap junctions and desmosomes, too. Large cytoplasmic areas of astroglial cells localized in the deepest portion of the glial layer protrude into the neuropil of the molecular layer, giving rise to several radiate processes, which extend deeper into the cortex. The second, heterogeneous group of cell bodies is characterized by elongated nuclei, ovoid or irregular in outline, which are smaller than those of astroglial cells, and contain blocks of condensed chromatin; a thin cytoplasmic rim generating a few appendages surrounds the nucleus. The first sub-type is characterized by a nucleus with large chromatin blocks bordering the inner nuclear membrane and a medium-dense cytoplasmic matrix. The second sub-type displays smaller chromatin condensations at the inner nuclear membrane and many microtubules are scattered throughout an electron-lucent cytoplasm.

Adult↗