Organic anion transport across the choroid plexus

Several organic anion transport systems have recently been identified and localized at the apical and basolateral plasma membrane domains of choroid plexus epithelial cells. These organic anion transporters include (1) indirectly coupled Na(+)/dicarboxylate cotransport and dicarboxylate/organic anion exchange, which is represented on the molecular level by a member of the "kidney"-type organic anion transporter (OAT) family at the apical plasma membrane domain; (2) the organic anion transporting polypeptide 1 (Oatp1) and Oatp2, which both mediate typical "liver"-like organic anion transport activities at the apical and basolateral plasma membrane domains, respectively; and (3) the multidrug resistance protein Mrp1/MRP1 at the basolateral plasma membrane domain, and the P-glycoprotein Mdr1/MDR1 at an apical and subapical membrane vesicle compartment. This cellular transport polarity can account, at least in part, for the previously suggested physiologic transport properties of the choroid plexus epithelium and provides a framework for the identification and localization of additional organic anion transporters involved in the absorption and/or excretion of drugs and drug metabolites at the choroid plexus.     

Quantitative microscopy reveals 3D organization and kinetics of endocytosis in rat hepatocytes

In order to demonstrate the power of quantitative microscopy, the endocytic apparatus of rat hepatocytes was reexamined using in situ liver and short term cultured hepatocyte couplets that were allowed to internalize endocytic markers for various time intervals. Correlative confocal light and electron microscopy demonstrate a tubulovesicular reticulum representing the endocytic apparatus. Volume and membrane area account for 2% of cell volume and 30% plasma membrane surface. Colocalization analysis demonstrated that pathway-specific ligands and fluid-phase markers enter EEA1-positive vesicles, the early endosomal compartment, immediately after internalization. These vesicles are translocated rapidly from basolateral to perinuclear and apical locations. Ligands are sorted within 5 min to their respective pathways. Sequential colocalization of an asialoglycoprotein-pulse with rab7 and lamp3 demonstrates that early endosomes change into or fuse with late endosomes and lysosomes. Alternatively, markers are sequestered into the common endosome consisting of rab11-positive, long tubules that originate from early endosomes and show an affinity for the transcytotic marker pIgA and its receptor. This compartment mediates transcytosis by delivering the receptor-ligand complex to the subapical compartment, a set of apical, rab11-positive vesicles, which are connected to the tubular reticulum. We conclude that vesicular traffic between preexisting compartments, maturation or fusion of endocytic organelles, and transport in tubules act in concert and together mediate transport between compartments of a tubulovesicular endocytic apparatus. In addition, we show that quantitative microscopy using high resolution data sets can detect and characterize kinetics of various parameters thus adding a dynamic component to 3D information.     

Mapping cholesteryl ester analogue uptake and intracellular flow in Paramecium by confocal fluorescence microscopy

In Paramecium primaurelia the uptake and intracellular flow of cholesteryl ester was studied by fluorescence confocal laser scanning optical microscopy and by the fluorescent analogue cholesteryl‐BODIPY^® FL C₁₂ (BODIPY‐CE). The BODIPY FL fluorophore has the characteristic of emitting green fluorescence, which is red‐shifted as the probe concentrates. In cells incubated with 25 µm BODIPY‐CE for 30 s, fluorescence is found in vesicles located around the cytopharynx in the posterior half of the cell. Successively, the lipid is internalized by food vacuoles, the fluorescent vesicles are distributed throughout the cell and the intracellular membranes are labelled. The food vacuole number is maximum after 10–15 min of continuous labelling, then it decreases until no food vacuoles are found in 30‐min fed cells. BODIPY‐CE accumulates in red‐labelled cytoplasmic droplets located in the anterior half of the cell. When food vacuole formation is inhibited by trifluoperazine, fluorescence is found on cellular membranes and in small green‐labelled vesicles at the apical pole. The inhibition of clathrin‐mediated endocytosis does not interfere in P. primaurelia with BODIPY‐CE intracellular flow: intracellular membranes and storage droplets in the cell anterior part are dyed. Conversely, the use of sterol‐binding drugs prevents the lipid accumulation in droplets, stopping the lipid within the cytoplasmic membranes. Furthermore, the cells treated with monensin and cytochalasin B show a labelling of the cellular membranes and lipid droplets, whereas NH₄Cl reduces the lipid storage. Low temperature (4 °C) does not prevent the internalization of BODIPY‐CE that, however, is localized at the cytoplasmic membrane level and does not accumulate in storage droplets. In addition, BODIPY‐CE inhibits phagocytosis, as evidenced by comparing the kinetics of food vacuole formation of control cells, only fed with latex particles, with that of cells fed with latex particles and BODIPY‐CE. In conclusion, this study points out that in P. primaurelia the cholesteryl ester enters the cell via food vacuoles and through the plasma membrane and, inside the cell, it alters cell functions.     

A general variance predictor for Cavalieri slices

A general variance predictor is presented for a Cavalieri design with slices of an arbitrary thickness t ≥ 0. So far, prediction formulae have been available either for measurement functions with smoothness constant q = 0, 1, … , and t ≥ 0, or for fractional q ∈ [0, 1] with t = 0. Because the possibility of using a fractional q adds flexibility to the variance prediction, we have extended the latter for any q ∈ [0, 1] and t ≥ 0. Empirical checks with previously published human brain data suggest an improved performance of the new prediction formula with respect to the hitherto available ones.     

A new approach for high-pressure freezing of primary culture cells: the fine structure and stimulation-associated transformation of cultured rabbit gastric parietal cells

A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H⁺/K⁺‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H⁺/K⁺‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.     

A study of recrystallization in single-phase aluminium using in-situ annealing in the scanning electron microscope

In‐situ annealing experiments were performed in the scanning electron microscope on a single‐phase Al?0.13Mg alloy cold rolled to different strain levels. Once the validity of the technique had been verified by comparison of the recrystallization kinetics and final grain size with bulk annealed samples, the method was used in combination with electron back‐scattered diffraction (EBSD) to study the potential mechanisms for recrystallization in this alloy. During annealing of material rolled to moderate strains (?_t < 0.7), the primary mechanism was strain‐induced boundary migration (SIBM). In material rolled to higher true strains (?_t > 1.4), recrystallization occurred extensively along pre‐existing cube bands and EBSD measurements showed that the mean size of cells within the cube bands was larger than for all other orientations measured, suggesting a size advantage was responsible for the strengthening of cube texture during recrystallization. SIBM was shown to occur concurrently with the nucleation along cube bands but this contributed a lower proportion of nucleation sites during recrystallization.     

Structural analyses of Nd-doped CeO2 thin films deposited by pulsed laser deposition

CeO₂ thin films doped with neodymium oxides for application to gas sensors have been elaborated by the pulsed laser deposition technique. The films were deposited on orientated Si (100) substrates with variable deposition times (t = 90, 180 and 360 s) and molar fractions of Nd₂O₃ (0, 6.5, 15, 21.5 and 27 at.%). The resulting Nd–CeO₂ thin films were characterized by means of X‐ray diffraction analysis, scanning electron microscopy and transmission electron microscopy equipped with EDS (Energy Dispersive Spectrometer) microanalysis. From X‐ray diffraction analyses, it is clearly established that the texture is modified by Nd additions. The preferred (111) orientations of the CeO₂ crystals change into the (200) orientation. The morphology of the CeO₂ grains changes from triangles, for pure CeO₂ thin films, to spherical grains for Nd‐doped films. In addition, cell parameter analyses from X‐ray diffraction data show that a partial chemical substitution of Ce by Nd should occur in the face‐centred cubic lattice of ceria: this should give rise to Ce_1‐xNd_xO_2?z phases with oxygen non‐stoichiometry.     

Dentin bond strength and nanoleakage of the adhesive interface after intracoronal bleaching

This study evaluated dentin bond strength (BS) and nanoleakage of non‐ and pre‐etched dentin immediately (T₀,), 7 days (T₇), and 14 days (T₁₄) after bleaching. Bovine incisors (150) were selected and half of them submitted to intrapulpal dentin etching (e). Non‐ and pre‐etched dentin were subjected to the following (n = 15): no bleaching/control (C); 35% carbamide peroxide (CP); 35% hydrogen peroxide (35% HP); 25% hydrogen peroxide (25% HP); and sodium perborate (SP). Bleaching agents were applied to the pulp chamber four times within a 72‐h interval. Afterwards, pulp chamber dentin was prepared for the BS test at different evaluation times (n = 5): T₀, T₇, and T₁₄. Composite blocks were built on pulp chamber and sectioned in slices. Slices were reduced to an hour‐glass shape with a cross‐sectional area of 0.8 mm² and submitted to microtensile BS test. Two additional specimens for each group were prepared for nanoleakage evaluation by transmission electron microscopy (TEM). Results were analyzed by ANOVA (two‐way) and Dunnett's test (p < .05). BS decreased immediately after intracoronal bleaching for both sound and pre‐etched dentin (p < .05). At T₁₄, the BS of non‐etched bleached dentin increased for all groups, whereas the pre‐etched SPe group presented BS similar to the Ce. Nanoleakage within the hybrid layer was perceptible immediately after bleaching, although a decrease in nanoleakage was observed for all groups at T₁₄. Adhesive restorations should be performed 7–14 days after bleaching, according to the bleaching agent used. Intracoronal bleaching should be performed preferably with sodium perborate if previous dentin etching is applied.     

Morphological studies on the translocation of tubulovesicular system toward the intracellular canaliculus during stimulation of the gastric parietal cell

The gastric parietal has two characteristic membrane systems. One is the intracellular canaliculus, which is specialized networks of enfolded luminal membrane channels lined with numerous microvilli. The other structures common to all parietal cells are the tubulovesicles or the tubulovesicular membranes, a system of tubules and vesicles. The tubulovesicular compartment is drastically depleted during maximal gastric acid secretion and this is coincident with an increase in the canalicular cell surface membrane. A plausible explanation for this redistribution is the fusion and transfer of tubulovesicular membranes to the plasma membrane. However, for many years there was no convincing evidence of connections between these two membrane systems. The mechanism of the transformation of tubulovesicular membrane into the plasma membrane without demonstrable connections has been an enigma to electron microscopists. Using a recently developed fixation technique for parietal cells [Sugai et al. (1995) Acta Anat Nippon 74:S101], we have investigated the organization of the cytoplasmic membrane systems in the rat resting and tetragastrin stimulated stomachs by ultra-high-resolution scanning electron microscopy (SEM). Gastric mucosae were microwave-fixed in a cacodylate buffer, (334 milliosmoles/kgH(2)O (mOsm)), to which 1.0% glutaraldehyde and 0.5% formaldehyde were added. Specimens examined by TEM of thin sections revealed the cytoplasm packed with tubular membranes similar to images detected by rapid-freeze/freeze-substitution fixation. To render the cytoplasmic membranes visible by SEM, fixed mucosae were treated by the aldehyde-osmium-DMSO-osmium maceration procedure. With much of the cell matrix and filaments removed, SEM revealed numerous 30-60-nm tubules, which formed a meshwork with small cisternae. Vesicles or isolated tubules were not found in adequately macerated parietal cells. The cytoplasmic surface of the intracellular canaliculus was smooth except for round openings representing the bases of macerated microvilli. In favorable sites, connections of the tubular membranes to the canaliculi were clearly visible. Stereo pair views were particularly useful to demonstrate these continuities. Connections between these two membrane compartments suggest the probability of rapid membrane transposition. In this article, the form and distribution of membrane systems of parietal cells in the resting state and after tetragastrin stimulation will be presented and discussed. Special emphasis is made to demonstrate connections between the tubulovesicular system and the intracellular canaliculus.     

Time-gated total internal reflection fluorescence spectroscopy (TG-TIRFS): application to the membrane marker laurdan

A novel setup for total internal reflection fluorescence microscopy with spectral and temporal (nanosecond) resolution was used to measure the emission spectra of the membrane marker laurdan either selectively within the plasma membrane or in whole living cells, depending on the incident angle of the excitation light. With increasing temperature, the intensity of the fluorescence band around 490 nm increased in comparison with the band around 440 nm, which has previously been assigned to a phase transition of membrane lipids from gel to liquid crystalline phase. For a better separation of the overlapping spectral bands, time‐gated detection with a delay of 10–15 ns with respect to the exciting laser pulse was used. As a parameter of membrane dynamics the so‐called generalized polarization GP = (I₄₄₀ ? I₄₉₀)/(I₄₄₀ + I₄₉₀) was evaluated at temperatures between 24 and 41 °C and variable angles of the incident light permitting to excite laurdan molecules either within the plasma membrane or in the whole cell. A decrease of the GP values by ≈ 0.2 units between 28 and 41 °C indicated an increase in membrane fluidity or a decrease in membrane stiffness with increasing temperature. In addition, higher GP values were observed for the plasma membrane as compared with intracellular membranes, probably due to a higher amount of cholesterol. Because properties of the plasma membrane have a large influence on the uptake or release of certain pharmaceutical agents or metabolites, the direct assessment of the dynamics of the plasma membrane by total internal reflection fluorescence spectroscopy appears to be important for pharmacology.     

About the role of the various types of secondary electrons (SE1; SE2; SE3) on the performance of LVSEM

The relative weight, δ_Β, of the yield of secondary electrons, SE₂, induced by the backscattered electrons, BSE, with respect to that, δ_P, of secondary electrons, SE₁, induced by the primary electrons, PE, is deduced from simple theoretical considerations. At primary energies E₀ larger than E_M (where the total SE yield δ = δ_P + δ_B is maximum), the dominant role of the backscattering events is established. It is illustrated in SEM by a direct comparison of the contrast between SE images and BSE images obtained at E₀ ～ 5 keV and E₀ ～ 15 keV on a stratified specimen. At energies E₀ less than E_M, the dominant role of SE₁ electrons with respect to SE₂ (and SE₃) is established. It is illustrated by the better practical resolution of diamond images obtained with an in‐lens detection in low voltage SEM E₀ ～ 0.2–1 keV range compared with that obtained with a lateral detector. The present contribution illustrates the improved performance of LVSEM in terms of contrast and of practical resolution as well as the importance of variable voltage methods for subsurface imaging. The common opinion that the practical lateral resolution is given by the incident spot diameter is also reconsidered in LVSEM.     

Third-order aberration theory of Wien filters for monochromators and aberration correctors

Third-order aberrations at the first and the second focus planes of double focus Wien filters are derived in terms of the following electric and magnetic field components – dipole: E₁, B₁; quadrupole: E₂, B₂; hexapole: E₃, B₃ and octupole: E₄, B₄. The aberration coefficients are expressed under the second-order geometrical aberration free conditions of E₂ = −(m + 2)E₁/8R, B₂ = −mB₁/8R and E₃R²/E₁ − B₃R²/B₁ = m/16, where m is an arbitrary value common to all equations. Aberration figures under the conditions of zero x- and y-axes values show very small probe size and similar patterns to those obtained using a previous numerical simulation [G. Martínez & K. Tsuno (2004) Ultramicroscopy, 100 , 105–114]. Round beam conditions are obtained when B₃ = 5m²B₁/144R² and (E₄/E₁ − B₄/B₁)R³ = −29m²/1152. In this special case, aberration figures contain only chromatic and aperture aberrations at the second focus. The chromatic aberrations become zero when m = 2 and aperture aberrations become zero when m = 1.101 and 10.899 at the second focus. Negative chromatic aberrations are obtained when m < 2 and negative aperture aberrations for m < 1.101. The Wien filter functions not only as a monochromator but also as a corrector of both chromatic and aperture aberrations. There are two advantages in using a Wien filter aberration corrector. First, there is the simplicity that derives from it being a single component aberration correction system. Secondly, the aberration in the off-axis region varies very little from the on-axis figures. These characteristics make the corrector very easy to operate.     

Analysis of force patterns and tool life in milling operations

The paper describes the practical effects of the operating parameters in the milling operation. Experiments have been conducted to measure cutting force and tool life under dry conditions. Based on the experimental results, three mathematical models have been developed: Force, TLife and Force/TLife. Further analyses have been conducted on the cutting force patterns: seasonal pattern and nonlinear trend. A process optimisation that is based on the minimum production cost has been applied to relate Force model, TLife model and machinability criteria, such as power consumption, cutting parameters and surface roughness.Nomenclature C _w cost of workpiece ($) - C _s set-up cost ($) - C _m machining cost ($) - C _o overhead cost ($) - C _r tool replacement cost ($) - C _t tool cost ($) - D diameter of the cutter (inch) - d depth of cut per pass (inch) - d ₀ required depth (inch) - e _t random error attth sample - F cutting force (N) - f feedrate (ipm) - L length of workpiece (inch) - N spindle speed (r.p.m.) - n number of teeth - P power of the motor (h.p.) - R surface roughness (µm) - R _e real part of a complex function - T tool life (min) - t sample number - t _m machining time (s) - t ₀ overhead time (s) - t _r tool replacement time (s) - t _s set-up time (s) - U _i unit cost of itemi ($/unit)v - v cutting speed (i.p.m.)     

Resistive loss compensation in the power supply system of the toroidal field winding in the TUMAN-3M tokamak

In the tokamak, in which the winding generating the toroidal field with induction B _t is powered from an inductive energy storage, resistive losses are the main factor that causes B _t to decrease during a discharge. Upgrading of the power supply circuit of the toroidal magnetic field winding (TFW) in the TUMAN-3M tokamak is aimed, primarily, at increasing B _t in the injection heating phase and, second, at maintaining B _t quasi-stationarity during the whole tokamak discharge. To do this, an additional two-section capacitive storage commuted by two thyristor switches has been introduced into the available circuit. Either section of the storage is characterized by a charging voltage of 0.25 kV, a capacitance of 4.32 F, and an energy capacity of 135 kJ. The maximum discharge current of the section is 40 kA. The upgraded circuit compensates for the resistive loss in the TFW and ensures thereby a 50% increase in the magnetic field during injection heating relative to the old circuit: B _t = 1.0 T instead of 0.68 T. In this case, the circuit maintains a TFW current of 110 kA with an accuracy of 10% for ∼60 ms.     

Dependence of the Detectability of Flaws on the Distribution of the Magnetizing Field in the Vicinity of a Flaw

It is shown that the flaw detectability of the magnetic-powder inspection method depends on the ratio of the normal H _n and tangential H _t components of the field in the flaw's vicinity and that H _n/H _t < 3 is a condition that ensures the detectability of surface flaws.     

Quasi-static axial compression of thin-walled circular aluminium tubes

This paper presents further experimental investigations into axial compression of thin-walled circular tubes, a classical problem studied for several decades. A total of 70 quasi-static tests were conducted on circular 6060 aluminium tubes in the T5, as-received condition. The range of D/t considered was expanded over previous studies to D/t=10–450. Collapse modes were observed for L/D10 and a mode classification chart developed. The average crush force, F_AV, was non-dimensionalised and an empirical formula established as F_AV/M_P=72.3(D/t)^0.32. It was found that test results for both axi-symmetric and non-symmetric modes lie on a single curve. Comprehensive comparisons have been made between existing theories and our test results for F_AV. This has revealed some shortcomings, suggesting that further theoretical work may be required. It was found that the ratio of F_MAX/F_AV increased substantially with an increase in the D/t ratio. The effect of filling aluminium tubes with different density polyurethane foam was also briefly examined.     

Influence of glass substrate thickness in laser scribing of glass

The thickness of the glass substrate used in liquid crystal displays continues to be decreased from its original thickness of 1.1 mm for the purpose reducing size and weight. The aim of this study was to clarify the influence of the glass substrate thickness during laser scribing with crack propagation caused by laser heating followed by quick quenching. The laser scribe conditions for soda-lime glass substrates with thickness equal to or less than 1.1 mm were obtained in laser irradiation experiments. Two-dimensional thermal elasticity analysis was conducted with a finite element method based on the scribable conditions obtained in the experiment. The laser scribable conditions can then be estimated by the upper limit of the maximum surface temperature, T_max, and the lower limit of the maximum tensile stress, σ_tmax, in the cooling area, regardless of the glass substrate thickness. There is a substrate thickness with which the maximum tensile stress σ_tmax becomes the largest under each scribe condition. The substrate thickness with which σ_tmax becomes the largest is obtained at a faster scribe velocity for thinner glass substrate and at slower scribe velocity for thicker glass substrate. Owing to these relations, the crack depth also has almost the same tendency as σ_tmax.     

Solute-enriched surface layers and X-ray microanalysis of thin foils of a commercial aluminium alloy

Thin foils of the commercial Al alloy 7075 were subjected to X-ray microanalysis in a Jeol 100 CX TEMSCAN using a modified double-tilting stage. The ratio of the Cu K_x and Al K_x peaks, I_cu/I_Al, was found to decrease rapidly with increasing foil thickness, t, in regions thinner than about 200 nm. This was attributed to the formation of surface layers enriched in Cu during electropolishing, consistent with the findings of other investigators. An equation predicting the variation of I_B/I_A with t in an A-rich alloy in which B (or additional solute elements) is dilute is derived for a sample with surface layers enriched in B. The derivation predicts that I_B/I_A should vary linearly with t^?1, and that the intercept of such a plot should be positive. It is suggested that consistency with this prediction serves as a criterion for true surface enrichment. Our data on Cu obey these predictions, and further analysis indicates that the layers on both surfaces are enriched by a factor of about 5.6. The ratio of the intensities of the Zn K_x and Al K_x X-ray peaks was found to be nearly independent of foil thickness to t?400 nm. This result, combined with additional analysis, indicates that the apparent enrichment observed by other investigators is most likely due to the influence of instrumental factors.     

Study of two tungstates Ca0.5Cd0.5WO4 and Ca0.2Cd0.8WO4 by transmission electron microscopy

To better understand the role of crystal structures and local disorder in the photonic properties of the system (1 ? x)CaWO₄ ? xCdWO₄ with 0 < x < 1, two specific phases with compositions x = 0.5 (scheelite phase) and 0.8 (wolframite phase) have been studied by scanning and transmission electron microscopies. High‐resolution electron microscopy images and image simulations, associated with X‐ray diffraction data, allowed confirming the lattices and space groups I4₁/a and P2/c of the two scheelite and wolframite phases, at the local scale. The electron microscopy data show the existence of a high degree of crystallization associated with statistical distribution of Ca or Cd atoms on a Ca_1?xCd_x site in each lattice.     

Determining the Current-Compensation Factor for a High-Current Relativistic Electron Beam in the Presence of an External Liner

The current-compensation factor f _m of a high-current relativistic electron beam (REB) with an energy E _e = 1.1 MeV, a current I _b = 24 kA, and a pulse duration t _p = 60 ns was investigated. The beam traveled in a metallic drift pipe filled with air at a pressure of 1 to 760 Torr. The dependence of the current compensation of the REB on the resistance of an external liner and the gas pressure was determined by two methods: using Rogowski loops placed around the transport channel or resistive back-current shunts inserted in the breaks of the drift pipe. It was ascertained that f _m of the high-current REB slightly varied with the gas pressure and was virtually independent of the resistance of the external liner. It was shown that the current compensation could be correctly determined by measuring the total current with the Rogowski loops placed inside the drift pipe and not with resistive shunts.