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* Context.--Histopathology is the current standard to diagnose skin disease. However, biopsy may not always be feasible, such as in patients with multiple nevi, a patient with a lesion on an aesthetically significant site, or in children. Recently, noninvasive techniques, including reflectance confocal microscopy (RCM), optical coherence tomography, and Raman spectroscopy, have enabled dermatologists to manage skin lesions in real time without the need for biopsy. Objective.--To report the updated diagnostic accuracy of RCM for equivocal skin lesions. Design.--In this study, we retrospectively reviewed our data of clinically suspicious lesions from 2010 to 2017 that were evaluated by RCM. Results.--Our results showed an overall sensitivity of 98.2% and specificity of 99.8%. Conclusions.--In conclusion, RCM is a noninvasive real-time tool with the potential to diagnose skin lesions with high accuracy and without biopsy. doi: 10.5858/arpa.2018-0124-OA), Histopathology is the current standard to diagnose skin disease. However, biopsy may not always be feasible, such as in patients with multiple nevi, a patient with a lesion on an [...]

* Context.--Rapid advances in the fields of biophotonics, computer science, and instrumentation have allowed for high-resolution imaging of biologic tissues. Objective.--To evaluate the quality of images from an optimized confocal fluorescence microscopy (CFM) platform for rapid evaluation of small fragments of tissue, compared with hematoxylin-eosin staining. Design.--Tissue fragments (up to 1.0 X 0.3 cm) were stained with 0.6 mM acridine orange for 60 seconds and imaged using a CFM platform at 488-nm and 785-nm wavelength. The imaged tissues were then fixed in formalin and processed to generate hematoxylin-eosin-stained tissue sections. The quality of CFM images was scored on a scale of 0 to 3 on the basis of the percentage of the CFM images with recognizable tissue architecture (0, 0%; 1, 50%). The diagnoses made using CFM images were compared with those made using histopathologic analysis of the hematoxylin-eosin-stained tissue sections. Results.--We imaged 118 tissue fragments obtained from 40 breast, 23 lung, 39 kidney, and 16 liver surgical excision specimens. We acquired CFM images in 2 to 3 minutes; 95.8% (113 of 118) of images showed a quality score of 3, and 4.2% (5 of 118) had a score of 2. We achieved a sensitivity of 95.5%, specificity of 97.3%, positive predictive value of 95.5%, and negative predictive value of 97.3%. Conclusions.--Our results demonstrate the suitability of the CFM platform for rapid and accurate evaluation of small tissue fragments in surgical pathology practice. 10.5858/arpa.2018-0352-OA), In recent years, rapid advances and developments in the fields of computer science and biophotonics, which is the science and technology of the interaction of photons within and on biologic [...]

Byline: Sevil. Savas, Esma. Yuksel, Asl?. Erdemir, Mehmet. Gurel, Cem. Leblebici Coexistence of two types of malignant neoplasms, called 'a collision tumor,' is relatively uncommon. Basal cell carcinomas (BCCs) are [...]

Small ubiquitin modifier 1 (SUMO1) is shown to regulate K2P1 background channels in the plasma membrane (PM) of live mammalian cells. Confocal microscopy reveals native SUMO1, SAE1, and Ubc9 (the enzymes that activate and conjugate SUMO1) at PM where SUMO1 and expressed human K2P1 are demonstrated to colocalize. Silent K2P1 channels in excised PM patches are activated by SUMO isopeptidase (SENP1) and resilenced by SUMO1. K2P1-Lys274 is crucial: when mutated to Gin, Arg, Glu, Asp, Cys, or Ala, the channels are constitutively active and insensitive to SUMO1 and SENP1. Tandem mass spectrometry confirms conjugation of SUMO1 to the e-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Single-particle TIRF microscopy shows that wild-type channels in PM have two K2P1 subunits and assemble with two SUMO1 monomers. Although channels engineered with one Lys274 site carry just one SUMO1 they are activated and silenced by SENP1 and SUMO1 like wild-type channels. KCNK1 | TWIK1 | SAE1 | Ubc9 | SENP1 doi/ 10.1073/pnas.1004712107

Thiamine is an important cofactor of metabolic enzymes, and its deficiency leads to cardiovascular dysfunction. First, we characterized the metabolic status measuring resting oxygen consumption rate and lactate blood concentration after 35 days of thiamine deficiency (TD). The results pointed to a decrease in resting oxygen consumption and a twofold increase in blood lactate. Confocal microscopy showed that intracellular superoxide (~40%) and [H.sub.2][O.sub.2] (2.5 times) contents had been increased. In addition, biochemical activities and protein expression of SOD, glutathione peroxidase, and catalase were evaluated in hearts isolated from rats submitted to thiamine deprivation. No difference in SOD activity was detected, but protein levels were found to be increased. Catalase activity increased 2.1 times in TD hearts. The observed gain in activity was attended by an increased catalase protein level. However, a marked decrease in glutathione peroxidase activity (control 435.3 [+ or -] 28.6 vs. TD 199.4 [+ or -] 30.2 nmol NADPH x [min.sup.-1] x [ml.sup.-l]) was paralleled by a diminution in the protein levels. Compared with control hearts, we did observe a greater proportion of apoptotic myocytes by TdT-mediated dUTP nick end labeling (TUNEL) and caspase-3 reactivity techniques. These results indicate that during TD, reactive oxygen species (ROS) production may be enhanced as a consequence of the installed acidosis. The perturbation in the cardiac myocytes redox balance was responsible for the increase in apoptosis. catalase; glutathione peroxidase; superoxide dismutase doi: 10.1152/ajpheart.00820.2009.

Exercise training enhances agonist-mediated relaxation in both control and collateral-dependent coronary arteries of hearts subjected to chronic occlusion, an enhancement that is mediated in part by nitric oxide. The purpose of the present study was to elucidate exercise training-induced adaptations in specific cellular mechanisms involved in the regulation of endothelial nitric oxide synthase (eNOS) in coronary arteries of ischemic hearts. Ameroid constrictors were surgically placed around the proximal left circumflex coronary artery (LCX) of adult female Yucatan miniature swine. Eight weeks postoperatively, animals were randomized into sedentary (pen-confined) or exercise training (treadmill run; 5 days/ wk; 14 wk) protocols. Coronary artery segments (~1.0 mm luminal diameter) were isolated from collateral-dependent (LCX) and control (nonoccluded left anterior descending) arteries 22 wk after ameroid placement. Endothelial cells were enzymatically dissociated, and intracellular [Ca.sup.2+] responses (fura 2) to bradykinin stimulation were studied. Immunofluorescence and laser scanning confocal microscopy were used to quantify endothelial cell eNOS and caveolin-1 cellular distribution under basal and bradykinin-stimulated conditions. Immunoblot analysis was used to determine eNOS, phosphorylated (p)eNOS, protein kinase B (Akt), pAkt, and caveolin-1 protein levels. Bradykinin-stimulated nitrite plus nitrate (NOx; nitric oxide metabolites) levels were assessed via HPLC. Exercise training resulted in significantly enhanced bradykinin-mediated increases in endothelial [Ca.sup.2+] levels, NOx levels, and the distribution of eNOS-to-caveolin-1 ratio at the plasma membrane in endothelial cells of control and collateral-dependent arteries. Exercise training also significantly increased total eNOS and phosphorylated levels of eNOS ([pSer.sup.1179]) in collateral-dependent arteries. Total eNOS protein levels were also significantly increased in collateral-dependent arteries of sedentary animals. These data provide new insights into exercise training-induced adaptations in cellular mechanisms of nitric oxide regulation in collateral-dependent coronary arteries of chronically occluded hearts that contribute to enhanced nitric oxide production. coronary artery disease; coronary circulation; nitrate; nitrite; porcine doi: 10.1152/ajpheart.00754.2009.

Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 [micro]m (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated ceils (1.71 [micro]m, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 [micro]m, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data. two-photon imaging; Monte-Carlo simulation; magnetic resonance imaging; confocal microscopy; ventricular tissue slice doi: 10.1152/ajpheart.00481.2009.

Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health (1). High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective (2). In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. Here we report the discovery of 287 human host cell genes influencing influenza A virus replication in a genome-wide RNA interference (RNAi) screen. Using an independent assay we confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the current pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. Notably, a subset of these common hits was also essential for replication of a highly pathogenic avian H5N1 strain. In-depth analyses of several factors provided insights into their infection stage relevance. Notably, SON DNA binding protein (SON) (3) was found to be important for normal trafficking of influenza virions to late endosomes early in infection. We also show that a small molecule inhibitor of CDC-like kinase 1 (CLK1) (4) reduces influenza virus replication by more than two orders of magnitude, an effect connected with impaired splicing of the viral M2 messenger RNA. Furthermore, influenza-virus-infected [p27.sup.-/-] (cyclin-dependent kinase inhibitor 1B; Cdkn1b) mice accumulated significantly lower viral titres in the lung, providing in vivo evidence for the importance of this gene. Thus, our results highlight the potency of genome-wide RNAi screening for the dissection of virus-host interactions and the identification of drug targets for a broad range of influenza viruses., During the course of infection, the influenza virus encounters numerous bottlenecks, constituted by host cell functions essential or inhibitory for viral propagation (5). Comprehensive knowledge of such critical host cell [...]

It has been well-established that polarized migrating cells exhibit a stable and transient gradient of intracellular calcium concentration ([[Ca.sup.2+.sub.i]), increasing from front-to-rear, that is thought to be responsible for rear retraction. The paradox that arises is how calcium at the front of a cell catalyzes critical high-threshold calcium-dependent processes during cell migration and particularly in decision-making for a cell to turn. In this brief review, we discuss the recent discovery of flickering high-[[Ca.sup.2+.sub.i] microdomains ('calcium flickers') at the front of migrating fibroblasts and their common role in transducing local membrane mechanical stress (via TRPM7, a stretch-activated calcium-permeating transient receptor potential channel) and chemoattractant-elicited signals (via type 2 inositol 1,4,5-trisphosphate receptor in the endoplasmic reticulum). Furthermore, we present a new model for patterned calcium flicker activity as the mechanism for steering the turning of a migrating cell. Key words: calcium microdomains, calcium flickers, cell migration, TRPM7, inositol-1,4,5-trisphosphate receptor, confocal microscopy. Il est clairement etabli que les cellules migrantes polarisees presentent des gradients de concentrations de calcium intracellulaire ([[Ca.sup.2+.sub.i] stables et transitoires, lesquels seraient responsables de la retraction de l'arriere des cellules. Le paradoxe soulevee est lie; a la maniere dont le calcium situee a l'avant de la cellule catalyse les processus dependants de fortes concentrations de calcium durant la migration cellulaire et, particulierement, durant le changement de direction de la cellule. Dans cette breve synthese, nous discutons de la recente decouverte de microdomaines a forte concentration en calcium [[Ca.sup.2+.sub.i] (<< calcium flickers >>) a l'avant des fibroblastes migrants, et de leur role en tant que transducteur de la tension meecanique a l'inteerieur de la membrane (par le biais du TRPM7, un canal TRP permeable au calcium active; par retirement) et des signaux deeclenchees par les substances chimiotactiques (par l'intermeediaire du recepteur de l'inositol-1,4,5trisphosphate de type 2 dans le reticulum endoplasmique). De plus, nous presentons un nouveau modeele d'activitee des microdomaines de calcium pour guider le mouvement directionnel d'une cellule migrante. Mots-cles: microdomaines de calcium, microdomaines a forte concentration en calcium, migration cellulaire, TRPM7, recepteur de l'inositol-1,4,5-trisphosphate, microscopie confocale. [Traduit par la Redaction], Introduction Directional cell movement at the guidance of environmental cues is fundamental to embryogenesis, organogenesis, and wound healing, as well as the development of atherosclerosis and the metastasis of tumors. [...]

We report a differential fluorescence speckle confocal microscope that acquires an image in a fraction of a second by exploiting the very high frame rate of modern digital micromirror devices (DMDs). The DMD projects a sequence of predefined binary speckle patterns to the sample and modulates the intensity of the returning fluorescent light simultaneously. The fluorescent light reflecting from the DMD's 'on' and 'off' pixels is modulated by correlated speckle and anticorrelated speckle, respectively, to form two images on two CCD cameras in parallel. The sum of the two images recovers a widefield image, but their difference gives a near-confocal image in real time. Experimental results for both low and high numerical apertures are shown. [c] 2010 Optical Society of America OCIS codes: 180.1790, 180.2520, 110.2990, 030.6140, 230.4685.

Perry C, Baker OJ, Reyland ME,Grichtchenko II. PKC[alpha][beta][gamma]- and PKC[delta]-dependent endocytosis of NBCel-A and NBCel-B in salivary pasrotid Acinar cells. Am J Physiol Cell Physiol 297: C1409-C1423, 2009. First Published September 30, 2009; doi: 10.1152.ajpcell.00028.2009.--We examined membrane trafficking of NBCe1-A and NBCe1-B variants of the electrogenic [Na.sup.+]-HC[O.sup.3.sub.-] cotransporter (NBCe1) encoded by the SLC4A4 gene, using confocal fluorescent microscopy in rat parotid acinar cells (ParC5 and ParC10). We showed that yellow fluorescent protein (YFP)-tagged NBCe1-A and green fluorescent protein (GFP)-tagged NBCe1-B are colocalized with E-cadherin in the basolateral membrane (BLM) but not with the apical membrane marker zona occludens 1 (ZO-1). We inhibited constitutive recycling with monensin and W13 and detected that NBCe1-A and NBCe1-B accumulated in vesicles marked with the early endosomal marker early endosome antigen-1 (EEA1), with a parallel loss from the BLM. We observed that NBCe1-A and NBCe1-B undergo massive carbachol (CCh)-stimulated redistribution from the BLM into early endosomes. We showed that internalization of NBCe1-A and NBCe1-B was prevented by the general PKC inhibitor GF-109203X, the PKC[alpha][beta][gamma]-specific inhibitor Go-6976, and the PKC[delta]-specific inhibitor rottlerin. We verified the involvement of PKC[delta] by blocking CChinduced internalization of NBCe1-A-cyan fluorescent protein (CFP) in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKC[delta]-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKC[alpha][beta][gamma]) and by novel PKC[delta] in rat epithelial cells. To help develop a more complete model of the role of NBCe1 in parotid acinar cells we also investigated the initial phase of the secretory response to cholinergic agonist. In an Ussing chamber study we showed that inhibition of basolateral NBCe1 with 5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1- carboxamide (tenidap) significantly decreases an initial phase of luminal anion secretion measured as a transient short-circuit current ([I.sub.sc]) across ParC10 cell monolayers. Using trafficking and functional data we propose a model that describes a physiological role of NBC in salivary acinar cell secretion. internalization; model of saliva secretion; Ussing chamber; SLC4A4 doi: 10.1152/ajpcell.00028.2009

Improved differential confocal microscopy is proposed to improve axial resolution and to enhance disturbance resistibility of confocal microscopy. The subtraction and sum values of the two defocusing detected signals are divided as the response function. Both ultrahigh signal-to-noise ratio (SNR) and wide range can be selectively obtained by controlling the defocusing amount of the two differential detectors more tightly with the reflectance disturbance resistibility. Since the detecting sensitivity of the proposed confocal microscopy is unrelated to the energy loss of the reflected beam, the multiplicative mode disturbance can be used to measure microstructures made of hybrid materials and overcome the power drift of a laser source during long scanning. In the case of ultrahigh SNR, the axial resolution reaches 1 nm when NA = 0.75 and [lambda] = 632.8 nm. OCIS codes: 180.1790, 180.3170, 180.5810.

A study was conducted to examine the relationship between occlusal loading and the marginal quality of cervical wedge-shaped restorations composed of resin-based composite. It is argued that occlusal loading does not affect the marginal behavior of restorations. Findings indicate that occlusal loading leads to a significant increase in gap formation.

Wetting phenomena in reversed-phase liquid chromatographic (RPLC) stationary phase particles strongly influ. ence the retention of solutes with high water-content mobile phases. To better understand these phenomena, this work reports the spectroscopic observation of the wetting of the interior of individual [C.sub.18]-silica stationary phase particles with acetonitrile-water mobile phase solutions by confocal Raman microscopy. It was found that the pores of dry [C.sub.18] silica do not wet when the concentration of acetonitrile is below O. 12 mol fraction (28% by volume). It was also found that there is a wetting hysteresis, where particles that had been previously exposed to aeetonitrile solutions above the wetting transition remain filled with solution even at much lower concentrations of acetonitrile in the surrounding solution. Contact angles of acetonitrile-water solutions were measured at a planar [C.sub.18]-modified silica surface and used to predict the capillary wetting of the particles based on the Young-Laplace equation. The solution composition at the wetting transition detected by Raman microscopy is higher in acetonitrile concentration than predicted by the Young-Laplace equation, which may be due to the presence of a vapor or air gap at the interface between the hydrophobic pores and aqueous solution. Further evidence of this behavior is found in water porosimetry results, which show wetting pressures ~5 times greater than predicted by the Young-Laplace equation and are consistent with only 50% of a water interface being in contact with the [C.sub.18] surface. This fraction increases to 80% at an acetenitrile concentration of 0.12 mol fraction, leading to spontaneous and irreversible wetting of the hydrophobic pores.

We visualized insulin uptake in vivo across the apical membrane of the rat proximal tubule (PT) by confocal microscopy; we compared it with in vitro findings in a rat PT cell line (WKPT) using fluorescence microscopy and flow cytometry. Surface tubules were observed in vivo with a 633-nm single laser-illuminated real-time video-rate confocal scanning microscope in upright configuration for optical sectioning below the renal capsule. Fields were selected containing proximal and distal tubules; Cy5-labeled insulin was injected twice (the second time after 140 min) into the right jugular vein, and the fluorescence signal (at 650-670 nm) was recorded. Fluorescence was detected almost immediately at the brush-border membrane (BBM) of PT cells only, moving inside cells within 30-40 min. As a measure of insulin uptake, the ratio of the fluorescence signal after the second injection to the first doubled (ratio: 2.11 [+ or -] 0.26, mean [+ or -] SE, n = 10), indicating a 'priming,' or stimulating, effect of insulin on its uptake mechanism at the BBM. This effect did not occur after pretreatment with intravenous lysine (ratio: 1.03 [+ or -] 0.07, n = 6; P < 0.01). Cy2-or Cy3-1abeled insulin uptake in a PT cell line in vitro was monitored by 488-nm excitation fluorescence microscopy using an inverted microscope. Insulin localized toward the apical membrane of these cells. Semiquantitative analysis of insulin uptake by flow cytometry also demonstrated a priming effect (upregulation) on insulin internalization in the presence of increasing amounts of insulin, as was observed in vivo; moreover, this effect was not seen with, or affected by, the similarly endocytosed ligand [beta]2-glycoprotein. intravital confocal microscopy; fluorescence; kidney

Confocal microscopy is a potentially powerful technique for obtaining equation-of-state (EOS) data for fluids in a diamond anvil cell. Unlike conventional microscopy, a confocal microscope scans the cell in three dimensions. From the intensity profile of the reflected laser light, we calculated the index of refraction and optical thickness of the sample contained in the cell. These measurements, combined with the cross-sectional area of the sample, enabled us to calculate the volume. As a test of the experimental technique and analysis, we produced a pressure-volume curve for liquid water at 300 K. The results agree with published EOS data within experimental error. OCIS codes: 000.2170, 000.6850, 010.2940, 010.7340, 160.4760, 180.1790.

Lipoxin [A.sub.4] ([LXA.sub.4]) is a biologically active eicosanoid produced in human airways that displays anti-inflammatory properties. In cystic fibrosis and severe asthma, [LXA.sub.4] production has been reported to be decreased, and, in such diseases, one of the consequences of airway inflammation is disruption of the tight junctions. In the present study, we investigated the possible role of [LXA.sub.4] on tight junction formation, using transepithelial electrical resistance (TER) measurements, Western blotting, and immunofluorescence. We observed that exposure to [LXA.sub.4] (100 nM) for 2 days significantly increased zonula occludens-1 (ZO-1), claudin-1, and occludin expression at the plasma membrane of confluent human bronchial epithelial 16HBE14o- cells. [LXA.sub.4] (100 nM) stimulated the daily increase of the 16HBE14o- cell monolayer TER, and this effect was inhibited by boc-2 ([LXA.sub.4] receptor antagonist). [LXA.sub.4] also had a rapid effect on ZO-1 immunofluorescence at the plasma membrane and increased TER within 10 min. In conclusion, our experiments provide evidence that [LXA.sub.4] plays certainly a new role for the regulation of tight junction formation and stimulation of the localization and expression of ZO-1 at the plasma membrane through a mechanism involving the [LXA.sub.4] receptor. protein kinase C; immunofluorescence quantification; confocal microscopy

The pancreatic islets of Langerhans are highly vascularized micro-organs that play a key role in the regulation of blood glucose homeostasis. The specific arrangement of endocrine cell types in islets suggests a coupling between morphology and function within the islet. Here, we established a line-scanning confocal microscopy approach to examine the relationship between blood flow and islet cell type arrangement by real-time in vivo imaging of intra-islet blood flow in mice. These data were used to reconstruct the in vivo 3D architecture of the islet and time-resolved blood flow patterns throughout the islet vascular bed. The results revealed 2 predominant blood flow patterns in mouse islets: inner-to-outer, in which blood perfuses the core of [beta] cells before the islet perimeter of non--[beta] cells, and top-to-bottom, in which blood perfuses the islet from one side to the other regardless of cell type. Our approach included both millisecond temporal resolution and submicron spatial resolution, allowing for real-time imaging of islet blood flow within the living mouse, which has not to our knowledge been attainable by other methods., Introduction Irregularities in pancreatic islet blood flow have been postulated to play a major role in islet pathophysiology as well as in the failure of islet transplants (1-5). Pancreatic islets [...]

Compartmentalization of proteins into subcellular organelles in eukaryotic cells is a fundamental mechanism of regulating complex cellular functions. Many proteins of Plasmodium falciparum merozoites involved in invasion are compartmentalized into apical organelles. We have identified a new merozoite organelle that contains P. falciparum rhomboid-1 (PfROM1), a protease that cleaves the transmembrane regions of proteins involved in invasion. By immunoconfocal microscopy, PfROM1 was localized to a single, thread-like structure on one side of the merozoites that appears to be in close proximity to the subpellicular microtubules. PfROM1 was not found associated with micronemes, rhoptries, or dense granules, the three identified secretory organelles of invasion. Release of merozoites from schizonts resulted in the movement of PfROM1 from the lateral asymmetric localization to the merozoite apical pole and the posterior pole. We have named this single thread-like organelle in merozoites, the mononeme. malaria

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar ceils stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosollc dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types. pancreatic acinar cells; confocal microscopy; patch clamp; cholecystokinin

Acid [beta]-glucosidase (GCase) is a soluble lysosomal enzyme responsible for the hydrolysis of glucose from glucosylceramide and requires activation by the small nonenzymatic protein saposin C (sapC) to gain access to the membrane-embedded glycosphingo-lipid substrate. We have used in situ atomic force microscopy (AFM) with simultaneous confocal and epifluorescence microscopies to investigate the interactions of GCase and sapC with lipid bilayers. GCase binds to sites on membranes transformed by sapC, and enzyme activity occurs at loci containing both GCase and sapC. Using FRET, we establish the presence of GCase/sapC and GCase/ product contacts in the bilayer. These data support a mechanism in which sapC locally alters regions of bilayer for subsequent attack by the enzyme in stably bound protein complexes. atomic force microscopy | confocal microscopy | FRET | interfacial catalysis | lipid storage disease

In sensitive cells, verotoxin 1 (VT1) utilizes a globotriaosylceramide receptor-dependent retrograde transport pathway from the cell surface to the Golgi/endoplasmic reticulum (ER). The VT1 A subunit (VTA) is an RNA glycanase. Although translocation of VTA from the ER to the cytosol is considered the route for protein synthesis inhibition, cell-based evidence is lacking. A dual-fluorescent-labelled VT1 holotoxin was constructed to simultaneously monitor VTA and VT1 B subunit (VTB) intracellular transport. By confocal microscopy, VTA/VTB subunits remained associated throughout the retrograde transport pathway without cytosolic staining. However, in [[sup.125]I][VT.sub.1]-treated cells, the selective cytosolic translocation (4%) of the activated form of VTA, [VTA.sub.1], was demonstrated for the first time by monitoring [[sup.125]I][VTA.sub.1] release after plasma membrane permeabilization by streptolysin O (SLO). Lactacystin, a proteasome inhibitor, increased cytosolic [VTA.sub.1] and enhanced VT1 cytotoxicity. VT1 ER arrival coincided with cytosolic [VTA.sub.1] detection. Brefeldin A and 16 [degrees]C, conditions which inhibit VT1 retrograde transport to the Golgi/ER, prevented [VTA.sub.1] cytosolic translocation; however, these treatments did not completely prevent VT1-induced protein synthesis inhibition. Thus, efficient cytosolic translocation of VTA1 requires transport to the Golgi/ER, but alternative minor escape pathways for protein synthesis inhibition may operate when transport to the Golgi/ER is prevented. Inhibition of protein synthesis was time and dose dependent, and not necessarily a valid index of subsequent cytopathology. Only protein synthesis inhibition following >3 h VT1 exposure correlated with eventual cell cytotoxicity. Extrapolation of translocated cytosolic [VTA.sub.1] values indicates that about one molecule of translocated [VTA.sub.1] per cell is sufficient to inhibit protein synthesis and kill a cell.

In this paper, we present a novel 2-D microelectromechanical systems (MEMS) scanner that enables dual-axes confocal microscopy. Dual-axes confocal microscopy provides high resolution and long working distance, while also being well suited for miniaturization and integration into endoscopes for in vivo imaging. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer (a silicon wafer bonded on a SOI wafer) and is actuated by self-aligned vertical electrostatic combdrives. Maximum optical deflections of [+ or -] 4.8[degrees] and [+ or -] 5.5[degrees] are achieved in static mode for the outer and inner axes, respectively. Torsional resonant frequencies are at 500 Hz and 2.9 kHz for the outer and inner axes, respectively. The imaging capability of the MEMS scanner is successfully demonstrated in a breadboard setup. Reflectance images with a field of view of 344 [micro]m x 417 [micro]m are achieved at 8 frames/s. The transverse resolutions are 3.94 and 6.68 [micro]m for the horizontal and vertical dimensions, respectively. [2006-0218] Index Terms--Dual-axes confocal microscopy, electrostatic actuation, micromirror, self-alignment, vertical comb actuators, 2-D microelectromechanical systems (MEMS) scanner.

Two proteins that mediate bile acid export from the ileal enterocyte, organic solute transporter (OST)-[alpha] and -[beta], have recently been identified. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interactions, transport functions, and membrane localization of human (h)OST-[alpha] and -[beta] proteins were examined. The results demonstrated that coexpression of hOST-[alpha] and -[beta] in transfected cells resulted in a three- to fivefold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and nontransfected ceils. Confocal microscopy demonstrated plasma membrane colocalization of hOST-[alpha] and -[beta] proteins in cells cotransfected with hOST-or and -[beta] cDNAs. Protein-protein interactions between hOST-[alpha] and -[beta] were demonstrated by mammalian two-hybrid and coimmunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST-[alpha] abolished its interaction with hOST-[beta] and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST-[alpha] still interacted with hOST-[beta], and majority of this cytoplasmic tail-truncated protein was expressed on the basolateral membrane when it was stably cotransfected with hOST-[beta] protein in Madin-Darby canine kidney cells. In summary, hOST-[alpha] and -[beta] proteins are physically associated. The intracellular carboxyl-terminal domain of hOST-[alpha] is not essential for this interaction with hOST-[beta]. The extracellular amino-terminal fragment of hOST-[alpha] may contain important information for the assembly of the heterodimer and trafticking to the plasma membrane. bile acid transporter doi: 10.1152/ajpgi.00457.2006