Search results for "Fluorescence"
showing 10 items of 2463 documents
Human macrophages simultaneously express membrane-C1q and Fc-receptors for IgG
2005
Membrane C1q (mC1q) of macrophages (MPhi) is a precursor of the IgG-binding serum protein C1q. Thus, mC1q potentially provides one of several Fcgamma binding sites of mature MPhi and we analyzed whether simultaneous expression occurs of established receptors for IgG, FcgammaRI, II, and III, and mC1q during in vitro differentiation of MPhi. Using flow cytometry, immunoprecipitation combined with Western blotting and Northern blot analysis mC1q was hardly detected in freshly isolated blood monocytes, but increasingly in developing monocyte-derived MPhi. Laser scanning fluorescence microscopy confirmed the membrane localization of mC1q. Two-color-staining flow cytometry experiments indicated t…
Identification of a Dynein Interacting Domain in the Papillomavirus Minor Capsid Protein L2
2006
ABSTRACT Papillomaviruses enter cells via endocytosis (H. C. Selinka et al., Virology 299:279-287, 2002). After egress from endosomes, the minor capsid protein L2 accompanies the viral DNA to the nucleus and subsequently to the subnuclear promyelocytic leukemia protein bodies (P. M. Day et al., Proc. Natl. Acad. Sci. USA 101:14252-14257, 2004), suggesting that this protein may be involved in the intracytoplasmic transport of the viral genome. We now demonstrate that the L2 protein is able to interact with the microtubule network via the motor protein dynein. L2 protein was found attached to microtubules after uncoating of incoming human papillomavirus pseudovirions. Based on immunofluoresce…
Dissection of human papillomavirus type 33 L2 domains involved in nuclear domains (ND) 10 homing and reorganization
2003
Abstract We have recently shown that the minor capsid protein L2 of human papillomavirus type 33 (HPV33) recruits the transcriptional repressor Daxx into nuclear domains (ND) 10 and causes the loss of the transcriptional activator Sp100 from these subnuclear structures (Florin et al., 2002b) . In order to dissect L2 domains involved in nuclear translocation, ND10 homing, loss of Sp100, and recruitment of Daxx, a detailed deletion mutagenesis of L2 was performed. Using immunofluorescence and green fluorescent protein fusions, we have identified two nuclear localization signals (NLS) in the central and C-terminal part of L2, respectively, homologous to previously identified NLS in HPV6B L2 (S…
A Protein-Interaction Array Inside a Living Cell
2013
Cell phenotype is determined by protein network states that are maintained by the dynamics of multiple protein interactions.1 Fluorescence microscopy approaches that measure protein interactions in individual cells, such as by Forster resonant energy transfer (FRET), are limited by the spectral separation of fluorophores and thus are most suitable to analyze a single protein interaction in a given cell. However, analysis of correlations between multiple protein interactions is required to uncover the interdependence of protein reactions in dynamic signal networks. Available protein-array technologies enable the parallel analysis of interacting proteins from cell extracts, however, they can …
Cilvēka aizkuņģa dziedzera organoīdu raksturošana: vizualizācijas un kvantificēšanas metožu pielāgošana
2022
Organoīdi ir pašorganizējošas 3D šūnu struktūras, kuru uzbūve un šūnu hierarhija atgādina to izcelsmes audus. Šī līdzība ļauj tos izmantot dažādos pētījumos. Darba mērķis ir raksturot aizkuņģa dziedzera organoīdus, izmantojot mikroskopēšanas metodes un pielietot attēlu kvantificēšanas programmas attēlu analīzei. Organoīdu attēlu analīze ir netriviāla, jo organoīdiem ir atšķirīgas formas un izmēri, bet šūnas tajos atrodas dažādās plaknēs. Salīdzinot veselu organoīdu iezīmēšanas metodi ar imūnfluorescenci uz kriotoma griezumiem metodi, kvalitatīvāku informāciju sniedz veselu organoīdu iezīmēšanas metode. Neskatoties uz to, abas pieejas apstiprina izveidoto aizkuņģa dziedzera kanāliņu fenotipu…
Self-Organization Pathways and Spatial Heterogeneity in Insulin Amyloid Fibril Formation
2009
At high temperature and low pH, the protein hormone insulin is highly prone to form amyloid fibrils, and for this reason it is widely used as a model system to study fibril formation mechanisms. In this work, we focused on insulin aggregation mechanisms occurring in HCl solutions (pH 1.6) at 60 degrees C. By means of in situ Thioflavin T (ThT) staining, the kinetics profiles were characterized as a function of the protein concentration, and two concurrent aggregation pathways were pointed out, being concentration dependent. In correspondence to these pathways, different morphologies of self-assembled protein molecules were detected by atomic force microscopy images also evidencing the prese…
Fluorescence labelling of organic acidic compounds with 4-bromomethyl-7-methoxycoumarin (Br-Mmc)
1977
4-Bromomethyl-7-methoxycoumarin (Br-Mmc) is introduced as a fluorescence marker for aromatic and heterocyclic acids. To investigate the applicability of this method on substances of different chemical classes, screening experiments with 110 compounds were carried out using a microrefluxer. Most aromatic and heterocyclic monocarboxylic acids gave Mmc-esters which are fluorescent on thin-layer plates, like the Mmc-esters of fatty acids, which have been previously investigated. Strong acids, alcohols, amides and most amines did not react, whereas certain cyclic amines such as piperidine gave strongly fluorescent derivatives. Mmc-phenyl ethers shows only weak fluorescence. A new standard proced…
Incorporation of membrane proteins into lipid surface monolayers: Characterization by fluorescence and electron microscopies
2007
The preparation of oriented protein samples is an attractive goal, e.g., to gain more detailed information from spectroscopic experiments. Our approach towards this aim was to prepare monolayers of phospholipids at the air-water interface and to incorporate the proteins into these ordered structures. Subsequently, we used the Langmuir-Boldgett (LB) transfer technique to obtain samples of oriented proteins on solid supports. — Incorporation was achieved by spreading the proteins from a detergent solution onto a prespread lipid monolayer on the water surface. We characterized successful incorporation by in situ fluoresence microscopy and by electron microscopy, and investigated the topology o…
Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria.
2003
A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising techniq…
The intraoral device of overlaid disk-holding splints as a new in situ oral biofilm model
2014
Objectives: To design a device that allows the formation of in situ oral biofilm with similar characteristics to those from the dental plaque, overcoming the limitations of previous devices. Study Design: The Intraoral Device of Overlaid Disk-holding Splints (IDODS) was designed and manufactured. To test its validity, five healthy adult volunteers wore them for two and four days allowing the biofilm to grow without any type of distortion. After each period, the thickness, vitality and structure of the formed biofilm were measured with a Confocal Laser Scanning Microscope (CLSM) in combination with a dual fluorescence solution. All volunteers filled out a Likert-type questionnaire to evaluat…