RESUMO
There is increasing prevalence of digital diagnostic imaging in veterinary medicine with a progressive need to use medical imaging software. As Digital Imaging and Communications in Medicine (DICOM)-viewers for veterinary use do not require medical device approval in many countries, freeware viewers might be a practical alternative. The aim of this study was to identify and evaluate free DICOM-viewer software for veterinary purposes. The functionality and user-friendliness of various DICOM-viewers from the internet were analyzed and compared. Inclusion criteria for the evaluation were free availability, PACS (picture archiving and communication system)-connectivity, and stand-alone and client-based software. Based on this, eight viewers were found: Ginkgo CADx, Horos, K-PACS, MAYAM, MITO, OsiriX Lite, RadiAnt, Synedra personal. In these DICOM-viewers, 14 core tools were tested and rated on a score from 1 to 10 by multiple observers with different levels of training, using studies of four imaging modalities. Criteria were functionality and user-friendliness. For each viewer, the total number of a predefined set of 47 important tools was counted. The ranking based on functionality and user-friendliness of 14 core tools (mean score in brackets) was as follows: 1. Horos/OsiriX Lite (8.96), 2. RadiAnt (8.90), 3. K-PACS (8.02), 4. Synedra (7.43), 5. MAYAM (6.05), 6. Ginkgo CADx (5.53), 7. MITO (3.74). The DICOM-viewers offered between 20 and 44 tools of the predefined important tool set and are sufficient for most veterinary purposes. An increasing number of tools did not necessarily impair user-friendliness, if the user interface is well designed. Based on the results of this study, veterinarians will find suitable free DICOM-viewers for their individual needs. In combination with PACS-freeware, this allows veterinary practices to run a low-budget digital imaging environment.
Assuntos
Sistemas de Informação em Radiologia , Processamento Eletrônico de Dados , Humanos , Radiografia , Software , Tomografia Computadorizada por Raios XRESUMO
Blocking is an important step before an enzyme-linked immunosorbent assay (ELISA) can be performed. It reduces non-specific binding to the microtiter plate to a minimum. For detecting food allergens by means of ELISA, the problem with protein blocking solutions is obvious. The blocker might interfere with the antibodies of the assay and leads to false positive results. Therefore, other blocking solutions are greatly needed. There are some alternatives like synthetic blockers or carbohydrates. Comparisons of these different blocking agents, namely proteins, carbohydrates, and synthetic blockers, were made at different reaction conditions. The incubation periods and temperatures were varied, as well as the pH. The best combinations were evaluated and compared, in respect of their blocking efficiency. The two best non-proteinaceous blockers, i.e. polyvinylalcohol and Ficoll, were subsequently applied to ELISA tests for the determination of alpha-casein and peanut. The study showed that Ficoll and PVA did as well as BSA in buffer solution. Therefore, they can be considered as alternative blocking reagents for ELISA, especially for the detection of food allergens.
Assuntos
Alérgenos/análise , Alérgenos/imunologia , Produtos Biológicos/análise , Ensaio de Imunoadsorção Enzimática/métodos , Hipersensibilidade Alimentar/imunologiaRESUMO
Mitochondrial fission ensures organelle inheritance during cell division and participates in apoptosis. The fission protein hFis1 triggers caspase-dependent cell death, by causing the release of cytochrome c from mitochondria. Here we show that mitochondrial fission induced by hFis1 is genetically distinct from apoptosis. In cells lacking the multidomain proapoptotic Bcl-2 family members Bax and Bak (DKO), hFis1 caused mitochondrial fragmentation but not organelle dysfunction and apoptosis. Similarly, a mutant in the intermembrane region of hFis1-induced fission but not cell death, further dissociating mitochondrial fragmentation from apoptosis induction. Selective correction of the endoplasmic reticulum (ER) defect of DKO cells restored killing by hFis1, indicating that death by hFis1 relies on the ER gateway of apoptosis. Consistently, hFis1 did not directly activate BAX and BAK, but induced Ca(2+)-dependent mitochondrial dysfunction. Thus, hFis1 is a bifunctional protein that independently regulates mitochondrial fragmentation and ER-mediated apoptosis.
Assuntos
Apoptose/fisiologia , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Respiração Celular/fisiologia , Análise Mutacional de DNA , Fibroblastos/citologia , Fibroblastos/ultraestrutura , Humanos , Proteínas de Membrana , Camundongos , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Permeabilidade , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/deficiência , Proteína X Associada a bcl-2/deficiênciaRESUMO
The spider venom alpha-latrotoxin (alpha-LTX) induces massive exocytosis after binding to surface receptors, and its mechanism is not fully understood. We have investigated its action using toxin-sensitive MIN6 beta-cells, which express endogenously the alpha-LTX receptor latrophilin (LPH), and toxin-insensitive HIT-T15 beta-cells, which lack endogenous LPH. alpha-LTX evoked insulin exocytosis in HIT-T15 cells only upon expression of full-length LPH but not of LPH truncated after the first transmembrane domain (LPH-TD1). In HIT-T15 cells expressing full-length LPH and in native MIN6 cells, alpha-LTX first induced membrane depolarization by inhibition of repolarizing K(+) channels followed by the appearance of Ca(2+) transients. In a second phase, the toxin induced a large inward current and a prominent increase in intracellular calcium ([Ca(2+)](i)) reflecting pore formation. Upon expression of LPH-TD1 in HIT-T15 cells just this second phase was observed. Moreover, the mutated toxin LTX(N4C), which is devoid of pore formation, only evoked oscillations of membrane potential by reversible inhibition of iberiotoxin-sensitive K(+) channels via phospholipase C, activated L-type Ca(2+) channels independently from its effect on membrane potential, and induced an inositol 1,4,5-trisphosphate receptor-dependent release of intracellular calcium in MIN6 cells. The combined effects evoked transient increases in [Ca(2+)](i) in these cells, which were sensitive to inhibitors of phospholipase C, protein kinase C, or L-type Ca(2+) channels. The latter agents also reduced toxin-induced insulin exocytosis. In conclusion, alpha-LTX induces signaling distinct from pore formation via full-length LPH and phospholipase C to regulate physiologically important K(+) and Ca(2+) channels as novel targets of its secretory activity.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Exocitose/fisiologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Receptores de Peptídeos/metabolismo , Venenos de Aranha , Animais , Cálcio/metabolismo , Linhagem Celular , Cricetinae , Vesículas Citoplasmáticas/metabolismo , Exocitose/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/citologia , Potenciais da Membrana/fisiologia , Camundongos , Técnicas de Patch-Clamp , Peptídeos/metabolismo , Ratos , Receptores de Peptídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venenos de Aranha/genética , Venenos de Aranha/metabolismo , Venenos de Aranha/farmacologia , Fosfolipases Tipo C/metabolismoRESUMO
Constitutive expression of the transcription factor Snail was previously shown to trigger complete epithelial-mesenchymal transition (EMT). The aim of this study was to determine whether inducible expression of Snail could modify epithelial properties without eliciting full mesenchymal conversion. For this purpose, we expressed mouse Snail (mSnail) cDNA in Madin-Darby canine kidney (MDCK) cells under the control of a doxycycline-repressible transactivator. Inducible expression of Snail did not result in overt EMT but induced a number of phenotypic alterations of MDCK cells, the most significant of which was the absence of fluid-filled blisterlike structures called "domes." To understand the mechanisms responsible for dome suppression, we assessed the effect of mSnail expression on epithelial barrier function. Although mSnail did not alter tight junction (TJ) organization and permeability to uncharged solutes, it markedly decreased transepithelial electrical resistance. In light of these findings, we evaluated the ability of MDCK cell monolayers to maintain ionic gradients and found that expression of mSnail selectively increases Na+ and Cl- permeability. Analysis of the expression of claudins, transmembrane proteins that regulate TJ ionic permeability, showed that mSnail induces a moderate decrease in claudin-2 and a substantial decrease in claudin-4 and -7 expression. Together, these results suggest that induction of mSnail selectively increases the ionic permeability of TJs by differentially modulating the expression of specific claudins.
Assuntos
Regulação da Expressão Gênica/fisiologia , Transporte de Íons/fisiologia , Junções Íntimas/metabolismo , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular , Cães , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Fatores de Transcrição da Família Snail , Tetraciclina/farmacologia , Fatores de Transcrição/biossínteseRESUMO
Vascular endothelial cells are thought to be the main source of plasma tissue-type plasminogen activator (t-PA) and von Willebrand factor (VWF). Previous studies have suggested that both t-PA and VWF are acutely released in response to the same stimuli, both in cultured endothelial cells and in vivo. However, the subcellular storage compartment in endothelial cells has not been definitively established. We tested the hypothesis that t-PA is localized in Weibel-Palade (WP) bodies, the specialized endothelial storage granules for VWF. In cultured human umbilical vein endothelial cells (HUVECs), t-PA was expressed in a minority of cells and found in WP bodies by immunofluorescence. After up-regulation of t-PA synthesis either by vascular endothelial growth factor (VEGF) and retinoic acid or by sodium butyrate, there was a large increase in t-PA-positive cells. t-PA was exclusively located to WP bodies, an observation confirmed by immunoelectron microscopy. Incubation with histamine, forskolin, and epinephrine induced the rapid, coordinate release of both t-PA and VWF, consistent with a single storage compartment. In native human skeletal muscle, t-PA was expressed in endothelial cells from arterioles and venules, along with VWF. The 2 proteins were found to be colocalized in WP bodies by immunoelectron microscopy. These data indicate that t-PA and VWF are colocalized in WP bodies, both in HUVECs and in vivo. Release of both t-PA and VWF from the same storage pool likely accounts for the coordinate increase in the plasma level of the 2 proteins in response to numerous stimuli, such as physical activity, beta-adrenergic agents, and 1-deamino-8d-arginine vasopressin (DDAVP) among others.