Digital Charge-coupled Device Research Articles

REGISTRATION OF OPTICAL IMAGES OF A VACUUM ARC DISCHARGE WITH AN ELECTRON-OPTICAL CONVERTER WITH NANOSECOND TIME RESOLUTION

Abstract—The process of switching a short vacuum gap by an auxiliary discharge over the dielectric surface was studied via recording of images of discharge plasma radiating in the optical spectral range. A two-channel electro-optical (EO) image-recording system based on an EO converter (EOC) was used. Each channel contained a unit for generating an EOC-photocathode gating pulse, a transposing objective lens, and an EOC, which was joined to a digital CCD (charge-coupled device) camera for reading information from the EOC screen. Based on the analysis of the obtained experimental data, it was suggested that the radiation of a cathode spot and a cathode flame in the UV range play a significant role in the formation of a current channel in the discharge.

Quantitative assessment of the conjunctival microcirculation using a smartphone and slit-lamp biomicroscope

PurposeThe conjunctival microcirculation is a readily-accessible vascular bed for quantitative haemodynamic assessment and has been studied previously using a digital charge-coupled device (CCD). Smartphone video imaging of the conjunctiva, and haemodynamic parameter quantification, represents a novel approach. We report the feasibility of smartphone video acquisition and subsequent haemodynamic measure quantification via semi-automated means. MethodsUsing an Apple iPhone 6 s and a Topcon SL-D4 slit-lamp biomicroscope, we obtained videos of the conjunctival microcirculation in 4 fields of view per patient, for 17 low cardiovascular risk patients. After image registration and processing, we quantified the diameter, mean axial velocity, mean blood volume flow, and wall shear rate for each vessel studied. Vessels were grouped into quartiles based on their diameter i.e. group 1 (<11 μm), 2 (11–16 μm), 3 (16–22 μm) and 4 (>22 μm). ResultsFrom the 17 healthy controls (mean QRISK3 6.6%), we obtained quantifiable haemodynamics from 626 vessel segments. The mean diameter of microvessels, across all sites, was 21.1μm (range 5.8–58 μm). Mean axial velocity was 0.50mm/s (range 0.11–1mm/s) and there was a modestly positive correlation (r 0.322) seen with increasing diameter, best appreciated when comparing group 4 to the remaining groups (p < .0001). Blood volume flow (mean 145.61pl/s, range 7.05–1178.81pl/s) was strongly correlated with increasing diameter (r 0.943, p < .0001) and wall shear rate (mean 157.31 s−1, range 37.37–841.66 s−1) negatively correlated with increasing diameter (r − 0.703, p < .0001). ConclusionsWe, for the first time, report the successful assessment and quantification of the conjunctival microcirculatory haemodynamics using a smartphone-based system.

The Accuracy of 3D Optical Reconstruction and Additive Manufacturing Processes in Reproducing Detailed Subject-Specific Anatomy

3D reconstruction and 3D printing of subject-specific anatomy is a promising technology for supporting clinicians in the visualisation of disease progression and planning for surgical intervention. In this context, the 3D model is typically obtained from segmentation of magnetic resonance imaging (MRI), computed tomography (CT) or echocardiography images. Although these modalities allow imaging of the tissues in vivo, assessment of quality of the reconstruction is limited by the lack of a reference geometry as the subject-specific anatomy is unknown prior to image acquisition. In this work, an optical method based on 3D digital image correlation (3D-DIC) techniques is used to reconstruct the shape of the surface of an ex vivo porcine heart. This technique requires two digital charge-coupled device (CCD) cameras to provide full-field shape measurements and to generate a standard tessellation language (STL) file of the sample surface. The aim of this work was to quantify the error of 3D-DIC shape measurements using the additive manufacturing process. The limitations of 3D printed object resolution, the discrepancy in reconstruction of the surface of cardiac soft tissue and a 3D printed model of the same surface were evaluated. The results obtained demonstrated the ability of the 3D-DIC technique to reconstruct localised and detailed features on the cardiac surface with sub-millimeter accuracy.

Actinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors

Dendritic spines are dynamic, actin-rich protrusions in neurons that undergo remodeling during neuronal development and activity-dependent plasticity within the central nervous system. Although group 1 metabotropic glutamate receptors (mGluRs) are critical for spine remodeling under physiopathological conditions, the molecular components linking receptor activity to structural plasticity remain unknown. Here we identify a Ca(2+)-sensitive actin-binding protein, α-actinin-4, as a novel group 1 mGluR-interacting partner that orchestrates spine dynamics and morphogenesis in primary neurons. Functional silencing of α-actinin-4 abolished spine elongation and turnover stimulated by group 1 mGluRs despite intact surface receptor expression and downstream ERK1/2 signaling. This function of α-actinin-4 in spine dynamics was underscored by gain-of-function phenotypes in untreated neurons. Here α-actinin-4 induced spine head enlargement, a morphological change requiring the C-terminal domain of α-actinin-4 that binds to CaMKII, an interaction we showed to be regulated by group 1 mGluR activation. Our data provide mechanistic insights into spine remodeling by metabotropic signaling and identify α-actinin-4 as a critical effector of structural plasticity within neurons.

Silicon-Nitride-Based Integrated Optofluidic Biochemical Sensors Using a Coupled-Resonator Optical Waveguide

Silicon nitride (SiN) is a promising material platform for integrating photonic components and microfluidic channels on a chip for label-free, optical biochemical sensing applications in the visible to near-infrared wavelengths. The chip-scale SiN-based optofluidic sensors can be compact due to a relatively high refractive index contrast between SiN and the fluidic medium, and low-cost due to the complementary metal-oxide-semiconductor (CMOS)-compatible fabrication process. Here, we demonstrate SiN-based integrated optofluidic biochemical sensors using a coupled-resonator optical waveguide (CROW) in the visible wavelengths. The working principle is based on imaging in the far field the out-of-plane elastic-light-scattering patterns of the CROW sensor at a fixed probe wavelength. We correlate the imaged pattern with reference patterns at the CROW eigenstates. Our sensing algorithm maps the correlation coefficients of the imaged pattern with a library of calibrated correlation coefficients to extract a minute change in the cladding refractive index. Given a calibrated CROW, our sensing mechanism in the spatial domain only requires a fixed-wavelength laser in the visible wavelengths as a light source, with the probe wavelength located within the CROW transmission band, and a silicon digital charge-coupled device (CCD) / CMOS camera for recording the light scattering patterns. This is in sharp contrast with the conventional optical microcavity-based sensing methods that impose a strict requirement of spectral alignment with a high-quality cavity resonance using a wavelength-tunable laser. Our experimental results using a SiN CROW sensor with eight coupled microrings in the 680nm wavelength reveal a cladding refractive index change of ~1.3 × 10^-4 refractive index unit (RIU), with an average sensitivity of ~281 ± 271 RIU-1 and a noise-equivalent detection limit (NEDL) of 1.8 ×10^-8 RIU ~ 1.0 ×10^-4 RIU across the CROW bandwidth of ~1 nm.

Homeostatic Competition between Phasic and Tonic Inhibition

The GABAA receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABAA receptors mediate phasic inhibition, whereas extrasynaptic GABAA receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABAA receptors through various molecular manipulations. Overexpression of extrasynaptic α6β3δ-GABAA receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6β3δ subunits. Interestingly, blocking all GABAA receptors after overexpressing α6β3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6(GBS)β3δ(GBS) receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6β3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5β3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABAA receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.

Epidermal Growth Factor (EGF)-induced Corneal Epithelial Wound Healing through Nuclear Factor κB Subtype-regulated CCCTC Binding Factor (CTCF) Activation

Epidermal growth factor (EGF) plays an important role in corneal epithelial migration and proliferation to improve the wound healing process. This study aimed to understand the role of NFκB in EGF-induced corneal epithelial wound healing through regulation of CTCF activity, which plays important roles in cell motility and migration to promote wound healing. The effect of NFκB p50 on corneal epithelial wound healing was investigated by comparing the eyes of wild-type and p50 knockout mice. We found that there was a significant retardation in corneal epithelial wound healing in the corneas of p50 knockout mice. Wound closure rates were measured in human corneal epithelial cells transfected with an NFκB activation-sensitive CTCF expression construct to demonstrate the effect of human CTCF expression under the control of EGF-induced NFκB activation on wound healing. EGF stimulation activated NFκB, which directly triggered the expression of the exogenous human CTCF in transfected cells and, subsequently, promoted human corneal epithelial cell motility, migration, and wound healing. Overexpression of CTCF in corneal epithelial cells and mouse corneas significantly enhanced the wound healing process. Furthermore, the effect of overexpressing NFκB p50 in corneal epithelial cells on the promotion of wound healing was abolished by knockdown of CTCF with CTCF-specific shRNA. Thus, a direct regulatory relationship between EGF-induced NFκB p50 and CTCF activation affecting corneal epithelial wound healing has been established, indicating that CTCF is, indeed, a NFκB p50-targeted and effective gene product in the core transcriptional network downstream from the growth factor-induced NFκB signaling pathway.

First Place: Renal Oxygenation During Robot-Assisted Laparoscopic Partial Nephrectomy: Characterization Using Laparoscopic Digital Light Processing Hyperspectral Imaging

Abstract Background and Purpose: Digital light processing-based hyperspectral imaging (DLP(®)-HSI) was adapted for use during laparoscopic surgery by coupling the spectral illumination source with a conventional laparoscopic light guide and incorporating a customized digital charge-coupled device camera for image acquisition. The system was used to characterize renal oxygenation during robot-assisted laparoscopic partial nephrectomy (RALPN) in humans. After Institutional Review Board approval, laparoscopic DLP-HSI was performed in consecutive patients undergoing RALPN at our institution. Time trends in relative tissue oxygen saturation (%HbO2) were descriptively analyzed. Associations between %HbO2 and patient age, comorbidities, and estimated glomerular filtration rate (eGFR) were investigated using the Kendall tau test. Laparoscopic DLP-HSI was performed in 18 patients between May 2011 and February 2012. Median (interquartile range; IQR) age was 55.9 (49-67.5) years. Of the patients, 10/18 (56%) were men and 12/18 (66.7%) had a history of hypertension, diabetes, and/or tobacco use. Median (IQR) %HbO2 before, during, and after ischemia was 60.8% (57.9-68.2%), 53.6% (46.8-55.1%), and 61.5% (54.9-67.6%), respectively. Baseline %HbO2 was inversely associated with preoperative eGFR (τ=-0.38; P=0.036), and eGFR at most recent follow-up (τ=-0.38; P=0.036). Baseline or ischemic %HbO2 did not correlate with hypertension, diabetes, and/or tobacco history. Younger patients (<56 years) had a lower median baseline %HbO2 (P=0.07) and a higher median preoperative eGFR (P=0.038), than their older counterparts. The laparoscopic HSI system successfully characterized dynamic changes in renal oxygenation during RALPN. Intraoperative laparoscopic HSI outcomes have the potential to predict postoperative individual kidney function.

Digital radiography with computerized conventional monitors compared to medical monitors in vertical root fracture diagnosis.

Vertical root fracture (VRF) is a complication which is chiefly diagnosed radiographically. Recently, film-based radiography has been substituted with digital radiography. At the moment, there is a wide range of monitors available in the market for viewing digital images. The present study aims to compare the diagnostic accuracy, sensitivity and specificity of medical and conventional monitors in detection of vertical root fractures. In this in vitro study 228 extracted single-rooted human teeth were endodontically treated. Vertical root fractures were induced in 114 samples. The teeth were imaged by a digital charge-coupled device radiography using parallel technique. The images were evaluated by a radiologist and an endodontist on two medical and conventional liquid-crystal display (LCD) monitors twice. Z-test was used to analyze the sensitivity, accuracy and specificity of each monitor. Significance level was set at 0.05. Inter and intra observer agreements were calculated by Cohen's kappa. Accuracy, specificity and sensitivity for conventional monitor were calculated as 67.5%, 72%, 62.5% respectively; and data for medical grade monitor were 67.5%, 66.5% and 68% respectively. Statistical analysis showed no significant differences in detecting VRF between the two techniques. Inter-observer agreement for conventional and medical monitor was 0.47 and 0.55 respectively (moderate). Intra-observer agreement was 0.78 for medical monitor and 0.87 for conventional one (substantial). The type of monitor does not influence diagnosis of vertical root fractures.

Imaging Properties of Scintillation Screens for High Energetic Ion Beams

The imaging properties of various scintillation screens were investigated for ion beams extracted from the heavy ion synchrotron SIS 18 at GSI. Different ion beams such as C, Ne, Ar, Ta and U with initial kinetic energy of about 300 MeV/u were applied to various scintillation screens with particle fluxes ranging from 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">4</sup> to 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">9</sup> particles per pulse typically within 0.3 s pulse length. The scintillation process was observed with a digital Charge Coupled Device (CCD) camera. The performed study compares the light output, the beam profiles, and the image deformation of various scintillating screens for different ion beams. The light output scales linearly with respect to the ion beam flux over five orders of magnitude for the sensitive scintillation screens and ceramics like Al <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> , Al <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> O <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> :Cr. The highest light output was observed for CsI:Tl, and the lowest was recorded for Herasil. At higher beam intensities, non linear behavior of light output was observed for Mg and Y doped ZrO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> samples. The recorded beam profiles showed differences in width up to 50%, depending on the screen material.

Impaired Manganese Metabolism Causes Mitotic Misregulation

Manganese is an essential trace element, whose intracellular levels need to be carefully regulated. Mn(2+) acts as a cofactor for many enzymes and excess of Mn(2+) is toxic. Alterations in Mn(2+) homeostasis affect metabolic functions and mutations in the human Mn(2+)/Ca(2+) transporter ATP2C1 have been linked to Hailey-Hailey disease. By deletion of the yeast orthologue PMR1 we have studied the impact of Mn(2+) on cell cycle progression and show that an excess of cytosolic Mn(2+) alters S-phase transit, induces transcriptional up-regulation of cell cycle regulators, bypasses the need for S-phase cell cycle checkpoints and predisposes to genomic instability. On the other hand, we find that depletion of the Golgi Mn(2+) pool requires a functional morphology checkpoint to avoid the formation of polyploid cells.

Cholesterol Modulates the Rate and Mechanism of Acetylcholine Receptor Internalization

Stability of the nicotinic acetylcholine receptor (AChR) at the cell surface is key to the correct functioning of the cholinergic synapse. Cholesterol (Chol) is necessary for homeostasis of AChR levels at the plasmalemma and for ion translocation. Here we characterize the endocytic pathway followed by muscle-type AChR in Chol-depleted cells (Chol(-)). Under such conditions, the AChR is internalized by a ligand-, clathrin-, and dynamin-independent mechanism. Expression of a dominant negative form of the small GTPase Rac1, Rac1N17, abolishes receptor endocytosis. Unlike the endocytic pathway in control CHO cells (1), accelerated AChR internalization proceeds even upon disruption of the actin cytoskeleton. Under Chol(-) conditions, AChR internalization is furthermore found to require the activity of Arf6 and its effectors Rac1 and phospholipase D. The Arf6-dependent mechanism may constitute the default endocytic pathway followed by the AChR in the absence of external ligands, membrane Chol levels acting as a key homeostatic regulator of cell surface receptor levels.

A novel endoscopic fluorescent clip for the localization of gastrointestinal tumors

Accurate tumor localization is essential for gastrointestinal surgery, especially in cases of early cancer. This study was designed to develop a novel fluorescent clip for rapid and exact visualization of tumor sites. A transparent polymer matrix containing highly bright fluorochromes was coated on the front end of endoscopic clips. The fluorescent clips were placed on the mucosal surface of a porcine colon and stomach, and the operator then attempted to identify the fluorescent clips from the outer serosal side of the colon and stomach. A 532-nm diode laser and filter glass were used for visualizing the fluorescence signals through the colonic tissue. A 650-nm diode laser and a digital charge-coupled device (CCD) camera equipped with a bandpass emission filter were used for the imaging of the fluorescent clips through the thick stomach tissue. When a green light from a 532-nm diode laser (power density=0.35 mW/cm2) was applied on the serosal surface of the porcine colon, we could identify all clips that had been placed endoscopically on the mucosal surface of the inner colonic wall. By using the light from a 650-nm diode laser (power density=0.7 mW/cm2), we identified all fluorescent clips through the stomach wall in real time. Similar results were also obtained with the filtered xenon lamp. An endoscopic fluorescent clip can be useful for the rapid and exact localization of tumors, and this technique can also be useful during laparoscopic surgery.

Spectral Imaging of the Human Retina and Computationally Determined Optimal Illuminants for Diabetic Retinopathy Lesion Detection

An ophthalmic fundus camera was modified for spectral imaging of the human retina. Spectral channels were separated using 30 narrow bandpass interference filters in the visual range from 400 to 700 nm. A monochrome digital charge-coupled device camera was used as a detector. Using this system, spectral fundus images were recorded from 72 voluntary human subjects: 55 diabetic patients and 17 healthy control subjects. Furthermore, using the gathered spectral data, optimal illuminations' spectral power distributions for the detection of different lesions of diabetic retinopathy and retinal landmarks (e.g., macula) were calculated. Optimized illuminations were found to enhance the visibility and contrast of retinal features. Computational example images are presented.

Acute effects of triclosan, diclofenac and carbamazepine on feeding performance of Japanese medaka fish ( Oryzias latipes)

The toxicity of three pharmaceutical and personal care products (PPCPs) – carbamazepine (CBMZ), diclofenac (DCF), and triclosan (TCS) – was examined by measuring their effects on feeding behavior and swimming speed of adult Japanese medaka fish ( Oryzias latipes). Medaka were exposed to 6.15 mg L −1 CBMZ, 1.0 mg L −1 DCF, 0.17 mg L −1 TCS, or no PPCP (control) for 9 d. Fish behaviors were monitored during days 5–9 of the exposure period. Feeding behavior (time to eat midge larvae, TE) and swimming speed (SS) of individual exposed and control fish were tracked in two dimensions, using an automated system with a digital charge-coupled device camera. As a result, feeding behavior was affected by exposure to CBMZ and DCF, while SS was altered by exposure to CBMZ and TCS. Thus, TCS, DCF and CBMZ appear to affect fish behaviors through different mechanisms. Overall, the results suggest that behavioral changes may provide a sensitive indicator for assessing the toxicity of PPCPs to aquatic organisms.

Use of laser speckle flowgraphy in ocular blood flow research

Laser speckle flowgraphy (LSFG) allows for the quantitative estimation of blood flow in the optic nerve head, choroid, retina and iris in vivo. It was developed to facilitate the non-contact analysis of ocular blood flow in living eyes, utilizing the laser speckle phenomenon. The technique uses a fundus camera, a diode laser, an image sensor, an infrared charge-coupled device (CCD) camera and a high-resolution digital CCD camera. Normalized blur (NB), an approximate reciprocal of speckle contrast, represents an index of blood velocity, and shows a good correlation with tissue blood flow rates determined with the microsphere method in the retina, choroid or iris, as well as blood flow rates determined with the hydrogen gas clearance method in the optic nerve head. The square blur ratio (SBR), another index for quantitative estimation of blood velocity, is proportional to the square of the NB. The SBR is theoretically a more exact measurement which is proportional to velocity, whereas the NB is an approximation. Normalized blur was calculated in earlier versions of LSFG because of technical limitations; the SBR is used in current versions of the LSFG instrument. As these values are in arbitrary units, they should not be used to make comparisons between different eyes or different sites in an eye. Clinical protocols, calibration, evaluation procedures and possible limitations of the LSFG technique are described and the results of ocular blood flow studies using LSFG are briefly summarized. The LSFG method is suitable for monitoring the time-course of change in the tissue circulation at the same site in the same eye at various intervals, ranging from seconds to months. Unresolved issues concern the effect of pupil size on measurement results, the effects of various stimulations, and how to measure choroidal and retinal blood flow velocity separately without using the blue-component of argon laser.

Kinesin Adapter JLP Links PIKfyve to Microtubule-based Endosome-to-Trans-Golgi Network Traffic of Furin

JIPs (c-Jun N-terminal kinase interacting proteins), which scaffold JNK/p38 MAP kinase signaling modules, also bind conventional kinesins and are implicated in microtubule-based membrane trafficking in neuronal cells. Here we have identified a novel splice variant of the Jip4 gene product JLP(L) (JNK-interacting leucine zipper protein) in yeast-two hybrid screens with the phosphoinositide kinase PIKfyve. The interaction was confirmed by pulldown and coimmunoprecipitation assays in native cells. It engages the PIKfyve cpn60_TCP1 consensus sequence and the last 75 residues of the JLP C terminus. Subpopulations of both proteins cofractionated and populated similar structures at the cell perinuclear region. Because PIKfyve is essential in endosome-to-trans-Golgi network (TGN) cargo transport, we tested whether JLP is a PIKfyve functional partner in this trafficking pathway. Short interfering RNA (siRNA)-mediated depletion of endogenous JLP or PIKfyve profoundly delayed the microtubule-based transport of chimeric furin (Tac-furin) from endosomes to the TGN in a CHO cell line, which was rescued upon ectopic expression of siRNA-resistant JLP or PIKfyve constructs. Peptides from the contact sites in PIKfyve and JLP, or a dominant-negative PIKfyve mutant introduced into cells by ectopic expression or microinjection, induced a similar defect. Because Tac-TGN38 delivery from endosomes to the TGN, unlike that of Tac-furin, does not require intact microtubules, we monitored the effect of JLP and PIKfyve depletion or the interacting peptides administration on Tac-TGN38 trafficking. Remarkably, neither maneuver altered the Tac-TGN38 delivery to the TGN. Our data indicate that JLP interacts with PIKfyve and that both proteins and their association are required in microtubule-based, but not in microtubule-independent, endosome-to-TGN cargo transport.

Modulation of Yeast Sln1 Kinase Activity by the Ccw12 Cell Wall Protein

The yeast Sln1p sensor kinase is best known as an osmosensor involved in the regulation of the hyperosmolarity glycerol mitogen-activated protein kinase cascade. Down-regulation of Sln1 kinase activity occurs under hypertonic conditions and leads to phosphorylation of the Hog1p mitogen-activated protein kinase and increased osmotic stress-response gene expression. Conditions leading to kinase up-regulation include osmotic imbalance caused by glycerol retention in the glycerol channel mutant, fps1 (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367). The hypothesis that Sln1p kinase activity is responsive to turgor was first suggested by the increased Sln1p kinase activity in mutants lacking Fps1p in which glycerol accumulation leads to water uptake. Also consistent with the turgor hypothesis is the observation that reduced turgor caused by treatment of cells with nystatin, a drug that increases membrane permeability and causes cell shrinkage, reduced Sln1p kinase activity (Tao, W., Deschenes, R. J., and Fassler, J. S. (1999) J. Biol. Chem. 274, 360-367; Reiser, V., Raitt, D. C., and Saito, H. (2003) J. Cell Biol. 161, 1035-1040). The turgor hypothesis is revisited here in the context of the identification and characterization of the cell wall gene, CCW12, as a determinant of Sln1p activity. Results of this analysis suggest that the activity of the plasma membrane localized Sln1p is affected by the presence or absence of specific outer cell wall proteins and that this effect is independent of turgor.

Up-regulation of the Angiotensin II Type 1 Receptor by the MAS Proto-oncogene Is Due to Constitutive Activation of Gq/G11 by MAS

Coexpression of the MAS proto-oncogene with the angiotensin II type 1 (AT(1)) receptor in CHO-K1 cells has been reported to increase the number of [(3)H]angiotensin II-binding sites, although MAS does not bind [(3)H]angiotensin II. In HEK293 cells stably expressing AT(1) receptor-cyan fluorescent protein (CFP), MAS-yellow fluorescent protein (YFP) expression from an inducible locus caused strong up-regulation of AT(1) receptor-CFP amounts and [(3)H]angiotensin II binding levels. The time course of AT(1) receptor-CFP up-regulation was also markedly slower than that of induction of MAS expression. These effects were not mimicked by induced expression of I138D MAS-YFP, a mutant unable to cause constitutive loading of [(35)S]guanosine 5'-O-(thiotriphosphate) onto the phospholipase Cbeta-linked G protein Galpha(11). Protein kinase C (PKC) inhibitors and the selective Galpha(q)/Galpha(11) inhibitor YM-254890 fully blocked MAS-induced up-regulation of AT(1) receptor-CFP amounts, whereas the PKC activator phorbol 12-myristate 13-acetate produced strong up-regulation of AT(1) receptor-CFP without induction of MAS-YFP expression and in the presence of I138D MAS-YFP. The C-terminal tail of the AT(1) receptor is a known target for PKC-mediated phosphorylation. In cells stably expressing a C-terminally truncated version of the AT receptor, induction of MAS expression did not up-regulate the truncated construct levels. These data demonstrate that the ability of MAS to up-regulate AT(1) receptor levels reflects the constitutive capacity of MAS to activate Galpha(q)/Galpha(11) and hence stimulate PKC-dependent phosphorylation of the AT(1) receptor.

Detectability of experimental peri‐implant cancellous bone lesions using conventional and direct digital radiography

The aim of this study was to compare the diagnostic performance obtained from direct digital radiographic images (in their original form and after applying a grey-scale inversion) and conventional film in the detection of artificial peri-implant cancellous bone lesions. Four titanium implants were placed into the cancellous bone of a dry mandible and increasingly larger bone defects were created in their approximal sites. Radiographs were taken using conventional film and a digital charge-coupled device sensor. Twelve observers estimated three series of images (conventional, digital original, digital inverse) on a 5-point confidence scale. Data were evaluated statistically by analysis of variance and the sensitivity, specificity and accuracy of the three imaging modalities were calculated. Total mean observer confidence scores increased as the size of the defect also increased. No statistically significant differences were found among the three images for the absence of defect and the defect that corresponds to the smallest bur size. Significant differences were found for larger bur sizes between the conventional image and the two digital images and for the largest bur size between the digital inverse and the other two images. Specificity was high and sensitivity relatively low. Peri-implant bone lesions must exceed a certain size to be confidently detected but the lesion absence is detected equally well with all three imaging modalities.